Advances in Environmental Research, Volume 13

ADVANCES IN ENVIRONMENTAL RESEARCH
ADVANCES IN ENVIRONMENTAL
RESEARCH.
VOLUME 13
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ADVANCES IN ENVIRONMENTAL
RESEARCH.
VOLUME 13
JUSTIN A. DANIELS
EDITOR
Nova Science Publishers, Inc.

New York
Copyright 2011 by Nova Science Publishers, Inc.

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CONTENTS
Preface
vii
Short Commentary
The Global Extent of Black C in Soils: Is It Everywhere?
Evelyn Krull, Johannes Lehmann, Jan Skjemstad,
Jeff Baldock and Leonie Spouncer
Research and Review Studies

Chapter 1
Chapter 2
Chapter 3
Chapter 4
Chapter 5
Current and Emerging Microbiology Issues of Potable Water in

Developed Countries
William J. Snelling, Catherine D. Carrillo, Colm J. Lowery
John E. Moore, John P. Pezacki, James S. G. Dooley
and Roy D. Sleator
Vermiculture Biotechnology: The Emerging Cost-Effective and
Sustainable Technology of the 21st Century for Waste and Land
Management to Safe and Sustainable Food Production
Rajiv K. Sinha, Sunil Herat, Gokul Bharambe, Swapnil Patil,
Uday Chaudhary, Priyadarshan Bapat, Ashish Brahambhatt,
David Ryan, Dalsukh Valani, Krunal Chauhan, R. K. Suhane
and P. K. Singh
11
41
Human Waste - A Potential Resource: Converting Trash into

Treasure by Embracing the 5 Rs Philosophy for Safe and
Sustainable Waste Management
Rajiv K. Sinha, Sunil Herat, Gokul Bharambe, Swapnil Patil,
Pryadarshan Bapat, Krunal Chauhan and Dalsukh Valani
111
Effective Removal of Low Concentrations of Arsenic and Lead and

the Monitoring of Molecular Removal Mechanism at Surface
Yasuo Izumi
173
On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead)

Loads in Mink Accompanying Parasitic Infection by the Giant
Kidney Worm (Dioctophyme Renale)
Glenn H. Parker and Liane Capodagli
187
vi
Contents
Chapter 6
Aerobically Biodegraded Fish-Meal Wastewater as a Fertilizer

Joong Kyun Kim and Geon Lee
219
Chapter 7
Equity of Access to Public Parks in Birmingham, England

Andrew P. Jones, Julii Brainard, Ian J. Bateman
and Andrew A. Lovett
237
Chapter 8
An Idea for Phenomenological Theory of Living Systems

Svetla E. Teodorova
257
Chapter 9
A New Trait of Gentoo Penguin: Possible Relation to Antarctica

Environmental State?
Roumiana Metcheva, Vladimir Bezrukov, Svetla E.Teodorova
and Yordan Yankov
Chapter 10
Assessing Population Viability of Focal Species Targets in the

Western Forest Complex, Thailand
Yongyut Trisurat and Anak Pattanavibool
Chapter 11
Protection of Riparian Landscapes in Israel

Tseira Maruani and Irit Amit-Cohen
Chapter 12
Hydraulic Characterization of Aquifer(s) and Pump Test Data

Analysis of Deep Aquifer in the Arsenic Affected Meghna River
Floodplain of Bangladesh
Anwar Zahid, M. Qumrul Hassan, Jeff L. Imes and David W. Clark
Chapter 13
Application of DNA Microarrays to Microbial Ecology Research:

History, Challenges, and Recent Developments
John J. Kelly
271
285
305
325
357
Chapter 14
Food Safety in India: Challenges and Opportunities

Wasim Aktar
Chapter 15
Impact of Pesticide Use in Indian Agriculture Their Benefits and

Hazards
Wasim Aktar
423
Ozone Decomposition by Catalysts and its Application in Water

Treatment: An Overview
J. Rivera-Utrilla, M. Snchez-Polo and J. D. Mndez-Daz
433
Chapter 16
Chapter 17
Index
Use of Microarrays to Study Environmentally Relevant Organisms:

A UK Perspective
Michael J. Allen, Andrew R. Cossins, Neil Hall, Mark Blaxter,
Terry Burke and Dawn Field
385
465
481
PREFACE
Short Communication - The latest projections of the Intergovernmental Panel on Climate
Change (IPCC) estimate a 3C increase in global temperatures within the next 100 years
(IPCC 4th Assessment Report, 2007), and global warming is seen as a major driver in
accelerating decomposition of soil organic matter, resulting in increased production of CO2
(e.g. Davidson and Janssens, 2006). The 2007 IPCC report recommended coupling models of
terrestrial biogeochemical and atmospheric and oceanic processes in order to improve general
circulation models and to recognize the quantitative value of soil organic carbon (SOC) in the
global carbon cycle.
Estimates of CO2 emissions from soil rely on predictions of the response of different
SOC pools to global warming and correct estimation of the size of these pools. In comparison
to the pool representing the most stable and biologically unreactive fraction, commonly
referred to as passive or inert organic carbon (IOC), the decomposition of labile C is expected
to be faster as a response to temperature increase. IOC is a fraction of the SOC pool that is not
readily available for microbial decomposition and has turnover times exceeding 100 years
(e.g. Krull et al., 2003).
Black C (BC) is usually considered the most abundant form of IOC and is defined as the
carbonaceous residue of incomplete combustion of biomass and fossil fuels (Schmidt and
Noack, 2000). BC is important to several biogeochemical processes; for example, BC
potentially modifies climate by acting as a potential carbon sink for greenhouse gases
(Kuhlbusch, 1998) and leads to increasing solar reflectance of the Earths atmosphere, but
also to a heating of the atmosphere (Crutzen and Andreae, 1990). BC production from fossil
fuel combustion contributes to aerosol C, decreasing surface albedo and solar radiation (IPCC
4th Assessment Report). Due to its condensed aromatic structure, BC has a low biochemical
reactivity. 14C ages of BC in soils vary between 1160 and 5040 years (e.g. Schmidt et al.
2002).
Chapter 1 - Water is vital for life; for commercial and industrial purposes and for leisure
activities in the daily lives of the worlds population. Diarrhoeal disease associated with
consumption of poor quality water is one of the leading causes of morbidity and mortality in
developing countries (especially in children <5 years old). In developed countries, whilst
potable water is not a leading cause of death, it can still pose a significant health risk. Water
quality is assessed using a number of criteria, e.g. microbial load and nutrient content which
affects microbial survival, as well as aesthetic factors such as odor. In water systems, the
presence of disinfectant, low temperatures, flow regimes and low organic carbon sources do
not appear to be conducive to microbial persistence. However, frequently this is not the case.
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Justin A. Daniels
A variety of human pathogens can be transmitted orally by water and in the developed world
water quality regulations require that potable water contains no microbial pathogens.
Chlorine dioxide is a safe, relatively effective biocide that has been widely used for
drinking water disinfection for 40 years. Providing the water is of low turbidity, standard
chlorination procedures are sufficient to prevent the spread of planktonic bacteria along water
mains. However, despite this, bacterial contamination of water distribution systems is well
documented, with growth typically occuring on surfaces, including pipe walls and sediments.
Rivers, streams and lakes are all important sources of drinking water and are used routinely
for recreational purposes. However, due to fouling by farm and wild animals, these sources
can be contaminated with microbes, e.g. chlorine resistant Cryptosporidium oocysts, no
matter how pristine the source or well maintained the water delivery system. The high
incidence of Cryptosporidium in surface water sources underlines the need for frequent
monitoring of the parasite in drinking water. The use of coliforms as indicator organisms,
although considered relevant to most cases, is not without limitations, and is thus not a
completely reliable parameter of water safety, e.g. Campylobacter contamination cannot be
accurately predicted by coliform enumeration. Furthermore, the presence of biofilms and
bacterial interactions with protozoa in water facilitate increased resistance to antimicrobial
agents and procedures such as disinfectants and heating, e.g. Legionnaires disease caused by
Legionella pneumophila.
The high cost of waterborne disease outbreaks should be considered in decisions
regarding water utility improvement and treatment plant construction. The control of human
illnesses associated with water would be aided by a greater understanding of the interactions
between water-borne protozoa and bacterial pathogens, which until relatively recently have
been overlooked.
Chapter 2 - A revolution is unfolding in vermiculture studies (rearing of useful
earthworms species) for multiple uses in environmental management and sustainable
development. (Martin, 1976; Satchell, 1983; Bhawalkar and Bhawalkar, 1994; Sinha et.al
2002; Fraser-Quick, 2002). Vermiculture biotechnology promises to provide cheaper
solutions to following environmental and social problems plaguing the civilization
Management of municipal and industrial solid wastes (organics) by biodegradation and
stabilization and converting them into useful resource (vermicompost) THE VERMICOMPOSTING TECHNOLOGY (VCT) ;
Treatment of municipal and some industrial (food processing industries) wastewater,
purification and disinfection - THE VERMI-FILTRATION TECHNOLOGY (VFT);
Removing chemical contaminations from soils (land decontamination) and reducing soil
salinity while improving soil properties- THE VERMI-REMEDIATION TECHNOLOGY
(VRT);
Restoring and improving soil fertility and boosting crop productivity by worm activity
and use of vermicompost (miracle growth promoter) while eliminating the use of destructive
agro-chemicals - THE VERMI-AGRO-PRODUCTION TECHNOLOGY (VAPT);
Vermi-composting, vermi-filtration, vermi-remediation and vermi-agro-production are
self-promoted, self-regulated, self-improved and self-enhanced, low or no-energy requiring
zero-waste technology, easy to construct, operate and maintain. It excels all bio-conversion,
bio-degradation and bio-production technologies by the fact that it can utilize organics that
otherwise cannot be utilized by others. It excels all bio-treatment technologies because it
achieves greater utilization than the rate of destruction achieved by other technologies. It
Preface
ix
involves about 100-1000 times higher value addition than other biological technologies.
(Appeholf, 1997).
About 4,400 different species of earthworms have been identified, and quite a few of
them are versatile waste eaters and bio-degraders and several of them are bio-accumulators
and bio-transformers of toxic chemicals from contaminated soils rendering the land fit for
productive uses.
Chapter 3 - Waste is being generated by the human societies since ancient times.
Ironically waste was not a problem for the environment when men were primitive and
uncivilized. Waste is a problem of the modern civilized society. Materials used and waste
generated by the traditional societies were little and simple while those by the modern
human societies are large and complex. With modernization in development drastic changes
came in our consumer habits and life-style and in every activity like education, recreation,
traveling, feeding, clothing and housing we are generating lots of wastes. The world today
generate about 2.4 billion tones of solid waste every year in which the Western World alone
contributes about 620 million tones / year.
Discarded products arising from all human activities (cultural and developmental) and
those arising from the plants and animals, that are normally solid or semi-solid at room
temperature are termed as solid wastes. Municipal solid waste (MSW) is a term used to
represent all the garbage created by households, commercial sites (restaurants, grocery and
other stores, offices and public places etc.) and institutions (educational establishments,
museums etc.). This also includes wastes from small and medium sized cottage industries.
We are facing the escalating economic and environmental cost of dealing with current
and future generation of mounting municipal solid wastes (MSW), specially the technological
(developmental) wastes which comprise the hazardous industrial wastes, and also the health
cost to the people suffering from it. Developmental wastes poses serious risk to human health
and environment at every stage from generation to transportation and use, and during
treatment for safe disposal. Another serious cause of concern is the emission of greenhouse
gases methane and nitrous oxides resulting from the disposal of MSW either in the landfills or
from their management by composting.
Dealing with solid household waste in more sustainable ways involves changes not only
to everyday personal habits, consumerist attitudes and practices, but also to the systems of
waste management by local government and local industry and the retailers.
This chapter reviews the causes and consequences of escalating human waste, the
increasing complexity of the waste generated, and the policies and strategies of safe waste
management. It also provides food for thought for future policy decisions that government
of nations may have to take to reduce waste and divert them from ending up in the landfills,
drawing experiences from both developed nation (Australia) and a developing nation (India).
Chapter 4 - New sorbents were investigated for the effective removal of low
concentrations of arsenic and lead to adjust to modern worldwide environmental regulation of
drinking water (10 ppb). Mesoporous Fe oxyhydroxide synthesized using dodecylsulfate was
most effective for initial 200 ppb of As removal, especially for more hazardous arsenite for
human's health. Hydrotalcite-like layered double hydroxide consisted of Fe and Mg was most
effective for initial 55 ppb of Pb removal.
The molecular removal mechanism is critical for environmental problem and protection
because valence state change upon removal of e.g. As on sorbent surface from environmental
water may detoxify arsenite to less harmful arsenate. It is also because the evaluation of
Justin A. Daniels
desorption rates is important to judge the efficiency of reuse of sorbents. To monitor the low
concentrations of arsenic and lead on sorbent surface, selective X-ray absorption fine
structure (XAFS) spectroscopy was applied for arsenic and lead species adsorbed, free from
the interference of high concentrations of Fe sites contained in the sorbents and to selectively
detect toxic AsIII among the mixture of AsIII and AsV species in sample.
Oxidative adsorption mechanism was demonstrated on Fe-montmorillonite and
mesoporous Fe oxyhydroxide starting from AsIII species in aqueous solution to AsV by
making complex with unsaturated FeOx(OH)y sites at sorbent surface. Coagulation
mechanism was demonstrated on double hydroxide consisted of Fe and Mg from the initial 1
ppm of Pb2+ aqueous solution whereas the mechanism was simple ion exchange reaction
when the initial Pb2+ concentrations were as low as 100 ppb.
Chapter 5 - Patterns of metal uptake and accumulation in mink living under conditions of
environmental pollution and simultaneously inflicted with the invasive giant kidney worm
(Dioctophyme renale) parasite have not been examined, nor is the combined effect of these
dual insults on the health and physical condition of the animal known. Using animals
collected within the influence of the long-active ore-smelters at Sudbury, Ontario, an
examination was made of toxic metal (Cd, Ni and Pb) levels and their tissue distributions
within adult male mink bearing different intensities of parasite infection. Higher metal
burdens were indicated within infected specimens than those uninfected. Combined renal and
hepatic nickel and lead burdens were highest for mink with multiple worm infections,
although only lead accumulations reached statistical significance. Cadmium accumulated to
the greatest extent in the hypertrophied left kidney and liver, whereas nickel and lead were
deposited more readily in the bony spicule of the parasitized right kidney cyst. The relative
distribution of cadmium among renal, hepatic and renal cyst tissues (cast, spicule, worms)
remained unchanged subsequent to D. renale infection, while the proportions of nickel and
lead deposited in hepatic tissue were reduced. Metal burdens in female D. renale were threefold higher than those of male worms, with the difference being attributable to the
substantially greater size of the females. Canonical Correlation Analyses of condition
measures and body metal burdens failed to indicate a direct relationship between infection
intensity and body fat deposits but did confirm a positive association between metal loads and
increased fat levels, along with enhanced gonad weights, neck circumference and reduced
spleen weights. Such associations may be productive aspects for future investigation into the
combined effects of increased metal loads and parasitic infection on the host system.
Chapter 6 - Reutilization of fish-meal wastewater (FMW) as a fertilizer was attempted,
and aerobic biodegradation of the FMW were successfully achieved by microbial consortium
in a 1-ton bioreactor. During the large-scale biodegradation of FMW, the level of DO was
maintained over 1.25 mgl-1, and a strong unpleasant smell remarkably disappeared in the
end. Although the level of total amino acids and the concentrations of N, P and K in the
biodegraded FMW were relatively lower than those in two commercial fertilizers, the
concentrations of noxious components in the biodegraded FMW were much lower than the
standard concentrations. The phytotoxicity of the biodegraded FMW was almost equal to that
of the commercial fertilizers. The fastest growth in hydroponic cultures of red bean and
barley was achieved at 100-fold and 500-fold dilution, respectively, the growth of which was
comparable to those of 1,000-fold diluted commercial-fertilizers. From all above results, it
was concluded that a large-scale biodegradation of FMW was successful and the properties of
Preface
xi
FMW were acceptable. This is the first study to demonstrate aerobic production of liquidfertilizer from FMW.
Chapter 7 - Provision of public parks has long been advocated as an equalising measure
between different elements of society. This study assesses equity of park provision for
different ethnic and income-status populations in the urban area of Birmingham in central
England. Parks in Birmingham were categorized into a two group typology of green areas
suited for more solitary and passive activities (amenity parks) or open spaces designed more
for informal sports or other physical and group activities (recreational parks). Using a
geographical information system, measures of access to these green spaces were computed
for populations of different ethnicities and levels of material deprivation, derived from data
from the 2001 UK Census and the 2004 Index of Multiple Deprivation. Distance-weighted
access scores were calculated and compared for five population groups ranked by relative
deprivation, and for five ethnic groups; Bangladeshis, blacks, Indians, Pakistanis and whites.
Statistical analysis found that there were strong disparities in access with respect to
deprivation whereby the most income-deprived groups were also the most deprived with
regard to access to public parks. There was little evidence of unequal access between ethnic
groups. The implications of these findings are discussed.
Chapter 8 - An idea for developing of new science field, biodynamics, as biophysical
macroscopic theory is propounded. The functioning of living organism as an organized
entirety is the main specificity of the life. Hence it is quite reasonable to describe behaviour
of biological systems in terms of own theoretical basis. In this article a new state variable
vitality as integral characteristic of biological object and measure unit bion are stated. A
quantity biological energy is introduced as energy form related to biological selfregulation.
Quantity synergy is suggested as measure of selfregulation quality. Biological principle for
maximum synergy in healthy living systems is stated. On the basis of variational principle an
equation describing recovery process of a biological object after disturbance is obtained. The
quantity optimal vitality, related to homeostasis, decreases in lifespan scale and its evolution
is described by ordinary differential equation. The potential lifespan maximum of several
species at different life conditions may be calculated at different parameters of the equation.
A wide range of environmental influences on the living organism could be promptly
and easily assessed in the terms of the biodynamics approach. Such an approach could be
used for a simple estimation of a patients health status.
Chapter 9 - Some morphological traits of Antarctic animals could be considered in the
context of a trend to more direct relation between environmental conditions and possible
adaptive mechanisms of animals organism. Penguins are excellent object for biomonitoring.
Here a preliminarily report is presented regarding a new trait, a spot-like coloration (yellow
spot) of the bill, observed on the upper mandible of Gentoo penguin (subspecies Pygoscelis
papua ellsworthii). The spot varied in size and colour. It was recorded among chicks over two
months old and adult birds (normal and molting). The trait had no significant relationship to
the animal' s sex. Among all inspected females 31% and among all inspected males 27%
exhibited beak spot. Three breeding colonies were investigated at three different geographical
locations at the Antarctic Peninsula Livingston Island, South Shetlands (6238 S), Wiencke
Island (64o52 S), and Petermann Island (6510 S). Yellow spot was found with different
frequencies at these three locations: 20%, 36%, and 30%, respectively. All spotted penguins
from the three colonies were 32% when compared to all non-spotted. The possible reasons for
the spot-like coloration are discussed. The trait could be a phenotypic characteristic. It could
xii
Justin A. Daniels
play some role in the mate choice. A possible connection to carotenoid pigments content does
not be eliminated in the context of Gentoo diet. A probable cause for the beak spot
appearance seems to be the increasing of ozone depletion above Antarctica. The ultraviolet
radiation enhances free radical production. Other studies reported an increase of the flux of
transsulfuration pathway as a defense reaction, resulting in elevated level of cysteine. When
cysteine concentration is raised, an attaching of cysteine to melanin synthesis pathway occurs
and this results in formation of reddish pigments.
Chapter 10 - The Western Forest Complex (WEFCOM) in Thailand covers
approximately 19,000 km2. This protected area complex comprises 11 national parks and 6
wildlife sanctuaries. During 1999-2004, the Danish Government provided financial support to
the Royal Forest Department to manage this forest complex through the ecosystem
management approach. The WEFCOM Project employed rapid ecological assessment (REA)
to determine the current distribution statuses of wildlife species, develop a Geographic
Information System (GIS), and define habitat uses of wildlife. This paper is based upon the
achievements of the WEFCOM Project. It aims to define suitable habitats of selected key
wildlife species in the WEFCOM and to assess the current and desired statuses under a
population viability estimate for those species. The focal wildlife species were sambar
(Cervus unicolor), gaur (Bos gaurus), banteng (Bos javanicus), Asian elephant (Elephas
maximus), and tiger (Panthera tigris. The authors used logistic multiple regression to
determine habitat uses of wildlife and employed minimum dynamic area and landscape
matrix surrounding suitable habitats as criteria to assess population viability. The results
indicate the current suitable habitat mainly remains in Huai Kha Khaeng and Thung Yai
wildlife sanctuaries. In addition, the current viability condition is good for sambar, fair for
gaur, elephant and tiger; and poor for banteng. However, landscape matrices outside the
suitable habitats for all species range from moderate to high connection of native vegetation.
If the project aims to upgrade the viabilities to the next level in the next 10 years, park rangers
and multi-stakeholders have to increase the amount of suitable habitats for all species from
12,630 km2 or 67% of the WEFCOM to 16,750 km2 or 89%. By doing this, the number of
suitable patches would significantly decrease and the mean patch size would increase
substantially, thereby indicating less fragmentation.
Chapter 11 - Riparian landscapes are natural habitats of unique ecological and scenic
values, which are highly sensitive to human intervention and impact. Yet, due to their
qualities, and especially the presence of water, they are also usually attractive for recreation
purposes. This is more so in arid and semi-arid zones like Israel. Nevertheless, in the past, the
importance of riparian landscapes in Israel did not receive adequate attention in policy and
planning. As a result, over the years they were exposed to various negative impacts, including
pollution by industrial and agricultural effluents, exploitation of water for agricultural and
other purposes, and land use conflicts. Although in recent years with the growing awareness
of their ecological and recreational potential, considerable efforts are being invested in the
rehabilitation of deteriorated riparian landscapes, their protection is still deficient.
This chapter reviews and examines policy tools used for the protection of riparian
landscapes in Israel, based mainly on regulations, reports and existing literature. It concludes
by offering some lessons for policy-making in general and suggestions for improving the
protection of riparian landscapes in Israel in particular.
Chapter 12 - To determine the hydraulic characteristics of aquifers and development
potential of deep aquifer for sustainable long-term use, study was undertaken by assessing
Preface
xiii
water levels of different aquifer formations and conducting pumping test in deep aquifer
under Meghna floodplain area of southeastern Bangladesh. Because of arsenic contamination
in shallow groundwater, characterization of deeper aquifers and assess their hydraulic
connectivity is now an important issue in Bangladesh. Study shows that groundwater
pumping for irrigation and other uses cause large seasonal water level fluctuations that is
between 2 and 4.5m, 6.5 and 11m and 6.5m in the shallow, main and deep aquifers,
respectively. The trend of groundwater level fluctuations supports the hydraulic connectivity
of these aquifers. Aquitards separating aquifers are not continuous regionally. This implies
that uncontrolled development of deep aquifers may cause leakage of arsenic from
contaminated shallow depths to aquifers below. Water levels dropping below sea level for
over withdrawal may also cause saline water intrusion as well. However, during the constantdischarge pumping test for deep aquifer, water levels in observation wells open to the shallow
and main aquifers showed no noticeable effect from pumping i.e. under conditions of
moderate groundwater use for public supply, arsenic-rich groundwater in the shallow aquifer
are not likely to be drawn into the deep aquifer. The transmissivity values of the aquifer is
generally favorable for groundwater development and ranged from about 1,070 m2/day to
2,948 m2/day at a distance of 44 m from the pumped well. Transmissivity ranged between
1,570 m2/day and 2,956 m2/day at a distance of 120m. Transmissivity was calculated as
2,385 m2/day using recovery data. Estimated storage coefficient values ranged between
0.0000375 and 0.00268, indicates that the aquifer is confined to leaky-confined or semiconfined in nature.
Chapter 13 - During the last three decades, molecular methods have dramatically
expanded the author view of microbial diversity in natural and engineered systems. A variety
of molecular approaches, including both PCR-based and hybridization-based techniques, have
been applied extensively to the analysis of complex microbial communities and have yielded
new insights. However, the majority of molecular methods that are widely used in microbial
ecology are limited in their ability to encompass the incredible diversity of microbial
communities. DNA microarrays, which were first introduced in the early 1990s, are one of
the fastest growing technologies in biology, and they offer tremendous potential for microbial
ecologists. DNA microarrays consist of nucleic acids spotted within a very small area on
some solid support, and they enable the immobilization and simultaneous hybridization of
hundreds of thousands of nucleic acids. This represents a dramatically higher degree of
multiplexing than is possible with other widely used technologies. In addition, microarrays
offer the advantages of increased speed of detection, low cost, and the potential for
automation. Microarray technology has been used extensively for measuring gene expression
in a wide variety of organisms, including human cells, plants, yeast, and bacteria, but its
application to microbial ecology has been more limited. There are significant challenges to
the use of microarrays in microbial ecology studies, including optimization of specificity and
sensitivity and quantification of targets. However, in recent years several research groups
have made significant progress in overcoming these challenges, and microarrays are
beginning to be applied more frequently to microbial ecology studies in a variety of systems
including terrestrial soils, wetland sediments, and freshwater and marine ecosystems. This
article will provide a brief history of the use of molecular methods in microbial ecology, and
will then review the development of microarray technology, the challenges that exist for
application of microarrays to microbial ecology, the available strategies for overcoming these
challenges, and some recent applications of microarrays to studies in microbial ecology.
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Justin A. Daniels
Chapter 14 - Rising incomes and urbanization, an expanding domestic consumer base

concerned about food quality and safety, and rapidly growing agricultural exports have been
important drivers for the increased attention to food safety in India. But the development of
effective food safety systems is hampered by a number of factors, including: restrictive
government marketing regulations, weak policy and regulatory framework for food safety,
inadequate enforcement of existing standards, a multiplicity of government agencies
involved, weak market infrastructure and agricultural support services. The small farm
structure further limits farmer capacity to meet increasing domestic and export food safety
and SPS requirements. Addressing food safety concerns in India will require adoption of
appropriate legislation, strengthening capacity to enforce rules, promoting adoption of good
agricultural, manufacturing and hygiene practices, greater collective action, and some
targeted investments. Implementing these actions will require joint efforts by the government
and the private sector.
Developing countries are paying increased attention to food safety, because of growing
recognition of its potential impact on public health, food security, and trade competitiveness.
Increasing scientific understanding of the public health consequences of unsafe food,
amplified by the rapid global transmission of information regarding the public health threats
associated with food-borne and zoonotic diseases (e.g. E. coli and salmonella, bovinespongiform encephalopathy (BSE), severe acute respiratory syndrome (SARs) and H5N1
avian flu) through various forms of media and the internet has heightened consumer
awareness about food safety risks to new levels globally (Lindsay 1997, Unnevehr 2003,
Buzby and Unnevehr 2003, Kafersteing 2003, Ewen et al. 2006, Bramhmbatt 2005).
Increased understanding of the impact of mycotoxins, which can contaminate dietary staples
such wheat, maize, barley and peanuts, has further raised food security and public health
concerns in many developing countries (Dohlman 2003, Bhat and Vasanthi 2003, Unnevehr
2003).
As developing countries seek to expand agricultural exports especially to OECD
countries, many are receiving a wake-up call on the challenges of meeting both government
and private sanitary and phyto-sanitary (SPS) standards in export markets (Otsuki et al. 2001,
Henson 2003, Unnevehr 2003, World Bank 2005a). Private standards or supplier protocols
have grown in prominence over the past decade as a means to further ensure compliance with
official regulations, to fill perceived gaps in such regulations, and/or to facilitate the
differentiation of company or industry products from those of competitors. Trends in private
standards increasingly tend to blend food safety and quality management concerns (i.e. the
recent creation of ISO 22000), or to have protocols which combine food safety,
environmental, and social (child labor, labor conditions, animal welfare) parameters (Willems
et al. 2005, World Bank 2005). At the same time, increasing globalization of trade introduces
greater risks of cross-border transfer of food-borne illnesses. Recent cases of disease episodes
in the United States resulting from imported food produce, such as cyclospora from
raspberries, hepatitis A from strawberries and salmonella from cantaloupe (Calvin 2003),
illustrate to developing countries the potential food safety challenges that can arise in a more
globalized market.
Weaknesses in food safety systems can have a high cost to society and the global
economy. The World Health Organization (WHO) estimates that 2.2 million people
worldwide die from diarrheal diseases caused by a host of bacterial, viral and parasitic
organisms, which are spread by contaminated water (WHO 2006a). In India, it is estimated
Preface
xv
that 20% of deaths among children under five are caused by diarrheal disease (WHO 2006b).
The SARs outbreak in 2003 in East Asia is estimated to have caused an immediate economic
loss of about 2% of the Regions GDP in the second quarter of that year, even though only
800 people died from the disease (Brahmbatt 2005). The Lowy Institute for International
Policy (2006) estimates that a mild global outbreak of the avian flu can cost the world 1.4
million lives and close to 0.8% of GDP (US$330 billion) in lost economic output. At the
same time, country reactions to protect its citizens from food safety risks can also have large
consequences for exporting countries. Otsuki et al (2001) examined the projected impact of
the EUs new harmonized aflatoxin standard on the value of trade flows to 15 European
countries from 9 African countries and found that it could decrease African exports by 64%
(US$670 million).
Food safety concerns are getting widespread attention in India. The countrys rural
development strategy, for which a key element is the promotion of increased agricultural
exports as a means to foster rural growth and poverty reduction, is coming up against
tightening food safety and SPS standards in prospective markets (World Bank 2006a, 2006b).
From a domestic perspective, the large national market of 1.2 billion people is undergoing
rapid change. Increasing incomes, a growing middle class, increased urbanization and
literacy, and a population highly tuned to international trends fueled by the information
technology boom are creating a large consumer base giving increasing value to food quality
and safety. Improving food safety systems, to meet domestic and export requirements,
however, face a number of policy, regulatory, infrastructural and institutional obstacles.
Chapter 15 - The term pesticide covers a wide range of compounds including
insecticides, fungicides, herbicides, rodenticides, molluscicides, nematicides, plant growth
regulators and others. Among these, organochlorine (OC) insecticides, used successfully in
controlling a number of diseases, such as malaria and typhus, were banned or restricted after
the 1960s in most of the technologically advanced countries. The introduction of other
synthetic insecticides organophosphate (OP) insecticides in the 1960s, carbamates in 1970s
and pyrethroids in 1980s and the introduction of herbicides and fungicides in 1970s - 1980s
contributed greatly in pest control and agricultural output. Ideally a pesticide must be lethal to
the targetted pests, but not to non-target species, including man. Unfortunately, this is not, so
the controversy of use and abuse of pesticides has surfaced. The rampant use of these
chemicals, under the adage, if little is good, a lot more will be better has played havoc with
human and other life forms.
Chapter 16 - Ozone has recently received much attention in water treatment technology
for its high oxidation and disinfection potential. The use of ozone brings several benefits but
has a few disadvantages that limit its application in water treatment, including: i) low
solubility and stability in water, ii) low reactivity with some organic compounds and iii)
failure to produce a complete transformation of organic compounds into CO2, generating
degradation by-products that sometimes have higher toxicity than the raw micropollutant. To
improve the effectiveness of ozonation process efficiency, advanced oxidation processes
(AOPs) have recently been developed (O3/H2O2, O3/UV, O3/catalysts). AOPs are based on
ozone decomposition into hydroxyl radicals (HO), which are high powerful oxidants. This
chapter offers an overview of AOPs, focusing on the role of solid catalysts in enhancing
ozone transformation into HO radicals. Catalytic ozonation is a new way to remove organic
micropollutants from drinking water and wastewater. The application of several homo- and
heterogeneous ozonation catalysts is reviewed, describing their activity and identifying the
xvi
Justin A. Daniels
parameters that influence the effectiveness of catalytic systems. Although catalytic ozonation
has largely been limited to laboratory applications, the good results obtained have led to
investigations now under way by researchers worldwide. It is therefore timely to provide a
summary of achievements to date in the use of solid materials to enhance ozone
transformation into HO radicals.
Chapter 17 - Historically, the majority of microarray work has been restricted to welldefined model organisms. This was primarily due to the limited availability of genomic or
transcriptomic sequence data and the then high cost involved in developing microarrays.
However, recent technological developments have greatly enhanced the speed of generating
the underpinning sequence data for non-model species, and have opened up more costeffective approaches for microarray production to make them far more affordable for
researchers at the lower end of the budget range. These developments have been seized upon
by the environmental genomics community within the UK. The creation of a network of
closely integrated facilities for sequencing, microarray printing and bioinformatics has
opened the gateway for the study of environmentally relevant organisms. Here, the authors
describe the infrastructure for microarray development within the UK, and the diverse
applications for which they are currently being used.
Versions of these chapters were also published in Environmental Research Journal,
Volume 3, Number 1, published by Nova Science Publishers, Inc. They were submitted for
appropriate modifications in an effort to encourage wider dissemination of research.
SHORT COMMENTARY
In: Advances in Environmental Research, Volume 13

Editor: Justin A. Daniels
ISSN: 2158-5717
2011 Nova Science Publishers, Inc.
THE GLOBAL EXTENT OF BLACK C IN SOILS:

IS IT EVERYWHERE?
Evelyn Krull, Johannes Lehmann, Jan Skjemstad,
Jeff Baldock and Leonie Spouncer
THE ROLE OF BLACK CARBON IN GLOBAL CLIMATE MODELS
The latest projections of the Intergovernmental Panel on Climate Change (IPCC) estimate
a 3C increase in global temperatures within the next 100 years (IPCC 4 th Assessment Report,
2007), and global warming is seen as a major driver in accelerating decomposition of soil
organic matter, resulting in increased production of CO2 (e.g. Davidson and Janssens, 2006).
The 2007 IPCC report recommended coupling models of terrestrial biogeochemical and
atmospheric and oceanic processes in order to improve general circulation models and to
recognize the quantitative value of soil organic carbon (SOC) in the global carbon cycle.
Estimates of CO2 emissions from soil rely on predictions of the response of different
SOC pools to global warming and correct estimation of the size of these pools. In comparison
to the pool representing the most stable and biologically unreactive fraction, commonly
referred to as passive or inert organic carbon (IOC), the decomposition of labile C is expected
to be faster as a response to temperature increase. IOC is a fraction of the SOC pool that is not
readily available for microbial decomposition and has turnover times exceeding 100 years
(e.g. Krull et al., 2003).
Black C (BC) is usually considered the most abundant form of IOC and is defined as the
carbonaceous residue of incomplete combustion of biomass and fossil fuels (Schmidt and
Noack, 2000). BC is important to several biogeochemical processes; for example, BC
potentially modifies climate by acting as a potential carbon sink for greenhouse gases
(Kuhlbusch, 1998) and leads to increasing solar reflectance of the Earths atmosphere, but
also to a heating of the atmosphere (Crutzen and Andreae, 1990). BC production from fossil
fuel combustion contributes to aerosol C, decreasing surface albedo and solar radiation (IPCC
4th Assessment Report). Due to its condensed aromatic structure, BC has a low biochemical
reactivity. 14C ages of BC in soils vary between 1160 and 5040 years (e.g. Schmidt et al.
2002).
Evelyn Krull, Johannes Lehmann, Jan Skjemstad et al.
Figure 1. Distribution of soilsused for the prediction of BC in surface soils and global NPP.
Figure 2. Distribution of BC%SOC and %SOC in surfaces soils across latitudes.
Baldock (2007) reported that BC constitutes up to 60% of SOC, indicating that BC can
make up a significant part of SOC affecting the response of SOC to temperature changes and
the overall turnover time of SOC. Thus, the effect of such a large and unreactive C pool must
be effectively integrated into global C cycle and climate models. However, the latest IPCC
report regards BC only as an aerosol and not as part of SOC, despite an earlier
recommendation to the IPCC to better gauge the influence of BC on the global carbon
cycle as the result would ensure a more accurate global black C budget and a better
understanding of the role of BC as a potential sink in the global C cycle. (2006 IPCC
guidelines for national greenhouse gas inventories; appendix 1). The lack of incorporating
The Global Extent of Black C in Soils: Is it Everywhere?
soil BC in global climate change assessments may be largely due to the lack of global
databases on BC in soils and sediments and the understanding of factors and processes that
may influence BC contents in soils (climate, soil texture, primary productivity and fire
abundance).
In order to provide reliable projections of future CO2 emissions from soils, due to global
warming, it is important to consider the global distribution of soil BC. Through an initial
assessment of the World Soil Archive (http://library.wur.nl/isric/) we demonstrate the
variability and trends of global soil BC distribution between different climates and soil types
and discuss the implications of this chemically recalcitrant form of C on the global C cycle.
By doing this, we also demonstrate that current methods exist to routinely analyse BC and in
the future to develop a global BC map.
ARE THERE ROUTINE METHODS TO GENERATE A GLOBAL BC MAP?

We have developed a rapid fourier-transform infrared-based technique that provides
predictive capability for major soil properties, including BC content. BC was quantified by a
novel mid-infrared (MIR) method coupled with partial least squares (PLS) (Janik et al., 2007)
that allowed rapid analysis of a large number of samples, not feasible by other published
methods (Hammes et al., 2007). This method was originally developed and calibrated on
Australian soils (Janik et al., 2007); however, subsequently, we were able to verify that the
predictive capacity of the MIR technique for soils from other parts of the world was robust in
most cases.
BC IN WORLD SOILS: WHAT DRIVES BC PRODUCTION

AND DECOMPOSITION?
Our initial assessment took advantage of the large collection of soils that constitute
part of the ISRIC world soil information database (http://library.wur.nl/isric/). We obtained
over 400 samples that span all major climate zones and soil types across most parts of the
world (Fig. 1). Utilising the MIR/PLS technique, we developed a database that illustrates for
the first time the predicted proportion of BC in this large soils dataset. We hope that this
initial dataset may become an incentive for the rigorous establishment of a global BC map,
data from which may then be incorporated into future IPCC reports and C cycle models.
Figure 2 shows the proportion of BC (as % of total SOC: BC%SOC) as well as total
SOC contents across northern and southern hemispheres. These data show that BC in surface
soils (A and A/B horizons) is ubiquitous in large parts of the world and occurs in the majority
of the sampled locations. The variability was large, resulting in BC%SOC varying from over
50% to almost 0%. The soils richest in BC occur in latitudes of 20-30 in both hemispheres,
situated mostly in central and South America and southern parts of Africa. These areas
correspond to tropical climates with a pronounced dry season in winter (Aw in the Koeppen
classification). In higher latitudes in the northern hemisphere (60-70), BC contents were also
high and were associated with an increase in total SOC (Fig. 2). These areas are classified as
Evelyn Krull, Johannes Lehmann, Jan Skjemstad et al.
humid-temperate, constantly moist climates (Cf in Koeppen classification). Higher SOC

content at high latitudes results from cool climates and low decomposition rates, which
promotes accumulation of organic matter.
Despite the high spatial variability of BC%SOC, soils with a high proportion of BC fell
largely in the soil group of Vertisols. Vertisols occur worldwide in seasonal climate zones and
in lower relief positions (Richardson and Vepraskas, 2000). Such areas of deposition would
favour accumulation of BC through erosion and deposition. Another factor contributing to the
high amounts of BC%SOC in Vertisols could be the comparably high proportion of clay
(known to stabilize soil organic matter) and the type of clay (smectitic). The MIR-predicted
data showed that Vertisols were amongst the soil types with the highest clay contents
(average 44%). High clay content may promote retention of fine BC. However, clay alone is
not a determining factor for high BC%SOC. Oxisols and Ultisols, having the highest clay
content (from MIR prediction) of all analysed soil types (45%), had the lowest BC %SOC
values. Oxisols, and to a lesser degree Ultisols, are mostly found in high-rainfall tropical
climates, lacking seasonal rainfall (Af in Koeppen classification) and having a lower fire
activity. While fire activity and BC content are both high in the southern hemisphere this
relationship is not consistent and does not concur with findings in parts of the northern
hemisphere where fire activity is lower, yet BC%SOC is still high (Carmona-Moreno et al.,
2005).
Thus, a combination of factors, such as climate (the necessity of a pronounced dry
season to ensure fire occurrence), position in the landscape (areas of accumulation) and
mineralogy (high amount of expansive clays) may be instrumental in promoting the formation
and/or retention of BC%SOC in soils. As illustrated in Figure 1, net primary productivity alone
did not appear to be a significant factor globally as it is highest in equatorial regions where
BC%SOC was lowest.
While we show here that BC contents in surface soils are often significant, BC can
also accumulate deeper in the soil. In addition, soil erosion and transport of BC may result in
significant accumulation of BC in rivers, estuaries and off-shore sediments (Krull et al.,
2006).Thus, the inclusion of BC in global climate models will require a thorough assessment
of BC contents not only in surface but in deeper soil horizons as well as aquatic sediments.
The analyses conducted in this study indicate that methods exist to accomplish BC
measurements for large areas. However, the high variability of the data indicates that broad
empirical measurements and extrapolation over large areas are not sufficient for the aim of
producing a global BC map. Thus, the next steps in a comprehensive assessment of global BC
stocks and distribution have to include a detailed and consistent sampling format as well as a
thorough assessment of the processes that control sources and sinks of BC.
ACKNOWLEDGMENTS
We thank Janine McGowan for assistance in the analyses of the samples and both her and
Ryan Farquharson for review of an earlier version of the manuscript. We thank CSIRO Land
and Water for supporting the analyses of this work and ISRIC for access to samples from the
World Soils Database.
The Global Extent of Black C in Soils: Is it Everywhere?
REFERENCES
Baldock, J.A. Composition and cycling of organic carbon in soils (2007), in Marschner, P.
and Rengel, Z. (eds.), Nutrient Cycling in Terrestrial Ecosystems. Springer, Berlin.
Carmona-Moreno, C., Belward, A., Malingreau, J.P., Hartley, A., Garcia-Alegre, M.,
Antonovskiy, M., Buchshtaber, V., Pivovarov, V. (2005), Characterizing Interannual
Variations in Global Fire Calendar Using Data From Earth Observing Satellites, Global
Change Biology 11, 1537-1555.
Crutzen, P.J. and Andreae, M.O. (1990), Biomass burning in the tropics: impact on
atmospheric chemistry and biogeochemical cycles, Science 250, 1669-1678.
Davidson, E.A. and Janssens, I.A. (2006), Temperature sensitivity of soil carbon
decomposition and feedbacks to climate change, Nature 440, 165-173.
Hammes, K. et al. Comparison of quantification methods to measure fire-derived
(black/elemental) carbon in soils and sediments using reference materials from soil,
water, sediment and the atmosphere. Global Biogeochemical Cycles 21, GB3016,
doi:10.1029/2006GB002914 (2007).
IPCC 4th Assessment report (2007), Working Group I: The physical science basis of climate
change, http://ipcc-wg1.ucar.edu/wg1/wg1-report.html
2006 IPCC Guidelines for National Greenhouse Gas Inventories, vol. 4, Agriculture, Forestry
and other Land use, Appendix 1, http://www.ipcc-nggip.iges.or.jp/public/2006gl/
vol4.htm
Janik, L.J., Skjemstad, J.O., Shepherd, K.D., and Spouncer, L.R. (2007) The Prediction of
Soil Carbon Fractions Using Mid-Infrared-Partial Least Square Analysis. Australian
Journal of Soil Research 45, 73-81.
Krull, E.S., Baldock, J.A., and Skjemstad, J.O. (2003), Importance of Mechanisms and
Processes of the Stabilisation of Soil Organic Matter for Modelling Carbon Turnover,
Functional Plant Biology 30, 207-222.
Kuhlbusch, T.A.J. (1998), Black carbon and the carbon cycle. Science 280, 1903-1904.
Richardson, J.L. and Vepraskas, M.J. (2000), Wetland Soils: Genesis, Hydrology,
Landscapes, and Classification. CRC Press, Florida.
Schmidt, M.W.I. and Noack, A.G. (2000), Black carbon in soils and sediments: Analysis,
distribution, implications, and current challenges, Global Biogeochemical Cycles 14,
777-793.
Schmidt, M.W.I., Skjemstad, J.O., and Jger, C. (2002), Carbon isotope geochemistry and
nanomorphology of soil black carbon: Black chernozemic soils in central Europe
originate from ancient biomass burning, Global Biogeochemical Cycles 16, 1123
doi:10.1029/2002GB001939.
RESEARCH AND REVIEW STUDIES

ISSN: 2158-5717
Chapter 1
CURRENT AND EMERGING MICROBIOLOGY ISSUES

OF POTABLE WATER IN DEVELOPED COUNTRIES
William J. Snelling1, Catherine D. Carrillo2, Colm J. Lowery1,
John E. Moore3, John P. Pezacki4, James S. G. Dooley1
and Roy D. Sleator5
1
Centre for Molecular Biosciences, School of Biomedical Sciences,

University of Ulster, Cromore Road, Coleraine, Co., Londonderry,
Northern Ireland, United Kingdom, BT52 1SA,
2
The Steacie Institute for Molecular Sciences, National Research Council of Canada,
100 Sussex Drive, Ottawa, Ontario, Canada K1A OR6.
3
Bureau of Microbial Hazards, Health Canada,
251 Sir Frederick Banting Driveway, Ottawa, ON, Canada, K1A 0L2
4
Department of Bacteriology, Northern Ireland Public Health Laboratory,
Belfast City Hospital, Belfast, United Kingdom, BT9 7AD
5
Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland
ABSTRACT
Water is vital for life; for commercial and industrial purposes and for leisure
activities in the daily lives of the worlds population. Diarrhoeal disease associated with
consumption of poor quality water is one of the leading causes of morbidity and mortality
in developing countries (especially in children <5 years old). In developed countries,
whilst potable water is not a leading cause of death, it can still pose a significant health
risk. Water quality is assessed using a number of criteria, e.g. microbial load and nutrient
content which affects microbial survival, as well as aesthetic factors such as odor. In
water systems, the presence of disinfectant, low temperatures, flow regimes and low
organic carbon sources do not appear to be conducive to microbial persistence. However,
frequently this is not the case. A variety of human pathogens can be transmitted orally by
Correspondence: Dr Roy Sleator, Alimentary Pharmabiotic Centre, University College Cork, Ireland. Phone: 00
353 21 490 1366, Fax: 00 353 21 490 3101, email: r.sleator@ucc.ie
12
William J. Snelling, Catherine D. Carrillo, Colm J. Lowery et al.

water and in the developed world water quality regulations require that potable water
contains no microbial pathogens.
Chlorine dioxide is a safe, relatively effective biocide that has been widely used for
drinking water disinfection for 40 years. Providing the water is of low turbidity, standard
chlorination procedures are sufficient to prevent the spread of planktonic bacteria along
water mains. However, despite this, bacterial contamination of water distribution systems
is well documented, with growth typically occuring on surfaces, including pipe walls and
sediments. Rivers, streams and lakes are all important sources of drinking water and are
used routinely for recreational purposes. However, due to fouling by farm and wild
animals, these sources can be contaminated with microbes, e.g. chlorine resistant
Cryptosporidium oocysts, no matter how pristine the source or well maintained the water
delivery system. The high incidence of Cryptosporidium in surface water sources
underlines the need for frequent monitoring of the parasite in drinking water. The use of
coliforms as indicator organisms, although considered relevant to most cases, is not
without limitations, and is thus not a completely reliable parameter of water safety, e.g.
Campylobacter contamination cannot be accurately predicted by coliform enumeration.
Furthermore, the presence of biofilms and bacterial interactions with protozoa in water
facilitate increased resistance to antimicrobial agents and procedures such as disinfectants
and heating, e.g. Legionnaires disease caused by Legionella pneumophila.
The high cost of waterborne disease outbreaks should be considered in decisions
regarding water utility improvement and treatment plant construction. The control of
human illnesses associated with water would be aided by a greater understanding of the
interactions between water-borne protozoa and bacterial pathogens, which until relatively
recently have been overlooked.
INTRODUCTION
Currently, over 1 billion people worldwide have no access to safe drinking water (CDC,
2007a). In the developed world water quality regulations require that potable water does not
contain any microbial pathogens (Percival and Walker, 1999). Residents of affluent nations
are remarkably lucky to have high-quality, safe drinking water supplies that most residents of
modem cities enjoy, particularly when considered in contrast to the toll of death and misery
that unsafe drinking water causes for most of the world's population (Hrudey and Hrudey,
2007). The supply of clean drinking water is a major, and relatively recent, public health
milestone (Berry et al., 2006). However, some may presume that drinking-water disease
outbreaks are a thing of the past, but complacency can easily arise (Hrudey and Hrudey,
2007). Increasing human populations and urbanisation have placed burdens on water sources,
i.e. rivers, streams and lakes, used to provide potable water to most metropolitan areas
(Calderon et al., 2006). Excreta from humans, pets, livestock and wildlife, e.g. foxes, present
in these source waters have the potential to harbour hundreds of pathogenic microorganisms
of public health concern (Leclerc et al., 2002). In developed countries, major outbreaks of
waterborne infections have fuelled widespread public concern regarding the microbiological
quality of potable water (De Paula et al., 2007; Pankhurst and Coulter, 2007).
Potable water can be a source of various potentially infectious microorganisms, which in
the majority of cases are contracted by ingestion, but can also be contracted via inhalation of
aerosol droplets or by direct dermal exposure (Stojek and Dutkiewicz, 2006). Rivers, streams
and lakes, which are not only important sources of drinking water but are also routinely used
in the pursuit of human leisure interests, are often contaminated with microbes, e.g. chlorine
Current and Emerging Microbiology Issues of Potable Water in Developed Countries 13

resistant Cryptosporidium oocysts, no matter how pristine the source, or well maintained the
water delivery system (Snelling et al., 2006). Fecal contamination of surface waters can occur
via wastewater discharge, farming activities and fouling by wild animals. Perhaps the best
known potential pathogens are certain strains of Escherichia coli and related Gram-negative
species of fecal origin belonging to the Enterobacteriaceae family, usually referred to as
coliforms, which are commonly used as sanitary indicators of potable water quality (Stojek
and Dutkiewicz, 2006). Important agents of waterborne infections include various genera of
Gram-negative bacteria such as Campylobacter, Escherichia coli O157:H7, Legionella,
Salmonella, Shigella, Yersinia, Vibrio cholerae, mycobacteria (Gram positive), enteroviruses
and intestinal protozoa (Giardia, Cryptosporidium) (Stojek and Dutkiewicz, 2006).
Potable water distribution systems generally present a hostile environment for growth of
microorganisms due to low nutrient levels as well as the presence of disinfection residuals
(Lngmark et al., 2007; Momba et al., 2007). Yet despite this, biofilms form ubiquitously in
environments that have been subjected to a range of disinfection processes including
chlorination and ultra-violet (UV)-treatment (Momba et al., 1998). Most of the bacteria in
drinking water distribution systems, e.g. M. avium, L. pneumophila, and E. coli, are
associated with biofilms. In biofilms, their nutrient supply is better than in water, and biofilms
can provide shelter against disinfection (Lehtola et al., 2007). Pathogenic bacteria and viruses
entering water distribution systems can survive in biofilms for at least several weeks, even
under conditions of high-shear turbulent flow, and may be a risk to water consumers (Lehtola
et al., 2007). Also, considering the low number of virus particles needed to result in an
infection, their extended survival in biofilms must be taken into account as a risk for the
consumer (Lehtola et al., 2007). Recent inquiries into the microbial ecology of distribution
systems have found that pathogen resistance to chlorination is affected by microbial
community diversity and interspecies relationships (Berry, et al., 2006). Research indicates
that multispecies biofilms are generally more resistant to disinfection than single-species
biofilms (Berry, et al., 2006).
Control of microbial growth in drinking water distribution systems, often achieved
through the addition of disinfectants, is essential to limiting waterborne illness, particularly in
immunocompromised subpopulations (Berry et al., 2006). Whilst disinfection residuals
within distribution systems reduce the growth of autochthonous and allochthonous pathogens
and reduce the potential re-growth of heterotrophic bacteria, they do not insure the sterility of
distribution waters (Payment et al., 1993). The omnipresence of heterotrophs does not in itself
constitute a potential health hazard, they can be used to evaluate the microbiological quality
of the water and the efficacy of water treatment and distribution processes, as well as the
biological stability of distributed waters (Lngmark et al., 2007).
Waterborne diseases occur worldwide, and outbreaks caused by the contamination of
community water systems have the potential to cause disease in large numbers of consumers
(Table 1) (Karanis et al., 2007). These cases create a lack of confidence in potable water
quality and in the water industry in general; waterborne outbreaks have economic
consequences far beyond the cost of health care for affected patients, their families and
contacts. In addition to outbreaks caused by contaminated potable water, there is also a risk
associated with the accidental ingestion of recreational (or other) waters. National statistics on
outbreaks linked to contaminated water have been available in the USA since 1920, and since
1971, the Centers for Disease Control (CDC), the US Environmental Protection Agency
(USEPA), and the Council of State and Territorial Epidemiologists have maintained a
Table 1. Summaries of Notifiable Diseases in the United States in 2005 (McNabb et al., 2007)
16
collaborative surveillance system for collecting data pertaining to the occurrence and causes
of outbreaks of waterborne disease (Karanis et al., 2007). In Europe during 198696, 277
outbreaks associated with drinking and recreational water were reported from 16 European
countries (Kramer et al. 2001).
VIRUSES
Viruses are possibly the most hazardous of all enteric pathogens and have relatively low
infectious doses (Santamara and Toranzos 2003). Viruses are found in very low
concentrations in treated water due to the effect of dilution and to the potabilization process
(Gutirrez et al., 2007). For this reason, it has been proposed that large amounts of water need
to be collected in order to detect them. A negative result might not be meaningful, while a
positive result is important, particularly when viruses are detected in small water samples
(Gutirrez et al., 2007).Thus, the paucity of data regarding waterborne viruses makes it
difficult to determine the true risk they represent and precludes the development of plans to
prevent viral transmission through contact with environmental water (De Paula et al., 2007).
It has been proposed that more than 140 different types of viruses, apparently not
eliminated by massive purification treatments, can be found in drinking water (Gutirrez et
al., 2007). The main source for water contamination is human excreta (Gutirrez et al., 2007).
In fact, viruses infiltrate the ground, penetrating to depths greater than 67 m and can remain
latent there for several months, as long as the temperature remains low and the environment
humid (Gutirrez et al., 2007). Under these conditions they can easily reach aquifers. Enteric
pathologies due to the presence of Rotavirus (RV), Norovirus (NV), Astrovirus (HAstV),
Adenovirus (Ad), Hepatitis A (HAV), polio, Coxsakie, and echo type Enteroviruses, among
others, have been reported to be associated with consumption of fresh water (Gutirrez et al.,
2007).
Enteroviruses are a group of viruses including the polioviruses, coxsackieviruses,
echoviruses, and others (CDC, 2007c). In addition to the three different polioviruses, there are
62 non-polio enteroviruses that can cause disease in humans: 23 Coxsackie A viruses, 6
Coxsackie B viruses, 28 echoviruses, and 5 other enteroviruses (CDC, 2007c). Non-polio
enteroviruses are second only to rhinoviruses - causative agent of the "common cold" as the
most common viral infectious agents in humans (CDC, 2007c). The enteroviruses cause an
estimated 10-15 million or more symptomatic infections per year in the United States (CDC,
2007c). All three types of polioviruses have been eliminated from the Western Hemisphere,
as well as Western Pacific and European regions, by the widespread use of vaccines (CDC,
2007c). Most people who are infected with an enterovirus exhibit no clinical manifestations
of disease. Infected individuals who do exhibit clinical signs of disease usually develop either
mild upper respiratory symptoms (a "summer cold"), a flu-like illness with fever and muscle
aches, or illness with an associated rash (CDC, 2007c). A less common, though none the less
possible, manifestation of the illness is "aseptic" or viral meningitis (CDC, 2007c). Rarely,
infected individuals may develop an illness that affects the heart (myocarditis) or the brain
(encephalitis) or causes paralysis (CDC, 2007c).
Human noroviruses (NoVs) are a significant cause of non-bacterial gastroenteritis
worldwide with contaminated drinking water a potential transmission route (Bae and Schwab,

2008). As members of the Caliciviridae family, NoVs (previously known as Norwalk-like
viruses) are small (27 nm), icosahedral, non-enveloped human enteric viruses that cause
acute gastroenteritis (Bae and Schwab, 2008). Due to their non-enveloped structure which is
similar to other human enteric viruses such as poliovirus (PV), coxsackievirus, and echovirus,
NoVs are presumed to be as resistant to environmental degradation and chemical inactivation
as the aforementioned viruses (Bae and Schwab, 2008).
The most common vehicles for Hepatitis A virus (HAV) transmission are ingestion of
contaminated water, consumption of contaminated foods and contact with infected
individuals (De Paula et al., 2007). Hepatitis E virus (HEV) is an emerging food borne
pathogen in developing countries, e.g. Asia, Africa and Latin America (Skovgaard, 2007).
Transmitted by the fecaloral route, infection which results in liver inflammation, is linked to
the consumption of swine; the primary source of the virus for humans. The incidence of
sporadic cases has begun to increase recently in developed countries, but it is uncertain
whether these cases were food- or water borne (Skovgaard, 2007). The incidence of hepatitis
E infection is highest in adults between the ages of 15 and 40. Children are also succeptable
to infection but are less likely to become symptomatic. Mortality rates are generally low as
Hepatitis E is a self-limiting disease; symptoms generally dissipate by themselves and the
patient usually recovers. It is spread mainly through fecal contamination of water supplies or
food; person-to-person transmission is uncommon. Outbreaks of epidemic Hepatitis E most
commonly occur following disruption to water supplies caused by heavy rainfalls and
monsoons.
Viruses can be inactivated in the environment by the breakage of their capsid and the
liberation of the contained nucleic acid, which can be easily degraded when deprived of the
capsid protection (Gutirrez et al., 2007). Environmental degradation of viruses can result
from extremes in pH, thermal inactivation, sunlight and predation or release of virucidial
agents from endogenous microorganism in environmental water (Hurst 1988; Yates etal.,
1985). Chlorine, the most commonly used drinking water disinfectant, can also inactivate
enteric viruses if sufficient dose and contact time are provided (Ellis, 1991). Despite some
opinions to the contrary, this means that detection of viral protein (VP) in laboratory assays is
not a sufficient criterion as to the infectious nature of the virus (Gassilloud et al., 2003).
TOXIGENICITY
Microcystis aeruginosa is a gas vacuolate, bloom-forming cyanobacterium that is of
interest from a water quality perspective because of its ability to produce a range of toxic
compounds (Saker et al., 2005). The cyclic peptides, also known as microcystins, are
produced by several species of cyanobacteria including M. aeruginosa, and have been
implicated in the death of humans as well as domestic and wild animals (Saker et al., 2005).
These compounds inhibit protein phosphatases 1 and 2A in a similar manner to okadaic acid,
and have been linked to liver cancer in humans (Saker et al., 2005). To date, more than 60
microcystin variants have been identified and chemically characterized (Saker et al., 2005). In
recognition of their toxic properties, the World Health Organization has adopted a maximum
allowable concentration in drinking water of 1gl-1 (WHO 1998). With appropriate water
treatment, maximum exposure to total microcystins is probably less than 1 gl-1, based on the
18
above data. While average exposure is generally well below this level (WHO, 1998), not all
water supplies are treated by filtration or adsorption; many are untreated or simply
chlorinated (WHO, 1998). In addition to microcystins, many strains of Microcystis are known
to produce other peptides including aeruginosins, anabaenopeptilides, cyanopeptolins,
anabaenopeptins and microginins, which show a diverse range of bioactivities (Saker et al.,
2005).
FOODBORNE CONTAMINATING BACTERIA

Several foodborne pathogens, e.g. Salmonella typhimurium, Campylobacter jejuni,
Yersinia enterocolitica, Mycobacteria, Escherichia. coli O157:H7 and Cryptosporidium, are
common intestinal contaminants frequently found in both farmed animals and wildlife (Doyle
and Erickson 2006), and in several cases are carried asymptomatically (Snelling et al., 2005;
Thompson et al., 2007). These pathogens are generally shed in feces in large populations and
can be transmitted to surface water, both directly and indirectly (Doyle and Erickson 2006).
Soil as a recipient of solid wastes is able to contain enteric pathogens in high concentrations,
which can then be spread to surface water (Figure 1) (Santamara and Toranzos, 2003).
Although most E. coli strains are harmless, E. coli O157:H7 produces a powerful toxin
that can cause severe human illness (CDC, 2007b). E. coli O157:H7 has been found in the
intestines of livestock (CDC, 2007b), is part of the natural microbiota of the soil, and is
therefore also associated with manure, crops, minimally processed ready to eat foods and
surface water (Selma et al., 2007; Himathongkham et al., 2007). Infections often lead to
bloody diarrhea, and in some individuals, particularly children under 5 years of age and the
elderly, the infection can cause haemolytic uremic syndrome (HUS), in which the red blood
cells are destroyed and the kidneys fail (CDC, 2007b). E. coli O157:H7 is a major global
cause of foodborne illness (CDC, 2007b). Based on a 1999 estimate, 73,000 cases of infection
and 61 deaths occur in the United States each year. People can become infected with E.coli
O157:H7 in a variety of ways (CDC, 2007b). Though most illness has been associated with
eating undercooked, contaminated ground beef, people have also become ill from eating
contaminated bean sprouts or fresh leafy vegetables such as lettuce and spinach (CDC,
2007b). In addition, infection can occur after drinking raw unpasturized milk and after
swimming in or drinking sewage-contaminated water (CDC, 2007b).
Salmonella are enteric bacterial pathogens, of which mainly S. enterica and S.
typhimurium cause a variety of food and water-borne diseases ranging from gastroenteritis to
typhoid fever (Ly and Casanova 2007). Previous investigations have found that sewage
effluent regularly contain Salmonella and Campylobacter (Kinde et al., 1997). Globally, C.
jejuni is the major cause of human bacterial diarrhoeal illness and is by far the most common
Campylobacter species associated with human illness. The major C. jejuni reservoir is poultry
(Snelling et al., 2005). However, C. jejuni has been linked to several drinking water-related
epidemics in Finland (Lehtola et al., 2006). C. jejuni is not normally able to multiply in
drinking water or in biofilms, although it may survive in biofilms and/or within protozoa
(Lehtola et al., 2006; Snelling et al., 2006).
Figure 1. Diagram showing the different, most important cycles of transmission for maintaining Giardia
and Cryptosporidium. As well as direct transmission, water and food may also play a role in
transmission. Question marks indicate uncertainty regarding the frequency of interaction between
cycles (Hunter and Thompson, 2005).
Shigellosis is a global human diarrhoeal disease leading to dysentery and is caused by S.

dysenteriae, S. flexneri, S. boydii and S. sonnei (Niyogi et al., 2005). Shigella dysenteriae type
1 produces severe disease and may be associated with life-threatening complications (Niyogi
et al., 2005). The symptoms of shigellosis include diarrhoea and/or dysentery with frequent
mucoid bloody stools, abdominal cramps and tenesmus (Niyogi et al., 2005). Transmission
usually occurs via contaminated food, e.g. ready to eat salads (Ghosh et al., 2007), water,
livestock manure, or through person-to-person contact (Niyogi et al., 2005). Of the estimated
165 million cases of Shigella diarrhoea that occur annually, 99% occur in developing
countries with 69% of episodes occuring in children under 5 years of age (Kotloff et
al.,1999).
Mycobacterium is a genus of Actinobacteria, given its own family. Mycobacteria are
aerobic and generally non-motile bacteria, that are characteristically acid-alcohol fast (Ryan
and Ray, 2004). Environmental mycobacteria are common heterotrophic bacteria in soils and
natural waters, especially in boreal drinking water systems (Torvinen et al., 2004).
Mycobacteria are classified as an acid-fast Gram-positive bacterium due to their lack of an
outer cell membrane (Ryan and Ray, 2004). All Mycobacterium species share a characteristic
cell wall, thicker than in many other bacteria, making a substantial contribution to the
hardiness of this genus (Ryan and Ray, 2004). Therefore, compared to most other bacterial
species, mycobacteria are exceptionally resistant to chlorination and heating (Torvinen et al.,
2004). Some thermotolerant species, including the important mycobacterial pathogens M.
avium complex (MAC) and M. xenopi, tolerate heating and may even survive in hot water
(>60) (Torvinen et al., 2004). Thus, they may survive chemical treatments at waterworks and
enter the distributed water and finally tap water, where their occurrence has been known since
the beginning of the 20th century.
20
Tuberculosis (TB) affects one-third of the world's population, claiming a life every 10
seconds and global mortality rates are increasing, especially in developing countries
(Sacchettini et al., 2008; Tabbara, 2007). The incidence of tuberculosis is increasing with the
increase in the HIV infected population and increased strain drug resistance (Tabbara, 2007).
Complex interactions involving humans, domestic animals, and wildlife create environments
favorable to the emergence of new diseases (Palmer, 2007). Today, reservoirs of M. bovis
subsp. paratuberculosis (MAP), the causative agent of tuberculosis in animals and a serious
zoonosis, exist in wildlife (Palmer, 2007). The presence of these wildlife reservoirs is the
direct result of spillover from domestic livestock, especially cows, in combination with
anthropogenic factors such as translocation of wildlife, supplemental feeding of wildlife and
wildlife populations reaching densities beyond normal habitat carrying capacities (Palmer,
2007).
Toxigenic Vibrio cholerae, the causative agent of cholera, is a native inhabitant of the
aquatic environment (rivers, estuaries, and coastal waters) which is transmitted through
drinking water and still remains a leading cause of morbidity and mortality in many
developing countries, especially in Asia and Africa (Chomvarin et al., 2007). In aquatic
environments V. cholerae associates with the chitinous exoskeletons of copepod molts, which
serves as a surface of nutrients thus facilitating biofilm formation and induces competence for
natural transformation (Blokesch and Schoolnik, 2007). Despite more than a century of
investigation, much remains to be discovered about how pathogenic strains of V. cholerae
interact with the human host and how the biology of disseminating stool V. cholerae drives
devastating cholera outbreaks (Nelson et al., 2007). The V. cholerae species encompasses
more than 200 serogroups (Blokesch and Schoolnik, 2007). Cholera is an ancient secretory
diarrheal disease caused by the O1 and O139 serogroups of V. cholerae (Nelson et al., 2007).
Despite the dramatic reduction of mortality rates due to the development of oral rehydration
solution, the emergence of multiple drug-resistant V. cholerae may reduce the efficacy of
antimicrobial treatment and alter the dynamics of outbreaks (Nelson et al., 2007). V. cholerae
is a bacterial pathogen of the gastrointestinal tract and the secreted toxin is largely responsible
for the massive fluid loss that may reach between 0.5 and 1.0 liter per hour (Nelson et al.,
2007).
Yersinia (family Enterobacteriaceae) are faculatative anerboic bacteria, of which Y.
enterocolitica are foodborne pathogens which are mostly associated with human disease. Y.
enterocolitica is responsible for outbreaks of acute human gastroenteritis and chronic sequela,
e.g. reactive arthritis, and livestock morbidity, e.g. mastitis (Meusburger et al., 2007;
Rudwaleit et al., 2000; Shwimmer et al., 2007). Y. pseudotuberculosis (food-borne route) and
Y. pestis (flea-borne zoonotic disease) can also cause disease in humans. Rodents are the
major reservoirs of Y. enterocolitica; and while humans are the primary hosts other mammals
may also be infected. Infection may occur either through blood (in the case of Y. pestis) or
occasionally via consumption of food products (especially vegetables, milk-derived products
and meat) contaminated with infected urine or feces. An important property of Yersinia is its
ability to multiply at temperatures close to 0 C (Skovgaard, 2007). The minimum infection
dose of Yersinia is relatively high (>100,000), and in many countries, e.g. Denmark, Yersinia
interest and concern have declined (Skovgaard, 2007).
PROTOZOAN PARASITES
At least 325 water-associated outbreaks of parasitic protozoan disease have been reported
(Karanis et al., 2007). North American and European outbreaks accounted for 93% of all
reports and nearly two-thirds of outbreaks occurred in North America (Karanis et al., 2007).
Over 30% of all outbreaks were documented from Europe, with the UK accounting for 24%
of outbreaks, worldwide (Karanis et al., 2007). Giardia duodenalis and C. parvum account for
the majority of outbreaks (132; 40.6% and 165; 50.8%, respectively), Entamoeba histolytica
and Cyclospora cayetanensis have been the aetiological agents in nine (2.8%) and six (1.8%)
outbreaks respectively, while Toxoplasma gondii and Isospora belli have been responsible for
three outbreaks each (0.9%) and Blastocystis hominis for two outbreaks (0.6%) (Karanis et
al., 2007). Balantidium coli, the microsporidia, Acanthamoeba and Naegleria fowleri were
responsible for one outbreak each (0.3%) (Karanis et al., 2007).
Waterborne parasites produce transmission stages which are highly resistant to external
environmental conditions, and to many physical and chemical disinfection methods routinely
used as bacteriocides in drinking water plants, swimming pools or irrigation systems
(Gajadhar and Allen, 2004). Resistant stages include cysts of amoebae, Balantidium, and
Giardia, spores of Blastocystis and microsporidia, oocysts of Toxoplasma gondii, Isospora,
Cyclospora and Cryptosporidium and eggs of nematodes, trematodes, and cestode(Gajadhar
and Allen, 2004). These exogenous transmission stages are microscopic in size and of low
specific gravity, which facilitate their easy dissemination in fresh water, or seawater
(Gajadhar and Allen, 2004). The exogenous stages of waterborne parasites possess outer
surfaces capable of withstanding a variety of physical and chemical treatments (Gajadhar and
Allen, 2004). The resistant surfaces are comprized of multiple polymeric layers of lipids,
polysaccharide, proteins or chitin (Gajadhar and Allen, 2004). Examples of these are the two
protein layers of coccidian oocysts derived from the coalescence of wall-forming bodies, the
chitinous wall of microsporidian spores, the multi-layered (inner lipid/protein-, middle
protein/chitin-, outer protein/mucopolysaccharide) shell of Ascaris eggs, and the impermeable
embryophore of the Echinococcus egg which is constructed of polygonal blocks of keratinlike protein held together by a cement substance (Gajadhar and Allen, 2004).
The intestinal protozoan parasites Cryptosporidium (Apicomplexan) and Giardia (G.
duodenalis) are major global causes of diarrhoeal disease in humans (Smith et al., 2007).
Significantly, normal concentrations of chlorine and ozone used in mass water treatment are
not adequate to kill these microbes (Smith et al., 2007), which have life cycles suited to both
waterborne and foodborne transmission (Smith et al., 2007). Giardia causes intestinal
malabsorption and diarrhoea (giardiasis) in humans and other mammals worldwide (Smith et
al., 2007). Giardia is one of the most prevalent pathogens that should be removed from
drinking water (Smith et al., 2007). In developing countries, the prevalence of human
giardiasis is on average 20% (443%), compared with 5% (37%) in developed countries,
where it is associated mainly with travel and waterborne outbreaks (Smith et al., 2007). G.
duodenalis assemblages A and B have been found in humans and most mammalian orders
(Smith et al., 2007). Giardiasis is a disease with economic ramifications due to the large
impact on domestic animals such as cattle and sheep (Smith et al., 2007). The role of animal
transmission of human giardiasis is unclear, but the greatest risk of zoonotic transmission
seems to be from companion animals such as dogs and cats. Interestingly, the capacity of
22
Acanthamoeba to predate Cryptosporidium oocysts has recently been demonstrated (GmezCouso et al., 2007). Free-living-amoeba (FLA) may act as environmentally resistant carriers
of Cryptosporidium oocysts and, thus, may play an important role in the transmission of
cryptosporidiosis (Gmez-Couso et al., 2007).
Zoonotic Cryptosporidium parvum and anthroponotic C. hominis are the major cause of
human cryptosporidiosis, although other species including C. meleagridis, C. felis, C. canis,
C. suis, C. muris and two corvine genotypes of Cryptosporidium have been associated with
human gastroenteritis (Xiao and Ryan 2004; Caccio et al. 2005). Cryptosporidium can
survive for months in a latent form outside hosts, as its oocysts retain their infectivity for
several months in both salt and fresh water (Fayer et al. 1998; Sunnotel et al., 2006a).
Cryptosporidium causes self-limited watery diarrhoea in immunocompetent subjects, but has
far more devastating effects in the immunocompromized and in some cases can be lifethreatening due to dehydration caused by chronic diarrhoea (Caccio 2005, Chen et al. 2005).
Cryptosporidiosis is responsible for significant neonatal morbidity in farmed livestock and
causes weight loss and growth retardation, leading to significant economic losses (McDonald
2000). Over the last two decades, increasing numbers of cryptosporidiosis (in particular water
related) outbreaks have been recorded in developed countries (Craun et al. 2005). In 1993, the
largest Cryptosporidium outbreak was registered in Milwaukee, Wisconsin, USA where
403,000 people were infected through contaminated drinking water (MacKenzie et al. 1994).
This outbreak was caused by C. hominis (Peng et al. 1997), and the total cost of outbreakassociated illness was estimated at >$96 million in both medical costs and productivity losses
(Corso et al. 2003).
Cryptosporidium has also been associated with treated water in swimming and wading
pools (Craun et al. 2005). Shellfish have the ability to filter large amounts of water and
concentrate oocysts within their gills (Gomez-Couso et al. 2006). Thus, despite prevention
measures, e.g. standard UV depuration treatment, the consumption of raw or undercooked
shellfish, may still be a potential health risk (Sunnotel et al., 2007). Also, the use of surface
water for irrigation can indirectly cause human infection via the consumption of contaminated
fresh produce, e.g. lettuce (Shigematsu et al., 2007). Outbreaks have been reported in
healthcare facilities and day-care centres, within households, among bathers and water sports
enthusiasts in lakes and swimming pools, and in municipalities with contaminated public
water supplies or people served by private water supplies. In 2005 the European Basic
Surveillance Network (BSN) recorded 7,960 human cases of cryptosporidiosis from 16
countries.
Microsporidial gastroenteritis; a serious disease of immunocompromized people, can
have a waterborne etiology (Graczyk et al., 2007). Microsporidia are obligate intracellular
eukaryotes parasitizing a wide range of invertebrates and vertebrates with over 1,200 species,
of which 14 are opportunistic human pathogens, with Encephalitozoon intestinalis, E. hellem,
E. cuniculi, and Enterocytozoon bieneusi being the most common (Graczyk et al., 2007).
Currently, Microsporidia are on the Contaminant Candidate List of the U.S. Environmental
Protection Agency due to their unknown transmission routes, technologically challenging
identification and the difficult treatment of human infections (Graczyk et al., 2007).
Considerable evidence indicates involvement of water in the epidemiology of
microsporidiosis, however, this link has not been conclusively substantiated (Graczyk et al.,
2007). Risk factor analysis for encephalito zoonosis has previously suggested groundwater as
a source of infection, and a massive outbreak of microsporidiosis was epidemiologically

linked to a drinking water distribution system (Cotte, et al., 1999). More accurate
Microsporidial epidemiological data is urgently required to accurately assess the importance
of this emerging pathogen.
EMERGING AND OPPORTUNISTIC PATHOGENS

Opportunistic microbes are a subset of the emerging pathogens and include species of
both fecal and environmental origin. Treated potable water contains a variety of microbes that
are not well characterized (Stelma et al., 2004). Many of these organisms grow slowly and
require nutrient-poor media for culturing (Stelma et al., 2004). Although there is evidence that
these microbes are generally not hazardous to the general healthy population, there is a
possibility that some of them may be opportunistic pathogens and may be capable of causing
adverse health effects in individuals with impaired body defences (Stelma et al., 2004).
Examples of opportunistic bacterial pathogens of potable water origin include Aeromonas
hydrophila, L. pneumophila, M. avium, and Pseudomonas aeruginosa. There is no reason to
assume that the currently known opportunistic pathogens are the only opportunistic pathogens
indigenous to potable water (Stelma et al., 2004). Many cases of respiratory infections and
digestive system infections are still of unknown etiology and it is possible that some of them
could be due to pathogens that are currently unknown (Stelma et al., 2004).
Opportunistic bacterial pathogens have the potential to reproduce in the natural
environment, particularly in the presence of increased temperatures and nutrients, often when
associated with free-living protozoa (Lngmark et al., 2007). Free-living protozoa are in most
cases non-parasitic, however, some species are known to cause human illness (Lngmark et
al., 2007). The potential significance of free-living protozoa, e.g. amoeba, as potential
environmental reservoirs for aquatic pathogens has been recognized for more than twenty
years (King et al., 1988), as has the often complex interspecies relationships between mixed
biofilm populations, e.g. bacteria and protozoa (Snelling et al., 2006; Lngmark et al., 2007).
Yet despite this, with the exception of L. pneumophila, relatively little attention has been
payed to studying and understanding interactions between free-living protozoa and other
smaller human pathogens (Brown and Barker, 1999; Snelling et al., 2006). Some
microorganisms have evolved to become resistant to protozoa digestion and these amoebaresistant microorganisms include established bacterial pathogens, such as Legionella spp.,
Chlamydophila pneumoniae, M. avium, P. aeruginosa, and C. jejuni, and emerging pathogens
such Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal
pathogens (Snelling et al., 2006).
The fate of internalized bacteria can be divided into three main outcomes;
i)
Bacteria which multiply and cause lysis of amoebal cells (FLA), e.g. Legionella and
Listeria monocytogenes.
ii) Bacteria which multiply without causing cell lysis, e.g. Vibrio cholera.
iii) Bacteria which survive without multiplication, e.g. mycobacteria (Snelling et al.,
2006).
24
FLA feed on bacteria, fungi and algae and as such are ubiquitous predators that control
microbial communities while at the same time acting as reservoirs for many human
pathogens. FLAs are thus often regarded as the Trojan horses of the microbial world
(Chang and Jung, 2004; Greub and Raoult 2004; Molmeret et al., 2005). Dictyostelium
discoideum is an established host model for several pathogens including, P. aeruginosa,
Mycobacterium spp., and L. pneumophila (Snelling et al., 2006). Significantly, numerous
bacterial species survive disinfection treatments when internalized within protozoa, compared
to the exposed and thus more sensitive planktonic state which forms the basis of current
disinfection policies (Snelling et al., 2005b; King et al., 1998; Whan et al., 2006). Interactions
such as these are unaccounted for in current disinfection models (Berry, et al., 2006).
FLA provide habitats for the environmental survival of L. pneumophila, which have been
observed undergoing binary fusion within intracellular vacuoles of amoeba (Atlas, 1999;
Colbourne et al., 1984). To date it has never been shown that Legionella can multiply in its
own natural environment outside protozoa (Atlas, 1999; Colbourne et al., 1984). Legionella
pneumophila are fastidious bacteria which develop mostly in water, causing
legionnairesdisease [(LD), legionellosis, or atypical pneumonia/flu-like illness] in humans
(Stout et al. 1992; Stojek and Dutkiewicz, 2006). In environmental water bodies L.
pneumophila proliferates intracellularly in more than 15 protozoan genera, e.g.
Acanthamoeba, Hartmannella, Vahlkamphia and Ehinamoeba (Steinert et al., 1994).
Infection of humans, generally the elderly and immunocompromized, occurs after inhalation
of aerosolized bacteria from contaminated water sources (Steinert et al., 1994). The infectious
particle is unknown, but may be excreted as legionellae-filled vesicles, intact legionellaefilled amoebae, or free legionellae that have lysed their host cell, resulting in L. pneuomphila,
not amoeba, persisting within the respiratory tract (Steinert et al., 1994).
The processing of L. pneumophila by A. castellanii shows many similarities to monocyte
phagocytosis, e.g. the uptake of L. pneumophila by coiling phagocytosis (Steinert et al.,
1994). Phagosomelysosome fusion is prevented based upon L. pneumophila expressing
dot/icm genes that code for a putative large membrane complex which forms a type IV
secretion system that is used to alter the endocytic pathway (Steinert et al., 1994). Genes such
as hfq appear to play a major role in exponential phase regulatory cascades of L. pneumophila
(McNealy et al., 2005; Solomon and Isberg, 2000). Globally, potable water supplies which
harbour L. pneumophila are important sources of community acquired LD (Stout et al. 1992).
LD accounts for an estimated 8,000 to 18,000 cases of hospitalized community-acquired
pneumonia in the United States annually (Phares et al., 2007). Individuals are most often
infected with Legionella by inhaling bacteria-laden aerosol droplets, e.g. via bathing, but can
also become infected by the oral route through drinking water or via traumatized skin or
mucous membranes (Stojek and Dutkiewicz, 2006). Approximately 35% of all LD cases
reported to the Centers for Disease Control and Prevention (CDC) are acquired in health care
facilities (Phares et al., 2007).
M. avium is a common cause of systemic bacterial infection in patients with AIDS
(Miltner and Bermudez, 2000; Snelling et al., 2006). Infection with M. avium, commonly
occurs through the gastrointestinal tract and has been linked to bacterial colonization of
domestic water supplies, where A. castellanii may serve as an environmental host for M.
avium AIDS (Miltner and Bermudez, 2000). M. avium can also survive within A. polyphaga
cyst outer walls and bacterial growth occurs in cocultures (Steinert et al., 1998; Snelling et al.,
2006). M. avium enters and replicates in A. castellanii, and similar to mycobacteria within

human macrophages, inhibits lysosomal fusion and replicates in vacuoles that are tightly
juxtaposed to the bacterial surfaces within amoebae (Cirillo et al., 1997).
Growing M. avium in amoebae enhances invasion and intracellular replication of the
bacterium in human macrophages, the intestinal epithelial cell line HT-29, as well as in mice
(Miltner and Bermudez, 2000), and also increases resistance to rifabutin, azithromycin, and
clarithromycin, which might have significant implications for prophylaxis of M. avium
infection in AIDS patients (Miltner and Bermudez, 2000).
INDICATOR METHODS
For more than thirty years the measurement of bacteria of the coliform group has been
extensively relied on as an indicator of water quality (Yez et al., 2006). Among the
coliforms, the specific determination of E. coli contamination can be performed as one of the
best means of estimating the degree of recent fecal pollution (Edberg et al., 2000). The
European Drinking Water Directive 98/83/EC (Anon, 1998) defines that the isolation of
coliforms using Lactose TTC agar with Tergitol (Tergitol-7 agar) by membrane filtration
(Anon, 2000) be the reference method for the enumeration of total coliforms, including E.
coli in drinking water (Yez et al., 2006). However, numerous outbreaks, e.g.
Crytosporidium, Campylobacter, and viruses, have made it clear that the presence of bacterial
indicators of fecal contamination does not consistently correlate with pathogen levels (De
Paula et al., 2007).
Alternatively, a number of instrumental methods for the rapid determination of
microorganisms based on their metabolic activity have been developed, but not really utilized,
for example, impedance (Madden and Gilmour, 1995), conductance (Gibson, 1987),
chemiluminescence and fluorescence (Van Poucke and Nelis, 2000). Different chromogenic
and/or fluorogenic culture media have also been developed in recent years (Manafi, 2000).
One is TBX agar medium, a modification of Tryptone bile agar medium where the substrate
5-bromo-4-chloro-3-indolyl-b-d-glucuronide (BCIG) is added. This substrate is cleaved by
the enzyme -d-glucuronidase (GUD) that is produced by approximately 95% of the E. coli
strains investigated (Adams et al., 1990), and the released chromophore produces easy to read
blue-green coloured E. coli colonies. Furthermore, the TBX agar method complies with the
ISO/DIS Standard 16649 for the enumeration of E. coli in food and animal foodstuffs (Anon,
2001). In addition, different chromogenic media such as Chromocult Coliform agar (CC
agar), and coli ID agar have been developed for the simultaneous detection of total coliforms
and E. coli, based on the presence of chromogenic substrates for the enzymes galactosidase
(Lac) and -D-glucuronidase (GUD) (Geissler et al., 2000). Results from the evaluation of
these two media have been reported previously (Finney et al., 2003). In spite of their
advantages, the main drawback of chromogenic media is the relatively high cost for routine
analysis laboratories where a great number of analysis are performed (Yez et al., 2006).
The results obtained with the combination of the two media, Tergitol agar and TBX agar, for
the enumeration of total coliforms and E. coli have been comparable to those obtained with
the individual chromogenic media assayed, CC agar and coli ID (Yez et al., 2006).
However, the combined method has the advantage of reduced analytical cost since the TBX
agar is used only for total coliform-positive samples (Yez et al., 2006). In addition, using
26
this small modification, the enumeration of E. coli in total coliforms-positive samples can be
performed in only two hours, maintaining the advantages of rapid turn-around time and the
specificity of the individual chromogenic media (Yez et al., 2006). The combination of
Tergitol agar with TBX fulfils the European Drinking Water Directive where the ISO
Standard 9308 is indicated for the analysis of total coliforms and E. coli (Yez et al., 2006).
Moreover, this small modification provides a simple and comparatively cost-effective method
that is as specific and rapid as the chromogenic media, and which can be used easily by
laboratories dedicated to routine analysis of water (Yez et al., 2006).
RAPID DETECTION METHODS

Many bacterial pathogens are routinely grown on selective/differential agar or broth, e.g.
Shigella (Niyogi et al., 2005). Infection with E. coli O157:H7 is diagnosed by detecting the
bacterium from stool samples (CDC, 2007b). About one-third of laboratories that culture
stool still do not test for E. coli O157:H7, so it is important to request that the stool specimen
be tested on sorbitol-MacConkey (SMAC) agar for this organism (CDC, 2007b). All
individuals presenting with bloody diarrhoea should have their stool tested for E. coli
O157:H7 (CDC, 2007b). However, a major limitation of enrichment methods is the length of
time required to complete the testing, since at least two days are needed for the complete
identification of bacterial typical colonies (Yez et al., 2006). To overcome this problem,
new techniques are continually being developed, and in particular, molecular biology
methods appear to be interesting alternatives for rapidly detecting pathogens in water samples
(Yez et al., 2006). To maximise the useful impact of epidemiological data, it is important to
be able to rapidly detect and identify low numbers of oocysts from different sample types,
including water, and if possible to ascertain if they are viable (Sunnotel et al., 2006a;
Sunnotel et al., 2006b). It is important to be able to accurately differentiate between nonpathogenic and pathogenic species of Cryptosporidium, and between different
Cryptosporidium isolates of the same species (Sunnotel et al., 2006a; Sunnotel et al., 2006b).
Also, the rapid and accurate detection and identification of E. coli O157:H7 and V. cholerae
O139 pathogens is crucial for diagnosis, treatment and eventual control of the contagious
disease outbreak (Jin et al., 2007).
Accurate and reliable epidemiological and molecular typing techniques are crucial for
tracing sources of pathogen infection, the recognition of which is important for the
implementation of preventive measures (Fendukly et al., 2007). Methods include ribotyping,
amplified fragment length polymorphism analysis (AFLP), pulsed field gel electrophoresis
(PFGE), restriction fragment length polymorphism analysis (RFLP), restriction endonuclease
analysis (REA), and arbitrary primed PCR (Fendukly et al., 2007). Nevertheless, molecular
techniques are unfortunately still incompatible with most routine water laboratories because
of the expense and the need for trained personnel (Angles d'Auriac et al., 2000).
Disappointingly, despite the steady improvement in modern molecular biology techniques,
the epidemiology of many infections remains unclear (Snelling et al., 2005b). This confusing
epidemiological evidence is partly because of the lack of standard global typing methods and
communication between laboratories (Wassenaar and Newell 2000). However, these

problems are being addressed via initiatives like CAMPYNET (Wassenaar and Newell 2000)
and PulseNet for standard molecular typing.
Nucleic acid based detection methods such as PCR and real-time PCR are more rapid and
sensitive than traditional culturing techniques. However, they are limited by the number of
targets that can be detected in a single assay. PCR products can be multiplexed, but typically
not more than six targets can be assayed at a single time, and optimizing such a complex
reaction can be challenging. For example, primers must not interact with one another to form
primer-dimers, and amplification of each target must proceed with equal efficiency. Analysis
of the products of the reaction requires size separation by electrophoresis, thus amplicons
must be of significantly different size in order to be easily distinguished. Real-time PCR
(qPCR) is more rapid as it monitors the amount of PCR products as they are being produced
and does not require size separation (Kubista et al. 2006; Zhang and Fang, 2006). In addition
qPCR is more sensitive than traditional PCR methods and can estimate the initial
concentration of nucleic acids. As with standard PCR methods, multiplexing qPCR is limited
by the difficulty in optimizing reactions so that all targets are amplified with similar
efficiencies. Moreover, the number of assays that can be multiplexed is limited by the number
of fluorescent dyes that can be distinguished in a single reaction (typically no more than 4,
depending on the specifications of the instrument being used).
Saker et al., (2007) found that PCR can be used to detect inocula for cyanobacterial
populations and therefore provides a useful tool for assessing which conditions particular
species can grow into bloom populations. PCR methods were particularly useful when the
concentration of the target organism was very low compared with other organisms (Saker et
al., 2007).
The European Working Group for Legionella Infections (EWGLI) has implemented the
AFLP and more recently the sequence-based typing (SBT) to define its collection of L.
pneumophila serogroup 1 strains (Fendukly et al., 2007). However, the SBT has not been
widely used for genotyping non-serogroup 1 L. pneumophila isolates (Fendukly et al., 2007).
Of the many phenotypical, immunological and molecular typing methods which have been
implemented for the characterization and epidemiological typing of L. pneumophila, only the
AFLP and the SBT techniques have gained acceptance by the EWGLI. Both methods were
shown to have good reproducibility and accuracy and obviated the need to submit Legionella
strains between microbiological laboratories in different countries (Fendukly et al., 2007).
The AFLP gel images were, however, more difficult to interpret (Fendukly et al., 2007). This
method allows for comparison of the allelic profile of an isolate to previously assigned allele
numbers of 6 genes in the following order: flaA, pilE, asd, mip, mompS and proA (Fendukly
et al., 2007).
Some enteric viruses grow poorly in cell culture, which constitutes a problem when
investigating strategies for virus control and prevention (Atmar and Estes, 2001; De Paula et
al., 2007). Current methods of detecting such viruses in environmental water samples rely on
genome amplification using molecular techniques such as qualitative and quantitative realtime reverse-transcription polymerase chain reaction (RTPCR) (De Paula et al., 2007).
Worldwide, virus detection in environmental and potable water samples is becoming an
important strategy for preventing outbreaks of infection with waterborne viruses, e.g. HAV
(De Paula et al., 2007).
To evaluate the public health threat posed by V. cholerae occurring in water, a rapid and
accurate method for detection of toxigenic V. cholerae is essential (Chomvarin et al., 2007).
28
The culture method (CM), which is routinely used for assessing water quality, has not proven
as efficient as molecular methods because this notorious pathogen survives in water mostly in
the viable but non-culturable state (VBNC) (Chomvarin et al., 2007). The isolation and
identification of V. cholerae by CM is expensive, time-consuming, labour intensive, and
unable to precisely distinguish between toxigenic V. cholerae and non-toxigenic V. cholerae
(Chomvarin et al., 2007). Fluorescent monoclonal antibody combined with molecular genetic
based methods can demonstrate the presence of toxigenic V. cholerae in the aquatic
environment (Chomvarin et al., 2007).
The last three decades have witnessed exponential progress in our understanding of the
biology of pathogens. The complete sequencing of the human genome and of many microbial
pathogens associated with potable water have rapidly fuelled the development of gene array
technology, improved pathogen detection systems and analysis methods. A host of different
molecular techniques for studying the transmission dynamics and detection of drug resistant
pathogens have been developed (Katoch et al., 2007).
DNA microarrays (or gene arrays) consist of segments of DNA called probes or reporters
arrayed on a solid surface (Call, 2005; Lemarchand, 2004). DNA spots are typically very
small, on the micrometer length scale, and can be arrayed in high density with hundreds to
thousands of spots present on a single array. Detection using microarrays is based on the
hybridization of fluorescently labelled nucleic acids (RNA or DNA) isolated from the test
matrix to these probes, and subsequent measurement of fluorescence at each spot.
Microarrays allow the simultaneous detection of thousands of targets in a single assay. Thus,
a single array could be used for the detection of a wide range of pathogens, virulent strains of
a particular pathogen, antimicrobial resistant strains, and even molecular typing. Microarrays
have been applied to microbial quality monitoring of water (Lee et al., 2006; Lemarchand et
al., 2004; Maynard et al., 2005). However, methods involving direct hybridization onto
microarrays are inherently lower in sensitivity relative to PCR because of the lack of
oligonucleotide amplification. For example, Lee et al. (2006) were only able to detect
pathogens in wastewater at levels of 2 x 108 copies of the target gene, using direct
hybridization of DNA isolated from wastewater. Straub et al. (2005) have developed a
microarray-based system for analysis of RNA isolated from microbial communities. This
system shows great promise, as detection of RNA could be linked to the presence of viable
cells; however, the sensitivity of this system is currently too low to be useful for pathogen
detection.
Sensitivity can be greatly improved by PCR amplification of the target, before
hybridization to the array. However, production of multiple PCR products can be labour
intensive and slow. Amplification of universal genes (i.e. rRNA genes) gets around this
problem as a single primer set can be used to amplify a segment from a gene that is conserved
among the pathogens of interest. Species-specific sequence variability within the amplicon is
detected by hybridization of the PCR products to a microarray. Using this method, with the
23s rRNA gene, Lee et al. (2006) demonstrated an exponential increase in the sensitivity of
their array to 20 copies of the target gene. Maynard et al. (2005) used a similar strategy, but
improved discriminatory power of their array by the inclusion of multiple genes (cpn60, 16s
rRNA and wecE ). They were able to detect 100 copies of a target gene, but this detection
level was decreased when the genomic DNA of interest was in a background of complex
DNA mixtures. PCR amplification favors the amplification of the most abundant bacterial
species and low-level targets may be underrepresented, or undetectable on the array. This is

an important problem, as pathogens are likely to represent a small proportion of the microbial
communities in water supplies. Kostic et al. (2007) were able to overcome this problem to a
degree by using a unique labelling method (sequence-specific end-labelling of
oligonucleotides) and inclusion of competitive oligonucleotides during labelling. They were
able to detect pathogens which comprised 0.1% of the total microbial community analyzed.
Wu et al. (2006) used a method of total DNA amplification (multiple displacement
amplification) to non-specifically amplify total genomic DNA isolated from contaminated
groundwater prior to microarray hybridization. This method seems promising, as it appears to
eliminate the bias of PCR based amplification, while increasing the sensitivity of the
microarray-based assay.
Microarrays have proven to be useful tools for monitoring microbial communities in
water and environmental samples. However, there are practical limitations in the use of
microarray-based approaches including the requirement of specialized equipment and trained
personnel. Automation of array systems would contribute significantly to the ease of
deployment of these systems. Suspension arrays, such as the Luminex 100 system, are
particularly amenable to automation (Baums et al., 2007; Gilbride et al., 2006). These arrays
work on similar principles to planar microarrays, but they are rapid, cost effective, flexible
and provide high reproducibility. In these systems, probes are immobilized onto solid surfaces
of microspheres that are labelled with fluorophores (up to 100 colours are available) to
facilitate their identification. After hybridization, the beads are analyzed in a flow cytometer.
A red laser identifies the bead, and a green laser registers if a target has been captured. This
system was found to be effective for detecting six different fecal indicating bacteria in
environmental samples (Baums et al., 2007). Straub et al. (2005) have developed a
comprehensive system for sample preparation and pathogen detection called BEADS
(biodetection enabling analyte delivery system). This system incorporates immunomagnetic
separation to capture cells for concentration and purification of the analytes, a flow through
thermal cycler and detection of PCR products on a suspension array. Using this system with
river water, Straub and colleagues were able to detect 10 cfu of E. coli. When multiplexed
with Shigella and Salmonella, sensitivity was reduced, but 100 cfu of each organism could be
detected.
Biosensor technologies offer the promise of portable devices delivering reliable results
within short periods of time. Biosensors can be defined as analytical devices incorporating a
biological material such as specific antibodies or DNA probes to confer specificity, integrated
within a transducing microsystem (optical, electrochemical, thermometric, piezoelectric,
magnetic or micromechanical) (Lazcka et al., 2007; Gilbride et al., 2006). Biosensors can be
incorporated into miniaturized microfluidic devices commonly referred to as lab-on-a-chip
devices. Typical Lab-on-a-chip devices contain wells for samples and storage of reagents,
and microchannels for distribution of the samples and reagents to reaction chambers. They are
connected to an instrument for control and detection of reactions by biosensors and to a
computer for data analysis. The use of such devices reduces reagent costs, increases speed of
analysis, minimizes handling steps, and provides portability, capability for parallel operations
and the automation of complex assays. Several biological assays have been miniaturized and
automated, including PCR, qPCR, DNA electrophoresis and microarrays, and immunoassays
(Chen et al., 2007). A hand held device comprising electrochemical biosensors was
successfully used to screen environmental water samples for 8 pathogens, within 3-5 h
(LaGier et al., 2007). The detection limit of the device was equivalent to 10 cells of Karenia
30
brevis when purified genomic DNA was assayed with the device. The scale of such devices,
in terms of the number of targets that can be analyzed at a time, is still limited. The
incorporation of high-density microarrays into integrated microfluidic devices containing
biosensors has the potential to address the important issues of sensitivity and assay
automation while maintaining the high information content of a microarray. Such systems
may enable the reliable real-time monitoring of microbial communities, and detection of
pathogens in water systems. While current devices show promise, there is a need to improve
sensitivity, to improve specificity when dealing with complex samples, to provide portability
and low cost in order to gain widespread use at water treatment facilities.
CONCLUSION
In most developed countries, the removal or inactivation of the majority of pathogens,
e.g. Cryptosporidium oocysts, can be accomplished by conventional water treatment
technology, which generally includes flocculation, coagulation, sedimentation, filtration and
chlorination (Betancourt and Rose 2004). Conventional treatment consists of
coagulation/flocculation, sedimentation, and filtration (Sunnotel et al., 2006a).
Implementation of multiple barriers to safeguard drinking water is recommended as pathogen
loads vary during the season, with peak loads during early spring and late autumn (Ferguson
et al. 2003). The high occurrence of many important human pathogens in surface water
sources underlines the need for frequent monitoring of the parasite in drinking water (Frost et
al. 2002). The high cost of waterborne disease outbreaks should be considered in decisions
regarding water utility improvements and the construction of treatment plants (Morgan-Ryan
et al. 2002). The integration of watershed and source water management and protection,
scientific management of agricultural discharge and run-off, pathogen or indicator organism
monitoring for source and treated water, outbreak and waterborne disease surveillance is
needed to reduce waterborne transmission of human diseases (Ferguson et al. 2003). Water
treatment facilities employing second-line treatment practices such as UV irradiation and
ozone treatment can alleviate the danger imposed by chlorine resistant pathogens, e.g.
Cryptosporidium (Keegan et al. 2003).
However, outbreaks of waterborne illness can still occur in developed countries, because
of malfunction or mismanagement of water treatment facilities (Ferguson et al. 2003). In
these cases, hazard analyses protocols (microbiological hazards based on fecal coliforms (FC)
and turbidity (TBY) as indicators) for critical control points (CCPs) within each facility may
help to minimise the risk of contaminated water distribution in cases of system component
failure, where CCPs include raw resource water, sedimentation, filtration and chlorinedisinfection (Jagals and Jagals 2004).
Without knowing the occurrence of pathogens in water it is difficult to determine what
risk they present to consumers of contaminated potable water (Karanis et al., 2007).
Standardized methods are required to determine the occurrence of both established and
emerging pathogens in raw untreated water abstracted for potable water, water treatment
systems, potable water, and also recreational waters (Karanis et al., 2007). Also, because of
the potential for pathogens to interact within protozoa and/or biofilms, the total populations of
potable water must be assessed and monitored, e.g. how do populations vary with seasonality

and from one country to another? An improved understanding of the microbial ecology of
distribution systems is necessary to design innovative and effective control strategies that will
ensure safe and high-quality drinking water (Berry, et al., 2006).
Better education and increased awareness by the general public and pool operators could
potentially reduce the number and impact of swimming pool and other recreational waterrelated outbreaks (Robertson et al. 2002a). Improved detection methods, with the ability to
differentiate species, can be useful in the assessment of infection and identification of
contamination sources. These will also provide vital data on the levels of disease burden due
to zoonotic transmission (Fayer 2004; Sunnotel et al., 2006a). The roles of humans, livestock
and wildlife in the transmission of microbial pathogens remain largely unclear for many
different areas. The continued and improved monitoring (using appropriate molecular
methods) of pathogens in surface water, livestock, wild life and humans will increase our
knowledge of infection patterns and transmission of pathogens in potable water (Fayer 2004;
Sunnotel et al., 2006a).
For zoonotic pathogens which are spread both directly and indirectly to potable water, a
wide array of interventions have been developed to reduce the carriage of foodborne
pathogens in poultry and livestock, including genetic selection of animals resistant to
colonisation, treatments to prevent vertical transmission of enteric pathogens, sanitation
practices to prevent contamination on the farm and during transportation, elimination of
pathogens from feed and water, additives that create an adverse environment for colonization
by the pathogen, and biological treatments that directly or indirectly inactivate the pathogen
within the host (Doyle and Erickson, 2006; Sunnotel et al., 2006a). To successfully reduce the
carriage of foodborne pathogens, it is likely that a combination of intervention strategies will
be required (Doyle and Erickson, 2006). Collaborative efforts are needed to decrease
environmental contamination and improve the safety of produce. There is a very real need for
studies that monitor the quality of the water as well as for policies and investments that focus
on sanitation (De Paula et al., 2007).
Unfortunately, in developed countries, the currently adopted short-term and blinkered
strategy of simply detecting pathogens, e.g. a positive/negative Cryptosporidium result,
without further species and isolate information impedes any significant or meaningful
epidemiological analysis and/or effective implementation of intervention strategies. Because
of the current necessity to minimise costs, current molecular methods (outlined previously)
and future nanotechnology based methods are not being routinely employed, and thus may
never fulfil their maximum potential.
This situation demands significant and immediate attention and collaborative input from
governments and water industries world-wide resulting in a more concerted effort from both
scientists and policy makers to standardise the global epidemiological response. Currently,
due to the significant amount of work involved in sample collection and statistical analysis,
there is a time lag of approximately 2-3 years in attaining up to date pathogen data (see CDC
table 1). A global data base and collective goals for biological contaminant loading are vital
for achieving safe potable water. More funding is needed to speed up this process, and to
better educate the general public of the advantages of effective intervention strategies.
Globally, rapid and accurate monitoring and species typing must be carried out routinely, not
just when outbreaks occur. There is also a need for much more accurate and conclusive
information on the overall microbial populations present in water and their relationships to
one another symbiotic, parasitic or otherwise (Snelling et al., 2006). Once successfully
32
introduced in developed countries, then and only then, can this template be effectively applied
to the developing world.
If successful the substantial economic cost (clinical costs and lost working hours) of
water borne pathogens might, in time, be reimbursed through improved epidemiological data
collection which, with proactive intervention, will dramatically reduce the health and
economic burden of waterborne disease.
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ISSN: 2158-5717
Chapter 2
VERMICULTURE BIOTECHNOLOGY:
THE EMERGING COST-EFFECTIVE AND
SUSTAINABLE TECHNOLOGY OF THE 21ST CENTURY
FOR WASTE AND LAND MANAGEMENT TO SAFE AND
SUSTAINABLE FOOD PRODUCTION
Rajiv K. Sinha1, Sunil Herat 2 , Gokul Bharambe 3, Swapnil Patil3,
Uday Chaudhary3, Priyadarshan Bapat3, Ashish Brahambhatt3
David Ryan3, Dalsukh Valani3, Krunal Chauhan3, R. K. Suhane4
and P. K. Singh4
1
Visiting Senior Lecturer, School of Engineering (Environment),

Griffith University, Nathan, Campus, Brisbane, QLD-4111, Australia
2
Senior Lecturer, School of Engineering (Environment), Griffith University
3
School of Engineering (Environment), Griffith University
4
Scientists, Rajendra Agriculture University, Bihar, India
Keywords: Vermicomposting; Vermifiltration; Vermiremediation; Vermi-agro-production;
Vermitreatment-Self-Promoted Odor-Free Process; Earthworm Gut A Bioreactor;
Earthworms Reduce Greenhouse Gases; Earthworms Stimulate Microbial Degradation;
Vermicompost Rich in Minerals and Hormones; Vermifiltered Water - Chemicals and
Pathogen Free and Nutritive.
1. INTRODUCTION
A revolution is unfolding in vermiculture studies (rearing of useful earthworms species)
for multiple uses in environmental management and sustainable development. (Martin, 1976;
Principal and Corresponding Author: Rajiv.Sinha@griffith.edu.au
42
Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al.
Satchell, 1983; Bhawalkar and Bhawalkar, 1994; Sinha et.al 2002; Fraser-Quick, 2002).
Vermiculture biotechnology promises to provide cheaper solutions to following
environmental and social problems plaguing the civilization
1) Management of municipal and industrial solid wastes (organics) by biodegradation
and stabilization and converting them into useful resource (vermicompost) THE
VERMI-COMPOSTING TECHNOLOGY (VCT) ;
2) Treatment of municipal and some industrial (food processing industries) wastewater,
purification and disinfection - THE VERMI-FILTRATION TECHNOLOGY
(VFT);
3) Removing chemical contaminations from soils (land decontamination) and reducing
soil salinity while improving soil properties- THE VERMI-REMEDIATION
TECHNOLOGY (VRT);
4) Restoring and improving soil fertility and boosting crop productivity by worm
activity and use of vermicompost (miracle growth promoter) while eliminating the
use of destructive agro-chemicals - THE VERMI-AGRO-PRODUCTION
TECHNOLOGY (VAPT);
Vermi-composting, vermi-filtration, vermi-remediation and vermi-agro-production are
self-promoted, self-regulated, self-improved and self-enhanced, low or no-energy requiring
zero-waste technology, easy to construct, operate and maintain. It excels all bio-conversion,
bio-degradation and bio-production technologies by the fact that it can utilize organics that
otherwise cannot be utilized by others. It excels all bio-treatment technologies because it
achieves greater utilization than the rate of destruction achieved by other technologies. It
involves about 100-1000 times higher value addition than other biological technologies.
(Appeholf, 1997).
About 4,400 different species of earthworms have been identified, and quite a few of
them are versatile waste eaters and bio-degraders and several of them are bio-accumulators
and bio-transformers of toxic chemicals from contaminated soils rendering the land fit for
productive uses.
Vermiculture Biotechnology: The Emerging Cost-Effective
43
2. EARTHWORMS:
THE GREAT WASTE AND LAND MANAGERS ON EARTH
The earthworms have over 600 million years of experience in waste and land
management. No wonder then, Charles Darwin called them as the unheralded soldiers of
mankind and friends of farmers; and the Greek philosopher Aristotle called them as the
intestine of earth, meaning digesting a wide variety of organic materials including the waste
organics, from earth. (Darwin and Seward, 1903).
Versatile Waste Eaters and Decomposers

Earthworms are versatile waste eaters and decomposers. They feed lavishly on the
organic waste, and also on the microorganisms (bacteria, fungi and the actinomycetes) that
invade and colonize the waste biomass. Most earthworms consume, at the best, half their
body weight of organics in the waste in a day. Eisenia fetida is reported to consume organic
matter at the rate equal to their body weight every day.(ARRPET, 2005).
Earthworm participation enhances natural biodegradation and decomposition of organic
waste from 60 to 80 %. Study indicates that given the optimum conditions of temperature
(20-30 C) and moisture (60-70 %), about 5 kg of worms (numbering approx.10,000) can
vermiprocess 1 ton of waste into vermi-compost in just 30 days (ARRPET, 2005). Upon
vermi-composting the volume of solid waste is significantly reduced from approximately 1
cum to 0.5 cum of vermi-compost.
Earthworms can also treat and purify municipal wastewater (sewage) and also some
industrial wastewater from the food processing industries significantly reducing the BOD and
COD loads, the total dissolved and suspended solids (TDSS). It does this by the general
mechanism of biodegradation of organics in the wastewater, ingestion of heavy metals and
solids from wastewater and also by their absorption through body walls. What is more
significant is that, there is no sludge formation in the process and the resulting vermifiltered water is clean, nutritive and disinfected enough to be reused for irrigation in farms,
parks and gardens.
Bio-Accumulators of Toxic Soil Chemicals and Contaminants

Earthworms have been found to bioaccumulate heavy metals, pesticides and lipophilic
organic micropollutants like the polycyclic aromatic hydrocarbons (PAH) from the soil
(Contreras-Ramos et. al, 2006). After the Seveso chemical plant explosion in 1976 in Italy,
when vast inhabited area was contaminated with certain chemicals including the extremely
toxic TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) several fauna perished but for the
earthworms that were alone able to survive. Earthworms which ingested TCDD contaminated
soils were shown to bio-accumulate dioxin in their tissues and concentrate it on average 14.5
fold. (Satchell, 1983). E. fetida was used as the test organisms for different soil contaminants
and several reports indicated that E. fetida tolerated 1.5 % crude oil (containing several toxic
organic pollutants) and survived in this environment. (OECD, 2000; Safwat et al., 2002).
44
Earthworms also tolerate high concentrations of heavy metals in the environment. The species
Lumbricus terrestris was found to bio-accumulate in their tissues 90-180 mg lead (Pb) / gm of
dry weight, while L. rubellus and D. rubida it was 2600 mg /gm and 7600 mg /gm of dry
weight respectively.(Ireland, 1983).
Earthworm Species Suitable for Waste Management (Waste Biodegradation)

Long-term researches into vermiculture have indicated that the Tiger Worm (Eisenia
fetida), Red Tiger Worm (E. andrei), the Indian Blue Worm (Perionyx excavatus),the African
Night Crawler (Eudrilus euginae),and the Red Worm (Lumbricus rubellus) are best suited for
vermi-composting of variety of organic wastes and vermifiltration of both municipal and
industrial wastewater under all climatic conditions. (Graff, 1981). E. fetida and E. andrei are
closely related. Our study has indicated that E. fetida is most versatile waste eater and
degrader and an army of the above 5 species combined together works meticulously.
Earthworm Species Suitable for Land Remediation (Soil Decontamination)

Certain species of earthworms such as Eisenia fetida, Aporrectodea tuberculata,
Lumbricus terrestris, L. rubellus, Dendrobaena rubida, D. veneta, Eiseniella tetraedra,
Allobophora chlorotica have been found to tolerate and remove wide range of chemicals from
soil. Our study also indicate that E. fetida is most versatile chemical bio-accumulators.
(Ireland, 1983).
Several of above species are common to India and Australia, both nations being
biogeographically very close. (Sinha et. al,.2002).
3. THE BIOLOGY AND ECOLOGY OF EARTHWORMS

Earthworms are long, narrow, cylindrical, bilaterally symmetrical, segmented animals
without bones. The body is dark brown, glistening and covered with delicate cuticle. They
weigh over 1400-1500 mg after 8-10 weeks. On an average, 2000 adult worms weigh 1 kg
and one million worms weigh approximately 1 ton. Usually the life span of an earthworm is
about 3 to 7 years depending upon the type of species and the ecological situation.
Earthworms harbor millions of nitrogen-fixing and decomposer microbes in their gut.
They have chemoreceptors which aid in search of food. Their body contains 65 % protein
(70-80 % high quality lysine rich protein on a dry weight basis), 14 % fats, 14 %
carbohydrates and 3 % ash. (Gerard, 1960; ARRPET, 2005).
Enormous Power of Reproduction and Rapid Rate of Multiplication

Earthworms multiply very rapidly. They are bisexual animals and cross-fertilization
occurs as a rule. After copulation the clitellum (a prominent band) of each worm eject lemon-
45
shaped cocoon where sperms enter to fertilize the eggs. Up to 3 cocoons per worm per week
are produced. From each cocoon about 10-12 tiny worms emerge. Studies indicate that they
double their number at least every 60 days. Given the optimal conditions of moisture,
temperature and feeding materials earthworms can multiply by 28 i.e. 256 worms every 6
months from a single individual. Each of the 256 worms multiplies in the same proportion to
produce a huge biomass of worms in a short time. The total life-cycle of the worms is about
220 days. They produce 300-400 young ones within this life period. (Hand, 1988). A mature
adult can attain reproductive capability within 8 12 weeks of hatching from the cocoon. Red
worms takes only 4-6 weeks to become sexually mature. (ARREPT, 2005). Earthworms
continue to grow throughout their life and the number of segments continuously proliferates
from a growing zone just in front of the anus.
Table 1. Reproductive Capacity of Some Environmentally Supportive Worms
Species
E. fetida
E. eugeniae
P. excavatus
D. veneta
Sexual
maturity
time (days)
53-76
32-95
28-56
57-86
No. of
cocoon.
3.8
3.6
19.5
1.6
Cocoons
hatching
time (days)
32-73
13-27
16-21
40-126
Egg
maturity
days
85-149
43-122
44-71
97-214
%
hatching
83.2
81.
90.7
81.2
No. of
hatchlings
3.3
2.3
1.1
1.1
Net
reproduction
rate/week
10.4
6.7
19.4
1.4
Source : Edwards (1988).
Sensitive to Light, Touch and Dryness

Earthworms are very sensitive to touch, light and dryness. They tend to migrate away
from light. Cold (low temperature) is not a big problem for them as the heat (high
temperature). Their activity is significantly slowed down in winter, but heat can kill them
instantly. They temporarily migrate into deeper layers when subjected to too cold or too hot
situations. It seems worms are not very sensitive to offensive smell as they love to live and
feed on cattle dung and even sewage sludge. However, offensive smell can persist only for a
short while in any environment where worms are active. They arrest all odour problems by
killing anaerobes and pathogens that create foul odour.
4. VERMICULTURE : A GLOBAL MOVEMENT

The movement was started in the middle of 20th century and the first serious experiments
for management of municipal / industrial organic wastes were established in Holland in 1970,
and subsequently in England, and Canada. Later vermiculture were followed in USA, Italy,
Philippines, Thailand, China, Korea, Japan, Brazil, France, Australia and Israel
(Edward,1988). However, the farmers all over the world have been using worms for
composting their farm waste and improving farm soil fertility since long time.
In UK, large 1000 mt vermi-composting plants have been erected in Wales
(Frederickson, 2000). The American Earthworm Technology Company started a 'vermi-
46
composting farm' in 1978-79 with 500 t /month of vermicompost production (Edward, 2000).
Collier (1978) and Hartenstein and Bisesi (1989) reported on the management of sewage
sludge and effluents from intensively housed livestock by vermiculture in USA. Japan
imported 3000 mt of earthworms from the USA during the period 1985-87 for cellulose waste
degradation (Kale,1998). The Aoka Sangyo Co. Ltd., has three 1000 t /month plants
processing waste from paper pulp and the food industry (Kale,1998). This produces 400 ton
of vermicompost and 10 ton of live earthworms per month. The Toyhira Seiden Kogyo Co. of
Japan is using rice straw, municipal sludge, sawdust and paper waste for vermicomposting
involving 20 plants which in total produces 2-3 thousands tons of vermicompost per month
(Edward, 2000). In Italy, vermiculture is used to biodegrade municipal and paper mill sludge.
Aerobic and anaerobic sludge are mixed and aerated for more than 15 days and in 5000 cum
of sludge 5 kg of earthworms are added. In about 8 months the hazardous sludge is converted
into nutritive vermicompost (Ceccanti and Masciandaro, 1999). In France, 20 tons of mixed
household wastes are being vermi-composted everyday using 1000 to 2000 million red tiger
worms (Elsenia andrei) in earthworm tanks. (ARRPET, 2005). Rideau Regional Hospital in
Ontario, Canada, vermi-compost 375 - 400 kg of wet organics mainly food waste everyday.
The worm feed is prepared by mixing shredded newspaper with the food waste. (ARRPET,
2005). In Wilson, North Carolina, U.S., more than 5 tons of pig manure (excreta) is being
vermi-composted every week. (NCSU, 1997). In New Zealand, Envirofert is a large
vermicomposting company operating in over 70 acre site in Auckland converting thousands
of tons of green organic waste every year into high quality compost. (www.envirofert.co.nz).
Almost all agricultural universities in India are now involved in vermiculture and a
movement is going across the sub-continent especially involving the poor rural women with
dual objectives of making wealth (food and fertilizer) from waste and combating poverty
while cleaning the environment and combating pollution of land and soil. Earthworms have
enhanced the lives of poor in India. It has become good source of livelihood for many.
(White, 1994; Hati, 2001).
Vermiculture is being practiced and propagated on large scale in Australia too as a part of
the 'Urban Agriculture Development Program' (to convert all the municipal urban wastes into
compost for local food production) and Diverting Waste from Landfills Program (for
reducing landfills in Australia).
5. THE VERMICOMPOSTING TECHNOLOGY (VCT)

Vermicomposting is a rapid biological degradation process (aided by earthworms and soil
microbes) in which several kinds of organic materials are converted from unstable product
(which is likely to decompose further, creating objectionable odors, generating greenhouse
gas methane and producing environmental insanitation) to an increasingly more stable
product whose value is upgraded as nutritive materials for the soil and that can remain in the
environment without creating any environmental problem. All organic wastes by its very
nature (chemical composition) is bound to disintegrate anaerobically in environment and
generate greenhouse gas methane (CH4). Only if they are allowed to degrade under aerobic
conditions (which is readily facilitated by earthworms) that this can be prevented.
47
5.1. Wastes Suitable for Vermicomposting

Earthworms can physically handle a wide variety of organic wastes from both municipal
(domestic and commercial) and industrial (livestock, food processing and paper industries)
streams. Earthworms are highly adaptable to different types of organic wastes (even of
industrial origin), provided, the physical structure, pH and the salt concentrations are not
above the tolerance level. Another matter of considerable significance is that the earthworms
also partially detoxify (by bio-accumulating any heavy metals and toxic chemicals) and
disinfect (by devouring on pathogens and killing them by anti-bacterial coelomic fluid) the
waste biomass while degrading them into vermi-compost which is nearly sterile and odorless.
(Pierre et al., 1982).
Some Industrial Wastes Suitable for Vermi-Composting

Solid waste including the wastewater sludge from paper pulp and cardboard industry,
brewery and distillery, sericulture industry, vegetable oil factory, potato and corn chips
manufacturing industry, sugarcane industry, aromatic oil extraction industry, logging and
carpentry industry offers excellent feed material for vermi-composting by earthworms. (Kale,
et al., 1993; Kale and Sunitha, 1995; Seenappa et al.,1995; Gunathilagraj and Ravignanam,
1996; Lakshmi and Vijayalakshmi, 2000; ARRPET, 2005.)
Worms Can Even Vermicompost Human Excreta (Feces)
Bajsa et al. (unpublished data) studied vermicomposting of human excreta (feces). It was
completed in six months, with good physical texture meeting ARMANZ (1995) requirements,
odourless and safe pathogen quality. Sawdust appeared to be the best covering material that
can be used in vermicomposting toilets to produce compost with a good earthy smell, a
crumbly texture and dark brown colour. There was no re-growth of pathogens on storing the
compost for longer period of time and the initial pathogen load did not interfere in the die off
process as the composting process itself seemed to stabilize the pathogen level in the system.
Waste Materials Preferably to be Avoided for Vermi-composting
1) Heavily salted products unless soaked in water for 24 hours;
2) Excess citrus (orange and lemon peels and crushes) and onions wastes (might reduce
pH and impair worm activity);
3) Feces of pets (may carry viral or bacterial toxins);
4) Fresh green grasses and foliages (green waste) creates high temperature and can
harm worms;
5) All non-biodegradable wastes;
6) Meat and dairy products and slaughterhouse waste are to be avoided in the initial
stage till the number of earthworms become high enough in the composting bed (it
may invite maggots and also create bad odor for sometimes).
However, Nair et al (2006) studied that thermophilic aerobic composting of food waste
for 7 days followed by vermicomposting can eliminate the need for screening off of citrus
and other acidic wastes like onion peels from vermicomposting. It comprises a short period
48
of high temperature (around 55 C) followed by a period of lower temperature. This also

leads to reduction of pathogens in much shorter time. (See table 9 below).
The Envirofert, New Zealand, which is vermicomposting thousands of tons of green
waste every year, also practice putting the green waste first to a lengthy thermophilic cooking,
and then to vermicomposting by worms after cooling. Cooking of green waste also help
destroy the weeds and pathogens which may come from the feces of pets in grasses. They
claim that each worm eat the cooked green waste at least 8 times leaving an end product
which is rich in key minerals, plant growth hormones, enzymes, and beneficial soil microbes.
(www.envirofert.co.nz).
5.2. Vermicomposting of Municipal Solid Waste

Millions of tons of municipal solid wastes (MSW) from homes and commercial
institutions, cattle and farm wastes are generated in the human society and are mostly ending
up in the landfills everyday, creating extraordinary economic and environmental problems for
the local government to manage and monitor them (may be up to 30 years) for environmental
safety (emission of greenhouse and toxic gases). Construction of engineered landfills incurs
20-25 million US dollars before the first load of waste is dumped.
5.3. Mechanism of Worm Action in Vermicomposting

Worms Reinforce Microbial Population and Act Synergistically Promoting Rapid
Waste Degradation
Earthworms promotes the growth of beneficial decomposer aerobic bacteria in waste
biomass and this they do by improving aeration by burrowing actions. They also act as an
aerator, grinder, crusher, chemical degrader and a biological stimulator. (Dash, 1978; Binet et
al., 1998 and Sinha et al., 2002). Earthworms hosts millions of decomposer (biodegrader)
microbes in their gut (as they devour on them) and excrete them in soil along with nutrients
nitrogen (N) and phosphorus (P) in their excreta (Singleton et al., 2003). The nutrients N and
P are further used by the microbes for multiplication and vigorous action. Edward and
Fletcher (1988) showed that the number of bacteria and actinomycetes contained in the
ingested material increased up to 1000 fold while passing through the gut. A population of
worms numbering about 15,000 will in turn foster a microbial population of billions of
millions. (Morgan and Burrows, 1982). Singleton et al. (2003) studied the bacterial flora
associated with the intestine and vermicasts of the earthworms and found species like
Pseudomonas, Mucor, Paenibacillus, Azoarcus, Burkholderia, Spiroplasm, Acaligenes, and
Acidobacterium which has potential to degrade several categories of organics. Acaligenes can
even degrade PCBs and Mucor can degrade dieldrin.
Under favorable conditions, earthworms and microorganisms act symbiotically and
synergistically to accelerate and enhance the decomposition of the organic matter in the
waste. It is the microorganisms which breaks down the cellulose in the food waste, grass
clippings and the leaves from garden wastes. (Morgan and Burrows, 1982; Xing et al. 2005)
Vermicomposting of organic waste involves following actions of worms
49
1) The waste feed materials ingested is finely ground (with the aid of stones in their
muscular gizzard) into small particles to a size of 2-4 microns and passed on to the
intestine for enzymatic actions. The gizzard and the intestine work as a bioreactor;
2) The worms secrete enzymes proteases, lipases, amylases, cellulases and chitinases in
their gizzard and intestine which bring about rapid biochemical conversion of the
cellulosic and the proteinaceous materials in the waste organics. Earthworms convert
cellulose into its food value faster than proteins and other carbohydrates. They ingest
the cellulose, pass it through its intestine, adjust the pH of the digested (degraded)
materials, cull the unwanted microorganisms, and then deposit the processed
cellulosic materials mixed with minerals and microbes as aggregates called
vermicasts in the soil. (Dash, 1978).
3) Only 5-10 percent of the chemically digested and ingested material is absorbed into
the body and the rest is excreted out in the form of fine mucus coated granular
aggregates - the vermicasts rich in nitrates, phosphates and potash.
4) The final process in vermi-processing and degradation of organic matter is the
humification in which the large organic particles are converted into a complex
amorphous colloid containing phenolic materials. Only about one-fourth of the
organic matter is converted into humus. The colloidal humus acts as slow release
fertilizer. (ARRPET, 2005).
5.4. Experimental Studies on Vermicomposting

a). Study of Biodegradation Abilities of Individual Worm Species
This study was underataken by Sinha et al. (2002) at University of Rajasthan, Jaipur,
India. About 150 mixed species of composting worms Eisinia fetida, Eudrilus euginae, and
Perionyx excavatus were added to 1 kg each of buffalo dung, garden and kitchen wastes in
different containers. In another three containers, each of these wastes and the three individual
species of worms were used separately to assess their individual feeding and biodegradation
abilities. (See table 2).
Table 2. Vermicomposting of Cattle Dung, Kitchen Wastes and Garden Wastes by
Individual Composting Worms
Waste Materials
1 kg of each
Cattle dung
(Week old &
semi-dry)
Raw food wastes
(Spinach stems,
cauliflower &
eggplant rejects)
Garden wastes
(Grasses &
dry leaves)
Time Taken in Vermicomposting & Complete Degradation (in days)

E. fetida
E. euginee
P. excavatus
Mixed Species
59
44
62
45
78
61
83
70
89
69
91
80
Source: Sinha et al., (2002): The Environmentalist, UK.
50
Findings and Results

Results clearly showed that cattle dung is accepted quickly and composted rapidly by
earthworms. Dung is partially degraded materials and the worms love to feed on them as they
are rich in microbes. Raw food waste and garden wastes which contain cellulosic materials
are accepted slowly as they are hard to degrade. Performance of Eudrilus euginae was best
among the three species followed by Eisinia fetida.
b). Study of Composting with Worms (Vermicomposting) and without Worms
(Conventional Aerobic Composting) to Assess the Efficiency of Two Processes
This study was made by Sinha et al. at University of Rajasthan, Jaipur, India on different
cooked and raw kitchen wastes. In each container, 100 mixed species of composting worms
(Eisinia fetida, Eudrilus euginiae and Perionyx excavatus) were added. A control pot was
also organized for conventional aerobic composting without worms. Experiments were
continued throughout the year in both summer, rainy and winter seasons to assess the effect of
temperature variation, humidity and climate change on worm activity. (See table 3).
Table 3. Biodegradation of Raw and Cooked Kitchen Wastes With and Without Worms
Source: Sinha et al., (2002) : The Environmentalist, UK. (Data is for warmer climate in India - MayJuly 1998: Temperature Min. 20C ; Max. 42C; Values in brackets are for cold periods Dec.1998 Feb. 1999 : Temperature Min. 8 C; Max. 18C) Key: EP = Earthworms Present
(Vermicomposting); EA = Earthworms Absent (Aerobic Composting) ; NA= Never Achieved
Within the Period of Study.

Study confirmed beyond doubt that vermicomposting by worms is most efficient process
(nearly 4-6 times faster) over aerobic composting which involves only microbial
decomposition. Worms act both ways carry out enzymatic degradation and also enhance
microbial decomposition. Worm activity is at still greater threshold during warmer periods
than in cold.
c). Study of Vermicomposting of Raw and Cooked Food Wastes to Assess the Food
Preferences of Earthworms and the Time Taken in Degradation of Each Food
Component
The study was made by Sinha et al. (2005) and Patil (2005) at Griffith University,
Brisbane, Australia. Vermi-composting was carried out in a specially fabricated plastic
vermiculture bin (Figure 1) sold in major hardware stores all over Australia. There is
adequate provisions for aeration from side walls and top is open with cover lid on it. The
51
vermicomposting bed was prepared with a thick layer of wet newspaper and about 4 inches of
garden soil spread over it. About 250 numbers of earthworms were released in this soil bed.
Food wastes were collected from homes and restaurants. About 100 gm of each of the food
waste components were used. Mixed species of Elsenia fetida, Eudrilus eugeniae and
Perionyx excavatus were used. Worms ranged between 3-6 cm in length and 0.1-0.2 cm in
thickness. Moisture content was maintained at around 60 % by regular spray of water.
Temperature within the bin was maintained at around 22-24 C as in the glass-house.
No of Days taken for complete

Biodegradation
100
90
80
70
60
50
40
30
20
10
0
E. fetida,
E. euginae,
P. excavatus
Mixed Species
Earthworm Species Used

Cattle dung
Kitchen w astes
Garden w astes
Figure 1. Time Taken To Complete Biodegradation of Different Food Waste by Various Earthworm
Species.
A control VC bin was organized with all above features except for the earthworms to
compare the rate of waste biodegradation without worms (but by aerobic process), odor
problems etc., and to exactly determine the role and efficiency of earthworms in waste
biodegradation. Results of vermicomposting have been given in tables 4 and 5.
120
16
% Reduction
12
80
10
60
8
6
40
4
20
Raw potato
cuts &
peels
Crushed
egg shells
Cooked
chicken,
lamb & beef
(curry)
Indian
chapatti
Cooked
potato,
cauliflower
etc.
Raw
rejects of
cauliflower
etc.
Baked
potato,
tomato etc.
Apple,
pear, kiwi
fruit etc.
Raw
cabbage,
lettuce etc.
Rice,
noodles &
pasta
Bread,
buns &
naans
Food Waste components

% Biodegradation With Worms
% Biodegradation WithoutWorms
Figure 2. Rate of Biodegradation of Food Waste Components.
Weeks
Duration (Weeks)
14
100
52

Worms can eat and degrade all food components including the cooked meat wastes
(except the bones) conveniently according to their food preferences. They prefer softer ones
first. But they are not comfortable with egg-shells, raw onions and raw potato rejects.
Table 4. Rate of Biodegradation of Raw and Cooked Food Wastes With and Without
Earthworms Under Identical Climatic Conditions (Food waste components about 100
gm each with 250 worms)
Source: Sinha et al. (2005)

Keys: * 100 % degradation was achieved within the study period (Week 14) though with foul odor
and heavy invasion of fungus;
** 100 % degradation could never be achieved, had odor problem and badly invaded by fungus
(blue, black and green moulds) and had to be terminated;
*** Terminated in week 4 because of maggots, heavy fungus and offensive smell;
Table 5. Rate of Biodegradation of Fried and Oily Food Wastes With and Without
Earthworms Under Identical Climatic Conditions (Food waste used was about 9.5 kg
with 500 worms)
Source: Patil (2005)

Key: ND = Not detectable
* Mild foul odor
** Highly offensive foul odor T= Terminated.
% Biodegradation
53
100
90
80
70
60
50
40
30
20
10
0
2
10
12
14
Time (Weeks)
% Biodegradation With Worms of Crushed Food
% Biodegradation With Worms of Intact Food
% Biodegradation Without Worms of Crushed Food
% Biodegradation Without Worms of Intact Food
Figure 3. Rate of Biodegradation of Fried and Oily Food Wastes With and Without Earthworms Under
Identical Climatic Conditions
Figure 4. The Vermiculture Bin
Figure 5. Fried chicken nuggets and fish, calamari

rings, potato fries.
Figure 6. Worm size before vermicomposting.
Figure 7. Worm size and health after

vermicomposting.
54
Figure 8. Food Waste in VC Bin (Half Crushed

and Half Intact) on Day 1 (30.8.05).
Figure 9. Biodegraded (Vermicomposted) food

waste on Day 29 after week 8 (26.10.05).

Worms slowly accept the fried greasy and oily foods although reluctantly in the
beginning, and then more faster, after adapting with new food products. They prefer crushed
food materials.
5.4. Vermicomposting of Sewage Sludge : Some Preliminary Studies

Sludge is an inevitable hazardous and odorous byproduct from the conventional water
and wastewater treatment plants which eventually require safe disposal either in landfills or
by incineration incurring heavy cost. When the sludge is dewatered and dried they are termed
as biosolids. Management of the biosolids remains problematic due to the high cost of
installing sewage sludge stabilization reactors and dehydration systems. Vermicomposting
has been successfully used for treating and stabilizing municipal (water and wastewater
treatment plants) as well as industrial (paper mill, dairy and textile industry) sludge, diverting
them from ending up in the landfills. (Elvira et al. 1998; Lotzof, 1999; Ndegwa and
Thompson 2001; Fraser-Quick, 2002; Contreras-Ramos et al., 2005). The quality of compost
is significantly improved in terms of nutritional and storage value by worms. (Klein et al.,
2005).
The chemical and biological composition of sewage sludge is unpredictable as they may
contain chemicals and pathogens from industry and agriculture. They are potential health
hazard as they have been found to contain high numbers of cysts of protozoa, parasitic ova,
fecal pathogens like Salmonella, Shigella and E. coli and also heavy metals such as zinc (Zn),
cadmium (Cd), mercury (Hg) and copper (Cu). However, they also contains organics and
essential plant nutrients like nitrogen (N), phosphorus (P), potassium (K) and various trace
elements. Sludge is stabilized to reduce or eliminate pathogens and heavy metals, eliminate
offensive odor, and reduce or eliminate the potential for putrefaction. Stabilized sewage
sludge can become good source of organic fertilizer and soil additive for beneficial reuse in
farms. (McCarthy, 2002). Earthworms feed readily upon the sludge components, rapidly
convert them into vermicompost, reduce the pathogens to safe levels and ingest the heavy
55
metals. Volume is significantly reduced from 1 cum of wet sludge (80 % moisture) to 0.5 cum
of vermicompost (30 % moisture). (Eastman, 1999).
Contreras-Ramos et al. (2005) studied the vermicomposting of biosolids (dried sewage
sludge) from various industries but mainly from textile industries and some households
(municipal) mixed with cow manure and oat straw. 1,800, 1,400 and 1000 gms of aerobically
digested biosolids were mixed with 800, 500 and 200 gms of cow manure and 200, 100 and 0
(zero) gms of oat straw in triplicate set up. A control was also kept with only biosolids. Cow
manure was added to provide additional nutrients and the oat straw to provide bulk. 50
earthworms (weighing 40 gm live weight) were added in each sample and the species used
was Eisenia fetida. They were vermicomposted at three different moisture contents 60 %,
70 % and 80 % for two months (60 days). The best results were obtained with 1,800 g
biosolids mixed with 800 g of cow manure and no (0) straw at 70 % moisture content.
Volatile solids of the vermicompost decreased by 5 times, heavy metals concentrations and
pathogens (with no coliforms) were below the limits set by USEPA (1995) for an excellently
stabilized biosolid. Carbon content decreased significantly due to mineralization of organic
matter, and the number of earthworms increased by 1.2 fold.
Vermiprocessing of sludge (biosolids) from the sewage and water treatment plants is
being increasingly practiced in Australia and as a result it is saving large landfill spaces every
year in Australia. The Redland Shire in Queensland started vermi-composting of sludge
(biosolids) from sewage and water treatment plants with the aid of Vermitech Pty. Ltd. in
1997. The facility receives 400-500 tons of sludge every week with 17 % average solid
contents and over 200 tons of vermicast is produced every week by vermicomposting.
(Vermitech, 1998).
The Hobart City Council in Tasmania, Australia, vermicompost and stabilize about 66
cum of municipal biosolids (from sewage sludge) every week, along with green mulch
diverting them from landfills. Zeolite mixed with the biosolids helps balance the pH and also
in absorbing ammonia and odor. About two-third of this volume (44 cum) becomes vermicompost which is sold to public. The City Council is saving AU $ 56,000 per year just from
avoiding landfill disposal (transport and tipping fees etc.). They are earning an equal amount
as revenue from the sale of vermi-compost. (Datar et al., 1997).
5.4.1. Mechanism of Worm Action in Vermicomposting of Sludge : Worms Act as

Sludge Digester
The basic mechanism of worm action is same as it is in the degradation and composting
of general organic waste components of MSW. However, vermistabilization of sludge
involves very complex mechanical, chemical and biological transformation processes and the
resultant product has higher stabilization and soil supplement value than traditional
composting that relies on mechanical incorporation of sludge with green waste in large
compost heaps. It decompose the organics in the sludge, mineralise the nutrients, ingest the
heavy metals and devour the pathogens (bacteria, fungus, nematodes and protozoa) found in
sludge making them chemicals and pathogen free ready to be reused as soil additive and
organic fertilizer. Essentially, it works as a sludge digester which is accomplished in the
following steps1) The sludge is softened by the grume excreted in the mouth of the earthworms and
from there it goes to the esophagus;
56
2) In the esophagus the softened sludge components is neutralized by calcium (Ca)

(excreted by the inner walls of esophagus) and passed on to the gizzard and the
intestine for further enzymatic action.
5.4.2. Experimental Study of Vermicomposting of Sewage Sludge

Brahambhatt (2006) studied the vermicomposting of sewage sludge. The sludge was
obtained from the Oxley Wastewater Treatment Plant in South Brisbane and earthworms were
obtained from Bunnings hardware. It contained mixed species of Eisenia fetida, Perionyx
excavatus and Eudrillus eugeniae. Cow dung was obtained from cattle farm in Ipswich. Both
sewage sludge and the cow manure was partially air dried for 5 days to prevent any methane
and hydrogen sulfide generation that might harm the worms. Vermicompost was obtained
from our vermiculture lab in the School of Environmental Engineering. Five sets of
experimental bins (40 litre HDPE containers) with biosolids were prepared with one as
CONTROL and were studied for morphological (color and texture), biological (pathogens)
and chemical (heavy metals) changes over the 12 weeks period.
Table 6. Status of Coliforms in the Untreated (Control), Vermicomposted and
Microbiologically Composted Biosolids After 12 Weeks
Source: Brahambhatt (2006).

Key: VC = Vermicomposting; * Microbial Composting.
% of stabilisation
% of stabilisation
100.00%
80.00%
60.00%
40.00%
20.00%
0.00%
Biosolids
(CONTROL)
VC Biosolids
by
Earthw orms
VC Biosolids
*Composted
by
Biosolids by
Earthw orms + Cow Dung
Cow Dung
*Composted
Biosolids by
Organic Soil
Nature of samples tested
Figure 10. Status of Coliforms in the Untreated (Control), Vermicomposted and Conventionally
Composted (By Microbial Degradation) Biosolids after 12 Weeks.
57

Results clearly shows that the earthworms significantly reduce or almost eliminates the
pathogens from the digested (composted) sludge. Sludge treated with earthworms (with or
without feed materials) only showed negative results by the Colilert test under the UV lamp.
And this was achieved in just 12 weeks.
It also infers that under the microbial composting systems the pathogens remains in the
sludge for longer time even after treatment until it is completely dry with all food and
moisture exhausted making them difficult to survive.
Table 7. Status of Heavy Metals Cadmium (Cd) and Lead (Pb) in the Untreated
(Control), Vermicomposted and Microbiologically Composted Biosolids (mg/kg of soil)
in 12 Weeks
Source: Brahambhatt (2006).

Key: * Conventional Composting by Microbial Degradation; NC = No Change in Value.
90
80
2.5
70
60
50
1.5
40
30
20
0.5
Lead (mg/kg of soil)
Cadmium (mg/kg of soil)
10
0
Biosolids
(CONTROL)
VC Biosolids VC Biosolids
*Composted
*Composted
by
by
Biosolids by
Biosolids by
Earthw orms
Earthw orms + Cow Dung
Organic Soil
Cow Dung
Nature of sample tested
Cadmium
Lead
Figure 11. Status of Heavy Metals Cadmium (Cd) and Lead (Pb) in the Untreated (Control),
Vermicomposted and Conventionally Composted Biosolids after 12 Weeks.

Results clearly show that the earthworms reduce the heavy metals cadmium (Cd) and
lead (Pb) from the digested sludge. There was no change in the values of heavy metals
between the untreated sludge (Bin 1) and those treated by adding cow dung and by organic
soil (Bins 4 and 5) to enhance microbial composting. Although biosolids can be slowly
58
stabilized by microbial degradation over a period of time, the heavy metals will remain in the
system for quite sometimes after which it may leach into soil or get bound with soil organics
by chemical reactions occurring in the soil.
% Reduction in Cadmium
% Reduction in lead
70%
% Reduction
60%
50%
40%
30%
20%
10%
0%
Biosolids
(CONTROL)
VC Biosolids
VC Biosolids
*Composted
by Earthw orms by Earthw orms
Biosolids by
+ Cow Dung
Cow Dung
*Composted
Biosolids by
Organic Soil
Nature of sample te ste d
Figure 12. Percentage Reduction in Cadmium and Lead in the Untreated (Control), Vermicomposted
and Conventionally Composted Biosolids after 12 Weeks.
Though there was not very significant removal of heavy metals by earthworms in the 12
weeks of experiment yet their role in heavy metal removal cannot be undermined. Providing
additional feed materials (Bin 3) enhanced worm activity and also their number (stimulating
reproduction) and led to greater removal of heavy metals. It infers that over a period of time
and with enhanced worm activity the heavy metals can be completely removed from the
biosolids.
5.5. Critical Factors Affecting Optimal Worm Activity and VermiComposting

1. Moderate Temperature: In general earthworms prefers and tolerates cold and moist
conditions far better than the hot and dry ones. Most worms involved in vermicomposting require moderate temperature between 20 30 C. Heat causes more
problems in vermi-composting than the cold. Red worms are reported to become
inactive above 29 C. They are at the highest levels of both waste degradation and
reproduction activity as the weather cools in the fall and warms in the spring.
2. Adequate Moisture: Earthworms requires plenty of moisture for growth and survival
ranging from 60 to 70 %. The bed should not be too wet as it may create anaerobic
condition adversely affecting worm activity.
3. Adequate Aeration: Vermi-composting is an aerobic process and adequate flow of air
in the waste biomass is essential for worm function. Worms breath through their skin
and need plenty of oxygen in the surrounding areas. Although worms constantly
aerate their habitat by burrowing actions, periodical turning of waste biomass can
improve aeration and biodegradation.
59
4. Neutral pH (7.0): Earthworms are sensitive to pH. The decomposition of organic

matter produces organic acids that lower the pH of the bedding soil and impair
worm activity. Although the worms can survive in a pH range of 4.5 to 9 but
functions best at neutral pH of 7.0. Although worms can lower pH of its medium by
secreting calcium (Ca), it is suggestive to add ground limestone (calcium carbonate)
powder to the bedding periodically. This would serve two purposes- maintain neutral
pH and also supply the much needed calcium (Ca) to the worms for its metabolism.
5. Carbon / Nitrogen (C/N) Ratio of the Feed Material: High C/N ratio above 30:1 in
waste biomass can impair worm activity and vermi-composting. Although
earthworms help to lower the C/N ratio of fresh organic waste, it is advisable to add
nitrogen supplements such as cattle dung or pig and goat manure or even kitchen
waste (which are rich in nitrogen contents) when waste materials of higher C/N ratio
exceeding 40:1 such as the green cellulosic wastes (grass clippings) are used for
vermi-composting.
6. Adequate Supply of Calcium (Ca): Calcium appears to be important mineral in worm
biology (as calcarious tissues) and biodegradation activity. Although most organic
waste contains calcium, it is important to add some additional sources of calcium for
good vermi-composting. Egg shells are good source of natural calcium. Occasionally
limestone powder should be added.
5.6. Advantages of Vermi-Composting

Earthworms have real potential to both increase the rate of aerobic decomposition and
composting of organic matter and also to stabilize the organic residues in the MSW and
sludge removing the harmful pathogens and heavy metals from the compost. The quality of
compost is significantly better, rich in key minerals and beneficial soil microbes. In fact in the
conventional aerobic composting process which is thermophilic (temperature rising up to 55
C) many beneficial microbes are killed and nutrient especially nitrogen is lost (due to
gassing off of nitrogen).
Vermicomposting by earthworms excels all other bio-degradation and bio-conversion
technologies by the fact that - it can utilize waste organics that otherwise cannot be utilized by
others; achieve greater utilization (rather than the destruction) of materials that cannot be
achieved by others; and by the fact that it does all with enzymatic actions and enzymes are
biological catalysts giving pace and rapidity to all biochemical reactions even in minute
amounts. It also keeps the system fully aerated with plenty of oxygen available to aerobic
decomposer microbes. Aerobic processes are about 10 times faster than the anaerobic.
1. Nearly Odor-free Process

Earthworms create aerobic conditions in the waste materials by their burrowing actions,
inhibiting the action of anaerobic micro-organisms which release foul-smelling hydrogen
sulfide and mercaptans.
60
2. Destroy Pathogens in the End Product (Compost) Making Them Pathogen Free
The earthworms release coelomic fluids that have anti-bacterial properties and destroy all
pathogens in the waste biomass. (Pierre et al.,1982). They also devour the protozoa, bacteria
and fungus as food. They seems to realize instinctively that anaerobic bacteria and fungi are
undesirable and so feed upon them preferentially, thus arresting their proliferation. More
recently, Dr. Elaine Ingham has found in her research that worms living in pathogen-rich
materials (e.g. sewage and sludge), when dissected, show no evidence of pathogens beyond 5
mm of their gut. This confirms that something inside the worms destroys the pathogens, and
excreta (vermicast) becomes pathogen-free (Appelhof, 2003).
In the intestine of earthworms some bacteria and fungus (Pencillium and Aspergillus)
have also been found (Singelton et. al, 2003). They produce antibiotics and kills the
pathogenic organisms in the sewage sludge making it virtually sterile. The removal of
pathogens, faecal coliforms (E.coli), Salmonella spp., enteric viruses and helminth ova from
sewage and sludge appear to be much more rapid when they are processed by E. fetida. Of all
E.coli and Salmonella are greatly reduced. (Bajsa et al., 2003).
Bajsa et al. (2004 and 2005) studied the pathogen die-off in vermicomposting of sewage
sludge spiked with E.coli, S.typhimurium and E.faecalis at the 1.6-5.4 x 106 CFU/g , 7.25 x
105 CFU/g and3-4 x 10 4 CFU/g respectively. The composting was done with different
bulking materials such as lawn clippings, sawdust, sand and sludge alone for a total period of
9 months to test the pathogen safety of the product for handling. It was observed that a safe
product was achieved in 4-5 months of vermicomposting and the product remained the same
quality without much reappearance of pathogens after in the remaining months of the test.
Eastman (1999) and Eastman et. al., (2001) also studied significant human pathogen
reduction in biosolids vermicomposted by earthworms.
Lotzof (2000) also revealed that the pathogens like enteric viruses, parasitic eggs and
E.coli were reduced to safe levels in sludge vermicast. Cardoso and Remirez (2002) reported
a 90 % removal of fecal coliforms and 100 % removal of heliminths from sewage sludge and
water hyacinth after vermicomposting.
Contreras-Ramos et, al., (2005) also confirmed that the earthworms reduced the
population of Salmonella spp. to less than 3 CFU/gm of vermicomposted sludge. There were
no fecal coliforms and Shigella spp. and no eggs of helminths in the treated sludge. (Eastman
et al.,2001; Kumar and Sekaran, 2004). Our studies, Brahmbhatt (2006) has also confirmed
complete removal of coliforms by earthworms.
In conventional aerobic composting system, which is thermophilic process, pathogens
are killed due to high temperature (beyond 55 C). Wu and Smith (1999) studied that for
efficient composting and pathogen reduction a temperature of 55 C must be maintained for
15 consecutive days. But this also kills several beneficial microbes. If this high temperature is
not achieved which could be the case in small scale composting pathogen die off will not be
effective. Vermicomposting is a mesophilic composting system where temperature does not
increase beyond 30 C and the harmful microbes (pathogens) are killed selectively by the
worms through biological (physiological), microbial and enzymatic actions. Nair et al. (2006)
studied a combination of thermophilic followed by vermicomposting and observed that the
combination of the process leads to faster reduction of pathogens than the same period of
thermophilic composting (21 days) and after 2 and 3 months (Table 9). It was also noticed
that 21 days of a combination of thermocomposting and vermicomposting produced compost
61
with acceptable C:N ratio and good homogenous consistency of a fertilizer. However the E.
coli and E. faecalis was greater than 110 MPN/g) while S. typhimurium was undetectable.
The optimum period to obtain pathogen-free compost was 9 days of thermophilic composting
followed by 2.5 months of vermicomposting (Nair et. al., 2006).
The study also indicated that vermicomposting leads to greater reduction of pathogens
after 3 months upon storage. Whereas, the samples which were subjected to only thermofilic
composting, retained higher levels of pathogens even after 3 months.
Table 8. Removal of Pathogens (E. coli and E. faecalis) in Thermophilic Composting
Vis-a-Vis Vermicomposting Processes
Source: Nair et. al., (2006)

Key: dT = days of Thermophilic composting; dV = days of Vermicomposting
N.B. E.coli and E. faecalis was tested using the Most Probable Number (MPN) per gram of compost
(Standards Australia, 1995 a and b respectively).
3. Vermicompost Is Free of Toxic Chemicals

Several studies have found that earthworms effectively bio-accumulate or biodegrade
several organic and inorganic chemicals including heavy metals, organochlorine pesticide
and polycyclic aromatic hydrocarbons (PAHs) residues in the medium in which it feeds.
(Nelson et al., 1982; Ireland, 1983).
4. Low Greenhouse Gas Emissions by Vermi-composting of Waste
Emission of greenhouse gases carbon dioxide (CO2), methane (CH4) and nitrous oxide
(N2O) in waste management programs of both garbage and sewage has become a major
global issue today in the wake of increasing visible impacts of global warming.
Biodegradation of organic waste has long been known to generate methane (CH4). Studies
have also indicated high emissions of nitrous oxide (N2O) in proportion to the amount of food
waste used, and methane (CH4) is also emitted if the composting piles contain cattle manure.
N2O emission is relatively high at the beginning of the conventional aerobic composting
process, but declines after two days. N2O is mainly formed under moderate oxygen (O2)
concentration. High emission of N2O at the beginning might be due to the metabolism of the
microbial community coming from food waste, as food waste have been found to generate
N2O even stored at 40 C. (Toms et, al. 1995; Wu et, al. 1995; Wang et, al., 1997; and Yaowu
et. al., 2000).
In theory, vermicomposting by worms should provide some potentially significant
advantages over conventional composting with respect to GHG emissions. Worms
significantly increase the proportion of aerobic to anaerobic decomposition in the compost
62
pile by burrowing and aerating actions leaving very few anaerobic areas in the pile, and thus
resulting in a significant decrease in methane (CH4) and also volatile sulfur compounds which
are readily emitted from the conventional (microbial) composting process.(Mitchell et al.,
1980). Molecule to molecule, methane is 20-25 times more powerful GHG than the CO2.
Earthworms can play a good part in the strategy of greenhouse gas reduction and mitigation
in the disposal of global organic wastes as landfills also emit methane resulting from the slow
anaerobic decomposition of waste organics over several years.
Recent researches done in Germany has found that earthworms produce a third of nitrous
oxide (N2O) gases when used for vermicomposting (Frederickson, 2007). Molecule to
molecule N2O is 296 to 310 times more powerful GHG than carbon dioxide (CO2). However,
analysis of vermicompost samples has shown generally higher levels of available nitrogen (N)
as compared to the conventional compost samples made from similar feedstock. This implies
that the vermicomposting process by worms is more efficient at retaining nitrogen (N) rather
than releasing it as N2O. This needs further studies and is being done at Griffith University,
Brisbane, Australia (Middleditch, 2008).
5. More Homogenous End Products Rich in Nutrient

The advantages for employing vermi-composting to process and stabilize organic waste
including sewage sludge over conventional composting by microbial action are that the end
product are more homogenous, rich in plant nutrients and the levels of contaminants are
significantly reduced. Lotzof, 1999). McCarthy (2002) asserts that vermicomposting of
sewage sludge has converted them into an end product that is safe for agricultural use.
Vermicompost has very high porosity, aeration, drainage and water holding capacity
and also contains plant-available nutrients. The resulting product appears to retain more
nutrients for longer period of time and also greatly increases the water holding capacity of the
farm soil (Hartenstein and Hartenstein, 1981; Appelhof, 1997).
Table 9. Initial and Final Nutritional Quality of the Vermicomposted Sludge
Source: Bajsa et al., 2005

Key: SL Sludge; LC Lawn clippings; SD Sawdust; S Sand
The chemical analyses of casts showed two times more available magnesium, 15 times
more available nitrogen and seven times more available potassium compared to the
surrounding soil (Kaviraj and Sharma, 2003). This is an excellent biofertilizer and soil
conditioner (Jensen, 1998).
6. No or Low Energy Use in Vermi-composting Process

Conventional microbial composting requires energy for aeration (constant turning of
waste biomass and even for mechanical airflow) and sometimes for mechanical crushing of
63
waste to achieve uniform particle size. Vermi-composting do not involve such use of energy.
Occasionally turning may be required for aeration which can be done manually.
7. Production of Earthworm Biomass : A Nutritive Earthworm Meal

Large scale vermi-composting of sludges would result into tons of earthworms biomass
every year as under favorable conditions earthworms can double their number at least every
60 70 days. Potentially large quantities of worm biomass will be available as pro-biotic
food for the cattle and fish farming, after the first year of composting. This can be a good
source of nutritive worm meal rich in essential amino acids lysine and methionine. It is
superior to even fish meal.
8. Mineralization of Nutrients from the Sludge
Earthworms accelerates the decomposition of the sludge and mineralization of the
organic compounds in it. Most important is that earthworms mineralize the nitrogen (N) and
phosphorus (P) in the sludge to make it bio-available to plants as nutrients. They ingest
nitrogen from the sludge and excrete it in the mineral form as ammonium and muco-proteins.
The ammonium in the soil is bio-transformed into nitrates. Phosphorus (P) contents increased
in the vermicomposted sludge treated with earthworms but decreased in the samples without
earthworms, while the nitrogen (N) content did not show much difference (Parvaresh, et. al.,
2004). Elvira et, al,. (1998) reported increase in the potassium (K) content of the sludge
vermicompost.
9. Decrease in Total Organic Carbon (TOC) and Lowering of C/N Ratio of Sludge
This has significance when the composted sludge is added to soil as fertilizer. Plants
cannot absorb and assimilate mineral nitrogen unless the carbon to nitrogen (C/N) ratio is
about 20:1 or lower. Mineralization of organic matter in the sewage sludge by earthworms
lead to significant decrease in total organic carbon (TOC) content thus lowering the C/N ratio.
This they do by consuming and breaking carbon compounds during respiration. Elvira et al.,
(1998) found that vermicomposting of paper-pulp-mill sewage sludge for 40 days decreased
carbon (C) content by 1.7 fold. Contreras-Ramos et al., (2005) found that carbon content
decreased by 1.1 to 1.4 fold in two months. Our work (Brahambhatt, 2006) also indicated that
earthworms reduce TOC from composted sludge.
10. Reduction in Volatile Solids (VS) from Sludge
Maximum reduction of the volatile solids (VS) is the goal of any sludge stabilization
system and reduced VS is an indicator of stabilized sludge. Study found that Elsenia fetida
increases the rate of volatile solid sludge (VSS) destruction when present in aerobic sludge
and this reduces the probability of putrefaction occurring in the sludge due to anaerobic
conditions. (Loehr, et al,1998). Earthworms creates and maintains good aerobic conditions in
the sludge due to its burrowing actions and this enhances the process of VSS destruction.
Hartenstein and Hartenstein (1981) reported a 9 % reduction in volatile solids over 4 weeks of
sludge vermicomposting by earthworms, which was higher than that of control by almost
one-third. Fredrickson et al., (1997) found a VS reduction of 30 % in compost after 4 months
of conventional composting, whereas, the reduction was 37 % after only 2 months of
64
vermicomposting. Higher decrease means more stable product and earthworms plays
important role.
6. THE VERMIFILTRATION TECHNOLOGY (VFT)

Vermifiltration of wastewater using waste eater earthworms is a newly conceived novel
technology with several advantages over the conventional systems. Earthworms body work as
a biofilter and they have been found to remove the 5 days BOD (BOD5) by over 90 %, COD
by 80-90 %, total dissolved solids (TDS) by 90-92 % and the total suspended solids (TSS) by
90-95 % from wastewater by the general mechanism of ingestion and biodegradation of
organic wastes, heavy metals and solids from wastewater and also by their absorption
through body walls. Suspended solids are trapped on top of the vermifilter and processed by
earthworms and fed to the soil microbes immobilized in the vermifilter.
The most significant part is that there is no sludge formation in the process as the
worms eat the solids simultaneously and excrete them as vermicast. (Huges et. al., 2005).
This plagues most councils in world as the sludge being a biohazard requires additional
expenditure on safe landfill disposal. This is also an odor-free process and the resulting
vermi-filtered water is clean enough to be reused in industries as cooling water and also
highly nutritive to be reused for farm irrigation.
6.1. Vermifiltration of Municipal Wastewater (Sewage)

Sewage is the cloudy fluid of human fecal matter and urine, rich in minerals and organic
substances. Water is the major component (99 %) and solid suspension amounts to only 1 %.
The biochemical oxygen demand (BOD) and oxygen consumption (OC) values are extremely
high demanding more oxygen by aerobic microbes for biodegradation of organic matter.
Dissolved oxygen (DO) is greatly depleted. Low oxygen in water leads to anaerobic
decomposition of organic contents and emission of obnoxious gases like hydrogen sulfide,
methane and carbon monoxide. The nitrogen (N) and phosphorus (P) contents are very high
and there are heavy metals like cadmium (Cd) and significant amount of coliform bacteria.
The total suspended solids (TSS) is also very high like the BOD and nutrients and this often
leads to a high anaerobic microenvironment in the sediments.
BOD, COD and SS Values of Raw Sewage and the Acceptable Values of Treated
Sewage
The average BOD value of the raw sewage ranged between 200 400 mg/L, COD ranged
between 116 -285 mg/L, the TSS ranged between 300 350 mg/L and the pH ranged between
6.9 7.3. There is great fluctuation in these values depending upon catchment area, flow rate
and season. Sewage from industrial areas can have high COD values, very low or high pH,
due to accidental mixing of industrial wastewater. The normal acceptable values for BOD in
treated wastewater is 1-15 mg/L, COD is 60-70 mg/L, TSS 20-30 mg/L and pH around 7.0.
65
6.2. The Mechanism of Worm Action in Vermifiltration

1) The twin processes of microbial stimulation and biodegradation, and the enzymatic
degradation of waste solids by worms as discussed above simultaneously work in the
vermifiltration system too.
2) Vermifilters provide a high specific area up to 800 sq m/g and voidage up to 60 %.
Suspended solids are trapped on top of the vermifilter and processed by earthworms
and fed to the soil microbes immobilized in the vermifilter.
3) Dissolved and suspended organic and inorganic solids are trapped by adsorption and
stabilized through complex biodegradation processes that take place in the living
soil inhabited by earthworms and the aerobic microbes. Intensification of soil
processes and aeration by the earthworms enable the soil stabilization and filtration
system to become effective and smaller in size.
4) Earthworms intensify the organic loadings of wastewater in the vermifilter soil bed
by the fact that it granulates the clay particles thus increasing the hydraulic
conductivity of the system. They also grind the silt and sand particles, thus giving
high total specific surface area, which enhances the ability to adsorb the organics
and inorganic from the wastewater passing through it. The vermicast produced on the
soil bed also offers excellent hydraulic conductivity of sand (being porous like sand)
and also high adsorption power of clay. This is ideal for diluted wastewater like
sewage. (Bhawalkar, 1995).
5) Earthworms also grazes on the surplus harmful and ineffective microbes in the
wastewater selectively, prevent choking of the medium and maintain a culture of
effective biodegrader microbes to function.
6.3. Critical Factors Affecting Vermifiltration of Wastewater

1. Worm Population and Density (Biomass) in Vermifilter Bed (Soil)
As the earthworms play the critical role in wastewater purification their number and
population density (biomass) in soil, maturity and health are important factors. This may
range from several hundreds to several thousands. There are reports about 8-10,000 numbers
of worms per square meter of the worm bed and in quantity (biomass) as 10 kg per cubic
meter (cum) of soil for optimal function.
2. Hydraulic Retention Time (HRT)
Hydraulic retention time is the time taken by the wastewater to flow through the soil
profile (vermifilter bed) in which earthworms inhabits. It is very essential for the wastewater
to remain in the vermifiltration (VF) system and be in contact with the worms for certain
period of time. HRT depends on the flow rate of wastewater to the vermifiltration unit,
volume of soil profile and quality of soil used. HRT is very critical, because this is the actual
time spent by earthworms with wastewater to retrieve organic mater from it as food. During
this earthworms carry out the physical and biochemical process to remove nutrients,
ultimately reducing BOD, COD and the TDSS. The longer wastewater remains in the system
in contact with earthworms, the greater will be the efficiency of vermi-processing and
66
retention of nutrients. Hence the flow of wastewater in the system is an important

consideration as it determines the retention of suspended organic matter and solids (along
with the chemicals adsorbed to sediment particles). Maximum HRT can results from slower
rate of wastewater discharge on the soil profile (vermifilter bed) and hence slower
percolation into the bed. Increasing the volume of soil profile can also increase the HRT. The
number of live adult worms, functioning per unit area in the vermifilter (VF) bed can also
influence HRT.
HRT of vermifiltration system can be calculated as
HRT = ( x Vs) / Q wastewater
where;
HRT = theoretical hydraulic retention time (hours)
Vs = volume of the soil profile (vermifilter bed), through which the wastewater flow and
which have live earthworms (cum)
= porosity of the entire medium (gravel, sand and soil) through which wastewater flows
Q wastewater = flow rate of wastewater through the vermifilter bed (cum / hr)
Thus the hydraulic retention time (HRT) is directly proportion to the volume of soil
profile and inversely proportion to the rate of flow of wastewater in the vermifilter bed.
3. Hydraulic Loading Rate (HLR)

The volume and amount of wastewater that a given vermifiltration (VF) system
(measured in area and depth of the soil medium in the vermifilter bed in which the
earthworms live) can reasonably treat in a given time is the hydraulic loading rate of the
vermifilter (VF) system. HLR can thus be defined as the volume of wastewater applied, per
unit area of soil profile (vermifilter bed) per unit time. It critically depends upon the number
of live adult earthworms functioning per unit area in the vermifilter bed. The size and health
of the worms is also critical for determining the HLR.
HLR of vermi-filtration system can be calculated as
HLR = V wastewater / (A X t)
whereLR = Hydraulic Loading Rate (m / hr)
V wastewater = volumetric flow rate of wastewater (cum).
A = Area of soil profile exposed (sqm).
t = Time taken by the wastewater to flow through the soil profile (hr).
High hydraulic loading rate leads to reduced hydraulic retention time (HRT) in soil and
could reduce the treatment efficiency. Hydraulic loading rates will vary from soil to soil. The
infiltration rates depend upon the soil characteristics defining pore sizes and pore size
67
distribution, soil morphological characteristics, including texture, structure, bulk density, and
clay mineralogy.
6.4. Some Preliminary Studies on Vermifiltration of Sewage

A pilot study on vermifiltration of sewage was made by Xing et al,. (2005) at Shanghai
Quyang Wastewater Treatment Facility in China. The earthworm bed which was 1m (long) x
1m (wide) x 1.6m (high), was composed of granular materials and earthworms. The worms
number was kept at about 8000 worms/sqm. The average chemical oxygen demand (COD)
value of raw sewage used was 408.8 mg/L that of 5 days biological oxygen demand (BOD5)
was 297 mg/L that of suspended solids (SS) was 186.5 mg/L. The hydraulic retention time
varied from 6 to 9 hours and the hydraulic loading from 2.0 to 3.0 m3 / (m2.d) of sewage. The
removal efficiency of COD ranged between 81-86 %, the BOD5 between 91-98 %, and the SS
between 97-98 %.
Gardner et. al.,(1997) studied on-site effluent treatment by earthworms and showed that it
can reduce the BOD and COD loads significantly. Taylor et. al., (2003) studied the treatment
of domestic wastewater using vermifilter beds and concluded that worms can reduce BOD
and COD loads as well as the TDSS (total dissolved and suspended solids) significantly by
more than 70-80 %. Hartenstein and Bisesi (1989) studied the use of earthworm for the
management of effluents from intensively housed livestock which contain very heavy loads of
BOD, TDSS and nutrients nitrogen (N) and phosphorus (P). The worms produced clean
effluents and also nutrient rich vermicompost. Bajsa et. al., (2003) also studied the
vermifiltraion of domestic wastewater using vermicomposting worms with significant results.
6.5. Experimental Study of Vermifiltration of Sewage

Chaudhry (2006) studied the vermifiltration of sewage using earthworms. The raw
sewage was obtained from the Oxley Wastewater Treatment Plant in South Brisbane. The
study was carried out in the same vermicomposting (VC) bin which was made to work as the
vermifiltration (VF) kit. This was located in PC2 lab of Griffith University, as a hazardous
material (raw sewage) was to be used. The temperature in lab was maintained at 21.50C, with
50% humidity. The Vermifiltartion kit contained about 30-40 kg of gravels with a layer of
garden soil on top. This formed the vermifilter bed. It has provisions to collect the filtered
water at the bottom in a chamber which opens out through a pipe fitted with tap. Above the
chamber lies the net of wire mesh to allow only water to trickle down while holding the
gravels above. The bottommost layer is made of gravel aggregates of size 7.5 cm and it fills
up to the depth of 25 cm. Above this lies the aggregates of 3.5 to 4.5 cm sizes filling up to
another 25 cm. On the top of this is the 20 cm layer of aggregates of 10-12 mm sizes mixed
with sand. The topmost layer of about 10 cm consists of pure soil in which the earthworms
were released.
The worms were given around one week settling time in the soil bed to acclimatize in the
new environment. As the earthworms play the critical role in wastewater purification their
number and population density (biomass) in soil, maturity and health are important factors.
This may range from several hundreds to several thousands. There are reports about 8-10,000
68
numbers of worms per cubic meter of the worm bed and in quantity (biomass) as 10 kg per
cubic meter (cum) of soil for optimal function. (Komarowski, 2001).However, in this
experiment we started with 500 worms in 0.025 cum of soil bed, which comes to about
20,000 worms per cum of soil. This means even lesser number of worms could have done the
job.
The Control Kit without Earthworms: Assessing the Precise Role of Worms
A control kit (exact replica of vermifilter kit but devoid of earthworms) was also
organised for reference and comparison. It is important to note that the soil and sand particles
and the gravels in the kit also contribute in the filtration and cleaning of wastewater by
adsorption of the impurities on their surface. They provide ideal sites for colonization by
decomposer microbes which works to reduce BOD, COD and the TDSS from the wastewater.
As the wastewater passes through, a layer of microbial film is produced around them and
together they constitute the geological and the microbial (geo-microbial) system of
wastewater filtration.
With more wastewater passing through the gravels there is more formation of biofilms
of decomposer microbes. Hence it is important to have a control kit in order to determine the
precise role of earthworms in the removal of BOD, COD and the TDSS.
Experiences, however, have shown that the geo-microbial system gets choked after
sometimes due to slow deposition of wastewater solids as sludge and becomes unoperational whereas, the vermifiltration system with earthworms continues to operate.
The Experimental Procedures
Around 5 6 litre of municipal wastewater (sewage) was kept in calibrated 10 litre
capacity PVC drum. These drums were kept on an elevated platform just near the vermifilter
kit, as shown in figure 1. The PVC drums had tap at the bottom to which an irrigation system
was attached. The irrigation system consisted of simple 0.5 inches polypropylene pipe with
holes for trickling water that allowed uniform distribution of wastewater on the soil surface
(vermifilter bed).
Figure 13. Vermifilter Kits : Regulated flow of wastewater from storage drums determining 12 hrs
HRT.
Figure 14.
Figure 15.
Figure 16.
Figure 17.
69
Figures 14, 15, 16. Different sizes of gravels used in preparation of the vermifilter bed.
Figure 17. Soil bed with earthworms and the device of wastewater discharge and distribution through
perforated PVC pipes.
Wastewater from the drums flowed through the irrigation pipes by gravity. The
wastewater percolated down through various layers in the vermifilter bed passing through the
soil layer inhabited by earthworms, the sandy layer and the gravels and at the end was
collected in a chamber at the bottom of the kit. Next day this treated wastewater from both
kits were collected and analysed for BOD5 , COD and the TSS. The hydraulic retention time
(HRT) was kept uniformly between 1-2 hours in all experiments.
Key Parameters Studied in Sewage Vermifiltration

a) Biochemical Oxygen Demand (BOD)
The biodegradable organic matter in wastewater is expressed as BOD (Biochemical
Oxygen Demand). It is the amount of oxygen needed in a specified volume of wastewater to
decompose organic material by the aerobic microbes. The unit of BOD is ppm or mg / L.
b) Chemical Oxygen Demand (COD)
Many organic substances, which are difficult to oxidize biologically by aerobic microbes,
or are toxic to micro-organisms, such as lignin, can be oxidized chemically by using strong
oxidizing agent like dichromate (Cr2O7) in acidic media. The unit of COD is ppm or mg / L.
70
Earthworms can ingest and remove several organic substances from the wastewater
which otherwise cannot be oxidized by microbes and thus bring down the COD values
significantly.
c) Total Suspended Solid (TSS)

Solids in wastewater consist of organic and inorganic particles and they can either be
suspended or dissolved. They provide adsorption sites for chemical and biological
contaminants. As suspended solids degrade biologically, they can create toxic by-products.
TSS can be represented as mg/L or ppm
d) Turbidity
Turbidity is measured as the cloudiness of water and this is caused by suspended and
colloidal particles, such as clay, slit, finely divided organic matter. Turbidity is measured in
NTU ( Number of Transfer Units).
e) pH Value
pH is a symbol that represents negative base -10 logarithm of the effective concentration
of hydrogen ions (H+) in moles per Lit.
The Analytical Methods Used in Laboratory Study
The untreated sewage that was fed to the vermifilter kit and treated sewage which was
collected at the bottom of the kit in a chamber were analyzed to study the biological oxygen
demand (BOD), chemical oxygen demand (COD), (TSS), turbidity and the pH value.
Following standard methods of analysis were adopted,
Table 10. Standard Methods adopted to study specified parameters
SR. No.
1
2
3
4
5
Description
Biological Oxygen Demand (BOD)
Chemical Oxygen Demand (COD)
Total Suspended Solids (TSS)
Turbidity
PH
Standard Methods
405.1
5220C
160.2
180.1
D1293-99
The Experimental Results and Discussion

a) Removal of BOD5
Results shows that the earthworms can remove BOD (BOD5) loads by over 98 % or
nearly complete at hydraulic retention time (HRT) of 1-2 hours (Chowdhary, 2006). BOD
removal in the control kit (where only the geological and microbial system works) is just
around 77 %. (Table 11).
71
Table 11. BOD5 Removal of Municipal Wastewater (Sewage) Treated by Earthworms

(Vermifiltered) and Without Earthworms (Control) (HRT: 1- 2 hrs)
Expt
No
Untreated
Raw Sewage
BOD5 (mg/L)
Treated Sewage BOD5 (mg/L)

With Worms
Without Worms
(Vermifiltered) (CONTROL)
1
2
3
4
5
309
260
316
328
275
1.97
4.00
6.02
2.06
4.25
86.3
45.8
63.3
83.2
63.5
% Reduction
in BOD5 by
Earthworms
(Vermifiltered)
99.4
98.5
98.1
99.4
98.5
% Reduction in
BOD5 without
Earthworms
(CONTROL)
72.1
82.4
80.0
74.6
76.9
% Reduction in BOD5
Av. = 98.78 % Av. = 77.2 %.

Source: Chowdhary (2006).
100
90
80
70
60
50
40
30
20
10
0
1
Experim ent No
% Reduction in BOD5 by Earthworms (Vermifiltered)

% Reduction in BOD5 without Earthworms (CONTROL)
Figure 18. % Reduction in BOD5 of swage Water Treated With and Without Earthworms.
b) Removal of COD
Results shows that the average COD removed from the sewage by earthworms is over 45
% while that without earthworms (only the geological and microbial system in the control kit)
is just over 18 %. (Chowdhary, 2006). (Table 12). COD removal by earthworms is not as
significant as the BOD, but at least much higher than the microbial system.
Table 12. COD Removal of Municipal Wastewater (Sewage) Treated by Earthworms
(Vermifiltered) and Without Earthworms (Control) (HRT : 1 - 2 hrs)
Expt
No
Untreated
Raw Sewage
COD (mg/L)
Treated Sewage COD (mg/L)

With Worms
Without Worms
(Vermifiltered)
(CONTROL)
1
2
3
4
5
293
280
280
254
260
132
153
128
112
139
Av. = 45.7 % Av. = 18.4 %.

245
235
201
217
227
% Reduction
in COD by
Earthworms
(Vermifiltered)
54.9
45.4
54.3
55.9
46.5
% Reduction in
COD without
Earthworms
(CONTROL)
16.4
16.1
28.2
14.6
12.7
72
% Reduction in COD
100
90
80
70
60
50
40
30
20
10
0
1
Experim ent No
% Reduction in COD by Earthworms (Vermifiltered)

% Reduction in COD without Earthworms (CONTROL)
Figure 19. % Reduction in COD of Sewage Wastewater Treated With and Without Earthworms.
c) Removal of TSS
Results shows that the earthworms can significantly remove the suspended solids from
the sewage by over 90 %, which in the control kit (where geological and microbial system
works together) is over 58 % only. (Chowdhary, 2006). (Table 13).
Table 13. TSS Removal Efficiency of Sewage Wastewater Treated by Earthworms
(Vermifiltered) and Without Earthworms (Control) (HRT 1 - 2 hrs)
Expt
No
Raw Untreated
Wastewater
TSS (mg/L)
Treated Wastewater TSS (mg/L)

With Worms
Without Worms
(Control)
1
2
3
4
5
390
374
438
379
407
28
24
22
27
25
116
190
184
179
168
% Reduction
in TSS by
Earthworms
(Vermifiltered)
92.82
93.58
94.97
92.87
93.85
% Reduction in
TSS without
Earthworms
(CONTROL)
70.25
49.19
57.99
52.77
58.72
Av. = 92.97 % Av. = 58.34 %. Source: Chowdhary (2006).
Table 14. Turbidity Removal of Municipal Wastewater (Sewage) Treated by

Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT : 1- 2 hrs)
Expt
No
1
2
3
4
5
Untreated Raw
Sewage Turbidity
(NTU)
112
120
74
70
100
Treated Sewage Turbidity (NTU)

With Worms
(Vermifiltered)
1.5
0.6
1.1
1.2
1.1
Without Worms
(CONTROL)
3.6
1.5
1.2
1.8
2.0
Av.= 98.78 % Av.= 97.88 %. Source: Chowdhary (2006).
% Reduction in
Turbidity by
Earthworms
(Vermifiltered)
98.7
99.5
98.5
98.3
98.9
% Reduction in
Turbidity Without
Earthworms
(CONTROL)
96.8
98.8
98.4
97.4
98.0
73
% Reduction in BOD5
d) Turbidity Removal
Results indicates that the average reduction in turbidity by earthworms is over 98 %
while that without earthworms in the control kit is also significantly high and over 97 %.
(Chowdhary, 2006). (Table 14).
100
90
80
70
60
50
40
30
20
10
0
1
Experim ent No
% Reduction in TSS by Earthworms (Vermifiltered)

% Reduction in TSS without Earthworms (CONTROL)
Figure 20. % Reduction in TSS of Sewage Wastewater Treated With and Without Earthworms.
% Reduction in Turbidity
100
80
% Reduction
in Turbidity by
Earthworms
(Vermifiltered)
60
40
20
0
1
% Reduction
in Turbidity
Without
Earthworms
(CONTROL)
Experiment No
Figure 21. % Reduction in Turbidity of sewage treated with and without Earthworms.
e) Improvement in pH Value of Treated Sewage

Results indicates that the pH value of raw sewage is almost neutralized by the
earthworms in the vermifilter kit. pH value of treated sewage without earthworms also
improved but it was not consistent in all experiments. (Table 15).
74
Table 15. Improvement in pH Value of Municipal Wastewater (Sewage) Treated by

Earthworms (Vermifiltered) and Without Earthworms (Control)
Expt. No
Untreated Raw Sewage pH
1
2
3
4
5
6.58
5.76
6.65
6.67
6.05
Treated Sewage pH
With Worms
Without Worms
(Vermifiltered)
(CONTROL)
7.05
6.62
7.35
6.05
7.00
7.47
7.25
6.95
7.06
7.15
Figure 22. Appearance of Sewage After and Before Treatment. Bottle A : The clear vermifiltered
sewage water; Bottle B: The hazy water from the controlled kit; Bottle C : The turbid and cloudy
sewage water.
6.5. Significance and Advantages of Vermi-filtration Technology Over

Conventional Wastewater Treatment Systems
Vermi-filtration system is low energy dependent and has distinct advantage over all the
conventional biological wastewater treatment systems- the Activated Sludge Process,
Trickling Filters and Rotating Biological Contactors which are highly energy intensive,
costly to install and operate and do not generate any income. Since the conventional
technologies are mostly the flow-processes and have finite hydraulic retention time (HRT) it
always results into a residual stream of complex organics and heavy metals (while only the
simple organics are consumed) in the sludge that needs further treatment (requiring more
energy) before landfill disposal. This becomes unproductive. In the vermifilter process there
75
is 100 % capture of organic materials, the capital and operating costs are less, and there is
high value added end product (vermicompost).
1) Vermifilter Treatment of Wastewater is an Odorless System

There is no foul odor as the earthworms arrests rotting and decay of all putrescible
matters in the wastewater and the sludge. They also create aerobic conditions in the soil bed
and the waste materials by their burrowing actions, inhibiting the action of anaerobic microorganisms which release foul odor.
2) Vermi-filtration is Low Energy System
Some energy may be required in pumping the wastewater to the vermi-filtration unit if
gravity flow is not adequate.
3) Synchronous Treatment of Wastewater and the Solids : End Products Become
Useful Resource for Agriculture and Horticulture
Earthworms decompose the organics in the wastewater and also devour the solids (which
forms the sludge) synchronously and ingest the heavy metals from both mediums. This
stabilized solids is discharged in the vermifilter bed as excreta (vermicompost) which is
useful soil additive for agriculture and horticulture.
4) Vermifiltered Wastewater is Free of Pathogens
As the earthworms devour on all the pathogens (bacteria, fungus, protozoa and
nematodes) in the medium in which they inhabit the resulting filtered water becomes free of
pathogens. The celeomic fluid secreted by worms which has anti-bacterial properties (Pierre
et, al., 1982) further eliminate the pathogens.
5) Vermifiltered Wastewater is Free of Toxic Chemicals (Heavy Metals and Endocrine
Disrupting Chemicals)
As earthworms have the capacity to bio-accumulate high concentrations of toxic
chemicals including heavy metals in their tissues the resulting wastewater becomes almost
chemical-free. Earthworms have also been reported to bio-accumulate endocrine disrupting
chemicals (EDCs) from sewage. Markman et al. (2007) have reported significantly high
concentrations of EDCs (dibutylphthalate, dioctylphthalate, bisphenol-A and 17 - estrdiol)
in tissues of earthworms (E.fetida) living in sewage percolating filter beds and also in garden
soil.
6.6. Decentralized Sewage Treatment by Vermifiltration at Source of

Generation
All above results were obtained with approximately 500 worms in the vermifilter bed
made in about 0.032 cubic meter (cum) of soil. This comes to approximately 16,000 worms
per cum of soil. The initial number of worms must have increased substantially over the
period of 7-8 weeks of experiment.
76
If a vermifilter bed of 0.3 cum soil is prepared with approximately 5000 worms (over 2.5
kg) to start with, it can easily treat 950 - 1000 L of domestic wastewater / sewage generated
by (on an average) a family of 4 people with average BOD value ranging between 300 - 400
mg/L, COD 100 300 mg/L, TSS, 300 350 mg/L everyday with hydraulic retention time
(HRT) of the wastewater in the vermifilter bed being approximately 1 - 2 hours. Given that
the worms multiply and double its number in at least every 60 days under ideal conditions of
temperature and moisture, even starting with this number of earthworms a huge population
(biomass) of worms with robust vermi-filtration system can be established quickly within few
months which will be able to treat greater amount of wastewater generated in the family. An
important consideration is the peak hour wastewater generation which is usually very high
and may not comply with the required HRT (1 - 2 hrs) which is very critical for sewage
treatment by vermi-filtration system. To allow 1 - 2 hrs HRT in the vermifilter bed an onsite
domestic wastewater storage facility will be required from where the discharge of wastewater
to the vermifilter tank can be slowly regulated through flow control.
7. THE VERMIREMEDIATION TECHNOLOGY (VRT)

Large tract of arable land is being chemically polluted due to mining activities, heavy use
of agro-chemicals in farmlands, landfill disposal of toxic wastes and other developmental
activities like oil and gas drilling. No farmland of world especially in the developing nations
are free of toxic pesticides, mainly aldrin, chlordane, dieldrin, endrin, heptachlor, mirex and
toxaphene. There are over 80,000 contaminated sites in Australia, over 40,000 in the US;
55,000 in just six European nations, over 7,000 in New Zealand, and about 3 million
contaminated sites in the Asia-Pacific. They mostly contain heavy metals cadmium (Cd), lead
(Pb), mercury (Hg), zinc (Zn) etc. and chlorinated compounds like the PCBs and DDT.
Cleaning them up mechanically by excavating the huge mass of contaminated soils and
disposing them in secured landfills will require billions of dollars.
Earthworms in general are highly resistant to many chemical contaminants including
heavy metals and organic pollutants in soil and have been reported to bio-accumulate them in
their tissues. Vermiremediation (using chemical tolerant earthworm species) is emerging as a
low-cost and convenient technology for cleaning up the chemically polluted / contaminated
sites / lands in world.
Earthworms Efficiently Remove Toxic Organic and Inorganic Chemicals from Soil:
Several species of earthworms but more particularly Eisenia fetida, Lumbricus terrestris and
L. rubellus, have been found to remove significant amounts of heavy metals, pesticides and
lipophilic organic micropollutants like the polycyclic aromatic hydrocarbons (PAH) from the
soil (Contreras-Ramos et. al, 2006). Several studies have found definite relationship between
organochlorine pesticide residues in the soil and their amount in earthworms, with an
average concentration factor (in earthworm tissues) of about 9 for all compounds and doses
tested. (Ireland, 1983). Studies indicate that the earthworms bio-accumulate or biodegrade
organochlorine pesticide and polycyclic aromatic hydrocarbons (PAHs) residues in the
medium in which it lives. (Nelson et al., 1982; Ireland, 1983).
Earthworms especially E. fetida can bio-accumulate high concentrations of metals
including heavy metals in their tissues without affecting their physiology and this particularly
77
when the metals are mostly non-bioavailable. Studies indicate that they can take up and
bioaccumulate in their tissues heavy metals such as cadmium (Cd), mercury (Hg), lead (Pb),
copper (Cu), manganese (Mn), calcium (Ca), iron (Fe) and zinc (Zn). They can ingest and
accumulate in their tissues extremely high amounts of zinc (Zn) and cadmium (Cd). Of all the
metals Cd and Pb appears to bio-accumulate in most species of earthworms at greater level.
They can particularly ingest and bio-accumulate extremely high amounts of cadmium (Cd)
which is very mobile and may be readily incorporated into soft and non-calcareous tissues of
earthworms. Cadmium levels up to 100 mg per kg dry weight have been found in tissues.
Contreras-Ramos et, al.,(2005) also confirmed that the earthworms reduced the
concentrations of chromium (Cr), copper (Cu), zinc (Zn) and lead (Pb) in the vermicomposted
sludge (biosolids) below the limits set by the USEPA in 60 days. Malley et. al., (2006) also
studied bioaccumulation of copper (Cu) and zinc (Zn) in E. fetida after 10 weeks of
experiment. (Table 16).
Table 16. Concentration of Cu and Zn in E. fetida Tissues Initially and After 10 Weeks
Initial sample
Control
Dosage 1
Dosage 2
Dosage 3
Cu (mg/Kg) SD
17.292
20.34 4
104.58 47
158.95 10
213.07 22
Zn (mg/Kg) SD
108.224
127.548
137.528
132.0316
138.5118
Source: Malley et al., (2006).
Some metals are bound by a protein called metallothioneins found in earthworms which
has very high capacity to bind metals. The chloragogen cells in earthworms appears to mainly
accumulate heavy metals absorbed by the gut and their immobilization in the small spheroidal
chloragosomes and debris vesicles that the cells contain.
7.1. Mechanism of Worm Action in Vermiremediation : The Uptake of

Chemicals from Soil and Immobilization by Earthworms
Earthworms uptake chemicals from the soil through passive absorption of the dissolved
fraction through the moist body wall in the interstitial water and also by mouth and
intestinal uptake while the soil passes through the gut. Earthworms eat large volume of soil
with microbes and organic matter during the course of their life in soil. Earthworms
apparently possess a number of mechanisms for uptake, immobilization and excretion of
heavy metals and other chemicals. They either bio-transform or biodegrade the chemical
contaminants rendering them harmless in their bodies.
a) Biotransformation of Chemical Contaminants in Soil

Heavy metals in earthworms are bound by a special protein called metallothioneins
which has very high capacity to bind metals. Ireland (1983) found that cadmium (Cd) and
lead (Pb) are particularly concentrated in chloragogen cells in L. terrestris and D. rubidus,
where it is bound in the form of Cd-metallothioneins and Pb-metallothioneins respectively
78
(i.e. bio-transformed) with small amounts deposited in waste nodules. The chloragogen cells
in earthworms appears to mainly accumulate heavy metals absorbed by the gut and their
immobilization in the small spheroidal chloragosomes and debris vesicles that the cells
contain.
b) Biodegradation of Chemical Contaminants in Soil: Earthworms Promote Microbial

Activity for Biodegradation
Ma et al., (1995) found that earthworms biodegrade organic contaminants like phthalate,
phenanthrene and fluoranthene. It may be noted that several soil microorganisms especially
bacteria and fungi also biodegrade several categories of chemicals including hydrocarbons in
soil. However, when earthworms are added to the soil they further stimulate and accelerate
microbial activity by increasing the population of soil microorganisms and also through
improving aeration (by burrowing actions) in the soil, and in totality enhance the rate of
biodegradation. The earthworms excrete the decomposer (biodegrader) microbes from their
gut into soil along with nutrients nitrogen (N) and phosphorus (P). These nutrients are used
by the microbes for multiplication and enhanced action.
Reinforcement of microbial activity by earthworms for biodegradation of chemical
contaminants in soil have been reported by Binet et al., (1998). Edward and Lofty (1972)
showed that the number of bacteria and actinomycetes contained in the ingested material by
earthworms increased up to 1000 fold while passing through the gut. A population of worms
numbering about 15,000 can in turn foster a microbial population of billions of millions.
7.2. Experimental Study of Vermiremediation of PAHs Contaminated Soils

Ryan (2006) studied the vermiremediation of PAHs contaminated soils by earthworms.
The soil was obtained from a former gas works site in Brisbane where gas was being
produced from coal. The initial concentration of total PAHs compounds in the soil at site was
greater than 11,820 mg/kg of soil. (Ryan, 2006). The legislative requirements for soil PAHs
concentration is only 100 mg/kg for industrial sites and 20 mg/kg for residential sites.
10 Kg of contaminated soil was taken in each of the four 40 litre black HDPE containers
and made into Treatments 1,2, 3 and 4. The first remained as control and no treatment was
done. In Treatment 2 approximately 500 earthworms (mixed species of E. fetida, Perionyx
excavatus and Eudrillus euginae) of varying ages and sizes were added to the soil. (Worms
were contained in about 2 kg of primary feed materials bought from Bunning Hardware). To
this was added 5 kg of semi-dried cow dung as secondary feed material. In Treatment 3, about
5 kg of kitchen waste organics were added as secondary feed material to the 500 worms. In
Treatment 4, only 5 kg of organic compost was added to the contaminated soil and no worms.
This was set up to assess the effect of only microbial action on the contaminated soil as any
organic compost is known to contain enormous amount of decomposer microbes.
In all the four treatments enough water was added time to time, to maintain the moisture
content between 70-80 % and were allowed to stand for 12 weeks. They were kept under
shade thoroughly covered with thick and moist newspapers to prevent any volatilization or
photolysis of the PAH compounds in the soil.
79
Thus, Treatments 2 and 3 had total of 17 kg materials (10 kg contaminated soil + 2 kg of

primary feed materials with worms + 5 kg of secondary feed material added additionally).
Treatment 4 had 15 kg (10 kg soil + 5 kg compost). Due to addition of feed materials (cow
dung and kitchen waste) and compost in the contaminated soil significant dilution of PAH
compounds are expected to be made and was taken into consideration while determining the
impact of earthworms and the microbes in the removal of PAH compounds.
The results have been shown in tables 17 and 18 and figures 23 and 24. Mauli
Table 17. Removal of Some PAH Compounds from Contaminated Soil by Earthworms
Provided With Different Feed Materials (10 Kg Contaminated Soil + 500 Worms* in 2
kg Feed Materials With Additional Feed Materials Cow Dung (5kg) and Kitchen Waste
(5kg) for 12 Wks) and compost (5Kg)
Source: Ryan (2006).

*Mixed species of worms (E.fetida, Perionyx excavatus and Eudrillus eugeniae) were used.
Table 18. Percent Removal of Some PAH Compounds from Contaminated Soil by
Earthworms Provided With Different Feed Materials (10 Kg Contaminated Soil + 500
Worms* in 2 kg Feed Materials With Additional Feed Materials Cow Dung (5kg) and
Kitchen Waste (5kg) for 12 Wks)
Source: Ryan (2006).

(%) Values within bracket are those after taking the dilution factor (due to mixing of feed materials)
into account. This is just in 12 weeks period.
80
100.00%
Set 2 Soil +
Worms + Cow
Dung
% Removal
80.00%
60.00%
Set 3 Soil +
Worms +
Kitchen w aste
40.00%
20.00%
A
ve
ra
ge
hr
ys
en
F
B
e
lo
en
u
zo
ra
n
(k
th
)
e
F
ne
lo
ur
a
B
n
en
th
en
zo
e
(a
B
en
)
P
zo
yr
en
(g
e
,h
,i)
py
re
ne
(b
)
Set 4 Soil +
Compost (No
Worms)
B
en
B
en
zo
zo
(a
)
an
th
r
ac
en
e
0.00%
Extracte d PAH Com pounds
100.00%
80.00%
Set 2 Soil +
Worms +
Cow Dung
60.00%
40.00%
20.00%
(a
)a
nt
hr
ac
en
e
Be
C
nz
hr
o
ys
(b
en
)F
e
Be
lo
ur
nz
a
o
nt
(k
he
)F
ne
lo
ur
D
ib
an
en
th
zo
en
e
(a
,
h
Be
)P
nz
yr
o
en
(g
e
,h
,i )
py
re
ne
Av
er
ag
e
0.00%
Be
nz
o
% Removal (Considering dilution factor)
Figure 23. Percent removal of PAH from contaminated soil by earthworms, provided with different
feed materials.
Set 3 Soil +
Worms +
Kitchen
waste
Set 4 Soil +
Compost
(No Worms)
PAH Com pounds
Figure 24. Percent removal of some PAH compounds from contaminated soil by earthworms provided
with different feed materials, after taking the dilution factor (due to mixing of feed materials) into
account.

Results confirm the decisive role of earthworms in PAHs removal which can be by both
activities bioaccumulation, and by promoting microbial activities. When microbial activity
in the soil is enhanced alone (without aided by earthworms) by adding microbe rich organic
compost to the soil (Treatment 4) the removal rate of PAHs are not very significant. This
indicates that earthworms acts in a different manner and contributes decomposer microbes for
hydrocarbons which otherwise is normally not available in soil or in ordinary compost.
Singleton et al. (2003) has reported some uncultured bacterial flora tightly associated with
81
the intestine of the earthworms. Some of them, such as Pseudomonas, Acaligenes and
Acidobacterium are known to degrade hydrocarbons.
Providing the earthworms with additional feed materials in the form of cow dung and
kitchen waste (Treatments 2 and 3) must have played important role in raising worm activity
and in its reproductive behavior, and also in stimulating the soil microbial activity. There was
not much significant difference in the impact of two types of feeds. Ma et al. (1995) showed
an increase in PAH loss from polluted soil when the worms were denied of any additional
source of food. They concluded that earthworms increase oral intake of soil particles when
driven by hunger stress and consequently ingested more PAHs polluted soil. While this may
be a temporary phenomenon in short time (9 weeks of study by authors) it can never be a
long-term strategy because any bioremediation treatment of polluted soil is time-taking in
months and years. Worms would starve and die by then. And, adding organic feed materials
to the polluted soil has several other advantages. It promotes microbial activities in soil and
when the worms ingest them and excrete the excreted products are nutrient rich organic
fertilizers.
7.3. Advantages of Vermiremediation Technology: Earthworms Improves

Total Quality of Soil in Terms of Physical, Chemical and Biological
Properties
Significantly, vermiremediation leads to total improvement in the quality of soil and land
where the worms inhabit. Earthworms significantly contribute as soil conditioner to improve
the physical, chemical as well as the biological properties of the soil and its nutritive value.
They swallow large amount of soil everyday, grind them in their gizzard and digest them in
their intestine with aid of enzymes. Only 5-10 percent of the chemically digested and ingested
material is absorbed into the body and the rest is excreted out in the form of fine mucus
coated granular aggregates called vermicastings which are rich in NKP (nitrates, phosphates
and potash), micronutrients and beneficial soil microbes including the nitrogen fixers and
mycorrhizal fungus. The organic matter in the soil undergo humification in the worm
intestine in which the large organic particles are converted into a complex amorphous colloid
containing phenolic materials. About one-fourth of the organic matter is converted into
humus. The colloidal humus acts as slow release fertilizer in the soil.
During the vermi-remediation process of soil, the population of earthworms increases
significantly benefiting the soil in several ways. A wasteland is transformed into
wonderland. Earthworms are in fact regarded as biological indicator of good fertile soil
(Neilson, 1951). One acre of wasteland when transformed into fertile land may contain more
than 50,000 worms of diverse species. Bhawalkar and Bhawalkar (1994) experimented and
concluded that an earthworm population of 0.2 1.0 million per hectare of polluted land /
wasteland can be established within a short period of three (3) months.
82
8. THE VERMI-AGRO-PRODUCTION TECHNOLOGY (VAPT)

Vermiculture biotechnology promises to usher in the Second Green Revolution through
Organic Farming doing away with the destructive agro-chemicals which did more harm than
good to both the farmers and their farmland during the First Green Revolution of the 1960s.
Earthworms restore and improve soil fertility and boost crop productivity by the use of their
metabolic product - vermicast / vermicompost (Tomati and Galli, 1995). They promote soil
fragmentation and aeration, and brings about soil turning and dispersion in farmlands. On an
average 12 tons / hectare / year of soil or organic matter is ingested by earthworms, leading to
upturning of 18 tons of soil / year, and the world over at this rate it may mean a 2 inches of
fertile humus layer over the globe (Bhawalkar and Bhawalkar, 1994). Earthworms excrete
beneficial soil microbes, and secrete polysaccharides, proteins and other nitrogenous
compounds into the soil from their body to improve the physical, chemical and the biological
properties of soil.
8.1. Earthworms Vermicast / Vermicompost : A Miracle Growth Promoter

Vermicompost produced by biodegradation of MSW by earthworms is a nutritive plant
food rich in NKP (2 - 3 % nitrogen, 1.85 2.25 % potassium and 1.55 2.25 % phosphorus),
micronutrients, beneficial soil microbes like nitrogen-fixing bacteria and mycorrhizal
fungi and are wonderful growth promoters (Buckerfield, et al.,1999). Kale and Bano (1986)
reports as high as 7.37 % of nitrogen (N) and 19.58 % phosphorus as P2O5 in worms
vermicast). Moreover, vermicompost contain enzymes like amylase, lipase, cellulase and
chitinase, which continue to break down organic matter in the soil (to release the nutrients and
make it available to the plant roots) even after they have been excreted. (Chaoui et al., 2003).
Vermicompost has very high porosity, aeration, drainage and water holding capacity
and also contains plant-available nutrients. Vermicompost appears to retain more nutrients
for longer period of time and also greatly increases the water holding capacity of the farm soil
(Hartenstein and Hartenstein, 1981; Appelhof, 1997).
a) High Level of Plant-Available Nutrients

Atiyeh et al. (2000) found that the conventional compost was higher in ammonium,
while the vermicompost tended to be higher in nitrates, which is the more available form of
nitrogen. The nitrogenous waste excreted by the nephridia of the worms is mostly urea and
ammonia. The ammonium in the soil is bio-transformed into nitrates. Patil (1993) found that
earthworm recycle nitrogen in the soil in very short time. The quantity of nitrogen recycled is
significant ranging from 20 to 200 kg N/ha/year.
Barley and Jennings (1959) reported that worms significantly improve soil fertility by
increasing nitrogen contents. Hammermeister et al. (2004) also found that vermicompost has
higher N availability than the conventional compost on a weight basis. They also found that
the supply of several other plant nutrients e.g. phosphorus (P), potassium (K), sulfur (S) and
magnesium (Mg), were significantly increased by adding vermicompost as compared to
conventional compost to soil.
83
b) High Level of Beneficial and Biologically Active Soil Microorganisms

Earthworms hosts millions of beneficial microbes (including the nitrogen fixers) in their
gut and excrete them in soil along with nutrients nitrogen (N) and phosphorus (P) in their
excreta i.e. vermicast (Singleton et al., 2003). The nutrients N and P are further used by the
microbes for multiplication and vigorous soil remediation action. The mycorrhizal fungi
encouraged by the earthworms transfer phosphorus by increasing solubilisation of mineral
phosphate by the enzyme phosphatase. Edward and Fletcher (1988) and Edwards (1999)
showed that the number of beneficial bacteria and actinomycetes contained in the ingested
material increased up to 1000 fold while passing through the gut. A population of worms
numbering about 15,000 will in turn foster a microbial population in billions. (Morgan and
Burrows, 1982).
c). Enhance Seed Germination
Worms castings used as a seed germinator produce rapid results with a superior strike
rate of unusually healthy seedlings
d) Rich in Growth Hormones: Ability to Stimulate Plant Growth

Researches show that vermicompost further stimulates plant growth even when plants are
already receiving optimal nutrition. Vermicompost has consistently improved seed
germination, enhanced seedling growth and development, and increased plant productivity
much more than would be possible from the mere conversion of mineral nutrients into plantavailable forms (Edwards and Burrows, 1988). Arancon (2004) found that maximum benefit
from vermicompost is obtained when it constitutes between 10 to 40 % of the growing
medium. Atiyeh et al. (2000) speculates that the growth responses of plants from
vermicompost appears more like hormone-induced activity associated with the high levels
of humic acids and humates in vermicompost rather than boosted by high levels of plantavailable nutrients. This was also indicated by Canellas et al. (2002) who found that humic
acids isolated from vermicompost enhanced root elongation and formation of lateral roots in
maize roots. Tomati et al. (1985) had also reported that vermicompost contained growth
promoting hormone auxins and flowering hormone gibberlins secreted by earthworms.
8.2. Earthworms Reduce Soil Salinity, Renew Soil Fertility and Improve
Crop Productivity
Earthworms not only help renew the natural soil fertility but also improve the soil pH and
reduce soil salinity. Hota and Rao (1985) reported that three tropical earthworm species viz.
Perionyx millardi, Octocheaetona surensis and Drawida calebi survived in saline solutions of
9.5 gm, 8.5 gm and 7 gm NaCl per liter (L) respectively. In a study made by Kerr and Stewart
(2006) at the US Department of Energy it was found that E. fetida can tolerate soils nearly
half as salty as seawater i.e. 15 gm / kg of soil. (Average seawater salinity is around 35 g/L).
Farmers at Phaltan in Satara district of Maharashtra, India, applied vermiculture (live
earthworms) on his sugarcane crop grown on saline soils irrigated by saline ground water.
The yield was 125 tones / hectare of sugarcane and there was marked improvement in soil
chemistry. Within a year there was 37 % more nitrogen, 66 % more phosphates and 10 %
84
more potash. The chloride content was less by 46 %. Farmer in Sangli district of Maharashtra,
India, grew grapes on eroded wastelands and applied vermicasting @ 5 tones / hectare. The
grape harvest was normal with improvement in quality, taste and shelf life. Soil analysis
showed that within one year pH came down from 8.3 to 6.9 and the value of potash increased
from 62.5 kg/ha to 800 kg/ha. There was also marked improvement in the nutritional quality
of the grape fruits (Bhawalkar and Bhawalkar, 1994).
8.3. Vermicompost Protects Plants Against Various Pests and Diseases

There has been considerable anecdotal evidence in recent years regarding the ability of
vermicompost to protect plants against various pests and diseases either by suppressing or
repelling them or by inducing biological resistance in plants to fight them or by killing them
through pesticidal action. Agarwal (1999),also reported less incidence of diseases in
vegetable crops grown on vermicompost.
Vermicompost also contains some antibiotics and actinomycetes which help in increasing
the power of biological resistance among the crop plants against pest and diseases. Pesticide
spray was reduced by 75 per cent where earthworms and vermicompost were used in
agriculture. The actinomycetes fungus excreted by the earthworms in their vermicast produce
chemicals that kill parasitic fungi such as Pythium and Fusarium.
a) Ability to Repel Pests

There seems to be strong evidence that worms varmicastings sometimes repel hardbodied pests (Anonymous, 2001; Arancon, 2004). Edwards and Arancon, (2004) reports
statistically significant decrease in arthropods (aphids, buds, mealy bug, spider mite)
populations, and subsequent reduction in plant damage, in tomato, pepper, and cabbage trials
with 20 % and 40 % vermicompost additions. George Hahn, doing commercial
vermicomposting in California, U.S., claims that his product repels many different insects
pests. His explanation is that this is due to production of enzymes chitinase by worms which
breaks down the chitin in the insects exoskelton (Munroe, 2007).
b) Ability to Suppress Disease
Edwards and Arancon (2004), also found statistically significant suppression of plantparasitic nematodes in field trials with pepper, tomatoes, strawberries and grapes. The
scientific explanation behind this concept is that high levels of agronomically beneficial
microbial population in vermicompost protects plants by out-competing plant pathogens for
available food resources i.e. by starving them and also by blocking their excess to plant roots
by occupying all the available sites. This concept is based on soil-foodweb studies
pioneered by Dr. Elaine Ingham of Corvallis, Oregon, U.S.(http://www.soilfoodweb.com).
Edwards and Arancon (2004) reported the agronomic effects of small applications of
commercially produced vermicompost, on attacks by fungus Pythium on cucumber,
Rhizoctonia on radishes in the greenhouse, by Verticillium on strawberries and by Phomposis
and Sphaerotheca fulginae on grapes in the field. In all these experiments vermicompost
applications suppressed the incidence of the disease significantly. They also found that the
ability of pathogen suppression disappeared when the vermicompost was sterilized,
85
convincingly indicating that the biological mechanism of disease suppression involved was
microbial antagonism. More studies is required to develop this potential of vermicompost as
a sustainable, non-toxic and environmentally friendly alternative to chemical pest control or
at least its application in farming practices can also lead to significant reduction in use of
chemical pesticides.
8.4. Vermiwash: The Nutritive Liquid Byproduct of Vermicomposting Rich

in Growth Promoting and Pesticidal Properties
The brownish-red liquid which collects at the base of vermcomposting unit due to high
moisture content in the compost pile should be collected. This liquid partially comes from the
body of earthworms too (as worms body contain plenty of water) and is rich in amino acids,
vitamins, nutrients like nitrogen, potassium, magnesium, zinc, calcium, iron and copper and
some growth hormones like auxins, cytokinins. It also contains plenty of nitrogen fixing
and phosphate solubilising bacteria (nitrosomonas, nitrobacter and actinomycetes).
Farmers from villages near Rajendra Agricultural University in Bihar reported growth
promoting and pesticidal properties of this liquid. They used it on brinjal and tomato with
excellent results. The plants were healthy and bore bigger fruits with unique shine over it.
Spray of vermiwash effectively controlled all incidences of pests and diseases, significantly
reduced the use of chemical pesticides and insecticides on vegetable crops and the products
were significantly different from others with high market value. These farmers are using
vermicompost and vermiwash in all their crops since last 2 years completely giving up the use
of chemical fertilizers. (Personal Communication With Farmers in Pusa, December 2006).
8.5. Experimental Studies on Agronomic Impacts of Earthworms and

Vermicompost on Crop Plants
1) Potted Wheat Crops
Bhatia (1998) and Bhatia et al. (2000) studied the agronomic impact of earthworms on
potted wheat crops at University of Rajasthan, Jaipur, India. Wheat seeds (Triticum aestivum
Linn) and earthworms were obtained from Rajasthan Agricultural Research Institute, Jaipur.
Three identical sets of pots with ten replicates of each were prepared from uniform soil of the
same stock, which was assumed to be near neutral, i.e., without any organic matter. Three sets
of 10 pots each were prepared.
1) Treatment 1 was kept as a control (without any input);
2) In Treatment 2, fifty (50) worms (mixed species of Eisinia fetida, Perionyx excavatus
and Eudrillus euginae) were added to each pot and 250 gm of one week old cattle
dung (allowing release of methane from fresh dung) was added as feed material for
the worms.
3) In Treatment 3, chemical fertilizers (104 gms urea, 0.2 gms potash and 0.75 gms
single super phosphate) were added;
86
4) In Treatment 4, about 250 gm of week old cattle dung was added to precisely
determine the role of earthworms in growth promotion as the dung also contain
nutrients and support growth after degradation and conversion into compost.
Ten seeds were sown in each pot (10 x 10 = 100 in ten pots) after mixing the soil with the
respective fertilizers (Urea was added in two identical doses, one at the time of sowing and
another 21 days).
Results are given in table 19.
Table 19. Agronomic Impacts of Earthworms Compared With Chemical Fertilizers on
Growth and Yield of Potted Wheat Crops (Triticum aestivum Linn.)
Parameters
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
Number of seed
germinated out of 100
Root length (Av. cm)
Shoot length (Av. cm)
Ear length (Av. cm)
Total height of plant (Av.
cm)
Leaf length (Av. cm)
Dry weight of ears
(Av. cm)
Number of seed grains per
ear (Average)
Chlorophyll content (mg/l)
Number of tillers per plant
Treatment 1
Control
Treatment 2
Soil Containing
Earthworms (50
Nos.) and Cattle
Dung (As feed
material)
Treatment 3
Soil With
Chemical
Fertilizers
(N=104 gm;
K=0.2 gm;
P=0.75 gm)
Treatment 4
Soil Containing
Cattle Dung
(250 gm) only
50
90
60
56
7.13
22.1
4.82
16.46
59.99
8.77
9.32
25.2
5.45
8.23
23.1
5.1
34.16
85.22
39.97
37.30
12.73
26.37
14.19
13.45
0.135
0.466
0.171
0.16
11.8
31.1
19.9
17.4
0.783
1
3.486
2-3
1.947
1-2
1.824
1-2
Scale
Source: Bhatia (1998) and Bhatia et al. (2000): Also in Sustainable Agriculture (Sinha, 2004).; Key:
Av. = Average.
100
90
80
70
60
50
40
30
20
10
0
Percentage of
seed
germination
Root length
(cm)
Shoot length Ear length (cm) Total height of

(cm)
plant (cm)
Leaf length
(cm)
Dry w eight of
ears (gm)
Number of
seed grains
per ear
Chlorophyll
Number of
content (mg/l) tillers per plant
Parameter
Set
Set
Set
Set
1
2
3
4
Control
Soil with Chemical Fertilizers
Soil Containing Live Earthworms & Cow Dung (as feed material)
Soil Containing Cattle Dung
Figure 25. Agronomic Impacts of Earthworms Compared With Chemical Fertilizers on Growth and
Yield of Potted Wheat Crops (Triticum aestivum Linn.).
87

The potted wheat crops with earthworms and added cattle dung as feed materials
(Treatment 2) made excellent progress from the very beginning of seed germination up to
maturation. They were most healthy and green, leaves were broader, shoots were thicker and
the fruiting ears were much broader and longer with average greater number of seed grains
per year. Significantly, they were much better even as compared to those grown on chemical
fertilizers (Treatment 3). Worms fed on cattle dung and excreted them as vermicast
(vermicompost) which worked as the miracle growth promoter. Wheat crops added with
cattle dung only (Treatment 4) were almost close to those grown on chemical fertilizers. The
dung was eventually converted into compost by soil microbes which worked as growth
promoter.
2) Potted and Farm Wheat Crops
Reena Sharma (2001) studied the agronomic impacts of vermiculture on the potted as
well as on the farm wheat crops at University of Rajasthan, Jaipur, India. In this experiment
live population of earthworms and vermicompost were applied separately to pot and farm
soil. Same mixed species of worms e.g. E. fetida, P. excavatus and E. euginae were used and
vermicompost was also prepared by using same species by composting kitchen waste and
cattle dung. The farm was divided into eight plots of 25 x 25 sqm size. Three treatments were
prepared:
1) Vermicompost : In the pots 30 gm of vermicompost was used while in the farm it
was used @ 2.5 tonnes / ha.
2) Chemical Fertilizers (NPK) : As urea (N), single super phosphate (P) and potash (K),
in one full dose and two reducing doses for the pots and one for farm. Vermicompost
(30 gm) were applied with both reduced doses of chemical fertilizers;
3) Earthworms : In each pot 50 numbers of earthworms were used, while 1000 worms
were released in the farm plot of 25 x 25 sq. mt. ; and
4) The fourth farm plot was kept as control (no inputs).
All the treatments were replicated twice. The wheat seed was grown @ 100 kg/ha.
Irrigation schedule was maintained as recommended for wheat. Chemical nitrogen (urea) was
applied in two split doses (first half at the time of sowing and second half dose after 21 days
of sowing) whereas the phosphate and potash fertilizers were applied as single dose at the
time of sowing. Results are given in tables 20 and 21.

In the pot experiments the best growth performance in respect of root, shoot and ear
length and their weight, weight of 1000 grains were observed where a combination of reduced
dose (3/4) of chemical fertilizer (NPK-90:75:60) and normal amount of vermicompost (30
gm) were applied. It was significantly much better than even where full doses of chemical
fertilizers were used. More significantly, both vermicompost and earthworms positively
influenced the total yield of the grains which is 19.2 and 19.1 grains / ear respectively.
88
Table 20. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers

on Potted Wheat Crops
Treatments
1.
2.
3.
4.
5.
6.
Vermicompost
Earthworms (50 Nos.)
NPK (120:100:80) Full Dose
NPK (90:75:60) Reduced Dose + VC
NPK (60:50:40) Reduced Dose + VC
CONTROL
Shoot
Length
(cm)
41.11
39.27
42.14
47.89
45.96
37.01
Ear
Length
(cm)
7.65
6.74
7.67
8.91
8.59
5.83
Root
Length
(cm)
15.5
8.35
16.35
20.2
18.3
7.18
Wt. Of 1000
grains (gm)
Grains/
Ear
33.38
32.41
30.2
40.58
39.29
27.28
19.2
19.1
17.1
18.67
18.91
15.4
Source : Sharma (2001); In Sustainable Agriculture (Sinha, 2004); Key : VC= Vermicompost.
Table 21. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers

on Farm Wheat Crops
Treatments
1.
3
4
5.
Shoot
Length
(cm)
Vermicompost (@ 2.5 t / ha)
83.71
NPK (90:75:60) (Reduced Dose) + VC 88.05
(Full Dose)
NPK (120:100:80) (Full Dose)
84.42
CONTROL
59.79
Ear
Length
(cm)
13.14
13.82
Root
Length
(cm)
23.51
29.71
Wt. Of 1000
grains (In
grams)
39.28
48.02
Grains /
Ear
14.31
8.91
24.12
12.11
40.42
34.16
31.2
27.7
32.5
34.4
Source : Sharma (2001): In Sustainable Agriculture (Sinha, 2004); Key: VC = Vermicompost.

Key: VC= Vermicompost; N = Urea; P = Phosphate; K = Potash.
60
50
Scale
40
30
20
10
0
Vermicompost
Live Earthw orms NPK (120:100:80) NPK ( 90:75:60 ) + NPK (60:50:40) +

(50 Nos.)
VC
VC
CONTROL
Parameters
Shoot Length (cm)
Root Length (cm)
Grains/Ear
Ear Length (cm)

Wt. of 1000 grains (gm)
Figure 26. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Potted Wheat
Crops.
89
100
90
80
Scale
70
60
50
40
30
20
10
0
Shoot Length
Ear Length
Root Length
Wt. of 1000 grains
Grains / Ear
Parameters
Vermicompost
Earthworms (1000 Nos.)
NPK (90:75:60) + VC
CONTROL
Figure 27. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Farm Wheat
Crops.

In the farm experiment the highest growth and yield was achieved where reduced doses
(3/4) of chemical fertilizer (NPK- 90:75:60) were applied with full dose of vermicompost (@
2.5 tons/ha). However, the total yield of the grain (grain / ear) as well as the ear length and the
weight of 1,000 grains of crops grown on vermicompost were as good as with those grown on
full doses of chemical fertilizers (NPK).
3). Farmed Wheat Crops
This facility was provided by Rajendra Agriculture University, Pusa, Bihar, India under a
collaborative research program with Griffith University, Brisbane, Australia. We studied the
agronomic impacts of vermicompost and compared it with cattle dung compost & chemical
fertilizers in exclusive application and also in combinations on farmed wheat crops. Cattle
dung compost was applied four (4) times more than that of vermicompost as it has much less
NPK values as compared to vermicompost. Results are given in table - 22
Table 22: Agronomic Impacts of Vermicompost, Cattle Dung Compost & Chemical
Fertilizers in Exclusive Applications & In Combinations on Farmed Wheat Crops
Treatment
Input / Hectare
Yield / Hectare
1). CONTROL
(No Input)
15.2 Q / ha
2). Vemicompost (VC)
25 Quintal VC / ha
40.1 Q / ha
3). Cattle Dung Compost (CDC) 100 Quintal CDC / ha
33.2 Q / ha
4). Chemical Fertilizers (CF) NPK (120:60:40) kg / ha
34.2 Q / ha
5). CF + VC
NPK (120:60:40) kg / ha + 25 Q VC / ha
43.8 Q / ha
6). CF + CDC
NPK (120:60:40) kg / ha + 100 Q CDC / ha
41.3 Q / ha
----------------------------------------------------------------------------------------------------------------------------
Suhane et. al., (2008): Communication of RAU, Pusa, Bihar, India

Key: N = Urea; P = Phosphate; K = Potash (In Kg / ha)
90
50
Control
45
40
Vermicompost (25
Q / ha)
35
30
Cattle Dung
Compost (100 Q /
ha)
25
20
Chemical Fertilizer
(NPK 120: 60:40)
15
10
CF (Full Dose) +
VC (25 Q / ha)
5
0
Yield (Quintal / hectare)
CF (Full Dose) +
CDC (100 Q / ha)
Figure 28: Agronomic Impacts of Vermicompost, Cattle Dung Compost & Chemical
Fertilizers in Exclusive Applications & In Combinations on Farmed Wheat Crops
Findings & Discussion

Exclusive application of vermicompost supported yield comparable to rather better than
chemical fertilizers. And when same amount of agrochemicals were supplemented with
vermicompost @ 25 quintal / ha the yield increased to about 44 Q / ha which is over 28 %
and nearly 3 times over control. On cattle dung compost applied @ 100 Q / ha (4 times of
vermicompost) the yield was just over 33 Q / ha. Application of vermicompost had other
agronomic benefits. It significantly reduced the demand for irrigation by nearly 30-40 %. Test
results indicated better availability of essential micronutrients and useful microbes in
vermicompost applied soils. Most remarkable observation was significantly less incidence of
pests and disease attacks in vermicompost applied crops.
4). Potted Corn Crops
Sinha & Bharambe (2007) studied the agronomic impacts of earthworms & its
vermicompost on corn plants at Griffith University, Brisbane, Australia. It had two parts A &
B. Part A was designed to compare the growth promoting abilities of vermicompost with
chemical fertilizers and also the earthworms when significantly present in the growth
medium. It had three (3) treatments with three replicas of each and a control. Treatment 1
with 25 number of adult worms only; Treatment 2, with chemical fertilizers; and Treatment 3,
with vermicompost and also containing same number of worms. Soluble chemical fertilizers
Thrive was used. Approx. 8 gm of chemicals was dissolved in 4.5 L of water. It had total
nitrogen (N) 15 %, total phosphorus (P) 4 %, total potassium (K) 26 % and a combination of
essential micronutrients. Three applications were made during entire growth period while the
worms and vermicompost was applied only onetime. Results are given in table 23 (a).
91
Table 23 (a). Agronomic Impacts of Earthworms, Worms With Vermicompost and

Chemical Fertilizers on Corn Plants (Added to 4 kg of soil; Av.Growth in cm)
Parameters Studied
CONTROL
(No Input)
Treatment 1
EARTHWORMS
Only (25 Nos.)
(Without Feed)
Treatment 2
Soluble
CHEMICAL
FERTILIZERS
Treatment 3
EARTHWORMS +
VERMICOMPOST
(200 gm)
Seed Sowing
Seed Germination
29th July 2007

9th Day
Do
7th Day
Do
7th Day
Do
7th Day
Avg. Growth in 4
wks
31
40
43
43
Avg. Growth in 6
wks
44
47
61
58
App. Of Male Rep.

Organ (In wk 12)
None
None
46
53
87
None
None
None
48
53
(App. Of Male
Rep. Organ)
88
95
None
None
None
New Corn
53
56
92
105
Pale & thin

leaves
Green & thin
Avg. Growth
In 12 wks
App. of Female Rep.
Organ (In wk 14)
Avg. Growth in
15 wks
App. Of New Corn
(in wk 16 )
Avg. Growth in
19 wks
Color & Texture of
Leaves
Male Rep.
Organ
Green & stout

leaves
Male Rep.
Organ
90
Female Rep. Organ
Green, stout &

broad leaves
Source: Sinha & Bharambe (2007)

Corn plants in pot soil with earthworms and vermicompost (Treatment 3 Pot D)
achieved good growth after week 4, and those with chemical fertilizers (Treatment 2 Pot C)
after week 6. After that, both maintained good growth over the other two (Control &
Treatment 1 Pots A & B) and attained maturity in 11 weeks (appearance of male spike).
Female reproductive organ, however, appeared in corn plants grown on vermicompost only in
14 weeks. The new corn appeared after 111 days (in week 16).
Until week 4, corn plants in all four treatments showed almost identical growth.
Ironically, the corn plants in pot soil with worms only (Treatment 1) could not make any
significant progress. Soil being completely devoid of organics could not be used by worms to
produce any vermicast. However, they were all greener and healthier than control. Another
significant finding was that pot soil with vermicompost demanded less water for irrigation as
compared to others.
92
120
100
CONTROL
Height in cm
80
EARTHWORMS (25)
Without Feed
60
Soluble CHEMICAL
FERTILIZERS (NPK)
40
EARTHWORMS (25) +
VERMICOMPOST (200
gm)
20
0
Avg. Growth Avg. Growth
in 4 wks
in 6 wks
Avg. Growth Avg. Growth Avg. Growth

in 12 wks
in 15 wks
in 19 wks
Figure 29 : Agronomic Impacts of Earthworms, Worms With Vermicompost and Chemical Fertilizers
on Corn Plants (In 19 Weeks Period)
(1). (Growth until 4 weeks)

Plants with chemical fertilizers (CF) and
vermicompost (VC) grew well; Male spikes
appeared in both plants in week 11.
Keys:

Plants with CF and VC maintained excellent
growth; Female reproductive organs appeared
in VC only in week 14 and new corn in week 16.
(A) Pot soil without any input (CONTROL)

(B) Pot soil with worms only
(C) Pot soil with chemical fertilizers
(D) Pot soil with worms and vermicompost (200gm)
Figure 6 (a). Photo showing growth of corn plants under the influence of earthworms, worms with
vermicompost and chemical fertilizers.
93
Part B
This study was designed to test the growth promoting capabilities of earthworms added
with feed materials and vermicompost, as compared to conventional compost. It had three
(3) treatments with three (3) replicas of each. The dose of vermicompost was doubled (400
gm) from previous study and same amount of conventional compost was used. Only one
application of each was made. Crushed dry leaves were used as feed materials (400 gm).
Results are given in table 23 (b).
Table 23 (b). Agronomic Impacts of Earthworms (With Feed), Vermicompost and
Conventional Compost on Corn Plants (Added to 4 kg of soil ; Av. Growth in cm)
Parameters Studied
Seed Sowing
Seed Germination
Avg. Growth in 3 wks
App. of Male Rep. Organ (in wk 6)
App. Of Female Rep. Organ (in wk
10)
App. of New Corn (in wk 11)
Color & Texture of Leaves
Treatment 1
Earthworms
(25) with Feed
(400 gm)
9th Sept. 2007
5th Day
41
49
None
57
64
Treatment2
Conventional
COMPOST
(400 gm)
Do
6th Day
42
57
None
70
72.5
Treatment 3
None
None
Female Rep. Organ
None
82
None
78
Green & thick
Light green & thin
New Corn
135
Deep green, stout,
thick & broad leaves
VERMICOMPOST
(400 gm)
Do
5th Day
53
76
Male Rep. Organ
104
120
Source : Sinha & Bharambe (2007)
160
Growth in cm
140
120
Earthworms (25) With

Feed (400 gm)
100
80
Conventional COMPOST
(400 gm)
60
VERMICOMPOST(400 gm)
40
20
0
Avg.
Avg.
Avg.
Avg.
Avg.
Growth in Growth In Growth In Growth Growth
3 wks
4 wks
6 wks In 9 wks in 14 wks
Figure 30: Agronomic impacts of Earthworms (with feed), Vermicompost and Conventional
Compost on Corn Plants (In 14 Weeks Period)
94

Corn plants in pot soil mixed with vermicompost (Treatment 3) achieved rapid and
excellent growth after week 4 and attained maturity very fast. Between weeks 6 to 11, there
was massive vegetative growth with broad green leaves. Male spike appeared in week 6 while
the female reproductive organs appeared in week 10 which bored the new corn in 11th
week. Corn plants in pot soil with worms only (Treatment 1) and conventional compost
(Treatment 2) could not keep pace with vermicompost. Corn plants with worms only,
however, were more green and healthy and took over those plants grown on conventional
compost finally in the 14th week. Female reproductive organs and new corn could not
appear until the completion of the study by 21st November 2007.
Keys:
(A) Pot soil with earthworms only

(B) Pot soil mixed with conventional compost (400 gm)
(C) Pot soil mixed with vermicompost (400 gm)
Figure 6 (b). Photo showing growth of corn plants under the influence of earthworms, conventional
compost and vermicompost.
95
Results of both studies (Part A & B) established beyond doubt that the vermicompost
(metabolic products of worms) works like miracle growth promoter and is biologically
(nutritionally) much superior to the conventional compost. This has also been found by other
researchers (Subler et al., 1998; Pajon (Undated); and Bogdanov, 2004). And what is most
significant is that when the dose of vermicompost is doubled (from 200 grams in Part A
study to 400 grams in Part B, all other conditions remaining same) it simply enhanced total
plant growth to almost two-fold (from average 58 cm on 200 gm VC to average 104 cm on
400 gm VC) within the same period of study i.e. 6 weeks. Corn plants with double dose of
vermicompost (400 gm) achieved maturity in much shorter time. Male reproductive organs
(spike) appeared after 81 days (in week 12) in plants grown on 200 gm of vermicompost,
while in those grown on 400 grams, it appeared just after 39 days (in nearly half of the time in
week 6). Similarly, the female reproductive organs and eventually the new corn appeared
after 96 days (in week 14) and 111 days (in week 16 ) respectively in plants grown on 200
grams of vermicompost, while it appeared only after 69 days (in week 10) and 75 days (in
week 11) respectively, in plants grown on 400 grams of vermicompost. It is also significant
to note that the corn plants with earthworms only, performed better over those grown on
conventional compost, once again establishing the role of earthworms as growth promoters.
Study also confirmed that vermicompost is superior over conventional compost as
growth promoter & in retaining soil moisture while also helping the plants to attain maturity
and reproduce faster, thus reducing the life-cycle of crops and also shortening the
harvesting time. Conventional compost fails to deliver the required amount of macro and
micronutrients including the vital NKP (nitrogen, potassium & phosphorus) to plants in
shorter time. The leaves and stems of corn plants grown on vermicompost was much greener,
broader and stouter than those grown on conventional compost (Sinha and Bharambe, 2007).
8.5.1 Current Experimental Study on Potted Wheat, Corn and Tomato

Plantys Under Progress: Some Striking Observations of Plants Under
Vermicompost
Valani, Dalsukh and Chauhan, Krunal (2008) is currently studying the agronomic impacts of
vermicompost vis--vis conventional cow dung compost and chemical fertilizers on wheat,
corn and tomato plants. Some very exciting results have started appearing in the very
beginning of the study.
1). High Strike Rates of Wheat Seed Germination

The striking rates of wheat seeds were very high under vermicompost. They germinated
nearly 48 hours (2 days) ahead of others and the number of seeds germinated were also high
by nearly 20 %. The seedlings are also much greener and healthier and have more offshoots..
2). Rapid Growth of Corn Seedlings
There was not much difference in the striking rates of seed germination but once germinated
the seedlings grew at a much faster rate almost doubling in size (from 22 cm to 42 cm) in just
two days. Seedlings are more greener, stouter and healthier than others.
96
3). More Flowering and Rapid Growth of Tomato Fruits

Flowering in tomato plants under vermicompost was delayed by 5-6 days but once started
there were greater numbers of flowers (average 7-8 per plant as compared to 5-6 in chemical
fertilizers) and subsequently fruit developments. Significantly, the fruits grew in size very
rapidly overtaking all others and are much greener.
One more striking observation is that the tomato plants on vermicompost are showing greater
adaptability to higher temperature (about 30 - 35 C) in the glasshouse.
CONCLUSIONS AND REMARKS

Vermiculture practices for waste and land management and for improving soil fertility to
boost crop productivity has grown considerably in recent years all over the world.
Vermiculture is in fact a technological revival of the traditional age-old methods practiced by
the ancient farmers for disposal of farm wastes and improvement of farm fertility by
earthworms. It is like getting gold from garbage (solid waste) by vermicomposting, silver
from sewage (wastewater) by vermifiltration; converting a wasteland (chemically
contaminated and saline lands) into wonderland (fertile land) by vermiremediation;
harvesting green gold (crops) by using brown gold (vermicompost) and all with the help of
earthworms which are abundant in soil all over the world but most prominently in the tropical
nations. The three versatile species E. fetida, E. euginae and P. excavatus performing wide
environmental functions occur almost everywhere. Earthworms are justifying the beliefs and
fulfilling the dreams of Sir Charles Darwin as unheralded soldiers of mankind. (Sinha and
Sinha, 2007).
9.1. The Vermicomposting Technology (VCT)

Earthworms have real potential to both increase the rate of aerobic decomposition and
composting of organic matter, and also to stabilize the organic residues in them, while
removing the harmful pathogens and heavy metals from the end products. Waste composting
by earthworms is proving to be environmentally preferred technology over the conventional
microbial composting technology and much more over the landfill disposal of wastes as it is
rapid and nearly odorless process, can reduce composting time significantly and above all,
there is no emission of greenhouse gas methane which plagues both these solid waste
management options. Vermicomposting of organic waste to divert massive domestic waste
from the landfills are gaining importance and many municipal council in Australia are
adopting this rapid and odorless vermicomposting technology (VCT) using waste eater
earthworms.(UNSW ROU, 2002(a) and 2002(b)).
Poor nations cannot even afford to construct and maintain a truly engineered landfill.
Developing countries needs more options for safe waste management, and also low-cost, as
they have several other social and developmental priorities with limited resources. Moreover,
in the rural communities of both developed and developing world, centralized waste
management system is never a good option. Individual households or a cluster of homes can
97
treat their wastes better and also recover some useful resources (compost). This will prove
more useful in the rural areas where farms are located.
The worm number and quantity (biomass) is a critical factor for vermi-composting of
organic wastes besides the optimal temperature and moisture which determines worm
activity. A minimum of about 100-150 adult worms per kg of waste would be ideal to start
with for rapid biodegradation and also odor-free process. Vermicomposting process driven by
the earthworms tends to become more robust and efficient with time as the army of degrader
worms grows and invade the waste biomass and further proliferating several battalions of
aerobic decomposer microbial army.
What is of greater significance is that earthworms accept and adapt to all kinds of new
food products (even the fried foods) of modern civilization (except a few) to which their
ancestors were never used to in history and readily degrade them converting into
vermicompost.
9.2. The Vermifiltration Technology (VFT)

Vermifiltration of wastewater is a logical extension of soil filtration which has been
used for sewage silviculture (growing trees) since ancient days. Healthy soil is a biogeological medium acting as an adsorbent of organics, inorganic, pathogens and parasites.
Vermifiltration technology (VFT) can be a most cost-effective and odor-free process for
sewage treatment with efficiency, economy and convenience. Any non-toxic wastewater from
the households, commercial organizations or industry can be successfully treated by the
earthworms and the technology can also be designed to suit a particular wastewater. It can be
used in a de-centralized manner in individual industry or cluster of similar industries so as to
reduce the burden on the wastewater treatment plants down the sewer system. It can treat
dilute (less than 0.1 % solids) as well as concentrated wastewater. It has an in-built pH
buffering ability and hence can accept wastewater within a pH range of 4 to 9 without any pH
adjustment. Though significant removal of BOD, COD and the TDSS is achieved by the geomicrobial system unaided by earthworms (as shown from our study in the control kit) the
system fails to work after some time as it is frequently choked due to the formation of sludge
and also colonies of bacteria and fungi (in the vermifilter bed) in the absence of the
earthworms which constantly keep devouring on them. Presence of earthworms in the system
also improve the adsorption properties of the geological system (sands and soils) by
grinding them in their gizzard.
Vermifiltration process driven by the earthworms also tends to become more robust and
efficient with time as the army of degrader worms grows, further proliferating microbial
population (army of aerobic decomposers). It is also a compact biological wastewater
treatment system as compared to other non-conventional system such as the constructed
wetland system which often suffer from limitations of oxygen for the decomposer aerobic
microbes to act efficiently. Wetland based technologies involve mainly treatment and low
utilization of waste materials and hence can be wasteful.
BOD, COD, TSS and turbidity removal efficiency generally increases with increase in
HRT up to a certain limit and is positively affected by the number of earthworms
(earthworms biomass) per cubic meter (cum) in the vermifilter bed (soil profile). It can reduce
the small BOD loadings of sewage (200-400 mg/L) within 30 40 minutes of HRT. Worms
98
have been found to remove very high BOD loads (10,000 1,00,000 mg/L often found in
wastewater from food processing industries) within 4 to 10 hours of HRT. If the population of
worms were to be higher, the same efficiency of BOD, COD, TSS and turbidity removal
could have been achieved at lower HRT.
However, in case of municipal wastewater (sewage) treatment the objectives are not only
to remove BOD, COD and TDSS, but also to remove the toxic chemicals including the heavy
metals and pathogens from the wastewater. The presence of endocrine disrupting chemicals
(EDCs) in sewage which was discovered recently with the development of new instruments
(Markman et. al., 2007) is causing great concerns these days. And what is the matter of more
serious concern is that they cannot be removed by the conventional sewage treatment
methods. They can only be removed by the reverse osmosis methods of membrane filtration
technology (MFT) which is cost-prohibitive at present and all nations cannot afford it. The
cost-effective vermifiltration technology (VFT) assumes great significance in the removal of
EDCs from wastewater. Hence greater hydraulic retention time (1-2 hours) is allowed so that
the worms can ingest (bio-accumulate) the toxic chemicals and also devour upon the
pathogens completely. Greater interaction with wastewater components also provides better
opportunity for the worms to eat all the solids and prevent any sludge formation.
Vermi-filtration of wastewater must be started with higher number of earthworms, at
least over 15,000-20,000 worms per cubic meter (cum) of soil in the vermifilter bed for good
results. It is also important that they are mostly adult and healthy worms. In vermicomposting of solid waste, which is a continuous process (in days and weeks) the worms
have to act gradually in phases while their population (new army of bio-degraders) keeps on
building up to intensify the biodegradation process. In vermifiltration of wastewater, the
worms have to act instantly as the wastewater flow past their body (degrading the organics,
ingesting the solids and the heavy metals). That is why the wastewater has to be retained
(HRT) in the vermifilter bed for some appropriate period time (which has to be in hours and
not in days) while the worms act on the wastewater.
9.3. The Vermiremediation Technology (VRT)

Earthworms have great potential in removing hydrocarbons and many other chemicals
from contaminated soil, even the PAHs like benzo(a)pyrene which is very resistant to
degradation. They are extremely resistant to toxic PAHs and tolerate concentrations normally
not encountered in the soil. It is important to mention here that nearly 80 % removal (60-65 %
if the dilution factor is taken into account) of seven important PAH compounds was achieved
in just 12 weeks period and that too with only about 500 worms (of both mature and juvenile
population) in 10 kg of soil (50 worms/kg of soil). And this was during the winter season in
Brisbane (March May 2006) when the biological activities of worms are the lowest ebb.
Increasing the number of worms per kg of contaminated soil to about 100 mature adult worm
/kg of soil, and the time of remediation up to 16 weeks could have completely (100 %)
removed the PAH compounds. Contreras-Ramos et. al, (2006) studied with 10 worms / 50
gms of contaminated soil (which is equivalent to 200 worms / kg of soil) in about 11 weeks
and got 50-100 % removal of some PAHs.
Many soils contain abundance of pores with diameters of 20 nm or less. Such pores are
too small to allow the smallest bacterium (1 um), protozoa (10 um) or root hairs (7 um) to
99
penetrate and attack the chemicals. A chemical contaminant residing in such fine pores in soil
is thus completely protected from attack by a microbe in the soil for biodegradation action. In
other words such chemical contaminants are not bio-available for any biological action.
Earthworms play a very important and critical role here by enlarging the pores through
continuous burrowing actions in the soil, thus allowing the microbes to enter into the pores
and act on the contaminants. It also stimulate the population of decomposer microbes to
several folds for enhanced biodegradation action. The gizzard in the earthworms helps to
grind the food very thoroughly with the help of tiny stones swallowed by the worms into
smaller particles 2-4 m in size. This grinding action may serve to make PAHs or any other
chemical contaminants sequestered in the soil bio-available to decomposer microbes for
degradation.
Vermi-remediation may prove very cost-effective and environmentally sustainable way
to treat polluted soils and sites contaminated with hydrocarbons in just few weeks to months.
With the passage of time, the remedial action is greatly intensified. As the worms multiply at
an enormous rate it can quickly achieve a huge arsenal for enhanced degradation of PAHs in
much shorter time. Comparing the cost incurred in mechanical treatment by excavation of
contaminated soils and their safe dumping in secured landfills (as hazardous wastes), this
technology is most economic. More study will be needed on the PAH removal activities of
earthworms with and without additional feed materials and upon different categories and
doses of organic feed.
9.4.The Vermi-Agro-Production Technology (VAPT)

Earthworms when present in soil inevitably work as soil conditioner to improve the
physical, chemical as well as the biological properties of the soil and its nutritive value for
healthy plant growth. This they do by soil fragmentation and aeration, breakdown of organic
matter in soil and release of nutrients, secretion of plant growth hormones, proliferation of
nitrogen-fixing bacteria, increasing biological resistance in crop plants, and all these worm
activities contribute to improved crop productivity. (Barley, 1959).
Studies have found that if 100 kg of organic waste with say, 2 kg of plant nutrients (NPK)
are processed through the earthworms, there is a production of about 300 kg of fresh living
soil with 6 % of NPK and several trace elements that are equally essential for healthy plant
growth. This magnification of plant nutrients is possible because earthworms produce extra
nutrients from grinding rock particles with organics and by enhancing atmospheric nitrogen
fixation. Earthworms activate this ground mix in a short time of just one hour. When 100 kg
of the same organic wastes are composted conventionally unaided by earthworms, about 30
kg compost is derived with 3 % NPK. This usual compost thus has a total NPK of only about
1 kg. Rest one kg nutrient might have been leached or volatilized during the process of
composting. (Bhawalker and Bhawalkar, 1993 and 1994).
The metabolic products (vermicast / vermicompost) of worm activities (feeding and
excretion) works like a miracle growth promoter. Vermicompost is agronomically much
superior to conventional microbial compost sold in the market and can play very important
role in promoting growth in crop plants, even competing with chemical fertilizers as our
studies have shown. It is mainly due to the growth promoting factors (hormones and
enzymes) that is present in the vermicompost. Study shows that doubling the dose of
100
vermicompost almost double the growth of corn plants within the same period and can help
attain maturity much earlier.
Worms and its vermicompost have tremendous crop growth promoting potential while
maintaining soil health and fertility and significantly reducing (by over 80 %) the use of
chemical fertilizers and pesticides, and in some crops can even replace them completely. This
is what being termed as sustainable agriculture.
Use of vermicompost in farm soil eventually leads to increase in the number of
earthworm population in the farmland over a period of time, as the baby worms grow out
from their cocoons contained in the vermicast. In Argentina, farmers who use vermicompost
consider it to be seven (7) times richer than conventional composts in nutrients and growth
promoting values (Pajon (Undated); Munroe, 2007). No wonder, Sir Charles Darwin, the
great visionary scientist of 19th Century, called the earthworms as friends of farmers and
unheralded soldiers of mankind working day and night under the soil.
Tribute to the Earthworms

Earthworms are justifying the beliefs and fulfilling the dreams of Charles Darwin. He
wrote a book in which he emphasized that there may not be any other creature in world that
has played so important a role in the history of life on earth (Bogdanov, 2004). One of the
leading authorities on earthworms and vermiculture studies Dr. Anatoly Igonin of Russia has
said Nobody and nothing can be compared with earthworms and their positive influence on
the whole living Nature. They create soil and everything that lives in it. They are the most
numerous animals on Earth and the main creatures converting all organic matter into soil
humus providing soils fertility and biospheres functions: disinfecting, neutralizing,
protective and productive. (Appelhof, 2003). There can be little doubt that mankinds
relationship with worms is vital and needs to be nurtured and further expanded.
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110
Dr. Rajiv Sinha with his vermiculture team at Griffith University, Australia.

ISSN: 2158-5717
Chapter 3
HUMAN WASTE - A POTENTIAL RESOURCE:

CONVERTING TRASH INTO TREASURE BY
EMBRACING THE 5 RS PHILOSOPHY FOR SAFE
AND SUSTAINABLE WASTE MANAGEMENT
Rajiv K. Sinha1, Sunil Herat2, Gokul Bharambe3, Swapnil Patil3,
Pryadarshan Bapat3, Krunal Chauhan3 and Dalsukh Valani3
1
School of Engineering (Environment), Griffith University, Nathan Campus, Brisbane,

QLD-4111, Australia
2
3
Keywords: Culture of Consumerism; Culture of Disposables; Packaging Culture; Australians

and Americans as Superconsumers and Waste Generators; Waste A Misplaced
Resource; Traditional Societies Recycling Societies; Modern Society Throwaway
Society; Waste Source of Greenhouse Gases; Vermiculture Movement for Efficient and
Cost-effective Waste Management.
1. INTRODUCTION
Waste is being generated by the human societies since ancient times. Ironically waste was
not a problem for the environment when men were primitive and uncivilized. Waste is a
problem of the modern civilized society. Materials used and waste generated by the
traditional societies were little and simple while those by the modern human societies are
large and complex. With modernization in development drastic changes came in our
consumer habits and life-style and in every activity like education, recreation, traveling,
feeding, clothing and housing we are generating lots of wastes. The world today generate
Corresponding Author: Rajiv.Sinha@griffith.edu.au
112
about 2.4 billion tones of solid waste every year in which the Western World alone
contributes about 620 million tones / year.
Discarded products arising from all human activities (cultural and developmental) and
those arising from the plants and animals, that are normally solid or semi-solid at room
temperature are termed as solid wastes. Municipal solid waste (MSW) is a term used to
represent all the garbage created by households, commercial sites (restaurants, grocery and
other stores, offices and public places etc.) and institutions (educational establishments,
museums etc.). This also includes wastes from small and medium sized cottage industries.
We are facing the escalating economic and environmental cost of dealing with current
and future generation of mounting municipal solid wastes (MSW), specially the technological
(developmental) wastes which comprise the hazardous industrial wastes, and also the health
cost to the people suffering from it. Developmental wastes poses serious risk to human health
and environment at every stage from generation to transportation and use, and during
treatment for safe disposal. Another serious cause of concern is the emission of greenhouse
gases methane and nitrous oxides resulting from the disposal of MSW either in the landfills or
from their management by composting.
Dealing with solid household waste in more sustainable ways involves changes not only
to everyday personal habits, consumerist attitudes and practices, but also to the systems of
waste management by local government and local industry and the retailers.
This chapter reviews the causes and consequences of escalating human waste, the
increasing complexity of the waste generated, and the policies and strategies of safe waste
management. It also provides food for thought for future policy decisions that government
of nations may have to take to reduce waste and divert them from ending up in the landfills,
drawing experiences from both developed nation (Australia) and a developing nation (India).
2. CITIES AS THE CENTERS OF MOUNTING MUNICIPAL WASTES

Cities have become major centers of consumption and waste generation all over the
world. In fact a city consumes as well as produce. This is called urban metabolism. City
use some 75 % of world resources and release a similar proportion of wastes. According to
UN Population Fund Report (1990), a city with one million population consumes 2000 tones
of food and 9,500 tones of fuel, generating 2000 tones of solid wastes (garbage and excreta)
and 950 tones of air pollutants; consumes 6,25,00 tones of pure water and secrete 5,00,000
tones of sewage. (UNEP, 1996).
United Nation Environment Program (UNEP) worked out the urban metabolism of
London city. Greater London with a population of 7 million consumed 2,400,000 tones of
food; 1,200,000 tones of timber; 2,200,000 tones of paper; 2,100,000 tones of plastics;
360,000 tones of glass; 1,940,000 tones of cement; 6,000,000 tones of bricks, blocks, sand
and tarmac; 1,200,000 tones of metals every year and produced 11,400,000 tones of industrial
and demolition wastes; 3,900,000 tones of household, civic and commercial wastes and
7,500,000 tones of wet, digested sewage sludge. Everyday London dispose off some 6,600
tones of household wastes. (UNEP, 1996).
Human Waste - A Potential Resource: Converting Trash into Treasure
113
3. MODERN CULTURE OF CONSUMERISM: THE ROOT OF WASTE

PROBLEM
The root of waste problem is the culture of consumerism and is directly proportional to
the affluency of the human societies. To this has added the culture of disposables. Large
number of goods in the society are being manufactured for only one time use, and to be
discarded as waste after use. Modern urban culture of using canned and bottled foods,
frozen foods, take-away foods, exchange of greeting cards on all occasions, using
disposable home equipments (spoons, cups, plates, tumblers and safety razors), medical
instruments (syringes, sharps and needles), office equipments (writing pens and utilities), and
plastic bags in all grocery shopping has escalated the solid waste problems.
People all over the world consume food and the 5 Ps (paper, power, petrol, potable water
and plastics) in their daily life without realizing the environmental consequences and costs of
the waste generated in their production, distribution, and consumption. Every commodity
processed from natural resources, and every consumer product, from shampoo to
champagne has an environmental cost and generally causes some damage to the
environment, before use, as a source of pollution during production, and after use, as
waste (Eklington and Hailes, 1989).
3.1. Waste Generation Is Proportional to Resource Consumption

and the Way We Use Resources
Consuming resources and generating wastes are two sides of the same coin. The way
we use resources to maintain our quality of life, assumes as much significance in waste
generation as the sheer amount of resources that we use. For instance, one kilogram of steel
might be used in a construction that lasts hundreds of years or in the manufacture of several
cans thrown away just after a few uses. A few kilograms of PVC plastic materials might be
molded into durable home and office furniture, water and sewer pipes, and remain useful for
decades or might be used to manufacture plastic bags to be used just once or twice and then
thrown away as enduring waste.
Packaging Culture Proliferate Waste Generation

Packaging materials have become part of our modern culture and generate huge amounts
of waste. Manufacturers and retailers see packaging as a way to attract purchasers. Everything
needs fine packaging today, from cornflakes to computers, from gifts to garments, from
flowers to foods. Life today cannot be imagined without plastic bags, glass bottles, paper
boxes, tins and cans. Plastics are versatile, convenient and light weight good packaging
material but ultimately end up as a non-biodegradable waste in the landfills to remain
intact for centuries.
On average each European Union citizen is currently responsible, directly or indirectly,
for the generation of some 172 kg of packaging waste every year. Packaging waste
generation increased by 10 % in the EU between 1997 and 2002. Per capita consumption of
plastics increased by almost 50 % from 64 kg / year in 1990 to 95 kg / year in 2002. Only UK
114
managed to actually reduce, and Austria stabilize the generation of packaging waste since
1997. (GEO, 2006).
Plastic consumption in Australia has increased from negligible quantities in the early
1940s to enormous quantities today. Plastics made up around one-third of all rubbish
collected on Clean Up Australia Day (Clean Up Australia 2004). Even though Australians
reduced their use of plastic shopping bags by around one-fifth between 2002 and 2004, each
person was still using almost one bag per day (EcoRecyle 2006). Australians use 6 billion
plastic bags every year much of that end up in landfills. These bags form litter, infest and
block waterways, kill animals.
The Ecological Footprint of Global Human Population

The Measure of Resource Consumption and Waste Generation. An ecological footprint is
an estimate of the average area of productive land and water required to maintain a given
populations resource consumption and waste generation. (Table 1). In this instance we
simply use it to indicate the comparable cost and sheer significance of waste generation.
Australia with a small population of just 19.5 million, is part of the North developed world,
and Australians are super-consumers and super-waste makers.
Table 1. Ecological Footprints of Human Population on Earth Reflecting Resource
Consumption Vis--Vis Waste Generation (2002)
Total
Population
(millions)
World
High income countries
Middle income countries
Low income countries
Australia
United Kingdom
China
Asia Pacific (regional)
Canada
USA
6 225.0
925.6
2 989.4
2 279.8
19.5
59.3
1302.3
3448.4
31.3
291.0
Total
Ecological
Footprint
(global ha/person)
2.2
6.4
1.9
0.8
7.0
5.6
1.6
1.3
7.5
9.7
Total
Energy Footprint
(global ha/person)
1.2
4.1
0.9
0.3
4.0
3.6
0.7
0.6
4.6
6.3
Total
Biocapacity*
(global
ha/person)
1.8
3.4
2.1
0.7
11.3
1.6
0.8
0.7
15.1
4.7
Source: Global Footprint Network (2005).

*Biocapacity includes cropland, grazing land, forest and fishing ground.
High income countries includes Australia.
In developing Asian countries, consumption and waste generation is growing at an

unprecedented pace and populous countries like China and India are becoming consumerist.
The consuming class in India is estimated at around 100 million, and in China at 200
million; traditional conservative Indians believing in modesty, simple living and saving, are
gradually giving way to rich generations highly influenced by the consumerist North (UNEP,
2006). China manufactures, packages, transports, distributes nationally and for export over 60
billion pairs of disposable wood chopsticks which use up over 32 million trees, harvested
115
unsustainably, indeed China risks being without forests within ten years.(<http://blog.
bcchinese.net/bingfeng/archive/2006/01/25/51815.aspx>).
4. WASTE GENERATED IN THE RICH VIS-A-VIS IN THE POOR

SOCIETIES OF WORLD
The United Nations Environment Program (1992) carried out a study of per capita waste
generation in the low, middle and the high-income countries and found it to be 0.5, 1.5 and
3.5 kg. per day per person respectively. The solid wastes generated in the rich affluent
societies of the developed nations are exceptionally large in quantity and varied in quality
(components). Of these, a considerable part is hazardous waste.
4.1. Waste Generated in the Rich Developed Countries of World

The World Watch Institute (1991), Washington, reported that 14 out of 16 members of
OECD countries showed increase in generation of MSW per person between 1980 85. In
the US, each sunset sees a new mountain of nearly 410,000 tones of garbage. (Toth, 1990).
The countries of the European Community (EC) throws away an estimated 2 billion tones of
solid waste each year. Only Japan and West Germany produced less waste, but after
unification MSW in Germany skyrocketed. Americans, Canadians and Australians are great
waste makers. They generate roughly twice as much garbage per person as West Europeans
or Japanese do. In his /her lifetime an average American wear and discard 250 shirts and 115
pairs of shoes; use and discard 27,50 newspapers, 3900 weekly magazines and 225 pounds of
phone directories; consume 12,000 paper grocery bags, use 28,627 aluminum cans weighing
1022 pounds, use 69,250 pounds of steel and 47,000 pounds of cement. The Scandinavian
nations generate much less waste than the Americans and Europeans. (WWI, 1991; UNEP,
1996).
Table 2. Average Per Capita Municipal Solid Waste (MSW) Generated by Some
Developed Nations Country Waste Generation Per Day (in Kg)
Country
USA
Japan
France
Singapore
Germany
Italy
Waste Generation Per Day (in Kg)

1.80 2.60
1.38 - 2.10
1.10 1.90
0.87 1.37
0.75 1.85
0.69 1.75
Source: WWI (1990):State of the World (These are 1990 Values which must have increased).
116

Table 3. Urban Waste Generated in Some Developed (Rich) Countries
Urban Waste Generated in Some Developed (Rich) Countries

Country
Annual Generation (In tones)
USA
20,00,00,000
Canada
1,26,00,000
Australia
1,00,00,000
Spain Netherlands
89,28,000 54,00,000
Belgium Sweden
30,82,000 25,00,000
Switzerland
21,46,000
Denmark
20,46,000
Norway New Zealand
17,00,000 15,28,000
Finland
12,00,000
Source: Dorling Kindersley Blueprint for Green Planet ;London (1987).
4.2. Waste Generated in the Poor Developing Countries of World

In the low and middle income developing countries of Asia, Asia-Pacific and Africa,
waste is a luxury, only produced by the wealthy minority which of course is increasing with
the growing economy and the exploding population. What is most concerning is that the
waste is not regularly collected by the municipal authorities and often becomes a horrible site
of piled and rotting waste on street corners with stray animals (dogs, pigs and cows) feeding
on the scraps. Domestic waste heaps often becomes sites of defacation and discharge of
human excreta and illegal dumping of hazardous wastes by scrupulous industries with
potential health threats. Municipal waste services often swallow between a fifth and a half of
city budgets, yet much solid waste is not removed. Even if municipal budgets are adequate for
collection, safe disposal of collected wastes often remains a problem. (Holmes, 1984).
Another ugly feature in these countries are that waste is often picked up by poor people
called rag-pickers for whom waste reuse and recycling is a way of life, and many poor
societies survive here by scouring the garbage of the rich for valuable scraps. They collect
recyclable wastes (mainly papers, plastics, glasses and metals) from the street corners and
even the dumpsites and sell them to the recycling industries to earn for their livelihood.
Table 4. Average Per Capita Municipal Solid Waste (MSW) Generated by Some
Developing (Poor) Nations Country Waste Generation Per Day (in Kg)
Country
Pakistan
Indonesia
India
Nigeria
Waste Generation Per Day (in Kg)

0.25 0.60
0.33 0.55
0.15 0.51
0.16 0.46
Source:WWI (1990);State of the World (These are 1990 Values which must have increased).
117
4.3. Typical Waste Components in the MSW Generated in Both Rich

Developed and Poor Developing and Underdeveloped Nations
Several waste components have been identified in the municipal solid waste (MSW) of
both the rich and poor societies of world. It ranged from twelve (12) to fourteen (14) and a
considerable portion of this waste is Organic while others are Inorganic. Ironically, there
is relatively high amount of food waste in poor developing and underdeveloped nations as
compared to the rich developed nations. In contrast, there is high amount of paper and garden
wastes in rich nations and low in poor nations. However, very little (or none) food waste
finally reach the waste dump-sites in poor nations as they are scoured and scavenged by stray
animals pigs, dogs and cattle and even by the poor street beggars. Paper, cardboard, plastics,
leather, wood and metals also do not reach the dump-sites and are picked up by rag-pickers.
(WHO, 1976).
Table 5. Solid Waste Components in MSW of Low, Middle and High Income Countries
(In % age)
Waste Components
Organic
Food Waste
Paper
Cardboard
Plastics
Textiles
Rubber
Leather
Garden Wastes
Wood
Inorganic Glass
Tin cans
Aluminum
Other Metals
Dirt, Ash etc.
Low-Income
Middle-Income
High-Income
40 85
1 10
15
15
15
15
1 10
15
1 40
20 65
8 30
26
2 10
14
1 10
1 10
15
1 30
6 30
20 40
5 15
28
26
02
02
10 20
14
4 12
28
01
14
0 10
Source: Tchobanoglous et al, Integrated Solid Waste Management; McGraw-Hill (1995).

Low Income = Underdeveloped African, Asian and Pacific Nations;
Middle Income = Developing Asian, African, Pacific and South American Nations.
High Income = Developed European and North American Nations and Australia.
Table 6. Typical Solid Waste Components in the MSW of an European Society Waste
Components Percentage (%)
Waste Components
1. Paper and paper products
2. Metals
3. Glass
4. Plastics
Percentage (%)
Germany
19.9
8.7
11.6
6.1
Switzerland
26.6
5.6
11.5
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Table 6 Continued
5. Textiles
6. Minerals
7. Wood, leather, bones, rubber
8. Compounded materials
9. Sieving fractions (0-12 mm)
10. Sieving fractions (12-50 mm)
11. Residue
12. Total unidentified fraction
1.5
2.9
2.3
0.8
8.6
15.6
26.8
51.0
2.8
1.1
3.1
0.7
9.2
8.1
27.1
44.4
Table 7. Typical Solid Waste Components in the MSW in U.S. Society (in % age)
Organic
1 Food Waste
2. Paper
3. Cardboard
4. Plastics
5. Textiles
6. Rubber
7. Leather
8. Yard waste
9.0
34.0
6.0
7.0
2.0
0.5
0.5
8.5
Inorganic
10. Glass
11. Tin cans
12. Aluminum
13. Other Metal
14. Dirt, Ash etc.
8.0
6.0
0.5
3.0
3.0
Total = 100.00
Source: Tchobanoglous et al, Integrated Solid Waste Management; McGraw-Hill (1995)
5. CHANGING CHARACTER OF THE MSW IN MODERN SOCIETY:

INCREASING AMOUNTS OF TOXIC MATERIALS
The character and composition of the municipal solid wastes (MSW) are changing in the
modern human society. Significant changes have occurred in the composition of municipal
solid waste (MSW) ever since the technological revolution of the 20th century. They are no
longer only organic waste, as it used to be in the earlier societies. The technological
development which mainly influenced the character of the MSW was the fossil fuel driven
industrial revolution and the agro-chemicals driven green revolution. Waste components
that have an important influence on the composition of the MSW are food waste, paper and
plastic wastes, the white goods and the hospital wastes. What is the matter of more serious
concern is that all living organisms including the human beings have become exposed to
chemicals for which there has been no evolutionary adaptation and experience. The chemicals
in the hazardous wastes mixed up with the MSW are completely foreign to living organism.
(Sinha and Sinha, 2000).
119
5.1. Changing Quality and Quantity of Food Wastes in the MSW

The quantity and quality of residential food waste has changed significantly over the
years as a result of technical advances in food growing (use of agro-chemicals) and food
processing and packaging (use of chemical preservatives), and public attitudes towards food
procurement i.e. relying more on processed and packed takeaway foods rather than cooking
food at home from raw materials. However, due to education and awareness about the
nutritive values of home cooked food, now people are moving back again towards home
cooking.
Two technological developments that have had a significant effect on generation of food
waste are the development of the food processing and packaging industries and the use of
kitchen food waste grinders in modern homes. Because of kitchen grinders the grinded food
wastes are delivered directly into the sewer systems rather than being disposed as MSW. In
these modern homes, the percentage of food wastes, by weight, has decreased from about 14
% in the early 1960s to about 9 % in 1992. In the packed and takeaway food culture, the
generation of food wastes in homes have reduced, but it has increased significantly in the
food processing industries and the food outlets. In homes, and the food outlets there are more
paper and plastic wastes due to over-packaging of processed foods, than food wastes itself.
5.2. Proliferating Plastic Wastes in the MSW

Percentage of plastics in MSW has also increased tremendously during the last 50 years.
The use of plastic has increased from almost non-measurable quantities in the 1940s to
between 7 - 8 %, by weight, in 1992. It is anticipated that the use of plastics will continue to
increase, but at a slower rate than during the past 25 years. (UNEP, 1996).
5.3. Escalating Electronics Waste in MSW: Heading for E-waste Tsunami?

Electronic waste is a growing concern as technology changes and new generations of
electronic products and equipments more sophisticated, improved and upgraded versions
continue to invade the market and the minds of consumers. The unfortunate part is that the
price of the new models and upgraded versions is continuously falling giving more temptation
to the consumers for discarding the old ones and replacing with the new.
The UNEP working group on Sustainable Product Design described the e-waste
essentially as a chemical waste. Electronics industry uses several hazardous chemicals
including toxic heavy metals (lead, cadmium, mercury, chromium, barium etc.), acids and
plastics, chlorinated and brominated compounds in production process. Developers of
electronic products are introducing chemicals on a scale which is totally incompatible with
the scant knowledge of their environmental or biological characteristics. (O, Rourke, 2004).
More than 2 million tons of e-waste ends up in landfills every year and there is serious threat
of leaching lead (Pb) and other heavy metals that may seep into groundwater supplies.
Incineration results into emission of dangerous dioxins and furans as e-waste contain
considerable amounts of plastics, brominated and chlorinated compounds. (Sinha, et. al.,
2006).
120
Recent study indicate that e-waste make up approximately 1 % of the MSW waste stream
in all developed nations and mercury (Hg) from the e-waste has been cited as the main source
of this heavy metal in the general MSW. E-waste in MSW is creating serious health and
environmental problems for the MSW landfills and the waste workers, and for the MSW
incinerators. In Europe the e-waste is growing at three times the rate of other MSW and
mixing with it. USA today is virtually sitting on a mountain of obsolete PCs. A report
produced by the Silicon Valley Toxics Coalition (a grassroots coalition that performs research
and advocacy on health and environmental issues related to electronics industries in the U.S.)
in 2001 suggest that if all the consumers decided to throw out their obsolete computer at the
same time, the country would face a tsunami of e-waste scraps between 2006 and 2015. The
report called Poison PCs and Toxic TVs was released by another grassroots organization
California Against Waste (CAW). (Anonymous, 1999).
Developing Countries as the Electronic Junkyards of U.S. and Industrialized Nations :

An Untenable Choice Between Poverty and Poison
There are reports about Asia, mainly India, China, Taiwan, Vietnam, Singapore and
Pakistan, being made as the high-tech dumping ground of U.S. An estimated 20 million
computers become obsolete each year in the U.S. and an estimated 200 tons of these
computers end up in these countries in the name of reuse and recycling. Low labor cost
and weak environmental regulations have made these countries dumping grounds of e-waste
destined for recycling and final disposal in landfills. A pilot program that collected electronic
scrap in San Jose, California estimated that it was10 times cheaper to ship CRT monitors to
China than it was to recycle them in the U.S. It is still legal in the U.S., despite international
law (The Basel Convention, 1989) to the contrary, to allow export of hazardous waste without
controls. (It may be recalled that U.S. has not yet signed the Basel Convention (1989) which
prohibits trans-boundary movement of hazardous wastes). Industry insiders indicate that
about 80 % of the e-waste goes to Asia and of that 90 % ends up in China. (Puckett et. al,
2002)
A report by Basel Action Network and the Silicon Valley Toxics Coalition Exporting
Harm: The Techno-Trashing of Asia asserts that 50 to 80 % of e-waste collected for
recycling in the U.S. is exported to developing nations. BAN produced a film on the report
which shows the Guiyu village in Guangdong province in China as electronics junkyard.
Some 100,000 men, women and children make US $1.50 a day dismantling e-waste by bare
hands to retrieve the valuable metals and materials. Circuit boards are melted over coal grills
to release valuable metals giving highly toxic dioxin fumes. Riverbank acid baths are used to
extract gold. Lead-containing cathode ray tubes from monitors and televisions are not of
much market value and hence are dumped in some wastelands. Toner cartridges are pulled
apart manually, sending clouds of toner dust into the air. Soil and drinking water at Guiyu are
contaminated by lead much above WHO limits- soil by 200 times and water by 2,400 times.
Water has to be trucked from 30 km away. At one point of time both China and India were
willing to take the e-waste for almost free. For poor countries of the world it is an untenable
choice between poverty and poison. (Puckett et. al, 2002)
In November, 2002 officials from eight Asian nations met in Tianjin, China, under the
auspices of Basel Convention (1989) to prevent their nations from being made the dumping
grounds of hazardous e-waste in the name of free trade (export and import) for recycling
discarded electronic products. It was represented by India, Malaysia, the Philippines,
121
Singapore, Sri Lanka, Thailand, and Vietnam. Resource persons came from Canada, Japan,
the U.S. and the Secretariat of the Basel Convention. Financial support was provided by
Australia, Japan and Canada. China has now banned and India also follows. (Sinha, et. al.,
2006).
5.4. The White Goods and Bulky Items in MSW

There are bulky items which include large worn-out or broken household, commercial
or industrials goods such as furniture, lamps, bookcase, filing cabinets, etc. There are
consumer electronics wastes which include worn-out, broken, and no longer wanted items
like stereos, radios, computer and television sets. There are white goods as waste which
include worn-out and broken household, commercial and industrial appliances like stoves,
refrigerators, dishwashers, cloth washers and driers. The rejected auto-parts like tires,
batteries and accessories also constitute important constituents of MSW. About 230 to 240
million rubber tires are disposed off annually in landfills or in tire stockpiles. (UNEP, 1996).
5.5. The Biomedical Wastes in MSW

The biomedical waste from hospital and clinics and slaughterhouses contain about 85 %
as general refuse, but 10 % is hazardous wastes contaminated with infectious pathological
agents, and 5 % is non-infectious but potentially toxic (chemicals) and radioactive and hence
hazardous. Dressing and swabs contaminated with blood and body fluids; syringes, needles
and sharps; surgically removed placenta, tissues, tumors, organs or limbs are potentially
infected wastes from hospitals. There are several disposable items made of PVC and
thermocol now being used in medical organizations. Hospital wastes are of special category
and require special care for final disposal. WHO has provided strict guidelines for their safe
disposal. (WHO, 1976).
6. THE COMPLEX SYNTHETIC WASTES INVADING HUMAN

ENVIRONMENT
Human ingenuity has created some new and synthetic materials in the wake of
technological revolution. They contain both organic and inorganic chemicals and resins and
creates more complex type of waste after being discarded. Nature do not possess any
organism and mechanism to biodegrade them.
The processing of some new materials discovered by technology such as
semiconductors, optical fibers, new class of ceramics, and composites requires the use
of large amount of toxic chemicals which eventually ends up as hazardous wastes in the
MSW. These technological wastes are often toxic and are posing danger not only for the
environment, but also for the human health. It has already caused several accidents, deaths
and disabilities among the municipal waste workers.
122
6.1. The Non-Biodegradable Wastes: Potential to Remain Long in Human

Ecosystem
The synthetic wastes are non-biodegradable because they cannot be decomposed and
can remain in the human ecosystem for years and decades polluting the environment.
Common examples are all forms of plastics, x-ray films, celluloid films, cells and batteries,
several chemicals and all synthetics.
What nature cannot do, human beings are trying to do through the knowledge of
environmental biotechnology. Genetically tailored bacteria are being created which would
possess the necessary enzymes to degrade the synthetic wastes. Some strains of bacteria and
fungi have been identified in nature too, which has the arsenal to degrade some of the
complex organic chemicals. Efforts are also being made to create biodegradable synthetics
using starch as the raw material. They can be degraded in 4-6 weeks. (Sinha and Sinha,
2007).
6.2. The Hazardous Wastes: Permeating the Human Society

Wastes containing toxic chemicals, radioactive substances and infectious materials which
poses potential risk to human health and environment are categorized as hazardous wastes.
Toxicity, radioactivity, flammability, chemical reactivity, corrosivity, nonbiodegradability, carcinogenicity, mutagenicity, infectiousness, oxidizing and leachating are
some of the characteristics of hazardous wastes. (WHO, 1983).
Many primary and manufacturing industries using toxic chemicals generate hazardous
waste (solid or liquid) in the production process. They can be referred as industrial hazardous
wastes (IHW). Many of our favorite cultural activities depend on products the manufacture
of which creates industrial hazardous waste. Glaring examples are glass and metal, paper
and plastic, leather and textile, painting and dyeing, printing and publication and
photography and dry cleanings. Consumer industries today use a variety of chemicals to
produce consumer goods a number of which are now disposables. When these items are
consumed and discarded by society, they eventually end up as hazardous wastes in our
homes. This can be referred as household hazardous waste (HHW). Prime examples are
torch dry-cells and batteries, pesticides / disinfectant cans and bottles, fluorescent tubes and
electric bulbs, detergents and shampoos, lead-acid car batteries, auto tires and waste oils, and
the expired medicines.
As we enjoy the benefits of consumer goods (furniture and fixtures, white goods,
electrical and electronic goods, automobiles, processed and packed food and drinks etc.)
produced by consumer industries, we also generate considerable amount of hazardous wastes
as by-products when we discard them after use or change the old version with new. They
can be referred as consumer hazardous waste (CHW). Industries producing products that
sustain our modern life-style and living habits generate tremendous amount of hazardous
wastes. Glaring examples are the agro-chemical industries (to boost our food production)
and the petroleum industries (which drives our automobiles). (Raghupati, 1994; Sinha and
Herat, 2004).
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Household Hazardous Wastes (HHW): The Poison in Our Homes

Household hazardous wastes are either solids, semi-solids, or liquids. In addition trace
chemical compounds can exist as a solute within a liquid solvent, as a gas adsorbed onto a
solid, or as a component of the gaseous emissions from MSW.
Plastics contain organochlorine compounds, organic solvents in PVC; paints contain
heavy metals, pigments, solvents and organic residues; home pesticides contain
organochlorine and organophosphates compounds; oil and gasoline contain phenols and
organic compounds, heavy metals, salt acids, ammonia and caustics; textiles may have metal
dyes and organochlorine compounds; carpets can contain chemical stain resisters, pesticides,
solvent-heavy glues, VOCs laden underlayer; curtain treated with stain resisters and backing
can give off VOCs; dry-cleaned clothes give off VOCs; chip board and plywood furniture
releases formaldehyde; fibre-glass based insulation of ceiling cavity gives off VOCs; window
sealants give off toxic fumes; bathroom air fresheners release VOCs; and the carcinogenic
benzene can be formed in the garage from the car exhaust. Virtually all of the mercury in
MSW is due to the disposal of household dry cell batteries (mercury, alkaline, and carbonzinc types). A smaller amount of mercury may come from the disposal of broken home
thermometers. (Sinha, et. al., 2005 a)
Several toxic chemicals are commonly used today in modern homes that also results into
generation of HHW. They are usually mixed and disposed with the MSW. Some glaring
examples of toxic chemicals used in modern homes that contributes in the generation of
household hazardous wastes (HHW) are1) Perfumes and cosmetics used by the women contain some 884 neurotoxic chemical
compounds. (Report of National Institute of Occupational Safety and Health in the
US).
2) Paper whitener is toxic. The chemical used in it has potential to kill.
3) Cadmium is present in food processing equipment, kitchenware enamels, pottery
glazes and plastics and relatively high levels in the sea foods.
4) Lead is present in paints and dyes, toys and newspapers, solder and batteries, lead
water pipe;
5) The highly toxic polychlorinated biphenyls (PCBs) are added to paints, copying and
printing paper inks, adhesive and plastics to improve their flexibility. Fish food
contain generally higher levels of PCBs. High levels of PCBs were reported from
the breakfast cereals in Sweden and Mexico as a result of contamination by
packaging materials.
6) Containers of paints and enamels used in homes and in automobiles. They contain
dangerous chemicals like glycol, ether, ammonia, benzene and formaldehyde and
continue to give out toxic fumes at least for 7 years.
7) Containers of pesticides, insecticides, herbicides and fungicides are available in
modern homes to eradicate pests and insects in garden plants, cockroaches and
spiders in kitchens and storerooms.
8) Many relatively innocuous items, such as plastics, glossy magazines, and flashlight
batteries used in homes, contain metallic elements.
9) Metals like cadmium (Cd), chromium (Cr), mercury (Hg), and lead (Pb) are present
in several household items. After combustion with MSW metals are either emitted as
124

particulate matter or vaporized into air. Mercury pose a particular problem because it
volatilizes at a relatively low temperature, 675 F. (UNEP, 2004).
Very little is known about the amount of HHW generated in various countries. UNEP
(2006) reported that The Netherlands generate 41,000 tonnes of HHW every year. The
University of Arizona, US, made a survey and found that about 100 hazardous items
(containers) are discarded per household each year. Australian study made in Melbourne in
1990-92 also found 89,576 kg of hazardous wastes from households. (CSIRO, 1996).
Mercury in the MSW from Household Wastes Has the Potential to Kill : A Case Study
from U.S.
It is interesting to note that if the tons of household batteries generated in California is
calculated for whole of U.S., the total amount would be 160,000 tons per year. Given that a
typical household battery weighs 50 grams, the corresponding number of batteries is
2,910,000,000. It is estimated that more than 2,700,000,000 battery units were purchased in
the US in 1990. If half the household batteries were alkaline, and assuming that each battery
contained about 1200 mg of mercury (Hg), then, based on the data reported above, 1923 tons
of mercury (Hg) would enter the environment each year in California alone. This mercury is
enough to kill 8,730,000,000 people based on a lethal dose of 200 mg per person
(Tchobanoglous et. al., 1993).
Such situation exist in all metropolitan cities of world, in both developingand the
developed countries. Clearly, proper disposal of these household batteries in the MSW is an
important issues that must be addressed. If not collected separately, all these household
batteries get mixed up with MSW and is disposed in the ordinary sanitary landfills instead
of the secured landfills.
The Hazards of Disposable Baby Nappies in the MSW
More than 20 billion disposable baby nappies (equivalent to some 2.7 million tons of
solid waste) are ending up in the landfills every year the world over. They contain hazardous
chemical sodium polyacrylate (a super water absorbent) responsible for several medical
conditions in infants, including hampering of genital growth in male child. On an average
disposable nappies occupy landfill space of 0.40 m2 per child per year in Australia. They are 2
% by weight in total solid waste but occupy 3.5 % of total landfill space. They are also
disturbing the natural microbial biodegradation processes of organic wastes in the closed
landfills by absorbing all water internally. (Brahambhatt and Saeed, 2005).
6.3. The Nuclear Waste : Radioactive Substances Invading the Human

Environment
Nuclear wastes are the result of our urge to generate nuclear energy without emission of
greenhouse gases (which is in fact a myth as it requires 18 years of CO2 producing fossil fuel
energy (in uranium mining and enrichment, building reactors etc.) to produce one calories of
nuclear generated energy. (Report of Friends of Earth, 2000). Nuclear waste are produced
regardless of whether nuclear fission is controlled (such as for energy generation in reactors),
125
or occurs explosively, as in the atom bomb. The resulting fission products, isotopes of
approximately 30 elements, have mass numbers in the range of 72 to 162, are for the most
parts solids, and emit beta particles, together with electromagnetic reaction (gamma rays)
which are exceedingly penetrating. The chemical separation of fission products and their
conversion to nuclear fuel are the most important sources of radioactive wastes. The
radioactive wastes can be in all the three forms solid, liquid and gaseous and two categories
of radioactive wastes are mostly encountered- the Low Level Radioactivity Waste (LLRW)
and the High Level Radioactive Wastes (HLRW). (IAEA, 1991).
Eight tons of liquid radioactive waste result per year from the typical average-size,
nuclear reactor. The nuclear reactors mostly generate HLRW in the form of plutonium-239
(Pu239). Other two most significant fission products are strontium-90 (Sr90) and cesium-137
(Cs137) with half-life of 19.9 and 33 years respectively. They are routinely emitted from the
reactors and continue to release radiation energy over long periods of time (several
generations of the human race). Even dismantling (decommissioning) of retiring nuclear
reactors produce enormous amount of radioactive wastes and contaminate vast land area.
There are 439 nuclear power reactors in operation around the world mostly in France and
Japan. They would all retire in years to come.
Uranium mining and processing (enrichment) produces huge amount of solid and liquid
radioactive wastes which is highly hazardous. The extraction of uranium from the earth crust
leaves vast quantities of wastes as tailings which contain up to 80% of the original
radioactivity of the extracted ore. After mining uranium is further enriched to produce
nuclear fuel. Depleted uranium hexafluoride (DU) is a radioactive waste by-product of
enrichment. For every 1000 tones of processed uranium fuel , 100,000 tones of mined wastes
as tailings and 3,500,000 liters of liquid waste is produced. They migrate into the
environment through air, soil and water. Processing of uranium ores produces considerable
volumes of alpha emitters, mainly radium-226. The half-life of Ra226 is 1600 years and gives
rise to a toxic gas radon. (IAEA, 1991).
7. WASTE : POTENTIAL SOURCE OF GREENHOUSE GASES

(METHANE AND NITROUS OXIDE)
So far, not much attention was paid towards this aspect of waste generation. Marked
increases in the amount of waste generated has also contributed to emission of greenhouse
gases carbon dioxide, methane and nitrous oxides. A major issue of concern today is emission
of greenhouse gas methane (CH4) resulting from disposal of MSW in landfills and this may
be between 45 60 %. This is mainly due to anaerobic degradation of the organic waste
components in the landfills as oxygen becomes deficient due to compaction. Methane is 2025 times more powerful GHG than carbon dioxide (CO2) in absorbing the infrared solar
radiation.
Studies have also indicated high emissions of nitrous oxide (N2O) in proportion to the
amount of food waste. N2O is mainly formed under moderate oxygen (O2) concentration.
(Yaowu et. al., 2000). Molecule to molecule N2O is 296 times more powerful GHG than
carbon dioxide (CO2). Yaowu et. al., (2000) has studied the emission of both methane and
nitrous oxides from aerated food waste composting.
126
In fact, improved recycling of waste can significantly contribute to abatement of GHG

emissions. However, convention microbial composting (biological recycling) of organic
wastes also emits methane (Wang et, al., 1997) due to anaerobic sites appearing in the inner
layers of compost piles. However, it can be reduced significantly by improving aeration in
the waste biomass by periodical turning or through mechanical aerating systems. (Toms et.
al., 1995). Significantly, vermicomposting of waste by waste eater earthworms decrease the
proportion of anaerobic to aerobic decomposition, resulting in a significant decrease in
methane (CH4). Earthworms can play a good part in the strategy of greenhouse gas reduction
and mitigation in the disposal of global organic wastes. Currently we are studying the
potential of GHG emissions by various systems of biodegradation of wastes (Aerobic &
Anaerobic Composting & Vermicomposting by Earthworms). (Chauhan & Valani, 2008).
8. SAFE MANAGEMENT OF WASTE : A TECHNO-ECONOMIC

PROBLEM
Both rich developed and the poor developing nations of world have become conscious
towards safe waste management in the changing situation where the human waste is no longer
simple and organic to be salvaged by nature in course of time but getting more complex and
even hazardous, and threatening to remain in the human ecosystem for long time. Safe
management of all waste becomes imperative for the safety and security of the society and it
require the input of knowledge of diverse disciplines of material science, political science,
economics, geography, sociology, demography, urban planning, public and environmental
health, communication, conservation, and civil and mechanical engineering.
Waste management has been termed as Cradle- to- Grave management i.e. from point
of generation to final disposal, involving safe storage, transport and treatment- and in all these
steps, the generator owes the main responsibility. The collection and disposal of waste
involves huge expenditures in the development of landfills, waste collecting vehicles,
precious fuel (petrol and diesel) and labour costs.
8.1. The Natures Technology at Work: Salavaging the Organic Wastes

The organic wastes in the MSW are biodegradable and are decomposed in nature by
diverse microorganisms bacteria, fungi, actinomycetes and the protozoa. Among the
biodegradable wastes some are rapidly degraded while others are slowly degraded over
time. It may take time from few days to several months and years to degrade the organic
wastes, but it does happen ultimately. Some organic materials in the waste decomposes
rapidly (3 months to 5 years), while others slowly (up to 50 years or more). The relative ease
with which an organic waste is biodegraded (decomposed) depends on the genetic makeup
of the microorganisms present and the chemical makeup of the organic molecules (mainly
carbon structure that the organisms use as a source of energy and biodegrade in the process).
Carbons in sugars, lipids and proteins are easily decomposed than the carbon in lignin, while
the carbon in plastics are not at all biodegraded.
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The biodegradability depends to a large extent on the lignin content (present in wood
fibers) of the waste. Lesser the lignin content, rapid will be the biodegradation rate of that
organic material. Nature has those decomposer microorganisms (mainly bacteria and fungi)
in soil, air and water which perform the task. This is how the natural ecosystems on earth has
been operating since life evolved. Had there been no decomposer organisms, the earth would
have been full of animal and human excreta, animal carcasses and vegetable matters (leaves
and twigs) and dirt, and life impossible. The biodegradable waste include all plant, animal
and human products, the kitchen waste in every home and restaurants, wastes from the
agriculture farm, food processing industries, slaughter houses, fish and vegetable markets,
and paper and cotton wastes. All these wastes mainly contain organic matters.
The process of biodegradation (decomposition) in nature can be enhanced to several
times by introducing decomposer organisms such as the earthworms or even the bacterial
biomass directly into the waste biomass. This is being done these days to dispose the
mounting organic wastes rapidly. The process is called composting and the byproduct is
NKP rich biofertilizer.
Table 8. Rapidly and Slowly Biodegradable Organic Constituents in MSW
Rapidly Biodegradable
Food Waste
Newspaper
Office Paper
Cardboard
Yard Waste (Leaves and Grass Trimmings)
Slowly Biodegradable
Textiles
Rubber
Leather
Yard Waste (Woody portions)
Wood
Misc. Organics
Table 9. Biodegradability of Some Organic Components in the MSW

Component
Food Waste
Newspaper
Office Paper
Cardboard
Yard Waste
Lignin Contents (% of volatile solid)

0.4
21.9
0.4
12.9
4.1
Biodegradability (% of vs)
82
22
82
47
72
8.2. Sanitary Landfills - The Ultimate Graveyard for Wastes: An Economic

and Environmental Burden
Sanitary landfills constitute the ultimate graveyard for the safe burial of all human waste
in the womb of mother earth. There is an enormous range of materials found in landfills.
Todays landfill is tomorrows time capsule, writes Blatt (2005). Experience have shown
that modern landfills although made with great engineering skills are unable to contain the
toxic landfill gases and the leachate discharge into the environment. They are proving to
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be a techno-economic burden and a curse in disguise. The cost involved in landfill

construction is not only up-front, but also in its monitoring and maintenance, for controlling
landfill gases and the leachate collection etc., which is long term, often up to 30 to 50 years.
The up-front development costs for new landfills in U.S. varied from US $ 10 million to $ 20
million in 1992, before the first load of waste was placed in the landfill. This must have gone
up substantially by now. (Tchobanoglous, 1995).
Health and Environmental Concerns of Waste Landfills : The Uncontrolled Release of

Greenhouse and Toxic Gases into the Environment
Methane (CH4), carbon dioxide (CO2), carbon monoxide (CO), hydrogen (H2), oxygen
(O2) and nitrogen (N2) are the principal landfill gases. Methane and carbon dioxide are known
greenhouse gases and molecule to molecule methane (CH4) is 20 25 times more powerful
greenhouse gas than carbon dioxide in absorbing the infrared solar radiation. Methane can
cause explosion when present in air in concentrations between 5 and 15 %. Ammonia (NH3),
hydrogen sulfide (H2S) and the Volatile Organic Compounds (VOCs) are in trace amounts.
They are products of biochemical reactions occurring in the landfills. Landfill gases migrate
horizontally and migration distances greater than 300 m have been observed with 40 %
concentration.
Gas samples collected from over 66 landfills in UK showed the presence of 116 organic
compounds, many of which are classified as VOCs. (UNEP, 1996). VOCs are mostly evolved
from the newly placed MSW and specially which also contains hazardous wastes (HW). The
increasing quantities of household hazardous wastes(HHW) in the modern society and their
disposal along with the MSW, is posing a serious problem for the landfill engineers.
Table 9. Typical Hazardous Chemical Constituents Found in MSW Landfill Gases
Chemical Constituents
Methane
Carbon dioxide
Nitrogen
Oxygen
Sulfides, disulfides, mercaptans, etc.
Ammonia
Hydrogen
Carbon monoxide
Trace constituents (In VOCs)
% (By Dry Volume Basis)

45 60 (Greenhouse gas)
40 60 (Greenhouse gas)
25
0.1 1.0
0 1.0
0.1 1.0
0 0.2
0 0.2
0.01 0.6
Table 10. Concentrations of Toxic Trace Compounds Found in MSW Landfill Gases
Compound
Acetone
Benzene
Chlorobenzene
Chloroform
Mean Value in Parts Per Billion (ppb) by Volume

6, 838
2, 057
82
245
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Table 10.- Continued

Compound
1,1 Dichloroethane
Dichloromethane
1,1 Dichlorethene
Diethylene chloride
trans- 1,2-Dichloroethane
Ethylene dichloride
Ethyl benzene
Methyl ethyl ketone (MEK)
1,1,1-Trichloroethane
Trichloroethylene
Toluene
1,1,2,2-Tetrachloroethane
Tetrachloroethylene
Vinyl chloride
Styrenes
Vinyl acetate
Xylenes
Mean Value in Parts Per Billion (ppb) by Volume

2, 801
25, 694
130
2, 835
36
59
7, 334
3, 092
615
2, 079
34, 907
246
5, 244
3, 508
1,517
5,663
2,651
Some hazardous chemicals like vinyl chloride is going to the landfills by way of plastic
bags. Residents are throwing their kitchen wastes mostly packed in grocery plastic bags.
Earlier, there was also a practice to dispose some industrial solid wastes (ISW) with MSW
in the landfills, which have now been banned. However, to minimize the emission of VOCs, a
vacuum is applied and air is drawn through the completed portions of the landfill.
Trace gases although present in small amounts, can be toxic and pose grave risk to public
health and environment. Trace compounds may carry carcinogenic and teratogenic
compounds into the surrounding environment. Half-lives of various trace compounds in the
VOCs have been found to vary from fraction of a year to over a thousand years. (Heath,
1983).
The Uncontrolled Release of Leachate and the Threat of Contamination of Groundwater and Surface Water
The liquid (waste juice) that collects at the bottom of the landfill is known as leachate.
It is the result of percolation of precipitation, uncontrolled runoff, and irrigation water into the
landfill. Leachate can also include water initially contained in the waste as well as infiltrating
groundwater. Leachate seeps downward to the base of the landfill by gravity and poses a
potential health risk to public as it can percolate into the groundwater aquifer and contaminate
it. Concern is growing worldwide about wastes leaching heavy metals that may seep into
groundwater supplies. Leaching into soil and groundwater will occur regardless of whether
the landfill is sealed or not. It has become a common knowledge that all landfills leak. Even
the best state of the art landfills are not completely tight throughout their lifetimes and a
certain amount of chemicals and metal leaching will occur.
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Landfill leachate contains a variety of chemical constituents including heavy metals (Pb,
Cu, Ni, Cr, Zn, Cd, Fe, Mn, Hg, Ba, Ag), arsenic, cyanide, fluoride and selenium, and organic
acids derived from the solubilization of the materials deposited in the landfill and from the
products of chemical and biochemical reactions occurring in the landfills. (Tchobanoglous et,
al.,1995). Disposal of consumer electronics mixed with MSW accounts for 40 % of lead (Pb)
in the landfills. Mercury (Hg) will leach when circuit breakers are destroyed, PCBs will leach
when condensers are destroyed. When plastics with brominated flame-retardants or cadmium
containing plastics are landfilled, both PDBE and cadmium (Cd) may leach into the soil and
groundwater. (Miller, 2004).
8.3. Thermal Destruction (Incineration) of Waste

High temperature incineration of wastes especially the hazardous chemical and
biomedical wastes is considered to be the safest remedy to get rid of it. It enables
detoxification of all combustible carcinogens, mutagens and teratogens. Destruction is done
in stages. In the first stage the waste is thermally decomposed at 800 C in a refractory lined
chamber to produce ashes and volatiles. The ashes are removed and the volatiles with gases
are led to second stage where they are heated to around 1200C with additional air. It is the
only environmentally acceptable means of disposing some complex organic wastes like
chlorinated hydrocarbons, polychlorinated biphenyls (PCBs) and dioxins. These chemicals
are persistant, non-biodegradable and highly toxic.
However, the conventional incinerators emit dangerous dioxins, furans and other
highly toxic pollutants when inorganic chemical dyes in plastics are incinerated. Dangerous
dioxins may also be formed if some organic materials are incinerated at too low temperatures.
But, the modern, well-regulated incinerators have dramatically reduced such toxic emissions.
It has been observed that injection of lime and activated carbon significantly remove the
dioxins and mercury from the gases by 95 %. However, people in most countries are
opposing incinerators and landfills in their neighborhood.
Plasma Arc Incineration System

High temperature incineration using plasma arc furnaces is a growing technology for
the management of hazardous chemical and biomedical wastes. In plasma arc system a
thermal plasma field is created by directing an electric current through a low-pressure gas
stream. Plasma fields can reach temperature from 5000 C to 15,000 C. This intense high
temperature zone can be used to dissociate the wastes into its atomic elements in the
combustion chamber. Heat generated from the plasma torch can melt and vitrify solid wastes.
Organic components can be vaporized and decomposed by the intense heat and ionised by the
air used as the plasma gas. Oxygen may also be added in the primary chamber to enhance
combustion. Metal-bearing solids are vitrified into a monolithic non-leachable mass.
(Hasselriss, 1995).
The system is hermetically sealed and operated below atmospheric pressure to prevent
leakage of process gases. Dioxin formation is prevented. The vented gas is held in the tank
and recycled into the furnace. The clean gases are released into the atmosphere through an
exhaust stack. The destruction and removal efficiency (DREs) of organic compounds are
131
greater than 99.99 %. There is less public opposition to such incineration system. Plasma
technologys inherent ability to eliminate risks of future liabilities from waste disposal
through a single step treatment is an added advantage. A mobile plasma arc system would
also reduce or eliminate public health risks associated with the hazards of accidents or spills
resulting from the transportation of toxic or hazardous wastes by road or rail.
Plasma Arc Technology has proved to be an ideal and economically viable method to
successfully treat even the carcinogenic asbestos waste, the nuclear power plant wastes and
for safe disposal of arms and military waste worldwide. Temperatures in the order of 1,000
C are necessary to irreversibly transform asbestos into a non-hazardous material. Plasma
treatment transformed the asbestos waste into a harmless vitrified slag.
Japan has been using the plasma arc technology on large scale to vitrify its municipal
solid wastes (MSW) to reduce the volume of its MSW going to the landfills. Land starved
Japan cannot afford to have large number of landfills on its island. The slag produced by the
plasma arc pyrolysis is recycled into glassy bricks used as construction materials in buildings.
General cost of waste treatment by plasma arc technology range between US $ 400 and $
2,000 per ton, depending on the characteristics of the waste. PAT is however, a capital
intensive technology due to its initial equipment costs. A new plasma waste processing plant
can cost between US $ 3 million and $ 12 million depending on size, the hazardous nature of
the waste and the complexity of the treatment process. (Hasselriss, 1995).
Combining Incineration with Energy Generation : Killing two Birds in One Shot
Although waste incineration has been discredited worldwide particularly due to emission
of dioxins and furans, most European nations have waste incinerator plants combined with
energy recovery plants, and several categories of wastes (with high calorific value) including
the hazardous wastes are used as fuel to achieve the dual objectives of waste disposal and
electricity generation. The waste-to-energy movement started with the oil crisis in the
Middle East and the increased cost of oil in the 1970s. Denmark and Sweden are leaders.
They incinerate 65 % and 55 % of their MSW respectively and also produce thermal
electricity from steam generation. Of the 12 MSW incinerator plants in Netherlands, 5
generate thermal electricity. Some large German cities operate combined incinerator plants
providing up to 5 10 % of the total electricity demand. U.S. has few incinerators and
incinerates 16 % of its MSW currently, but recovers energy from almost 80 % of the plants.
The U.S. National Energy Strategy (1991), projected 7 fold increase in the electricity
generation from MSW incineration plants by 2010. Nearly three quarters of the Japans 1900
MSW incinerators, recover energy. (UNEP, 1996).
9. EMBRACING THE 5 RS PHILOSOPHY OF WASTE MANAGEMENT

THROUGH WASTE AND CONSUMER EDUCATION
As the generation of waste has increased in volume and also become more complex in
nature (due to mixing of non-biodegradable plastic wastes and household hazardous wastes)
traditional waste management systems have proved inadequate and inappropriate. Landfills
are not the lasting solution to the waste problem. Worldwide consensus is emerging to
discourage incineration and landfill disposal of wastes. In accordance with the EU Landfill
132
Directive of 2001, the UK government is now phasing out the landfills for waste disposal.
Several U.S. states have banned landfill disposal of e-waste.
9.1. Waste and Consumer Education for the Wasteful Modern Society
Environmental education about consumption and waste is a kind of re-education of the
already educated but ignorant modern wasteful society. Waste generation involves the entire
population, so broad cooperation is necessary for sustainable and efficient waste
management. Partnerships are required between people and governments to deal with waste
collection and disposal. Waste and consumer education aims to raise public awareness about
the stresses on municipal councils and enable communities to understand their role and share
in the financial responsibilities needed for efficient management of solid wastes.
Educational programs must involve consciousness-raising on these fundamentals:
Waste is an inevitable by-product of all individual and social activities and is

proportionate to daily consumption.
Waste is a potential resource and every individual needs to commit to help recover
waste by reuse and recycling.
Waste generation can be minimized it can never be eliminated through
changing the patterns of production and consumption of goods and materials either
used most and/or that are used once then end up as waste.
Judicious consumption of food and the 5 Ps paper, plastic, power, petrol and
potable water can help minimize waste generation on a large scale.
However, there are series of issues that consumers have no control over and that require
government interventions at the level of production and distribution, involving manufacturers
and retailers.
The Consumer Power of Acceptance / Rejection Can Positively Influence Consumer

Industries Counteracting Vested Commercial Interest : India Showed the Way in 1940s
Consumer power can change things provided they are aware. By accepting or rejecting
consumer goods for sale, buyers can positively influence the production process, conveying
messages to industries and retailers to reduce waste at source both in quantity as well as in
quality (hazardous). Consumers can also influence government and industrialists policies to
manufacture goods that are durable, and also re-useable and recyclable after one use.
Consumer boycotts, which represent the kind of non-violence preached by the great Indian
saint, Mahatma Gandhi, back in the 1940s, counteract the vested commercial interest of
manufacturing industries promoted by media for commercial gain.
Consumer Education: Women Holds the Key
Consumerism is a growing human culture all over the world. Shopping is a key activity in
market based modern societies and grocery shopping for domestic consumption is done
mostly by women all over the world who are keenly aware of the various shelf products. They
133
also observe the invasion of diverse consumer products in the market through media and
assess their values for family consumption.
However, buying less and narrowing choices are seen as infringements on peoples
rights. Individuals and householders who care about the environment can feel powerless.
Therefore policy makers, program coordinators, and educators need to address a series of
complex issues associated with educating about consumerism and waste generation.
A news poll survey conducted in mid-2005 found that five out of ten women compared
with three out of ten men were saying No to plastic bags and only two in ten women but
more than a third of the men surveyed preferred to use plastic bags rather than reusable ones
(Clean Up Australia 2006) ; (Also on <http://www.noplastics.org.au>).
International Efforts and Role of UNEP, WWF and WWI

The United Nation Environment Program (UNEP) and the World Wide Fund for Nature
Conservation (WWF) and the Washington based World Watch Institute (WWI) are playing
important roles in educating people at an international level, creating mass awareness about
waste problems as well as other environmental issues. UNEP sponsored the first Clean Up the
World Campaign, 17-19 September 1993. Several million people, from 79 countries, actively
participated in that campaign, which combined dual objectives, environmental sanitation and
resource generation. The campaign followed up on the first World Clean Up Movement in
Sydney, Australia, initiated by Australian yachtsman, Ian Kiernan, on 8 January 1986.
Kiernan gathered around 40,000 Sydney siders to collect more than 5000 tons of rubbish from
different parts of the city. The first Clean Up Australia Day, 1990, involved almost 300,000
volunteers.
Programs have been developed to influence consumers and to encourage them to rethink
about their patterns of consumption which can reduce waste. The Japanese Environmental
Agency (JEA) has launched a scheme called Household Eco-Account Books that
encourages the citizens to live sustainabily in an environmentally friendly manner and also
save money by consuming resources judiciously and by embracing the philosophy of 3 Rs
for waste reduction, reuse and recycling. In Norway NGOs run a program that builds
householders awareness on environmental impacts of consumer products, especially on its
contribution to piling municipal solid waste (MSW) and also the household hazardous wastes
(HHW) as several consumer products in modern homes may contain hazardous chemicals and
materials.
9.2. Educating about the Golden Rules of 5 Rs: Refusal, Reduction, Reuse,
Recycling and Responsible Behavior
People and policy makers need to embrace the 5 Rs environmental philosophy for
sustainable waste management. In the waste management hierarchy waste refusal and
reduction is the best option, waste reuse and recycling the better option than the waste
disposal in landfills. (Sinha et. al. 2005 c). It is imperative to educate the masses to 1. Refuse to accept articles and materials that generate / create more waste, especially
those that generate toxic and hazardous wastes;
134

2. Reduce waste at source by consuming resources as little as possible and which is
enough to enjoy a minimum quality of life;
3. Reuse articles and materials several times and as much as practicable before
discarding them as waste and insist on buying durable, reusable and recyclable
articles and products and repair household goods before rejection as waste;
4. Recycle / recover / retrieve new materials / energy from discarded waste products
and assist the recycling industries by separating the recyclable waste from the nonrecyclables faithfully at source; and to behave with
5. Responsibility (for both consumer societies and producer industries) with regard to
judicious use of resources and reduction in waste generation in everyday activities.
If people (society) and producers (industries) embrace the 5 Rs golden rule majority of
the waste will be taken care of and very little will be left for treatment or to contain them
and finally to dispose them in landfills.
The environmental organization EcoRecycle of Victoria, Australia (2006) proposed a
waste management hierarchy which emphasizes that waste reduction / avoidance should
be the first option and disposal the last. Waste reuse and recycling comes after reduction. If
this management plan is sincerely implemented, there will be very little waste left to be
disposed finally. (Figure 1).
Figure 1. Source: EcoRecycle 2006.
135
The Power of Refusal : It Can Prevent Misuse of Resources

In the 1980s, the Women Environmental Network (WEN) of the UK campaigned against
over-packaging of food products and refused to buy those products that were heavily wrapped
with plastics and papers. Women groups went into supermarkets, ripping off all the
unnecessary extra layers of paper and plastics and handed them back to the managers. WEN
has campaigned against several environmentally unfriendly consumer products such as paper
handkerchiefs and toilet rolls made from newly produced paper refusing to accept them.
(Belamy 1995).
Way back in the 1940s the great Indian philosopher and educator M.K. Gandhi, used this
power of refusal against the mighty British Empire (although in a different context) to
generate awareness among the masses.
Box 1. Responsibility of Consumers: Check Before Shopping and Refuse to Accept
Articles That Can Generate More Waste Upon Use
1.
2.
3.
Refuse to accept non-biodegradable plastic bags for grocery and other shopping
force manufacturers and retailers to offer environmentally friendly alternatives.
Refuse to accept any plastic or paper bag for small or just a few articles that you can
carry instead in your pockets or personal bags always carry easily tucked away
cloth or alternative bags for shopping.
Refuse to accept articles over-packed in plastic and paper, which have no eco-label
(are not produced through clean methods of production), and have the potential to
generate more waste when used.
The Wisdom of Waste Reduction / Prevention: It Conserves Material Resources

This is the best option in the waste management hierarchy. Preventing waste is like
preventing a social disease to occur. Waste reduction means conserving resources, that
otherwise constitute waste, with further economic and ecological benefits, including:
conserving valuable raw geo-chemical and biological resources, water and energy (fossil
fuels) used in the manufacture of products; saving money otherwise spent on constructing and
maintaining waste landfills; reducing health and environmental impacts of air, water and soil
pollution and global warming. Science and technology, the industry and the society all have
to play critical role towards waste reduction. If people judiciously use and reduce the
consumption of 5 Ps (paper, plastic, power, petrol and potable water) in daily life, it would
dramatically reduce all the three wastes- the solid, liquid and the gaseous from the
environment.
Society has to begin the process of reducing waste at source the home, office, or
factory- so that fewer materials will become part of the disposable solid wastes of a
community. Efforts must be made to reduce the quantity of materials used in both packaging
and obsolescent goods. Waste reduction may also occur at the household, commercial, or
industrial facility through selective and judicious buying and consuming patterns and the
reuse of products and materials. Source reduction is an option that will conserve resources
and also has economic viability.
136
Technology Has Improved Efficiency of Resource Use and Reduced Waste Generation
by Consumers at Source
Technological advancement has undergone a process of dematerialization for reducing
the consumption of resources (metals, plastics, glasses etc.) in manufacturing products, with
consequent reduction in waste generation. Many packaging items (cans and bottles),
consumer electronic goods and even the automobiles have become lighter, slikker and
smaller. Since 1977, the popular 2-liter PET plastic soft drinks bottles have been reduced
from 68 grams each to 51 grams, a 25 % reduction in material used per bottle. One hundred
12 fluid ounce aluminum cans which weighed 4.5 pounds in 1972, only weighed 3.51 pounds
in 1992, a 22 % reduction in material use. Steel beverage cans have also been downsized and
are now 40 % lighter than they were in 1970. This means that people can still enjoy a good
quality of life while consuming smaller amounts of resources from the environment and
generating lesser amount of waste. Lesser resource use would also mean lesser energy
consumption and lesser waste generation, thus benefiting the environment in every way.
(WWI, 1991).
The Wisdom of Waste Reuse: It Extends the Life of Material Resources
There are several articles like bottles and jars made of glasses and tough plastic materials,
large tins, metallic cans and cansisters which can remain in our economy and ecosystem for
very long time (if not discarded as waste) just by simple cleaning and washing.
Box 2. Recipe and Responsibility of Consumers to Reduce and Re-use Waste in Daily
Life
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
Buy in bulk and concentrates.

Buy durable not disposable products.
Buy products packed in reusable and refillable containers.
Chose products without or as little packaging as possible.
Take Your Own Bags, Box or Basket (TYOB). Insist on reusable cotton bags or durable
synthetic bags for grocery shopping; keep a few used shopping bags in your car.
Select paper bags/wrap only if needed and avoid plastic bags/wrap.
Reuse paper/envelopes only printed/photocopied on one side printed.
Use rechargeable batteries.
Use refillable ink fountain pens and biros.
Avoid using greeting/invitation cards.
Avoid wrapping, or sparsely wrap, presents etc.
Avoid paper napkins carry cotton handkerchiefs.
Save electronic copies of data or print and photocopy on both sides of paper.
Prepare meals at home (not take-away/convenience foods).
Avoid frequent use of canned, bottled, packed, processed and preserved foods and eat
raw, fresh foods (good for human and environmental health).
Avoid peeling fruits and vegetables to reduce kitchen wastes (and preserve nutrients for
digestion) and compost any food scraps and leftovers.
Carry food and edibles to school/workplace/friends place in reusable boxes.
Repair clothes and garments, toys, tools and appliances.
Go to garage sales rather than throwing away old things or buying new ones.
Use safe retreated car tyres.
137
Box 3. Responsibility of Producers (Industries) to Reduce Waste

1.
2.
3.
Producers of consumer goods and products MUST embrace the philosophy and
principles of cleaner production, which emphasize producing more with less,
thus conserving resources and reducing waste.
Producers MUST embrace the ethical principle of producing durable, re-usable
and recyclable goods, which can remain in use for longer periods of time even
through changes in the purposes of their uses before being discarded.
Producers MUST inform consumers about the recycling potential of their products
and be committed to take back products after use for recycling, to recover
maximum useful materials from them to reduce the amount of final waste.
It do not involve any complex industrial processing, use of chemical and energy for their
reuse. They might have been originally made for some other use, but now can be reused for
different purpose- especially for storing and packaging. It should rather be termed as
resource reuse.
The Wisdom of Waste Recycling : Retrieving New Materials / Energy from Waste and
Conserving Virgin Raw Materials and Natural Resources
Waste recycling is a technological process to reuse waste materials involving physical,
chemical and biological processing to recover useful materials from them. It converts waste
into a resource, conserves primary and virgin raw materials from environment (geo-chemical
and bio-chemical natural resources), saves tremendous amount of water and energy and
protect the environment. Hidden environmental protection values of recycled goods, include
the energy and water saved, the pollution and deforestation prevented.
Government and Peoples Support is Paramount for Recycling to Succeed
Recycling combines social, economic, and ecological values. The opportunity to recycle
provided by local and state governments has seen an almost fourfold increase in the Victorian
recycling industry in Australia in the years 19932003 (EcoRecycle, 2006). EcoRecycle
estimates that re-processors have saved: water equivalent of filling 17,500 Olympic sized
swimming pools; greenhouse gas equivalent to that produced by 580,000 cars; and, enough
energy to power every household in Victoria (Australia) for 7 months (EcoRecycle, 2006).
Consumers can support recycling by buying goods which bear recycled stickers from
their manufacturers. People can indirectly support and promote recycling industries by
separating the recyclable from non-recyclable wastes and can directly participate by recycling
domestic, e.g. kitchen, wastes through composting. Policy makers could encourage or
regulate for all recycled goods to bear a tag recording the origin and life history of the goods,
i.e. identifying from which waste it was produced and how it has saved damage to the
environment. Such initiatives allow consumers to confidently reuse and recycle products. A
demand for recycled goods in society can be created by education.
138

Box 4. Responsibilities of Consumers (Society) in Promoting Waste Recycling
1.
2.
3.
Separate recyclable wastes glass bottles and jars, aluminum and steel cans, plastic
bottles, milk cartons, paper, cardboard, magazines and phone books from other
household wastes faithfully for collection by councils or deliver them to recycling
centers / industries.
Buy recycled goods. This will encourage promotion of recycling efforts by the
government and industries.
In order to make recycling an effective strategy, people must understand more about
the qualities of materials in everyday items. For example, several vitreous materials
that look like, and shatter like, glass are not recyclable. Even a minute amount (such
as 5 grams per ton) of these vitreous materials found in some ceramic mugs and
plates, cups and crockery, mirror, broken drinking glasses, flower vases, light globes
and laboratory glass, can contaminate a load of recyclable glass and render it useless.
Box 5. Responsibilities of Producers (Consumer Industries) Towards Waste Recycling

1.
2.
3.
Producer industries MUST produce / manufacture consumer goods that are

potentially recyclable. 2. Producers should have a policy to take back their own
products to recycle them;
Producers should provide necessary information to its prospective buyers on the
matter of using, handling, conservation, disposal and recycling potentialities of its
products.
In designing new products, the industry must assess its potential and even suspected
adverse impact on its consumers health and the environment.
10. WASTE RECYCLING : A GROWING GLOBAL BUSINESS WHERE

WASTE (TRASH) IS TURNED INTO RESOURCE (TREASURE)
Waste is no longer considered as a discarded product to be disposed off from the human
ecosystem. Wastes are in fact now considered as a misplaced resource to be brought back
into the human ecosystem through the reuse and recycling technologies. Recycling means that
otherwise wasted items are returned back to the countrys economy to make either the same
product again, another product or other products. This saves cost on landfill disposal, save
landfill space, and prolongs the life of the primary resources used to make the product.
(Fairlie, 1992).
Recycling can involve mechanical / biological / chemical / thermal processing of waste
using some energy, water and chemicals to effectively and efficiently reconvert the waste into
secondary raw materials to manufacture new products or recover energy from them in the
form of fuel gas or heat. Several of the items of domestic, industrial (non-hazardous) and
commercial wastes viz. paper, leather, rubber, cotton rags, metals and glasses, wood and
plastics can be recycled in the material recovery and reprocessing industries to get valuable
new products.
Science and technology has provided a tool in the hands of mankind to renew all those
non-renewable resources on earth which otherwise cannot be renewed by natures
139
mechanism. This would also save tremendous energy, preserve forest, prevent soil erosion
and pollution and above all arrest emissions of greenhouse gases (GHGs) and reduce global
warming.
Some economies in the developed nations of Europe and America, and also in Australia,
is retrieving back 80 to 85 % of the materials from the waste as useful products for societal
consumption or to be reused in developmental activities and only less than 20 % are going to
the landfills for final disposal. (Goldoftas, 1989).
10.1. Global Trade and Exchange for Recycling

The old saying one persons trash is another persons treasure is becoming true as
waste trade and exchange between nations for recycling is entering into new era both
economically, ecologically and politically. Japan, Germany, France, Hong Kong, U.K., U.S.
and also Australia is recycling their wastes on large scales. Japan recycles 65 % of its MSW
into usable products and 40% of its waste paper into high quality paper.
A computerized waste exchange register has been prepared to link waste producers
with potential waste users and diverse waste items like discarded aluminum cans, paper and
card-boards, wooden crates and off-cuts, steel plate off-cuts, plastic products, saw dust, eggshells, caustic soda and chemicals have been listed in this register. The OECD countries have
established a central system for moving the non-hazardous recyclable wastes across
international borders under the rules of normal trade goods listed in the green list of Basle
Convention (1989). In these countries several C and F agents have come up with list of waste
available and waste wanted. The buyers benefit by the reduced cost of raw materials, while
the sellers benefit by the reduced cost of treatment and disposal of wastes.
Trade in waste between two countries is economically and politically justified if it is
based on ethics. Faced with rising cost of safe waste disposal and recycling at home, many
industrialized nations prefer to pass their hazardous wastes along with the recyclable wastes
on to the poor Third World countries where there are facilities, cheaper manpower and
infrastructure for waste recycling. This has happened greatly in case of hazardous electronics
wastes. U.S. has dumped huge pile of e-waste in China and India for recycling.
Trade-in Program for Recycling Electronic Waste

Recycling is a good option for the extremely old generation computers such as the PrePentium generation, or the computers (specially the monitors) which are broken. According to
the International Association of Electronics Recyclers (IAER) more than 1.5 billion pounds of
electronics equipment are recycled annually and is likely to grow by a factor of 4 or 5 by the
end of this decade. Eleven countries currently have mandatory electronics recovery laws on
the books. These are Denmark, The Netherlands, Norway, Sweden, Switzerland, Japan,
Belgium, Taiwan, Portugal and South Korea. Some EU nations have very strong system for ewaste collection, such as the SWICO system in Switzerland and the Netherlands Association
for Disposal of Metalectro Products (NVMP). NVMP collect 80 % of e-waste. About 77 % of
TVs and 64 % of other small brown goods are recovered for reuse and recycling. (Cui and
Forssberg, 2003)
Most major computer manufacturers in world e.g. Dell, Hewlett-Packard (HP), Compaq,
Gateway have begun to address e-waste problems with their own end-of-life management
140
programs which offers a combination of trade-in, take-back and recycling programs. Dell and
Gateway lease out their products thereby ensuring they get them back to further upgrade and
lease out again. Dell offers both reuse and recycling programs. Dell purchases it computers
for a nominal $15 fee. They will recycle both Dell and non-Dell computers, but for corporate
customers only. Dell Europe, however, recycles individual consumers computers. Gateway
provides $25-50 cash refund for new PC and donate the customers used computers. HP and
Compaqs trade-in program provides a refund cheque for the value of the used computer if it
is traded-in on the purchase of a new HP/Compaq computer. HP has been very active in
recycling programs. Since 1987, it has recycled over 500 million pounds of materials from the
e-waste and pledge to achieve 1 billion pounds by 2007. HP entered into a joint venture with
Micro Metallics in 1996 to recycle materials recovered internally and to recover parts from
products returned by customers and reported US $ 72.3 billion as revenue from the recycling
program. IBM accepts any type of PC (even other brands) but would charge $ 29.99 to take it
back. (Cui and Forssberg, 2003).
10.2. Economics of Recycling: Value Addition When Garbage Becomes Gold

Any waste has negative economic and environmental value and is a big techno-economic
problem for the local government and involves an economic and environmental cost in safe
disposal. But if the same waste is converted into an useful new material its economic and
environmental value goes to the positive. (Sinha, 1994).The Brisbane City Council in
Queensland, Australia process over 60,000 tones of recyclable materials every year and add $
20 million to their economy. (BCC, 2002).
What was the value of flyash before technology founds its use to reconvert it into
building material ? It was an environmental a hazard. What is the value of food scraps and the
human excreta ? It is a human waste to be safely disposed off every day and involves cost.
Composting technology can convert them into a high value end product i.e. compost (a
nutritive organic fertilizer for the farms). Biogas technology can now retrieve methane- a
clean burning fuel for power generation. (Sinha, 1994; Goldstein, 1995). Recycling also
reduces the cost of construction and siting of new landfills and closing it after use. It
reduces the economic and environmental cost of incineration of several categories of wastes.
Recycling economy is a closed loop in which consumers, manufacturers and waste
collectors and haulers, all have a critical sustaining role to play. Many waste articles in the
society are potentially recyclable, but they may not actually be recycled unless there is a
practical way to do so and there is a demand / market for the recycled goods. The waste
haulers would be encouraged to collect the recyclable wastes if there is a demand by the
recycling industries, and the recycling industries will buy these wastes (as secondary raw
materials for processing) only if it is less expensive (economically cheaper) than the primary
(virgin) raw material. The consumers will buy recycled items only if it is as good as the
product made from virgin materials and still less expensive. None of them are bothered about
the high environmental cost of the procurement of the primary raw materials from earth or
the products made from them.
When recycled materials have a high social and economic value despite the cost of
collecting and processing, they find a ready market. Much of the gold, silver and other
precious metals that were ever mined and extracted from their primary ores centuries ago is
141
still circulating (recycling) in our economy (human ecosystem). Materials with low social and
economic value relative to the cost of collecting and processing do not find a ready market.
The grocery and kitchen wastes, agriculture, dairy and slaughter house wastes and the
sewage sludge, with greater organic contents can be biologically recycled to get fuel (biogas)
and fertilizer (compost). Even the human hair which are protein materials can be recycled
for getting amino acids and making newer and cheaper proteins. It is like getting gold from
the garbage and silver from the sewage.
Recycling of hazardous industrial wastes also minimizes the cost and risk of transporting,
storing, treating and disposing of hazardous wastes to distant places. US and Japan recycles a
great part of its hazardous wastes. Several recyclable hazardous wastes are exchanged
among nations under strict rules of Basel Convention (1989). The recycling potential of
wastes from the pharmaceutical industries is 95 %, paints and allied products 40 %, organic
chemicals 25 %, petroleum refinery 10 % and small industrial machinery 20 %. Used and
discarded products from the automobiles e.g. the lead-acid batteries (LABs), the waste oil and
the auto tires are also recycled.
In some cases the waste may have to undergo some modifications, such as dewatering,
in order to become recyclable and salable product. An aluminum die-casting firm developed a
market for a by-product of their production process- the fumed amorphous silica. After
much researches into uses for the product it was found to be a valuable additive to concrete.
The firm marketed the waste and now sells all the fumed amorphous silica it generates to
cement plants. This is bringing an income of US $ 1 million every year for the company and
also saves the enormous cost of disposal. (Noll et. al., 1985).
An x-ray film manufacturer in U.S. generates a salable waste product. The company
installed equipment that flakes and bales waste polyester coated film stock which is sold as
raw material input to another firm. Over 9 million kg of film stock is exchanged each year.
This saves US $ 200,000 annually which would have incurred in collection, transport, and
disposal cost. Above that, there is annual profit of US $ 150,000 to the x-ray film
manufacturing firm from the sale of the recyclable materials. (Noll et. al., 1985).
10.3. The Environmental Significance of Recycling

Nature possess tremendous capacity of recycling of several categories of wastes by
biodegradation (biological recycling) aided by the decomposer organisms on earth. The
earth provides all the necessary resources for development of mankind and also assimilate
all the wastes generated by them in the process of those developmental activities. This is
defined as the carrying capacity of Earth. Unfortunately, due to the growing consumerism of
resources and the consequent increase in the quantity and concentration of human wastes on
earth, this carrying capacity is threatened to be jeopardized with dangerous consequences for
the global ecological balance and grave risk of poisoning of the life support systems on earth.
The addition of non-biodegradable technological wastes has aggravated the problem. They
can remain in the human environment for centuries as nature has not evolved the mechanism
to degrade and recycle these new man-made materials.
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Table 10. Environmental Benefits of Recycling of Papers, Metals and Glasses
Waste
Recyled
1. Iron & Steel Scraps
2. Aluminum Cans
3. Papers Wastes
4. Glass Wastes
Energy
Savings
60-70 %
90-95 %
60-65 %
30-32 %
Pollution
Control
Reduction in
Solid Waste
Water
Savings
Forest
Protection
30 %
95 %
95 %
20 %
95 %
100 %
100 %
60 %
40 %
46 %
58 %
50 %
100 %
100 %
100 %
-
Source: WWI (1984) State of the World.
By resorting to recycling technologies we can actually renew and sustain the natural
carrying capacity of earth ecosystems and not only the natural human wastes can be
reconverted into a resource, but also the man-made synthetic and hazardous wastes.
Recycling of some municipal and industrial wastes such as papers, metals, glasses can accrue
several environmental benefits by way of water and energy saving (consequent reduction of
greenhouse gas emission), control of air pollution and forest protection.
Figure 2. Explaining the Economic and Environmental Significance of Recycling.
10.4. Need for Appropriate Recycling Technologies

Several municipal as well as industrial solid wastes have potential to be recycled. They
only need appropriate technologies for recycling. New recycling techniques are being
developed constantly to maximize recovery of useful materials and minimize the amount of
waste going to the landfills. Essentially two types of recycling technologies are being
developed for waste processing to get valuable end products mechanical and biological.
Mechanical technology involves thermal and chemical processing of waste (both organic and
inorganic fractions), while biological technology involves biological processing of waste
143
(organic fraction only) by microbes and earthworms. While mechanical technologies incur
expenditure of energy, the biological technologies may generate energy.
10.5. Biological Recycling of Wet Organic Wastes into Fertilizer and Fuel:
Diverting the Major Part (70- 80 %) of MSW from Landfills
Biological recycling involves processing of wet and dry organics such as the food waste
from homes and commercial institutions, green garden and farm wastes, cattle and farmyard
wastes and the waste organics from the food processing industries). The traditional
composting methods are essentially a biological recycling technology which is being revived
and improved with new knowledge in microbiology and environmental biotechnology. Other
biological recycling methods developed are technology to retrieve biogas and bio-alcohol
(cleaner energy sources) from organic wastes.
MSW contain 70-80 % by weight of organic materials and the waste biomass is rich in
carbon (C) and nitrogen (N) with other valuable minerals like phosphorus (P) and potassium
(K) and have potential to be biologically recycled to recover fertilizer and fuel (energy).
However, waste with high organic components should preferably be recycled to produce
fertilizers (composts) and not fuel. (White, 1996).
Table 11. Useful Materials Recovered from Waste by Recycling Technologies
Recyclable Materials from MSW
Useful Materials Recovered / Typical Uses
WET RECYCLABLES
Organic Fraction of MSW (70-80 %)
Food waste, yard & garden waste etc.
Recovery of fertilizer (compost) and

fuel (methane and ethanol) ;
DRY RECYCLABLES
Paper
Old Newspaper
Corrugated Cardboard
High-grade Paper
Plastics
Polyethylene Terephthalate (PET)
High-density Polyethylene (HDPE)
Polyvinyl Chloride (PVC)
Low-density Polyethylene (LDPE)
Polypropylene (PP)
Polystyrene (PS)
Newsstand and home-delivered newspaper

Bulk packaging, fiberboard & roofing material
Computer paper, white ledger paper
Soft drink bottles, salad dressing and vegetable oil
Bottles; photographic film
Milk jugs, water containers, detergent and cooking
oil bottles.
Home landscaping, irrigation pipes, some food
packaging, and bottles
Thin-film packaging and wraps; dry cleaning film
bags; other film material
Closures and labels for bottles and containers,
battery casings, bread and cheese wraps, cereal
box liners
Packaging for electronic and electrical component,
144

Table 11 Continued
Multilayer and other mixed plastics

Glass
Metals
Ferrous Metals (Iron & Steel)
Non-ferrous Metals
Textiles
Construction & Demolition Wastes
foam cups, fast-food containers, tableware and

microwave plates;
Packaging, ketchup and mustard bottles
Clear, green and brown glass bottles & containers;
Tin cans, white goods, reinforcing bars
Aluminum cans, copper and lead products
Wiping rags
Soil, asphalt, concrete, wood, drywall, shingles
HAZARDOUS WASTES
Auto Tires
Household Batteries
Automobile Batteries
Incinerator Residues
Engine Oil and Lubricants
Road building materials, paving and tire-derived

fuel;
Recovery of zinc, mercury and silver
Recovery of acid, plastic and lead
Concrete, road construction material
Refined Oil
Fertilizer Production : Retrieving Nutrients from Organic Wastes

i) Compost from Waste (Getting Gold from Garbage)
There is always greater economic as well as ecological wisdom in converting as much
waste into compost (a nutritive fertilizer for farms), so that less and less waste (the noncompostable and non-recyclable residues) finally go to the landfills. The organic fraction of
MSW (70-80 %) is rich in nitrogen, potash and phosphorus (NKP) and all macro and
micronutrients. This can be easily recycled through microbial biodegradation process called
composting, to retrieve the nutrients from them. If residents all over the world, practice home
composting and backyard composting of their kitchen and garden wastes, this will
significantly reduce burden on the councils and very little MSW will be left for the landfills.
Even councils should practice composting of waste on commercial scale instead of sending
them to the landfills, earn money by selling them to the farmers, rather than spending money
on their landfill disposal.
The components that constitute the organic fraction of MSW are food wastes, paper,
cardboard, plastics, textiles, rubber, leather, yard wastes, and wood. Yard waste may contain
even higher percentage of organic matter. All of these waste materials can be recycled, either
separately or as a commingled waste. Certain industrial wastes such as those from the food
processing, agricultural and paper-pulp industries are mostly organic in composition and
can also be composted. Controlling the environmental conditions i.e. the biological, physical
and the chemical factors can significantly improve and enhance the composting process
without the emission of foul odor and pollution of the environment and without the loss of
essential nutrients from the compost. (Epstein, 1997).
The conventional composting technology has now been significantly improved with our
modern scientific knowledge in microbiology and biotechnology to biodegrade all kinds
of organic wastes including the municipal solid wastes (MSW)containing sufficient organic
components under a completely controlled environmental conditions. We have innovated
145
some new, cost-effective and more efficient methods of converting organic waste into
compost which besides providing the macro and micronutrients also provide beneficial soil
microorganisms to the soil and work as soil conditioner to prevent soil erosion. (Haug,
1993; Epstein, 1997).
The relative ease with which an organic material is biodegraded (composted) depends on
the genetic makeup of the microorganisms present and the chemical makeup of the
organic molecules. Carbons in sugars, lipids and proteins are easily decomposed than the
carbon in lignin. A small portion of the carbon is converted to new microbial cells, while a
significant portion is converted to carbon dioxide and lost to the atmosphere. The new cells
that are produced become part of the active biomass (decomposer microbes), to further
enhance and multiply the biodegradation and composting process and on death ultimately
become part of the compost.
European cities and societies are developing and adopting ambitious technologies for
recycling and composting of wastes to divert them from landfills. Some cities have installed
sophisticated equipments for composting and resource recovery. Austrian, French and Swiss
cities have taken the lead in installing waste recycling and composting systems. Twenty seven
(27) composting plants with a combined annual capacity of 60,000 tones of compost from
city trashes are currently under construction in German towns and cities (UNEP, 2004).
Vermicomposting: Using Waste Eater Earthworms for Rapid and Odorless Composting
of Municipal and Industrial Organic Wastes
Vermicomposting is rapid and odorless process triggered by earthworms through
enzymatic breakdown of waste organics and also enhancing the microbial degradation by
proliferating the microbial population in the waste biomass. Vermicompost is completely free
of pathogens but rich in beneficial decomposer microbes including nitrogen fixing bacteria,
mycorrhizal fungi and actinomycetes. It is rich in NKP, trace elements, enzymes, growth
promoting hormones (gibberlins and auxins) and readily works as soil conditioner.
Long-term researches into vermiculture have indicated that the Tiger Worm (Elsenia
fetida), Red Tiger Worm (E. andrei), the Indian Blue Worm (Perionyx excavatus),the African
Night Crawler (Eudrilus euginae),and the Red Worm (Lumbricus rubellus) are best suited for
vermi-composting of variety of organic wastes including some of the hazardous wastes like
the sewage sludge (biosolids) from the sewage treatment plants and the fly-ash from the
coal power plants.
Vermiculture was started in the middle of 20th century for management of municipal /
industrial organic wastes in Holland in 1970, and subsequently in England, and Canada. Later
vermiculture were followed in USA, Italy, Philippines, Thailand, China, Korea, Japan, Brazil,
France, Australia and Israel. However, the farmers all over the world have been using worms
for composting their farm waste and improving farm soil fertility since long time. In UK,
large 1000 mt vermi-composting plants have been erected in Wales. The American
Earthworm Technology Company started a 'vermi-composting farm' in 1978-79 with 500 t
/month of vermicompost production. Japan imported 3000 mt of earthworms from the USA
during the period 1985-87 for cellulose waste degradation. The Aoka Sangyo Co. Ltd., has
three 1000 t /month plants processing waste from paper pulp and the food industry. This
produces 400 ton of vermicompost and 10 ton of live earthworms per month.
The Toyhira Seiden Kogyo Co. of Japan is using rice straw, municipal sludge, sawdust
and paper waste for vermicomposting involving 20 plants which in total produces 2-3
146
thousands tons of vermicompost per month. In Italy, vermiculture is used to biodegrade

municipal and paper mill sludge. Aerobic and anaerobic sludge are mixed and aerated for
more than 15 days and in 5000 cum of sludge 5 kg of earthworms are added. In about 8
months the hazardous sludge is converted into nutritive vermicompost. In France, 20 tons of
mixed household wastes are being vermi-composted everyday using 1000 to 2000 million red
tiger worms (Elsenia andrei) in earthworm tanks. Rideau Regional Hospital in Ontario,
Canada, vermi-compost 375 - 400 kg of wet organics mainly food waste everyday. In Wilson,
North Carolina, U.S., more than 5 tons of pig manure (excreta) is being vermi-composted
every week. (Edward, 1998; Frederickson, 2000; Fraser-Quick, 2002; Sinha et. al., 2005 b).
ii) Fertilizer Pellets from Sludge Cake (Getting Silver from Sewage)
A technology has been developed in U.S. by Massachusetts Water Resources Authority
to recycle the dewatered sewage sludge into fertilizer pellets. The sludge is shipped from
sewage treatment plant and pumped directly from barges into the storage tank and then
transferred to belt-filter press where water is mechanically squeezed out. The sludge cake is
moved through the conveyer belts to rotating heat dryers and heated to convert into small
hard pellets. This also destroys the foul smell and harmful bacteria. The pellets are low-grade
fertilizers, but if blended with other synthetic nutrients can form a complete fertilizer. Cities
all over the US is operating sludge processing plant like MWRA.
Fuel Production : Retrieving Cleaner Sources of Energy from Organic Waste
The organic municipal solid wastes enriched with biomass and materials with high
calorific value (combustible) can be recycled to yield either gaseous fuel methane (biogas)
or liquid fuel ethanol by fermentation. The wastes can be directly incinerated and the heat
liberated is used for steam generation and electricity production. The new idea is to use the
wastes a source of fuel in cement kilns in cement industries to replace the costly fossil fuels.
The emission problems accompanying incineration is also minimized to a great extent. But
only wastes with high calorific value is useful for energy recovery. (Parker and Roberts,
1985; Porter and Roberts, 1985).
i) Biogas
There is always greater ecological wisdom in generating biofuel (biogas and
bioethanol) from the MSW rather than generating thermal energy from them by combustion
technology, with accompanying release of toxic gases especially dioxins and residual
ashes which needs landfill disposal.
The biogas technology by the use of anaerobic metanobacteria utilizes the organic
wastes rich in cellulosic materials with high carbon and nitrogen (C/N) ratio. It produces both
fuel and fertilizer. Each ton of organic waste by dry weight yields about 36 cum of biogas and
350 kg of biomanure. Methane is a clean burning substance and on combustion yields 550
BTU of heat per cft of its volume. Even the sewage sludge rich in organic matter and high
C/N ratio is efficiently recycled to yield methane.
ii) Bio-diesel
Bio-diesel is emerging as an alternative cleaner fuel for diesel engines brewed from waste
organic feed-stocks, such as animals waste fats (tallows), lard and waste cooking oils. This is
147
being produced in Australia and other countries on commercial scales. Bio-diesel is also
being produced from offal at a turkey-processing plant in the U.S. Any vegetable oil can form
the feedstock to produce bio-diesel. It can produced by recycling the waste oil from fast-food
restaurants and the deep friers of French fries which generate huge amount of waste vegetable
oils.It can now be produced from household waste and used auto tires.
Bio-diesel is completely non-toxic and biodegradable, almost free of sulfur and
aromatics, and have lower CO2 emissions than the mineral oil derived diesel. It can be used in
existing fuel engines without any modifications. The emissions from bio-diesel are 100 %
lower in sulfur, 96 % lower in total hydrocarbons (HC), 80 % lower in polycyclic
hydrocarbon (PAH), 45 % lower in carbon monoxide (CO) and 28 % lower in suspended
particulate matters (SPM). (UNEP, 2006).
iii) Bio-alcohol (Ethanol)

Waste biomass rich in starch and cellulosic materials provides good raw material for
ethanol production by enzymatic fermentation carried out by organisms yeast and some
bacteria. Bagasse, the waste from sugarcane industry is most appropriate raw material. 6000
kg of bagasse upon fermentation yields 1000 litres of ethanol of 95 % strength. It is a cleaner
auto-fuel and Brazil is already using it as an auto-fuel since 1975.
New bioconversion technologies could open the gate to the cost-effective use of a wide
variety of feed-stocks including agricultural waste products like corn stalks, rice and wheat
straws and perennial grasses to produce bio-fuel ethanol
iv) Coal Briquetts
Technology to recycle the agricultural wastes into a non-polluting fuel has been
developed. The dried biomass is crushed and pre-heated to a temperature of 100-120 C and
then compacted.
v) Fuel Pellets
A technology has been developed for converting garbage into non-polluting fuel pellets.
The garbage is first shredded and blown dry in rotary kiln. It is then blended with combustible
wastes like saw dust and is then pelletized. The pellets have calorific value around 4,000 kcal
/kg and there is no harmful emission upon combustion.
10.6. Mechanical Recycling of Dry Recyclables into Original or New

Products
Mechanical recycling involves processing of dry organic and inorganic recyclables such
as papers and cardboards, plastic cans and bottles, metal cans, glass jars and bottles.
Mechanical methods include thermal and chemical processing of waste materials and also
consumes considerable amounts of water and energy. Now new mechanical technologies is
being developed to recycle even hazardous industrial wastes.
Metals, glasses, papers and plastics, wood and rubbers are some common domestic and
commercial wastes which have high recycling potential to recover goods of mass
consumption through mechanical technologies. (WWI, 1984).
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Recycling Potential of Metallic Wastes

Iron, aluminum, lead, tin and copper are metals of mass consumption by the society.
Their production from their virgin ores by mining activities requires huge amount of energy
and water and also cause blatant environmental pollution, solid waste generation (as tailings)
and deforestation. Metals like iron and aluminum can be effectively recycled for ever with
least impact on environment while conserving huge amount of energy. (AEN, 2000).
a) Ferrous Metals (Iron and Steel)
The ferrous scraps include autos, household appliances, equipment, bridges, cans and
other iron and steel products. The largest amount of recycled steel has traditionally come
from large items like road unworthy cars and appliances. It can be reclaimed from the
automobile bodies and engines, disused household or industrial equipment and building
materials. The shipyards, railyards and the automobile junkyards offer vast amount of waste
metallic products to be recycled. They can alone meet more than 50 % of the worlds metal
requirements. Marine ship break is a continuous process throughout the world as the ships
lose sea worthiness in 25 to 30 years.
Recycling of steel cans is also becoming very popular. They can easily be separated from
the mixed recyclables or MSW using large magnets. To protect them from corrosion, all steel
cans are coated with a thin layer of tin that must be removed in the recycling process.
Immersing the sheets of steel in alkaline bath and transmitting an electric current completes
the detinning process.
Iron and steel has high recycling potential and can be recycled again and again without
reducing the quality of the end products. Nearly half of the iron and steel which has already
entered into the human ecosystem is now being used through recycling. World steel
production alone consumes as much energy annually as Saudi Arabia produces. Making steel
from recycled materials uses only a quarter of the energy needed to make steel from iron ores.
(AEN, 2000). Iron scraps costs little more than iron ore but can be converted into steel with
much lower economic and environmental cost. Using coke for iron ore reduction produce
copious particulate matters including carcinogenic benzopyrene. Recycling of iron reduces
this emission by 11 kg/metric tons of steel produced and also cuts iron ore waste and coal
mining wastes by 1100 kg/metric tons recycled. Iron and steel scraps are baled into bricks at
the material recycling facility (MRF) and melted at 1700 C in the smelters to produce ingots.
b) Non-ferrous Metals
Non-ferrous scrap metals include aluminum, copper, lead, tin, and precious metals.
Recyclable nonferrous metals are recovered from common household items (outdoor
furniture, kitchen cookware and appliances, aluminum cans, ladders, tools, hardware); from
construction and demolition projects (copper wire, pipe and plumbing supplies, light fixtures,
aluminum siding, gutters and downspouts, doors, windows); and from large consumer,
commercial and industrial products (appliances, automobiles, boats, trucks, aircraft,
machinery)
Aluminum: It has high recycling potential. The amount of aluminum which has already
entered into the human ecosystem is sufficient to cater the needs of the society through
recycling and there is no need to process it from the virgin ores. Aluminum cans are baled
into bricks and melted at 700 C in rotary furnaces. The molten aluminum is cast into ingots
149
and remade into cans or processed into other aluminum products like saucepans and homewares.
Copper: It can be recycled from the boilers of hot water systems, old car radiators and
copper pipes. Electric cabling and wiring contains copper and aluminum which can be
recycled.
Lead: Lead can be recycled from old car batteries and old lead pipes. Lead is recycled in
high rate because it is highly toxic and processing from its ore is highly damaging to both
man and environment.
Silver can also be recovered from silver-plating industries through recycling. A silverplating plant in the US spends about US $ 120,000 a year on waste treatment, of which US $
60,000 is returned as credit for silver recovered from the waste. Silver and even gold is
recovered from electrical industries.
Table 12. Metal Consumption Procured through Recycling in USA
(1). Lead
(4). Aluminum
(7). Tungsten
73 %
45 %
29 %
(2). Copper 60 %
(5). Zinc
43 %
(8). Nickel 26 %
( 3). Iron & Steel

(6.) Tin
(9). Chromium
56 %
38 %
21 %
Source : WWI, Washington D.C. State of the World (1989).
Environmental and Economic Benefits of Recycling Metallic Wastes

World steel production alone consumes as much energy annually as Saudi Arabia
produces. Making steel from recycled materials uses only a quarter of the energy needed to
make steel from iron ores. Iron scraps costs little more than iron ore but can be converted into
steel with much lower economic and environmental cost. Using coke for iron ore reduction
produce copious particulate matters including carcinogenic benzopyrene. Recycling of iron
reduces this emission by 11 kg/metric tons of steel produced and also cuts iron ore waste and
coal mining wastes by 1100 kg/metric tons recycled.
Recycling aluminum uses only 5 % of the energy needed to produce new aluminum from
its ore bauxite. Recycling one aluminum beverage can, saves energy enough to run TV for 3
hours. The energy needed to make 1 ton of virgin aluminum from bauxite could be sufficient
to recycle 20 tons of aluminum from its scrap. It takes about 4 tons of bauxite to produce 1
ton of finished aluminum. Recycling aluminum reduces air pollution including the toxic
fluoride by 95 %, and cut millions of tones of greenhouse gases (mainly CO2). (AEN,
2000).
Recycling Potential of Paper and Cardboard Wastes
About 30 % of the paper products which we use today are made from recycled papers and
cardboards. Three common grades of paper recycled are corrugated cardboard, high grade
office paper and old newsprint. Waste papers and card boards make excellent pulp for making
different grades of paper to be used for stationary, magazine and newspapers, game boards,
ticket stubbs, cereal and cake mix boxes, grocery bags, tissue papers, paper towels, egg boxes,
cards and packaging materials. If cotton rags are mixed with them they make excellent pulp
for making other kinds of papers too. Office papers are recycled to manufacture computer
papers, writing and printing papers.
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However, infinite recycling of paper is not possible because the fibers become shorter
and shorter and the quality of papers declines. Also the tissue papers and the wax coated
papers cannot be recycled. The printed papers also need to be de-inked before recycling as it
may contaminate the entire fibre stock.
Environmental and Economic Benefits of Recycling Paper Wastes

Recycling of waste papers saves green trees, large amount of energy and water and
prevent the use of chemicals. Recycling 1 ton of waste paper saves 13 trees, 2.5 barrels of oil,
4100 kWh of electricity and 31,780 litres of water. About 64 % less energy and 58 % less
water is needed to make papers from recycled fibers than to make from virgin pulp obtained
from the plants. Recycling half of the papers used in the world today would meet almost 75 %
of the demand for the new paper and would liberate 8 mha of forest from clear-felling for
plantation for paper production. According to one estimate the energy required to produce one
ton of paper from the virgin wood pulp is 16, 320 kWh, while only 6000 kWh is needed to
obtain the same amount of paper through recycling from paper waste. Reduction in energy
use (oil or electricity generated from fossil fuels) proportionately reduce the emissions of
greenhouse gas CO2 and other pollutants which would have occurred otherwise. (UNEP,
2004).
Recycling of Paper Industry Waste
All paper mills recycle and recover their intermediate and untreated effluents called
white water (water passing through a wire screen upon which paper is formed) and thus
reduces the volume of spray and wash water it uses. Other wastes like sulfite waste-liquor
by-product have been found to have multiple uses. Their complete evaporation produces fuel
and other salable by-product used in making core binder, road-binder, road bank stabilizer,
cattle fodder, fertilizer, insecticides and fungicides, linoleum cement, ceramic hardener,
insulating compounds, boiler-water additives, flotation agents, and in the production of
alcohol and artificial vanillin. The liquor may be fermented to produce ethyl alcohol.
About 40 liters of alcohol can be produced per ton of dry solids. Acetone and butyl alcohol
can also be produced from the liquor waste. Another product of fermenting the liquor is yeast
for cattle feed.
The spent cooking liquor produced in the sulfate process (kraft mills) contain
recoverable quantities of sodium salts, resins and fatty acids. Caustic soda (Na2CO3) is
recovered from them. The resin and fatty acids are further refined and have a variety of
applications in industry. In chemical precipitation with lime as coagulant, lignin is
precipitated which is used both as a fuel and in the manufacture of plastics, production of
tannins, as an anti-scale or antifoam agent in boilers. Wastewater from the paper mills have
also been found to be good for irrigation of number of crops.
Recycling Potential of Glasses
Glasses are 100 % recyclable and can be effectively recycled for ever. The recyclable
glasses in MSW are container glass (the clear, green and brown bottles and jars for food and
beverages), flat glass (e.g. window panes), and pressed amber or green glass. Glasses to be
recycled is often separated by color into categories of clear, green and amber. Broken glasses
are known as cullet which are valuable raw material in the production of new glasses.
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Manufacturer adds about 40 tons of cullet to every 100 tons of raw materials silica sand to
produce glass.
However, several vitreous materials may look and shatter like glasses, but are NOT
recyclable. They do not melt like glass. Even a minute amount of these materials can
contaminate the whole load of recyclable glasses and render them useless. These are ceramic
mugs and plates, cups and crockery such as pyrex and corning ware, mirror, broken drinking
glass and flower vase, light globes and laboratory glass. Contamination as little as 5 grams
per ton is harmful. One tiny fragment of ceramic material in a load of glass cullet can cause
a weakness in the new glass which may crack or even explode when the bottle is filled or
opened.
Recycled glasses are being used as raw material in cement production. (Wong, 2000). A
British firm is building an industry to recycle the used wine bottles into green sand to be
used for filtering drinking water and purify sewage. When the recycling plant is fully
developed it can save the quarrying of high quality sand and use all the waste wine bottles.
The European Union and the UK Department of Environment, Food and Rural Affairs are
funding the project. (www.drydenaqua.com).
Environmental and Economic Benefits of Recycling Glass Wastes

The cullet (broken glass materials) melt at low temperature than the primary raw
materials and hence require 25-30 % less energy on addition and also extends the life of
melting furnaces. Every ton of cullet used saves the equivalent of 30 gallons of oil and
replaces 1.2 tons of virgin raw materials and proportionately reduce the emissions of
greenhouse gas CO2 and other pollutants which would have occurred upon use of oil and
other energy sources in the extraction of virgin materials from nature.
Recycling Potential of Plastic Wastes
Recycling of plastic wastes is not really a good option and is rather a hazard for the
human health and the environment. It gives out toxic fumes like dioxins during melting.
Technologies are not available for recycling of all categories of plastics. Plastics are recycled
by codes. Currently only plastic bottles made of PET (Code 1: soft drink, juice and water
bottles and some plastic jars); HDPE (Code 2: milk bottles, cream containers and juice
bottles); PVC (Code 3: detergent, shampoo and cordial bottles); PP (Code 5: ice cream
containers, takeaway food containers flower pots etc.); PS (Code 6: polystyrene cups, glasses
and meat trays), and bottles with R or please recycle symbols are accepted for recycling.
Two types of plastics most commonly recycled in world are PET (Polyethylene
Terephthalate) and HDPE (High Density Polyethylene). PET is recycled to make soft drink
bottles, deli and bakery trays, carpets, fibrefill and geotextile liners for the waste landfills.
HDPE is recycled to manufacture plastic milk, disinfectant and detergent bottles, recycling
bins, sleeping bags and pillow stuffing, roadside guide posts, irrigation pipes, eskies, water
meter boxes, air-conditioning and recycled layer in new PET plastic bottles, agricultural water
pipes, bags and motor oil bottles. LDPE (Low Density Polyethylene) is recycled to make new
plastic bags and films. PVC (Poly Vinyl Chloride) is recycled to manufacture drainage pipes,
non-food bottles and fencing posts. Recycled polypropylene (PP) is used in auto-parts, carpets
and geo-textiles. Recycled polystyrene (PS) is used to manufacture wide range of office
accessories, video-cassettes and cases.
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Plastic grocery bags made of LDPE or very thin HDPE cannot be recycled as they clog
the system. Some supermarkets take back plastic shopping bags, recycling them into bin
liners, and hospital waste bags. Other plastics like motor oil containers, plastic takeaway food
containers and disposable nappies also cannot be recycled. Also different plastics cannot
always be recycled together. The recycled plastic is often hard and brittle. To overcome this
problem a compatibilser molecule that sticks together the different plastic molecules have
been developed. Such commingled plastic with much gloss and sturdiness as the originals can
be used to make car bumpers and fence posts.
Environmental and Economic Benefits of Recycling Plastic Wastes

Recycling of plastics at least diverts thousands of tons of plastic materials from ending up
in landfills every year and besides saving landfill space also arrest emissions of some very
toxic gases and leachate discharge from the landfills.
Recycling Potential of Waste Wood
It is a waste from the timber industries, forestry and agricultural activities and from
building construction and demolitions. Technology found its use in the manufacture of
plywood and medium density fiber board (MDF). They have properties like natural wood
with additional advantage of not being flammable and absorbing moisture, resistant to pest
attack and low cost. They are also shredded and processed as wood chips for fuel or
landscaping cover.
Recycling Potential of Construction and Demolition (CD) Wastes
CD wastes results from construction, renovation, and demolition of buildings; road
repaving; bridge repairs; and the cleanup after natural disasters. Typically they are made of
about 40-50 % rubbish (concrete, asphalt, bricks, blocks, and dirt), 20-30 % wood and related
products (pallets, stumps, branches, forming and framing lumber, treated lumber, and
shingles), and 20-30 % miscellaneous wastes (painted or contaminated lumber, metals, tarbased products, plaster, glass, white goods, asbestos and other insulation materials, plumbing,
heating and electrical parts).
The principal materials that are now recovered from the CD wastes are asphalt, concrete,
wood, drywall, asphalt shingles, and ferrous and non-ferrous metals. Reinforcing steel used in
foundations, slabs, and pavements are usually recovered and sold as scraps for recycling.
Concrete and asphalt are processed for road base, aggregate in asphalt pavement, and as
substitute for gravel aggregate in new concrete. Many landfills use the rubbles for road
building and as daily cover material for the compacted waste in the landfill. (Hamilton,
2000).
10.7. Recycling Potentials of Some Hazardous Industrial Wastes (IHW)

Technologies have been developed to recycle several categories of hazardous industrial
wastes. Several hazardous industrial wastes are now finding new use and applications in
construction industries through recycling. (Noll et. al., 1985).
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New Use of Phosphogypsum from Phosphatic Fertilizer Industry

Radioactively contaminated calcium sulphate slurry called phosphogypsum
(CaSO4.2H2O) is a waste byproduct of these industries. For every ton of phosphoric acid
produced, 5 tons of phosphogypsum (PG) must be removed. It is now being recovered and
reused to manufacture partition panel, ceiling tiles/boards, walling blocks etc. They save
cement and steel and minimize the use of timber. Direct reuse of PG, however, presents the
potential problem of incorporating radioactivity into building or road products.
Several firms in the US and UK is processing the PG into useful cement or plaster. The
quality of cement compares favourably with limestone-based cement and is used in all classes
of building construction and civil engineering. It possess a compressive strength 3 to 4 times
that of Portland cement (1100 kg / cm2 as compared to 300-400 for Portland cement).
Moreover the cost of cement production from PG was US $10 / ton as compared to $ 30- $ 40
/ ton for Portland cement. The PG is chemically processed into hemihydrate powder to use
in cement manufacture. Some South African fertilizer plants disposes about 25 % of its PG as
soil conditioner, cement clinker and as cement retarder. (Noll et. al., 1985).
Any recovery and reuse of phosphogypsum (PG) would also free up reclaimable land
resources for productive purposes by the industry or privately.
New Use of Fly-ash from Waste Slurry in Coal-fired Power Plants
Fly-ash is a waste byproduct of coal combustion in coal-fired power plants generally
consisting of very fine particles. It pollutes water bodies. The wastewater usually contain
6750 gm / liter of fly-ash. A common method of fly-ash removal from the steam power-plant
flue gases is by the use of wet scrubbers, electrostatic precipitators, or cyclone
separators. Major chemical component of fly-ash are silica (30-50 % by weight as SiO2) and
alumina (20-30 % by weight as Al2O). Other materials are sulfur trioxide (SO3), carbon (C)
and boron (B). It is now being reused to manufacture clay bonded bricks / blocks, fly-ash
ceramics, aerated light weight cellular blocks and slabs, precast blocks for footpath,
butiminous concrete for road surfacing and cement concrete etc. Fly-ash bricks have replaced
baked earthen bricks and have saved million tones of fertile soil from getting eroded.
Technology has found several new uses for fly-ash:
1)
2)
3)
4)
5)
6)
7)
8)
As a pozzolana ingredient in Portland cement;

As a pozzolana in soil stabilization;
As a soil conditioner in agriculture;
As a grout in oil wells;
In asphalt roofing and siding materials;
As a cement replacement in concrete;
As a mineral filler in asphalt pavement;
As a coagulant in sewage treatment.
New Use of Red Mud from Aluminum Industry

Red Mud is a waste from the aluminum industry being generated in million tones every
year. It is a bauxite residue and clay like silt consisting of undissolved minerological
components. It is being reused in cement industries both as component of cement raw mix as
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well as additives. Red mud light roofing sheets has been developed as an alternative to the
dangerous asbestos roofs.
New Use of Metallic Slag from Iron and Steel Industries It is a waste from the Iron and
Steel industries and an excellent secondary raw material for cement production.
10.8. Thermal Recycling of Hazardous Wastes to Retrieve Thermal Energy

Several hazardous industrial wastes such as paint thinners, degreasing solvents, solvents
from ink and printing industries, dry-cleaning fluids, chemical by-products from
pharmaceutical industries, waste papers, waste oils and waste auto tires, sewage sludge and
municipal solid waste can be used as fuel in industries.
Cement Kiln Using Hazardous Industrial Wastes as Fuel and Replacing Fossil Fuels The
manufacture of cement from limestone require high kiln temperatures (about 1500 C) and
long residency times. This create an excellent opportunity for hazardous waste to be used as
fuel and also get destroyed in the process. As much as 40 % of the fuel requirement of a well
operated cement kiln is saved by the use of hazardous wastes.. It is like killing two birds in
one shot. Further, as the environment inside the kiln is alkaline due to the presence of lime,
the acidic gases and the hydrogen chloride generated from the chlorinated wastes (which is
poses problem in conventional incinerators) are neutralized. Combustion of wastes in a
commercial incinerator also produces ash which needs to be disposed off. Here there is no
ash and the only by-product is the dust which is recycled. Any incombustible material such
as metals in the waste, becomes vaporized and incorporated in the product. Up to 95-99 % of
the chloride and over 99 % of the lead entering the kiln are retained by the process solids.
When the kilns are operating properly dioxins and furans and the particulate matters
(which are emitted in the conventional incinerators) are significantly cut down and there is no
risk to human health and the environment. U.S. uses about 1 million tons of hazardous wastes
as a fuel in cement kilns every year. Reduction in use of fossil fuels as the source of energy in
cement plants proportionately reduce the emissions of greenhouse gas CO2 and other
pollutants which would have occurred otherwise. (Jones and Herat, 1984).
10.9. Recycling Potential of Some Hazardous Consumer Wastes (CHW)

Appropriate technologies are now available to recycle several categories of hazardous
wastes including the hazardous electronics wastes (e-waste) generated by society.
Recycling of E-Waste A Hazardous and Costly Process: Reuse A Better Option

Millions of tones of e-waste all over the world are ending up in landfills. According to
the Silicon Valley Toxics Coalition in the U.S. cost of recycling obsolete and discarded
computers is estimated to be at US $ 10 to $ 60 per unit. And if poorly handled during the
clean-up cost of toxic materials from the e-waste, it could go higher. SVTC estimates the
minimum costs for recycling and proper disposal of the remaining non-recyclable e-waste in
US will reach US $ 10.8 billion by 2015 (Schneiderman, 2004). This could be the
approximate cost of recycling of e-waste in all developed nations including the European
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Union, Canada and Australia where labor cost is high. Local governments and councils in
these nations have neither the technical ability nor the financial resources to tackle this
gigantic techno-economic problem at their own.
Reuse is a better option for several categories of e-waste. The term reuse would mean
using any old and obsolete electronic product or equipment with or without minor repair and
reasonable upgrading, if possible. The best way of reuse is that computers can be sold to the
employees of the organizations or the students of institutions at very reasonable price or
donated to charitable organizations, schools, orphanage centers, old people homes, women
asylums etc. Institutions and organizations in the rich developed nations (where computer
models are changing fast) should develop a system based on ethics for donating their old
computers to the needy organizations in the developing countries.
The term recycling of e-waste would mean to dismantle the equipment or a product and
retrieve the valuable components / materials from it for their reuse in other equipment or remanufacture a new equipment / product. The difficulty with electronic waste and many other
end of life electronic products is that they are made from a huge range of component
materials that are useless for further manufacture until the product is dismantled and the
component materials are separated often a very difficult and expensive process.
Recycling may be a good option for the extremely old generation computers such as the
Pre-Pentium generation, or the computers (specially the monitors) which are broken.
According to the International Association of Electronics Recyclers (IAER) more than 1.5
billion pounds of electronics equipment are recycled annually and is likely to grow by a factor
of 4 or 5 by the end of this decade. Eleven countries currently have mandatory electronics
recovery laws on the books. These are Denmark, The Netherlands, Norway, Sweden,
Switzerland, Japan, Belgium, Taiwan, Portugal and South Korea. Some EU nations have very
strong system for e-waste collection, such as the SWICO system in Switzerland and the
Netherlands Association for Disposal of Metalectro Products (NVMP). NVMP collect 80 %
of e-waste. About 77 % of TVs and 64 % of other small brown goods are recovered for reuse
and recycling. (Monchamp, 2000; Cui and Forssberg, 2003; Mathew et. al., 2004)
Recycling Potential of Lead-Acid Car Batteries

Billions of lead-acid car batteries are used and replaced every year across the world, 70 to
80 millions in US alone. The average battery contains about 18 pounds of recoverable lead
and the worldwide recycling rate is now 90 %. Batteries are crushed and then the lead, plastic,
and sulfuric acid are separated. In an innovative recycling process developed in Italy in the
1980s, batteries are crushed in a hammer mill and the components are separated on a
vibrating screen. The acid / lead paste slurry is neutralized, the lead oxide is separated, and
the reusable sodium hydroxide and sulfuric acid are recovered from the solution by
electrodialysis. Lead oxides are reduced by electrolysis and combined with metallic
components, then melted at 400-500 C and cast into ingots. (Vaysgant, et. al., 1995).
Recycling Potential of Household Batteries
Billions of household dry-cell batteries are used and discarded worldwide, 2.5 billion in
the US alone. These batteries are discarded with the general household waste. These batteries
contain mercury, cadmium, lead, and other metals, which become toxic contaminants in the
landfill leachate or in the air emissions if MSW are incinerated. Study reveals that household
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batteries are the source of more than 50 % of the mercury and cadmium in the MSW.
Recycling of these batteries are difficult. Cylindrical 6-volt and 9-volt alkaline and carbonzinc batteries are not recyclable. Only nickel-cadmium cylindrical cells or mercuric oxide and
silver oxide button cells can be recycled. Mixed button batteries are difficult to sort out and
may present a storage hazard in the MSW waste bin due to mercury vapor emissions.
Recycling Potential of Auto Tires

Several millions of auto tires are rejected every year all over the world which turn as
hazardous wastes. Earlier they were reused after retreading, but with the advent of steelbelted radials and cheaper new tires most are discarded as waste. They cannot be landfilled as
they occupy large volume and tend to come to the surface.
Auto tires present problem for safe recycling as they are made of hazardous materials.
The best option is to reuse them as fuel resource in cement kilns. About 70 % of the waste
auto tires in the US is being used as source of energy in cement kilns thus also reducing use
of coal and emission of greenhouse gases. Power plants, paper and pulp mills and the cement
kilns commonly use the shredded tires as fuel. Whole tires are also used to create artificial
reefs for erosion control and as highway crash barriers. Split and punched tires are used to
make muffler hangers, belts, gaskets, and floor mats.
Recycling Potential of Waste Oils and Lubricants from Automobiles Industries
Billions of gallons of petroleum-derived waste oil are produced worldwide mostly from
the automobiles and some from the industries. Automotive oils include crancase oil, diesel
engine oil, transmission, brake and power steering fluids. Waste oil often contains metals like
arsenic, cadmium, barium, chromium, and zinc; chlorinated solvents and organic compounds
like benzene and naphthalene.
Used lubricants can be recycled in two ways- by reprocessing and by re-refining.
Reprocessing is done by water and bottom sediment removal of suspended material and ash
by gravity settling or chemical treatment to produce partially cleaned fuel oil. Heat is
sometimes used to decrease viscosity and improve gravity settling. Distillation is also done to
evaporate light fuel fractions. Re-refining produces a clean oil but it is very expensive affair.
This is done by solvent treatment / vacuum distillation / hydrotreatment; by acid clay,
chemical cleaning / demetalling etc.
Recycled lubricating oils are used as transformer oil, equipment oil, motor oil, cutting oil,
soluble oil, diesel oil, gasoline, antifreeze, brake fluids and hydraulic oils. Waste oils are
mainly recycled to be used as fuel in cement kilns, in commercial, industrial, and marine
boilers, and for space heating. Waste oils can also be used as fuel in cement kilns, in
commercial, industrial, and marine boilers, and for space heating.
11. SOME POLICY MEASURES ON WASTE MANAGEMENT BY

GLOBAL SOCIETY
Considerations of environmental sanitation, health and hygiene, contributing to safety
and security of society are key factors which has prompted to enact suitable legislation for
management of all kinds of waste by the global society. Safe waste management is not only a
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technological pursuit, but also require legal and administrative, economic and ecological
planning.
Governments can develop policies to encourage and support the manufacture of durable
and recyclable goods as well as take-back programs by producers to recycle their products.
If required to take-back and recycle their products, manufactures will be compelled to
produce durable and recyclable items, thus reducing waste for consumers.
11.1. The European Unions Packaging and Packaging Waste (PPW)

Directive (1994)
On average each EU citizen is currently responsible, directly or indirectly, for the
generation of some 172 kg of packaging waste every year. (UNEP, 2006). As early as in
1994 it issued this directive to prevent packaging waste. Yet, the packaging waste (PW)
generation increased by 10 % in the EU-15 between 1997 and 2002, in close line with the
12.6 % growth in GDP. Per capita consumption of plastics increased by almost 50 % from 64
kg / year in 1990 to 95 kg / year in 2002. Only UK managed to actually reduce, and Austria
stabilize the generation of PW since 1997.
The review of the EU-15 PPW in 2005 however, showed that the both the producers
(companies) and the consumers (society) made good progress in recycling PW. The EU target
of recycling 25 % of packaging waste in 2001 significantly increased to 54 % in 2002 in the
15 member countries then. (UNEP, 2006).
11.2. EU Directives on Extended Producer / Manufacturer Responsibility

(EPR) for Reducing Electronic Waste (2001)
In Europe, e-waste is projected to reach 12 million metric tones by 2010 (Schneiderman,
2004). In 2001, the European Union adopted a system called Extended Producer /
Manufacturer Responsibility that requires the electronics manufacturers to take-back their
used products and assume full responsibility for the production of cleaner electronics items,
phasing out of hazardous materials in production process, and also dismantling the e-waste
products more easily for recycling at the end of their useful life by trading-in the products for
recycling.
In January 2003, the EU parliament enacted two directives. The first - Waste Electrical
and Electronics Equipment (WEE) is based on the concept of Extended Producer
Responsibility (EPR) which requires the industries to take-back all their used and obsolete
electronic products for safe recycling. The second directive Restriction on Hazardous
Substances (RoHS), called for phasing out of heavy metals Hg, Cd, Pb and Cr VI in all
electronics items by July 2006 (with a number of exemptions) to reduce hazardous waste
when they are discarded. This has now come into effect. (Adam, 2005).
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11.3. The International Legal Instruments for Combating the Problem of

Hazardous Wastes at Global Level
The United Nations General Assembly initiated to review the international environmental
laws in 1981 at Montevideo. The laws were to cover among other things, the transport,
handling and disposal of toxic and hazardous wastes. Real negotiations to impose curb on the
production, storage and transport of hazardous chemicals and wastes began soon after the
Basel Disaster (Switzerland) in 1986 which resulted into the adoption of Basel Convention
in 1989.
The Basel Convention on the Control of Transboundary Movement of Hazardous

Wastes and Their Safe Disposal (1989)
The Convention was adopted in 1989 unanimously by 116 States in the Swiss city of
Basel, to reduce the global generation of hazardous wastes and chemicals to a minimum and
to prohibit / regularize the illegal traffic in hazardous wastes and to fix the responsibilities of
the parties involved. It entered into force on 5 May, 1992 and now have 131 Parties to the
Convention except the U.S. It outlines the general obligations of the hazardous waste
generating nations for moving their wastes across their boundaries either for disposal or for
exchange for recycling. It also outlines the principles of international co-operation to improve
and achieve environmentally sound management of all kinds of hazardous wastes.
Trade in hazardous waste that does not comply with the terms of the Basel Convention or
its control system is illegal, and considered to be criminal. Parties are obliged to enact
stringent national laws to prevent and punish the illegal trafficators in hazardous waste. The
Convention also cooperates with the Interpol over illegal traffic. The first 10 years of the
Convention (1989 1999) concentrated on consolidating its control system, legal framework
and operation through improving the classification of wastes and refining the work on their
hazard characterization.
Cooperation to Developing Countries
Under the Convention international cooperation is to be extended to developing countries
in:
1. Transferring technology and management systems for hazardous waste;
2. Developing and implementing new environmentally sound low-waste technologies,
and improving existing ones to eliminate the generation of waste, as far as
practicable, and studying the economic, social and environmental effects of adopting
the new technologies;
3. Developing and promoting environmentally sound management of hazardous and
other wastes;
4. Promoting public awareness about hazardous waste.
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12. SOME INNOVATIVE IDEAS TOWARDS SUSTAINABLE WASTE

MANAGEMENT
12.1. The Australian Innovations on Landfill Gas Utilization for Energy
Generation While Reducing Emission of Greenhouse Gases
An innovative technology to convert the MSW landfills into a bioreactor is being
experimented in Australia. In the traditional landfills the anaerobic decomposition of waste
is highly retarded and rather inhibited, as the waste has very little moisture. Daily covering
and heavy compaction of waste exclude the moisture content. Normal waste typically
contains 10-30 % moisture. The rate of waste biodegradation is a function of moisture
content. Bioreactor technology significantly reduces the decomposition time and the
production of gas begins soon.
Bioreactor is a large anaerobic digester, in a specially designed void in landfills which
receives municipal solid wastes and the also the biosolid (sludge) from the sewage treatment
plants. In the landfill bioreactor, the waste is not heavily compacted and sufficient moisture
content (45 60 %) is maintained through extensive networks of pipes that re-circulate the
nutrient rich leachate and inject additional water such as the stormwater and the run-off.
Additional microbial inoculum is added to promote rapid anaerobic microbial decomposition
and biogas (60 % methane and 40 % carbon dioxide) production is augmented with 98 %
collection efficiency and conversion to energy. Each kilogram of waste can generate up to
220 liters of methane. In case there is more paper content in the waste, the methane
production is 365 litre / kg. A typical 4000,000 tones per year landfill bioreactor will
produce about 7,500 cum of methane per hour, with a generating capacity of 25 MW. This
will also save greenhouse gas equivalent to taking 175,000 cars off the road.
When biogas generation in the bio-cell declines, the residual partially degraded organic
materials can be excavated and used as a soil conditioner or feedstock for composting.
Besides, generating biofuel and biofertilizer, the recycling technology will reduce the
emission of greenhouse gas (methane) from the landfills. Fugitive emissions of VOCs is
decreased to as low as 0.7 %. It would also reduce the environmental risk period of landfills
to 5-10 years from 30-50 years.
Power Generation from Landfill Gas

The ReOrganic Energy Swanbank in Ipswich, Brisbane, Australia is generating electricity
from the landfill biogas. Natural biodegradation processes in the landfills are enhanced in a
special configuration to accelerate the production of biogas (methane). This is supplied to the
Swanbank Power Plant for electricity generation displacing coal. By end of April 2002, some
20,000 cubic meters per day of biogas was produced and utilized and over 2,300 MWh of
electricity generated. It is envisaged that over this period beginning from 2002 to 2016, some
90,000 cubic meters of biogas per day will be utilized, 500,000 MWh of electricity will be
generated, while some 250,000 tones of coal will be displaced and approximately 3 million
tones of carbon dioxide equivalent greenhouse gas (methane and carbon dioxide) will be
arrested from going to the atmosphere. The $ 4 million project took some 20 months to
complete.
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After successful utilization of landfill gases for power generation at Ipswich, another $ 5
million landfill gas power generating facility was launched at Rochedale Landfill site in
Brisbane in 2004, generating 3 MW of power. This is expected to reduce greenhouse gases by
20,000 tones a year and also supply electricity to 5000 homes. Since the closure of the landfill
at Roghan Road, Fitzgibbon in March 2000, BCC has also started generating 2 MW
electricity from the methane produced in the old landfill. New waste landfills in Brisbane are
now purpose designed to be used as bioreactors for biogas generation and electricity
production, killing two birds in one shot (www.thiess-services.com.au).
12.2. Molok Waste Bins: Odor Free, Less Space, Holds More Waste and
Emptied Less Often: An Australian Innovation
The Molok Pty. Ltd. Of NSW in Australia has invented a new waste collection bin (40 x
120 L size) 60 % of which lies underground and only 40 % is visible. It is installed to a depth
of 1.5 meter in ground and takes 80 % less surface area than the conventional waste bins. It
comes in 300 L, 1300 L, 3000 L and 5000 L sizes suiting to all locations in residential and
commercial areas. The containers are water tight and the waste drop hole opening is
approximately 1.1 m above ground level. Within the PVC container is suspended the lifting
waste bag made of double layered textile material. While emptying the bag is simply lifted by
the hydraulic lifting arm and hoisted into the truck container. Emptying 5000 L Molok bin
takes about 3 mins and is one man job.
The key advantage of the vertical bin is that gravity forces the old waste to compact as
the new waste is added, and the oldest waste materials at the bottom of the container is kept
cool because the earth underground is naturally cool due to evaporation. The lowering of
temperature at the bottom reduces microbial activity arresting any odor problem. It is also
likely that there will be lesser emission of greenhouse gas methane.(www.molok.com.au).
12.3. The Innovative Bio-Bin System of Cleanway for Kerbside Composting

of Green Waste
Cleanaway is an enterprise of Brambles Industries Limited established in 1970. It
operates across Australia and around the world. It provides services in waste recycling, safe
hazardous waste disposal, site remediation, and landfill operations. It operates 8 waste
recycling plants in four Australian states. Its co-mingled bin recycling system services over
770,000 Australian households and has participation rate over 85 %. Cleanaway has
introduced a new and innovative concept of Bio-insert system. The Bio-Bins break down
green organic waste on the kerbside and reduces the weight of the waste. It promotes
oxygen-flow around and through the organic waste, triggering the aerobic decomposition
process. Problems like odor, leachate production and greenwaste sticking to the bottom of the
bin are eliminated because of oxygenation. The contents of the bio-bins are less compacted,
drier, lighter and more uniform than those in the standard bins. It forms good feed-stock
material for the compost facility operators. A collection frequency of 4 weeks is
recommended for green organics, while 2 weeks for the food organics. It is also inexpensive
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to integrate the bio-bin system into the existing waste management services and would save
the city councils considerable expenses in collection and landfill costs. Bio-Bins are widely in
use in Europe and North America for the past ten years. A trial of bio-bins is being conducted
in the City of Mooney Valley, Melbourne, Victoria with very encouraging results.
12.4. The Innovative DiCOM Aerobic-Anaerobic-Aerobic Hybrid System of

Bioconversion of MSW into Clean Biofuel and Biofertilizer
A new and innovative hybrid biological system for composting of MSW was developed
in Australia in 2000 by AnaeCo which process the MSW and produces a finished product in
less than half the time required for normal aerobic process of microbial composting without
any odor problem. The end products are green energy (biogas methane) and compost. This
has been termed as DiCOM bioconversion process.
The facility was installed at the cost of $ 5 million and is situated in Perth, Western
Australia. It receives commingled municipal solid wastes (MSW) from the local councils,
removes the non-compostable inert materials (the dry recyclable) such as the metals, glasses
and plastics from the waste and then subject the organic materials to a Three (3) Stage
Processing that involves an Anaerobic Digestion Phase in between the initial and final
Aerobic Composting period. The methane (CH4) gas generated from the anaerobic digestion
phase is captured and used as biogas fuel for electricity generation at the facility. The
separated recyclable materials are sent to MRF. (www.anaeco.com).
The company has patented this technology. The initial bioconversion capacity was
17,000 tons of MSW per annum which increased to 55,000 tons per annum by 2005. The
whole plant has small ecological foot print requiring small operational area and with
potential for decentralization of waste treatment to multiple sites close to the sources of
community waste generation, thus significantly reducing the cost of waste transportation and
pollution.
12.5. The Innovative Total Waste Management System (TWMS) in

Australia: No Waste to Landfills
Berrybank Piggery in Victoria Shows the Way Charles Integrated Farming Enterprises
Private Limited company in Victoria developed an innovative concept of Total Waste
Management System (TWMS) for its Berrybank Piggery. in Victoria, Australia. The piggery
produces on an average 275,000 L of effluents (sewage) a day with solid contents
approximately 2 %. The company found that the traditional farming philosophy of wasting
nothing and waste from one part of a farm is the input in another makes a good business
sense. On regular inventory program the management of piggery found that half of the feed
consumed by the animals is actually utilized, and the remaining half goes as waste. The
company considered this generation of waste as poor return on investment.
The TWMS is a seven-stage process which salvages all the waste byproducts in the form
of fuel, fertilizer and flush water. Since the introduction of TWMS, the Berrybank Piggery
now daily produces
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Seven (7) tones of waste solids which are utilized as fertilizer for farms;
100,000 L of flush water (recycled water) to be used for flushing toilets;
100,000 L of mineralized water rich in essential micro and macro-nutrients which is
utilized as liquid fertilizer for farm irrigation; and
1,700 cum of biogas fuel (methane) which is used for power generation on farm with
daily output of 2,900 kW of electricity.
The capital cost of the Berrybank Farm project was approximately AU $ 2 million which
was paid back in about 6 years by way of fuel, fertilizer and usable water. From a $ 2 m
investment made in the TWMS for the Berrybank Piggery, the Charles IFE brings in AU $
435,000 return every year. Other environmental benefits was that the odor problem and the
risk of groundwater contamination (due to sewage) was completely eliminated and it
dramatically reduced the consumption of freshwater
(www.environment.gov.au/settlement/industry/corporate/eecp/casestudies/charlesife.html)
12.6. The Indian Innovation on Dumpsite Composting of Commingled MSW:

A Cheaper and Safer Alternative to Costly Landfills?
An innovative technology to compost the un-segregated municipal solid waste (MSW)
biomass on ordinary waste dump-site was developed in India in the 1990s and is giving
excellent results for managing the solid wastes. No prior segregation of commingled waste is
required. Segregation of commingled wastes at source or before composting imposes the
biggest obstacle in any composting technology because it is highly labour intensive job to
segregate the often sticky and wet biodegradable (compostable) matters from the dry nonbiodegradable ones. It becomes much easier after composting.
The technology was developed by Ms. Excel Industries, Mumbai, India and can suit to all
countries and is also flexible for 150-700 MT of waste per day. The largest plant with an
installed capacity to bioprocess 500 tones /day of MSW is operating in Mumbai. The process
can recycle all organic wastes from the households, restaurants and hotels, dairy, agriculture
and agro-processing industries, brewing industries and slaughter houses. (Ranjwani,
1996;.Sinha and Herat, 2002 b).
The Bioconversion Process and the Composting Mechanism

The dump site is leveled and either cemented or paved with bricks on the bottom to
prevent leachate and for easy movement of waste carrying vehicles. Long windrows, about 5
meters wide and 2-3 meters high (deep) are erected and the MSW is then stacked and heaped
in the windrows. A microbial consortium slurry containing active decomposer bacteria and
enzymes are then added to the windrows to initiate rapid aerobic decomposition of the waste
biomass. The slurry is spread on the surface of the garbage and inside the heaps in the
windrows with help of probes, so that it reaches deep and in every pocket of the heap.
Microbial culture of active decomposer bacteria is prepared from the sewage sludge which
contains active decomposer bacteria in millions/gram of the sludge. (Ranjwani, 1996).
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The microbial culture is known as Celrich Substrate DF BC-01. It is prepared after

analyzing the composition of the waste and identifying the predominant materials such as
celluloses, hemicelluloses, lignins, proteins and fats etc. The microbes produce enzymes such
as cellulase, lipase, amylase, protease, pectinase and phospholipase to breakdown the long
chain complexes of the substrates. About 1 kg of the consortium in the colloidal emulsion
form is mixed with 20 litres of water to be used for spraying on about 3 cubic meter of solid
waste and for one ton of waste 200 litres of water is needed. Recycled water can also be used.
The waste heaps are turned around once in 7 to 10 days for proper aeration and the inoculant
slurry is sprayed in each turning to enhance decomposition and maintain the proper moisture
level which is usually 45 - 55 %. The process is exothermic and the windrows reach a
temperature of 70-75 C within 24-36 hours, killing the harmful pathogens and repelling all
birds, stray animals, flies and mosquitoes from the dump site. (Ranjwani, 1996).
Recovery of Compost
The entire process of aerobic decomposition of garbage is completed within 4-6 weeks
and as the decomposition is complete the temperature comes down to normal. It recovers over
90 % of the organic matter in the form of compost which may be 25-30 % of the raw waste on
dry weight basis. Recovery of compost depends upon the presence of organic matter in the
garbage. There will be greater recovery of compost in developed countries as much higher
amount of organic wastes reaches the dump-sites (tips) in every city.
Retrieving the Dry Recyclables
The decomposed waste biomass is passed through rotary and vibratory screens to sieve
out the compost. The soft decomposed powdery materials gets easily separated from the
plastics, metals, stones and pebbles. About 20-25 % are dry recyclable materials and the rest
about 20-25 % are inert materials which are disposed in ordinary land-fills.
Same Land for Dumpsite Can be Reused and No Need of Engineered Landfill
The same dump-sites can be used again and again after excavating the biodegraded mass
(compost) and there is no need of additional land for making more dump-sites. Also the need
for engineered land-fills are greatly reduced because very little is left to be disposed off
after retrieving the compost and the recyclable materials.
Low Emission of Greenhouse Gas Methane
The problem of emission of landfill gas methane (CH4) is significantly minimized as the
waste is turned constantly and the waste biomass is thoroughly aerated throughout the
composting period.
Foul Odor at Waste Dumpsite Disappear Soon Giving Relief to Waste Workers
Emission of ammonia and hydrogen sulfide which are mainly responsible for foul odor at
the waste dump-site (tips), and the leachate discharge is also greatly reduced. The foul odor at
the waste dump-site disappear within 2-3 days of sanitization by the microbial culture giving
great relief to the waste workers and the local residents.
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13. CONCLUSION AND RECOMMENDATIONS

An assessment of waste generation not only involves the production and distribution of
commodities and services but also its actual history of use/reuse/recycling. How people act as
consumers, re-users and recyclers is as important as how a thing is made and sold to
consumers.
Any government policy, any waste reducing and recycling technology and strategy
cannot succeed unless every member of society is aware and behave responsibly. Sometimes
policing becomes essential to change societal behavior. Random check of waste bins by
councils and refusal to pick up waste not disposed according to council directives, can force
people to behave. The strategy has worked well in some Canadian cities. Economic
instruments also seem to affect human behavior. Landfill taxes in Denmark, Austria,
Ireland, Italy and the UK, and charging people for plastic bags in supermarkets in Denmark
and Ireland (and in France from late 2005 onward) have changed the human behavior in these
nations. Manufacturers of consumer goods hold great responsibility in reducing waste in
modern consumerist society. Governments of nations need to come out with a policy of
durable and recyclable goods by manufacturers, and also take back policy of their products
for recycling. It will also be a disincentive for them to change their product versions too
frequently to lure people.
Society has to play very critical role in all waste management programs. The traditional
societies were basically recycling societies. They made best use and reuse of all materials
several times before discarding them. The modern society is basically a throwaway society
which discard most materials after short use. Modern human societies have to revive some of
the traditional cultures of resource conservation, resource recycling and their ethical use.
Waste recycling is essential for economic stability, ecological sustainability, environmental
safety and survival of the global human society.
Human society has to begin the process of recycling at the source the home, office, or
factory- so that fewer materials will become part of the disposable solid wastes of a
community. A consumer education program is needed to educate the society about the
hidden environmental value (the energy and water it has saved, the pollution and
deforestation it has prevented) of the recycled goods. All recycled goods should have
recycled tag telling about the origin and life history of the goods (from which waste it was
produced) and how it has saved the environment. It would develop a sense of civic pride
among the consumers that he / she is helping the environment. There is always greater
economic and ecological wisdom in reducing waste at source, and diverting more and more of
the waste to the mechanical recycling, biological recycling (composting) and thermal
recycling (combustion) processes to recover some useful materials and energy from them
and reduce their volume, so that less and less waste is left for final disposal in landfills.
Government, industries, science and society all have to join hands in fighting the menace
of mounting wastes. Industries producing consumer goods have to play more responsible role
in waste management program because they have potential to generate waste twice in the lifecycle of the goods produced. First at the production level when they process the virgin raw
materials and generate industrial wastes. Second at the consumption level when the goods
produced by them are used and discarded by the society as municipal solid waste (MSW).
Policy makers have carrot and stick options to encourage or enforce industry to contribute to
165
reduce waste. Industrialists also owe moral obligation to provide necessary information to its
prospective buyers on the matter of using, handling, conservation, disposal and recycling
potentialities of its products. In designing new products, the industry must assess its potential
and even suspected adverse impact on its consumers health and the environment.
We need more and more waste to be converted into resource (by recycling) to sustain our
growing population as the several natural resources are either on decline or becoming scarce
or are unavailable and beyond our capability to exploit them sustainabily with present
technology and within the ecological limits. Government must encourage and promote the
recycling industries using waste as raw materials by way of reduced taxation, reduced cost of
water and electricity supply etc. Given current technology, not all the municipal or industrial
wastes can be readily recycled. Nor do all the waste materials have qualities that currently
make them a valuable commodity in the recycling marketplace.
Hazardous waste is growing in U.S. industries and very little is being done to reduce or
recycle them at source. Most hazardous wastes are being exported to poor developing
countries either for dumping or for recycling. (Duke, 1994). Wrong policy decisions of the
U.S. government has aggravated the problem. Study made by an environmental organization
in the U.S. indicates that subsidies given to the timber, mining, oil, energy, and waste disposal
industries undermine and discourage waste recycling industries. These subsidies lower the
cost of products made from new and virgin materials, giving them a competitive advantage
over those made from recyclable materials. Fifteen government subsidies given to these
industries in the U.S. amounted to as much as US $ 13 billion over the next 5 years. It was a
great setback for the waste recycling industries in the U.S. They include indirect subsidies
such as cheap water and energy supply to these industries.(UNEP, 2006).
Life-cycle assessments is also important to determine the recycling potential of a waste
product. A number of life-cycle assessments have found that fully recycled paper is not
always the most environmentally friendly choice. In some countries paper produced from
local agricultural wastes, such as rice straw, may be environmentally more sustainable than
that produced from recyclable paper wastes shipped from overseas or transported from distant
locations in the same country. One study in Australia found that if the recyclable paper waste
is transported more than about 20 km by road, the energy balance (fuel used and pollution
generated during transport) is not in favor of recycling.
Safe disposal of the radioactive wastes which are accumulating exponentially in the
human environment cannot be guaranteed at all even after huge expenditure. A huge pile of
radioactive wastes (most in the U.S., France and Japan) remains to be disposed safely. It was
just 84,000 tones in 1990 and must have crossed 100,000 tones limit by now. According to
the celebrated geologist Konrad No scientist or engineer can give an absolute guarantee
that radioactive waste will not some day leak in dangerous quantities from even the best of
repositories. The word safe seems to be incompatible with radioactive wastes.
However, waste prevention through cleaner production is considered to be even more
wiser and sustainable idea of waste management so that any treatment or recycling is not at
all needed and tremendous cost can be saved. Waste prevention would also reduce the cost of
construction and maintenance of secured landfills because some residual wastes are always
left after treatment or recycling which has to be safely dumped in the landfills.
Educating the environmentally illiterate society is a big challenge. People need to
understand the importance of their activities in the context of global consequences, for
instance, the links between waste generation, greenhouse gas emission and climate change.
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Waste education needs to take place in a continuous way through schools and universities,
through mass and diffuse media and community education programs run by governments at
all levels as well as non-government organizations, businesses and industries.
Waste education of society should become an integral part of all waste management
programs. We have to mend our ways, change our behavior and attitude of life, re-order our
priorities, simplify our life-style, and then only the gigantic problem of mounting solid waste,
which literally threatens to bury the mankind alive, can be overcome. Only a resource
conserving, waste reducing and waste recycling society would be the sustainable human
societies of the future and the throwaway wasteful societies would perish.
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171

ISSN: 2158-5717
Chapter 4
EFFECTIVE REMOVAL OF LOW CONCENTRATIONS OF

ARSENIC AND LEAD AND THE MONITORING OF
MOLECULAR REMOVAL MECHANISM AT SURFACE
Yasuo Izumi
Department of Chemistry, Graduate School of Science, Chiba University
Yayoi 1-33, Inage-ku, Chiba 263-8522, Japan
ABSTRACT
New sorbents were investigated for the effective removal of low concentrations of
arsenic and lead to adjust to modern worldwide environmental regulation of drinking
water (10 ppb). Mesoporous Fe oxyhydroxide synthesized using dodecylsulfate was most
effective for initial 200 ppb of As removal, especially for more hazardous arsenite for
human's health. Hydrotalcite-like layered double hydroxide consisted of Fe and Mg was
most effective for initial 55 ppb of Pb removal.
The molecular removal mechanism is critical for environmental problem and
protection because valence state change upon removal of e.g. As on sorbent surface from
environmental water may detoxify arsenite to less harmful arsenate. It is also because the
evaluation of desorption rates is important to judge the efficiency of reuse of sorbents. To
monitor the low concentrations of arsenic and lead on sorbent surface, selective X-ray
absorption fine structure (XAFS) spectroscopy was applied for arsenic and lead species
adsorbed, free from the interference of high concentrations of Fe sites contained in the
sorbents and to selectively detect toxic AsIII among the mixture of AsIII and AsV species
in sample.
Oxidative adsorption mechanism was demonstrated on Fe-montmorillonite and
mesoporous Fe oxyhydroxide starting from AsIII species in aqueous solution to AsV by
making complex with unsaturated FeOx(OH)y sites at sorbent surface. Coagulation
mechanism was demonstrated on double hydroxide consisted of Fe and Mg from the
initial 1 ppm of Pb2+ aqueous solution whereas the mechanism was simple ion exchange
reaction when the initial Pb2+ concentrations were as low as 100 ppb.
yizumi@faculty.chiba-u.jp
174
Yasuo Izumi
INTRODUCTION
Recently, global environment induces even serious debate, e.g. the Novel Prize 2007 for
Piece to "An Inconvenient Truth" by Al Gore [1]. The environmental problem is not only the
global warming as the major claim in this movie/book by Gore. Contamination of water and
soil is one of traditional, major environmental problems and directly affects the human health,
e.g. carcinogenic risk via drinking water [2 4]. Cadmium contaminated in rice [5], copper
contaminated in environmental water from mine, mercury contaminated in fish [6 8], and
arsenic contaminated in powdered milk are most notorious environmental tragedies occurred
in Japan between 1890 and 1960. These accidents are all related to contamination of water
derived from human activity (industry) [2].
The health risk of poisonous elements in water has been studied and is becoming clear.
Recent environmental regulation sets the minimum level of Mn, Cu, Cd, Pb, Cr, As, and Hg
to 400, 125, 5, 10, 50, 10, and 0.5 ppb, respectively. Among these elements, lead is less
focused and less intensively studied to adjust to the regulation. Arsenic can be contaminated
in ground water not only anthropogenically but from the earth naturally [2 4].
Unfortunately, mines containing As mainly distribute in contact with the ground water in
developing countries, e.g. Bangladesh, East Bengal, Argentina, Chile, and Vietnam [9].
Especially in Bangladesh, the shallow ground water with arsenic concentrations up to 1 ppm
is often used pumped from tube wells without adequate treatment for drinking and thus
leading to serious health problem. The arsenic release into ground water is related to
microbial metabolism of organic matter [10]. The high capital and maintenance costs of piped
water supply are still not acceptable in Bangladesh, especially most affluent villages
compared to present individual tube wells [10]. Lead was used as gasoline additive and
automobile tail pipes and may be included in drinking water from water supply pipes made of
lead, soil contamination, and toys/tools made in developing countries [11, 12].
This chapter reviews recent development of sorbents for low concentrations of Pb and As
to adjust to modern environmental regulation for drinking water and monitoring of the
molecular removal process by selective spectroscopy in water [13]. The monitoring of surface
uptake of Pb [14] and As includes local coordination structure and electronic structure
changes in the inner sphere reaction.
METHODS
This chapter focuses on the removal of Pb and As by sorption because sorption is
economic process to be applicable in developing countries and the reuse is possible by
desorption. The concentrations of Pb and As in test aqueous solutions were set between 55
ppb and 32 ppm in this chapter. Various sorbents were evaluated to maintain the
concentrations of Pb or As less than 10 ppb or not.
Fe-montmorillonite was prepared by mixing 0.43 M ferric nitrate solution with Namontmorillonite (Kunipia F; Na1.5Ca0.096Al5.1Mg1.0Fe0.33Si12O27.6(OH)6.4) [15]. A 0.75 M
sodium hydroxide solution was added dropwise to the mixture until the molar ratio Fe3+
added and hydroxide reached 1:2. Iron cations and/or FeOx(OH)y nanoparticles were inserted
between negatively-charged montmorillonite clay layers. Recently, some chemical forms of
Effective Removal of Low Concentrations of Arsenic and Lead
175
FeIII species formed between montomorillonite layers were spectroscopically analyzed [16].
Monomeric and/or dimeric FeIII species was active in oxidative dehydrogenation of propane.
In contrast, polymeric FeOx(OH)y nanoparticles were effective for arsenic sorption and
unselective propane combustion.
FeOx(OH)y porous material was prepared by mixing 0.10 M ferrous chloride with 0.070
M sodium dodecylsulfate followed by the addition of 0.25 M H2O2 [17]. Obtained FeOx(OH)y
material was mixed with 0.050 M sodium acetate in ethanol for anion exchange or with pure
ethanol for washing. The micro/mesoporous FeOx(OH)y material was characterized by X ray
diffraction (XRD), specific surface area measurements and pore volume determination by N2
adsorption/desorption, high-resolution transmission electron microscope (TEM), Fouriertransformed infrared absorption (FT-IR), inductively coupled plasma (ICP) combined with
optical emission spectroscopy (OES), electron probe microanalysis (EPMA), thermogravimetric differential thermal analysis (TG-DTA), and Fe K-edge X-ray absorption fine
structure (XAFS). Based on these analyses, detailed structural transformation was clarified
for the sorbents as depicted in Figure 8 of Ref [17].
Hydrotalcite-like (pyroaurite) layered double hydroxide Mg6Fe2(OH)16(CO3)3H2O was
synthesized via the procedure described in Ref 18. The carbonate anions are sandwiched
between positively-charged [Mg3Fe(OH)8]+ layers.
To monitor low concentrations of Pb and As, XAFS spectroscopy is most appropriate
technique. For several spectroscopic techniques of structural analysis in the application to
nanotechnology, the advantage and drawback were summarized in Table 1 [19]. For noncrystalline or hybrid samples, EXAFS (extended X-ray absorption fine structure) gives direct
structural information for X-ray absorbing local element sites. XANES (X-ray absorption
near-edge structure) is a part of EXAFS spectrum near the X-ray absorption edge region
ranging up to 100 eV and gives electronic and (indirectly) structural information [20].
Thus, XAFS spectroscopy (EXAFS, XANES) is essentially single technique for local
structure analysis accompanied with valence and coordination symmetry information of
nanoparticles and micro/mesoporous materials. The Pb and As adsorbed from low
concentrations of aqueous solutions in this chapter are typical examples of nanoparticles and
micro/mesoporous material samples.
Further, this chapter combines X-ray fluorescence (XRF) spectrometry with the XAFS
spectroscopy [21 24]. Simply, XRF spectra support valence state information deduced from
XAFS. Essentially, high-energy-resolution XRF spectrometry is able to discriminate valence
state of Pb and As. In this chapter, the XRF signals originating from PbII, AsIII, and AsV were
monitored independently in the XAFS measurements to obtain each coordination structure of
PbII, AsIII, and AsV (state-selective XAFS). The experimental setup and measurement
conditions for state-selective XAFS were depicted and described in Refs 21, 23, and 24. In
brief, XRF spectra and state-selective XAFS spectra measurements were performed at
Undulator beamline 10XU of SPring-8 (Sayo, Japan) by utilizing a homemade high-energyresolution Rowland-type fluorescence spectrometer equipped with a Johansson-type Ge(555)
crystal (Saint-Gobain) and NaI(Tl) scintillation counter (Oken). The monochromator of
beamline used Si(111) double crystal and the X-ray beam intensity in front of sample was
monitored using ion chamber (Oken) purged with N2 gas.
176
Yasuo Izumi
Table 1. Various Analytical Methods for Nano Structure Classified Based on Directness
of the Information and the Target to Be Analyzed
Method
Directness
Target
TEM (Transmission electron microscope)
Direct
Local
XRD (X-ray diffraction)
Direct
Local
EXAFS (Extended X-ray absorption fine structure)
Direct
Bulk
XANES (X-ray absorption near-edge structure)
Indirect
Bulk
Raman
Indirect
Bulk
Small angle scattering
Indirect
Bulk
NMR (Nuclear magnetic resonance)
Indirect
Local
Mssbauer
Indirect
Local
SPM (Scanning probe microscope)
Indirect
Local (surface)
Reflectivity
Indirect
Local (surface)
RESULTS AND DISCUSSION

Arsenic Problem. Adsorption isotherms of arsenite and arsenate at 290 K for 12 h on Femontmorillonite in batch setup are depicted in Figure 1. The Fe-montmorillonite was superior
to -FeO(OH) (gthite > 95%) both for arsenite and arsenate sorptions. Fe-montmorillonite
consisted of 2-dimensionally distributed Fe3+ ions and FeOx(OH)y nanoparticles between clay
layers [16].
The saturated amount of As adsorbed was evaluated to 8.0 and 76 mgAs gsorbent1 for
arsenite and arsenate, respectively, on Fe-montmorillonite. The equilibrium adsorption
constant was 1.4106 ml gAs1 for arsenite on Fe-montmorillonite. Even better adsorption
capacity was found on acetate-exchanged microporous FeOx(OH)y as depicted in Figure 2 for
arsenite. The saturated amount and the equilibrium adsorption constant of As adsorbed were
evaluated to 21 mgAs gsorbent1 and 1.0107 ml gAs1, respectively. The high specific surface
area of microporous FeOx(OH)y (230 m2 g1) was advantageous compared to Femontmorillonite (100 m2 g1) with as much as 14 wt% of Fe. Utilizing template synthesis
technique for microporous and mesoporous materials, lower coordination FeOx sites were
effectively exposed to surface to complex with AsIII(OH)3 [17, 26]. The acetate-exchanged
and ethanol-washed FeOx(OH)y consist of 3-dimensionally distributed wormholes exposed
with coordinatively unsaturated FeO(OH) sites.
177
Figure 1. Adsorption isotherms of arsenite (A) and arsenate (B) at 290 K on Fe-montmorillonite (14.0
wt% Fe) (circles) and -FeO(OH) (triangles). Batch tests for 12h. Observed data were plotted as points
and the fits to first-order Langmuir equations were drawn as lines.
The surface uptake mechanism of most toxic arsenite was monitored by XRF and XAFS
spectroscopy. Arsenic was adsorbed on Fe-montmorillonite from 200 ppb test aqueous
solution of arsenite. The As K1 emission spectrum was depicted in Figure 3. The peak
energy position suggested that the adsorbed As state changed to V, not remained at III,
compared to the data for KH2AsVO4 and AsIII2O3 [15].
As K-edge XANES spectra for standard inorganic compounds of As0, AsIII, and AsV
consist of broad peak feature (Figure 4a c) and it is complicating to evaluate each valence
contribution to a spectrum for sample of mixed valence. In order to demonstrate directly the
oxidative adsorption of arsenite suggested above, the author of this chapter observed the
uptake of low concentrations of arsenite on Fe-montmorillonite by means of state-selective
XAFS. Note that the energy resolution of fluorescence spectrometer (1.3 eV; Figure 3) was
smaller than the core-hole lifetime width of As K level (2.14 eV) [27].
Figure 2. Adsorption isotherms of arsenite at 290 K on acetate-exchanged FeOx(OH)y previously heated

at 423 K (circles), ethanol-washed FeOx(OH)y previously heated at 423 K (squares), Fe-montmorillonite
(14.0 wt% Fe; diamonds), and -FeO(OH) (triangles) [25]. Batch tests for 12h. Observed data were
plotted as points and the fits to first-order Langmuir equations were drawn as lines.
178
Yasuo Izumi
Figure 3. Arsenic K1 emission spectrum for As adsorbed on Fe-montmorillonite (14.0 wt% Fe) from
200 ppb test solution of arsenite (points). A fit to data with pseudo-Voigt function (solid line) and the
energy resolution of fluorescence spectrometer (dotted line) were also drawn. The intensity ratio of the
Lorentzian and Gaussian components was fixed to 1:1 for the pseudo-Voigt function.
Figure 4. XANES spectra measured at 290 K in transmission mode for As metal (a), AsIII2O3 (b), and
KH2AsVO4 (c). Arsenic K1-selecting As K-edge XANES spectra measured at 290 K for As adsorbed
on Fe-montmorillonite (14.0 wt% Fe) (d f) from aqueous test solutions of 16 ppm of KH2AsVO4 (d),
16 ppm of AsIII2O3 (e), and 200 ppb of AsIII2O3 (f). Tune energy of fluorescence spectrometer was
10544.3 eV for spectra d f.
179
Based on the theory discussed in the Appendix section of Ref 24, the As K1-selecting
As K-edge XANES spectrum with the energy resolution of 1.3 eV would be shaper and more
resolved. The energy values of K absorption edge and first strong peak after the edge were
essentially identical for As adsorbed on Fe-montmorillonite from 200 ppb 16 ppm of AsIII
solutions (Figure 4e, f) and from 16 ppm of AsV solution (d). Thus, oxidative adsorption of
200 ppb 16 ppm of arsenite on Fe-montmorillonite was confirmed based on As K1selecting XANES and As K1 emission spectrum. Proposed molecular surface uptake
mechanism was illustrated in Figure 5 over acetate-exchanged microporous FeOx(OH)y. The
reaction formula was AsIII(OH)3 + FeO(OH) (FeO)2AsV(OH)2 + H2O.
In summary, oxidative adsorption of low concentrations (200 ppb 16 ppm) of arsenite
was found on coordinatively unsaturated FeOx(OH)y nanoparticles or micro/mesoporous
FeOx(OH)y partially covered with acetate anions. The oxidation to arsenate seems to be due to
lower coordination of surface FeOx(OH)y species. The lower coordination was also the reason
to make the equilibrium sorption constant greater for acetate-exchanged FeOx(OH)y and Femontmorillonite [15, 17].
Lead Problem. Sorption tests for low concentrations (55 ppb) of lead in flow setup were
depicted in Figure 6 [18]. The superiority of Mg6Fe2(OH)16(CO3)3H2O was clearly
demonstrated to maintain the Pb2+ concentration less than modern environmental regulation
(10 ppb) compared to commercially available activated carbon.
Lead L1 emission spectrum for Pb adsorbed on Mg6Fe2(OH)16(CO3)3H2O from 100
ppb Pb2+ test solution was depicted in Figure 7. The peak energy was identical to that for
standard PbII compounds. The energy resolution of fluorescence spectrometer in this
measurement condition was 5.0 10.1 eV dependent on the measurement conditions of
fluorescence spectrometer (Figure 7) [24, 28]. Because the core-hole lifetime widths are 5.81
and 2.48 eV for Pb L3 and M5 levels, respectively [27], the width for L1 is 8.29 eV
comparable to the energy resolution of fluorescence spectrometer. Thus, sharper, more
resolved spectral feature was expected in Pb L1-selecting Pb L3-edge XANES spectrum if
the energy resolution of fluorescence spectrometer was smaller than 5.81 eV, similar to the
case of As K1-selecting XANES in previous section.
Figure 5. Proposed adsorption mechanism of low concentrations of arsenite on acetate-exchanged

FeOx(OH)y previously heated at 423 K.
180
Yasuo Izumi
Figure 6. Results of sorption on Mg6Fe2(OH)16(CO3)3H2O and on activated carbon from 55 ppb of

Pb2+ aqueous test solution. The space velocity was 150 min1.
Figure 7. Lead L1 emission spectrum for 0.12 wt% of Pb adsorbed on Mg6Fe2(OH)16(CO3)3H2O.

The solid line is the experimental data, and (wider) dotted line is a fit with a pseudo-Voigt function.
The intensity of the Lorentzian and Gaussian components was fixed to 1:1. The narrower dotted line is
the energy resolution of fluorescence spectrometer (10.1 eV).
181
Pb L1-selecting XANES spectra are shown in Figure 8a c. The spectral pattern of b

and c resembled each other. The two samples contained 0.30 0.12 wt% of lead adsorbed
from 100 ppb test aqueous Pb2+ solution. The Pb contents in samples were determined by
ICP. Compared to XANES spectra for standard inorganic Pb compounds (Figure 8d i) and
supported Pb species on zeolite [29] or on Fe2O3 [30], the spectra b and c resembled most
spectrum d measured for ion-exchanged PbY zeolite (d). Thus, surface uptake mechanism via
ion exchange reaction was proposed on Mg6Fe2(OH)16(CO3)3H2O from relatively low
concentration of 100 ppb divalent lead solutions (Figure 9). The reaction formula is
[Mg3Fe(OH)8]+ + Pb2+ [Mg3Fe(OH)7(OPb)]2+ + H+.
Pb L1-selecting XANES spectrum for Pb adsorbed from 1.0 ppm test Pb2+ solution is
depicted in Figure 8a. Compared to XANES spectra for standard inorganic Pb compounds
(Figure 8d i), the spectrum a resembled most spectrum g measured for 2PbCO3Pb(OH)2.
Thus, coagulation uptake mechanism was proposed on Mg6Fe2(OH)16(CO3)3H2O from
relatively high concentration of 1 ppm Pb2+ test solution. Thus-identified reaction formula
was Pb2+ + nCO32 + 2(1 n)OH nPbCO3(1 n)Pb(OH)2.
Figure 8. Lead L1-selecting Pb L3-edge XANES spectra measured at 290 K for Pb adsorbed on
Mg6Fe2(OH)16(CO3)3H2O (a c). Tune energy of fluorescence spectrometer was 10551.5 eV. The Pb
content was 1.0 wt% adsorbed from 1.0 ppm Pb2+ aqueous test solution (a) and 0.30 (b) and 0.12 wt%
(c) from 100 ppb Pb2+ test solution. XANES spectra measured in transmission mode (d i) for PbY
zeolite (d), PbO (e), Pb(NO3)2 (f), 2PbCO3Pb(OH)2 (g), Pb6O4(OH)4 (h), and PbCO3 (i).
With closer look of Figure 8a c, a shoulder feature appeared at 13049 eV. Similar
shoulder feature can be found in spectra for PbO, 2PbCO3Pb(OH)2, and Pb6O4(OH)4 (spectra
182
Yasuo Izumi
e, g, and h, respectively). However, intense peak at 13060.1 13060.5 eV for PbO or

Pb6O4(OH)4 did not appear in spectra a c. Thus, minor coagulation uptake mechanism was
suggested from 100 ppb Pb2+ solutions in addition to major ion exchange process (Figure 9).
Figure 9. Pb2+ adsorption mechanism on Mg6Fe2(OH)16(CO3)3H2O from Pb2+ 1.0 ppm and 100 ppb
aqueous solutions.
In summary, Pb2+ uptake mechanism on Mg6Fe2(OH)16(CO3)3H2O exhibited a switchover from coagulation to (major) ion exchange reactions as the Pb2+ concentration decreased
from 1.0 ppm to more environmentally plausible 100 ppb [28].
CONCLUDING REMARKS AND FUTURE PROSPECTS

This chapter focused on the removal of low concentrations (55 200 ppb) of arsenite and
lead by utilizing Fe-montmorillonite, micro/mesoporous FeOx(OH)y effectively porous due to
carboxyl-exchange method, and layered double hydroxide consisted of Fe and Mg
(pyroaurite). It is still open to discuss the systematic survey of the removal process of other
dilute toxic elements, e.g. Cr, Cu, Zn, Cd, or Hg. It is important to formulate the efficiency of
surface uptake with respect to critical factors, i.e. pH, chemical species of toxic elements
(naked cations, oxyanions, or oxyhydroxyl anions), initial concentrations (10 100 ppb) and
space velocity of aqueous solutions to be processed, and chemical combination of surface
versus chemical species (e.g. FeIIIO(OH) versus As(OH)3 and OH(clay surface)/CO32
(between layers) versus Pb2+).
In the analytical point of view to monitor the destiny of low concentrations of toxic
elements, selective XAFS technique, with which the author of this chapter has also
183
investigated surface catalytic sites of gold, platinum, tin, and vanadium [31 35], needs to be
combined with other technique with nanoscale spacial resolution. Spectroscopy with
nanoscale spacial resolution is under investigation, but not established to be applicable to
nanotechnology (Table 2).
At present, spacial resolution of X-ray microscopy is 1 m [36]. Several types of X-ray
microscopy/imaging are under investigation, e.g. microbeam XAFS, photoemission electron
microscope (PEEM), or phase contrast imaging [37, 38]. The spacial resolution of TEM is
already smaller than 1 nm if the sample nanoscopic condition matches to the high-resolution
measurement. To monitor the sorption between 2-dimensinal layers (e.g. montmorillonite,
hematite), in 2-dimensional mesopores (e.g. FSM-16, MCM-41), and in 3-dimensional
micro/mesopores (e.g. ZSM-5, acetate-exchanged FeOx(OH)y [17]), 3-dimensional TEM
images would be very helpful by taking series of TEM snapshots from various angles to
sample and organizing 3-dimensional image on computer [39].
Scanning probe microscope (SPM), especially scanning tunneling microscopy (STM) and
atomic force microscope (AFM), has an advantage of atomic resolution for well-defined
surface [40]. To utilize SPM technique to monitor the sorption from low concentrations of
toxic elements, the combination with element specific spectroscopy, e.g. XPS, XAFS, is
essential to describe the surface chemical mechanism. The author of this chapter is
developing this combination (AFM and XPS) based on temporal electron trap phenomena in
the metal nano-dots in the front of the AFM tip [41, 42].
Table 2. Spectroscopy Needed to Be Developed to Give Direct Spacial Information of
Surface Uptake Mechanism from Low Concentrations (10 100 ppb) of Toxic Metal
Elements
Probe
Method
Factor to be developed for this application
Refs
X-ray Microscope
Smaller X-ray beam (< 10 nm)
[36 38]
Electron Microscope
3-dimensional information
[39]
Tip Microscope
Scanning probe microscope to discriminate
[41, 42]
the kind of element

X-ray Diffraction
Surface-sensitivity between nano-layers or
[43]
in micro/mesospace
X-ray XAFS/XRF
On-site analysis in the field
[44, 45]
Surface-sensitive XRD may be applicable to monitor the application of water purification

[43]. The breakthrough is selectivity to internal surface of layered and micro/mesoporous
materials used as sorbents. Portable XRF/XAFS apparatus that has been investigated for
space science [44, 45] will be applicable to environmental on-site monitoring of the fate of
toxic elements in the field.
184
Yasuo Izumi
ACKNOWLEDGMENTS
This article is part 26 in the series of state-sensitive XAFS. The X-ray experiments were
performed under the approvals of the SPring-8 Program Review Committee and of the Photon
Factory Proposal Review Committee. The works included in this chapter were financially
supported by grants from the Grant-in-aid for Encouragement of Young Scientists
(B14740401, A12740376) and the Grant-in-aid for Basic Scientific Research (B13555230,
C17550073) from the Ministry of Education, Culture, Sports, Science, and Technology,
Yamada Science Foundation (2000), Toray Science Foundation (98-3901), and Research
Foundation for Opto-Science and Technology (2005 2006).
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ISSN: 2158-5717
Chapter 5
ON THE REDISTRIBUTION OF TISSUE METAL

(CADMIUM, NICKEL AND LEAD) LOADS IN MINK
ACCOMPANYING PARASITIC INFECTION BY THE
GIANT KIDNEY WORM (DIOCTOPHYME RENALE)
Department of Biology, Laurentian University,
Sudbury, Ontario, Canada, P3E 2C6
ABSTRACT
Patterns of metal uptake and accumulation in mink living under conditions of
environmental pollution and simultaneously inflicted with the invasive giant kidney
worm (Dioctophyme renale) parasite have not been examined, nor is the combined effect
of these dual insults on the health and physical condition of the animal known. Using
animals collected within the influence of the long-active ore-smelters at Sudbury,
Ontario, an examination was made of toxic metal (Cd, Ni and Pb) levels and their tissue
distributions within adult male mink bearing different intensities of parasite infection.
Higher metal burdens were indicated within infected specimens than those uninfected.
Combined renal and hepatic nickel and lead burdens were highest for mink with multiple
worm infections, although only lead accumulations reached statistical significance.
Cadmium accumulated to the greatest extent in the hypertrophied left kidney and liver,
whereas nickel and lead were deposited more readily in the bony spicule of the
parasitized right kidney cyst. The relative distribution of cadmium among renal, hepatic
and renal cyst tissues (cast, spicule, worms) remained unchanged subsequent to D. renale
infection, while the proportions of nickel and lead deposited in hepatic tissue were
reduced. Metal burdens in female D. renale were three-fold higher than those of male
worms, with the difference being attributable to the substantially greater size of the
females. Canonical Correlation Analyses of condition measures and body metal burdens
failed to indicate a direct relationship between infection intensity and body fat deposits
but did confirm a positive association between metal loads and increased fat levels, along
with enhanced gonad weights, neck circumference and reduced spleen weights. Such
associations may be productive aspects for future investigation into the combined effects
of increased metal loads and parasitic infection on the host system.
188
INTRODUCTION
Investigating metal contaminant levels in parasites has received considerable attention of
recent, with the goal of most studies being to identify useful bioindicators and/or biomonitors
of environmental pollutants. The use of parasites as indicators of metal contaminants within
fish and aquatic environments has been studied extensively, and has shown that
acanthocephalans and to a lesser extent cestodes, can accumulate extremely high
concentrations of metal pollutants (Sures et al., 1994 a,b,c; Sures and Taraschewski, 1995;
Sures et al., 1997 a,b,c; Siddall and Sures, 1998; Sures et al., 1999; Tenora et al., 1999a;
Turcekova and Hanzelova, 1999: Zimmermann et al., 1999; Tenora et al., 2000; Barus et al.,
2001b; Turcekova et al., 2002; Palikova and Barus, 2003; Sures, 2003; Williams and
Mackenzie, 2003). By comparison, studies focused on bird-parasite systems are relatively few
(Barus et al., 2001a; Tenora et al., 2001; Tenora et al., 2002). Virtually no work of this nature
has been done on nematode parasites in mammals, with the exceptions of Szefer et al. (1998)
who studied the bioaccumulation of trace elements in lung nematodes of harbour porpoises,
Greichus and Greichus (1980) and Sures et al. (1998) who reported on roundworms of the pig
and Tenora et al. (1999b) who examined metal levels in either gender of Toxocara canis and
Protospirura muricola specimens and their respective hosts. The extent to which parasites
affect metal uptake and distribution in host tissues is largely unknown and, for the most part,
was not investigated in the above studies. It is known from the literature, however, that
helminth infestation may affect the hosts sensitivity to toxic metals (Pascoe and Cram, 1977;
Poulin, 1992; Sures and Siddall, 1999). Since the giant kidney worm parasite, Dioctophyme
renale, produces pathophysiological changes in both the liver through which it migrates and
the kidney which it ultimately occupies, and these two organs represent the primary sites of
metal accumulation in the vertebrate body, one might expect measurable changes in the tissue
distribution of metals within the host animal following infection.
The development and life cycle of the giant kidney worm have been summarized by
Anderson (2000), and are depicted in Figure 1. Mink (Mustela vison) are the preferred
definitive host for D. renale, although other carnivorous species have occasionally been
infected (including otter Lontra canadensis, marten Martes americana, short-tailed weasel
Mustela erminea, long-tailed weasel M. frenata, wolverine Gulo luscus and Gulo gulo, coyote
Canis latrans, wolf C. lupus, dog C. familiaris, red fox Vulpes vulpes, bear Ursus
americanus, raccoon Procyon loto, and coati Nasua nasua). Adult kidney worms typically
occupy the right kidney (Figs. 2a and 2b) but on occasion have been observed unrestricted
within the abdominal cavity or entwined within the liver of their host (Figure 2d). At least one
male and one female worm (Figure 2f) must be present in the same kidney for a fertile
infection to occur and for the life cycle to continue. Fertilized eggs are released from the
female worm, passed down the ureter (which remains open post-infection) to the urinary
bladder and voided with the urine. If the eggs are released into water and reach appropriate
temperatures (approximately 14 to 30 C), they embryonate and are frequently consumed by
the aquatic oligochaete Lumbriculus variegatus. The larvae moult twice in this intermediate
host, reaching the infective third larval stage. Suitable paratenic hosts (frogs Rana
catesbeiana, R. clamitans and R. septentrionalis, pumpkinseed fish Lepomis gibbosus, brown
bullheads Ictalurus nebulosus and black bullheads I. melas) eat the parasitized oligochaete,
but no futher development of D. renale larvae takes place in these paratenic hosts.
189
Definitive Host
(final moult and growth to adult
worm occupying right kidney)
Incidental direct
transfer via infected
drinking water
Eggs released with

urine into water
Unembryonated
Eggs
Paratenic Hosts
Intermediate Host
(Lumbriculus variegatus)
(eggs hatch and develop to
infective 3rd stage larvae)
Figure 1. Development and transmission of the giant kidney worm, Dioctophyme renale.
Although it is possible for mink to become infected through the incidental consumption
of parasitized oligochaetes during feeding or drinking, the above-mentioned paratenic hosts
are frequent items in the minks diet, and thus typically serve as the vectors through which the
parasite is transferred to mink.
Once ingested, third-stage larvae penetrate the stomach wall of the definitive host and
develop to the adult stage before migrating through the liver. The adult worms make their
way to the right kidney, which they penetrate and ultimately excavate by destroying the
functional cortical and medullary tissues as they grow to full size. Only a tough thickened
capsule or cast (Figure 2c) remains of the right kidney, serving to encase the mature D. renale
worms and in most cases a bony spicule (or staghorn). The parasitized kidney with its
occupants and component structures is commonly referred to as a kidney cyst. The spicule is
usually embedded in the lumenal surface of the dorsal wall of the kidney cast; although size,
shape and colour have been shown to vary considerably (Dhaliwal and Taylor, 2000), the
basic form is a flat plate on the dorsal side, with finger-like projections or spicules radiating
ventrally (Figure 2c). Histological examination of the spicule has revealed osteoblasts within
lacunae and canaliculi making up the Haversian system of true replacement bone (McNeil,
1948; Mace, 1976). The plate edges and spicule projections have been found to contain
osteoclasts and consist of hyaline cartilage (McNeil, 1948). The left kidney of the infected
animal hypertrophies (50 to 100% by weight; Figure 2e) to compensate for the loss in
function of the right kidney.
190
Figure 2. Dioctophyme renale infection in mink: a) ventral view of kidneys showing right renal cyst of
infected animal b) cyst opened showing several worms present c) cast with bony spicule (staghorn)
embedded, after removing worms d) free-floating abdominal infection showing portions of 3 worms
present among visceral organs e) hypertrophied left kidney of infected animal (on right) vs control f) 3
male worms (top) and 3 female specimens (below) removed from renal cyst (bottom). Magnifications:
a), b) and d) = 0.75X actual size; c) and e) = 1.25X; f) = 0.38X.
In the Sudbury-area, the prevalence of D. renale infection in mink is approximately 50%

(N. Schaffner and G. Parker, unpublished data) and, due to local ore-mining/smelting
operations over the past 125 years, wildlife living within the influence of the area-wide
smelters are exposed to significantly elevated metal levels in their environment (Wren et al.,
1988; Capodagli and Parker, 2007).
Patterns of metal uptake and accumulation in mink living under these conditions of
environmental pollution and additionally inflicted with the invasive giant kidney worm
parasite are currently unknown. As part of an on-going program examining toxic metals and
endoparasites in Ontario wildlife species, the opportunity arose to investigate patterns of
metal distribution in wild Sudbury-area mink populations infected with the parasite. The
objectives of this investigation were as follows:
191
1) to determine the effects of D. renale infection (at high and low intensities) on the
uptake and renal-hepatic distribution of toxic metals (cadmium, nickel and lead) in
wild field-trapped mink.
2) to quantify the extent to which these toxic metals are accumulated within the right
kidney cyst and its component structures (namely the parasitic worms, bony spicule
and cast tissues) of the infected animal.
3) to compare the extent of metal uptake and accumulation in male versus female
specimens of the parasite.
4) to examine the extent to which metals are accumulated in the osseous spicule of the
infected animal relative to concentrations occurring in skeletal (femur) bone.
5) to determine the extent to which renal-hepatic metal levels and D. renale infection,
either singly or in concert, may influence the health and physical fitness of mink,
through an assessment of the effects of these perceived insults on selected
morphometric measures and fat reserves of the animal.
MATERIALS AND METHODS

Mink carcasses were obtained from local fur trappers in the Sudbury District of Northern
Ontario during the October to December trapping seasons of 1997, 1998 and 1999. Metal
fallout and environmental contaminant conditions prevailing within the Sudbury Basin, home
to intensive mining-smelting operations over the past 125 years, have been described
elsewhere (Capodagli and Parker, 2007). A total of 30 adult male specimens were selected
based on the intensity of giant kidney worm (Dioctophyme renale) infection present. Three
groups were formed; non-infected mink (n=14), those infected with a single worm (n=8) and
those infected with 4-6 worms (n=8).
Assignment to the adult (>1 yr) male cohort was based on visual inspection of the
genitals/gonads and the pattern of temporal muscle coalescence on the dorsal aspect of the
skull (E. Addison (pers. comm.). Prior to dissection, the animals were completely thawed and
morphometric measurements taken, including total peltless mass (to nearest 0.1 g), body
length (from nose to tail base), total body + tail length (nose to tip of tail) and neck
circumference (all to nearest 0.5 cm). Subcutaneous fat deposits in the groin and scapular
regions were subjectively rated as high (3), medium (2) or low (1).
During necropsy, the liver, heart, spleen, kidneys, gonads, omental fat, thoracic postcardinal fat deposit and abdominal mid-ventral fat deposit were removed and weighed (to
nearest 0.1 g) immediately upon removal to minimize the effects of dehydration. Livers,
femurs and kidneys (or their component parts in the case of infected animals) were retained
for determination of metal content. In the case of infected subjects, the right kidney cyst was
opened and each individual worm was removed, counted, weighed (to the nearest 0.1 g) and
measured (to the nearest mm). Individual D. renale worms were sexed by the
presence/absence of the prominent bell-shaped bursa at the caudal end and according to
differences in body length and diameter (females ranged from 16-50 cm in length and were
approximately 0.5 cm in diameter, while males ranged from 8-17 cm in length and were 0.25
cm in diameter) (Fyvie, 1971). Bony spicules were carefully dissected from the kidney cast of
infected mink and kept in small airtight plastic cups at 20 C. Kidneys, liver, femur, kidney
192
cast and worms were immediately packaged in individual polyethylene (Whirlpak-Nasco)

bags and restored at 20 C until metal analysis could be performed.
All glassware was washed using SparkleenTM dish detergent, rinsed, passed through a
20% HCl bath followed by two deionized distilled water baths and allowed to air dry before
use. The right kidney, left kidney, liver, kidney cast, shaft of the right femur, individual whole
worms and bony spicule were dehydrated in petri dishes in a drying oven at 60 C until
constant weight was attained (approximately 3 days). A 2.0 ( 0.005) g sub-sample of dried
liver, the shaft of the right femur and the entire sample of each of the remaining tissues were
weighed into ceramic crucibles and placed in a muffle furnace. The temperature in the
furnace was increased at a rate of 0.7 C/minute to 150 C and held for 10 hours, and then
raised by 0.3 C/minute to 500 C, where it was held for 15 hours. The samples were allowed
to cool to room temperature before digestion began.
The ash was then transferred to sterile 15 ml polypropylene centrifuge tubes and a three
ml aliquot of 2.5 N reagent grade HNO3 was added to the ash. The centrifuge tubes were
capped and vortexed, and the samples then placed in an oven at 60 C to digest for 20 hours.
The samples were then removed from the drying oven, vortexed again to break up any
precipitate and left to stand overnight to ensure complete digestion. The samples were then
centrifuged at full speed (approximately 5000 G) for five minutes and the supernatant was
extracted with disposable glass Pasteur pipettes and transferred into acid-washed test tubes.
Where necessary, the samples were diluted (5 fold) with 20% reagent grade HCl before being
analyzed by flame atomic absorption spectrophotometry for Cd, Ni and Pb content using a
Perkin Elmer Spectrophotometer (Model 703). Procedural blanks were included in each
sample run to control for metals introduced by the digestion process. Metal concentrations
were calculated and expressed as gg-1 dry weight. Tissue metal burdens (expressed as g)
were derived by multiplying metal concentration by the total tissue dry weight. To determine
recovery rates and assess the reliability of the analytical procedures, certified reference
materials (oyster tissue from the National Institute of Standards and Technology and citrus
leaves from the National Bureau of Standards) were also subjected to the above procedure. As
recovery rates (112 to 131%; n=3 per element) were generally deemed acceptable, tissue
concentrations were left unadjusted.
Where violations of normal distribution and/or homogeneity of variance in the data could
not be rectified with standard transformations, the data were statistically evaluated using both
parametric and non-parametric procedures. Organ weights and fat deposit measures were
standardized for body size using the formula:
Standardized measure = organ (or fat) weight x individual body length / mean body length
RESULTS
Body and Organ Weights
Dried weights of the primary organs of metal accumulation as well as standard body
weights for the three infection groups (uninfected mink, mink infected with one kidney worm
and mink infected with 4-6 worms) are reported in Table 1.
193
Table 1. Body weights and dried organ weights (g) in mink infected with D. renale
at different intensities (0, 1 or 4-6 worms). Values are means followed by standard
error in parenthesis
Infection Status
Organ
0 worms (n=14)
1 worm (n=8)
4-6 worms (n=8)
Liver
10.55
(0.43)
10.01
(0.35)
10.25 (0.72)
Left kidney
0.78
(0.02)
1.23
(0.08)
1.32
(0.05)
Right kidney/cyst
0.73
(0.03)
0.88
(0.15)
2.26
(0.23)
a) cast
0.34
(0.03)
0.75
(0.06)
b) spicule
0.10
(0.04)
0.19
(0.06)
c) worm(s)
0.44
(0.13)
1.32
(0.17)
Cyst components:
Body weight
746.68 (24.43) a
766.12 (22.70) a
715.27 (43.39) a
Within organs, mean values bearing the same letter are not significantly different (p > 0.05) as indicated
by a One-way Analysis of Variance and Duncans Multiple Range Test.
There were no significant differences in mean dry liver weights or overall body weights
among the three infection groups. However, substantial weight differences occurred among
the kidneys. The left kidneys of mink infected with either 1 worm or 4-6 worms were
approximately 58% and 69% heavier, respectively, than the left kidney of uninfected mink.
The right kidney cysts that developed in infected mink were likewise heavier than the intact
right kidney of non-infected animals. This increase in weight amounted to 21% and 210% in
the single worm and 4-6 worm infections, respectively. Despite the 2.6-fold weight difference
between single and multiple worm cysts, the contributions of individual structural
components were relatively uniform: cast tissue comprised 38.6 versus 33.2%, spicule 11.4
versus 8.4%, and worms 50.0 versus 58.4%, respectively.
Cadmium Levels
Cadmium burdens and concentrations in the liver, kidneys, kidney cast, spicule and giant
kidney worms of mink with varying intensities of D. renale infection are presented in Table
2. There was an overall increase in cadmium burden in infected mink over uninfected mink.
Together renal and hepatic burdens approximated 7 g in uninfected mink whereas infected
mink averaged 13 to 17 g. The elevated cadmium burdens in parasitized mink were
manifested in both the liver and the uninfected left kidney (Figure 3). On average, liver
burdens were elevated 2-fold and left kidney burdens by 3.6 fold over values seen in noninfected animals. Cadmium burdens present in the right kidney (non-infected animals) or its
component structures (cast, spicule and worms) in infected animals did not differ significantly
among the groups (Table 2). Thus the amount of cadmium that was present within the
194
confines of the renal cyst appeared to remain unchanged from that present in the right kidney
prior to infection, and was distributed among the component tissues/structures (Figure 3).
Table 2. Cadmium levels in body tissues of mink infected with D. renale at different
intensities. Values are means followed by standard error in parenthesis
CADMIUM BURDEN (g)
Infection Status
Organ
0 worms (n=14)
1 worm (n=8)
4-6 worms (n=8)
Liver
4.74
(0.55)
10.98
(2.61)
7.07
(1.08)
a,b
Left kidney
1.23
(0.23)
4.89
(1.41)
4.02
(0.88)
Right kidney/cyst
1.15
(0.21)
1.07
(0.18)
1.63
(0.25)
a) cast
0.40
(0.10)
0.35
(0.05)
b) spicule
0.18
(0.04)
0.26
(0.05)
c) worm(s)
0.48
(0.15)
1.02
(0.21)
Cyst components:
All above
organs/components 7.29
(0.99)
a
16.94
(4.06)
b
12.75 (2.43) a,b
combined
Within organs, mean values bearing the same letter are not significantly different (p 0.05) as
indicated by a One-way Analysis of Variance and Duncans Multiple Range Test.
CADMIUM CONCENTRATION (gg-1)
Infection Status
Organ
0 worms (n=14)
1 worm (n=8)
4-6 worms (n=8)
Liver
0.47
(0.07)
1.14
(0.30)
0.70
(0.10)
a,b
Left kidney
1.59
(0.33)
4.10
(1.30)
3.03
(0.66)
a,b
Right
kidney/cyst
1.62
(0.31)
1.66
(0.54)
0.72
(0.09)
a) cast
1.43
(0.57)
0.47
(0.07)
b) spicule
4.33
(1.36)
1.82
(0.23)
c) worm(s)
1.73
(0.55)
0.80
(0.14)
Cyst components:
Within organs, mean values bearing the same letter are not significantly different (p 0.05) as indicated
by Kruskal-Wallis One-way Analysis of Variance and Duncans Multiple Range Test.
195
Liver
20
Left kidney
Right kidney/cyst
Cyst components:
CADMIUM BURDEN (g)
15
Cast
Spicule
Worm(s)
10
B
A, B
0 worms
1 worm
4-6 worms
INFECTION STATUS
Figure 3. Cadmium burden (g) in organs of mink infected with D. renale at different intensities.
Within organs, values bearing a common letter are not significantly different (p 0.05) as indicated by
a One-way Analysis of Variance and Duncans Multiple Range Test. Bars indicate 1 S.E. of the
mean composite burden.
6
Liver
Left kidney
CADMIUM CONCENTRATION (g g-1)
Right kidney/cyst
Cyst components:
4
Cast
Spicule
Worm(s)
2
A,B
A
A B
B
B
A
A,B
A
0 worms
1 worm
4-6 worms
INFECTION STATUS
Figure 4. Cadmium concentration (gg-1 dry wt) in organs of mink infected with D. renale at different
intensities. Within organs, values bearing a common letter are not significantly different (p 0.05) as
indicated by a Kruskal-Wallis One-way Analysis of Variance and Duncans Multiple Range Test. Bars
indicate 1 S.E. of the mean.
196
While cadmium burdens of the left kidney increased 3 to 4-fold as a result of infection
(Table 2), organ hypertrophy (58 - 69%; Table 1) reduced mean tissue concentrations to
values of 3 - 4 gg-1 (Table 2). Nevertheless these values were significantly (1.9 to 2.6-fold)
higher than concentrations present in the left kidneys of non-infected subjects. Within the
right kidney cyst of infected animals, cadmium was most concentrated in the bony spicule
(mean = 4.33 1.36 and 1.82 0.23 gg-1; Figure 4). Worm concentrations averaged 1.73
0.55 and 0.80 0.14 gg-1 in the single and multiple worm groups, while cast tissues
averaged 1.43 0.57 and 0.47 0.07 gg-1 in these two respective groups. For all tissues
examined, mean cadmium concentration values tended to be higher among animals infected
with single versus multiple worms; however, only in the case of the right kidney cyst and cast
tissue did these differences reach statistical significance (p = 0.007 and 0.012, respectively).
Nickel Levels
Tissue nickel burdens and concentrations for the same three groups of mink are reported
in Table 3. Similar to cadmium, nickel burdens were likewise greater in the combined renal
and hepatic tissue of infected mink compared to non-infected mink. Unlike cadmium
however, the liver was not responsible for any of the increase in nickel burdens of the
parasitized animal. The elevated nickel burden was distributed within the renal tissues of the
animals, primarily the parasitized right kidney cyst. Approximately twice the nickel burden
was present in the left kidney of infected mink, whereas 4 and 8 times more nickel were
observed in the right kidney cyst of mink with 1 and 4-6 worm infections respectively, over
burdens present in respective uninfected kidneys (Figure 5).
Table 3. Nickel levels in body tissues of mink infected with D. renale at different
NICKEL BURDEN (g)
Infection Status
Organ
0 worms (n=14)
1 worm (n=7)
4-6 worms (n=8)
Liver
4.47
(0.28)
5.14
(0.67)
4.63
(0.50)
Left kidney
0.52
(0.07)
1.03
(0.21)
0.98
(0.10)
Right kidney/cyst
0.52
(0.08)
2.10
(0.50)
a,b
4.08
(0.51)
a) cast
1.16
(0.38)
1.44
(0.26)
b) spicule
0.71
(0.20)
1.03
(0.21)
c) worm(s)
0.60
(0.19)
1.78
(0.78)
7.71
(1.13)
a,b
10.19
(0.86)
Cyst components:
All above organs/components

5.49
combined
(0.33)
197
NICKEL CONCENTRATION (gg-1)

Infection Status
Organ
0 worms (n=14)
1 worm (n=7)
4-6 worms (n=8)
Liver
0.43
(0.02)
0.51 (0.05)
0.45
(0.03)
Left
kidney
0.65
(0.08)
0.83 (0.14)
0.75
(0.08)
Right kidney/cyst
0.72
(0.10)
2.21 (0.36)
1.88
(0.19)
a) cast
3.73 (1.50)
1.95
(0.32)
b) spicule
11.47 (2.30)
7.78
(1.21)
c) worm(s)
0.97 (0.19)
1.47
(0.22)
Cyst components:
12
Liver
Left kidney
10
Right kidney/cyst
Cyst components:
Cast
Spicule
Worm(s)
NICKEL BURDEN (g)
C
C
B
4
A
2
0 worms
1 worm
4-6 worms
INFECTION STATUS
Figure 5. Nickel burden (g) in organs of mink infected with D. renale at different intensities. Within
organs, values bearing a common letter are not significantly different (p 0.05) as indicated by a Oneway Analysis of Variance and Duncans Multiple Range Test. Bars indicate 1 S.E. of the mean
composite burden.
Mean hepatic nickel concentrations ranged between 0.43 0.02 and 0.51 0.05 gg-1,
and did not differ significantly among the three infection groups (Table 3). Similarly,
concentration values in the left kidney were comparable across the three infection groups,
indicating that the infection-induced increase in nickel loads within this organ were
198
proportional to the degree of hypertrophy undergone. Within the right kidney cyst, the
greatest concentration of nickel occurred within the spicules (mean = 11.5 2.3 and 7.8 1.2
gg-1 in single worm and multiple worm infections respectively; Figure 6).
14
Liver
Left kidney
NICKEL CONCENTRATION (g g-1)
12
Right kidney/cyst
Cyst components:
10
Cast
Spicule
Worm(s)
A
6
A
A
A A
B
B
A
A
A
B
0 worms
1 worm
4-6 worms
INFECTION STATUS
Figure 6. Nickel concentration (gg-1 dry wt) in organs of mink infected with D. renale at different
Worm concentrations averaged 0.97 0.19 and 1.47 0.22 gg-1 while cast
concentrations averaged 3.7 1.5 and 2.0 0.3 gg-1 respectively in the two infection
groups. Relative to non-infected animals, tissue nickel levels (burdens and concentrations) in
infected animals were more variable with few statistically significant differences occurring
between single-worm and multiple-worm groups.
Lead Levels
Lead burden and concentration values in mink from the three infection intensity groups
are presented in Table 4. Total lead burdens followed the same trend seen for both cadmium
and nickel in that combined kidney and liver lead loads were greater in mink infected with D.
renale when compared to uninfected mink. Elevations averaged 21% higher in mink infected
with 1 worm and 69% in those with 4-6 worms. As seen for nickel, the infection-induced
increase in lead burden was attributable to enhanced deposition in the renal tissues. For the
left kidney, lead burdens were elevated 1.6-fold (one worm infections) and 1.9-fold (4-6
worm infections) higher than values seen in the non-parasitized animal. The majority of the
increase in total lead burden arose from the right kidney components, which showed
199
elevations of more than 3-fold for 1-worm infections and 7-fold for 4-6 worm infections
(Figure 7). Although noticeably higher, lead concentrations in the liver and left kidney
followed the same trend as nickel in that mean tissue concentrations failed to differ
significantly across the three treatment groups (Table 4).
Table 4. Lead levels in body tissues of mink infected with D. renale at different
LEAD BURDEN (g)
Infection Status
Organ
0 worms (n=14)
1 worm (n=8)
4-6 worms (n=8)
Liver
10.28 (0.60) a
10.23
(0.44)
11.72
(1.18)
Left kidney
0.95
(0.05) a
1.54
(0.07)
1.83
(0.17)
Right kidney/cyst
0.93
(0.07) a
3.37
(0.73)
6.73
(1.03)
a) cast
0.54
(0.07)
1.11
(0.14)
b) spicule
2.19
(0.68)
3.44
(0.77)
c) worm(s)
0.64
(0.14)
2.15
(0.30)
Cyst components:
All above organs/components

12.50 (0.61) a
15.13
(0.87) b
21.15 (1.13) c
combined
LEAD CONCENTRATION (gg-1)
Infection Status
Organ
0 worms (n=14)
1 worm (n=8)
4-6 worms (n=8)
Liver
0.98
(0.04) a
1.02
(0.02)
1.13
(0.07)
Left
kidney
1.19
(0.05) a
1.26
(0.05)
1.40
(0.14)
Right kidney/cyst
1.29
(0.10) a
4.07
(0.71)
2.93
(0.29)
a) cast
1.68
(0.27)
1.43
(0.10)
b) spicule
40.50
(12.08) a
24.08
(2.84)
c) worm(s)
1.78
(0.19)
1.95
(0.10)
Cyst components:
200
Liver
Left kidney
20
Right kidney/cyst
LEAD BURDEN (g)
Cyst components:
Cast
Spicule
Worm(s)
15
C
B
10
A
5
1 worm
0 worms
4-6 worms
INFECTION STATUS
Figure 7. Lead burden (g) in organs of mink infected with D. renale at different intensities. Within
organs, values bearing a common letter are not significantly different (p 0.05) as indicated by a Oneway Analysis of Variance and Duncans Multiple Range Test. Bars indicate 1 S.E. of the mean
composite burden.
Liver
50
Left kidney
Right kidney/cyst
Cyst components:
LEAD CONCENTRATION (g g-1)
40
Cast
Spicule
Worm(s)
30
20
A
A
10
C
A
B
A
0 worms
1 worms
4-6 worms
INFECTION STATUS
Figure 8. Lead concentration (gg-1 dry wt) in organs of mink infected with D. renale at different
201
Among components of the right kidney cyst, the bony spicule concentrated lead to the
greatest extent with mean concentrations reaching 40 12.1 and 24 2.8 gg-1 for 1 worm
and 4-6 worm infections respectively (Figure 8). Concentrations in cast tissues and in worms
averaged less than 2 gg-1. Although burden values in the right kidney cyst were
substantially higher in the more heavily infected group, concentration values in the structural
components of the cyst did not differ significantly between 1 worm and 4-6 worm infection
groups.
Proportional Deposition among Tissues

To better control for variability in levels of uptake among individuals, the relative
distribution of body metal burdens between the liver and renal tissues/structures was
calculated and expressed on a percentage basis for both infected and non-infected animals
(Table 5). Data available from two mink infected with a single D. renale worm in the
abdomen (free-floating infection) and four mink with a single worm in the abdomen and one
in the right kidney (double infection) have been included in Table 5 to represent two
additional infection groups. The proportions of cadmium, nickel and lead deposited in renal
and hepatic tissues of mink with a single abdominal infection were similar to the proportions
deposited in non-infected mink. Of the total metal burden in mink with a free-floating
infection and in non-infected mink, liver tissue accumulated 67 and 68% of the cadmium, 79
and 82 % of the nickel and 83 and 85% of the lead, respectively. Sixteen percent of the total
cadmium, between 8 and 9% of the total nickel and 7-8% of the total lead was deposited in
each kidney of non-parasitized mink and mink with an abdominal infection.
Although there was a two-fold increase in total cadmium burden in mink infected with 1
worm over uninfected mink (Figure 3), the proportion deposited in the liver remained
constant at 65 to 68% (Table 5). Slightly less cadmium (59% and 57%) was deposited in the
livers of mink with double and 4-6 worm infections. Approximately 30% of the total body
cadmium was found in the functioning renal tissue, regardless of infection status. In noninfected and abdominally infected mink, this was equally distributed between the right and
left kidneys, while in mink parasitized in the right kidney, all 30% accumulated in the lone
functioning left kidney, regardless of the number of worms present. In single worm and
double worm renal infections, only 8 and 9% of the total cadmium was found in the resultant
kidney cyst whereas 14% was accumulated in the cyst of animals harbouring 4-6 worms. A
higher proportion of cadmium was deposited in worms from multiple worm infections (8%)
versus single worms (3%). At all infection intensities, 3% was deposited in the kidney cast
and 1-3% was sequestered in the bony spicule. When present, abdominal worms contained
only 1-2% of the cadmium burden.
In contrast to cadmium, nickel burdens in the liver did not differ with varying degrees of
infection intensity (Figure 5). However, the proportion of total nickel burden deposited in the
liver did change. In the non-parasitized group, approximately 82% of the total nickel burden
accumulated in the liver. Subsequent to infection, the proportion of total nickel found in the
liver dropped to 62% in mink infected with 1 renal worm, 44% in mink with a double
infection and 46% in mink infected with 4-6 worms.
202
Table 5. Relative distribution of metals among tissues, expressed as a percentage of total

renal-hepatic burden, in mink infected with D. renale at different intensities
Metal
Infection Status
0 worms
Organ
(n=14)
1 worm
Abdomen
(n=2)
2 worms
4-6 worms
Right kidney Abdomen + right Right kidney

(n=8)
kidney (n=4)
(n=8)
Cd
Liver
68 b
67 a,b
65 a,b
59 a,b
57 a
Left kidney
16 a
16 a
27 b
31 b
29 b
Right kidney/cyst
16 c
15 c
8a
9 a,b
Cyst components
a) cast
3a
3a
3a
b) spicule
1a
3a
2a
c) worm(s)
3a
3a
8b
Abdominal worm
82 c
79 c
62 b
44 a
46 a
14 b,c
Ni
Liver
Left kidney
9 a,b
9 a,b
12 b
7a
10 a,b
Right kidney/cyst
8a
8a
25 b
47 c
44 c
Cyst components
a) cast
10 a
27 b
14 a
b) spicule
10 a
7a
11 a
c) worm(s)
5a
13 a,b
19 b
Abdominal worm
85 b
83 b
68 a
67 a
58 a
Pb
Liver
Left kidney
8 a,b
7a
10 c
9 b,c
9 b,c
Right kidney/cyst
8a
7a
21 b
21 b
32 b
Cyst components
a) cast
4a
3a
5a
b) spicule
13 a
14 a
17 a
c) worm(s)
4a
3a
11 b
Abdominal worm
Within organs, values bearing the same letter are not significantly different (p 0.05) as indicated by a
One-way Analysis of Variance and Duncans Multiple Range Test.
203
The relative proportion of nickel deposited in the left kidney increased only slightly in
one worm infections, while that accumulating in the infected right kidney cyst reached as
much as 25% and 44% of the body burden in 1 worm and multiple worm animals
respectively. The left kidney of animals with an abdominal worm plus a right kidney worm
did not differ from that noted for non-infected mink, however 47% of the total nickel burden
was deposited in the right kidney cyst. In the case of single worm infections, 10% of the total
nickel was deposited in the kidney cast and 5% in the developing worm, whereas in mink
with 4-6 worms 14% was observed in the right kidney cast and 19% was taken up by the
worms. Mink with both an abdominal and right kidney worm accumulated 27% of the total
nickel in the cast and 13% in the right kidney worm. The spicule accounted for 10, 7 and 11
% of body nickel burdens in single, double and 4-6 worm infections respectively.
Relative proportions of lead deposited in renal and hepatic tissues paralleled those
observed for nickel. Eighty-five percent of the total body burden of lead was deposited in the
liver of uninfected mink, while only 68%, 67% and 58% were accumulated in the hepatic
tissue of mink with 1, 2 and 4-6 worm infections respectively. In response to infection,
proportions in the left kidney increased only slightly whereas total body proportions
accumulating in the infected right kidney cyst reached as much as 21% for both single and
double infections and 32% for the 4-6 worm infected animals. In each group, more than half
of the lead in the right kidney cyst was sequestered within the bony spicule. The worms
accounted for 4%, 3% and 11% and the cast tissues for 4%, 3% and 5% of the total lead
burden present in single worm, double worm and 4-6 worm infections respectively.
Gender Comparisons
A comparison of metal levels in male versus female D. renale taken from mink with
varying infection intensities is presented in Table 6. Dry weights for male and for female
worms did not vary with infection status as indicated by One-way Analyses of Variance
(males p = 0.46 and females p = 0.14). For multiple worm infections, metal levels in male and
in female worms occupying the same cyst were averaged and entered in a 2-way Analysis of
Co-Variance to assess the effects of infection status and worm gender while covarying for the
effects of total worm weight in the infected cyst. Total worm weight in each cyst was not
significantly correlated with levels of any of the three metals examined (p > 0.05), thus
indicating that competition between male and female parasites within the same cyst had not
affected metal uptake by the resident worms. For all three metals, overall mean burdens (i.e.
in all worms combined regardless of infection status) tended to be higher in female compared
to male worms by a factor of approximately 3 (Table 6). When differences in body mass were
factored in by calculating concentration values, significant gender differences were observed
only for lead, with males showing approximately 50% higher levels.
Femur versus Spicule Levels

The geometric means for metal concentrations in femur sub-samples and in the spicules
of parasitized animals were examined for the three infection groups and are presented in
Table 7.
204
Table 6. Metal levels in male and female D. renale taken from mink with varying
infection intensities. Values are means followed by standard error in parenthesis
Element
Infection Status
Variable
1 worm
( n=4, n=4)
4 worms a
56 worms a
( n=3, n=4) ( n=4, n=3)
Sex
All groups
combined
( n=10, n=11)
Cd
Burden (g)
Male
0.29 (0.09)
*
Female
0.07 (0.01)
**
0.15 (0.04)
*
0.18 (0.04)
*
0.69 (0.26)
0.34 (0.03)
0.57 (0.14)
0.53 (0.10)
Male
2.27 (0.85)
0.60 (0.13)
0.97 (0.25)
1.34 (0.37)
Female
1.20 (0.70)
0.62 (0.10)
1.14 (0.17)
0.97 (0.25)
0.11 (0.04)
0.16 (0.03)
0.27 (0.03)
0.19 (0.03)
Concentration
(gg-1)
Ni
Burden (g)
Male
**
Female
**
0.78 (0.20)
0.55 (0.06)
0.55 (0.16)
0.63 (0.09)
Male
0.82 (0.35)
1.36 (0.24)
1.94 (0.38)
1.43 (0.23)
Female
1.08 (0.25)
0.96 (0.10)
1.10 (0.28)
1.04 (0.11)
Male
0.29 (0.02)
0.29 (0.03)
0.35 (0.07)
0.31 (0.02)
Female
0.99 (0.09)
0.81 (0.07)
0.62 (0.006)
0.82 (0.06)
2.14 (0.17)
2.43 (0.21)
2.20 (0.24)
2.24 (0.12)
Concentration
(gg-1)
Pb
Burden (g)
**
**
Concentration
(gg-1)
Male
*
Female
a
1.41 (0.20)
1.42 (0.11)
**
1.30 (0.14)
1.38 (0.08)
In multiple-worm infections, values represent the average for all worms of same sex present within the
cyst. Gender differences assessed by 2-way Analysis of Variance while covarying for total worm
weight within cyst; p < 0.05 *; p 0.01 **.
205
Table 7. Metal concentrations (gg-1 dry wt) in femurs and spicules of mink infected
with D. renale at different intensities. Values are geometric means (presented as antilogs
of the log10 transformed data) followed by standard error in parenthesis
Tissue
Metal Infection Group
Cd
Ni
Pb
(n)
Femur
Spicule
0 worms
(12)
0.87
(+0.035, -0.033)
1 worm
(8 and 6)
0.94
(+0.038, -0.036)
<
2.14 (+0.49, -0.40)
4-6 worms
(8)
0.92
(+0.040, -0.039)
<
1.70 (+0.25, -0.22)
0 worms
(12)
3.39
(+0.12, -0.12)
1 worm
(8 and 6)
3.47
(+0.098, -0.094)
<
9.12 (+1.97, -1.62)
4-6 worms
(8)
3.55
(+0.12, -0.12)
<
7.24 (+1.25, -1.06)
0 worms
(12)
10.71
(+0.30, -0.29)
1 worm
(8 and 6)
11.22
(+0.29, -0.28)
<
23.99 (+2.68, -2.41)
4-6 worms
(8)
11.22
(+0.37, -0.36)
<
22.91 (+2.91, -2.59)
Equalities assessed by Paired-Samples T-Test; p < 0.01.
There were no differences in metal levels across the infection groups in either the femur
or the spicule for any of the three metals investigated as indicated by One-way Analyses of
Variance, and Analyses of Co-variance (p > 0.05) with spicule dry weight as the co-variant.
Thus, infection intensity had not significantly influenced the uptake of metals in either bone
tissue. The data were log10 transformed to accommodate for the non-linear relationship
between tissue metal concentration and spicule dry weight before a Paired-Samples T-test
was performed. Tissue comparisons revealed that the bony spicules of infected mink were
consistently higher in concentrations of cadmium, nickel and lead than were the femurs of the
same animals. Spicules from single worm infections concentrated 2.6 times more nickel, 2.3
times more cadmium and 2.1 times more lead than the femurs from the same group of mink.
Similarly, in multiple worm infections, spicule metal concentrations were approximately
twice those found in the femurs.
Effects on Body Condition

The Principal Component Analysis of the condition measures in mink for the three
infection groups (summarized in Table 8) is presented in Table 9. Four factors, explaining a
cumulative 73% of the variance, indicated shared sources of variance within the condition
measures. Abdomimal mid-ventral fat weight, thoracic post-cardinal fat weight and neck
circumference shared over 56% of their variance with Principal Component 1. These
measures were all correlated positively with the first component, indicating a positive
relationship with one another.
206
Table 8. Body measures (morphometric measures, organ weights and fat deposits) in
adult male mink infected with D. renale at different intensities. Values are means
followed by standard error in parenthesis
Infection Status
0 worms
(n=14)
Measure
1 worm
(n=8)
All mink
combined
(n=30)
4-6 worms
(n=8)
Morphometric measures:
Peltless body weight (g)
742.81 (22.42)
766.12
(22.70)
715.27 (43.39)
741.68 (16.48)
Total body + tail length (cm)
57.86
(0.69)
57.93
(0.48)
55.88
(0.85)
57.33 (0.44)
Body length (cm)
38.64
(0.55)
39.06
(0.49)
37.75
(0.62)
38.51 (0.34)
Neck circumference (cm)
11.88
(0.21)
12.04
(0.35)
11.75
(0.27)
11.89 (0.15)
Liver
43.19
(2.36)
37.67
(1.23)
40.40
(3.22)
40.97 (1.45)
Right kidney
3.61
(0.14)
4.20
(0.90)
14.20
(1.94)
6.59
(1.01)
Left kidney
3.74
(0.14)
5.69
(0.29)
6.04
(0.43)
4.87
(0.25)
Spleen
3.11
(0.40)
2.73
(0.28)
3.00
(0.38)
2.98
(0.22)
Heart
8.65
(0.33)
9.05
(0.48)
8.47
(0.54)
8.71
(0.24)
Gonads (combined)
0.38
(0.02)
0.66
(0.12)
0.54
(0.06)
0.51
(0.05)
Omental fat
3.54
(0.50)
3.11
(0.20)
4.08
(0.27)
3.55
(0.26)
Thoracic post-cardinal fat
0.20
(0.02)
0.16
(0.03)
0.19
(0.01)
0.19
(0.01)
Abdominal mid-ventral fat
0.64
(0.10)
0.58
(0.07)
0.59
(0.08)
0.61
(0.05)
Organ weights (g):
Fat measures (g):
Fat measures (1=low, 2=moderate, 3=high):

Subcutaneous fat
2.14
(0.14)
1.88
(0.23)
2.38
(0.18)
2.13
(0.10)
Groin fat
2.21
(0.15)
1.88
(0.23)
2.50
(0.19)
2.20
(0.11)
Accommodating for less variance but nevertheless indicating shared variance with the
first component were heart weight, body length, peltless body weight, scapular fat, groin fat
and spleen weight, which were also positively related. The second component presented
shared sources of variance for total body + tail length, body length, omental fat weight,
peltless body weight, total worm number and spleen weight. In this case, omental fat weight,
total worm number and spleen weight were negatively correlated to the second component
indicating that as total body + tail length, body length and peltless body weight increased,
omental fat weight, total number of D. renale worms and spleen weight decreased. The third
component accommodated for shared variance in liver weight, scapular fat, groin fat and
peltless body weight, which were all positively associated. The fourth and final component
207
indicated that right and left gonad weights, spleen weights and total worm number shared
common variance; however, spleen weight was negatively associated, and thus decreased as
gonad weight and total number of worms increased.
Table 9. Results of the Principal Component Analysis for body condition measures in
adult male mink with varying intensities of D. renale infection (n=21)
Variables
Correlation Coefficients
Component 1
Abdominal mid-ventral fat wt*
0.83
Thoracic post-cardinal fat wt*
0.80
Neck circumference
0.75
Heart wt*
0.48
Component 2
Total body + tail length

Body length
0.35
0.83
-0.71
0.46
Total worm number
0.58
0.50
-0.54
Liver wt*
0.49
0.86
Scapular fat
0.40
0.85
Groin fat
0.38
0.80
Right and left gonad wt *

Spleen wt*
Component 4
0.87
Omental fat wt*

Peltless body wt
Component 3
0.84
0.35
-0.48
-0.55
*Condition measures standardized for body size.
Table 10. Results of the Canonical Correlation Analysis on the Principal Components
for body condition measures in adult male mink and their total renal and hepatic
burdens (g) of cadmium, nickel and lead (n=21)
Correlation Coefficients for Root 1
Dependent variables:
Principal Component 1 (fat deposits, neck circumference)
0.69
Principal Component 4 (gonadal and spleen wts, total worm no.)
0.70
Independent variables:
Total Cd burden
0.64
Total Ni burden
0.49
Total Pb burden
0.79
208
These components were used as dependent variables in the subsequent Canonical

Correlation Analysis while total body (renal and hepatic) metal burdens of cadmium, nickel
and lead were used as independent variables. The results of the Canonical Correlation
Analysis are presented in Table 10. One root was significant at p < 0.05 with a Canonical
Correlation Coefficient (r) of 0.83. Principal Components 1 and 4 loaded on the dependent (y)
component of the root with correlation coefficients of 0.69 and 0.70 respectively. All three
total body metal burdens loaded moderately on the independent component (x) of the root.
Sixty-two per cent of the variance in total body lead, 41% of the variance in total body
cadmium and 24% of the variance in total body nickel burdens were shared with the
independent component of the root. In summary, the results of the Canonical Correlation
Analysis indicated that as the total body burdens of cadmium, nickel and lead increased, there
was an increase in abdominal mid-ventral and thoracic post-cardinal fats, neck circumference,
gonadal weight and total number of D. renale worms but the spleen weights decreased.
DISCUSSION
Giant kidney worm infection resulted in substantial weight changes among the kidneys,
but not the livers or peltless body weights, of mink. Kidneys infected with D. renale were
enlarged but the increase only reached significance for multiple worm infections where the
development of several worms along with the formation of a bony spicule had taken place
within the cyst. Hypertrophy of the left kidney to compensate for the loss in function of the
infected right kidney was indicated by the increase in dry organ weight over that seen in noninfected mink. McNeil (1948) and Mace (1976) similarly reported enlargements of both the
left kidney and the right kidney cyst in D. renale infected mink. The dry weight of the left
kidney in infected animals approximated the combined dry weights of right and left kidneys
in non-infected mink; thus the amount of functioning renal (and hepatic) tissue was
approximately equal for all infection groups. Consequently, any changes in tissue metal
burdens or concentration values noted in the parasitized animal can be deemed to be the result
of changes in metal uptake levels and/or the re-distribution of tissue metal loads.
Among the most interesting findings of this study was the increased renal plus hepatic
metal burden of parasitized (both single and multiple-worm infected) mink over that seen in
uninfected animals. Although quantitatively variable, this pattern was apparent for all three
metals examined. The nature of this metal-parasite association is of fundamental importance
and raises the question of whether the enhanced body metal loads had increased the
susceptibility of the animal leading to infection by the kidney worm or conversely whether it
was the invasion/presence of the D. renale worm(s) that had resulted in the increased levels of
metal uptake within these targeted tissues. The fact that more intense infections (4-6 worms
per animal) were generally associated with higher body metal burdens than seen in single
worm infections, although interesting, offers little towards answering this question. The latter
scenario, inferring a direct enhancing effect of the parasite on metal uptake and retention in
the host, appears more likely in this particular host-parasite relationship for a number of
reasons: D. renale is known to occur in areas devoid of point source metal emissions and
conversely is virtually absent in certain other locales heavily dominated by metal pollution
(e.g. Palmerton, Pennsylvania). Furthermore, juvenile and adult mink of the Sudbury area are
209
known to be infected at approximately equal frequencies (N. Schaffner and G.Parker,

unpublished data) despite the fact that the 5-6 month old juveniles, when trapped in the fall,
have had only brief exposures to prevailing environmental metal conditions and thus would
not be expected to have accrued significant metal burdens nor an accompanying increase in
susceptibility to the parasite. It is contended, therefore, that parasitic infection by the giant
kidney worms, whatever its primary predispositional cause(s) may have been, contributed
directly to the increased uptake and/or retention of tissue metals noted in this study. Future
laboratory studies investigating experimental infection intensities in captive mink exposed to
varying levels of metals within their diet would shed further light on this unresolved
contention.
Elevated metal burdens in the infected animals were most likely due to an increase in
metal uptake subsequent to parasitic infection. Although unconfirmed, it is certainly
conceivable that the energy demands imposed on the parasitized animal would be elevated, if
for no other reason than to provide the extra requirements needed in the growth and
development of the newly-developing cyst structures (namely the cast, worms and spicule),
the proliferation of actively hypertrophying tissues within the left kidney and the nutrients
necessary to sustain the resident parasites. Inherent in this supposition is the assumption that
increased levels of dietary intake would lead to enhanced exposure, uptake and retention of
metals by the infected animal.
Of the three elements sequestered by the parasitized animal, cadmium was the only one
to show significantly increased tissue levels in both the liver and the non-parasitized renal
tissue. Cadmium has a high affinity for the metal binding protein metallothionein (MT),
which is induced in the liver and to a lesser extent in the kidneys of the exposed animal
(Goering and Klaassen, 1984). It has been demonstrated that MT functions as a mechanism
for transport, storage and detoxification of metals (Fulkerson and Goeller, 1973).
Detoxification of cadmium in the vertebrate body occurs when the divalent form of the metal
(the most harmful form of most toxic metals) complexes with the low molecular weight
protein. The MT-cadmium complex is then transported to the kidneys for storage where it is
kept out of circulation. Once the levels of cadmium in the renal tissue reach a given threshold,
which varies among species, irreversible tubular damage results (Friberg et al., 1979). With
the loss in function of one kidney, the lone functioning left kidney and liver were responsible
for sequestering most of the cadmium taken up subsequent to parasitic infection. The
proportion of cadmium deposited in the left kidney of infected mink (27-31%; Table 5) was
roughly equivalent to the sum of the proportions deposited in both kidneys of the uninfected
animal (32%), and did not differ with increasing infection intensity. Also of interest here is
the fact that in infected animals the relative distribution of cadmium between the left
(hypertrophied) kidney and the liver remained at a ratio of approximately 3.5:1 a value
comparable to that seen in non-infected mink with both kidneys functional (Table 2). The
consistency of the above distribution ratios among infected and non-infected groups suggests
that cadmium was distributed between renal and hepatic tissues in a well regulated manner
and that the mechanisms and processes involved in the distribution and deposition of
cadmium in these two accumulator tissues had not been significantly altered as a result of
parasitic infection or increased metal loads. As parasitic burdens increased to 4-6 worms,
cadmium was consistently distributed among host tissues, with the exception of a slight
decrease in the proportion (57 vs. 65%; Table 5) deposited in the liver. This reduction may
have been due to the greater amount of worm tissue available for cadmium deposition and
210
which accounted for an increased proportion (8 vs. 3%; Table 5) of the total body burden of
the more heavily-infected animal, thus leaving less to be sequestered in the liver.
Whether the tissue damage induced by the invading D. renale parasite was directly
responsible for the augmented hepatic and/or renal cadmium levels noted in parasitized
animals is a matter of speculation. Lysis of the internal renal tissues of the right kidney by the
new-acquired invading worms might be expected to release bound cadmium from its MT
complex, thereby freeing it for incorporation into the growing worms, for reabsorption into
systemic circulation leading to increased production of MT followed by redistribution to other
host tissues or for elimination via the ureters which are known to remain open in the infected
animal (McNeil, 1948; Anderson, 2000).
Similarly, migration of the worms as third-stage larvae/subadults through the liver during
the initial infective process may have served as a physical or biochemical stimulus for the
production of additional MT in the liver. Mace and Anderson (1975) reported D. renale
larvae between the diaphragm and the right lobe of the liver in mink as late as 50 days postinfection. In addition, they found numerous lesions throughout the infiltrated liver containing
necrotic cells, fibrin, red blood cells, eosinophils, macrophages and lymphocytes. Visual
inspection and palpation of the livers of parasitized mink in the present study showed the
tissue to be abnormally firm and the ventral surfaces of the lobes to be uneven with evidence
of pathological damage. Fibres of connective tissue frequently attached the right kidney cyst
to the right lobe of the liver, as well as to other structures including the greater omentum,
duodenum and/or ventral surface of the abdominal wall, as has been noted by previous
investigators (McNeil, 1948; Mace, 1976). Cadmium, if leached from hepatic storage sites as
a result of the physical and/or pathological damage induced by the invading migratory larval
worms, could be expected to re-enter circulation, thus re-priming the production of MT
within host tissues. Evidence that the cadmium leaking out of necrotic liver tissue results in
increased kidney burdens of cadmium has been demonstrated in the mouse (Andersen et al.,
1985). The fact that abdominal infections not involving renal-hepatic tissue damage produced
little alteration in body cadmium burdens and their distribution confirms the central role of
parasite-induced tissue damage in prompting the substantial changes in cadmium
accumulation and distribution patterns seen in the more conventionally (i.e. right kidney)
infected mink.
Unlike the elevations noted in the liver and hypertrophied left kidney, cadmium levels in
the right kidney cyst of the parasitized animal remained unchanged from values observed for
kidneys prior to infection. On average, individual components within the cyst harboured
relatively low levels of cadmium, suggesting the absence of MT-complexing processes in
these tissues. The fact that only relatively small amounts of cadmium were taken up by the
resident worms may be explained by the absence of a well-developed excretory system, the
main site of MT-facilitated accumulation in most species, in these and similar nematodes
(Mace and Anderson, 1975).
Body burdens of nickel and lead were likewise elevated in response to parasitism, but
unlike the pattern noted for cadmium, most of the extra metal accumulated was confined to
the renal structures. Both nickel and lead are eliminated from the vertebrate body primarily
through the renal system (Friberg et al., 1979). There is no process comparable to the MTcadmium complex known to assist in the detoxification of either nickel or lead. With a
reduced ability to excrete these metals through the single remaining kidney, along with an
enhanced dietary intake as proposed earlier in this discussion, it would stand to reason that
211
some of the excess metals present within the infected animal would be localized within the
hypertrophied left kidney as indicated. By comparison, greater amounts of both lead and
nickel, however, were accumulated within the dysfunctional right kidney cyst of the
parasitized animal. Deposition into the various components of the cyst may have occurred
during the growth of these structures but is deemed to have been selective rather than
uniform, as concentrations of both lead and nickel within the spicule exceeded those present
in the cast tissue and resident worms by a factor of several-fold. The substantial levels
deposited within the component structures of the right renal cyst ultimately accounted for as
much as 25 to 47% and 21 to 32% of the total renal-hepatic nickel and lead burdens
respectively (Table 5) and appear to account for the markedly reduced proportions
sequestered within the liver of the parasitized animal.
Establishing trends in metal accumulation and distribution in response to infection
intensity as attempted here was fraught with several potential confounding factors. Sources of
variation that must be acknowledged include individual differences in host reaction to
parasitic infection and/or increased metal loads, variability in geographic origin of samples,
age-related variations in the host, age of the infection and inherent differences between the
sexes of the parasite. The biological response to a stress of any type varies from individual to
individual, and when trying to examine the response of individuals to multiple stresses, the
variability can be expected to magnify. The Sudbury region was chosen as an appropriate site
for this study because of its longstanding history of mining/smelting activity resulting in
substantial environmental contamination and because a 50% prevalence of D. renale
infections was known to exist within its wild mink populations. However, to collect sufficient
numbers, mink had to be taken at varying distances from the point source of metal pollution
a factor which undoubtedly contributed to the variability in metal loads among animals. Only
adult male mink were employed in an attempt to reduce variation in metal accumulation as a
function of age and/or gender. However, as the adult age class consisting of mink greater than
one year probably included some individuals ranging up to 4 or 5 years of age, the period of
exposure to metal contaminants cannot be considered uniform. Finally, due to substantial
sexual dimorphism in adult D. renale specimens, the gender of the infecting worm in the case
of single worm infections and the ratio of male to female worms in multiple worm infections
are factors which must be taken into consideration in establishing metal distribution patterns.
Female D. renale were consistently longer and heavier than male worms by a factor of 3,
which largely accounted for the approximate 3-fold elevation in cadmium, nickel and lead
burdens found in female worms versus male worms. However, when metal accumulations
were expressed on a per gram dry weight basis, male and female worms indicated nearequivalent concentrations, with the exception of lead which was 1.5 times higher in males.
While studies on Ascaris suum, a parasitic nematode of swine, failed to indicate genderdifferences in metal (Cd and Pb) uptake (Greichus and Greichus, 1980; Sures et al., 1998),
those involving other nematode species including Conracaecum rudolphii in cormorants
(Barus et al., (2001) and Anguillicola crassus in the European eel (Barus et al., 1999; Tenora
et al., 1999a) have shown a higher propensity for accumulation in males. In contrast, the
archiacanthocephalan Moniliformis moniliformis, experimentally introduced into the
gastrointestinal tract of rats and subsequently exposed through dietary supplements, indicated
higher Pb accumulations in female specimens a factor attributed to the substantial levels
sequestered within eggs (Sures et al., 2000). No attempt was made to localize the metal
accumulations within the giant kidney worms of the present study. Possible sites include the
212
cuticle, reproductive structures and/or the gastrointestinal tract, but not excretory tissues as
this nematode is not known to possess a structured excretory system (Mace and Anderson,
1975). Analytical methods sensitive enough to evaluate metal contaminants in very small
amounts of tissue may provide information identifying the sites of accumulation and
explaining differences in uptake between the two sexes.
The 2-fold higher concentrations of cadmium, nickel and lead in the developing bony
spicule relative to femur levels denote the spicule as an active site for deposition. Previous
studies have shown lead to be primarily deposited in bone and secondarily in soft tissue,
whereas nickel and cadmium accumulated primarily in soft tissues and secondarily in osseous
tissues (Friberg et al., 1979). The present results confirm this pattern in that lead presented the
highest concentrations in the spicule while cadmium showed the lowest concentrations. It is
tempting to conjecture that the deposition of toxic metals into the spicule structure may be
adaptive in allowing the host to store a portion of the increased metal load in a relatively nonexchangeable compartment of the body, thereby reducing the levels of toxic metals in
circulation or in storage within more vulnerable tissues. In the case of nickel, the proportion
deposited in the spicule was equivalent to the proportion in a single kidney of a nonparasitized mink (Table 5). Likewise, the spicule accounted for an equivalent proportion of
lead as seen in the entire renal system of a non-parasitized animal. This conjecture of adaptive
sequestration, if valid, could be expected to contribute only within limits as spicules seldom
exceeded 0.3 grams dry weight and generally accounted for less than 15% of the total renalhepatic metal burden (Table 5).
Few other studies investigating parasite-induced changes in host tissue metal distribution
were found in the literature. Palikova and Barus (2003) observed only low levels of mercury
accumulation in A. crassus, and concluded that the low efficacy of Hg (and of other elements
as reported by variuos authors) sequestering probably had little effect on tissue concentrations
in their host species, the eel. Evidence suggesting a significant role by tapeworms in
decreasing host tissue metal levels in several fish species has been presented by Turcekov and
Hanzelova (1999), Barus et al. (2001b) and Turcekova et al. (2002). Sures and Siddall (1999)
examined the effects of lead accumulation by adult intestinal worms (Pomphorhynchus
laevis) on the tissue metal levels of their definitive host, the chub (Leuciscus cephalus). The
presence of intestinal worms reportedly lowered lead accumulation in the intestinal wall
relative to that seen in uninfected fish. The authors suggested that the intestinal parasites had
extracted substantial lead from the diet of the host, thereby reducing the amount available for
absorption by the host tissues and accounting for the higher concentrations seen in worm
versus host tissues (unfortunately, burdens in host and parasite tissues were not reported). The
results of the present study in which D. renale infections were consistently associated with
elevated cadmium concentrations and unaltered lead and nickel levels within the renal and
hepatic tissues of the host are at variances with the above reported trends. The reason(s) for
the observed discrepancy is unclear, but may relate to differences in the source and route of
metal uptake (dietary versus extraction from the host tissue stores) and/or the level of
parasitic damage and host reaction incurred during the process of infection.
Assessment of the effects of both a parasitic stress and increased metal loads on mink
health revealed a positive relationship for two internal fat deposits, gonadal weight, neck
circumference and total number of D. renale worms with combined renal and hepatic burdens
of cadmium, lead and nickel. Spleen weight was negatively related to the total metal burdens.
These trends substantiate those reported by Capodagli and Parker (2007), in that higher levels
213
of fat deposition in both mink and muskrat (non-parasitized) were likewise observed among
animals bearing elevated tissue levels of cadmium and lead. If mink infected with the giant
kidney worm parasite were consuming more food to meet the increased energy demands of
parasitic infection, as suggested above, such dietary enhancement, if excessive, could explain
the increase in fat deposits noted for the abdominal mid-ventral and thoracic post-cardinal
sites. The extent to which the proposed increase in food consumption may have contributed to
the enhanced development of the neck musculature (as denoted by increased circumference),
and possibly an improved capacity for successfully capturing larger prey items, is open to
speculation. The positive relationship denoted between the intensity of infection (number of
worms present) and combined renal-hepatic metal burdens of the animal can best be
attributed to the level of tissue damage incurred combined with the nature of the host tissue
responses to the invading parasites as outlined earlier in this discussion.
The observed increase in gonad weight with increasing renal-hepatic metal levels,
although presently unexplained, may be indicative of direct alterations to reproductive
processes or indirectly through associated endocrinological functions. Testes have been
shown to be sensitive to the effects of cadmium, although low doses of the element have
generally been observed to induce necrosis, rather than hypertrophy (Fulkerson and Goeller;
1973). Given the possibility that accrued toxic metal burdens could adversely affect
reproductive status and productivity in this fur-bearing species, the metal-prompted increase
in testes weight may warrant further investigation including an assessment of histological
changes.
The negative relationship indicated between spleen weight and body metal burdens is
difficult to interpret. The spleen typically functions to breakdown and dispose of aged and
discarded red blood cells and has been shown to increase in size (splenomegaly) with
increased functional demand in rats experimentally exposed to lead (Ogilvie and Martin,
1981). At the present time, no explanation can be offered for the apparent decrease in fresh
spleen weights noted here.
It was initially hypothesized that destruction of the right kidney during D. renale
infection of the mink, particularly in geographic populations marked by elevated
environmental cadmium exposure, might result in critically elevated tissue metal levels
leading to functional impairment of the remaining contralateral. The findings of this study
support the view that metal levels were substantially elevated (2 to 2.5-fold) in the surviving
kidney of the parasitized animal. However, as damage in the human kidney generally does not
become apparent until renal cadmium levels reach values of approximately 350 gg-1 dry
weight (approximately 100 gg-1 wet weight) (Friberg et al., 1979) and individual values in
this study seldom exceeded 10 gg-1 dry weight, the risk to Sudbury-area mink would appear
to be only low to moderate. Although the animals had been simultaneously exposed to nickel,
lead and possibly other toxic elements within the Sudbury environment, there was no
evidence of increased concentrations of these elements within the left kidney tissue. The
necrotic effects of lead on renal tissue are well documented (Friberg et al., 1979) and the
possibility of synergistic toxicological effects during multi-elemental exposure, even in the
absence of tissue accumulations, cannot be ruled out.
214
CONCLUSIONS
Metal burdens in renal-hepatic tissues were higher in mink infected with the giant kidney
worm parasite than in non-infected mink. A trend of increasing metal burdens with greater
numbers of worms present was apparent for nickel and lead but not cadmium. Changes in the
proportional distribution of nickel and lead in host mink tissues were due to accumulations in
new tissue growth induced by parasitic infection. Cadmium consistently accumulated in renal
and hepatic tissue with minimal alteration in the proportional distributions subsequent to D.
renale infection.
The spicule was the tissue of preference for both lead and nickel deposition, whereas
cadmium accumulated more readily in the hypertrophying left kidney and the liver. The
substantial accumulation of metals (namely lead and nickel) in the bony spicules (but not in
the femurs) of infected animals may be an adaptive mechanism whereby the levels found in
systemic circulation and soft tissues are reduced and soft tissue damage thus avoided.
Female worms accumulated approximately 3-fold higher burdens of cadmium, lead and
nickel than did male worms. This gender difference is attributable to the larger mass of
females, as concentration values (with the exception of certain lead determinations) did not
differ between the sexes.
Although toxic metal burdens were enhanced in parasitized mink, concentration values in
the hosts soft tissues remained below levels known to elicit damage and disrupt function.
Nevertheless, a relationship among certain body measures and increased metal loads of
infected mink was noted. Abdominal mid-ventral and thoracic post-cardinal fat deposits, neck
circumference and combined gonad weight were positively related to combined renal and
hepatic metal burdens while spleen weights were negatively related. These trends substantiate
earlier findings reported for non-parasitized mink carrying elevated body metal burdens as a
result of residing within the immediate influence of active ore mining/smelting operations
(Capodagli and Parker, 2007). These associations may indicate a focal point for future
research on the impacts of increased metal loads and parasitic infection on the host system.
ACKNOWLEDGMENTS
We gratefully acknowledge the assistance of all members of the Sudbury Fur Trappers
Council who contributed carcasses from their traplines. Statistical advice was kindly provided
by M.A.Persinger. This work was financially supported through the Laurentian University
Research Fund, the Ontario Graduate Scholarship for Scientists and Technologists Program
and the Sudbury Game and Fish Protective Association.
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ISSN: 2158-5717
Chapter 6
AEROBICALLY BIODEGRADED FISH-MEAL

WASTEWATER AS A FERTILIZER
Joong Kyun Kim1 and Geon Lee2
1
Department of Biotechnology and Bioengineering,

Pukyong National University, Busan, Korea
2
Research Department, Samrim Corporation,
Kimhae, Kyongnam, Korea
ABSTRACT
Reutilization of fish-meal wastewater (FMW) as a fertilizer was attempted, and
aerobic biodegradation of the FMW were successfully achieved by microbial consortium
in a 1-ton bioreactor. During the large-scale biodegradation of FMW, the level of DO was
maintained over 1.25 mgl-1, and a strong unpleasant smell remarkably disappeared in the
end. Although the level of total amino acids and the concentrations of N, P and K in the
biodegraded FMW were relatively lower than those in two commercial fertilizers, the
concentrations of noxious components in the biodegraded FMW were much lower than
the standard concentrations. The phytotoxicity of the biodegraded FMW was almost
equal to that of the commercial fertilizers. The fastest growth in hydroponic cultures of
red bean and barley was achieved at 100-fold and 500-fold dilution, respectively, the
growth of which was comparable to those of 1,000-fold diluted commercial-fertilizers.
From all above results, it was concluded that a large-scale biodegradation of FMW was
successful and the properties of FMW were acceptable. This is the first study to
demonstrate aerobic production of liquid-fertilizer from FMW.
Keywords: fish-meal wastewater, fertilizer, microbial consortium, large-scale biodegradetion, phytotoxicity, hydroponic culture.
220
INTRODUCTION
The amount of fisheries waste generated in Korea is expected to increase with a steady
increase in population to enjoy taste of slices of raw fish. The fisheries waste is reduced and
reutilized through the fish meal production. The process, which uses fish wastes such as
heads, bones or other residues, is the commonest used in the Korean industries. The first step
of the fish-meal manufacturing processes is the compression and crushing of the raw material,
which is then cooked with steam, and the liquid effluent is filtered off in a filter press. The
liquid stream contains oils and a high content of organic suspended solids. After oil
separation, the fish-meal wastewater (FMW) is generated and shipped to wastewater
treatment place. FMW has been customarily disposed of by dumping into the sea, since direct
discharge of FMW can cause serious environmental problems. Besides, bad smell, which is
produced during fish-meal manufacturing processes, causes civil petition. Stricter regulations
for this problem also come into force every year in Korea. Therefore, there is an urge to seek
for an effective treatment to remove the organic load from the FMW; otherwise the fish meal
factories will be forced to shut down.
Biological treatment technologies of fish-processing wastewater have been studied to
improve effluent quality (Battistoni and Fava, 1995; Park et al., 2001). The common feature
of the wastewaters from fish processing is their diluted protein content, which after
concentration by a suitable method would enable the recovery and reuse of this valuable raw
material, either by direct recycling to the process or subsequent use in animal feed, human
food, seasoning, etc. (Afonso and Borquez, 2002). It has been also reported that the organic
wastes contain compounds, which are capable of promoting plant growth (Day and
Katterman, 1992), and seafood processing wastewaters do not contain known toxic or
carcinogenic materials unlike other types of municipal and industrial effluents (Afonso and
Borquez, 2002). Although these studies imply that FMW could be a valuable resource for
agriculture, potential utilization of this fish wastes has been limited because of its bad smell
(Martin, 1999). There is an increasing need to find ecologically acceptable alternatives to
overcome this problem.
Aerobic biodegradation has been widely used in treatment of wastewaters, and recently
references to the use of meso- and thermophilic microorganisms have become increasingly
frequent (Cibis et al., 2006). During the biodegradation, the organic matter is biodegraded
mainly through exothermic aerobic reactions, producing carbon dioxide, water, mineral salts,
and a stable and humified organic material (Ferrer et al., 2001). There have been few reports
that presented the reutilization of biodegraded waste products as liquid-fertilizer: a waste
product of alcoholic fermentation of sugar beet (Agaur and Kadioglu, 1992), diluted manure
streams after biological treatment (Kalyuzhnyi et al., 1999), and biodegraded fish-meal
wastewater in our previous studies (Kim et al, 2007; Kim and Lee, 2008). Therefore, aerobic
biodegradation is considered to be the most suitable alternative to treat FMW and realize a
market for such a waste as a fertilizer.
The growth of plants and their quality are mainly a function of the quantity of fertilizer
and water. So it is very important to improve the utilization of water resources and fertilizer
nutrients. The influence of organic matter on soil biological and physical fertility is well
known. Organic matter affects crop growth and yield either directly by supplying nutrients or
indirectly by modifying soil physical properties such as stability of aggregates and porosity
221
that can improve the root environment and stimulate plant growth (Darwish et al., 1995).
Incorporation of organic matter has been shown to improve soil properties such as
aggregation, water-holding capacity, hydraulic conductivity, bulk density, the degree of
compaction, fertility and resistance to water and wind erosion (Carter and Stewart, 1996;
Franzluebbers, 2002; Zebarth et al., 1999). Combined use of organic and inorganic sources of
nutrients is essential to maintain soil health and to augment the efficiency of nutrients (Lian,
1994).
Three primary nutrients in fertilizers are nitrogen, phosphate, and potassium. According
to Perrenouds report (1990), most authors agree that N generally increases crop susceptibility
to pests and diseases, and P and K tend to improve plant health. It has been reported that
tomato is a heavy feeder of NPK (Hebbar et al., 2004) and total nitrogen content is high in
leaves in plants having a high occurrence of bitter fruits (Kano et al., 2001). Phosphorus is
one of the most essential macronutrients (N, P, K, Ca, Mg, S) required for the growth of
plants, and the deficiency of phosphorus will restrict plant growth in soil (Son et al., 2006).
However, the excessive fertilization with chemically synthesized phosphate fertilizers has
caused severe accumulation of insoluble phosphate compounds in farming soil (Omar, 1998),
which gradually deteriorates the quality as well as the pH of soil. Different fertilization
treatments of a long-term field experiment can cause soil macronutrients and their available
concentrations to change, which in turn affects soil micronutrient (Cu, Fe, Mn, Zn) levels.
Application of appropriate rates of N, P and K fertilizers has been reported to be able to
increase soil Cu, Zn and Mn availabilities and the concentrations of Cu, Zn, Fe and Mn in
wheat (Li, et al., 2007).
It has been also reported that higher rates of fertilizers suppress microbial respiration
(Thirukkumaran and Parkinson, 2000) and dehydrogenase activity (Simek et al., 1999).
Recently, greater emphasis has been placed on the proper handling and application of
agricultural fertilizers in order to increase crop yield, reduce costs and minimize
environmental pollution (Allaire and Parent, 2004; Tomaszewska and Jarosiewicz, 2006).
Hydroponics is a plant culture technique, which enables plant growth in a nutrient
solution with the mechanical support of inert substrata (Nhut et al., 2006). Hydroponic culture
systems provide a convenient means of studying plant uptake of nutrients free of confounding
and uncontrollable changes in soil nutrient supply to the roots. Thus, it is fit for test of
fertilizing ability of liquid fertilizers. The technique was developed from experiments carried
out to determine what substances make plants grow and plant composition (Howard, 1993).
Water culture was one of the earliest methods of hydroponics used both in laboratory
experiments and in commercial crop production. Nowadays, hydroponics is considered as a
promising technique not only for plant physiology experiments but also for commercial
production (Nhut et al., 2004; Resh, 1993). The technique has been also adapted to many
situations, from field and indoor greenhouse culture to highly specialized culture in atomic
submarines to grow fresh vegetables for the crew (Nhut et al., 2004). Hydroponics provides
numerous advantages: no need for soil sterilization, high yields, good quality, precise and
complete control of nutrition and diseases, shorter length of cultivation time, safety for the
environment and special utility in non-arable regions. Application of this culture technique
can be considered as an alternative approach for large-scale production of some desired and
valuable crops.
In this study, a large-scale biodegradation was successfully carried out for three days in a
1-ton reactor using the FMW generated from fish-meal manufacturing processes, and the
222
properties of the biodegraded FMW, such as phytotoxicity, amino-acid composition,

concentrations of major and noxious components, and fertilizing ability on hydroponic culture
plants were determined to examine the suitability of the biodegraded FMW as a fertilizer.

Microorganisms and Media
The mixed microorganisms used in this study comprised seven microorganisms: Bacillus
subtilis, Bacillus licheniformis, Brevibacillus agri, Bacillus coagulans, Bacillus circulans,
Bacillus anthracis and Bacillus fusiformis. They were potential aerobically-degrading
bacteria, which were isolated from commercial good-quality humus and from compost and
leachate collected at three different sites of composting plants (Kim et al., 2007). There was
no potential bacterial antagonist among them. Each pure culture was maintained on 1.5%
Nutrient agar plate at 4until used, and transferred to a fresh agar plate every month. At the
same time, the potential degrading ability of each pure culture was also checked on 1% skim
milk agar, 3.215% spirit blue agar, starch hydrolysis agar (5 gl-1 of beef extract, 20 gl-1 of
soluble starch, 10 gl-1 of tryptose, 5 gl-1 of NaCl, and 15 gl-1 of agar, pH 7.4) and cellulose
agar (10 gl-1 of cellulose powder, 1 gl-1 of yeast extract, 0.1 gl-1 of NaCl, 2.5 gl-1 of (NH4)
-1
-1
-1
2SO4, 0.25 gl of K2HPO4, 0.125 gl of MgSO47H2O, 0.0025 gl of FeSO47H2O, 0.025
gl-1 of MnSO44H2O, and 15 gl-1 of agar, pH 7.2), respectively. All agar plates were
incubated at 450C until change of color or a clear zone around each colony appeared.
Liquid-Fertilization of FMW
A large-scale biodegradation of the original FMW was carried out in a 1-ton reactor for
its liquid-fertilization. The inside of the reactor was sterilized by hot steam for 30 min, and
then 600 l of the FMW obtained from a fish-meal factory was added into the reactor by a
peristaltic pump under the steaming. The FMW was characterized as: 115,00013,000 mgl-1
of chemical oxygen demand- dichromate (CODCr), 15,4001,300 mgl-1 of total nitrogen
(TN), 68,9007,600 mgl-1 of five days biological oxygen demand (BOD5), 2,800600 mgl-1
of NH4+-N, 0 mgl-1 of NO3--N and 0 mgl-1 of NO2--N. The salt concentration and pH of the
FMW were 0.60.1% and 6.50.2, respectively. After the steaming was quit, the reactor was
placed until temperature dropped down to 450C. Then, 30-l liquid broth of seven
microorganisms (on the same ratio of cell mass) grown in exponential phase of growth were
seeded into the reactor. For faster biodegradation, the inoculated cells were previously
acclimated for two days in the original FMW under an aerobic condition. The bioreactor was
operated at 42+40C, and air was supplied from two blowers (6.4 m3min-1 of capacity) into the
reactor through ceramic disk-typed diffusers. The aeration rate was 1,280 lmin-1. Ten-fold
diluted Antifoam 204 was used when severe foams occurred during biodegradation.
Samples from the reactor were collected periodically. The concentrations of dissolved oxygen
(DO), CODCr and TN were measured with oxidation-reduction potential (ORP) and pH.
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Seed Germination Test

According to the method of Wong et al.(2001), seed germination tests for 50-, 100-, 250-,
500-, and 1,000-fold diluted final broths, which were taken from the biodegradation of
original FMW in a 1-ton reactor, were carried out against control in order to evaluate the
phytotoxicity of the biodegraded FMW. Two commercial fertilizers, C-1 (A-company,
Incheon, Korea) and C-2 (N-company, Kimhae, Korea), frequently used in Korea were also
tested for the comparison of their phytotoxicity. Five milliliters of each sample were pipetted
into a sterile petri dish lined with Whatman #1 filter paper. Ten cress (Lepidium sativum)
seeds were evenly placed in each dish. The plates were incubated at 25 C in the dark at 75%
of humidity. Distilled water was used as a control. Seed germination and root length in each
plate were measured at 72 h. The percentages of relative seed germination (RSG), relative
root growth (RRG) and germination index (GI) after expose to the sample were calculated as
the following formula (Zucconi et al., 1981; Hoekstra et al., 2002):
RSG (%) =
Number of seeds germinated in the

biodegraded FMW
Number of seeds germinated in control
RRG (%) =
Mean root length in the

biodegraded FMW
Mean root length in control
GI (%) =
100
100
RS
G
RRG
100
Hydroponic Culture
To test the fertilizing ability of the biodegraded FMW produced in a 1-ton reactor, a
hydroponic culture system was applied to cultivate red bean and barley in a mini-hydroponic
culture pot (5128 cm3) against control. Tests were carried out on the biodegraded FMW at
various dilutions (50, 100, 250, 500 and 1,000-fold). The tests were also carried out on 1,000fold diluted commercial-fertilizers in order to compare with the fertilizing ability of the
biodegraded FMW. The hydroponic culture pot was composed of a glass vessel and a plastic
screen inside. In each pot, ten seeds of red bean or twenty-five seeds of barley were put on top
of the plastic screen, respectively, and approximately 300-ml solution of the biodegraded
FMW at various dilution ratios was filled underneath the plastic screen. After set-up of the
hydroponic culture system, each pot was initially covered with aluminum foil to keep seeds in
dark before seed germination. After seed germination, the pot placed by the window all day
long to provide the necessary sunlight for plants growth. Day and night temperatures of air
were maintained at 2220C and 182 by natural ventilation and heating. Water temperature
was 1530C, and the relative humidity in the room was 60% on the average. The fertilizer
solution was refreshed every four days, and the seeds were soaked all the time. After seed
224
germination, roots grew through the pore of the plastic screen. The growth of plants was
observed periodically, and height, thickness of stem, number of leaf, and length of leaf of
each plant were measured.
Analyses
The concentrations of cations (NH4+) and anions (NO2- and NO3-) were estimated by IC
(Metrohm 792 Basic IC, Switzerland). The columns used in these analyses were Metrosep
C2-150 and Metrosep Supp 5-150 for cation and anion, respectively. The concentrations of
CODCr and TN concentrations were analyzed by the Water-quality Analyzer (Humas, Korea).
The concentration of BOD5 was analyzed by the OxiDirect BOD-System (Lovibond,
Germany). The composition of amino acids, and concentrations of major and noxious
components in biodegraded FMW and commercial liquid-fertilizers were analyzed at Science
Lab Center Co., Ltd (Daejeon, Korea) by our request.

Liquid-Fertilization of FMW
To industrialize the biodegraded FMW as liquid-fertilizer, data obtained in laboratory
equipment should be transferred to industrial production. Problems of scale-up in a bioreactor
are associated with the behavior of liquid in the bioreactor and the metabolic reactions of the
organisms. Transport limitation is considered as one of the major factors responsible for
phenomena observed at large-scale. For this reason, a large-scale FMW biodegradation were
attempted in a 1-ton bioreactor, and the result is shown in Figure 1. As shown in Figure 1, DO
level in the reactor started to decrease after 5 h. This implies that active biodegradation of
FMW took placed after a lag phase by the mixed microorganisms used in this study, since
oxygen consumption has been known to be a general index of microbial metabolism (Tomati
et al., 1996). During two days biodegradation, the DO level was maintained over 2 mgl-1, but
it decreased further thereafter. The final DO level was 1.25 mgl-1. In aerobic processes,
oxygen is a key substrate and a continuous transfer of oxygen from the gas phase to the liquid
phase is decisive for maintaining the oxidative metabolism of the cells because of its low
solubility in aqueous solutions. Generally, it has known that DO level in a bioreactor should
be maintained over 1 mgl-1 for aerobic fermentation (Tohyama et al., 2000). Therefore, it
would seem that the supply of oxygen into the 1-ton reactor met the demand of oxygen by the
microorganisms during the biodegradation.
The pH was 6.99 at the beginning and decreased down to 6.15 after 20 h. Then the pH
was recovered slowly and its final value was 6.86. In our previous study (Kim et al., 2008),
pH had a tendency to increase during the biodegradation because of insufficient supply of
oxygen. These results imply that microorganisms had different metabolism, which was
dependent on the availability of dissolved oxygen. The value of ORP started at 18.2 mV. The
value of ORP decreased rapidly after 45 h and the final value reached to 0.6 mV. The
decrease in the ORP value resulted from the decrease in DO level. During the biodegradation,
the values of ORP maintained positive, and a strong unpleasant smell (mainly a fishy smell)
225
remarkably disappeared in the end. It seemed that a complete aerobic biodegradation might
take place to a certain extent with maintenance of ORP in a positive range, since unpleasant
odor can be easily produced under incomplete aerobic biodegradation (Zhang et al., 2004).
40
8.5
20
7.5
-20
7.0
-40
ORP (mV)
pH
DO (mg.l-1)
8.0
-60
6.5
-80
6.0
120000
18000
-1
CODcr (mg.l-1)
14000
TN (mg.l )
16000
100000
80000
12000
10000
60000
8000
6000
40000
4000
0
10
20
30
40
50
60
Time (h)
Figure 1. Changes of ORP( ), DO( ), pH( ), CODcr( ) and TN( ) during the biodegradation
in a 1-ton reactor.
From all the results, maintenance of DO level during biodegradation was found to be
very important and ORP could be a key parameter to operate biodegradation of FMW in a
large-scale. The ORP was reported to be used as a controlling parameter for regulation of
sulfide oxidation in anaerobic treatment of high-sulfate wastewater (Khanal and Huang,
2003), and on-line monitoring of ORP has been proved to be a practical and useful technique
for process control of wastewater treatment systems (Guo et al., 2007; Yu et al., 1997). As a
result, process optimization is required in a large-scale operation, especially aeration rate in
this case. After 52 h of biodegradation, the concentrations of CODCr and TN in original FMW
reduced to 25,100 and 4,790 mgl-1, respectively. The removal percentages of CODCr and TN
226
were 79.4 and 73.4%, respectively, with slight decrease of CODCr/TN ratio from 6.8 to 5.2. It
has been known that the COD/N ratio may influence biomass activity, and therefore on the
metabolic pathways of organic matter utilization (Ruiz et al., 2006). Based on this
information, the cell activity and metabolic pathways of the mixed microorganisms might be
maintained somewhat with their mutualism during the biodegradation.
Properties of Biodegraded FMW as Fertilizer

Since the FMW contains various compounds potentially useful for diverse plants, an
attractive application is its use as a fertilizer; however, this could have limitations due to some
toxic characteristics of the waste. For this reason, it is necessary to prove the non-toxic
properties and fertilizing ability of the final biodegraded FMW.
Phytotoxicity of Biodegraded FMW

Organic matters hold great promise due to their local availability as a source of multiple
nutrients and ability to improve soil characteristics. Sufficient aeration promotes the
conversion of the organic matters into nonobjectionable, stable end products such as CO2,
SO42-, NO3-, etc. However, an incomplete aeration may result in accumulation of organic acid,
thus giving trouble to plant growth if the fertilizer is incorporated into the soil (Jakobsen,
1995). The phytotoxicity of the biodegraded FMW produced in a 1-ton reactor was examined
at various dilution ratios, and compared with those of two commercial fertilizers (Figure 2).
Figure 2. Percentages of germination index (GI) at various dilution ratios of the biodegraded FMW
produced in a 1-ton reactor. The results are compared to those of 1,000-fold diluted commercial
fertilizers, C-1 and C-2.
227
As shown in Figure 2, the values of GI had a tendency to increase with increase of

dilution ratio of the biodegraded FMW. The GI values of the biodegraded FMW were less
than 20% at 50- and 100-fold dilution with low root elongation. The reduction in values of GI
indicates that some characteristics existed had an adverse effect on root growth. This may be
attributed to the release of high concentrations of ammonia and low molecular weight organic
acids (Fang and Wong, 1999; Wong, 1985), since cress (Lepidium sativum) used in this study
is known to be sensitive to the toxic effect of these compounds (Fuentes et al., 2004). At 250fold dilution, the GI value of the biodegraded FMW was close to 50%. A GI value of 50% has
been used as an indication of phytotoxin-free compost (Zucconi et al., 1985). According to
this GI criterion, the biodegraded FMW required more than 250-fold dilution to reach
stabilization of the organic matter to maintain the long-term fertility in soil. At 1,000-fold
dilution, the GI value was found to be close to 90%. This GI value was compared with those
of two commercial fertilizers at the same dilution, since 1,000-fold diluted liquid fertilizer
was used in horticulture for general purpose. The GI value of the biodegradative FMW was
much better than that of C-1 and comparable to that of C-2. This result implies that the
biodegradation of FMW in a 1-ton reactor was successfully carried out, and thus the
development of a liquid-fertilizer from the FMW was feasible.
Composition of Amino Acids of Biodegraded FMW

Amino acids are an essential part of the active fraction of organic matter in a fertilizer.
The growth of plants depends ultimately upon the availability of a suitable balance of amino
acids, and their composition might also be used as a means of assessing biodegradation. The
success of the scale-up process for the production of liquid fertilizer from the FMW can be
verified by determining the composition of amino acids. From this point of view, it is
necessary to analyze the amino-acid composition of the biodegraded FMW produced in a 1ton bioreactor. The analytical result was tabulated in Table 1 with those of two commercial
fertilizers. The level of total amino acids was 5.60 g100g sample-1, which was a little bit
better than that produced in our previous study (Kim and Lee, 2008). The higher content of
amino acids is probably due to the higher degree of mineralization of FMW, which indicates
release of more nutrients available for plants. However, the level of total amino acids in the
biodegraded FMW was lower, compared with those in two commercial fertilizers. This
implies that mineralization of FMW in a 1-ton reactor was not as great as that in a lab-scale
reactor (Bylund et al., 1998; Xu et al., 1999). Especially, the levels of aspartic acid, alanine
and lysine were low, whereas those of proline and glycine were relatively high. The
difference may be due to the composition of the original FMW, since it was found to be
dependent on the nature of the raw material processed in the factory (Kim et al., 2007).
Moreover, the composition of sulfur-containing amino acids, cysteine and methionine, was
relatively high in the biodegraded FMW. It has been reported that the sulfur-containing amino
acid, methionine is a nutritionally important essential amino acid and is the precursor of
several metabolites that regulate plant growth (Amir et al., 2002).
228
Table 1. Comparison of amino-acids composition of the biodegraded FMW with those of

commercial fertilizersa
Amino acid
Source of liquid fertilizer

Biodegraded FMW
Commercial C-1
Commercial C-2
Aspartic acid
0.49
0.90
0.58
Threonine
Serine
0.18
0.21
0.23
0.21
0.21
0.23
Glutamic acid
0.78
2.96
0.89
Proline
Glycine
Alanine
0.50
1.06
0.60
0.09
0.31
1.00
0.61
1.25
0.98
Valine
0.15
0.39
0.24
Isoleucine
0.14
0.28
0.13
Leucine
Tyrosine
0.24
0.07
0.42
0.17
0.26
0.05
Phenylalanine
0.19
0.22
0.18
Histidine
0.20
0.28
0.25
Lysine
0.29
1.54
0.53
Arginine
0.31
0.23
0.35
Cystine
0.04
n.d.c
0.04
Metionine
0.13
n.d.
0.01
Tryptophan
0.02
n.d.
0.02
Total
5.60
9.23
6.81
-1
composition of amino acids was based on dry weight (g100g sample ).

produced in a 1-ton reactor.
c
n.d. means not detected.
b
Concentrations of Major and Noxious Components in Biodegraded FMW

It is very important to improve the utilization of fertilizer nutrients, since the growth of
plants and their quality are mainly a function of the quantity of fertilizer. Organic matter
affects crop growth and yield directly by supplying nutrients (Darwish et al., 1995).
Combined use of organic and inorganic sources of nutrients is essential to augment the
efficiency of nutrients (Lian, 1994). For this reason, concentrations of three primary nutrients
(N, P, and K) in the biodegraded FMW were anaylized together with noxious components,
and compared with those in commercial fertilizers. The results were tabulated in Table 2. The
concentrations of N, P and K in the biodegraded FMW were 1.49, 0.28 and 0.41%,
respectively, and were much lower than those in commercial fertilizers.
229
Table 2. Comparison of concentrations of major and noxious components present in the

biodegraded FMW with those present in commercial fertilizers
Source of liquid fertilizer
Measurement
N, P, K
(%)
Noxious
Compounds
(mgkg-1)
Biodegraded FMWa
Commercial C-1
Commercial C-2
1.49
4.83
3.80
P2O5
0.28
2.86
2.83
K2O
0.41
2.10
3.04
Pb
n.d.b
0.63
0.31
As
n.d.
0.88
0.36
Cd
n.d.
0.08
0.03
Hg
0.01
n.d.
n.d.
Cr
0.20
3.52
3.44
Cu
n.d.
3.24
2.24
Ni
n.d.
1.54
2.32
Zn
1.61
4.39
3.51
produced in a 1-ton reactor.

n.d. means not detected.
However, the concentrations of noxious components in the biodegraded FMW were

much lower than those in commercial fertilizers. The noxious components, Pb, As, Cd, Cu
and Ni were not detected, and the other components were much less than the standard
concentrations provided by the law.
Hydroponic Culture on Biodegraded FMW

The fertilizing ability of the biodegraded FMW at various dilution ratios was tested in a
hydroponic culture system, which was applied to cultivate red bean and barley. The tests were
also carried out on 1,000-fold diluted commercial-fertilizers simultaneously. Results of
hydroponic culture of red bean on diluted biodegraded-FMW and commercial fertilizers were
tabulated in Table 3. As seen in Table 3, elongation of root was slow after seed germination
in all diluted FMW. However, roots elongated soon after development of root. The fastest
growth was achieved at 100-fold dilution, the growth of which was comparable to that of
1,000-fold diluted commercial-fertilizers. This result was somewhat surprising because the GI
test on the 100-fold diluted FMW showed low root elongation (Figure 2). The toxic effect of
the 100-fold diluted FMW might be not severe on red bean, although it was sensitive on
cress.
230
Table 3. Results of hydroponic culture of red bean on diluted biodegraded-FMW and

commercial fertilizers
Dilution of biodegraded FMW
Control (Water)
Measurement
Time (day)
50-fold
100-fold
250-fold
Time (day)
Time (day)
Time (day)
12 14 2
12 14 2
12 14 2
Height (cm)
1.5 3.0 6.0 -
1.0 2.0 -
2.0 4.0 6.0 9.0 -
1.0 3.0 4.5
Thickness of
stem (cm)
0.2 0.3 0.5 -
0.2 0.2 -
0.2 0.3 0.4 0.5 -
0.2 0.3 0.3
Number of leaf
Length of leaf (cm) -
3.0 -
1.0 2.0 -
1.5 4.0 -
1.0 1.5

Measurement
Height (cm)
Thickness of
stem (cm)
Number of leaf
Length of leaf (cm)
500-fold
Time (day)
12 14 2
1,000-fold, C-1
1,000-fold, C-2
Time (day)
Time (day)
1.0 3.0 4.0 -
1.0 2.0 4.0 5.5 1.0 4.0 4.5 5.0 8.0 -
2.0 3.0 4.0 6.0
0.2 0.3 0.4 -
0.2 0.2 0.3 0.4 0.2 0.3 0.3 0.4 0.5 -
0.2 0.3 0.4 0.5
1 2 1.0 1.5 -
Commercial fertilizer
1,000-fold
Time (day)
12 14
12 14 2
1 2 1.0 2.0 -
12 14 2
2 2 1.5 4.5 -
12 14
2 2
1.0 3.0
Table 4. Results of hydroponic culture of barley on diluted biodegraded-FMW and

commercial fertilizers
Control (Water)
Measurement
50-fold
Time (day)
Time (day)
1.7 5.8 8.3 8.5 -
1.2 2.6 4.4 5.2 -
2.1 4.1 6.5 7.5 -
2.4 4.3 6.7 7.6
Thickness of
stem (cm)
0.1 0.2 0.2 0.2 -
0.1 0.2 0.2 0.2 -
0.1 0.2 0.2 0.2 -
0.1 0.2 0.2 0.2
Number of leaf
12 14 2
0.7 1.3 3.1 3.5 -
Time (day)
Height (cm)
1.2 4.6 6.5 6.8 -
Time (day)
12 14 2
250-fold
Length of leaf (cm) -
100-fold
Height (cm)
Thickness of
stem (cm)
Number of leaf
Length of leaf (cm)
1.6 3.0 5.0 5.5 -

Measurement
12 14 2
12 14
Commercial fertilizer
500-fold
1,000-fold
1,000-fold, C-1
1,000-fold, C-2
Time (day)
Time (day)
Time (day)
Time (day)
12 14 2
1.9 3.0 5.0 5.6
2.9 5.5 7.8 8.1 -
1.5 3.9 5.6 6.3 -
12 14 2
2.8 5.6 7.1 7.7 -
12 14 2
1.8 4.8 8.6 9.0
0.1 0.2 0.2 0.2 -
0.1 0.2 0.2 0.2 -
0.1 0.2 0.2 0.2 -
0.1 0.2 0.2 0.2
1 2 2 2 2.4 4.2 5.7 6.1 -
1 2 2 2 0.9 2.7 3.8 4.2 -
1
2
1 2 2 2
1.3 3.8 6.6 7.0
2 2 2 4.3 5.3 5.7 -
12 14
231
In Figure 3, the change of red beans growth from the seed at 100-fold dilution is shown
against control. Germination from the seed was observed at day 8 with the 100-fold diluted
FMW, whereas the germination was observed at day 12 in control. After 14 days cultivation,
much better growth of red bean was observed with the 100-fold diluted FMW, compared with
that in control. At the same dilution of 1,000-fold, growth of red bean cultivated on the
biodegraded FMW was not as good as that cultivated on commercial fertilizers. A decreasing
order of growth in the culture of red bean was C-1 > C-2 > biodegraded FMW. According to
Perrenouds report (1990), N, P and K are primary nutrients and considered to improve plant
health. The deficiency of phosphorus restricts plant growth in soil (Son et al., 2006), although
the excessive fertilization with chemically synthesized phosphate fertilizers has caused severe
accumulation of insoluble phosphate compounds (Omar, 1998). Therefore, relatively lower
growth of red bean on the biodegraded FMW might be due to the shortage of NPK
components (Table 2).
Figure 3. Results of hydroponic culture of red bean in control (A) and 100-fold diluted FMW (B). Each
figure shows the growth of red bean from the seed along the cultivation time. (1) day 1; (2) day 2; (3)
day 5; (4) day 8; (5) day 12; (6) day 14 and (7) roots on day 14.
232
Growth indexes of barley during hydroponic culture were measured on the biodegraded
FMW at various dilution ratios, and the measurements were compared with those of
commercial fertilizers (Table 4). Even though the growth in root and stem of barley at 50-fold
dilution was lower than that of control, it had a tendency to improve with increase of dilution
ratio of the biodegraded FMW. The best growth was achieved at 500-fold dilution in which
the growth was seen evenly in root and stem of barley (Figure 4).
Figure 4. Results of hydroponic culture of barley in control (A) and 500-fold diluted FMW (B). Each
figure shows the growth of barley from the seed along the cultivation time. (1) day 1; (2) day 2; (3) day
5; (4) day 8; (5) day 12; (6) roots on day 12 and (7) day 14.
From the GI test, the biodegraded FMW at 500-fold dilution was found to be phytotoxinfree (Figure 2). With the 500-fold diluted FMW, germination from the seed was observed at
day 5, and the same result was observed in control. After 14 days cultivation, however, faster
growth of barley was observed with the 500-fold diluted FMW, compared with that in
control. This growth of barley with the 500-fold diluted FMW was comparable to that
cultivated on 1,000-fold diluted commercial-fertilizers. However, the effect of dilution on
barley was not as sensitive as that on red bean.
233
CONCLUSION
Seven thermophilic microorganisms, which had proteolytic, lipolytic and carbohydratedegrading functions, were used in order to reutilize fish-meal wastewater (FMW). With
coexistence of the seven microorganisms, a lab-scale aerobic biodegradation of FMW were
successfully achieved, and its data were transferred to a 1-ton bioreactor. Although the level
of DO was decreased during the large-scale biodegradation of FMW, it was maintained over
1.25 mgl-1. The decrease in the level of DO resulted in decrease in ORP value. However, the
ORP values were maintained positive, and a strong unpleasant smell remarkably disappeared
in the end. After 52 h of biodegradation, the concentrations of CODCr and TN in original
FMW reduced to 25,100 and 4,790 mgl-1, respectively with slight decrease of CODCr/TN
ratio.
Properties of the biodegraded FMW were determined to examine the suitability of the
biodegraded FMW as a fertilizer. The result of the phytotoxicity test showed that the
biodegraded FMW required more than 250-fold dilution to reach stabilization of the organic
matter to maintain the long-term fertility in soil. At 1,000-fold dilution, the GI value was
found to be close to 90%, which was comparable to those of commercial fertilizers. The level
of total amino acids in the biodegraded FMW was 5.60 g100g sample-1, which was lower
than those in two commercial fertilizers. Especially, the levels of aspartic acid, alanine and
lysine were low, whereas those of proline and glycine were relatively high. Moreover, the
composition of sulfur-containing amino acids, cysteine and methionine, were relatively high
in the biodegraded FMW. The concentrations of N, P and K in the biodegraded FMW were
1.49, 0.28 and 0.41%, respectively, which were lower than those in commercial fertilizers.
However, the concentrations of noxious components in the biodegraded FMW were much
lower than the standard concentrations. The fertilizing ability of the biodegraded FMW at
various dilution ratios was tested in a hydroponic culture system. The fastest growth in
culture of red bean was achieved at 100-fold dilution, the growth of which was comparable to
those of 1,000-fold diluted commercial-fertilizers.
In the hydroponic culture of barley, the best growth was achieved at 500-fold dilution in
which its growth was seen evenly in root and stem. This growth of barley with the 500-fold
diluted FMW was comparable to that cultivated on 1,000-fold diluted commercial-fertilizers.
From all above results, it was concluded that a large-scale biodegradation of FMW was
successful and the properties of FMW were acceptable as a fertilizer. Consequently, FMW
could be a potential industrial resource for liquid-fertilizer production.
ACKNOWLEDGMENTS
This research was supported by a grant (B-2004-12) from Marine Bioprocess Research
Center of the Marine Bio21 Center funded by the Ministry of land, Transport and Maritime
Affairs, Republic of Korea.
234
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ISSN: 2158-5717
Chapter 7
EQUITY OF ACCESS TO PUBLIC PARKS IN

BIRMINGHAM, ENGLAND
Andrew P. Jones, Julii Brainard, Ian J. Bateman
and Andrew A. Lovett
Centre for Social and Economic Research on the Global Environment,
The School of Environmental Sciences,
University of East Anglia, Norwich, Norfolk, UK.
ABSTRACT
Provision of public parks has long been advocated as an equalising measure between
different elements of society. This study assesses equity of park provision for different
ethnic and income-status populations in the urban area of Birmingham in central
England. Parks in Birmingham were categorized into a two group typology of green areas
suited for more solitary and passive activities (amenity parks) or open spaces designed
more for informal sports or other physical and group activities (recreational parks). Using
a geographical information system, measures of access to these green spaces were
computed for populations of different ethnicities and levels of material deprivation,
derived from data from the 2001 UK Census and the 2004 Index of Multiple Deprivation.
Distance-weighted access scores were calculated and compared for five population
groups ranked by relative deprivation, and for five ethnic groups; Bangladeshis, blacks,
Indians, Pakistanis and whites. Statistical analysis found that there were strong disparities
in access with respect to deprivation whereby the most income-deprived groups were also
the most deprived with regard to access to public parks. There was little evidence of
unequal access between ethnic groups. The implications of these findings are discussed.
Keywords: Environmental equity, accessibility, deprivation, ethnicity, urban parks.
Author for correspondence: Dr Andrew P Jones, School of Environmental Sciences, University of East Anglia,
Norwich, Norfolk, NR4 7TJ. Email: a.p.jones@uea.ac.uk Tel: 01603 593127, Fax: 01603 591327.
238
Andrew P. Jones, Julii Brainard, Ian J. Bateman et al.
INTRODUCTION
The establishment of urban public parks has its origins in the social ideal of providing
pleasant open space that is free at the point of access, and as such these places have always
been intended to be equally accessible for different social groups. Public urban parks were
originally established, in part, to provide a place where rich and poor could meet on an equal
footing (Young 1996). It is therefore somewhat surprising that relatively few studies
concerned with environmental social justice have examined the provision and accessibility of
open public space for different social groups.
The exact definition of equity of access is difficult to make, and often context specific.
Extended discussion of the general concept of equity is given by Harding and Holdren (1993).
Crompton and Wicks (1988), Marsh and Schilling (1994) and Wicks and Backman (1994)
discuss different approaches to defining or ensuring equity in a planning context. Crompton
and Lue (1992) and Nicholls (2001) consider how equity may be defined with relation to
urban park usage, and which definitions are likely to be practicable and preferred by the
public. We define equity simply as equal opportunity between social groups.
Previous research on equity of access to public parks is limited (see Wolch et al. 2005;
Estabrooks et al. 2003; Nichols 2001; Talen 1997), and we are not aware of any studies for
either a large city or for places outside the USA. Some research from the USA (Payne et al.
2002; Tinsley et al. 2002) reported that ethnic minorities tend to travel further to surveyed
parks, but did not question why this might be. In the UK, DTLR (2001) note that ethnic
minorities tend to visit urban parks less often than white people. The question arises as to
whether this is partly because non-white communities find it more difficult to travel to urban
parks. It is also important to consider whether access to parks is reduced for economically or
socially deprived populations.
The Greater London Authority acknowledge that socially deprived areas in London often
have relatively poor access to green spaces (GLA, 2001), and areas with less green space
seemed to have more benefit claimants and overcrowded households (GLA, 2003).
The relative dearth of work in the UK on the issue of access to urban parks is perhaps
surprising when it is considered how much they are a very visible and publicly-owned
environmental good. The work reported here focuses on differential access between ethnic
minorities and socio-economic groups with respect to urban parks. Our study area is the city
of Birmingham, where we had detailed information on parks, demographic statistics and the
transport network. Birmingham is located in central England (see Figure 1) and is the second
largest city in the UK with just under 1 million residents in the 2001 Census. Approximately
28% of the city population are non-white. In this research we examine equity of access in
terms of neighbourhood ethnicity and socio-economic deprivation.
239

Data Sources
Our analysis focused on urban rather than regional parks provision, and hence the
boundaries of the city of Birmingham in central England defined the study area. There were
977,087 residents in the city at the time of the 2001 Census.
Figure 1. Amenity and recreational parks in the study area, with visitor survey parks marked
(SF=Summerfield, PHPF=Perry Hall Playing Fields, KHRG=Kinghurst Recreation Grounds).
The project benefited from data derived from a wide variety of sources. These included
the 2001 UK Census, and an existing database of Ordnance Survey (OS) digital data
encompassing the city of Birmingham and the areas immediately beyond the city boundaries.
240
The Ordnance Survey dataset included property boundary features (OS Landline) and a
detailed road network (OSCAR).
This was augmented with information on the location and type of parks and green spaces
that we obtained from a number of sources. Social surveys were also undertaken to collect
information on park usage. This information was used to model the relationship between
frequency of visits and distance to park entrances.
Our analysis made use of the ArcGIS Geographical Information System (GIS) package.
The application of these various data sources in the research is discussed below.
Birmingham Road Network

We presumed that people would travel to parks via the city road network, either on foot,
by bicycle, or in a vehicle. The Ordnance Survey OSCAR digital map database was used to
extract road centrelines for the entire city and immediately adjacent areas. The data were
produced in early 2000, and hence coincide well with the 2001 Census.
Parks in Birmingham
What qualifies as a park is a subjective judgement. Examples of different typologies of
green and public open spaces are found in Kit Campbell Associates (2001) and DTLR (2002a
and 2002b). These typologies tend to include such open spaces as allotments, canal banks or
public squares. In our analysis we have consider only spaces that would be used for a
particular set of purposes, namely casual recreation. Parks and other green spaces within
Birmingham and up to 500m outside of the city boundaries were located with the use of a city
atlas (Geographers A-Z Map Co., 2000). Precise boundaries for each park area were located
and extracted from the Ordnance Survey digital Landline database. Following the labelling in
the A-Z atlas, four categories of park or green space were distinguished:
Amenity parks refers to leisure gardens, country parks, wildlife centres, woods, fish
ponds, public greens and commons, areas labelled as simply as park, and green
spaces not allocated for other specific undeveloped purposes (such as canal banks,
paved public squares or allotments).
Recreational parks are those designated as recreation grounds, playgrounds,
sports grounds and playing fields not attached to any specific school or
institution.
Specialist sports grounds denotes areas labelled as used for a specific sport, such as
tennis courts, hockey pitches, cricket grounds and golf courses.
Other facilities included cemeteries, school grounds and college or university
grounds.
We consulted both the Birmingham Open Spaces Forum and Birmingham City Council
in devising this typology and both organisations agreed with the manner by which we had
classified parks. Table 1 provides summary statistics for each type of park. Amenity and
recreational parks provide the majority of park provision in the city. Specialist sports grounds
241
and other parks were found to not be generally of open access to nearby communities. We
therefore focus the rest of our analysis on the former two types of green space. Figure 1
shows amenity and recreational parks in the study area.
The large scale of the OS data made it necessary to locate park entrances precisely, and
hence the locations of access points were identified for each park included in our analysis,
and these were digitised into the GIS. The A-Z map and the Landline database were jointly
consulted for this purpose. These sources indicate where parks border roads and where breaks
in surrounding buildings exist to apparently allow pedestrian access to the parks, but they do
not depict the presence of fencing. Moreover, we had no easy way of detecting informal
access routes and points (i.e., cut-throughs over rough ground, or holes in fencing).
Table 1. Main types of park included in each typological category
General Category
Amenity
Including:
Public open space/commons/greens
Ponds and reservoirs
Nature reserves/woods
Country parks
Park farms
Leisure gardens
Public playgrounds
School rough
Other parks not labelled as sporting areas
Number
59
50
31
5
3
2
2
1
89
2821 ha
Recreation grounds
Playing fields
Sports grounds
Paddling pool
105
98
36
1
1308 ha
Golf courses
Bowling greens/pavilions
Cricket grounds
Stadia
Tennis courts
Football grounds
Rugby grounds
Hockey pitch
Leisure centre
24
20
13
8
7
4
3
1
1
1175 ha
Total area
General Recreation
Total area
Specialist Recreation
Total area
Census area boundaries and 2001 population.
The Office of National Statistics (ONS) supplied data on population totals, populationadjusted geographical centroids (centre points), and geographical boundaries of Output Areas
(OAs). These are the smallest geographical units in the 2001 Census. There were 3127 OAs
within Birmingham city boundaries, containing an average of 312 persons and 125
households each. Output Area centroids and population totals for them were input to the
SurfaceBuilder computer programme (Martin 1996). SurfaceBuilder generates a surface
depicting the estimated number of persons resident in regularly spaced square areas, or cells.
242
A cell resolution of 20x20m was used. Because the resulting surface is partially a function of
original centroid locations, the resultant cell values are an imperfect measure of actual
population locations. Furthermore, the fine scale does not necessarily provide an accurate
measure of the number of residents in each individual cell. However, the production of the
surface enabled the easy estimation of accessibility measures that were weighted by relative
population sizes, and it was employed here in the estimation of population accessibility to
parks.
Deprivation and Ethnicity

Information on material deprivation and ethnicity was collected at the scale of Lower
Layer Super Output Areas (LSOAs). These contain small groups (typically 4-6 in number) of
contiguous OAs with similar social profiles. There are 641 LSOAs in Birmingham. The
LSOA level is appealing because of the availability of the Index of Multiple Deprivation
(IMD2004; ODPM 2004) for this geographical scale. The IMD2004 comprises of seven
domains describing different types of deprivation including income, access to housing and
services, education, employment, health and disability, skills and training, living
environment, and crime. For this research only the income domain of the IMD2004
(subsequently abbreviated to Inc-IMD2004) was used. The income domain relates to data
collected in 2001 and 2002 on the proportion of the population receiving various types of
means-tested income support, including benefits and tax credits. Hence areas scoring more
highly on Inc-IMD2004 are more materially deprived.
From 2001 Census data, statistics were derived on the percentage population composition
of various ethnic groups. The ethnic categorisations that we used (white, Bangladeshi,
Pakistani, Indian, and black,) were identical to those reported in the Census, and are based on
self-report. For the latter category we combined persons of Caribbean and African heritage
who identified themselves as black in the 2001 Census with persons of mixed white and
black heritage.
Quantification of the Effect of Distance on Accessibility

A number of factors will impact upon the decision to visit a park. Purpose of visit is
likely to shape decisions regarding which park a person might visit, and both seasonality and
time of day can also influence visit patterns (DTLR, 2002a; Scott, 1997). However, the
principal consideration for many is likely to be travel distance. Hence, in order to adequately
measure accessibility, information on the relationship between distance and park usage was
required.
We were unable to find previous research that provided information on distances that
users in Britain travel to visit urban parks. We therefore conducted our own survey. Visitors
to three parks in Birmingham were interviewed during the summer of 2001. Survey parks
were chosen by considering deprivation levels in the area around each. Census wards were
categorised according to their levels of deprivation into the 33% most deprived, 33% least
deprived, and the remaining 33%. One medium-size park was selected for survey in an area
243
dominated by each deprivation tercile: Summerfield Park (most deprived), Perry Hall Playing
Fields (least deprived) and Kingshurst Recreation Grounds (middle).
In total, 117 individuals were interviewed (a minimum of 35 at each park) and asked for
their mode of travel, the full postcode of their outset point, estimated one-way travel time to
reach the park, the number of persons in their visitor party, and reasons for being in the park
that day. Five respondents had to be excluded from subsequent analysis due to them
providing incomplete information (such as an invalid postcode). The outset origin postcode
supplied by each respondent was related to a 100m resolution reference (on the national grid
system) using the Central Postcode Directory held at Manchester University. The road
network distance between this origin and the nearest entrance to the park was then calculated
within the GIS.
Table 2 summarises the characteristics of each park and its visitors. Included are socioeconomic statistics for the origin areas from which visitors came. A trend is apparent where
Summerfield visitors tend to come from the most deprived areas, and Perry Hall park users
tend to originate from the least deprived areas. Otherwise, the most striking features are the
much higher proportion of visitors from predominantly white neighbourhoods to Kinghurst
Park, and an inverse association between the proportion of visitors travelling by car and the
deprivation levels surrounding each park.
Table 2. Summary statistics for the visitor survey locations
Deprivation tercile
Origin Inc-IMD2004 score, 10th90th %ile
(median)
Park size, ha
Median (mean) party size
Mode of travel:
Number of motorists (%)
Number of cyclists (%)
Number of pedestrians (%)
Travel distance:
Median (mean) road distance in metres,
all visitors
Median (mean) road distance in metres,
pedestrians only
Maximum stated travel time amongst nonmotorists (mins)
Maximum modelled travel distance amongst
non motorists (km)
Ethnic composition of neighbourhood
% White, 10th90th %ile (median)
% Bangladeshi, 10th90th %ile (median)
% Indian, 10th90th %ile (median)
% Pakistani, 10th90th %ile (median)
% Black, 10th90th %ile (median)
Summerfield
Kinghurst
Perry Hall
Most deprived
0.23-0.45 (0.39)
Middle
0.14-0.40 (0.27)
Least deprived
0.08-0.47 (0.13)
15.7
2 (3.37)
116
1 (2.26)
62.9
2 (2.36)
7 (20%)
0 (0%)
28 (80%)
11 (34%)
2 (5%)
25 (66%)
15 (38%)
3 (8%)
21 (54%)
311 (705)
420 (1232)
1420 (2194)
225 (395)
288 (814)
717 (923)
22
150
53
1.1
9.6
3.1
17.9-51.7 (42.0)
0-2.3 (1.1)
9.9-32.2 (19.2)
2.7-35.0 (13.9)
10.8-28.5 (18.6)
83.2-97.4 (94.1)
0-0 (0)
0-2.2 (0)
0-3.12 (0.5)
1.1-9.4 (4.5)
11.5-95.7 (36.7)
0-10.2 (1.01)
0-39.5 (16.7)
0-42.3 (6.5)
3.2-30.8 (12.1)
244
The survey data were used to model the relationship between travel distances and
frequency of visits. A model of the decay relationship between proximity and visit frequency
was developed for all parks. This was used to generate data indicating the level of park
accessibility from any single origin point in Birmingham. There are many possible ways to
derive an accessibility index for an environmental amenity and facility such as public parks.
Haynes et al. (2003) argued the case for a function to measure relative accessibility taking the
form:
P = C * exp (Decay_coefficient * Impedance)
{1}
where
P=Potential or accessibility index
C=Constant
Decay_coefficient = the rate at which accessibility declines per unit of impedance
Impedance = difficulty, usually measured in terms of travel time or distance
A common way to calculate P would be to predict the number of visitor parties from
origin zones as a per capita visitor rate. Unfortunately we did not know what percentage of
the total number of visitors we surveyed at each park. In the absence of this information we
used the number of visits reported by respondents during the preceding four weeks. This has
the advantage of not requiring information about the total number of visitors to the park in
any given period, yet visit frequency can be expected to decline with decreasing distance, and
to reflect, in large part, ease of access.
A distance decay relationship between journey origins and parks in Birmingham was
derived by comparing reported frequency with calculated travel distance in the GIS. The
relationship was generalised by grouping visitors into seven distance bands: 0-250m, 251500m, 501-750m, 751-1000m, 1001-1500m, 1500-2500m and 2500-3500m. Survey
respondents who were only in a park because they were en-route to another location were
excluded from this and further analysis. The average number of visits amongst respondents in
each distance band is plotted against the average calculated travel distance in Figure 2. A
strong and consistent decline is apparent in average visit frequency with increasing average
distance.
Using the functional form depicted in Equation 1, the average number of visits was
regressed against travel distance for Birmingham parks visitors, to produce;
P = 32.0085 * exp(-0.0006398 * distance)
{2}
The high R2 value obtained of 0.922 indicates a strong link between grouped visit
frequency and distance between home origin and the nearest park entrance, and this
relationship was used to model the way by which accessibility will decline with distance.
Based on the results of the above modelling, three accessibility 'potential' surfaces were
generated for recreational, amenity, and combined park areas. This process required the
identification of both designated outset and destination points. Potential population journey
origins were generated at 1420 road junctions throughout Birmingham. Destination points
were taken to be park entrances. Visits are known to increase with park size, but the
relationship between visit frequency and park size may not be linear. In a study of 516 urban
parks in Perth, Australia, Giles-Corti et al. (2005) found that visit frequency was best
245
predicted when they weighted their distance decay function by the natural logarithm of park
size (in ha) raised to the power of 0.85, or
Attractiveness=Distance_Decay_Function * log(size0.85)
{3}
Attractiveness as Giles-Corti et al. defined it equates to visit frequency in this work.

We therefore allocated to each entrance a supply weighting equal to the area (in ha) of that
park, raised to a power of 0.85 and then transformed by natural logarithm. For instance,
Phoenix Park to the south of the city centre measures 1.58 hectares and is classified as
entirely amenity park area. Phoenix has two entrances, each of which was given a supply
weighting of 0.388811 (=log(1.580.85)). Very small parks less than 1 ha were given a supply
weighting of 0.01 to prevent negative values being calculated. Only the nearest entrance for
each origin was considered as an access point, and all of the supply weighting was applied to
that.
No. visits in last 4 weeks
40
Average Number Of Visits in preceding four weeks
35
30
25
20
15
10
5
0
0
500
1000
1500
2000
2500
3000
3500
Distance (m)
Figure 2. Average number of visits in preceding four weeks plotted against average distance bands.
Within the GIS the distance in metres between each pair of origin and destination points
was calculated. Distances below 110 m were reset to a value of 110, to prevent spuriously
high potential being predicted very close to parks. 110m was chosen because of a natural
break in the original survey data at that point. Next, an interaction score between each origin
and destination was generated using the formula;
SCORE = [32.0085 exp(-0.0006398 * DISTANCE)] * log(HECTARES0.85)
{4}
Subsequently, scores were summed by origin, to yield a single value indicative of park
accessibility for that origin.
A triangulated irregular network (TIN) (Peuker et al. 1976) was generated to estimate
access scores for locations between origin points. The TIN data were sampled on a regular 20
x 20m grid to create potential surfaces. To facilitate interpretation, the potential values were
converted to Z-scores with a mean of zero and a standard deviation of one. The greater the
246
index value, the better the access to parks for persons travelling from that location. The
resulting values are mapped in Figure 3 for amenity and recreational parks, and Figure 4 for
both park types combined. Figure 3 shows that the northern-central area and the southwest of
the city are well-endowed with amenity type parks. There is a ring of high provision of
recreational type parks around the city centre, whilst the city centre itself and the furthest
suburbs, especially in the north, are relatively poorly provided with this type of park.
Figure 3. Calculated access scores for amenity (left) and recreational (right) parks in Birmingham.
Figure 4. Calculated access scores for amenity and recreational parks combined.
247
Combing the two types of park (Figure 4) the north eastern suburbs and the city centre
itself are most disadvantaged.
The next step was to overlay these surfaces with LSOA boundaries and the population
surface. This enabled us to calculate a population-weighted park access score for each
individual LSOA.
RESULTS
Equality of access to parks for different communities in Birmingham was examined with
respect to both income deprivation and ethnic composition. In order to determine how the
estimates of access were distributed across different populations, tests that compared the
distributions of access scores were undertaken, as discussed below.
Ethnicity and Equity of Access to Public Parks

The population of Birmingham was 70.35% white, 20.16% Asian/mixed Asian and
7.87% black/mixed black in the 2001 Census. To compare park access between these
populations, the percentage of the total Birmingham population in each ethnic group was
determined for each LSOA. For example, the LSOA labelled E01009276 had 166 persons
recorded as black. This equates to 0.216% of the total 76,930 black persons residing inside
the Birmingham city boundaries in 2001. Calculating this percentage of the total for each
LSOA allowed us to determine what proportion of each ethnic group had particular parks
access scores.
Table 3 shows the median access scores for each ethnic group. There are no striking
differences between ethnic groups for amenity parks access, although Pakistani populations
have the best access.
Median scores differ little. However, with respect to recreational or combined parks
access, whites seem to have more advantage over other groups, especially Bangladeshis.
The detailed distributions of park access scores for different ethnic groups were examined
by plotting cumulative frequency distributions against access scores. The resulting plot for
access to amenity parks is shown in Figure 5. The plots for access to recreational parks and
both types combined were similar and are not reproduced here. Park access scores for LSOAs
were divided into fifty categories. Each contains two percent of Birminghams total
population. The horizontal axis shows the upper limit of amenity or recreational access scores
in each category.
Table 3. Median parks access indices for ethnic groups in Birmingham.
Amenity
Recreational
Combined
Indian
Pakistani
Black
Bangladeshi
White
-0.052
0.069
0.145
0.107
0.109
0.193
-0.071
0.131
0.165
-0.101
-0.027
0.045
0.024
0.218
0.247
248
Cumulative frequency (%) .
100
90
80
70
60
50
40
30
White
Indian
Pakistani
Bangladeshi
20
Black
10
Amenity park access index

0.
46
0.
63
0.
80
1.
02
1.
24
1.
81
0.
32
0.
15
0.
00
.2
0
.0
8
-0
-0
.3
3
-0
.4
6
-0
.6
5
-0
.1
1
.8
8
-0
-1
-1
.4
6
Figure 5. Cumulative probability distributions for specified ethnic groups and access to amenity parks.
The vertical axis plots cumulative percentages. A perfectly straight diagonal line on the
plots would indicate that the given range of potential scores occurred equally often across that
ethnic group. This is because each category contains an equal proportion of the city
population and the horizontal axis is non-linear. Lines that deviate from a perfect diagonal
suggest a population displacement towards higher or lower access scores. Percentile lines
bulging to the left of a perfect diagonal indicate a population group with relatively lower than
average access, whilst lines pushed to the right suggest superior access. The greater the
vertical gaps between the cumulative percentile lines, the more likely it is that an inequality is
occurring between groups.
A robust way to test for differences in the data in cumulative distribution plots is to use
the Kolmogorov-Smirnov (KS) test (Connover, 1999). This test assesses the significance of
the relative distance between the cumulative frequency lines. A two-sample KS test was used
to examine the differences in population distribution between the various subgroups. Table 4
provides the resultant KS values, with the critical values at p=0.1, 0.05 and 0.01 levels.
Table 4. Kolmogorov-Smirnov statistics for two-sample tests comparing cumulative
probability distributions for ethnic groups and parks access
Amenity
White
Indian
Pakistani
Black
Indian
0.0776
Pakistani
0.0776
0.0996
Black
0.0704
0.0448
0.1230
Bangladeshi
0.1296
0.0775
0.1572
0.0884
249
Table 4 Continued
Recreational
Indian
0.0701
White
Indian
Pakistani
Black
Combined Amenity and Recreational

Indian
White
0.0610
Indian
Pakistani
Black
Pakistani
0.0823
0.0353
Black
0.0870
0.0535
0.0508
Bangladeshi
0.1375
0.0949
0.1153
0.0986
Pakistani
0.0514
0.0527
Black
0.0739
0.0694
0.0334
Bangladeshi
0.1381
0.0901
0.1010
0.1113
Critical KS values for p=0.1, 0.05 and 0.01 are 0.1725, 0.1923 and 0.2305 respectively.
The KS statistics confirmed the visual impression from the figures that there is no
significant inequity between ethnic groups and their relative access to different categories of
park type. The greatest differences occur between white and Bangladeshi populations, but
these do not reach statistical significance.
Deprivation and Equity of Access to Public Parks

Table 5 gives the median park access score for five deprivation groups (Inc-IMD2004
quartile groups and the most deprived decile) and by type of park. Figures 6 to 8 plot the
cumulative percentiles of population in each Inc-IMD2004 quartile (plus the top decile)
against amenity/recreational/combined parks access scores. Table 6 presents KS statistics for
the data in the figures.
The table and figures show that, for Amenity parks, there is some indication of disparities
in access between the different deprivation groups, with the poorest population cohorts
having worse access compared to other deprivation groups (p<0.01). For recreational parks,
and both park types combined these disparities are much stronger with the most deprived
population groups having significantly poorer access than the others. The middle two
deprivation quartiles tended to have better accessibility than the other groups, with the most
affluent quartile having comparatively average park access.
Table 5. Median access scores for each income deprivation group, by park type
Amenity
Recreational
Combined
Quartile 1
(least
deprived)
-0.030
0.027
0.248
Quartile 2
Quartile 3
0.268
0.422
0.423
0
0.3
0.270
Quartile 4
(most
deprived)
-0.079
-0.220
-0.043
Decile 10
(10% most
deprived)
-0.148
-0.505
-0.268
250
Table 6. Kolmogorov-Smirnov statistics for two-sample tests comparing cumulative

probability distributions for Inc-IMD2004 and park access
Amenity
Quartile 1 (least deprived)

Quartile 2
Quartile 3
Quartile 4 (most deprived)
Quartile 2
Quartile 3
Quartile 4
0.1509
0.1214
0.1878*
0.1778*
0.2349***
0.1086
Quartile 2
Quartile 3
Quartile 4
0.2573***
0.2261**
0.0968
0.2111**
0.4073***
0.3239***
Quartile 2
Quartile 3
Quartile 4
0.2416***
0.1960**
0.1470
0.2347***
0.3478***
0.2438***
Decile 10
(10% most deprived)
0.2037**
0.2589***
0.1545
0.0568
Recreational

Quartile 2
Quartile 3
Decile 10
(10% most deprived)
0.4101***
0.6574***
0.5961***
0.2853***
Combined Amenity and Recreational

Quartile 2
Quartile 3
Decile 10
(10% most deprived)
0.4302***
0.5806***
0.5048***
0.2610***
Amenity park access index
Quartile1
Quartile2
Quartile3
Quartile4
Decile10
-1
.4
6
-1
.0
4
-0
.7
1
-0
.4
6
-0
.2
8
-0
.1
2
0.
00
0.
20
0.
43
0.
63
0.
86
1.
17
1.
81
100
90
80
70
60
50
40
30
20
10
0
Cumulative frequency (%)
Critical KS values for p=0.1, 0.05 and 0.01 are respectively 0.1725 (*), 0.1923 (**) and 0.2305 (***).
Figure 6. Cumulative probability distributions for specified deprivation groups and access to amenity
parks.
251
Quartile1
Quartile2
Quartile3
Quartile4
Decile10
0.
00
0.
18
0.
32
0.
50
0.
66
0.
90
1.
28
2.
06
.1
7
-0
.3
5
-0
.5
3
-0
.8
2
Recreational park access index
-0
-1
.5
6
100
90
80
70
60
50
40
30
20
10
0
Quartile1
Quartile2
Quartile3
Quartile4
Decile10
0.
05
0.
23
0.
34
0.
48
0.
61
0.
82
1.
10
1.
76
.0
5
-0
.2
3
-0
.4
5
-0
.6
8
Combined park access index
-0
-1
.5
6
100
90
80
70
60
50
40
30
20
10
0
Figure 7. Cumulative probability distributions for specified deprivation groups and access to
recreational parks.
Figure 8. Cumulative probability distributions for specified deprivation groups and amenity and
recreational parks combined.
DISCUSSION
Relatively little previous research has addressed the question of the differential access to
green spaces amongst diverse communities residing in urban areas. Using a case study of the
252
city of Birmingham England, this study has sought to go some way to rectify this deficiency
by assessing the distribution of access to urban parks amongst a diverse urban population.
Our analysis found only weak evidence of disparities in access between different ethnic
groups in the population. However, we found very statistically significant indications of
unequal access to public parks with regard to income deprivation, whereby the most deprived
population had the poorest access to parks. Of course this cross sectional observation does
not, in itself, provide an insight into the reasons why these disparities may exist.
Pearce (2003) contended that environmental equity studies are often flawed because they
ignore a fundamental aspect of capitalism, that the rich have the ability to pay for a nicer
place to live. He took an economic perspective, arguing that equity could only be properly
assessed with respect to how much each community paid for a benefit (or to avoid a
disamenity), and whether their benefits or costs were commensurate with those payments.
Pearces arguments are supported by many North American studies (see Been and Gupta
1997; Lambert and Boerner 1997; Mitchell et al. 1999; Szasz and Meuser 2000; Yandle and
Burton 1996), which concluded that apparently unfair pollution burdens usually result from
economic not racist factors, particularly from a move-in of minority and poor social groups,
due to lower house prices for instance, after an environmental nuisance was created. Indeed, it
is quite possible that the residents of poorer communities in our study may receive other
benefits which compensate for their poorer access to urban parks. It is also noteworthy that
the most affluent populations did not have the best access, again suggesting that
compensatory mechanisms, possibly associated with the availability of private land, were
present. Nevertheless, it is important to note that the concept of equal access for all was one
of the principles upon which the public parks movement was founded.
It could be argued that, due to economic forces, improvements in park provision may
only achieve temporary gains for otherwise disadvantaged populations. As the environment in
an area improves, wealthier social groups may be attracted to it, making it difficult for the
original disadvantaged social group to remain in the now improved area. These exclusionary
processes may operate, for example, via rising housing costs or more general issues of social
prejudice. This perspective ignores, however, the value of short and medium term gains in
environmental quality in the lives of the original area inhabitants. Furthermore, house price
rises due to improved environmental conditions may enable existing home-owners in the area
to pass on more wealth to their children which is a permanent improvement for the next
generation.
As with any study, our results will in part be influenced by aspects of our study location
and chosen methodology. Our findings are based on a detailed analysis undertaken in a
socially and environmentally diverse, yet singular, urban area. We chose to study
Birmingham due to the interest generated by this diversity, coupled with availability of very
high quality data in the city. From our analysis it is not possible to determine whether similar
associations would be apparent elsewhere. However, our research has demonstrated the
application of a methodological framework that could be readily applied in other contexts.
We also assume that the distribution of households, with respect to deprivation and ethnicity,
within each LSOA is uniform. In reality, this will not be the case, although we chose to study
LSOAs because of their small size and hence relatively homogeneous nature. In our
development of a park typology, we attempted to select locations that would be available and
attractive for recreational use amongst the general population. However, any such typological
classification will be, at least in part, subjective. It is possible for example that in some areas,
253
patches or waste land or other types of green space may compensate for poor availability of
parks or enhance current levels of provision by providing some of the same functions.
In our study we have measured access in terms of road distances, and our only measure
of quality has been the size of the parks. In reality, of course, quality is multifaceted and
will be associated with a range of factors surrounding facility provision and standards of
maintenance. Research suggests that there is a national trend for deprived areas to contain
parks with relatively poorer maintenance, and since the 1960s, support for provision of open
spaces in urban areas has declined (ILAM Services and Harding 2000, Urban Parks Forum,
2001). The focus of investment and facility development has shifted instead to suburban and
rural country parks (Reeves, 2000). This tendency to suffer from under-investment has led to
a loss of perceived amenity value, most noticeably in inner city locations. Given that the
poorest populations and members of ethnic minorities in Birmingham (and most big cities)
tend to reside in inner city areas, this means that levels of disadvantage may be greater than
those apparent from the comparisons we have made if more subtle aspects of quality of
provision are considered.
The findings of this study reveal reduced access to a public environmental amenity
among certain social groups, in particular those living in the most deprived communities. The
lack of open-air sports facilities may well have consequences for general health levels in each
population, given that public open spaces are increasingly recognised as important places for
physical exercise (see Bedimo-Rung et al. 2005; Foster et al. 2005; Giles-Corti et al. 2005;
Hillsdon et al. 2006; Jones et al. 2007). Therefore, although our findings relate directly to
social inequities, they may have public health implications as well.
ACKNOWLEDGMENTS
This research was supported by the Programme in Environmental Decision Making at
CSERGE which is funded by the Economic and Social Research Council. We are grateful to
to Professor David Martin (University of Southampton) who assisted with the SurfaceBuilder
computer program and to Emma Woolf (BOSF) and Valerie Edwards (BCC) for providing
feedback on our typology scheme.
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ISSN: 2158-5717
Chapter 8
AN IDEA FOR PHENOMENOLOGICAL THEORY OF

LIVING SYSTEMS
Svetla E. Teodorova
Institute for Nuclear Research and Nuclear Energy,
Bulgarian Academy of Sciences, 72 Tzarigradsko chaussee,
1784 Sofia, Bulgaria
ABSTRACT
An idea for developing of new science field, biodynamics, as biophysical
macroscopic theory is propounded. The functioning of living organism as an organized
entirety is the main specificity of the life. Hence it is quite reasonable to describe
behaviour of biological systems in terms of own theoretical basis. In this article a new
state variable vitality as integral characteristic of biological object and measure unit bion
are stated. A quantity biological energy is introduced as energy form related to biological
selfregulation. Quantity synergy is suggested as measure of selfregulation quality.
Biological principle for maximum synergy in healthy living systems is stated. On the
basis of variational principle an equation describing recovery process of a biological
object after disturbance is obtained. The quantity optimal vitality, related to homeostasis,
decreases in lifespan scale and its evolution is described by ordinary differential equation.
The potential lifespan maximum of several species at different life conditions may be
calculated at different parameters of the equation. A wide range of environmental
influences on the living organism could be promptly and easily assessed in the terms
of the biodynamics approach. Such an approach could be used for a simple estimation of
a patients health status.
INTRODUCTION
XX century brought colossal discoveries in the field of molecular biology. In the same
time unified theory of living mater on phenomenological level is not created. Such a theory
could help study the behaviour of the native biological object (BO) in its entirety. This may
be a new step in exploration of living systems in the context of environmental condition
258
Svetla E. Teodorova
changes, diseases etc. The great complexity and different organization levels of BOs
embarrass a direct application of physical theories to biological systems. Notwithstanding,
serious attempts have been made to describe quantitatively different aspects of the life
phenomena on the basis of physical theories thermodynamics, electromagnetism,
hydrodynamics, quantum mechanics etc. Shrdinger (1944) suggested that the essence of the
life is that living organisms consume negative entropy (negentropy). Goodwin (1963) tried to
create a biological statistical mechanics and thermodynamics on the basis of kinetics of
synchronized biochemical oscillators. Rosen (1967) formulated optimal principles in biology.
Szent-Gyrgyi (1968) elucidated some mechanisms of cell regulation in the terms of
bioelectronics. Prigogine and Nicolis (1978) developed theory of dissipative structures, as an
extended irreversible thermodynamics, to explain self-organization processes in
nonequilibrium systems. Davidov (1979) gave a quantum-mechanical interpretation of some
biological phenomena. Thermodynamic approach was applied to the structure and functions
of macromolecules (Di Cera 2001). Kleidon and Lorenz (2005) presented a coherent study of
geosphere-biosphere couplings in the context of maximum entropy production principle.
Kurzynski (2005) proposed a thermodynamic approach combined with describing of
molecular machines. Many authors emphasized the great importance of information in the
functioning of BO. Quastler (1964) represented the problems of the emergence of biological
organization on the basis of biological aspect of information. Trincher (1965) tried to extend
thermodynamics via introducing of a concept of structural information in BO. Eigen (1971)
explained the evolution of macromolecular structures taking into account laws of information
exchange. Volkenstein (1994) considered life evolution in its informational aspects.
The great significance of all these considerations, however, does not abolish the need of a
general phenomenological approach to BOs. Phenomena as thermal conductivity, diffusion,
electric current etc. are not describable in the terms of mechanics and thus new fields as
thermodynamics and electrodynamics with their concepts and laws were created. On
thermodynamic level it is not possible to underline the essence of life. All attempts to
construct an extended thermodynamics of irreversible processes, including living matter,
remain artificial and not quite adequate. Blmenfeld (1967; 1974) noted that the true way to a
general life theory is not a creation of biological thermodynamics.
Really, the thermodynamic and electrodynamic laws are valid for different aspects of BO
functioning but they do not explain the essence of life selfregulation and behaviour of BO in
its entirety. A new science field BIODYNAMICS is needed.
Indeed, the biological regulation has been regarded in details in cybernetic aspect. Our
view is, however, that the biological selfregulation should be an object also of physics.
Although the life activity is based on biophysical and biochemical processes the perfect
co-ordination of these processes forms already a new quality of matter organization. Thus, it
is advisably to put in correspondence of BO behaviour new type quantities as integral
characteristics of BO on macroscopic level. Here we introduce the quantity vitality as a
basic biodynamic quantity. Thus, the following juxtaposition can be stated:
Classic dynamics mass
Electrodynamics charge, field intensity
Chromodynamics colour charge
Thermodynamics temperature, pressure, concentrations etc.
Biodynamics vitality
259
Naturally new type of measurements should be expected. The author presumes a creation
of a new device and believes that by means of such a device one will be able directly to
measure vitality in the future.
Such an approach will avoid the great complexity in the quantitative description of living
systems via thermodynamics, chemical kinetics etc.
The attention in this article is drawn on recovery processes of BO after disturbances
running in time intervals much shorter compared to BO lifespan. On the other hand, the
evolution of optimal vitality during the lifespan of BO is studied.
A POSSIBLE THEORETICAL BASIS OF BIODYNAMICS

We think BO as a macroscopic object, which saves its features and character only in its
integrity. The living system demonstrates a new level of material organization non-described
previously in physics.
We introduce a new quantity, vitality (V) uniquely determining the state of a given BO.
Thus V is an integral characteristic of BO functioning in its entirety. In thermodynamics the
temperature is a measure of kinetic energy of the molecules. In biodynamics the vitality
should be a measure of the health status of BO. Obviously, vitality is related to the
informational aspect of BO functioning. We propose the respective measurement unit of the
vitality in SI system to be called bion (b).
We assume that V could be in principle measured by a new type device vitalimeter.
Such an instrument should measure some electromagnetic wave emitting by BO, a wave (or
wave complex) being an integral characteristic of BO functioning. Electromagnetic waves of
different frequencies, generated by human, animal and plant organisms in their metabolic
activity were measured yet many years ago (Presman 1968). The explorations in this way
were continued (Godik and Guljaev 1991; Elizarov 1997). The recent development of
advanced technologies have been gone rather far and this fact promises well success in
searching of some device, reflecting resultant information about BO. A new constructed
apparatus, being able to detect some wave as integral BO characteristic, could be calibrated in
a manner that to show V in bions. For instance, one bion may be defined so that the excellent
standard of humans health corresponds to vitality of 100 bions.
Not only the frequency/length of the wave but also the wave amplitude gives information
for BO status. Thus, at least two new quantities should be defined. However, here for
simplicity we shall take into account only one quantity (understanding, for instance, the
wavelength). Here we are not interested in the incidence of the wave in the space. The very
value of the vitality at a certain moment is important.
We introduce also a quantity optimal vitality (W). That is the optimal vitality value,
corresponding to state of excellent health of BO and reflecting homeostasis characteristics
evolutionarily established for the respective species. W is genetically determined and in time
intervals much shorter compared to lifespan it may be regarded as a constant. During the life,
however, W decreases due to aging processes. After different transitory disturbances in BO
status V temporarily can deflect from optimal vitality, i. e. V W. Then a transitional process
is running and V W. That is a recovery process.
Svetla E. Teodorova
260
The total energy E of a natural system is sum of the mechanical energy EM, and internal
energy EI (Gyarmati 1970):
E = EM + E I
The internal energy contains heat energy, chemical energy etc. (Prigogine and Defay
1954):
dEI = dQ pdv + idni
It is reasonable to establish in biodynamics a respective specific energy. We shall cal it
biological energy (B). We could suppose that it is a function of V (t) and V (t) (where V is
the rate of change of the vitality in time):
B = B (V, V )
The biological energy should be a part of internal energy:
dEI = dQ pdv + idni + dB
B is the energy providing biological selfregulation (on the basis of enzyme synthesis,
resonance energy transfer between biological macromolecules, electric charge transfer,
immune response etc.). All these energies in macroscopic aspect could be denoted as
biological energy B.
We introduce also a state function synergy G (V), which uniquely determines the state of
a given BO:
dG
dB
V
(1)
as a measure of the quality of the BO selfregulation (therefore, of the biological organization).

G reflects the degree of coordination and synchronization of the regulatory links in BO. It
could be an indicator of BO health and youth. G is genetically determined. G increases in
organism growth as well as in training processes. In aging processes, in severe and chronic
diseases G decreases. In a mature and healthy BO the synergy G as almost constant G(V) =
G(W).
RECOVERY PROCESS AFTER DISTURBANCE

When a BO is disturbed (acute disease, trauma, intoxication, environmental influence
etc.) and its normal life parameters are violated, i. e. W V(t) 0, the genetic information
potential switches feedback control (repair, immune system reaction, enzyme synthesis etc.)
to restore BO to its physiological homeostasis. A recovery process starts. We assume that in
case of damages V(t) < W. Here we consider recovery processes, running in time interval
much shorter compared to the lifespan of BO.
Now we try to define the balance of biological energy in phenomenological aspect.
261
We assume the genome energy as composite of two parts: potential genome energy UW
and recovery energy UV:
U = UW + UV
(2)
The quantity UW = const is characteristic for a given species. The recovery energy UV, should
be proportional to the difference W V(t), i. e. UV = k (W V). UV should be a positive
function; hence we may present it as a positive determined quadratic form:
UV
1
K (W V ) 2
2
(3)
where K ([K] = [kg m2 b2 s2]) is the genome inductivity, representing the integral feedback
control strength. It is clear that UV has minimum at V(t) = W. Therefore, in a non-disturbed
state BO does not spend recovery energy.
We assume that the power of immune response P, expressed on phenomenological level,
should be proportional to the rate of change of the vitality. To be a positive function P should
be constructed as follows:
P MV 2
(4)
where M ([M] = [kg m2 b2 s1]) is coefficient of immune memory.

The immune response has a cumulative effect. The state of a BO at a given moment
depends not only on the synthesis of immune factors at that moment but on the summary
effect of immune response in all prior moments. Therefore the immune reaction energy Z in
the recovery process should have the form:
t
t0
t0
Z (t ) Pd MV 2 d
(5)
Because BO behavior is considered on phenomenological level, we are interested in the

total effect of immune response and here we do not differentiate cell and humoral immunity.
We assume that the constant M characterizes the total immune potential of BO.
The accomplishment of the recovery process may be embarrassed due to waste products
of metabolism (non-fully oxidized substances, macromolecules damaged by free radicals) and
toxicants (heavy metals, bacterial and virus toxins etc.) occurring in cell and decreasing the
efficiency of metabolic processes and cell selfregulation. Therefore, the total biological
energy B could decrease at expense of energy of metabolic resistance R. It is reasonable to
suppose that R is proportional to the rate of change of the vitality. To be a positive function R
could be defined in the form:
1 2
AV
2
where A ([A] = [kg m2 b2]) is coefficient of metabolic resistance.
(6)
Svetla E. Teodorova
262
One can write the following equation for the balance of biological energy B:
dB = dU + dZ dR
(7)
We define
dU
K (W V )
dV
and
dP
2 MV
dV
(8)
as biological force of feedback control and force of immune reactivity, respectively. Their
dimension is: [U] = [Z] = [kg m2 b1 s2].
We define also
dR
AV
dV
(9)
as impulse of metabolic resistance. Its dimension is [L] = [kg m2 b1 s1].

We assume that the functioning of the healthy living systems is based on the following
biological principle:
G(W) = max G(V)
(10)
It means that in its optimal, undisturbed state BO has a synergy maximum. G(W)
corresponds to excellent health. For recovery processes it follows from (10):
dG > 0
(11)
Taking into account (1), (2), (3), (4), (5), (6), (7), (8), (9) and (11) we can write:
dG
(dV LdV )
>0
V
(12)
where is the total recovery force
1
U Z
2
(13)
It follows from (12) that the expression
dV LdV > 0
is a condition for fully BO recovery.
(14)
263
One of the most profound concepts in theoretical physics is that the equations of motion
in different fields of physics can be obtained on the basis of integral variational principles.
The well known variational principle of Hamilton allows a common treatment of dynamic
problems in mechanics, electrodynamics, optics, thermodynamics, quantum mechanics etc.
By means of appropriately chosen Lagrangeans the basic equations in physics can be
introduced. This approach has a great heuristic concern. The presence of variation principles
in all physics fields clearly shows that a basic nature law takes place. This promises that in
fields, where no other approaches exist, a variational principle from the Hamilton type could
help to obtain adequate equations, describing the characteristic processes.
We propose the following integral principle:
T
(U Z R)dt max
(15)
t0
choosing the Lagrangean: L = L (V (t), V (t), t) = U + Z R,

where U, Z and R are determined by the equations (2), (3), (5) and (6).
After variation of (15)
( ) 0 0
(16)
where is an arbitrary parameter, the following biodynamic equation is obtained:
2M
K
KW
V
V
A 2 M (T t )
A 2 M (T t )
A 2 M (T t )
(17)
under initial conditions:
V (t0 ) V0
and
V (t0 ) V0
(18)
V0 is the state of disturbed BO from where the recovery process starts and V0 is the start
rate of time change of V. T is the time period of the recovery process. The equation (17) has a
physical sense and aperiodic solutions when the following conditions are valid:
A > 2MT
(19)
M2 > (A 2MT) K
(20)
Obviously, at a given value of M, in greater contamination of the organism (higher A

value) the time T of the recovery process will be longer.
The stationary solution of equation (17) is a stable knot.
Svetla E. Teodorova
264
Equation (17) is solved numerically. Some solutions at different values of the parameters
are displayed in Figure 1. The value of T = 10 days was arbitrary assumed for the recovery
process after some disturbance (for instance: influenza, poisoning, burn etc.). We stated that
the normal state of BO corresponds to W = 100 bions. We chose an arbitrary value for the
start of recovery process: V0 = 60 bions. In principle, on the basis of many empirical data one
can determine the real average recovery time periods for different diseases and damages and
real W for different species and ages. Using them the characteristic parameters in equation
(17) can be calculated.
Figure 1. Time courses of the quantity vitality during the recovery period as numerical solutions of
equation (17). The time interval of 10 days for the recovery period after some disturbance was arbitrary
chosen (T = 10). A value of 100 bions for optimal vitality was assumed (W = 100). The initial condition
was V0 = 60 bions. The displayed time courses were calculated at different values of characteristic
constants: A = 9, M = 0.4, K = 0.93 (over-shoot curve); A = 11, M = 0.5, K = 0.17; A = 12, M = 0.5, K =
0.13.
Time courses of several biological parameters, similar to these shown in Figure 1, are
very typical for different transitory processes in biological systems.
SINERGY AND ENTROPY CHANGES DURING THE LIFESPAN OF A

BIOLOGICAL OBJECT
In lifespan scale the following cases could be distinguished regarding the synergy G and
entropy S changes:
During development and growth
dG > 0
dS > 0
dG > dS
Near completion of development and growth

G = max
265
dS > 0
3) At mature age
dG 0
dS > 0
dG < dS
4) At aging
dG < 0
dS > 0
dG dS
5) Death
G=0
S = max
What means the inequality dG < dS at mature age? G can remain almost constant during
many years (maximum comfort conditions for BO) or decrease weakly (dG 0). But this
decrease is less compared to entropy increase (i. e. notwithstanding that the entropy increase
leads to age changes in macromolecules and structures, the integrity of the regulatory links
remains or changes slower than structure aging). The synergy is not reciprocal to entropy. The
inequality dG < dS shows the stability of BO selfregulation. For instance, the genetic
functions demonstrate a relative resistance against to the chronic action of damaging agents,
particularly heavy metals (Topashka-Ancheva et al. 2003). The difference in changes of
entropy and synergy once more indicates that via thermodynamics (in spite of its adjustment
to biological systems) one does not attain adequate description of the life processes.
EVOLUTION OF OPTIMAL VITALITY (W) DURING THE LIFESPAN OF A

BIOLOGICAL OBJECT
Up to now we considered the optimal vitality W as a constant quantity because our
attention was drawn only on processes much shorter compared to the lifespan. The optimal
vitality W, however, decreases in lifespan scale due to aging processes (genetic mutations,
alterations in the regulatory links of genetic apparatus and hence quantitative and qualitative
changes of proteins and attenuation of their self-renewal (Frolkis 1969). The simplest
quantitative assumption about W evolution during the life is that the rate of W change is
proportional to the time:
dW
qt
dt
(21)
where q ([q] = [b s2]) is a parameter reducing the optimal vitality. We call it aging factor.
The parameter q should be a time function. The temp of the aging is most intensive after
the age of 25 years (in human) and then it decreases with time (Strehler 1962). Quantitatively
this could be expressed by the following differential equation:
Svetla E. Teodorova
266
dq
q
dt
(22)
under initial condition

q0 = q(t0),
t0 = 25 years
(23)
where q0 ([q0] = [b s2]) and ([] = [s1]). The constant can be called aging correction,
because at higher values of the parameter q decreases faster with time and hence, as
follows from (21), the rate of aging decreases. Respectively, when q decreases slowly the
aging processes go faster.
The analytical solution of (22) is
q q0 e
(t t0 )
(24)
Taking into account (24) the equation (21) can be written as
dW
(t t0 )
q0 e
t
dt
(25)
The analytical solution of (25) is
W W0
q0
q
q
(t0 1) 0 te ( t t0 ) 02 e ( t t0 )
2
(26)
The constant may be determined from the solution (24) after taking a logarithm:
ln q = ln q0 (t t0)
(27)
The equation (27) is an equation of a straight line with angular coefficient . Using the
values of q, a plot of ln (q q0) as a function of t t0 could be constructed and determined.
The q-values could be calculated in the following way. Human individuals in perfect
(accordingly to the respective age) health could be selected in different age groups ranging by
five years (2530, 3035, 3540 etc.). Within these groups q may be considered
approximately constant. Then the analytical solution of equation (21) has the form:
W W0
1 2
qt
2
(28)
and respectively:
2(W0 W )
t2
(29)
267
W-values for the different age groups could be empirically measured (of course under a
reliable statistics) and on the basis of (29) values of q, specific for the respective age periods,
could be calculated. The value of q0 may be determined in individuals of 2426 years old. If
the constants q0 and were known W(t) would be drawn. Thus, we shall have a picture of a
normal aging, i. e. aging in individuals, who are not burden with chronic diseases. The same
procedure could be applied in people with certain chronic diseases, in smokers, etc. It is seen
from (29) that at a greather difference W0 W the parameter q has a higher walue. For
instance, at age 60 years W of a sick man will be less than W of a healthy man (Wsick <
Whealthy) and respectively W0 Wsick > W0 Whealthy. Therefore, qsick > qhealthy. This indicates a
faster aging process in the sick. In illness at young age the value of q0 may be higher than the
normal and this could reflect on the further life and aging pace.
In most cases the life mode can play a significant role for q modification. The healthful
nutrition, going in for sports, natural regimen etc. could increase -constant in large extent.
Consequently the rate of q decrease increases and hence the duration of the individual life
could increase.
Figure 2. Evolution of the optimal vitality W during the lifespan. The maximum value of optimal
vitality W is assumed of 100 bions. In human context it should correspond to the age of 25 years. A
value of 30 bions is assumed as Wcr. Three analytical solutions (26) of W (t) are drawn, calculated at
different values of the parameters (q0 = 0.01, = 0.0061; q0 = 0.01, = 0.0012; q0 = 0.015, = 0.0012).
The respective curves cross the dotted line Wcr in points corresponding to 150, 125, and 102 years.
These points could be considered as possible potential maximums of life duration at different
conditions.
In Figure 2 analitical solutions of equation (26) are presented. They are calculated under
initial conditions W0 = W(t0) = 100 bions and t0 = 25 years and at different values of the
parameters q0 and . The value Wcr corresponds to that value of vitality, under that the
selfregulation falls and life recovery processes are impossible. The values of X-axis,
corresponding to the points of the dotted line, indicate the lifespan of Homo sapiens in
different conditions. The points TWcr correspond to the absolutely life potential in the
respective conditions, not to the real end of the individual life. Usually the concrete lifespan is
268
Svetla E. Teodorova
(much) shorter than TWcr (often due to different fortuitous factors). In principle TWcr may be
considered as genetically determined possible lifespan maximum for a given species but it
could vary in a wide range depending on diseases, life conditions, and life mode.
CONCLUSION
It is impossible to deduce macro-characteristics of living systems on the basis of the
numerous processes on molecular level. Because of that it will be a great benefit to have a
measurable (one or a few) macro-characteristic(s) uniquely determining the status of BO as
an entire unit. Thus, the behaviour of BO would be explored and predicted. In exemplification
of this idea here we considered the simplest case of only one integral variable vitality V.
Such an approach is very important not only for practical purposes. It is also of great
theoretical significance. Biological selfregulation provides a new quality of matter. It is quite
reasonable to evaluate the state of living matter in terms of specific energy form. No kind of
energy known in physics can be put in correspondence to biological selfregulation in order to
explain its integrity. The health of BO essentially depends on selfregulation quality and it
seems to be very tempting to assess BO via adequate quantities. Thus, the creating of new
science field, biodynamics, could be a substantial step to a more profound study of living
matter.
The general responses of BO to environmental, therapeutic, and other influences as well
as to diseases could be effectively studied. Biodynamics approach will be of a great
importance in medicine for diagnosis and therapy. A simple and prompt assess of the healthy
status of the patients could be made and in patients files abreast of the other data could be
added data as vitality V , optimal vitality, and synergy G as important integral biomarkers.
Valuable evaluations could be carrid out about the aging rate and life duration via studing of
optimal vitality W evolution. Many empirical investigations could provide reliable data for
determining the characteristic lifespan TWcr for several species. Essential correlations could be
established between environmental conditions specificity and mean human lifespan.
Satisfactory values of the parameters introduced here (K, M, A, q0, and ) may be
determined via experiments, but it is also of essential interest to attempt to express these
constants as functions of some parameters of BO on molecular level. For this purpose many
targeted empirical research are needed.
Naturally, new device is needed to measure such quantities. The author is optimist
regarding the further technical development and the possibility to study the vital status of BO
via integral characteristics. The author hopes that the idea shared here may stimulate the
scientific thought for further efforts toward development of a phenomenological life theory.
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Bljumenfeld, L. A. (1967). Preface to the Russian edition. In: H. Quastler. The emergence of
biological organization 5-6. Moskow: Mir Publishing house.
Bljumenfeld, L. A. (1974). Problems of Biological Physics. Moskow: Naouka Publishing
House.
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Davidiv, A. (1979). Biology and Quantum Mechanics Kiev: Naukova Dumka Publishing
House.
Di Cera, E. (ed) (2001). Thermodynamics in Biology. Oxford-New York: Oxford University
Press.
Eigen, M. (1971). Molekulare Selbstorganistion und Evolution (Self organization of matter
and the evolution of biological macro molecules). Naturwisswnschaften 58 (10), 465-523.
Elizarov, A. A. (1997). Instrumental methods for investigating physical fields of biological
objects. Measurement Techniques (Springer) 40 (7), 700-707.
Frolkis, V. V. (1969) The Nature of Aging. Moskow: Naouka Publishing House.
Godik, E. E. and Guljaev, Yu. V. (1991). The human being through Eyes of Radiophysics.
J. Radio Engineering (Russian) 8, 51-62.
Goodwin, B. C. (1963). Temporal Organization in Cells. London: Academic Press Inc. Ltd.
Gyarmati, I. (1970). Non-equilibrium thermodynamics: Field theory and variational
principles. Berlin-Heidelberg-New York: Springer-Verlag.
Kleidon, A and Lorenz, R. D. (2005). Non-Equilibrium Thermodynamics and the Production
of Entropy. Life, Eearth, and Beyond. Berlin-Heiderlberg-New York: Springer.
Kurzynski, M. (2005). The Thrrmodynamic Machinery of Life. Berlin-Heiderlberg-New
York: Springer.
Nicolis, G. and Prigogine, I. (1978). Self-organization in Nonequilibrium Systems. From
Dissipative Structures to Order through Fluctuations. New York-London-SydneyToronto: John Wiley and Sons.
Presman, A. S. (1968) Electromagnetic fields and living nature. Moskow: Naouka Publishing
House.
Prigogine, I. and Defay, R. (1954). Chemical thermodynamics. London-New York-Toronto:
Longmans Green and Co.
Quastler, H. (1964). The Emergence of Biological Organization. New Haven and London:
Yale University Press.
Rosen, R. (1967). Optimality Principles in Biology. London: Butterworths.
Shrdinger, E. (1944). What is Life? Cambridge: University Press.
Strehler, B. L. (1962). Time, cells, and aging. New York and London: Academic press.
Szent-Gyrgyi, A. (1968). Bioelectronics. A Study in Cellular Regulation, Defense, and
Cancer. New York-London: Academic press.
Topashka-Ancheva, M., Metcheva, R., Teodorova, S. E. (2003). Bioaccumulation and
damaging action of polymetal industrial dust on laboratory mice Mus musculus alba II.
Genetic, cell, and metabolic disturbances. Environmental Research 54, 152-160.
Trincher, K. S. (1965). Biology and Information. Moskow: Naouka Publishing House.
Volkenstein, M. V. 1994). Physical Approaches to Biological Evolution. Berlin-Heidelberg:
Springer Verlag.

ISSN: 2158-5717
Chapter 9
A NEW TRAIT OF GENTOO PENGUIN:

POSSIBLE RELATION TO ANTARCTICA
ENVIRONMENTAL STATE?
Roumiana Metcheva1, Vladimir Bezrukov2, Svetla E.
Teodorova3, and Yordan Yankov4
1
Institute of Zoology, Bulgarian Academy of Sciences, 1,

Bd.Tzar Osvoboditel, 1000 Sofia, Bulgaria
2
Department of General and Molecular Genetics,
Taras Shevchenko National University of Kyiv,
64 Volodymyrska Str., Kyiv, 01033, Ukraine
3
Institute for Nuclear Research and Nuclear Energy,
Bulgarian Academy of Sciences, 72, Tzarigradsko shaussee,
4
Bulgarian Antarctic Institute, 15, Bd.Tzar Osvoboditel,
ABSTRACT
Some morphological traits of Antarctic animals could be considered in the context of
a trend to more direct relation between environmental conditions and possible adaptive
mechanisms of animals organism. Penguins are excellent object for biomonitoring. Here
a preliminarily report is presented regarding a new trait, a spot-like coloration (yellow
spot) of the bill, observed on the upper mandible of Gentoo penguin (subspecies
Pygoscelis papua ellsworthii). The spot varied in size and colour. It was recorded among
chicks over two months old and adult birds (normal and molting). The trait had no
significant relationship to the animal' s sex. Among all inspected females 31% and among
all inspected males 27% exhibited beak spot. Three breeding colonies were investigated
at three different geographical locations at the Antarctic Peninsula Livingston Island,
South Shetlands (6238 S), Wiencke Island (64o52 S), and Petermann Island (6510 S).
Yellow spot was found with different frequencies at these three locations: 20%, 36%, and
30%, respectively. All spotted penguins from the three colonies were 32% when
272
Roumiana Metcheva, Vladimir Bezrukov, Svetla E. Teodorova et al.

compared to all non-spotted. The possible reasons for the spot-like coloration are
discussed. The trait could be a phenotypic characteristic. It could play some role in the
mate choice. A possible connection to carotenoid pigments content does not be
eliminated in the context of Gentoo diet. A probable cause for the beak spot appearance
seems to be the increasing of ozone depletion above Antarctica. The ultraviolet radiation
enhances free radical production. Other studies reported an increase of the flux of
transsulfuration pathway as a defense reaction, resulting in elevated level of cysteine.
When cysteine concentration is raised, an attaching of cysteine to melanin synthesis
pathway occurs and this results in formation of reddish pigments.
INTRODUCTION
Antarctica environment is closely related to the global change of our planet and hence it
is a topic of increasing importance. Climatic features, atmospheric regime, and temperature
and water balance depend on the state of Antarctic continent. The investigation whether
Antarctica is influenced by anthropogenic pollution is of great interest. In this context the
biomonitoring could provide valuable data. Results reported by Metcheva et al. (2006)
suggest that due to annually molting penguin feathers are an excellent monitoring tool of
Antarctica environmental state, being precise indicator for detection of metal levels. This
allows assess possible contamination trends.
During the morphological investigations on Gentoo penguin (Pygoscelis papua
ellsworthii) a spot-like coloration (yellow spot) was observed at the base of the upper
mandible of some individuals. It varied from clear yellow-orange to reddish. Colour patterns
of terrestrial birds have been well studied, but there is little research on seabird coloration
(Jones and Hunter 1993; Jouventin et al. 2005).
Among the six genera belonging to family Spheniscidae, the genus Pygoscelis is the most
widely distributed (Del Hoyo et al. 1992). Three species belong to Pygoscelis: Chinstrap (P.
antarctica), Gentoo (P. papua) and Adelie (P. adeliae). Gentoo penguins breed on
subantarctic islands and on the Antarctic Peninsula (Stonehouse 1970). There are two
subspecies of Gentoo P. p. papua, J. R. Forster, 1781 and P. p. ellsworthii, Murphy, 1947.
The subspecies P. p. papua is distributed in sub Antarctic up to 60 S; P. p. ellsworthii
inhabits the Antarctic from 60 S up to 65 S (Del Hoyo et al. 1992). The subspecies P. p.
ellsworthii is of smaller size and bill proportion compared to P. p. papua (Martinez 1992).
Gentoo penguin populations inhabiting the Antarctic Peninsula have been studied with
respect to foraging behavior (Trivelpiece et al. 1986), morphometry (Stonehouse 1970), UV
reflectance (Jouventin et al. 2005) etc. However, this trait (yellow spot) observed has not
been described in the literature before.
Some authors (Stevenson and Anderson 1994; Siefferson and Hill 2005) that study
different particular colorations in birds tend to explain the function of these phenomena in
terms of health, mate choice behavior or intraspecific signaling. Saks et al. (2003) have
established that brighter yellow breast feathers in male greenfinches signals
immunocompetence and health status. Thus, females prefer more ornamental males, able to
provide parasite resistance genes for the offspring. Some of the colour patches are at the same
time also ultraviolet markings. Jouventin et al. (2005) have reported UV beak spots in King
penguin (Aptenodytes patagonicus) and Emperor penguin (Aptenodytes forsteri). They have
A New Trait of Gentoo Penguin: Possible Relation to Antartica
273
found UV peaks of reflectance, overlapping with spots of colour on both sides of the lower
mandible, which have appeared orange (with variations among individuals from yellow to
red). The authors suggested that UV beak spot could be an indicator of sexual maturity having
a possible role in pairing. UV reflective patches are of concern in the context of UV vision in
birds, which may be used also for foraging (Stari et al. 2002) and hunting (Koivula et al.
1997; Koivula et al. 1999).
Other interesting problems are whether such kind of markings (colour only or colour and
UV reflectivity) might appear as consequence of environmental changes or it depends on the
diet, biochemical disturbances and parasite infection. Most probably a complex reason might
take place. Similar traits could also be used for comparative population studies.
In the present study the distribution of spotted P. p. ellsworthii in three Antarctic
locations Livingston Island, South Shetlands (6238 S), Wiencke Island (64o52 S), and
Petermann Island (6510 S) is reported and probable reasons for the beak spot appearance are
discussed. Particularly, a possible reason related to the specific Antarctic environment is
outlined.

Populations Studied
Field measurements were carried out on adult, nonmoulting and molting Gentoo penguins
(P. p. ellsworthii), inhabiting Livingston Island (6238 S, 6024 W, South Shetland Islands),
Wiencke Island (64o52 S, 6330 W, Palmer Archipelago), and Petermann Island (6510 S,
6410 W). The birds were tested during the Antarctic summer seasons, from January to
March, at Livingston Island in years 2002 2005; at Petermann Island in 2002-2003, and at
Wiencke Island in 2003-2004. At Livingston Island the studied colony varied from 84 to 110
couples. At Petermann and Wiencke Islands the colonies were larger, about 1000 nests per
island. Additionally, at Livingston Island twelve marked couples of P. p. ellsworthii as well
as the offspring in the crche were observed in December 2005 - January 2006.
The penguins were captured using a hand net. After inspection the morphological traits
were measured according to the Commission for the Conservation of Antarctic Marine Living
Resources Ecological Monitoring Programme (CCAMLR EMP) recommendations
(CCAMLR 2004).
All the penguins were marked with glass-encapsulated TROVAN identification
electronic transponders. These transponders, implanted subcutaneously, have demonstrated to
be a reliable means to identify individual penguins. The TROVAN system enables long
lasting marking, automatic detection of birds and quick identification (Clarke an Kerry 1998).
The penguins were checked for the appearance of spot on the beak when they were marked.
Blood Samples for DNA Analysis

Blood sampling for sex identification and DNA analyses were performed by two different
ways according to the CCAMLR EMP Standard Methods Recommendations. Approximately
274
1 ml of blood was drawn from the cubital vein with a syringe and placed into tubes with
K3EDTA or into heparinzed tubes. DNA was extracted using a standard phenol-chloroform
method as described by Sambrook et al. (1989). Sex determination was performed by PCR as
described by Itoh et al. (2001).
Discriminant Analysis
Sex determination of Gentoo from the Petermann Island was carried out with
discriminant analysis, as described EMP Standard methods, Hobart (CCAMLR 2004) with
little modification. The equation was Z = 0.922*L + 3.885*H were L and H are the length and
high of the bill, respectively. The discriminate value was D = 103.302 instead of the
described 112.608, because the localization of the minimum at the male-female distribution of
D was at the 103.302 point. Data were verified with the values of body mass differences (only
for registered nesting couples) males were usually heavier than females. The results were
confirmed with PCR assay according Savov et al. (2004).
Statistical Treatment
The percentages of spotted individuals from Livingston Island, Wiencke Island, and
Petermann Island were compared and statistically analyzed for their significance.
Differences in the trait distribution between males and females were tested using the 2criterion for table 2 x 2. Trait distribution in the penguins at different locations was tested
with exact Fishers test (for two-tailed probability of the II type error) (Sokal and Rohlf 1995;
Dubrova 2000).
RESULTS
Gentoo penguins from Antarctic Peninsula (Figure 1) were tested for presence/absence of
beak spot (Figures 2 and 3) as follows: 157 individuals at Livingston Island (or about 81% of
the breeding colony), 114 at Wiencke Island (11% of the colony), and 201 at Petermann
Island (20% of the colony). The spot is located on the upper mandible of the bird (Figure 2).
It begins at the flashy core and extends onto the hardened part of the beak. In different
individuals the spot size ranges from very small (1 2 mm) to quite large (20 25 mm). The
largest spots spread over almost all the upper surface of the bill. We named the observed new
trait yellow spot because of its typical colour. However, the colour of the spot varied from
yellowish to red with yellow, orange, and pink intermediate forms in different birds. The
scheme in Figure 4 gives a picture regarding size and colour variations of the spot.
Other visible peculiarities in body or feather coloration of beak-spotted Gentoo penguins
were not recorded.
The trait was observed year-round, not only during molt. Over the time of exploration the
spots did not change their appearance.
275
The yellow spot was observed also in adults and chicks inspected. Among adult birds
the frequency of this trait was approximately two times higher than among chicks.
To prove preliminarily a possible heritable basis and to estimate the age-related process
of spot appearance, commencing with the hatching, we observed additionally 3 pairs with
spots (both of parents), 2 pairs without spots and 7 mixed pairs (with spot in only one of the
parents) at Livingston Island in December 2005 January 2006. From the beginning of
hatching to about 3 weeks of age no beak spot was found in the offspring (Figure 5).
Figure 1. Gentoo penguin distribution map: the white dots indicate the breeding areas of the subspecies
Pygoscelis papua papua and the black dots the breeding areas of the subspecies Pygoscelis papua
ellsworthii. 1 Livingston island; 2 Wienke island; 3 Petermann island.
Figure 2. Adult Gentoo penguin (Pygoscelis papua ellsworthii) with spot on the upper mandible.
276
Figure 3. Adult Gentoo penguin (Pygoscelis papua ellsworthii) without spot on the upper mandible.
Figure 4. A scheme of the Yellow spot, observed on the upper mandible of Gentoo penguins
(Pygoscelis papua ellsworthii): place of the spot on the mandible and spot size variations.
Figure 5. One-month Gentoo penguin (Pygoscelis papua ellsworthii) without spot on the upper
mandible.
277
Similar data were obtained at the other locations (Petermann and Wiencke Islands),
where recently hatched chicks with beak spot were not found. Spots were observed, however,
in two-month chicks (Livingston colony), when they have formed crche (Figure 6). Among
75 juveniles, 3 two-month chicks (4%) exhibited a small spot in the base of the upper
mandible.
Figure 6. Two-month Gentoo penguin (Pygoscelis papua ellsworthii) with an about 2 mm Yellow
spot on the base of the upper mandible.
The spot distribution among males and females is presented in Table 1. The DNA
analysis was carried out on 93 individuals in the Livingston colony and on 185 individuals in
Petermann colony. The analyses revealed that at Livingston Island 37 of the samples were
females and the rest 56 were males. Respectively, at Petermann Island 94 penguins were
females and 91 males. Among all inspected females 31% exhibited beak spot and among all
inspected males colour spot on the beak was observed in 27% of individuals. There was no
statistically significant difference in the frequency of spot appearance in both sexes (Table 1).
The distribution of the trait in the three Gentoo colonies (at Livingston, Wiencke, and
Petermann islands) for the penguins investigated during 2002 2005 (without those observed
in the summer season 2005/2006) is presented in Table 2.
All inspected birds were divided into two categories with spot and without spot.
The average frequency of all spotted individuals is 31.7%. The differences in the percentage
of spotted individuals among all the three colonies are statistically significant. The highest
frequency of the trait was found in the colony of Wiencke Island followed by the colonies of
Petermann and Livingston (Table 2).
278
Table 1. Distribution of the yellow spot between male and female individuals of
Pygoscelis papua ellsworthi
Location
Livingston
Island
Petermann
Island
All groups
n (all
inspected)
Sex
With beak spot
Without beak spot
Frequency in %
Male
56
15
27
41
Frequency
in %
73
Female
Total
Male
37
93
91
9
24
25
24
26
27
28
69
66
76
74
73
0,069
0.98
Female
Total
Male
Female
Total
94
185
147
131
278
31
56
40
40
80
33
30
27
31
29
63
129
107
91
198
67
70
73
69
71
0,66
0.78
0,37
0.93
Table 2. Distribution of the yellow spot in the three colonies of Pygoscelis papua
ellsworthi, inhabiting different Antarctic geographical areas. Frequencies (%) of spotted
individuals and Fishers test data are given
Population
Livingston
Island
Wiencke Island
Petermann
Island
All
Location
6238 S
6024 W
64o52 S
6330 W
6510 S
6410 W
Total
%
Spotted
n
With
beak spot
Without
beak spot
157
31
126
19.7
114
58
56
50.9
201
61
140
30.3
472
150
322
31.8
Statistical significance
(exact Fishers test)
Comparison
Twobetween
Tailed
populations
probability
Livingston 0.0014
Peterman
Peterman 0.0004
Wiencke
Livingston 0.00012
Wiencke
DISCUSSION
On the basis of the result that the distribution of the revealed beak spot in males and
females P. p. ellsworthii does not differ significantly (Table 1) one could safely state that the
yellow spot is not related to the sex of Gentoo penguin.
The yellow spot is a characterization both of non-molting and molting penguins.
This trait may be a polymorphic characteristic. In such case it will be of interest to study
its genetic architecture. The problem of Gentoo population analysis is important today
because Gentoo is one of the indicator species of Antarctic ecosystem (Zhu et al. 2005) and a
comparison of populations by trait frequencies could be useful for population studies and
monitoring programs of Gentoo penguin.
279
At the present stage of the study we have not yet collected data supporting an assumption
regarding a heritable basis of the trait. The fact that no spot was recorded in Gentoo chicks
bellow two months of age suggests a possible correlation between the spot appearance and
age, i. e. the spot appears in growing up offspring to play some role related to mature birds.
Such an assumption is reasonable taking into account that in the crche only 4% of the twomonth chicks exhibited a small spot and that in adult birds the frequency of the spot was
about five times higher than in juveniles.
Much attention has been paid to the significance of colour and UV phenomena in avian
intrasexual competition or intrasexual selection. Jouventin et al. (2005) have established that
recently paired King Penguins (Aptenodytes patagonicus) had shown higher UV reflectance
than courting ones. So, these authors consider the UV ornaments as a factor, which plays a
role in pairing of breeding males and females and could serve as an indicator of sexual
maturity. Also Mougeot and Arroyo (2006) have noted that UV signals play key roles in
social and sexual signaling in birds. European starlings (Sturnus vulgaris) (Benett et al.
1997), Blue tits (Parus caeruleus) (Anderson et al. 1998; Hunt et al. 1998), and Bluethroats
(Luscinia s. svecica) (Anderson and Amundsen 1997) use UV-reflecting plumage cues in
mate choice. On the other hand, there are studies indicating that UV vision in birds plays an
essential role in their foraging and hunting behaviour. So black grouses prefer UV-reflecting
berries (Stari and al. 2002), kestrels are attracted to the vole urine and faces marks that
reflect UV light (Viitala et al 1995; Koivula et al. 1999; Zampiga et al. 2006). The hunting
success of nocturnal owls is best during clear nights due to the visibility of the scent markings
in UV light (Koivula et al. 1997).
Endoparasitism is viewing as a cause for the coloration variety (McGraw and Hill, 2000).
It was found (Saks et al. 2003) that coccidian infection reduces the expression of plumage
coloration in greenfinches (Carduelis chloris) by creating a deficiency of carotenoids
available for deposition in ornamental feathers. Golemanski (2002) first described intestinal
coccidiosis in P. papua (Livingston Island).
One could suppose some relationship between spot and carotenoid pigments. Red, orange
and yellow coloration is usually related to these pigments. Saks et al. (2003) have established
that the plumage coloration in male greenfinches, signalizing their immunocompetence and
health status, is carotenoid-based. The combination of carotenoid pigments with proteins
generates many colors, from the brilliant yellow to red, in crustaceans, fish, and birds. The
general distribution and metabolic pathways of carotenoids have been investigated in details
(Katayama et al. 1971; Goodwin 1984; Davis 1985; Matsuno and Hirao 1989). We have not
analyzed biochemically Gentoo beak but it seems reasonable to suppose at least partial
carotenoid contribution in beak coloration because of Gentoo diet. It consists of about 50
80% crustaceans, mainly krill, which are a rich source of carotenoids (Berrow at all. 1999).
However, there are reports regarding the potential for melanins to produce yellow colors in
birds plumage (McGraw et al. 2004). These authors have not detected carotenoid pigments
in feathers of five avian species, including King Penguins (Aptenodytes patagonicus) and
Macaroni Penguins (Eudyptes chrysolophus). More over McGraw et al. (2004) suggested that
the yellow appearance of penguin and domestic chick feathers might be attributed to a new
form of plumage pigment, never before described from bird feathers.
A probable reason for appearance of the beak spot may be related to some aspects of the
changes in Antarctic environment and especially to the increased ozone depletion above
Antarctica. As known ozone layer protects the earth surface from the ultraviolet radiation.
280
Evidence is provided that UV radiation generates H2O2 (Peus et al. 1998). In presence of
H2O2 and under UV rays reactive oxygen species (ROS) are formed. What kind of way might
lead from ozone depletion and ROS to yellow spot?
The pigment melanin causes the black colour. It is an essential component in the bill
structure. Incorporation of melanin granules into the bill keratin increases the hardness of the
bill (Bonser and Witter 1993). Attaching of cysteine to DOPA-quinone after oxidative
cyclization and polymerization results in the formation of reddish pigments (Angelov et al.
1995). DOPA-quinone is the third step of the melanin synthesis pathway beginning from
tyrosine. Such a reaction is possible to occur in case of an increase of the concentration of
cysteine, taking part in desintoxication and antioxidant processes.
The low-molecular-weight thiols cysteine and especially glutathione are important
antioxidants acting in cells. In the biochemical anabolic pathway the cysteine is a precursor of
glutathione. Reduced glutathione (L-gamma-glutamyl-L-cysteinyl-glycine, GSH) is formed in
a two-step enzymatic process including, first, the formation of gamma-glutamylcysteine from
glutamate and cysteine, and second, the formation of GSH from gamma-glutamylcysteine and
glycine (Nikolov 1971; Franco et al. 2007). The production of GSH mainly depends on the
amount of available cysteine (van der Crabben et al. 2008).
The cysteine synthesis is involved in the transsulfuration pathway. The first step of this
pathway, from homocysteine to cystathionine, is catalyzed by cystathionine beta-synthase
(CBS). The enzyme transsulfurase (cystathionase) converts cystathionine to cysteine. Persa et
al. (2004), exploring some biochemical reactions in eye, showed that a transsulfuration
pathway is present in the lens and that oxidative stress of H2O2 could increase the flux of this
pathway activating the CBS enzyme. Thus, a lens under oxidative stress accumulates free and
protein-bound cysteine. They reported also that oxidative stress transiently up-regulates the
gene expression of CBS both in human lens epithelial cells and in pig lens.
It seems reasonable to suppose that transsulfuration pathway exists in the penguins bill
and likely the accumulation of ROS in the cells as a result of the enhanced UV radiation cooperate to an increase of CBS synthesis and CBS activity. The increase of cysteine production
and hence the GSH level may be considered as adequate adaptive reaction to the change of
environmental conditions. On the other hand, the elevated cysteine level plays a role for
involving of cysteine in melanin synthesis pathway via attaching to DOPA-quinine. The
formation of reddish pigments could reflect in beak spot coloration.
The frequency of the spot appearance in the three investigated geographical points did
not unambiguously show a cline in the trait distribution (Table 2). Still the lowest frequency
(19.7%) was found in the colony of the most north point (Livingston Island). The highest
percentage of spotted penguins was found at Wiencke Island, situated between Livingston
and Petermann Islands. Wiencke Island is by 2o14 southly from Livingston Island. The ozone
depletion increases in south direction, to the pole.
Longer studies and targeted investigations are required to determine precisely the most
probable cause (or cause complex) for the appearance of this trait and to clarify its possible
function.
\
281
ACKNOWLEDGMENTS
This work was supported by the grant INTAS-20010517 and grant of Ukrainian
Antarctic Center 04DF036-01(H/4-2004) and by grant B- 1615/2006 from the Bulgarian
National Scientific Fund.
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ISSN: 2158-5717
Chapter 10
ASSESSING POPULATION VIABILITY OF FOCAL

SPECIES TARGETS IN THE WESTERN FOREST
COMPLEX, THAILAND
Yongyut Trisurat1 and Anak Pattanavibool2
1
Faculty of Forestry, Kasetsart University,

Bangkok 10900, Thailand
2
Wildlife Conservation Society Thailand Program,
Nonthaburi 11120, Thailand
ABSTRACT
The Western Forest Complex (WEFCOM) in Thailand covers approximately 19,000
km2. This protected area complex comprises 11 national parks and 6 wildlife sanctuaries.
During 1999-2004, the Danish Government provided financial support to the Royal
Forest Department to manage this forest complex through the ecosystem management
approach. The WEFCOM Project employed rapid ecological assessment (REA) to
determine the current distribution statuses of wildlife species, develop a Geographic
Information System (GIS), and define habitat uses of wildlife. This paper is based upon
the achievements of the WEFCOM Project. It aims to define suitable habitats of selected
key wildlife species in the WEFCOM and to assess the current and desired statuses under
a population viability estimate for those species. The focal wildlife species were sambar
(Cervus unicolor), gaur (Bos gaurus), banteng (Bos javanicus), Asian elephant (Elephas
maximus), and tiger (Panthera tigris. We used logistic multiple regression to determine
habitat uses of wildlife and employed minimum dynamic area and landscape matrix
surrounding suitable habitats as criteria to assess population viability. The results indicate
the current suitable habitat mainly remains in Huai Kha Khaeng and Thung Yai wildlife
sanctuaries. In addition, the current viability condition is good for sambar, fair for gaur,
elephant and tiger; and poor for banteng. However, landscape matrices outside the
suitable habitats for all species range from moderate to high connection of native
vegetation. If the project aims to upgrade the viabilities to the next level in the next 10
years, park rangers and multi-stakeholders have to increase the amount of suitable
Email: fforyyt@ku.ac.th; Tel: 662-579-0176; Fax: 662-942-8107
286

habitats for all species from 12,630 km2 or 67% of the WEFCOM to 16,750 km2 or 89%.
By doing this, the number of suitable patches would significantly decrease and the mean
patch size would increase substantially, thereby indicating less fragmentation.
Keywords: Western Forest Complex (WEFCOM); population viability; habitat suitability;

landscape indices; GIS.
1. INTRODUCTION
The World Conservation Union (IUCN) defines a protected area as an area of land
and/or sea especially dedicated to the protection and maintenance of biological diversity, and
of natural and associated cultural resources, and managed through legal or other effective
means (IUCN, 1984). In Thailand, there were 282 protected areas, covering about 18% of
the countrys land area in 2004 (Trisurat, 2006).
Management of protected areas previously was limited to the boundary legally declared
in the National Gazette. Cooperation with park officials beyond the park boundary for
effective protection and management was relatively low and multi-stakeholders besides the
responsible agency have been rarely involved in management although the New Constitution
of Thailand of 1997 clearly defined their roles and responsibility in natural resources
management (DARUDEC, 1999). In the past four decades, protected areas have been selected
on a site-by-site basis in an ad-hoc way, and often based on the opportunity to preserve
remaining forest, as well as to promote tourism. These management practices are unlikely to
ensure the long-term protection of biodiversity and the ecological processes upon which
biodiversity depends as defined in protected areas management objectives (IUCN, 1984).
Therefore, the Royal Forest Department (RFD) and Danish Cooperation for Environment and
Development (DANCED) implemented the Western Forest Complex (WEFCOM) Project
during 1999-2004. The overall objective for this initiative was to maintain the health of the
ecological systems by using the Ecosystem Management Approach with community
participation.
The ecosystem approach is a strategy for management of land, water and living resources
that promotes conservation and sustainable use in an equitable way, which was adopted at the
Second Conference of the Parties of the Convention on Biological Diversity (Smith and
Maltby, 2003). This approach has recognized the need to incorporate ecological processes,
disturbances and biological population viability into the planning processes. By conserving
viable samples of the whole ecosystem, it is anticipated that all of the species contained
within them will, at least, have a fighting chance to survive in the long-term.
Viable population is defined as the minimum number in populations (MVP) of species
that can persist for long periods of time (Wielgus, 2002). There are many methods for
assessing population viability, ranging from the quantitative and data-gathering approaches to
more expert-driven and qualitative methods (Beissinger and McCullough, 2002). In practice,
there are relatively few species for which population viability analysis has been performed
because this technique is available only for a data-rich species. Thus, many scientists use the
50/500 rule of thumb as an effective population size over the short-term (Soule, 1987). For
instance, the British Columbia Ministry of Environment established a benchmark population
Assessing Population Viability of Focal Species Targets in the Western Forest
287
of grizzly bears ranging from 100-250 animals and determined the reserve sizes that can
accommodate these populations (Wielgus, 2002). More recent analyses suggest that an
effective population size of about 1,000 is needed to allow continued evolution and prevent
the accumulation of harmful mutation (Allendorf and Ryman, 2002). Based on MVP-related
research over the past 30 years, Traill et al. (2007) found that the median MVP for mammals
was 3,876 individuals. For practical implementation, there are increasingly fewer populations,
especially of large mammals, that would meet the effective population size of 1,000 or more.
Thus, a promising approach to maintaining MVP is to increase connectivity among
fragmented habitats. The common qualitative technique or expert opinion to assess population
viability is based on the criteria of species occurrence, habitat condition and landscape
context surrounding the occurrence (Groves, 2003). Recently, the methods for assessing the
viability of species that combine static habitat models to accommodate the minimum viable
population with population viability analyses has become a promising approach in regional
conservation plans, especially when digital data are available (Brito and Figueredo, 2003;
Leroux et al., 2007). This approach is named minimum dynamic area (MDA), which
describes the smallest area with natural disturbance regime, which maintains internal
recolonization sources, and hence minimizes extinction risk (Pickett and Thompson, 1978).
This paper is based on the achievements of the WEFCOM project. It aims to define
suitable habitats of selected key wildlife species in the WEFCOM and to assess the current
and desired status of population viability for these species.
2. STUDY AREA
The Western Forest Complex (WEFCOM) is situated along the Thailand-Myanmar
border. It comprises 17 contiguous protected areas, including six wildlife sanctuaries, nine
national parks and two proposed national parks (Figure 1). The toal land area of WEFCOM is
approximately 19,000 km2 or approximately 4% of the countrys land area. Among these 17
areas, Huai Kha Khaeng Wildlife Sanctuary is the largest protected area, covering 2,780 km2.
Salakpra Wildlife Sanctuary is the oldest protected area in this complex having been declared
as the first wildlife sanctuary in 1965. There are 162 ranger stations scattered in the
WEFCOM landscape mainly situated in the eastern buffer zone of the complex.
The WEFCOM is the largest intact forest area in Thailand and also within mainland
Southeast Asia. It is situated in six provinces in western Thailand. between latitudes 14 08 16 37 North and longitudes 98 11 - 99 32 East. The WEFCOM is part of the Tenassarim
Range extending southward along the Myanmar border. The landscape in the north and the
west is rugged highlands. The area slopes gently towards the south and is drained by the Huai
Kha Khaeng, which is the main perennial stream in this complex (WEFCOM, 2004).
Based on Landsat image interpretations and field assessments in 2000, approximately
90% of the total land area is under forest cover. Mixed deciduous forest is dominant in the
complex covering the highest percentage of the WEFCOM area (6,172 km2 or 32%).
Approximately 36% of the WEFCOM is comprised of various evergreen forests. Roughly one
third can be considered as relatively intact and undisturbed forest. Large tracts of intact and
healthy forest still occur in Thung Yai Naresuan and Huai Kha Khaeng Wildlife Sanctuaries
but the forest becomes more scattered and fragmented in other areas (WEFCOM, 2003,
288
2004). Remaining forest cover along the boundary and surrounding enclave communities is
threatened by agricultural encroachment. A socio-economic survey revealed that there were
112 communities situated inside the WEFCOM harboring approximately 5,400 households
and 27,700 individuals. Besides this, there were another 103 villages located within a 3-km
distance of the WEFCOM, mainly found along the eastern border (WEFCOM, 2002).
Figure 1. Protected area system of the Western Forest Complex in Thailand.
289
The WEFCOM has been nationally and internationally recognized as the key area of
terrestrial biodiversity in mainland Southeast Asia. It is located at the crossroads of three
plant geographies: Indo-Burma, Indo-China and Indo-Malaya; and two zoo-geographies:
India Indo-China and Sundaic Sub-region (Nakhasathien and Stewart, 1990; Smitinand,
1987). Based on Wikramanayake et al. (2000), the WEFCOM encompasses two important
ecoregions, namely the Kayah-Karen montane rain forests ecoregion and Tenasserim-South
Thailand semi-evergreen rain forests. This bio-geographical overlap provides a unique
assemblage of Asian species. Therefore, UNESCO designated Huai Kha Khaeng and Thung
Yai Naresuan Wildlife Sanctuaries, two of the largest protected area units in the core area of
the WEFCOM, as a Natural World Heritage Site in 1991. Some endangered species are tiger
(Panthera tigris), Asian elephant (Elephas maximus), gaur (Bos gaurus), banteng (Bos
javanicus), tapir (Tapirus indicus), great hornbill (Buceros bicornis), rufous-necked hornbill
(Aceros nipalensis), green peafowl (Pavo mulicus), and giant frog (Rana blythii).
3. METHODS
The general processes consisted of four steps, namely: 1) identify species conservation
targets; 2) estimate habitat suitability for selected species; 3) assess the current and desirable
condition of targets viability; and 4) delineate the congregation locations of target species in
the WEFCOM landscape. Activities for each step are summarized as follows:
3.1. Identify Species Conservation Targets

Conservation targets are those features or elements of biodiversity (ecosystem, species
and genetics) that planners seek to conserve within a system of conservation areas (Groves,
2003). In this study, they are those species that make the WEFCOM a globally important
conservation site. Wildlife specialists and the planning team comprising protected area staff,
scientists and NGO representatives, set four criteria to select the species targets, namely,
wide-range distribution, endangered status or ecosystem indicator, specialist availability and
public interest.
3.2. Develop Habitat Suitability Maps for Selected Species

The WEFCOM Project employed ecological rapid assessment (REA) (Sayre et al., 2000)
to gather wildlife occurrences in the study area. Wildlife signs and visual sightings were
recorded by park rangers who had been trained in map reading, Global Positioning System
(GPS) and identification of wildlife tracks and signs prior to actual survey. In addition, we
applied the logistic multiple regression model to generate the suitable habitats for selected
species (Trisurat 2003). The present and pseudo-absent data (chosen outside 1 km buffer from
present locations) were treated as dependent variables in the habitat suitability model.
The independent variables included biophysical and human disturbance factors. The
biophysical factors were vegetation type, distance to river and stream, slope, elevation and
290
aspect, while the human factors consisted of distance to ranger station and distance to
villages. The raster-based modeling in GIS ArcView 3.2 (ESRI, 1992) of 100 m resolution
was used to perform all spatial analysis functions. Seventy-five percent of wildlife
observations were used to develop the model and the remaining 25% were used to test the
model accuracy. According to Petdee (2000) and Prommakul (2003), sambar, banteng and
gaur are primary prey for tiger, therefore the suitability maps of these ungulate species were
aggregated to derive a prey coverage as an additional layer for tiger habitat.
The logistic multiple regression model is written as:
Zi
Prob
event
e
1 e
Zi
where Z i is the linear combination model of species i

The output maps produced from the predicted models were continuous probability values
ranking from 0.0 1.0. Later these probability values were categorized into 4 classes to
determine habitat suitability as follows:
Prob. Value 0.0 0.2
Prob. Value 0.2 0.4
Prob. Value 0.4 0.6
Prob. Value 0.6 1.0
=
=
=
=
Unlikely
Less Likely
Likely
Most Likely
3.3. Assess the Status of Species Targets Viability

We used the Five-S Framework developed by the Nature Conservancy (2000) to evaluate
the long-term viability of a focal conservation targets occurrence in the WEFCOM
landscape. In fact, this framework provides three criteria for assessing the persistence of focal
species: size, condition and landscape context. For the size criterion, we used the extent of
suitable habitats (likely and most likely) to support MVP or minimum dynamic area (MDA)
as a proxy indicator. In addition, we used habitat fragmentation (the percentage of native
vegetation surrounding the suitable habitats (2-km buffer) as a landscape context criterion.
Condition attributes of wildlife population (e.g. species composition, structure and biotic
interactions) were not taken into account due to limited population data.
The MVP size of each species was estimated according to the 50/500 rule, carrying
capacity and the recommendation of Thai wildlife experts. The MDA is simply derived by
multiplying the MVP and the home range of species. For instance, the home ranges of tiger in
the Thung Yai are between 50 and 200 km2 and its average coverage is 80 km2 (Prommakul,
2000) and the optimum MVP of tiger in the WEFCOM is 100 individuals. Therefore, the
estimated MDA is 8,000 km2. Meanwhile, a simple grading scale (very good, good, fair or
poor) was used to assess the current and desired conditions in the next 10 years. Table 1
elaborates the context of each viability class.
291
Table 1. Criteria for assessing viability of species conservation targets in the WEFCOM
Ranking 1/
Very good: functioning
at an ecologically desirable
status, and requires little
human intervention
Ecological indicators
Size 2/
The extent of likely or most
likely suitable habitats can
accommodate >80% of viable
population size.
Good: functioning
within its range of
acceptable variation; it may
require some human
intervention
Fair: lies outside its
range of acceptable variation
and requires human
intervention

accommodate 60-80% of viable
population size.
Poor: restoration or
preventing extirpation
practically impossible

accommodate <40% of viable
population size.

accommodate 40-60% of viable
population size.
Landscape context 2/
Highly connected, the
suitable habitats (patch > 2 km2)
are surrounded by intact natural
vegetation ( >60% of intact forest
within 2 km).
Moderately connected, the
are surrounded by moderately
intact natural vegetation (> 4060% of intact forest within 2 km).
Moderately fragmented, the
are surrounded by altered
vegetation (> 20-40% of intact
forest within 2 km).
Highly fragmented, the
are entirely or almost entirely
surrounded by altered vegetation
and human-induced land use (<
20% of intact forest within 2 km).
Remarks: 1/ described by TNC (2000); 2/ defined by the planning team and wildlife experts
3.4. Delineate the Congregation Areas of Target Species

The current congregation areas were simply derived from combining the likely and most
likely suitable habitats of all target species. The output grid map shows the current location of
suitable habitats of focal conservation target species. On the other hand, the extend of suitable
habitats for each species to desired level of population viability was obtained from expanding
the existing suitable habitat to meet the expected size based on the probability values as
defined in the previous step. Later, all desired suitability maps were aggregated as done for
the current status.
4. RESULTS AND DISCUSSION

4.1. Selected Target Wildlife Species
Five wildlife species were selected by wildlife scientists and a planning team as the
conservation targets. These species were tiger, Asian elephant, gaur, banteng, and sambar
based on the selection criteria and appropriate observations to run a logistic model
(Pattanavibool et al., 2003; Vanichbancha, 2001). The observations for elephant, sambar,
gaur, tiger, and banteng were 960, 700, 641, 224, and 131 points, respectively.
292
Tiger, elephant and banteng are classified as endangered species by IUCN (2000).
According to Lekakul and McNeely (1977), tigers are found in a very wide range of habitat
types; they only require sufficient prey species, water and shelter from the sun and occupy a
large home range (50-200 km2), the average size of which in Thung Yai is approximately 80
km2 (Prommakul, 2003). They also require good protection from tiger and prey poaching
pressures (Karanth et al., 2004). In addition, Simcharoen et al. (2007) used photographic
capture-recapture sampling to estimate tiger density in the core area of Huai Kha Khaeng.
The sample area yielded a density estimate of 3.98 tigers per 100 km2. Elephant is a top
herbivore and found in a variety of forested areas. The home range sizes of elephant herds are
between 105 and 320 km2 (Sukumar, 1989). Similarly, banteng lives in loose herds of 2-20
individuals. Prayurasiddhi (1997) reported that the annual home range size was 44 km2 for
banteng herds in Huai Kha Khaeng. Gaur is classified as a vulnerable species by IUCN
(2000). Gaur live in herds of 3-40 individuals and the home ranges of gaur herds are between
29.9 and 52.1 km2 (Conry, 1989). Sambar still exist in many protected areas in Thailand and
make a significant contribution to the long-term integrity and conservation values of the
WEFCOM. It is a preferred prey species of several carnivores, including tiger.
4.2. Suitable Habitat Models

The results of logistic multiple regressions indicated that three physical factors and two
anthropogenic factors were significantly related to the distributions of elephant, sambar, gaur,
tiger, and banteng. The environmental factors were elevation, slope, distance to stream,
distance to village, and distance to ranger station. The logistic regression models and overall
accuracy at cut-off of 0.5 are shown below.
Z
sambar
1.5191 0.0009Alt -0.0002Rst - 0.0136Slp
0.0003Str+0.0003Vil; overall accuracy 68.06%

Z
banteng =
-1.3795 - 0.0018Alt -0.0008Rst - 0.0939Slp
0.0005Str + 0.0020Vil; overall accuracy 91.97%

Z
gaur
-3.3434 - 0.0042Alt -0.0001Rst - 0.0946Slp

Z
elephant
1.4603 - 0.0013Alt -0.0001Rst -0.1109Slp
0.0003Str +0.0002Vil; overall accuracy 83.32%

Z
tiger
1.1335 + 0.0024Alt -0.0003Rst - 0.1327Slp

where
Alt = altitude (m);
Rst = distance to ranger station (m);
293
Slp = slope (%); Str = distance to stream (m);

Vil = distance to village (m).
The GIS habitat models indicated that most species prefer to inhabit low altitude, close to
ranger stations, low slope, close to streams and far from villages. These areas are safe from
illegal poaching and other human disturbances with near year-round water sources. The
overall accuracies for all species except sambar, were greater than 83%, being especially high
for banteng because its habitat is concentrated in small patches. For sambar, the model
indicates low accuracy because it uses various vegetation types. In addition, the pseudoabsent data may contain occurrence locations. Figure 2 shows the likelihood of habitat uses of
the five focal species in the WEFCOM, while Table 2 presents the coverage of each suitable
class. The distributions of each species are detailed below.
Suitable habitats of sambar (likely and most likely) are found in deciduous forest and
open areas in the core area and in the southern part of the WEFCOM, covering approximately
8,032 km2 or 43% (Figure 2a). Sambar is unlikely to be present in densely populated areas
and the steep terrain mainly in the northern and western parts of WEFCOM. Banteng or wild
ox is now restricted to small and fragmented populations congregated along Huai Kha
Khaeng stream. Other possible patches are in Khao Laem and Salakpra (Figure 2b). The
suitable habitats cover less than 4% of the complex. The main threats to this species are
poaching, habitat destruction and human encroachment, and overgrazing by domestic cattle
(Prayurasiddhi, 1997). Besides, the habitats of banteng have been degraded in recent years
due to the RFD and Department of national Park, Wildlife and Plant Conservation (DNP)
have implemented the policy of 100% forest fire prevention nationwide, especially in
protected areas. This misunderstood concept influenced vegetation community dynamics and
dependent fauna. Parts of open deciduous forests and grassland which are favorable habitats
for ungulate species have been invaded by pioneer species such as Cratoxylum spp. and
Eupatorium odoratum.
Table 2. Predicted suitable habitats for selected wildlife species in the WEFCOM
Species
Sambar
Banteng
Gaur
Elephant
Tiger
1/
Extent of Suitability Class (%/km2)

Unlikely
Less Likely
37.9
19.2
7,099
3,596
89.4
7.3
16,731
1,362
41.1
17.1
7,699
3,211
26.6
21.7
4,951
4,058
63.6
16.7
11,922
3,126
= Likely suitability + most likely suitability.
Likely
14.6
2,736
2.6
492
12.8
2,390
18.3
3,425
9.3
1,736
Most Likely
28.3
5,296
0.7
140
29.0
5,427
33.6
6,292
10.4
1,944
Suitable habitat 1/
42.9
8,032
3.4
632
41.7
7,817
51.9
9,717
19.7
3,680
Figure 2. Current suitable habitats of selected species in the WEFCOM based on probability values.
Figure 3. Comparison of congregation areas of suitable habitats for all species in current condition and desired condition.
Table 3. Assessing population viability in WEFCOM
Species
target
Sambar
Banteng
Good
Very good
Current
Rating 2/
Desired
Rating 3/
3,0004,500
4,5006,000
6,000-7,500
Very good
Very good
<20%
20-40%
40-60%
> 60%
Very good
NA 5/
<1,700
1,7002,600
2,6003,500
3,500-4,400
Poor
Fair
Category
Key attribute
Indicator
Size
Minimum
dynamic area to
support viable
population
(MVP = 3,000;
HR4/ = 1.7-4.0
km2 (Sankar
1994);
MDA = 7,500
km2)
Habitat
fragmentation
The extent of
likely or most
likely suitable
habitats in
km2
<3,000
% intact forest
surrounding 2km buffer of
suitable
habitats
The extent of
likely or most
likely suitable
habitats in
km2
Landscape
context
Viability Assessment 1/
Poor
Fair
Size
Minimum
dynamic area to
support viable
population
(MVP = 500;
HR4/ = 44
km2//herd
(Prayurasiddhi,
1997);
MDA = 4,400
km2)
Table 3 Continued
Species
target
Gaur
Category
Key attribute
Indicator
Poor
Fair
Good
Landscape
context
Habitat
fragmentation
<20%
20-40%
40-60%
Size
Minimum
dynamic area
to support
viable
population
(MVP = 2,000;
HR4/ = 29.9
52.1 km2/herd
(Conry, 1989);
MDA =
13,300 km2)
Habitat
fragmentation
% intact forest
suitable
habitats
The extent of
likely or most
likely suitable
habitats in km2
<5,320
5,3207,980
<20%
20-40%
Landscape
context
% intact forest
suitable
habitats
Current
Rating 2/
Desired
Rating 3/
> 60%
Very
good
NA
7,98010,640
10,64013,300
Fair
Good
40-60%
> 60%
Good
NA
Very good
Table 3 Continued
Species
target
Elephant
Poor
Fair
Category
Key attribute
Indicator
Size
Minimum
dynamic area
to support
viable
population
(MVP = 500;
HR4/ = 105320 km2/herd
(Sukumar,
1989);
MDA =
20,000 km2)
Habitat
fragmentation
The extent of
likely or most
likely suitable
habitats in km2
<8,000
% intact forest
suitable
habitats
<20%
Landscape
context
Current
Rating 2/
Desired
Rating 3/
16,00020,000
Fair
Good
> 60%
Very
good
NA
Good
Very good
8,00012,000
12,00016,000
20-40%
40-60%
Table 3 Continued
Species
target
Tiger
Poor
Fair
Category
Key attribute
Indicator
Size
Minimum
dynamic area
to support
viable
population
(MVP = 100;
HR4/ = 50-200
km2
(Prommakul,
2003);
MDA = 8,000
km2)
Habitat
fragmentation
The extent of
likely or most
likely suitable
habitats in km2
<3,200
% intact forest
suitable
habitats
<20%
Landscape
context
Current
Rating 2/
Desired
Rating 3/
6,4008,000
Fair
Good
> 60%
Good
NA
Good
Very good
3,2004,800
4,8006,400
20-40%
40-60%
1/ Viability assessment is based on measurable size and landscape context criteria; 2/ at present time; 3/ in the next 10 years; 4/ home range; 5/ Not applicable.
300
Figure 2c reveals that the distribution of gaur is more limited than that of elephant
(Figure 2d) because it is more sensitive to human pressure. The suitable habitats cover
approximately 42% of the WEFCOM area and it is most likely found in the core area. On the
other hand, the chance to observe gaur in other protected areas is minimal. In fact, elephant
can be found in a variety of habitats (Sukumar, 1989) but the GIS habitat suitability map
shows that the current suitable habitats are basically situated in the core area of WEFCOM
and areas along the Myanmar border (Figure 2d), covering approximately 9,717 km2. Areas in
the west, east, north and parts of southern landscapes are not suitable for elephant because
there are a lot of human settlements and they are easy accessible. Figure 2e shows that the
most suitable habitats for tiger can be found in Huai Kha Khaeng followed by Thung Yai East
and Thung Yai West. The total suitable areas cover approximately 20% of the WEFCOM.
The results are consistent with the studies of Prommakul (2003) and Simcharoen et al. (2007)
which revealed that the home range of tigers in Huai Khai Khaeng is smaller than in Thung
Yai due to higher abundance of prey species.
4.3. Current and Desired Conditions of Population Viability

The existing suitable habitats of sambar encompass approximately 8,000 km2, thus they
have potential to hold more than 3,000 individuals. On the other hand, the suitable habitat of
banteng is minimal and ranked as poor condition (Table 3). It can accommodate less than
40% of a viable population size (500 individuals). However, the largest suitable habitat patch
is located in the core area of Huai Kha Khaeng and surrounded by dry diptercarp forest and
well protected by park rangers. Even though the actual population size of gaur is not known,
the existing suitable habitats (7,800 km2) are assumed to accommodate approximately 1,0001,200 individuals (40-60% of viable population). In addition, the adjacent area within a 2 km
buffer is highly intact. Thus, the size criterion is classified as fairly viable, while landscape
context is in good condition.
The existing suitable habitat of elephant covers about 9,700 km2 and could hold
approximately 240-250 individuals. The size criterion is ranked as fair. However, the
landscape matrix is highly connected and has very high potential to facilitate elephant
movement in the WEFCOM landscape. The suitable habitats of tiger encompass approximate
3,700 km2. Based on the average home range size reported by Prommakul (2003), the current
suitable habitats are likely to accommodate approximately 40-50 tigers, thus, they are in fair
viability status but the landscape context outside occurrence patches is moderately intact with
natural vegetation.
The project aims to upgrade the viabilities of all species (except sambar) to the next level
in the next 10 years. In order to achieve this target, park rangers and multi-stakeholders have
to increase the amount of suitable habitats for banteng, gaur, elephant and tiger to an area
between 1,700-2,600 km2, 7,980-10,640 km2, 16,000-20,000 km2, and 6,400-8,000 km2,
respectively (Table 3).
301
4.4. Location of Congregation Areas of Population Viability

We overlaid the current suitable habitats of all five species using GIS method to derive
current congregation areas, and the desired suitable habitats to derive desired condition to
support viable populations. The results showed that the total area of current congregation
habitats cover approximately 12,630 km2 or 67% of the WEFCOM (Figure 3a).
Concentrations of five focal species are clustered in three places. The largest patch is located
in Huai Kha Khaeng and adjoining Thung Yai Naresuan. On the ground, the survey teams
observed that there are a number of migratory routes of the landscape species e.g. tiger,
elephant, gaur found in these areas. The second dominant area is located in the western part of
WEFCOM extending along the Myanmar border. The smallest patch can be found in the
north.
Meanwhile, the desired congregation habitats cover nearly 16,750 km2 or 89% (Figure
3b). Basically, these areas cover the whole of Huai Kha Khaeng, 80% of Thung Yai East and
West and extend to the Myanmar border. In the north, the size of small and fragmented
patches increases significantly. The desired areas could hold more wildlife population, in
addition to connect fragmented and patchy suitable habitats. However, areas along the
western boundary and in the south close to villages are still not feasible to support viable
populations.
CONCLUSION
The WEFCOM project aims to maintain the health of the ecological systems by using
species conservation targets. Five species were selected, namely sambar deer, banteng, gaur,
elephant and tiger. Their habitat suitability areas were determined using logistic multiple
regression method. The results of GIS habitat models indicated that most species prefer to
inhabit low altitude, close to ranger station, low slope, close to stream and far from village
locations. The suitable habitats (likely and most likely) of sambar deer, banteng, gaur,
elephant and tiger covered approximately, 43%, 3%, 42%, 52% and 20% of the WEFCOM
landscape, respectively.
The MVP of sambar, banteng, gaur, elephant and tiger in the WEFCOM defined by the
planning team and wildlife experts were 3,000, 500, 2,000, 500 and 100 individuals,
respectively. We used the MDA or suitable habitats to accommodate MVP population and
habitat fragmentation surrounding the suitable to assess the viability of target species. We
compared the MDA and predicted suitable habitat derived from the GIS habitat modeling and
found that the viability of sambar is in very good condition, while the viability status of gaur,
elephant and tiger is in fair condition. More importantly, the current viability status for
banteng is extremely poor. However, landscape matrices outside the suitable habitats are in
good or very good condition for all species. The planning team of WEFCOM would like to
upgrade the level of viabilities of these species to next level in the next 10 years. In order to
achieve these targets, park rangers and multi-stakeholders have to increase the congregation
habitats from 12,630 km2 or 67% of the WEFCOM to 16,750 km2 or 89%. By doing this, the
fragmented suitable patches would become more aggregated.
302
The results of GIS modeling may include omission errors (false negative) and
commission errors (false positive) derived from pseudo absence data and the grid-based
habitat suitability models. In addition, the predicted MDA may overestimate the actual
species extent. Nevertheless, the results of this initiative are of value and the approach can
become effective tool to implement ecosystem approach when demographic data is limited to
do perform traditional population viability analysis.
ACKNOWLEDGMENTS
We would like to thank the Danish Government for funding the Western Forest Complex
for Ecosystem Management (WEFCOM) Project. In addition, we are grateful to the
Superintendents and the staff of the WEFCOM Project for providing information and The
Nature Conservancy (TNC) for training on the Five-S Framework. Adrian Hillman is
acknowledged for his contribution to edit the manuscript.
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D.C.

ISSN: 2158-5717
Chapter 11
PROTECTION OF RIPARIAN LANDSCAPES IN ISRAEL

Department of Geography and Environment,
Bar-Ilan University, Ramat-Gan, Isral
ABSTRACT
Riparian landscapes are natural habitats of unique ecological and scenic values,
which are highly sensitive to human intervention and impact. Yet, due to their qualities,
and especially the presence of water, they are also usually attractive for recreation
purposes. This is more so in arid and semi-arid zones like Israel. Nevertheless, in the
past, the importance of riparian landscapes in Israel did not receive adequate attention in
policy and planning. As a result, over the years they were exposed to various negative
impacts, including pollution by industrial and agricultural effluents, exploitation of water
for agricultural and other purposes, and land use conflicts. Although in recent years with
the growing awareness of their ecological and recreational potential, considerable efforts
are being invested in the rehabilitation of deteriorated riparian landscapes, their
protection is still deficient.
This chapter reviews and examines policy tools used for the protection of riparian
landscapes in Israel, based mainly on regulations, reports and existing literature. It
concludes by offering some lessons for policy-making in general and suggestions for
improving the protection of riparian landscapes in Israel in particular.
Keywords: environmental policy, river corridors, conservation, open spaces.
INTRODUCTION
Riparian landscapes are natural habitats of unique ecological and scenic values, which are
highly sensitive to human intervention and impact (Goren, 2000). Yet, due to their qualities,
and especially the presence of water, they are also usually attractive for development as well
as for recreation purposes. This is more so in arid and semi-arid zones like Israel (Burmil,
306
1999; Patten, 1998). Nevertheless, in the past, the values of riparian landscapes in Israel did
not receive adequate attention in policy and physical planning. As a result, they were exposed
over the years to various negative impacts, including pollution by industrial and agricultural
effluents and household sewage, exploitation of water for agricultural and other purposes,
political disputes and land use conflicts (Amir-Shapira and Feldman, 1999; Gabbay, 1998). In
recent years, mainly since the year 2000, with the growing awareness of their ecological and
recreational potential, considerable efforts are being invested in the rehabilitation of
deteriorated riparian landscapes. Nonetheless, according to a recent report most of the rivers
in Israel and their surroundings still suffer from pollution and other negative impacts (AmirShapira, 2007).
This chapter reviews the protection of riparian landscapes in Israel. It presents the policy
tools used for protection of riparian landscapes up to the year 2000, examines their effectivity
and reveals embedded deficiencies. The methodology is based on the review and content
analysis of various sources, mainly laws, reports and literature. The first part of the chapter
introduces the importance of riparian landscapes in general, covering among others
ecological, environmental and scenic aspects. It than presents the subject of riparian
landscapes in Israel and the factors that affected policy and priorities concerning the use of
water in general and protection of riparian landscapes in particular. The second part reviews
and examines protection tools, focusing on legislation, institutional structure and physical
planning. The third and last part of the chapter discusses the deficiencies and weaknesses that
were revealed and proposes improvements to the existing state.
THE IMPORTANCE OF RIPARIAN LANDSCAPES

According to Naiman and Dcamps (1997) a riparian zone encompasses the stream
channel and that adjacent portion of the terrestrial landscape from the high water mark toward
the uplands, where vegetation might be influenced by elevated water tables or flooding. The
width of a riparian zone and the diversity of its functional attributes are related to the size of
the stream, its position within the drainage network, the hydrologic regime and the local
geomorphology (Gregory et al., 1991; Naiman and Dcamps, 1997). In other words, a
riparian zone may be regarded as a linear physical landscape entity composed of aquatic and
terrestrial components and the interface between them. Riparian landscapes are unique
ecosystems, highly important ecologically and environmentally, and they constitute a visually
and functionally outstanding component of the open space system. They form complex
habitats, richer than the average with species of plants, animals and microorganisms, thus
contributing to overall biodiversity (Gregory et al., 1991; Naiman et al., 1993). These habitats
and the richness of species they sustain are especially sensitive to various impacts and
interferences, including seasonal or other changes in water physical qualities (like
temperature, salinity or electric mobility) or quantities (Allan, 2004; Goren, 2000; Karr, 1994;
Naiman and Dcamps, 1997; Pollock et al., 1998; Shandas, 2007; Stauffer and Best, 1980).
They are considered the most diverse, dynamic and complex habitats (Goren, 2000; Gregory
et al., 1991; Naiman and Dcamps, 1997).
Riparian ecosystems perform a variety of environmental and ecological services (Allan,
2004; Brauman et al., 2007; Hale and Adams, 2007; Naiman and Dcamps, 1997; Patten,
307
1998). Many of these are considered life-supporting systems (De Groot, 1992; Naveh, 1997),
although they are difficult to evaluate in economic terms (Chavas, 2000; Constanza, 2000).
Due to their linear nature, riparian landscapes are natural ecological corridors, allowing
connectivity between habitats and patches in the landscape (Beier and Noss, 1998; Bentrup
and Kellerman, 2004; Jongman, 1995; Machtans et al., 1996; Naiman et al., 1993; Ndubisi et
al., 1995; Shkedi and Sadot, 2000; Taylor et al., 1995; Walmsley, 2006; Weber et al., 2006).
This is especially important in populated areas, where riparian landscapes may be the last
remnants of an open natural space within a built-up area. Another function that is especially
important in populated areas is their potential as catchement basins for floods, thus avoiding
damage to property and lives (Brody et al., 2007; McHarg, 1969; State Comptroller, 1993).
They are also attractive for recreation and leisure activities, and due to their linear form are
natural candidates for greenway planning, combining opportunities for recreation with
conservation of nature, landscape and heritage values (Ahern, 1995; Baschak and Brown,
1995; Bryant, 2006; Fbos, 2004, 1995; Jim and Chen, 2003; Lewis, 1964; Li et al., 2005;
Little, 1990). Open lands along rivers and streams are also often used for agriculture, because
of their fertility, due to embedded sediments, and relatively level plains (Bentrup and
Kellerman, 2004; Karr, 1994).
Riparian landscapes are extremely vulnerable to human impact. Surface water from all
over the drainage basin might carry with it soil particles and various pollutants, including
pesticides, fertilizers and agricultural wastes into the stream. Their potential for conservation,
recreation or agriculture is diminished by conflicts and liabilities from various sources.
Pollution by industrial and agricultural effluents and overflow of sewage from treatment plans
harms the landscape, disqualifies water for irrigation, prevents recreational water-related
activities (e.g. swimming and boating) and repels potential users in general. Exploiting river
water for irrigation decreases the availability of water for natural system functions. Though
riparian landscapes are attractive for development and recreation, they may be contradictory
to certain types of agricultural uses. Moreover, floods may cause damage to agriculture and
other uses within the flooded area.
Nevertheless, the presence of water is a potentially dominant factor that attracts
development. Unfortunately, often the resulting development ends up with the construction of
buildings and infrastructure too close to the water line, thus interfering with the ecological
functions of the riparian system.
Considering their uniqueness and attractiveness on the one hand and their vulnerability to
various impacts on the other, riparian landscapes are in need of effective protection from
inappropriate uses. In Israel, a hot and dry land, such landscapes are especially important. The
next section describes the factors that affect rivers and riparian landscapes in Israel.
THE RIVERS IN ISRAEL

The priorities concerning water and riparian landscapes in Israel stem from physical
conditions mainly geomorphology and climate combined with ideological and
geopolitical factors.
Israel is situated on the eastern side of the Mediterranean basin, with geomorphology and
climate changing both along the north-south axis and the east-west one. The north and center
308
of the country lie within the Mediterranean climate zone, characterized by a short rainy winter
and a long, hot and dry summer while the south of the country is more desert-like. The
mountainous northern areas are the rainiest with an average of 800-1000 millimeters per year,
decreasing in the central zone the a yearly average of 500-600 millimeters. Eastward and
southward the average decreases to 100 millimeters and less. These quantities may fluctuate
from year to year, and, in addition, there is high occurrence of droughts. All these factors add
up to a continual condition of water shortage (Menahem, 1999).
The area of the State of Israel is intersected longitudinally by a series of mountain ranges
that divide it into an eastern basin, where rivers flow towards the Jordan River, the Sea of
Galilee and the Dead Sea, along the natural eastern borders, and a western basin, where rivers
flow into the Mediterranean Sea. The western basins climate is milder and relatively rainy,
while the eastern basins is hot and dry. Due to the limited rain quantities, many river sections
are in fact seasonal streams, flowing during winter and spring, and drying out in the summer
until the next rainy season. In the dry south, the streams are characterized by short strong
flows following rain events, which dry out quickly. Only a few rivers that are fed by yearround springs have water flowing in all seasons.
Seasonal streams are exceptionally sensitive because of the fluctuations in water
availability for habitat performance in addition to the fact that some of them do not enjoy a
strong flow even in the rainy season. These characteristics affect their public conceptual
image as landscapes fit for conservation or suitable for recreation purposes. River sections
that run through areas declared as nature reserves or national parks are protected, along with
the whole area, under the National Parks and Nature Reserves Law of 1963. However, a river
or stream crossing an area that is not characterized by special scenic or nature values were
usually not grasped as deserving protection. Consequently, they are under pressures for
development, especially in areas of level topography, such as the inner and the coastal plains.
The most important rivers, considering water discharge amounts and landscape impact
are in Israels western basin, crossing the coastal plain where the majority of the population
(about 80%) is concentrated. These also have the highest potential for recreation. However,
their proximity to densely settled areas has resulted in their deterioration due to various
negative impacts, such as: trapping riparian waters for agricultural use and other purposes,
pollution by agricultural and industrial effluents, overflow of sewage from treatment
facilities, construction or agricultural cultivation close to the water line, neglect and garbage
disposal (Amir-Shapira and Feldman, 1999).
In addition to this, because of the scarcity of water resources and frequent droughts, the
national water policy focused on keeping control over all water sources and prioritizing the
use of water for agriculture and other uses. For example, since the mid 1950s, most of the
water from the Yarkon springs was captured and transported to agricultural fields in the
Negev (the southern region of the land) through the Yarkon-Negev pipeline; as a result, the
Yarkon River, the main river in the core, densely-populated area of Israel - once a wide deep
stream that the British soldiers had to cross by boats when conquering the land from the
Turks in the year 1917 deteriorated into a narrow and shallow stream, where most of the
flow consisted of industrial effluents and initially-treated sewage. The negative impacts on
riparian landscapes call for examination of existing protection and management tools and
their effectivity.
309
PROTECTION OF RIPARIAN LANDSCAPES IN ISRAELI LEGISLATION

The protection of landscapes by legislation is a universally accepted model for the
conservation of outstanding scenic or natural values (Maruani and Amit-Cohen, 2007).
Legislation is a source of authority and constitutes a strongly effective policy tool. Yet this
tool has its drawbacks, among them low flexibility, requiring a long, complicated
bureaucratic process whenever a change in legislation is desired. This is also a source of
conflict between conservationists and developers and landowners. On the other hand,
protection based on a statutory declaration is stronger than any other protection tool, and
provides the relevant authorities with enforcement measures.
In Israel, legislation concerning water resources, including rivers, is complex, involving a
multitude of laws and regulations as well as a multitude of authorities and organizations that
are meant to enforce them (Kaplan, 2004). This section presents three laws that are the most
relevant to the protection of riparian landscapes, as follows: the Water Law, the Drainage
Law and the Rivers Authorities Law.
The Water Law

The Water Law, enacted in 1959, should be understood in the context of the general
scarcity of water resources in Israel, the relative average low rainfall and the frequent
droughts, on the one hand, and the centralistic governance style and intensive involvement of
the State, on the other (Menahem, 1999). The main objectives of the law were to secure the
States sovereignty over all water sources and ensure their utilization for the benefit of Israeli
society. The law determines that the water sources in the State are owned by the public,
controlled by the State and designated for its population and development needs. The water
sources for that matter are the springs, the streams, the rivers etc. (Water Law, sec.1-2).
The law regulates the management of water sources, including allocation for users and
preservation of water quality. For these purposes, the law created several institutions, among
them a National Water Board, headed by the Water Commissioner. The law gave priority to
the agricultural sector, which at the time was regarded not only as a leading sector in the
national economy but also as the ideological and political elite of Israeli society (Menahem,
1999; Schiffman, 1999). This was reflected in various attributes of the law, among them its
subordination to the Minister of Agriculture, including the power to appoint the Water
Commissioner, who was to lead the design and implementation of water policy in Israel.
Thus, it is no wonder that the first seven elected commissioners were from the agricultural
sector, as were also most of the National Water Board members (Menahem, 1999). Only forty
years later, in the 1990s, following ideological, political and social change processes in Israeli
society, including the decrease in the economic and ideological importance of agriculture and
the acceleration of development (Maruani, 2005; Schiffman, 1999), the Water Law was
transferred to the authority of the Minister of National Infrastructures and some of its powers
were split among several other institutional structures. Laster (2004) claims that this split in
authorities is the reason for the decrease in water quality, in contrast to Western Europe where
a centralistic approach was adopted.
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The Water Commissioner, among others, used his powers collect water from springs and
rivers, and allocate it to consumers, thus reducing the natural flow, ignoring the ecological
consequences, which, in turn, also had a negative impact on the attractiveness and availability
of the surrounding area for recreation (Laster, 1995).
The only reference in the Water Law to the protection of land near water sources is the
available option to define buffer strips for purposes such as protection of water, of water
source , that are wide no more than is necessary for the purpose of the buffer strip
(sec.14-15). No references are made to purposes involved in the protection of landscape or
other values embedded in the terrestrial area outside the water itself.
The Water Law reflects a utilitarian approach towards water resources, and almost
completely ignores other values. Some years ago the law was amended, and to the list of uses
for water allocation was added the protection and rehabilitation of nature and landscape
values, including springs, rivers and wetland habitats (sec.6). However, there is no other
reference to such a goal anywhere else in the law, and its implementation is totally dependent
on the awareness and the good will on the part of the decision makers. It should also be noted
that the interests of the Head of the Water Authority (who replaced the original Water
Commissioner, following an amendment in 2006) and the Ministry of National Infrastructures
do not coincide with the interests of landscape conservation. This was even truer in the past,
when the main interest of the Water Commissioner and the Ministry of Agriculture was the
supply of water to the agricultural sector.
The Drainage Law

The Drainage Law (or in its full name: the Drainage and Flood Protection Law) was
legislated in 1957, after floods in the mid 1950s covered vast areas, mainly agricultural fields,
causing severe economic damage. The objective of the law was to prevent the recurrence of
floods by forming suitable institutions to manage drainage. This law, too, was subordinated to
the Minister of Agriculture and still is - since agricultural lands were conceived as being
most threatened by potential floods. The priority given to agriculture is expressed in the first
section of the law, which defines drainage as any operation intended to concentrate, to
store, to carry or to remove surface or any other water which harm or may harm agriculture,
public health etc.
The Drainage Law states that the Minister of Agriculture may establish a drainage
authority (Drainage Law, sec.11), but the establishment of such an authority is not
obligatory. The law also specifies the duties and powers of such an authority, including
regulating drainage and initiating drainage projects within its jurisdiction. The roles of a
drainage authority as they are specified and defined in the law do not refer to the protection of
the relevant riparian landscapes in any other way. In addition to drainage authorities, the law
determines the establishment of a National Drainage Board to advise the Minister on matters
of drainage, such as the declaration of drainage zones or the approval of drainage projects
(sec.2). This is an obligatory body, complementing the relevant institutional structure for
regulating drainage and preventing floods.
The Drainage Law defines some relevant terms for its purposes including channel and
buffer strip. A Channel is defined as river, stream and any other water route where
water is flowing or standing always or occasionally. A buffer strip is defined as strips of
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land along both sides of a channel (sec.1). The law prohibits agricultural cultivation or
construction within a buffer strip. However, according to the law, the overall width of buffer
strips on both sides should not exceed half the channels width, and no more than 5 meters
each (sec.5-6). It is needless to say that the potential protection of the riparian habitat is very
limited given such conditions.
The Drainage Law, like the Water Law, expresses the priority given to agriculture. Most
drainage projects during the 1960s and 1970s were intended to solve drainage problems in
agricultural and open areas, although in populated areas potential damage from floods to
property and persons is much higher. Moreover, since the legislation of the Drainage Law in
1957, the scope of development for residential, occupational and infrastructure uses increased
greatly, much of it was at the expense of open and agricultural lands where rains could have
previously penetrated the soil. As a result, the amounts and intensity of surface flow towards
rivers increased considerably, and with them the risk of floods. In spite of that, the Drainage
Law was not updated to include instructions regarding measures for the control of drainage in
new development plans. This neglect resulted in recurrent flood events in various areas,
causing millions dollars worth of damage (Laster, 2004; State Comptroller, 1993, 1999b).
The Rivers Authorities Law

The Rivers Authorities Law (RA Law: the full name being Rivers and Springs
Authorities Law) from 1965 complements the Water Law and the Drainage Law by referring
not only to the protection of the water but also to the land adjacent to it, thus expressing the
values of riparian landscapes for the first time in Israeli legislation. This expression is also
reflected in the ministerial subordination; when legislated, the RA Law was subordinated to
both the Ministry of Interior and the Ministry of Agriculture; however, it was transferred to
the Ministry of Environment after it was created in 1989.
The RA Law states that the Minister may establish an authority for a certain river
or part of it or impose on a drainage authority powers of a river authority (RA Law,
sec.2). That is to say, a river authority like a drainage authority is not obligatory. Among
the duties of a river authority, the law counts protection of the landscape and nature values
along the river on both sides but also regulation of the rivers water flow and drainage
within its jurisdiction (sec.3). This means that there is an overlap between the duties of a
Drainage Authority and that of a River Authority, albeit they are related to different
ministries. Laster (2004) claims that the law was intended to manage the rivers on a
watershed approach basis, but actually it did not enable that, since it demanded subordination
to the Water Law and the Water Commissioner. Moreover, the jurisdiction of a River
Authority does not necessarily cover a whole watershed. For example, the jurisdiction of the
Yarkon River Authority was limited to 20 meters on each side of the river.
The RA Law was implemented for the first time in the year 1988 23 years after its
legislation with the establishment of the Yarkon River Authority. It took six more years to
establish the Kishon River Authority, in 1994, which was the second, and, upto-now the
latest authority based on this law. Laster (2004) comments that the reason it took so long to
implement the law was the original subordination to two ministers. Another criticism of the
law relates to the absence of a national body, something like the National Drainage Board. In
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other words, the RA Law represents a particularistic approach, referring to each river
separately and lacking a broad vision on a national scale.
INSTITUTIONAL STRUCTURE
While legislation is a source of regulative powers, there is a need for institutions to use
these powers and enforce the regulations. In Israel, there are several institutions that have to
do with management and protection of riparian landscapes, the most important of them being
the Drainage Authorities, the River Authorities and the Rivers Administration. They differ by
their status, composition, main objectives, powers and budgeting.
Drainage Authorities
Drainage Authorities (DAs) may be established by the Minister of Agriculture, as already
mentioned, subject to the consent of the Minister of Interior and the relevant local
municipalities, which are also represented in its composition. The DA is responsible for
managing drainage zones within its jurisdiction, including initiating, developing and
maintaining drainage projects. The powers of a DA may contribute greatly to the protection
of the relevant riparian landscapes, since managing drainage requires, among other things,
restrictions on development along the water route.
Following the legislation of the Drainage Law, the Minister of Agriculture issued an
ordinance in 1960 establishing 26 DAs. The ordinance specified the jurisdiction allocated for
each DA, generally consisting of low-elevated plains, mainly agricultural lands (State
Comptroller, 1993). Laster (1995, 2004) comments that the large number of DAs was due to
political pressures rather than hydrological needs. Most of them actually operated as organs of
the relevant Regional Councils - which are the local municipalities in the rural zones and
were dominated by representatives of the agricultural sector, thus reinforcing its control over
water sources.
The recurrence of severe flood events implies the existence of shortcomings and
deficiencies in the Drainage Laws implementation in general and in DAs operation in
particular. Especially memorable are the floods of winter 1991-2, when river overflows
flooded large areas all around Israel, disrupting the course of everyday life: people had to be
temporarily evacuated from their homes, roads became impassable, large agricultural areas
were under water and there were even some fatal casualties. The overall direct and indirect
damage to households, businesses, public property and agriculture amounted to tens of
millions of dollars. Following those events, the State Comptroller (1993) issued a report that
pointed out various faults in the DAs functioning, stating among others that the drainage
infrastructure had been neglected over the years and the sums required for proper planning,
regulating and maintenance of many rivers were not allocated and invested as should have
been done.
In 1996, after studying the State Comptrollers report, the Minister of Agriculture issued
an ordinance reorganizing the DA system. Their number was decreased from 26 to 11, each
covering a jurisdiction overlapping a natural drainage basin, and together covering the whole
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country. Each DA is directed independently of the Regional Council system, intended to

apply a whole watershed approach rather than serve local interests (Laster, 2004). Although
since this reorganization floods have reccurred, it seems that - especially since 2000 - DAs are
taking more active measures to prevent flooding, revealing a more environmentally-oriented
approach, including the conservation of landscape and ecological values in the riparian zones,
in collaboration with other institutions and organizations, such as the Ministry of
Environment, the Nature and Parks Authority and the Society for the Protection of Nature
(Carmel Drainage Authority 2004).
Rivers Authorities
A River Authority (RA), with powers to limit and control development along the river, is
a potentially effective tool in the protection of riparian landscapes. However, this potential
cannot be fully realized, partly due to constraints imposed by the RA Law, such as the limited
size of the area under the RAs jurisdiction. The RAs effectivity is also limited by its
obligation to comply with the Water Authority (RA Law, sec.4), even in cases when their
interests are contradictory. An RAs composition is more complex than a DAs, including
several government officials. Thus, conflicts between government officials and local
municipalities representatives may also hamper the RAs functioning. Another potential
source of conflict is the existence of a DA in the same jurisdiction since the duties and
responsibilities according to the Drainage Law and the RA Law partly overlap.
The RA Law states that an RA is appointed to manage a particular river and is supposed
to operate independently, following its managements objectives and policies. However, there
is lack of a master organization on a national scale to design and lead a comprehensive policy
towards the protection of riparian zones. With the absence of national policy, the influence of
local interests increases, thus allowing development and infrastructure on or adjacent to
riverbanks that should have been preserved.
Nevertheless, an RA is still the only institutional structure that is essentially specified for
riparian nature and landscape protection in Israel. This may be exemplified by the Yarkon
River Authority (YRA), which since its establishment in 1988 has initiated a master plan for
the Yarkon River, seeking, among other goals, to restore the riparian ecosystem and
contribute to environmental quality, aesthetic values and climate amelioration (Rachmimov
1996). The YRA develops bicycle trails and areas for recreation along the river and is
currently monitoring water quality etc. The YRA was, in fact, a pioneer and a model in river
restoration in Israel. Regretfully, however, only one more RA was established till now: the
Kishon River Authority (in 1994). Both the Yarkon and the Kishon are large rivers, passing
through densely populated areas - the Yarkon in the Tel Aviv metropolis, in the center of the
State, and the Kishon in the northern Haifa metropolis - that were in a severely deteriorated
state due to pollution from multiple sources, water trapping, etc. However, several other large
rivers in a similar condition could have profited from a similar specified institutional structure
to manage and restore them.
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The Rivers Administration

The Rivers Administration (RAD) was established in 1993 as a mutual initiative of the
Ministry of Environment and the Jewish National Fund (JNF), the latter being a historical
non-governmental organization that is currently intensively involved in forestry and outdoor
recreation. Other institutions and organizations that take part in the RAD are the Nature and
Parks Authority, the Ministry of Interior, the Ministry of Agriculture, the Water Authority,
the Israel Government Tourist Corporation and the Society for the Protection of Nature which
is a non-governmental organization (Amir-Shapira and Feldman, 1999; Gabbay 1998). The
declared goals of RAD include: conservation of high-quality open spaces along river
channels, controlled development of recreation and tourism foci within riparian zones and
rehabilitation of rivers to a state enabling sustainability of unique habitats and landscape
values (Amir-Shapira and Feldman, 1999). RAD initiates and promotes plans for the
restoration of rivers, issues guidelines for existing and future River Authorities, supports the
establishment of local river administrations and is actually involved in many restoration
projects that are in progress, especially since 2000. For instance, the RAD takes part in the
steering committee of the successful restoration project for the Alexander River a large
coastal river in the Sharon region, and one of the first to enter a restoration program along
with the Yarkon River. This is done in collaboration with many other bodies, including the
Alexander Drainage Authority, the Emek Hefer Regional Council, the Emek Hefer Streams
Society, the Water Authority, the JNF, the Israel Government Tourist Corporation, the Nature
Conservation Research Institute, the Ministry of Environment, the Hadera Environmental
Association, the Nature and Parks Authority, the Society for the Protection of Nature, the
District Planning Commission, the Ministry of Interior and the Israel Lands Administration
(Cohen-Shoel 2003). It is clear that when so many organizations are involved, conflicts and
clashes of interest are unavoidable and may hamper conservation efforts. For example, the
Israel Lands Administration is supposed to manage all public lands, which in Israel amount to
93% of the area of the State. However, since the 1990s, it actually acts as the States main
developer, initiating large intensive development plans wherever possible, driven by
economic interests (Maruani, 2005). Such a development-oriented organization is bound to
confront conservation-oriented bodies like the Nature and Parks Authority and the Society for
Protection of Nature. The RAD, which is guided by a statewide vision regarding rivers and
riparian landscapes, can bridge such controversies and promote restoration and conservation
efforts.
Nevertheless, the protection offered by the RAD is limited since this is not a statutory
body, and consequently lacks regulative and enforcement powers. Whenever action against
polluters or other offenders of the riparian landscape is needed, it is the Ministry of
Environment that is supposed to interfere and take steps towards enforcement. Also, due to
lack of obligatory organizational procedures, RADs operations are spatially and temporally
not consistent in essence, scope and timing. Therefore, in many cases, riparian landscapes in
Israel are still subject to the arbitrariness of local authorities, and are not consistently regarded
as worthy of protection. In addition, the RADs potential effectiveness is reduced by budget
limitations, since budget allocated for riparian management is divided among too many
organizations, including DAs and RAs (Laster, 2004).
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The lack of a statutory basis is a source of instability for the RAD, since it may be
dismantled by a ministerial decision as quickly as it was established. Nonetheless, since its
establishment in 1993, the RAD is a dominant factor in river restoration in Israel.
PHYSICAL PLANNING AS A PROTECTION TOOL

Riparian landscapes are integral parts of the land. As such, physical planning of land uses
at national, regional and local levels is a potentially strong tool for their protection. In Israel,
land uses are determined by a statutory planning system, according to the Planning and
Building Law (PBL) of 1965 (which replaced former British mandatory legislation from
1936). The system is three-tiered, with Planning and Building Commissions at national,
district and local levels, each having the power to control lower-tier decisions. All planning
commissions may initiate outline plans within their jurisdiction.
National Planning
The Sharon Plan: The Sharon Plan was the first comprehensive national master plan
prepared after the declaration of statehood in 1948. The plan was initiated and prepared
within the (then) Planning Department of the Ministry of Labor and Construction, by a team
headed by architect Arie Sharon, and published in 1951. This plan was not statutory;
nevertheless it had significant long-range effects on spatial planning in Israel. The plan refers
to five main facets of planning: agriculture, industry, transportation, parks and new cities. The
agricultural plan is based on a national water policy that wishes to divert water out of
relatively rich sources among them rivers in the north and the Yarkon in the center of the
State and carry them to the dry south, thus enabling agricultural settlements that are
regarded in the plan as a key factor in development and economic independence (Sharon,
1951). In other words, the Sharon Plan again reflects the economic and ideological values
attached to agriculture, and grasps the water of the rivers as input for agriculture and not as a
landscape component to be protected.
The plan for the parks in the Sharon Plan proposed conservation of certain areas of
outstanding nature and landscape values. Four out of those were to be established
immediately as national parks, among them two mountainous landscapes: Mount Carmel and
Mount Jarmak in the Upper Galilee, and two riparian ones: the Falik Park proposed around
the Yarkon and Ayalon rivers in the Tel Aviv metropolis, and a park west of Jerusalem based
on the Sorek River (Sharon, 1951). However, although the Sharon Plan as well as the actual
implementation of the Carmel and the Jarmak parks significantly affected spatial planning for
years to come, the parks proposed around the rivers were ignored as was also the call for
using river corridors as buffers between built-up areas. The Sorek corridor area, for example,
was reduced over the years due to development (Amit-Cohen et al. 2005).
NOP 31: statutory planning in Israel at the national level is delineated in the National
Outline Plans (NOPs). Out of almost 40 NOPs prepared up to date, only two represent
comprehensive national planning. The first of the two was NOP 31, the Combined National
Outline Plan for Construction, Development and Immigration Absorption. NOP 31 was
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prepared and approved in the early 1990s, when Israel was facing a crisis stemming from a
sudden unanticipated increase in population due to mass immigration waves from the former
USSR (Alterman, 1995). This was the first comprehensive national statutory plan to
incorporate environmental considerations on a large scale, stating objectives like:
conservation of nature and landscape resources, preservation of surface water quality,
nurturing open spaces among them riparian landscapes - for recreation, and balancing
between development and conservation (Feitelson, 1993). The plan enhanced intensification
of metropolitan regions in order to preserve open spaces in the rural zone. Despite these good
intentions, it has been claimed that they triggered heavy development pressures in the
metropolitan core of Israel, even in areas designated for conservation (Maruani, 2005).
NOP 35: the Combined National Outline Plan for Construction, Development and
Conservation NOP 35 was approved in 2005, substituting NOP 31, which became obsolete in
1998. This plan, too, aspires to balance between development and conservation, taking this a
step further by dividing the whole country into zones defined as development-oriented or
conservation-oriented in varying degrees. Theplan specifically refers to river strips
including the water route and the banks 100 meters on each side and requires every
statutory plan to issue instructions concerning conservation of the river and its habitats,
protection of its drainage functions, bank stabilization and free access to the public. This
seems promising indeed, but it will take a number of years before its achievements can be
assessed. However, past experience teaches us that NOPs instructions are not always kept.
One example is the National Outline Plan for the Mediterranean coast, NOP 13, which
prohibited construction within 100 meters of the water line. That instruction did not always
stand up development pressures (State Comptroller, 1999a).
Sectorial NOPs: most national outline plans are sectorial, each dedicated to a specific
subject (e.g. roads, power plants, landfills, etc.). None of the plans that have been prepared
until now is designated for the protection of rivers and riparian landscapes, although in some
cases, there may be a reference to rivers where this is relevant to the main plans design. Such
is the case of the National Outline Plan for Nature Reserves and National Parks, NOP 8,
which offers protection to sections of riparian landscapes that are found within areas
designated and declared as a nature reserve or a national park. Yet, the plan does not ensure
protection along the whole water route. The National Outline Plan for Forests and
Afforestation proposes plantings along riverbanks (Kaplan, 1993), but only in small limited
areas, most of them outside of metropolitan zones where demand for recreation is especially
high. The National Outline Plan for Tourism, NOP 12, offers another example. It designates
some rivers as recreational spaces, among them the Sorek River (Amit-Cohen et al. 2005).
Yet, development attractive for tourism is usually intensive and could harm existing riparian
ecological and environmental values.
Regional Planning
District Outline Plans (DOPs), like NOPs, are essentially guiding plans, relating to the
district under the jurisdiction of a relevant District Commission or part of it. Israel is divided
into six administrational districts. Almost all of them had valid DOPs that by the early 1990s
were mostly out-of-date, and none of them conceived riparian landscapes as objectives for
conservation. An example for the disregard of riparian functions is the Ayalon Highway,
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crossing Tel Aviv through the route that once was the Ayalon River, leaving a rather narrow
channel for winter water flow. Planning of this highway started in the early 1970s and it was
opened for use at the beginning of the 1990s. In the winter of 1991/2, parts of it flooded, more
than 140 cars were trapped in the flood, and transportation along the highway had to be
stopped for some time (State Comptroller, 1993). This has since recurred several times, also
causing some casualties.
The Planning Administration in the Ministry of Interior promoted preparation of new
DOPs, some of which have already been approved since 2000. The new plans are much more
environmentally oriented than the old ones, including special references and instructions for
protection and conservation of rivers and riparian landscapes. For example, the new DOP
3/21 for the Central Area District designates river and its surroundings for conservation.
Still, there are considerable variations between DOPs in their definitions and conservation
instructions; some are guiding while others are obligatory, etc. (Maruani, 2005). In other
words, the protection offered by DOPs is not consistent in scope and intensity. Moreover,
since most current DOPs are relatively new, it will be some more time before their
effectiveness in riparian protection can be evaluated.
Local Planning
While national and district outline plans offer general guidelines, Local Outline Plans
(LOPs) are more detailed and serve as platform for issuing building permits. The permits are
approved and issued by Local Commissions, which are in fact organs of the relevant local
municipality, composed of the elected political representatives who constitute the municipal
board. Thus, Local Commissions are interested in the promotion of local economic
development, even when it is contradictory to conservation needs, and they tend to ignore a
broader regional vision. District Commissions control is supposed to minimize the influence
of local economic and political interests on land use decisions. However, since riparian
landscapes are attractive for housing, it is no wonder that initiatives for development within
riparian areas recur, assisted by interested Local Commissions which regardless of their
negative impact and potential flood risk (see for instance: Shechori, 1999; Shmul, 2000;
Vaserman-Amir, 1999). Development decisions are a major causal factor where floods are
concerned (Brody et al., 2007; State Comptroller, 1993). The recurrent flood events in Israel,
and their negative impacts and costs indicate that local development interests were not
restricted by the District Commissions.
DISCUSSION
The examination of legislative, organizational and planning tools used for protection of
riparian landscapes in Israel revealed various faults and deficiencies that may explain the still
distressing state of these landscapes. Despite restoration efforts undertaken, mainly since
2000, almost all rivers are still polluted and ecologically deteriorated with only limited
sections available and accessible for recreational uses.
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Table 1 that presents a comparative assessment of protection tools shows that no one tool
can be highly rated on all parameters. In addition, no parameter shows a consistently high
rating for all tools. One fundamental problem is that no tool with the possible exception of
RAD - is dedicated to the objective of protecting ecological, environmental and scenic values
embedded in riparian landscapes.
Table 1. Comparison of protection tools
Protection tool
Designation
Relevance
Power
Scale
Implementation
Water Law
Low
Medium
High
National
Low
Drainage Law
High
High
High
National
Low to medium
River
High
Authorities Law
High
High
National
Low to medium
Drainage
Authorities
Medium
High
Medium
Regional
Low
River
Authorities
High
High
Low to
medium
Regional
Low to medium
Rivers
Administration
Very high
Very high
Low
National
Medium
Physical
Sharon plan
Low
Low
Low
National
Low
planning
NOP 31
Low
Low
Medium
to high
National
Low
NOP 35
Low
Low
Medium
to high
National
Sectorial NOPs
Low
Low to
medium
Medium
to high
National
Low
DOPs
Low
Low
Medium
to high
Regional
Low
LOPs
Varies with
plan
Varies with
plan
High
Local
Varies with plan
Legislation
Institutional
structure
For example, each of the laws examined makes a partial contribution to this objective
although they differ in aims and scope. However, none of them regards the protection of
riparian landscapes as its main aim. In addition, none of these laws reflects a watershed
approach, theoretically or practically, albeit activities taking place anywhere in the watershed
area, especially development and various pollution generators, eventually affect the river and
the landscape along it. Moreover, partial overlap between the aims and directives of the
Drainage law and those of RA Law are a constant source of ambiguity and fuzziness as to
duties and responsibilities, on one hand, and inter-organizational and inter-personal frictions
and conflicts, on the other. This situation stands in the way of the conservation interest.
The different tools are interconnected. Legislation determines the structure,
responsibilities and operational procedures of the organization intended to implement it.
Subjects not covered by the law are left to the discretion of organizational decision makers.
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Hence, awareness of riparian values on the part of decision makers is an important factor in
effective protection. Statutory physical planning, too, relies on legislative power, and
therefore inter-relations between laws may affect organizational actions. For example, the
Drainage Law does not refer to statutory physical planning, while on the other hand the
Planning and Building Law does not require a preliminary examination of possible impacts
on drainage before the approval of a new development plan. The State Comptroller (1993)
pointed out that the planning system allowed development too close to water routes and
approved plans on large areas without ensuring suitable measures for infiltration of rains
within their boundaries. He argues that the severe floods that were the cause for his report
could have been prevented, had suitable instructions been embedded in the Planning and
Building Law, thus preventing construction within flood retention areas and conditioning plan
approval with proper infiltration and drainage solutions.
The protection of riparian landscapes as was formulated and implemented in Israel
expresses a particularistic approach, where each river is conceived as a discrete entity, with a
separate DA or RA. For comparison, nature and landscape values within areas that have been
declared as nature reserves or national parks all over the state are protected centrally and
managed by the Nature and Parks Authority, which is a national statutory institution,
established by the National Parks and Nature Reserves Law. It seems that centralistic
approach was efficient even when development pressures in Israel increased considerably
towards the end of the 20th century. The establishment of the RAD indicates a conceptual
change towards rivers as well but its lack of statutory position and the multitude of other
relevant organizations reduce its effectivity.
The protection of riparian landscapes also reflects the evolution of environmental
awareness in Israel (see also: Fletcher, 2000; Vogel, 1999). For example, the Water Law from
1959 almost completely ignores the environmental functions of water sources, including
rivers, while the RA Law from 1965 already regards the protection of riparian environmental
values as one of its main aims. The legislation also reflects the prevailing priorities in Israeli
society at the time, affected greatly by ideological and national security considerations. In the
past, agriculture and development were given priority; agriculture was conceived as a main
economic basis and development of spatially dispersed small agricultural settlements also
grasped as a tool for dominating national space while environmental needs were overlooked
(Hershcovich, 2006; Schiffman 1999). It should be noted that all three laws examined were
enacted before the global environmental revolution of the late 1960s. Moreover, as a
consequence of the Stockholm Convention in 1972 Israel was one of the first nations to
establish an environmentally designated institution in the form of the Environmental Service
that was established in 1973 within the Prime Ministers office, and later on as part of the
Ministry of Interior. However, the assimilation of environmental awareness was slow,
especially among decision makers, until the 1990s when development pressures increased
considerably, following a sudden increase of population due to large immigration waves,
threatening nature and landscape values, especially in the coastal area and in vicinity of water
bodies. This also was one of the triggers for initiation of RAD.
It should also be noted that split authoritative powers, multiplicities and deficiencies that
characterize the protection of riparian landscapes in Israel, tend to be typically characteristic
of environmental issues, which are naturally complex, interdisciplinary and bound up with
economic and social conflicts. In addition, since its independence in 1948, Israel has been
facing enormous challenges more than any other developed state, including an unstable
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geopolitical situation and recurrent wars, absorption of mass immigration waves and serious
social conflicts (Alterman, 1995; Fletcher, 2000; Vogel, 1999). Nevertheless, all this still does
not explain why former protection frameworks have not been reconstructed in spite of the
advances in environmental management and administration in general. For instance, the
Drainage Law that was mainly intended to prevent floods is still under the authority of the
Ministry of Agriculture although severe damage caused by floods in recent years was mainly
in urban areas and not in agricultural fields (State Comptroller, 1993). There is no doubt that
the present state of affairs calls for improvement.
CONCLUSION
Riparian landscapes constitute extremely vulnerable ecosystems. They need protection to
preserve the unique aquatic habitats with their biodiversity richness and ecological processes
as well as their value for scenic and recreational purposes. In spite of that, their protection in
Israel is defective, lacking comprehensive suitable legislation, a statutory institutional
structure on a national scale and a sectorially designated NOP. The existing state of affairs is
characterized by a complex array of authoritative powers, some split and others overlap, and
by deficiencies in formulation and implementation of policies. Only limited segments of
specific riparian landscapes may, in fact, be regarded as functional healthy ecosystems.
Several lessons can be drawn from the above discussion. We wish to focus here on those
that seem the most important and practical for the State of Israel in the immediate future.
First, there is need for a revision of present legislation, integrating together existing laws
mainly Drainage Law and RA Law rephrasing their aims and directives, and rearranging the
institutional structure and its powers according to updated environmental and other needs.
This should be done considering a whole watershed approach as has already been suggested
by Laster (1995).
Second, the prevailing, rather particularistic, approach should be replaced by a
comprehensive centralized one, based on a vision and needs on a national scale. This ought to
be reflected in all types of protection tools but especially in the institutional structure, as by
establishing a national statutory designated organization (similar to the Nature and Parks
Authority) or, alternatively, empowering the existing RAD by adequate legislation, including
broader definition of its powers and duties.
Third, increasing environmental awareness in general, and awareness of riparian
landscapes ecological, environmental and scenic aspects in particular, are a key element in
promoting protection and conservation of such landscapes. This is true for the general public,
as has already been claimed by Lowry (1998), but is even more important where decision
makers are concerned. Therefore, educational activities, formal and informal, carry great
significance for improving and intensifying riparian protection efforts.
Finally, there is hope for riparian landscapes, even when severely deteriorated, providing
suitable protection, restoration tools and a plan of action (see for instance: Harnik 2007, Kirk
2005, Tjallingii 2000). This is especially important in a small, densely populated and dry land
like Israel.
321
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ISSN: 2158-5717
Chapter 12
HYDRAULIC CHARACTERIZATION OF AQUIFER(S)

AND PUMP TEST DATA ANALYSIS OF DEEP AQUIFER
IN THE ARSENIC AFFECTED MEGHNA RIVER
FLOODPLAIN OF BANGLADESH
Anwar Zahid1,2, M. Qumrul Hassan1, Jeff L. Imes
and David W. Clark3
1
Department of Geology, University of Dhaka, Dhaka, Bangladesh

2
Bangladesh Water Development Board, Dhaka, Bangladesh
3
United States Geological Survey, National Drilling Company-USGS GroundWater Research Program, P.O. Box: 15287, Al Ain, UAE
ABSTRACT
To determine the hydraulic characteristics of aquifers and development potential of
deep aquifer for sustainable long-term use, study was undertaken by assessing water
levels of different aquifer formations and conducting pumping test in deep aquifer under
Meghna floodplain area of southeastern Bangladesh. Because of arsenic contamination in
shallow groundwater, characterization of deeper aquifers and assess their hydraulic
connectivity is now an important issue in Bangladesh. Study shows that groundwater
pumping for irrigation and other uses cause large seasonal water level fluctuations that is
between 2 and 4.5m, 6.5 and 11m and 6.5m in the shallow, main and deep aquifers,
respectively. The trend of groundwater level fluctuations supports the hydraulic
connectivity of these aquifers. Aquitards separating aquifers are not continuous
regionally. This implies that uncontrolled development of deep aquifers may cause
leakage of arsenic from contaminated shallow depths to aquifers below. Water levels
dropping below sea level for over withdrawal may also cause saline water intrusion as
well. However, during the constant-discharge pumping test for deep aquifer, water levels
in observation wells open to the shallow and main aquifers showed no noticeable effect
from pumping i.e. under conditions of moderate groundwater use for public supply,
arsenic-rich groundwater in the shallow aquifer are not likely to be drawn into the deep
E-mail: anwarzahid_b@yahoo.com, Tel-+880-2-7287176; +880-1819 105 871
Anwar Zahid, M. Qumrul Hassan, Jeff L. Imes et al.
326
aquifer. The transmissivity values of the aquifer is generally favorable for groundwater
development and ranged from about 1,070 m2/day to 2,948 m2/day at a distance of 44 m
from the pumped well. Transmissivity ranged between 1,570 m2/day and 2,956 m2/day at
a distance of 120m. Transmissivity was calculated as 2,385 m2/day using recovery data.
Estimated storage coefficient values ranged between 0.0000375 and 0.00268, indicates
that the aquifer is confined to leaky-confined or semi-confined in nature.
Keywords: Arsenic contamination, groundwater level, irrigation abstraction, fluctuation,

aquifer test, transmissivity, storage coefficient.
NOTATION
c
L
Q
r
s
S
S'
sm
D'/K': hydraulic resistance of the semi-pervious layer (day)

Tc: leakage factor (meter)
constant discharge rate at the well (meter3/day)
radial distance from the pumping well (meter)
drawdown at the well (meter)
aquifer storage coefficient
is the residual drawdown (meter)
maximum drawdown (meter) in a piezometer at distance r (meter) from the pumped
well
s the drawdown per log cycle of time (meter)
T transmissivity of the aquifer (meter2/day)
t time from the start of pumping (minutes)
t' time from the cessation of pumping (minutes)
t0 time, where the straight line intersects the zero-drawdown axis (minutes)
INTRODUCTION
Groundwater is the main source for drinking and irrigation water in the lower floodplain
areas of south-eastern Bangladesh and mainly withdrawn from shallow aquifer. The upper
aquifer system of the area can yield large quantities of water, however, is not completely
suitable for sustainable development because of quality problems. The arsenic contamination
of shallow (generally up to 50m depth) groundwater has changed the potentiality of its use.
Besides, high concentration of iron, manganese and salinity at different depth levels of main
aquifer makes it unsuitable for drinking use. Considering the increasing demand for
municipal and rural water supplies, agricultural, industrial and other uses and quality
problems in shallow and main aquifers, development of deep aquifer has already been started
in some areas. However, before large scale withdrawal of groundwater from deep aquifers,
understanding the natural distribution of groundwater for long term sustainability is very
important. Sustainable yield from aquifers, effective use of the water stored in aquifers,
preservation of water quality, and movement of water between different aquifer formations
into a comprehensive management system needs to be studied carefully. Rapid and
Hydraulic Characterization of Aquifer(s) and Pump Test Data Analysis
327
uncontrolled development of the deep aquifer could severely limit the usefulness and the
productive duration of the aquifer.
Groundwater levels of different aquifers were monitored throughout Kachua upazilla
(sub-district) area under Chandpur district and an aquifer test was conducted for the deep
aquifer at Sreerampur village of Kachua to determine the aquifer hydraulic characteristics,
potability of the aquifer system, and the response of the deep aquifer pumping to upper
aquifers and to development stresses. In particular, it is essential to understand whether
pumping stresses can induce arsenic contaminated water from the shallow aquifer or high
salinity water from depth or the shallow aquifer to migrate into the aquifer. The electric
conductivity (EC) of water pumped from the production well was also monitored during the
test to determine if higher salinity water was being captured by the well during the test. The
long-term constant-discharge pumping test was performed as the aquifer properties
determined from the analysis of constant-discharge pumping test represent the regional
hydraulic properties (Nury et al, 1998) in alluvial aquifers like Bengal Delta.
STUDY AREA
Most of Bangladesh including the study area lies within Bengal basin which began
forming during the Late Mesozoic as the continental landmass of Gondwana fragmented and
continued to form during the Tertiary when the Indian plate collided with the Eurasian plate
resulted in the formation of the Himalayan ranges. The Bengal basin contains a 15-km to 22km thick sequence of Cretaceous to Recent sediments and occupies some 100,000 km2 of
lowland floodplain and delta (DPHE-BGS 2001). Alluvial deposit carried by the GangesBramaputra-Meghna (GBM) river systems have gradually built up the delta and Meghna
estuary (Brammer 1996). Physiographically, the study area lies within Meghna estuarine
floodplain under Tippera surface (Morgan et al. 1959) that is bounded by the Meghna river in
the west, Lalmai hills in the east and Old Meghna estuary at its south. The total discharge of
the lower Meghna river was contributed from the eastern territory and the huge discharge hit
the western bank and caused erosion in the western bank and subsequent deposition in the
eastern bank towards the study area (Hussain and Huq 1998). The surface of the sequence,
composed of silt, silty clay, silty loam and grayish clay, has been assigned to the Chandina
Formation (Bakr 1977), while the status of the underlying medium sand aquifer is not well
known. The Geological Survey of Bangladesh (GSB) has dated the surface of the Chandina
deltaic plain as between 3,000 and 6,000 years. The underlying sands locally contain brackish
water and may belong to the Dupi Tila Formation.
Based on elevation and morphological features the area is divided into three units
(BWDB-USGS 2005). Southeastern part of the upazilla has been defined as Chandina
Alluvium High that always remains above normal flood level. The sediments mainly consist
of clayey silt, silty clay and very fine sand. Below this oxidized zone grey colored fresh sandy
silt and fine sand are present. The northwestern and southwestern part have been defined as
Chandina Alluvium Medium. This part normally remains under water for about 3 to 4
months. Maximum and minimum elevation of this unit is 6.0 and 4.6 m above mean sea level
(AMSL) respectively. The northeastern and central-western part of the area is defined as
Chandina Alluvium Low. The surface remains under water for longer periods than the other
328
units. In part of the southeast Bangladesh, multi-layered aquifer conditions exist. On a

regional basis BWDB-UNDP (1982) described three aquifers between Holocene and PlioPleistocene formations. In Kachua area aquifers may be classified as; (Figure 1), (a) the
shallow (1st) aquifer, extends down to 40 to 80m, below the 3 to 6 m thick upper clay and silt
unit.
Figure 1. Aquifer system under Kachua Upazila area (Zahid et al 2007).
The aquifer sediments are composed of sand with lenses of clay. Water of this unit is
severely contaminated by arsenic, (b) the main (2nd) aquifer, extends down to 250 to 350m
and is generally underlain and overlain by silty clay bed, and composed mainly of fine to
medium sand, grey to light brown in color, occasionally inter-bedded with clay lenses. It is
either semi-confined/leaky or consists of stratified interconnected, unconfined water-bearing
zones. Irrigation water in drawn predominantly from these strata, and (3) the deep (3rd)
aquifer, encountered to depths of 400m below a 10 to 15m thick silty clay bed. This aquifer is
composed mainly of grey to dark grey fine to medium sand that in places alternates with thin
sandy shale/clay lenses. This deeper water bearing unit is separated from the overlying main
aquifer by one or more clay layers of varied thickness.
METHODOLOGY OF THE STUDY

Observation of Water Levels in Different Aquifers
1998 to 2003 groundwater level data of 4 Bangladesh Water Development Board
(BWDB) monitoring wells (20-30m deep) installed in the shallow aquifer (Table 1), 2004-05
to 2005-06 data of 13 selected Department of Public Health Engineering (DPHE) hand
tubewells (180-225m deep) installed in the main aquifer (Table 2) and 2003 to 2004 data of 2
BWDB deep monitoring wells (336-352m deep) installed in the deep aquifer (Table 3) were
used to asses the response of groundwater level above or below mean sea-level (MSL)
considering recharge and withdrawal for different uses. Locations of monitoring wells are
shown in Figure 2.
329
Figure 2. Location of groundwater level monitoring wells.
Table 1. Information on BWDB observation wells screened in shallow aquifer

Location
Jagatpur
Machimpur
Sacher
Muradpur
Latitude
(North)
231555.0
231897.7
232649.6
232000.0
Longitude
(East)
910000.0
905532.1
905051.2
905000.0
Depth (m)
19.20
27.44
27.44
28.66
Measuring point (m)

Above MSL
12.70
6.48
5.36
6.53
Table 2. Tube wells selected for monitoring groundwater movement in main aquifer
Location
Nalua, Karaia
Sahedapur, Karaia
North Dumuria, Karaia
Hossainpur, Kachua South
Ghagra, Kachua South
Tetaia, Kachua North
Ujani, Kachua North
Hasimpur, Gohat North
Gobindapur, Gohat South
Singua, Sahadevpur West
Khilmeheb, Sahadevpur West
Kadla, Kadla
Chaumuhari, Kadla
Latitude
(North)
231827.3
231833.6
231906.1
232123.7
232134.5
232249.4
232219.0
232006.9
231724.1
232303.3
232342.0
231852.7
231946.9
Longitude
(East)
905440.7
905456.4
905240.9
905515.9
905222.8
905331.4
905522.0
905651.1
905638.8
904953.9
904942.8
905128.9
904946.6
Depth
(m)
215
215
220
220
218
213
211
191
218
223
220
210
225
measuring point
Above MSL (m)
6.288
6.376
5.848
7.016
6.335
6.317
6.28
6.342
6.649
5.992
6.236
5.987
7.188
330
Setting of Pumping Well and Observation Wells

For a well performance test, yield and drawdown are recorded to calculate the specific
capacity of the well. The aquifer test at Sreerampur village was conducted mainly to provide
data from which the principal factors of aquifer performance [transmissivity (T) and storage
coefficient (s)] can be calculated. An aquifer test with the setup of a 365-m deep pumping
well with a constant discharge of 708 gpm (gallons per minute), and 5 (five) observation
wells installed at different depth levels from 25 to 352 m (Figure 3, Table 3), were performed
to obtain a picture of the general hydraulic properties of the aquifer and also to predict the
effect of withdrawals on the aquifer system, the drawdown in the tested well with time, and
different discharges and the radius of the zone of influence for individual or multiple wells. In
a uniform and homogeneous aquifer the piezometer should be installed at about the same
depth as the middle of the well screen in the pumped well. Amongst observation wells, two
have been installed in the pumped aquifer and others are above the confining layer that
separates the two aquifer systems. The test continued for 98.5 hours and water levels were
measured in both the pumped well and five piezometers. The constant-discharge pumping test
was followed by recovery test. Important water quality parameters were also monitored and
samples were collected at different time intervals during the test.
Figure 3. (a) Location and (b) position of pumping well and observation well screens.
Table 3. Major features of pumping and observation wells

Well
ID
PW
P-1
P-2
P-3
P-4
P-5
Aquifer
Deep
Main
Deep
Deep
Shallow
Main
Depth
(m)
365
281
336
352
25
183
Screen length (m)

From
To
326
362
269
278
324
333
333
349
20
23
171
180
Diameter
of well (m)
0.35
0.076
0.076
0.076
0.076
0.076
Distance from
PW (m)
0
14.5
44.1
105
7.5
9.25
measuring point
(m) Above MSL
6.84
6.3
6.94
6.28
6.72
6.54
The flow rate of 3,860 m3/day was determined using standard tables for the discharge
pipe diameter considering a water height of 8.33 m in a manometer attached to the side of the
pumping-well discharge tube. The rate at which the discharge water stream dropped from the
331
horizontal was also measured. A flow rate of 3880 m3/day was determined using standard
tables that relate the rate of fall to discharge. The two methods were in good agreement and
an average value of 3870 m3/day flow was used to analyze the aquifer-test data.
The constant-rate test for the time duration of 98.5 hours was conducted at Sreerampur.
The pumping rate was estimated and monitored using two different methods. The circular
orifice weir was used to measure and monitor the discharge rate of the pump. The height of
the water column, the static water level just before the test started, time since the pump
started, pumping rate, dynamic groundwater levels at various intervals during the pumping
period, and time the pump stopped were recorded. Measurement of water levels after the
pump stopped (recovery data) were also measured as these are extremely valuable to verify
the aquifer storage coefficient calculated during the pumping phase of the test. The test was
started on November 13, 2003 at 12:00 noon. Before the test began, water level transducers
were placed in the pumping and observation wells. Drawdown was also recorded manually by
measuring tapes. To obtain better and more reliable results pumping continued till the
depression cone had reached a stabilized position. The depression cone continues to expand
until the recharge of the aquifer equals the pumping rate.
Best Fit Analytical Methods for Pump Test Data

Amongst different analytical methods, it is important to select a numerical solution which
is more appropriate to actual field conditions. Two analytical solutions were studied in detail
to determine the most appropriate solution to the deep observation well aquifer-test data. The
Papadopolous-Cooper (1967) solution for a confined aquifer with well-bore storage and the
Hantush-Jacob (1955) solution for a leaky confined aquifer were determined to be the best
choices. Besides, Jacob (1950) straight line plot was drawn manually and Jacob (1950), Chow
(1952) and Theis recovery methods were applied using computer programs developed by
Abdin (2004) for manually measured drawdown data.
The following assumptions and conditions were considered for analyzing aquifer-test
data using different methods valid for confined or leaky-confined aquifer.
All geologic formations are horizontal and the aquifer has a seemingly infinite areal
extent. In reality, hydrogeologic settings rarely have aquifers that can be considered
of infinite areal extent, rather they change laterally in grain size, shape or lithology
that affect the shape of a time-drawdown curve. Although such aquifers do not exist
in lower delta, many aquifers are of such wide extent that all practical purposes they
can be considered infinite.
The aquifer is homogeneous, isotropic and of uniform thickness over the area
influenced by the pumping test. Homogenous aquifers seldom occur in lower delta of
Bengal basin and most aquifers are stratified to some degree. As a result of this
stratification the drawdown observed at a certain distance from the pumped well may
be different at various depths within the aquifer, because of differences in hydraulic
conductivity in vertical and horizontal direction. These differences in drawdown
diminish with increased pumping itme.
Groundwater has a constant density and viscosity.
332
Prior to pumping, the piezometric surface were nearly horizontal over the area
influenced by the pumping test.
The aquifer was pumped at a constant discharge rate of about 3870 m3/day,
All changes in the position of the potentiometric surface were due to the effect of the
pumping well alone,
Pumping well was 100% efficient
Another important assumption is that the pumped well fully penetrations the aquifer and
thus receives water from the entire thickness of the aquifer by horizontal flow. If the well
only partially penetrates the aquifer, like Sreerampur study, the flow paths have a vertical
component to them. The flow paths are, therefore, longer and converge on a shorter well
screen, resulting in an increase in head loss (Driscill 1986). However, observation wells for
pumping tests were placed far enough away from the pumping well to avoid partial
penetration effects. If the observation well is partially penetrating and more than
1.5b(K h / K y )0.5 away from the pumping well, the effects are negligible (Hantush 1964)
(b is the saturated thickness, Kh and Kv are the horizontal and vertical hydraulic
conductivities). This condition is valid for Sreerampur aquifer. If this condition is not
satisfied, there will be an upward inflection in the response, similar to that obtained in the
leaky method or for some sort of recharge boundary (Domenico and Schwartz 1997).
Theis Method for Predicting Drawdown in a Confined Aquifer

A major advance was made by Theis (1935), who was the first to develop a nonsteadystate formula which introduces the time factor and the storage coefficient. Theis developed
the analytical solution for flow to a well in a confined aquifer. This is generally the base of
other methods applied to similar conditions. Theis noted that when a well penetrating an
extensive confined aquifer, is pumped at a constant rate, the influence of the discharge
extends outward with time. The rate of decline of head, multiplied by the storage coefficient
and summed over the area of influence, equals the discharge. Because the water must come
from a reduction of storage within the aquifer, the head will continue to decline as long as the
aquifer is effectively infinite. Since all sources of recharge are moving through a semiconfining unit, the flatness of the recharge effect on a Theis curve will continue in a
horizontal manner (Lohman 1979). However, when the semi-confining layer is saturated and
has a head higher than the head in the aquifer being pumped, this head differential may cause
the aquitard to release water from storage in an attempt to reach equilibrium (Weight and
Sonderegger 2000). As in the semi-confining scenario, the initial drawdown tends to follow
the confined Theis curve. As the aquifer becomes stresses and the head lowers in the pumping
well, the head change stimulates leakage from above and below to act as recharge to the
system. The result is a flattening of the curve rather than following the Theis curve.
Besides the assumptions mentioned above the following assumptions are also considered
for Theis solution.
Aquifer is fully confined and discharge is derived exclusively from storage in the
aquifer.
333
The flow to the well is in unsteady state, i.e. the drawdown differences with time are
not negligible nor is the hydraulic gradient constant with time.
The water removed from storage is discharged instantaneously with decline of head.
The storage in the well can be neglected.
The nonsteady-state or Theis equation which was derived from the analogy between the
flow of groundwater and the conduction of heat for predicting drawdown (s) at the well is as
follows:
Q e y dy
Q
Q
W (u ) and, consequently T
W (u )
4T u y
4T
4s
The well function W (u ) is the infinite series part of the analytical solution to the
nonsteady, radial groundwater flow equation that is approximated by:
W (u ) 0.577216 ln u u
where, u
u2
u3
u4
...
2 2! 3 3! 4 4!
r 2S
4Ttu
and, consequently S 2
4Tt
r
u determines the radius of a cone of depression (Theis 1940). The radius not only increases
with increasing time but, for a given time, is larger for decreasing values of storativity and
increasing values of transmissivity. Theis type curve u versus W(u) is unique only because it
pertains to a particular set of conditions at the pumped well and in the aquifer (Stallman
1976).

Water Level Fluctuations in Different Aquifers
Long-term groundwater hydrograph of four of BWDB wells, installed in the shallow
aquifer, show maximum depth to water table in dry season and in the monsoon it regains
(Figure 4). No permanent declining is observed. The average maximum and minimum water
level was observed as 6 and 0.5m above MSL respectively (Table 4). Seasonal groundwater
table fluctuation ranges in between 2 and 4.5m in the Upazila area. Generally, groundwater
withdrawal from shallow aquifer for domestic purposes and during dry period by shallow
irrigation wells is balanced with the vertical percolation of rain water and inflow from
surrounding aquifers in monsoon.
334
Table 4. Average groundwater levels above or below mean sea-level in different aquifers
Aquifer Formation
Nos. of
Wells
04
13
02
Shallow (1st)
Main (2nd)
Deep (3rd)
Depth of
Wells (m)
20-30
180-225
336-352
Groundwater Level (m)

Maximum Minimum
6
0.5
4
-8
4.5
-2
CM 014
CM020
7
Groundwater Elevation (m)
6
5
4
3
2
1
0
6
5
4
3
2
Jan-00
2003
1998
Aug-01
1/ 0/ 00
10/ 26/ 00
2003
1998
Year
Year
CM032
CM043
6
Fluctuation (m)
Maximum Minimum
4.5
2
11
6.5
6.5
6.5
1
1/ 0/ 00
10/ 26/ 00
2003
1998
Year
5
4
3
2
1
1/ 0/ 00
9/ 6/ 00
1998
2003
Year
Figure 4. Groundwater level hydrograph of four BWDB wells screened in shallow aquifer at Kachua
sub-district.
There is a clear difference between the amount of water that can potentially recharge the
aquifer system and the actual quantity. Actual recharge is the quantity of water when the
groundwater table has risen to the ground surface and no more water can enter the aquifer
system. Its quantity depends on the degree of depletion of this reservoir during the dry season.
335
Potential recharge is the maximum amount of recharge that can occur if enough storage
reservoir in the aquifer system is available. Any surplus rainfall is then rejected and
contributes to surface flooding. The ultimate limit on groundwater development is controlled
by the long-term average amount of potential recharge. Assessment done by WARPO (2000)
indicates that when the clay is relatively thin, but exhibits low vertical permeability, the
piezometric level may drop below the base of the clay. In this case the total gross quantity of
groundwater abstracted from the aquifer is balanced by infiltration through the upper clay.
When the upper clay varies in thickness, the piezometric level in the aquifer may locally drop
below the base of the clay, creating local unconfined conditions within the aquifer and
groundwater abstraction is partially balanced by leakage through the clay and partially by
unconfined storage change in the aquifer. The recharge to the aquifer, which is the leakage
during the period of no abstraction, may be less than the potential recharge, particularly when
the vertical permeability is low.
During the peak irrigation season in March and April, the hydrograph is fairly smooth
from year to year and steepest rise in the hydrograph is observed immediately after the
irrigation pumps are switched off (mid-April to mid-May) rather than at the start of the
monsoon (late June) as might be expected. During the monsoon, the hydrographs rise steadily
until the levels are within about 1-2m of the surface which indicates that a dynamic
equilibrium is established between the water table, deep rooted vegetation and surface water
bodies (Ravenscroft 2003). The aquifer full condition i.e. no more storage capacity is
attributed before the end of June.
A shallow aquitard is common in this region at depth about 75m. Below this aquitard all
deep irrigation wells are installed in the upper part while DPHE domestic and community
hand tubewells are screened in the deeper part of main aquifer. Groundwater pumping for
irrigation by deep irrigation wells causes large seasonal water level fluctuations (Figure 5),
ranges from 6.5 to maximum of 11m (Table 4).
The average maximum and minimum water level was observed as 4 and -8m above and
below MSL respectively. Withdrawal by shallow irrigation wells may also influence on huge
fluctuation of water level in the main aquifer. However, these levels in the deeper part get
recovered rapidly when seasonal pumping stops. This rapid recovery, parallel to water levels
in shallow aquifer, reveals that shallow and main aquifers are hydraulically connected and
rainfall also contributes recharge to main aquifer through shallow aquifer. However, with
intensive abstraction, groundwater levels may fall towards a permanent new equilibrium state.
At Sreerampur village a deep confining layer is encountered at 302 to 320m depth, which
is also common in surrounding areas of Meghna floodplain. Only two monitoring wells are
installed at entire Kachua area in this deep aquifer. The average maximum and minimum
water level of one hydrologic year was observed as 4.5 and -2m above and below MSL
respectively (Figure 6a).
Hence the seasonal fluctuation is 6.5m that is less compare to average water level
fluctuation of main aquifer (Figure 6b), but still high though the development stress in deep
aquifer is almost nil. This huge fluctuation indicates the influence of irrigation withdrawal in
upper aquifers.
336
Hydrographs for wells (2nd Aquifer) in Northern Kachua
Water levels (elevation) in meter
6
4
2
0
-2
KH-28
KH-40
KH-44
KH-45
KH-46
KH-63
-4
-6
-8
Mar-06
Feb-06
Jan-06
Dec-05
Nov-05
Oct-05
Sep-05
Aug-05
Jul-05
Jun-05
May-05
Apr-05
Mar-05
Feb-05
Jan-05
Dec-04
Nov-04
Oct-04
Sep-04
Aug-04
Jul-04
Jun-04
May-04
Apr-04
Mar-04
Feb-04
a
Hydrographs for wells (2nd Aquifer) in southern Kachua
Water level (elevation) in meter
6
4
2
0
-2
KH-22
KH-25
KH-34
KH-36
KH-55
KH-56
KH-60
-4
-6
-8
Mar-06
Feb-06
Jan-06
Dec-05
Nov-05
Oct-05
Sep-05
Aug-05
Jul-05
Jun-05
May-05
Apr-05
Mar-05
Feb-05
Jan-05
Dec-04
Nov-04
Oct-04
Sep-04
Aug-04
Jul-04
Jun-04
May-04
Apr-04
Mar-04
Feb-04
b
Figure 5. Groundwater level hydrographs of hand tubewells screened in the deeper part of main aquifer.
337
Hydrographs for wells (3rd Aquifer) at Srirampur

6
5
4
3
2
1
0
-1
Well (3rd Aquifer) 335 m
-2
-3
Nov-04
Oct-04
Sep-04
Aug-04
Jul-04
Jun-04
May-04
Apr-04
Mar-04
Feb-04
Jan-04
Dec-03
Nov-03
Oct-03
Sep-03
Aug-03
Jul-03
Jun-03
May-03
a
Hydrographs for wells (3 Aquifers) at Srirampur
6
4
2
0
-2
Well (2nd Aquifer) 280 m
Well (1st Aquifer) 25 m
Well (2nd Aquifer) 180 m
-4
-6
-8
Nov-04
Oct-04
Sep-04
Aug-04
Jul-04
Jun-04
May-04
Apr-04
Mar-04
Feb-04
Jan-04
Dec-03
Nov-03
Oct-03
Sep-03
Aug-03
Jul-03
Jun-03
May-03
b
Figure 6. (a) Groundwater level hydrograph of observation wells screened in the deep aquifer; (b)
Response of irrigation pumping on water level of different aquifers
In Figure 6b, water level hydrographs of five wells installed in all three aquifers within
500m2 area are drawn for comparison study. It is clear that maximum water level i.e. static
water level is almost same (about 4.5m above MSL) for three aquifers and during irrigation
period, all five wells response similarly with different declining intensity. Because of
continuous percolation of rain water through unsaturated zone, lowering of water level in
338
shallow aquifer is very low (about 1.0m) that is about 10.5m for main aquifer and about 6.5
for deep aquifer. As most of the irrigation wells are installed in the main aquifer, lowering of
water level is highest in this aquifer. However, the trend of water level fluctuations in
different aquifers support the hydraulic connectivity of these aquifer i.e. the aquitards
separating aquifers are not continuous regionally rather locally extended. This implies that
uncontrolled development of deep aquifers may cause both qualitative and quantitative
degradation of groundwater. Water levels dropping below sea level for over withdrawal in dry
season may eventually cause saline water intrusion as well as leakage of arsenic from shallow
aquifer to upper part of main aquifer.
Response of Groundwater Levels to Pumping

The water level in the pumping (deep) aquifer declined abruptly during the first 3 minutes
of pumping, then stabilized at about 12.5 to 13.0m drawdown for the duration of the test
(Figure 7a). The pumping phase of the aquifer test was terminated after 5,911 minutes (98.5
hours), and data was collected during the recovery phase of the test until 7,168 minutes (119.5
hours) after the test had begun. During the pumping test, water levels in observation wells
open to the shallow and main aquifers showed no noticeable effect from pumping in the deep
aquifer (Figure 7b).
The overall fluctuation of water levels (as caused by factors other than long-term declines
or barometric pressure changes) measured in P-1, P-4, and P-5 was less than about 0.04m
during the test. This indicates that the 12 to15m confining unit that separates the main and
deep aquifer retards the movement of ground water between the aquifers. Therefore, under
conditions of moderate groundwater use for public supply, arsenic-rich, iron-rich, and saline
ground water in the shallow aquifer are not likely to be drawn into the deep aquifer.
Groundwater levels in the deep aquifer observation wells responded to the withdrawal of
water from the pumping well. Water levels in P-2 (44 m from the pumping well) declined
about 1.1 m in response to pumping (Figure 7a). Water levels in P-3 (120m from the pumping
well) declined about 0.68m in response to pumping. Small fluctuations in the measured water
levels may be partly caused by variations in the pumping rate during the test as voltage in the
power supply fluctuated.
Water levels in both observation wells rose rapidly at the end of the aquifer test, and had
returned to within 0.05m of the pre-test water levels when data collection was stopped.
Comparison of water level altitudes in the shallow and deep observation wells shows that
water levels in the deeper aquifer were lower than water levels in the shallower aquifer in an
area about 100m radius centered on the pumping well after about 3,600 (60 hours) minutes
into the test.
Analysis of Drawdown Data

Gunther Theim (1962) published the first formula on the response equation for steady
radial flow (Ferris et al. 1962), based on the work of Darcy and Dupuit, and computed the
hydraulic characteristics of a water-bearing formation by pumping a well and observing the
effect of this pumping in a number of other wells. Now, most of the more or less complicated
339
flow problems can be solved by applying proper mathematical methods. Aquifer test analyses
may provide unrealistic estimates of hydraulic properties (Halford and Kuniansky. 2002).
However, it makes no difference in performing a test whether response curves are obtained as
analytical solutions or by other methods, e.g. flow nets, digital computers, etc (Stallman
1976).
P-3
P-2
P-1, P-4 and P-5
b
Figure 7. Monitoring of water level (drawdown) during aquifer test (a) Observation wells in deep (3rd)
pumping aquifer below aquitard; (b) Observation wells in upper aquifers.
The investigated deep aquifer under Sreerampur village may be considered as a confined
aquifer as it is overlain by a 10 m to 12 m thick nearly-impervious silty clay layer, 295m
340
below the surface. The lower boundary of the aquifer was not encountered above the
investigated depth of 362 m; however, from the depositional history of the region it may be
assumed that another impermeable formation can exist beneath the tested depth. A confined
aquifer is a completely saturated aquifer whose upper and lower boundaries are impervious
layers. However, completely impervious layers (aquatard) rarely exist in nature. A semiconfined or leaky aquifer, is a completely saturated aquifer that is bounded above by a semipervious layer and below by a layer that is either impervious or semi-pervious. Physically,
there is induced recharge during an aquifer test through the semi-pervious layer. The rate of
leakance is determined by the hydraulic conductivity of the aquitard and head difference
across the aquitard. In a semi-confining scenario, confining units may pinch out laterally and
part of the aquifer may become unconfined. This may true for the studied aquifer, too.
Hantush-Jacob Leaky Aquifer Model

For all practical purposes, the time-drawdown behavior before the inflection represents a
withdrawal of water from storage from the aquifer, no part of which was contributed from
other sources. This part of the curve, along with its straight line extension, may be analyzed
with methods discussed for time-drawdown behavior. There are several ways in which this
condition may be compromised in field conditions: direct recharge from streams, recharge
across bounding low permeable materials etc. The problem of leakage has been extensively
investigated by Hantush and Jacob (1955) and Hantush (1956, 1960, 1964). In a leaky
aquifer, the drawdown curve initially follows the nonleaky curve. However after a finite time
interval, the lowered hydraulic head in the aquifer induces leakage from the confining layer.
If there is storage in the confining layer, the rate of drawdown is slower than that in the case
where there is no storage in the confining layer (Hantush 1960).
In many aquifer tests, water is contributed from the less permeable confining units, in
addition to the aquifer that is pumped (Halford and Kuniansky 2002). Hantush and Jacob
(1955) presented a solution for drawdown in a pumped aquifer that has an impermeable base
and a leaky confining unit above. Conceptually, this would be a four layer system, from top to
bottom, a water table aquifer, a leaky confining unit, a confined aquifer, and an extremely low
permeability bed rock. During the early time of pumpage, water is coming out of storage from
the pumped aquifer and the leaky confining unit. Eventually, the discharge comes into
equilibrium with the leakage through the confining unit from the unstressed water-table
aquifer and the system is at steady-state.
The additional assumptions for the analysis are:
Aquifer is leaky, horizontal flow stressed aquifer, vertical flow through confining
unit.
Drawdown in the water table or unstressed aquifer is negligible.
Well storage can be neglected.
Water instantaneously comes out of storage in the aquifer.
Confining unit storage is negligible.
The equation is based on the drawdown of a well pumped at a constant discharge rate in a
leaky aquifer (Hantush and Jacob 1955).
341
Q
W (u, r )
B
4T
where, u
r 2S
is dimensionless time,
4Tt
K Z b'
T
KZ/b' is the leakance (1/T), where KZ is vertical hydraulic conductivity of the confining unit
(L/T) and b' is thickness of the confining unit (L).
Early drawdown data from the field curve match the non-leaky part of the curve, but they
soon deviate and follow one of the leaky r/B curves. The match point method yields values of
w(u, r/B), 1/u, t and s. In addition, the r/B curve followed by the field data is noted. T and S
are readily determined from
Q
r
4uTt
W (u, ) and S 2
4s
B
r
Unaware of the work done many years earlier by De Glee (1930, 1951), Hantush and
Jacob also derived the same equation, which expresses the steady-state distribution of
drawdown in the vicinity of a pumped well in a semi-confined aquifer in which leakage takes
place in proportion to the drawdown. Hantush (1956, 1964) noted that if r/L is small
(r/L0.05), the equation may, for practical purposes, be approximated by
sm
2.30Q
L
(log 1.12 )
2T
r
Thus a plot of sm against r on semi-logarithmic paper, with r on the logarithmic scale,

shows a straight-line relationship in the range where r/l is small. The slope of the straight
portion of the curve, i.e. the drawdown difference sm per log cycle of r, is expressed by
s m
2.30Q
2.30Q
, consequently T
2s m
2T
The Hantush-Jacob leaky aquifer solution was chosen to analyze the aquifer test data
because of the possibility that there was some induced leakage of ground water across the
confining unit during the test. With the advent of computer programs to analyze aquifer test
data, manually plot graphs and calculate aquifer parameters by hand is generally no longer in
use. This makes it much easier to analyze the data, but makes much less aware of the
assumptions behind the analytical solutions. Drawdown data for the individual deep
342
observation wells can be matched well to about 500 minutes using the Hantush-Jacob
solution. Drawdown from about 200 to 500 minutes becomes nearly constant at about 1 m for
P-2 and nearly constant at about 0.6 m for P-3. This response may be caused by equilibrium
between leakage across the confining unit and drawdown from well pumpage. After about
500 minutes, drawdown in both observation wells begins to increase in an approximately
linear manner, at a rate of about 0.00001 m/minute in P-2 and about 0.00002 m/minute in P-3.
This may indicate decreased leakage through the confining unit, draining of stored water in a
permeable sand lens, or the presence of a lateral barrier. Of the three possibilities, a lateral
barrier seems unlikely considering the geological setting.
The best fit curve match (Figure 8a) to P-2 yielded an aquifer storage of 0.0013, aquifer
transmissivity of 2,300 m2/day, vertical to lateral aquifer hydraulic conductivity ratio of
0.0044 (Kz/Kr), and a confining unit vertical hydraulic conductivity of 0.33 m/day (r/B =
0.1533). The best fit curve match (Figure 8b) to P-3 yielded an aquifer storage of 0.0022,
aquifer transmissivity of 2,956 m2/day, vertical to lateral aquifer hydraulic conductivity ratio
of 0.0044, and a confining unit vertical hydraulic conductivity of 0.43 m/day (r/B = 0.365).
Because the aquifer values are similar for the individual observation well data curve
matches, both sets of data can be approximately fit to a single set of values (Figure 8c). The
best fit curve match to both observation wells taken together yielded an aquifer storage of
0.0017, aquifer transmissivity of 2,386 m2/day, vertical to lateral aquifer hydraulic
conductivity ratio of 0.0044, and a confining unit vertical hydraulic conductivity of 0.35
m/day (1/B = 0.0035).
The parameter 1/B in the Hantush-Jacob solution is related to the confining unit vertical
hydraulic conductivity by the relation Kv' = (T*b')/2B, where T is the deep aquifer
transmissivity and b' is the confining unit thickness. The lateral hydraulic conductivity of the
deep aquifer is estimated as 23 m/day using Kh=T/b, where b is the aquifer thickness. The
vertical hydraulic conductivity of the deep aquifer as determined from the solution derived
ratio of the aquifer vertical hydraulic conductivity to aquifer lateral hydraulic conductivity
(Kz/Kr = 0.0044) is about 0.10 m/day, about 200 times smaller than the lateral hydraulic
conductivity of the deep aquifer. This may indicate that the cumulative hydraulic effect of
disseminated clay and silt in the aquifer is similar to the clay-rich confining unit.
During the long-term monitoring period from May 2003 to November 2003 water levels
in P-2 (deep aquifer) were from 0.15 m to 0.60 m higher than water levels in P-1. Therefore,
under natural flow conditions experienced during the summer months, the groundwater
gradient is upward from the deep aquifer to the shallow aquifer, and the specific discharge
through the confining unit is estimated to be from about 0.004 m/day to about 0.017 m/day.
Assuming a porosity of 0.30, the average velocity of water through the 12.2 m thick confining
unit is 0.014 m/day to 0.057 m/day. The time for groundwater to move through the confining
unit under these gradients at the estimated average velocities range from about 200 days to
about 850 days.
343
c
Figure 8. Best fit Hantush-Jacob solution to drawdow.n data in semilog scale collected from (a) P-2; (b)
P-3; (c) Average drawdown data for P-2 and P-3.
Papadopulos-Cooper Solution to Drawdown Data

A solution for drawdown in large diameter wells that takes into consideration the storage
within the well, which is assumed to be negligible in the Theis method, has been presented by
344
Papadopulous and Cooper (1967). Besides the general assumptions mentioned earlier, the
added conditions are
Storage in the well cannot be neglected as well diameter is relatively large.
The aquifer is confined.
The well losses are negligible.
The general flow equation inside a large diameter well is (Kruseman and Ridder, 1983),
sw
Q
F (u w, )
4T
where F (uw, ) is a function for which numerical values are given.

2
2
r S
rw S
and w 2
uw
4Tt
rc
The index w stands for at the pumped well and rc is the radius of the unscreened part of
the well.
The best visual fit to the complete data sets, using each observation well individually, is
the Papadopolous-Cooper solution (Figures 9a and 9b). However, the best match for each set
of observation well data required aquifer properties and well bore diameters that were not
reasonable.
A
Figure 9. (Continued)
345
c
Figure 9. Best fit Papadopulos-Cooper solution to drawdown data in semilog scale collected from (a) P2; (b) P-3; (c) Average drawdown data collected from both P-2 and P-3.
Aquifer storage values were extremely small (on the order of 10-9). The size of the well
bore needed to match the delayed drawdown response was much larger than the actual bore of
the pumping well for both data sets. Both observation well P-2 and P-3 have an actual casing
346
radius of 0.175 m. The predicted casing radius for P-2 was 1.735 m, and for P-3 that was
3.372 m. Also, the transmissivity value predicted from the data for P-2 (6921 m2/day) differed
considerably from the value predicted from the data for P-3 (8401 m2/day). Because the
transmissivity and well-bore radius values predicted from the individual observation well
curve matches differed considerably, the drawdown data cannot be matched simultaneously
for both observation well P-2 and P-3 using average transmissivity values (Figure 9c).
Jacobs Straight-Line Plot

The Cooper-Jacob (1946) method of time-drawdown and Jacob (1950) method of
distance-drawdown are a modification of the Theis equation and assume that u is small.
However, the conditions for its application are more restricted than for the Theis and Chow
method. Cooper-Jacob and Jacob methods are valid where the time (t) is sufficiently large or
radial distance (r) is sufficiently small. The Cooper-Jacob time-drawdown approach is
modified to plot drawdowns at various radial distances from the pumping well. By graphing
drawdown on a linear scale versus distance on a logarithmic scale, a straight line fit results
(Jacob 1950). Estimates of the hydraulic properties can also be made, if the assumption of u
being sufficiently small (0.02) is valid. The intercept where the straight-line fit crosses the
zero drawdown represents the range of influence of the cone of depression (Weight and
Sonderegger 2000).
The same assumptions apply to the Cooper-Jacob analytical solution as the Theis
solution, but the well function W(u) is calculated for u<0.01 in order to neglect all but the
first two terms of the infinite series of the well function. A straight-line approximation of
W(u) is adequate for most applications even where u is as great as 0.1 (Halford and
Kuniansky 2002).
In the Theis formula, the exponential integral can be expanded in a convergent series, so
that the drawdown (s) may be written as (Kruseman and Ridder 1983)
Q
u2
u2
s
(0.5772 ln u
)
4T
2.2! 3.3!
From u
r 2S
it will be seen that u decreases as the time of pumping t increases.
4Tt
Accordingly, for large values of t and/or small values of r the terms beyond ln u in the series
of the equation become negligible. So for small values of u (<0.01) the drawdown can be
expressed as
Q
r 2S
(0.5772 ln
)
4T
4Tt
After rewriting and changing into decimal logarithms this equation reduces to
347
2.30Q
2.25Tt
log 2
4T
r S
Therefore, a plot of drawdown versus the logarithm of t forms a straight line. If this line
is extended till it intercepts time-axis where s=0, the interception point will have the
coordinates s=0 and t=t0. Substitution of these values into the drawdown equation gives
2.25Tt 0
2.30Q
,
log
4T
r 2S
and because
2.25Tt 0
2.25Tt 0
2.30Q
1 or S
0 , it follows that
2
4T
r S
r2
If t/t0=10 and hence log t/t0 =1, s can be replaced by s, i.e. by the drawdown difference
per log cycle of time, and it follows that
2.30Q
.
4s
Jacob straight-line plot was applied to interpret the time-drawdown data for Sreerampur
test as time was sufficiently large and radial distance is small. Drawdown recorded manually
at selected intervals was used to create the hand-drawn data graph in Figure 10.
Figure 10. (Continued)
348
Figure 10. Jacob straight-line time-drawdown method for a confined aquifer plotted on semilog papers
for (a) P-2 and (b) P-3.
In this method, the field data are plotted on semi-logarithmic paper and a straight line is
drawn through the field data points and extended backward to the zero drawdown axis. This is
the distance at which the well is not affecting the water level. It may intercept the axis at
some positive value of time (t0). The value of drawdown per log cycle (S) is obtained from
the slope of the graph.
Drawdown of the observation wells installed in the deep aquifer were used to calculate
transmissivity and storativity values of the deeper formation. The rate of drawdown after
1000 minutes is negligible because the water table was nearly in the steady condition.
For P-2 two straight lines were drawn and transmissivity and storativity values were
calculated as 1,070.784 m2/day and 0.00095 (first cycle) and 2,078.496 m2/day and 0.00268
(third cycle) respectively. The average values are 1,574.64 m2/day and 0.001815 respectively
for transmissivity and storativity.
These values are more appropriate as this well (P-2) is within a reasonable distance from
the pumped well. Two straight lines were also drawn for P-3 and transmissivity and
storativity values were calculated as 1,570.464 m2/day and 0.00068 (second cycle) and
2,944.656 m2/day and 0.00010 (third cycle) respectively. The average values are 2,257.56
m2/day and 0.00039 respectively for transmissivity and storativity. Using computer program,
transmissivity and storativity were estimated as 2,468 m2/day and 0.000546 respectively
(Figure 11).
349
Figure 11. Jacob straight-line time-drawdown method for a fully confined aquifer using computer
program for P-2.
Chows Method
Chow (1952) developed a method which has the advantage of avoiding the curve fitting
of the Theis method and not being restricted to small values of r and large values of t as is the
Jacob method (Kruseman and Ridder 1983). The same assumptions and conditions are
generally satisfied as for the Theis method because this method is directly based on the Theis
equation.
Q
W (u )
4T
To find the values of W(u) and u corresponding with the drawdown s measured at a
certain moment t, Chow introduced the function
W (u )e u
F (u )
2.30
F(u) can be calculated from the drawdown (s) versus the corresponding time (t) on single
logarithmic paper (t on logarithmic scale).
Using a computer program, transmissivity and storativity were estimated as 2,948 m2/day
and 0.0000357 respectively (Figure 12).
350
Figure 12. Analysis of data with the Chow method for P-2.
Theis Recovery Data Analysis for Confined Aquifer

The analysis of recovery data involves the measurement of the rise in water levels, also
referred to as residual drawdowns, following the cessation of a period of pumping at a
constant rate. The field procedure requires a drawdown measurement at the end of the
pumping period (t) and recovery measurements during the recovery period (t'). The residual
drawdown is plotted on a linear axis and the value of t/t' on the logarithmic axis. The
analytical method is based on the Theis theory and applies to confined aquifers with fully
penetrating wells. The method relies on the theory of superposition in that the water level rise
after the test is assumed to be the combined response to an imaginary well recharging the
aquifer and continued pumping. Imaginary recharge occurs at an identical rate to the constant
discharge during the pumping test. The equation for residual drawdown after a pumping test
with constant discharge is:
s'
Q
W (u ) W (u ' )
4T
where, u
r 2S
r 2S
'
and u
4Tt
4Tt '
If u and u' are small, less than 0.01, then the above equation can be simplified to:
s'
2.3Q
t
log 10 '
4T
t
351
A semilog plot of s' versus t/t' will yield a straight line. The slope of which is:
s '
2.3Q
4T
where, s' is the change in residual drawdown in one log cycle of t/t'. The same assumptions
as for the Cooper-Jacob, straight-line method must be met, and the flow to the well is in an
unsteady state when t'>(25 r2S)T and u<0.01.
Using computer program, transmissivity was estimated as 2,385 m2/day (Figure 13).
Figure 13. Analysis of recovery data with the Theis recovery method for P-2.
Comparison of Results for Different Methods

The results of the pumping test and recovery data analysis using different methods are
presented in table 5. The estimated transmissivity of the aquifer using drawdown data
collected at P-2 varied between 1,070 m2/day for first cycle analysis using Jacobs straight
line method and 2,948 m2/day applying Chows method from constant-discharge test, to 6921
m2/day for Papadopulous-Cooper method. For P-3, the estimated transmissivity ranged
between 1,570 m2/day using Jacobs straight line method from second cycle analysis and
2,956 m2/day using Hantush-Jacob solution, to 8,401 m2/day using Papadopulous-Cooper
method. Because of solution match difficulties, the Papadopolous-Cooper solution was not
considered as the appropriate solution to the aquifer-test data.
Transmissivity was calculated as 2,385 m2/day using recovery data analyzed by the Theis
recovery method. As these are graphical methods of solution, there is often slight variation in
the results, depending upon the accuracy of the graph construction and subjective judgments
in matching field data to type curves (Fetter 1994).
352
Table 5. Aquifer properties determined from constant-discharge pumping test data

using different methods
Method
Papadopulous-Cooper
Hantush-Jacob
Jacob (Hand Drawn)
1st Cycle
2nd Cycle
3rd Cycle
Jacob
Chow
Theis Recovery
Transmissivity (m2/day)
P-2
P-3
6,921
8,401
2,300
2,956
Storage Coefficient
P-2
P-3
0.0013
0.0022
1,071
2,078
2,468
2,948
2,385
0.00095
0.00268
0.000546
0.0000357
-
1,570
2,945
-
0.00068
0.00010
-
The accuracy of the numerical values of the hydraulic characteristics of water-bearing

layers and less permeable strata determined during the graphical analyses and the accuracy of
the assumed boundary conditions play an important role in the reliability of the results
obtained by these methods.The transmissivity values estimated from the Theis curve method
and the Cooper-Jacob method are generally are comparable (Weight and Sonderegger 2000),
and the resulting answers are almost the same. Well losses and partial penetration have a
minimal effect on transmissivity values that are estimated using the Cooper-Jacob method.
Additional drawdown at later times is due to declining heads in the aquifer and the rate of
decline is controlled mostly by the transmissivity of the aquifer. Analyzing the change in
drawdown at later times negates the effect of a fixed offset due to well losses and partialpenetration on the determination of transmissiviy (Halford and Kuniansky 2002).
Estimated storage coefficient values ranged from 0.0000375 to 0.00268 for P-2 and from
0.00010 to 0.0022 for P-3. The estimated storage coefficients indicate that the aquifer is
confined to leaky-confined or semi-confined in nature. Bouwer (1978) and Fetter (1994)
suggest that storage coefficients for confined aquifer can vary from 0.00001 to 0.001, and
Weight and Sonderegger (2000) suggest that storage coefficients for leaky-confined or semiconfined aquifers can vary from 0.001 to 0.03.
The sorting of unconsolidated sediments largely controls the expected range of hydraulic
conductivity. Well-sorted sediment has a much larger hydraulic conductivity than poorlysorted sediment, because finer material fills the voids between coarser grains in poorly-sorted
sediment. The hydraulic conductivity of unconsolidated sediment can be estimated
empirically from the grain-size distribution (Vukovic and Soro 1992). Hydraulic conductivity
estimates from grain-size distributions typically have a greater uncertainty than estimates
from aquifer tests (Halford and Kuniansky 2002). Using the Hantush-Jacob solution, the
vertical to lateral aquifer hydraulic conductivity ratio was estimated at 0.0044, and the
confining unit vertical hydraulic conductivity was estimated at 0.35 m/day. Drawdown in
both observation wells becomes constant in the interval from about 200 to 500 minutes.
During the aquifer test, water levels in P-1 were about 0.4 m higher than water levels in
P-2. Therefore, under pumping stresses similar to those induced during the aquifer test, the
ground-water gradient is downward from the main aquifer to the deep aquifer, and the
groundwater flux through the confining unit is estimated at about 0.011 m/day. Assuming a
353
porosity of 0.30, the average velocity of water through the confining unit is 0.037 m/day. The
time for ground water to move through the confining unit under this gradient at the estimated
average velocity is about 330 days.
CONCLUSION
Three distinct aquifer units have been classified in the studied Meghna floodplain areas to
an investigated depth of about 400m. However, there is a complex aerial variability of the
hydrogeologic characteristics as a consequence of the floodplain depositional history of the
formations. Groundwater level in the shallow and main aquifers varies in places due to localscale lithologic variations, leakage, the impact of irrigation abstraction and partially by
unconfined storage change in the aquifer. During the dry irrigation period pumping water
mainly from the lower part of the shallow and upper part of the main aquifers, water levels in
all three aquifers response to the pumping stress. The trend of water level fluctuations in
different aquifer units supports the hydraulic connectivity of aquifer formations. The
aquitards separating aquifers are not continuous regionally but locally extended. During the
pumping test, water levels in observation wells open to the shallow and main aquifers showed
no noticeable effect from pumping in the deep aquifer that indicates at least local hydraulic
separation of aquifers and under conditions of moderate groundwater use for domestic and
municipal supply, arsenic and/or chloride-rich groundwater in the upper aquifers are not
likely to be drawn into the deep aquifer.
Aquifer test results as well as aquifer properties and characteristics of the deep aquifer
show that the deep aquifer can yield significant amounts of potable water. As different
hydrogeologic factors control the aquifer parameters and the graphical methods of solution
depend upon the accuracy of the graph construction and subjective judgments in matching
field data to type curves, solution results have varied for different method of analysis.
However, all results indicate that the aquifer is confined to leaky-confined or semi-confined
in nature. Slight deviations are not prohibitive to the application of different methods. When
greater deviations from the above assumptions occur, special flow problems may raise. So, it
is useful to evaluate data using as many methods as possible. Each may help to provide a
different perspective and aid in a better interpretation. The Hantush-Jacob solution for a leaky
confined aquifer was chosen as the most representative of the physical situation and this gives
better results considering field condition in the deltaic floodplain aquifers.
ACKNOWLEDGMENT
The authors are grateful to the respective officials of Ground Water Hydrology (GWH) of
Bangladesh Water Development Board (BWDB) and Bangladesh Arsenic Mitigation Water
Supply Project (BAMWSP) for undertaking such an extensive aquifer pumping test
component at Sreerampur. Gratitude goes to Mr. Alamgir Hossain, MA Karim, Satish C Das GWH, BWDB for supporting authors to participate during the pump test, Mr. M Zainal Abdin
to support data analysis using computer programs developed by him and to all field personnel
of BWDB participated in performing pump test.
354
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Ground Water Hydrology Division-I, BWDB.
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Domenico PA, Schwartz FW (1997) Physical and Chemical Hydrogeology, 2nd edition, John
Wiley and Sons Inc., New York, 506p.
DPHE-BGS (2001) Arsenic contamination of groundwater in Bangladesh, British Geological
Survey and Department of Public Health Engineering, Govt. of Bangladesh; rapid
investigation phase, Final Report.
Driscoll FG (1995) Groundwater and Wells, US Filter/Johnson Screens, St. Paul, MN 55112,
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Fetter CW (1994) Applied Hydrogeology, Third Edition: Macmillan, New York, 691 p.
Halford KJ and Kuniansky EL (2002) Documentation of spreadsheets for the analysis of
aquifer test and slug test data: U.S. Geological Survey, open-file report 02-197, 51 p.
Hantush MS (1956) Analysis of data from pumping tests in leaky aquifers: Transactions of
the American Geophysical Union, vl.37, 702-714.
Hantush MS (1964) Drawdown around wells of variable discharge. Journal of Geophysical
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Hantush MS (1960) Modification of the theory of leaky aquifer. Journal of Geophysical
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Wiley, New York, pp 321-386.
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Kruseman GP and Ridder NAD (1983) Analysis and evaluation of pumping test data:
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Journal of Environmental Geology, DOI 10.1007/s00254-007-0907-3.

ISSN: 2158-5717
Chapter 13
APPLICATION OF DNA MICROARRAYS TO

MICROBIAL ECOLOGY RESEARCH:
HISTORY, CHALLENGES, AND RECENT
DEVELOPMENTS
John J. Kelly
Department of Biology, Loyola University Chicago
6525 N. Sheridan Road, Chicago, IL 60626
ABSTRACT
During the last three decades, molecular methods have dramatically expanded our
view of microbial diversity in natural and engineered systems. A variety of molecular
approaches, including both PCR-based and hybridization-based techniques, have been
applied extensively to the analysis of complex microbial communities and have yielded
new insights. However, the majority of molecular methods that are widely used in
microbial ecology are limited in their ability to encompass the incredible diversity of
microbial communities. DNA microarrays, which were first introduced in the early
1990s, are one of the fastest growing technologies in biology, and they offer tremendous
potential for microbial ecologists. DNA microarrays consist of nucleic acids spotted
within a very small area on some solid support, and they enable the immobilization and
simultaneous hybridization of hundreds of thousands of nucleic acids. This represents a
dramatically higher degree of multiplexing than is possible with other widely used
technologies. In addition, microarrays offer the advantages of increased speed of
detection, low cost, and the potential for automation. Microarray technology has been
used extensively for measuring gene expression in a wide variety of organisms, including
human cells, plants, yeast, and bacteria, but its application to microbial ecology has been
more limited. There are significant challenges to the use of microarrays in microbial
ecology studies, including optimization of specificity and sensitivity and quantification of
targets. However, in recent years several research groups have made significant progress
in overcoming these challenges, and microarrays are beginning to be applied more
frequently to microbial ecology studies in a variety of systems including terrestrial soils,
Phone: 773.508.7097 ; Fax: 773.508.3646 ; E-mail: jkelly7@luc.edu
358
John J. Kelly
wetland sediments, and freshwater and marine ecosystems. This article will provide a
brief history of the use of molecular methods in microbial ecology, and will then review
the development of microarray technology, the challenges that exist for application of
microarrays to microbial ecology, the available strategies for overcoming these
challenges, and some recent applications of microarrays to studies in microbial ecology.
PURE CULTURE TECHNIQUES

One of the key innovations that spurred the development of the science of microbiology
was the pure culture technique, which was introduced by Robert Koch in the late 1800s
(Beck, 2000). The pure culture technique made it possible for the first time to isolate
individual microbial species in the lab and to study their physiological and biochemical
properties, and it enabled Koch in 1876 to prove the germ theory of disease by demonstrating
conclusively that Bacillus anthracis was the causative agent of anthrax (Koch, 1876).
Subsequently Koch used the pure culture technique to identify other disease-causing
microorganisms, including Mycobacterium tuberculosis and Vibrio cholerae (Beck, 2000),
thus revolutionizing our understanding of human and animal disease.
The pure culture technique also allowed microbiologists to explore the incredible
diversity of microorganisms in the natural world. In 1887 Sergei Winogradsky discovered the
process of chemolithotrophy by isolating the marine bacterium Beggiatoa and observing that
it could obtain the energy it needed for growth from the oxidation of an inorganic substrate
(hydrogen sulfide), and in 1890 he elucidated another chemolithotrophic process when he
isolated the first nitrifying bacteria from soil: Nitrosomonas and Nitrosococcus (Beck, 2000).
In 1888 Martinus Beijerinck isolated the first nitrogen fixing bacterium (Bacillus radicicola)
from the root nodules of the pea plant, and subsequently he isolated the first organism capable
of anaerobic respiration: the sulfate reducing Spirillum desulfuricans, which is now known as
Desulfovibrio desulfuricans (Beck, 2000).
More recently, Konneke et al. (2005) were the first to isolate an autotrophic ammoniaoxidizing Archaeon in pure culture. Autotrophic ammonia-oxidation was previously thought
to be limited to the Bacterial domain, but the possibility of Archaeal ammonia oxidation was
suggested by several recent studies: in a metagenomic analysis of the Sargasso Sea, Venter et
al. (2004) found a unique ammonia monooxygenase gene (ammonia monooxygenase
catalyzes the first step of ammonia oxidation) on an Archaeal-associated scaffold. In addition,
Treusch et al. (2005) found genes encoding a potential ammonia monooxygenase on a
metagenomic soil clone alongside an Archaeal ribosomal RNA operon. These studies
suggested that an Archaeon might be able to oxidize ammonia, but it was not until Konneke
et al. (2005) isolated the first pure culture of an Archaeal ammonia oxidizer, named
Nitrosopumilus maritimus, that the ability of an Archaean to catalyze this process was
conclusively proven.
Culture-based studies have thus made tremendous contributions to our understanding of
microbial diversity and microbial physiology, and they continue to be a vital component of
microbiology. However, culture-based studies, which require growth of microorganisms in
the laboratory, are severely limited by the fact that most microorganisms are difficult or
impossible to culture. Direct microscopic counts of bacteria in most habitats routinely exceed
culture-based counts (plate counts or most probable number counts) by several orders of
Application of DNA Microarrays to Microbial Ecology
359
magnitude (Amann et al., 1995). For example, Jones (1977) found that the culturable fraction
represented 0.25% of the total population in both freshwater and freshwater sediment, and
Torsvik et al. (1990) reported that culturable organisms represented only 0.3% of the total
community in soil. Staley and Konopka (1985) termed this phenomenon the great plate
count anomaly, and this anomaly represents a significant challenge to microbial ecologists,
as it indicates that culture-based studies provide an extremely restricted view of the diversity
of the natural microflora. Fortunately, molecular techniques, which do not rely on laboratory
culture, have led to significant advances in the study of microbial communities (Kelly, 2003).
SSU RRNA TECHNIQUES

The most widely used molecular methods for the analysis of microbial communities are
based on the small subunit ribosomal RNA (SSU rRNA) gene. The use of SSU rRNA as a
phylogenetic marker was first proposed by Carl Woese in 1977 (Fox et al., 1977). The SSU
rRNA molecule is a useful phylogenetic marker because it is present in all cells and because
its sequence is fairly well conserved across phylogenetic groups. Since the pioneering work of
Carl Woese, SSU rRNA gene sequences have been used to develop a comprehensive
phylogenetic framework for the analysis of microbial communities (Amann et al., 1995). The
most current version of the Ribosomal Database Project (release 9.56) contains 451,545
aligned and annotated SSU rRNA sequences (Cole et al., 2007).
Molecular techniques that analyze prokaryotic SSU rRNA (i.e. 16S rRNA) genes have
been widely applied to the study of microbial ecology (Macrae, 2000), and there are a wide
variety of methods that have been used. For example, a phylogenetic inventory of the
prokaryotic component of a microbial community can be assembled using the Polymerase
Chain Reaction (PCR) to amplify the 16S rRNA genes contained within the community,
followed by cloning and sequencing this collection of amplified genes. This approach, known
as clone library sequencing, was first applied by Giovannoni et al. (1990) for the analysis of
marine microorganisms from the Sargasso Sea. Phylogenetic inventories of this type can be
produced for the entire bacterial component of a microbial community by using universal
primers designed to amplify 16S rRNA genes from all of the bacteria within a sample (e.g.
Borneman et al., 1996; Kuske et al., 1997), or they can be produced for a specific
phylogenetic group by using primers designed to amplify only the 16S rRNA genes of the
group of interest. For example, Bruns et al. (1999) used primers specific for the 16S rRNA
genes of autotrophic ammonia oxidizers to create an inventory of ammonia oxidizing bacteria
from soil.
Clone library-based inventories have significantly increased our understanding of
microbial diversity by revealing much greater diversity than was detected by classical culturebased studies (Becker et al., 2000). However, clone library preparation and sequencing is time
consuming, labor intensive, and expensive. Although the cost and time required for large
scale sequencing projects are decreasing quickly, the incredible diversity of microbial
communities makes it impractical at this point to attempt to sequence every amplicon
produced from PCR amplification of an environmental sample. For example, soil from a
beech forest showed total bacterial counts of 1.5 x 1010 g soil-1 and approximately 4,000
unique bacterial genomes based on DNA reassociation analysis (Torsvik et al., 1990), and a
360
John J. Kelly
more recent study used reassociation kinetics to determine that a pristine soil contained more
than one million distinct genomes (Gans et al., 2005). Attempting to capture all of this
biodiversity by sequencing a clone library is beyond our current capabilities. Therefore,
although phylogenetic inventories of microbial communities based on cloning and sequencing
16S rRNA genes can provide a great deal of useful information, these inventories will
generally be incomplete, so populations comprising very small fractions of the overall
community may not be detected. In addition, clone library sequencing is too slow to be useful
for routine profiling and comparison of large numbers of environmental samples.
As an alternative to cloning and sequencing, techniques such as denaturing gradient gel
electrophoresis (DGGE; Muyzer et al., 1993) and terminal restriction fragment length
polymorphism analysis (T-RFLP; Liu et al, 1997) can be used to analyze the amplicons
produced by PCR amplification of DNA extracted from a microbial community. DGGE and
T-RFLP do not identify individual bacterial species. They instead generate profiles for
microbial communities based on differences in the gene sequences of their constituents, and
these profiles can be used to compare microbial communities and assess their degree of
similarity or difference. DGGE and T-RFLP have been applied to 16S rRNA amplicons
produced with universal PCR primers (e.g. Crump et al., 2003; Janus et al., 2005) and to
amplicons produced with group-specific PCR primers (e.g. Cebron et al., 2004; Rosch and
Bothe, 2005), and these approaches have been used to compare microbial communities from
different habitats (e.g. Ludemann et al., 2000), and to assess changes in microbial
communities over time (e.g. Duineveld et al., 1998), seasonal changes (e.g. Crump et al.,
2003), and changes resulting from different experimental treatments (e.g. Klamer et al.,
2002).
LIMITATIONS OF PCR-BASED METHODS

Clone libraries, DGGE, and T-RFLP are all useful techniques, but they are all reliant on
PCR amplification, and there are significant limitations to all techniques that are based on
PCR amplification of DNA extracted from environmental samples. First, PCR-based assays
cannot provide reliable quantitative information on microbial community composition due to
the potential for bias in PCR amplification. PCR bias can be caused by differences in DNA
extraction efficiency (Miller et al., 1999), differences in gene copy number (Farrelly et al.,
1995), and differences in the efficiency of the PCR reaction itself (Suzuki and Giovannoni,
1996). PCR bias can result in misrepresentation of phylogenetic diversity (Lueders and
Friedrich, 2003), and thus it can be problematic in microbial ecology studies that rely on
PCR. A second limitation of PCR-based assays is that DNA can persist in the environment
outside of a cell for significant periods of time. For example, free DNA can bind to soil
particles (Blum et al., 1997) and this binding can protect DNA from degradation (Demaneche
et al., 2001). Thus, when working with environmental samples, it is possible for PCR to
amplify DNA from cells that are no longer living, which would be a confounding factor in
studies of microbial community structure. Both of these limitations, PCR bias and DNA
persistence, should be considered when interpreting results from PCR-based community
analyses such as clone library sequencing, T-RFLP, and DGGE (Kelly, 2003).
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PROBE-BASED METHODS
As an alternative to PCR-based techniques, probe-based methods can be used to detect
specific target bacteria in environmental samples. DNA probes, which are composed of short
segments of DNA, typically 15 to 25 nucleotides in length, can be synthesized in the lab and
can be designed to target regions of the 16S rRNA gene that are unique to a particular
phylogenetic group (Stahl, 1995). Ribosomal RNA is a good target for probing because active
cells contain thousands to tens of thousands of copies of ribosomal RNA per cell, making it a
naturally amplified target which can often be detected without PCR amplification (Amann
and Ludwig, 2000).
DNA probes have generally been applied using either in-situ or membrane-based
hybridization techniques. The in-situ technique, also known as fluorescence in-situ
hybridization (FISH), hybridizes a fluorescently labeled probe to fixed whole cells. The
fixation process renders the cells permeable to the probe, so cells containing RNA matched
by the probe will be fluorescently labeled and can be observed and counted with an
epifluorescence microscope. FISH, which was first developed by DeLong et al. (1989), can
provide information on cell morphology as well as the abundance and spatial distribution of
targeted organisms (Amann et al., 1995). FISH can be especially useful for examining spatial
relationships between different phylogenetic groups of bacteria, as several unique probes can
be applied to a sample simultaneously if the probes are labeled with different fluorescent
markers (Mobarry et al., 1996).
DNA probes can also be utilized in a membrane hybridization format known as dot-blot
hybridization, which is similar to a conventional Southern hybridization. In dot-blot
hybridization, RNA isolated from a microbial community is immobilized on a nylon or
nitrocellulose membrane filter and exposed to a radiolabeled probe. Hybridization of a probe
to an immobilized target can be detected based on the radioactive tag and can provide
information on the presence or absence as well as relative abundance of the group targeted by
that probe. This approach was first applied to monitoring population changes in the rumen of
cattle (Stahl et al., 1988), and since then it has been used extensively for the analysis of
microbial communities from a variety of habitats (e.g. Raskin et al., 1996; Rooney-Varga et
al., 1997; Weber, 2001). However, membrane hybridization has several significant
limitations. Since the probes are labeled with radioactive tags, only one probe can be applied
per membrane, so a separate membrane must be prepared for each probe, which can become
quite cumbersome. In addition, membrane hybridization can provide information on relative
rRNA abundance, but relative rRNA abundance can not be directly translated into cell
numbers since cellular rRNA contents can vary significantly with growth rate (Amann et al.,
1995).
In-situ and membrane hybridization techniques are extremely useful tools for microbial
ecologists, and both methods have been widely applied. However, the scope of such studies is
often constrained by the fact that these hybridization techniques severely limit the number of
probes that can be applied simultaneously, thus limiting the amount of information that can be
acquired (Kelly, 2003). Microarrays offer the opportunity to overcome some of the limitations
of in-situ and membrane hybridization techniques.
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DNA MICROARRAY TECHNOLOGY

DNA microarrays (also known as DNA microchips or DNA chips) generally consist of a
set of nucleic acids spotted within a very small area on some solid support (Small et al.,
2001). The concept of microarray hybridization of nucleic acids was first proposed 20 years
ago by Bains and Smith (1988) as a method for sequence determination. In 1994 Pease et al.
built one of the first DNA microarrays using photolithography to synthesize a miniaturized
array of 256 densely packed oligonucleotide probes on a glass surface (Pease et al., 1994).
Pease et al. demonstrated that these surface immobilized oligonucleotides could be hybridized
in parallel to fluorescently labeled oligonucleotide targets, and hybridization could be
detected by epifluorescence microscopy. Pease et al. used their microarrays for sequencing by
hybridization, as had been proposed by Bains and Smith (1988). In 1995 Schena et al. took a
different approach and built a microarray by immobilizing 45 Arabidosis thaliana cDNAs on
poly-L-lysine-coated microscope slides using a custom-built arraying machine with a single
printing tip. All of the microarray-immobilized cDNAs were hybridized simultaneously with
a mixture of fluorescently labeled cDNAs produced from total Arabidopsis mRNA by a
single round of reverse transcription. Hybridization to the microarray-immobilized cDNAs
was quantified by measuring fluorescence with a laser scanner. This microarray was able to
quantify the relative expression of 45 Arabidopsis genes simultaneously (Schena et al., 1995).
Since the pioneering work of Pease et al. (1994) and Schena et al. (1995), microarrays have
been widely used: according to a search of Web of Science (Thompson Scientific,
Philadelphia, PA) on February 1, 2008, Pease et al., (1994) had received 674 citations and
Schena et al. (1995) had been cited 3,462 times. In addition, rapid developments in both
robotics and miniaturization technology have made it possible to immobilize higher numbers
of nucleic acids, from thousands to hundreds of thousands, in even smaller areas, thus
providing extremely high hybridization capacity (Gentry et al., 2006).
The main application of microarrays to date has been analysis of gene expression (Gentry
et al., 2006). In these studies, mRNAs are isolated from different samples or different
experimental treatments and each mRNA pool is then either directly labeled with different
fluorescent dyes (typically Cy3 and Cy5) (e.g. Taniguchi et al., 2001) or used to produce
differentially labeled cDNAs via reverse transcription (e.g. Lashkari et al., 1997).
Differentially labeled nucleic acid samples can then be hybridized simultaneously to a
microarray containing gene specific probes, and comparison of the intensities of the two dyes
provides relative expression levels of the genes contained on the microarray. This technique
allows for the analysis of the differential expression of thousands of genes in a single
experiment, and it has been used to analyze gene expression in a wide variety of organisms,
including human cell lines (DeRisi et al., 1996), plants (Schena et al., 1995), yeast (Lashkari
et al., 1997), and bacteria (de Saizieu et al., 1998). Microarrays have also been used for the
detection of single nucleotide polymorphisms (Hacia, 1999; Straub, 2002), the detection of
mutations (Cronin et al., 1996; Gerry et al., 1999), and the comparison of microbial genomes
(Cho and Tiedje, 2001; Murray et al., 2001).
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MICROARRAYS FOR MICROBIAL COMMUNITY ANALYSIS

David Stahls group first proposed the use of microarrays for the detection of microbes
within complex microbial communities (Guschin et al., 1997). The Guschin et al. approach
was essentially the reverse of the dot-blot membrane hybridization technique in that Guschin
et al. immobilized a set of oligonucleotide probes on a microarray, hybridized a fluorescently
labeled target to all of these immobilized probes simultaneously, and detected hybridization
via epifluorescence microscopy. Guschin et al. successfully demonstrated this approach by
fabricating a microarray that included a set of eight oligonucleotide probes targeting the 16S
rRNA genes of several groups of nitrifying bacteria. When this microarray was hybridized
with fluorescently labeled RNA from several reference strains, the immobilized probes were
shown to selectively capture RNA from the appropriate target organisms, thus providing
specific identification of the target organisms. Guschin et al. suggested that this approach
could be used for the detection of different microbial populations within complex
communities, and that the extremely high probe capacity of microarrays should allow for the
detection of hundreds to thousands of target organisms simultaneously. This would represent
much higher throughput than is possible with other probe hybridization formats (i.e.
membrane hybridization and FISH).
After the pioneering work of Guschin et al. (1997), several groups demonstrated the
effectiveness of microarrays for the detection of bacteria in complex communities based on
hybridization of microarrays with RNA isolated directly from environmental samples: Small
et al. (2001) detected Geobacter in soil samples; Koizumi et al. (2002) detected bacteria in
oil-contaminated marine sediments; El Fantroussi et al. (2003) detected Acidobacteria as well
as Alpha, Beta, and Gamma Proteobacteria in estuarine sediments; Kelly et al. (2005)
detected nitrifying bacteria in wastewater treatment plant samples; and Smoot et al. (2005)
detected bacteria in human saliva.
One significant advantage of microarrays for the analysis of complex microbial
communities via 16S rRNA sequences is that their extremely high probe capacity enables
researchers to create highly redundant and hierarchically nested probe sets. Redundant probes
are multiple distinct probes that target the same organism or group; for example, in Figure 1
probes 1 and 2 are redundant probes for organism A and probes 3 and 4 are redundant probes
for organism B. Hierarchically nested probes target organisms at multiple phylogenetic levels;
for example, in Figure 1 probes 1, 6, and 7 are hierarchically nested. Due to the fact that the
16S rRNAs include both highly conserved and highly variable regions, it is possible to design
hierarchically nested probes that target organisms at various phylogenetic levels (e.g. species,
genus, phylum and domain), with highly conserved sequences giving broad taxonomic
resolution and hypervariable sequences giving genus- and species-level resolution. For
example, SSU rRNA probes have been designed for the highest taxonomic level, the domains
Archaea, Bacteria, and Eukarya; for intermediate levels such as the alpha, beta, and gamma
subclasses of Proteobacteria; and for many genera, species, and subspecies (Amann et al.,
1995).
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John J. Kelly
Figure 1. Model of a redundant and nested probe set. The model shows the phylogenetic realtionships
of three hypothetical bacterial species (A, B, and C) as well as the coverage of hypothetical probes 1-7.
The use of redundant and hierarchically nested probes is not unique to microarray
technology. These approaches are often incorporated to some degree in FISH and membrane
hybridization experiments. However, the high probe capacity of microarrays enables
redundant and hierarchically nested probes to be used on an unprecedented scale. Microarrays
offer the possibility of including redundant and hierarchically nested probes for virtually
every target, which would enable the detection of target organisms to be confirmed by
multiple probes, thus greatly reducing false-positive errors. In addition, hierarchical nesting
can provide a more complete picture of the overall phylogenetic composition of a microbial
community than is possible if only genus- or species-specific probes are utilized, since
broadly targeted probes will include organisms not targeted by more specific probes. A
number of microarray studies have included both redundant and hierarchically nested probes
(Liu et al., 2001; Loy et al., 2002; Kelly et al., 2002; Loy et al., 2005; Sanguin, Herrera et al.,
2006).
MICROARRAY FORMATS
One interesting aspect of microarray technology is the fact that multiple formats are
available. The most widely-used format is the planar or 2-D array (Starke et al., 2006) in
which nucleic acids are immobilized on a glass slide in a single layer (e.g. Schena et al.,
1995). Planar arrays are widely available because the equipment required for fabricating these
arrays has been commercialized and is rapidly dropping in price due to competition between a
number of manufacturers, making this format accessible to many laboratories (Li and Liu,
2003). An alternative to planar arrays are gel-based microarrays (also known as 3-D arrays) in
which individual acrylamide gel pads or drops are arrayed on a glass slide, and nucleic acids
are covalently cross-linked to the acrylamide within these three-dimensional gel pads
(Yershov et al., 1996). Gel-pad microarrays have not been used as widely as planar arrays
because these arrays are not available commercially and currently they are only fabricated by
a limited number of research laboratories (Li and Liu, 2003). However, gel-pad microarrays
do offer some advantages over planar arrays. For example, the three dimensional nature of the
gel pads makes it possible to immobilize a much higher concentration of oligonucleotide
probes in the same microarray surface area: planar arrays can immobilize a maximum of
approximately 10 pmol/cm2, whereas gel-pad microarrays can immobilize several nmols/cm2
(Li and Liu, 2003). Higher probe concentration is significant as it can improve detection of
low abundance targets (Starke et al., 2006).
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One limitation of gel-pad microarrays is that they require targets to diffuse into the gel
pad in order to hybridize with probes located within the gel interior, so the pore size of the gel
will limit target access. Targets ranging in size from 20-150 nucleotides have been shown to
penetrate the gel effectively and hybridize to probes throughout the entire three-dimensional
gel pad (Starke et al., 2006). Therefore, when native RNAs or other large nucleic acids are
used as the target for gel-pad microarrays, fragmentation of the nucleic acids is necessary
prior to hybridization to allow access to the gel interior. Interestingly, fragmentation has also
been shown to result in stronger hybridization signals for planar arrays (Liu et al., 2007).
Fragmentation is beneficial in both gel-based and planar microarrays because it eliminates the
three dimensional structure of the target nucleic acids which can interfere with hybridization,
and it reduces steric hindrance during hybridization which can occur with large target
molecules (Liu et al., 2007).
Fragmentation of nucleic acids can be accomplished by treatment with divalent ions
(Chee et al., 1996), alkali (Proudnikov and Mirzabekov, 1996), or nucleases (Gunderson,
1998). In addition, Kelly et al. (2002) developed an effective fragmentation method based on
radical-generating coordination complexes. This method, which included simultaneous
fluorescent labeling of the targets, resulted in random fragmentation of nucleic acids and
effective hybridization of targets to DNA microarrays (Kelly et al., 2002). This method has
been used in several microarray studies of environmental microbial communities (El
Fantroussi et al., 2003; Smoot et al., 2005; Kelly et al., 2005; Siripong et al., 2006).
The gel used in manufacturing gel-pad microarrays can also be modified to increase its
porosity. Several new gel polymers with significantly larger pore sizes are under
development, which should help reduce the impact of the gel on target diffusion and should
allow for the hybridization of larger targets (Starke et al, 2006).
FUNCTIONAL GENE MICROARRAYS

Bacteria can be placed into taxonomic groups based on phylogenetic affiliations (species,
genera, etc.), but bacteria can also be grouped into functional guilds which can be defined by
some metabolic capability (e.g. ability to denitrify) (Kelly, 2003). In some cases bacteria
within a phylogenetically defined group share some metabolic functions. For example, among
the bacteria autotrophic ammonia oxidation is restricted to two monophyletic groups: the first
group belongs to the gamma subdivision of the Proteobacteria and includes one genus,
Nitrosococcus; the second group belongs to the beta subdivision of the Proteobacteria and
includes two genera: Nitrosomonas and Nitrosospira (Avrahami et al., 2002). In contrast,
some metabolic functions are more widely distributed across numerous phylogenetic groups.
An example is denitrification, which is found in about 50 bacterial genera (Rich et al., 2003).
Molecular approaches based on 16S rRNA genes can target microorganisms based on their
phylogenetic affiliation, which can provide functional information in some cases (e.g.
ammonia oxidizing bacteria). However, 16S rRNA approaches are not useful for the detection
of specific functional guilds when the function is widely distributed over the phylogenetic
tree (e.g. denitrification) (Braker et al., 2001).
Functional guilds can be targeted using so called functional genes that code for
enzymes critical to some metabolic process. Functional genes can be useful for detecting and
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John J. Kelly
monitoring functional guilds, and sequence variations within these functional genes can in
some cases be used to differentiate members of a functional guild (e.g. Purkhold et al., 2000).
Several prokaryotic functional genes involved in biogeochemical cycling processes have been
identified and widely studied. For example, amoA codes for the membrane-associated activesite polypeptide of ammonia monooxygenase, which catalyzes the first step in nitrification
(Rotthauwe et al. 1997); nirS and nirK code for structural variants of nitrite reductase, which
catalyzes a key step in denitrification (Braker et al. 1998); nifH codes for one of the subunits
of nitrogenase, the enzyme that catalyzes nitrogen fixation (Zehr et al., 2003) ; and dsrA and
dsrB code for the alpha and beta subunits of dissimilatory sulfite reductase, which catalyzes a
key step in sulfate reduction (Dahl et al., 1993). These functional genes have been used to
analyze microbial communities via many of the same molecular approaches discussed above,
including clone library sequencing (Zehr et al., 1998; Braker et al. 2000), T-RFLP (Ohkuma
et al ., 1999; Avrahami et al., 2002; Angeloni et al., 2006), and DGGE (Ibekwe et al., 2003).
A number of research groups have also applied microarray technology to the detection of
functional genes. For example, Wu et al. (2001) built a functional gene microarray targeting
different variants of several genes involved in the nitrogen cycle (amoA, nirK, and nirS). In
this array, large PCR products (0.76 kb) from each of the functional gene variants were
spotted on the array and used as hybridization probes. Taroncher-Oldenberg et al (2003)
designed a microarray that included smaller oligonucleotide probes (70-mer) targeting
different variants of amoA, nifH, nirK, and nirS. Zhou (2003) built a microarray that included
50-mer probes targeting genes involved in both nitrogen cycling (nirS, nirK, nifH, and amoA)
and sulfur reduction (dsrA and dsrB), and Zhang et al. (2007) built an oligonucleotide
microarray (15-25 mers) targeting variants of nifH.
CHALLENGES FOR MICROARRAY TECHNOLOGY: SPECIFICITY

The application of microarrays to the assessment of microbes in the environment poses a
number of technical challenges. One of the main challenges is the specific detection of target
nucleic acids against a complex background of non-target sequences. In any hybridization
format, the potential exists for non-specific hybridization, i.e. hybridization between a target
and probe that are not perfectly complementary. To achieve highly specific detection the
discrimination of perfect and imperfect duplexes is important, and for detection of specific
targets with short oligonucleotide probes this often requires the discrimination of single-base
mismatches. In membrane-based hybridizations, single base mismatch discrimination can be
achieved by optimizing the hybridization and wash conditions (buffer composition, salt
concentration, and temperature) for each oligonucleotide probe (Stahl et al., 1988). This
approach is effective for membrane hybridization since each membrane is hybridized with a
single probe. However, the challenge for microarrays is that a large number of probes, which
can vary in duplex stability due to differences in length and base composition, are hybridized
and washed simultaneously under the same conditions, making it impossible to optimize the
conditions for all of the probes on the microarray.
One approach to addressing this challenge is to design sets of probes such that all probes
on an array have nearly identical melting temperatures. In this way, the microarray can be
hybridized and washed under conditions (buffer composition, salt concentration, and
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temperature) that will minimize non-specific hybridizations for all of the probes. This
approach can be effective (e.g. Bodrossy et al., 2003; Zhang et al., 2007), but it places
significant constraints on probe design, which could make it difficult to build microarrays
with very large numbers of probes.
Another approach to optimizing specificity is the inclusion of tetramethylammonium
chloride (Maskos and Southern, 1993) or betaine (Rees et al. 1993) in the hybridization
buffer. These compounds equalize the melting points of oligonucleotides with different base
compositions by stabilizing AT base pairs (Peplies et al., 2003) so that a single wash
temperature can be used for all probes on a microarray (for an example of this approach see
Loy et al., 2002). This approach is reasonably effective and it is simple, but it does require
that all probes on the microarray be identical in length, which again places a constraint on
probe design.
Specificity on microarrays can also be optimized via non-equilibrium dissociation
kinetics, first demonstrated by Liu et al. (2001). In this approach, the microarray is hybridized
with the targets, a thermal platform is used to control the temperature of the microarray, and
the dissociation of targets from each of the probes is monitored simultaneously by measuring
the fluorescent signal for each probe as the temperature of the array is incrementally
increased. The resulting melting profiles (signal versus temperature) can be used to
discriminate target hybridizations from non-target hybridizations, since duplexes containing
one or more mismatches are generally less stable and dissociate at a lower temperature than
perfect-match duplexes (e.g. see Figure 2). Liu et al (2001) first demonstrated the
effectiveness of the non-equilibrium dissociation approach in gel-based microarrays, and Li et
al. (2004) and Wick et al. (2006) subsequently confirmed its effectiveness in planar
microarrays. The non-equilibrium dissociation approach has been applied to microarray
analysis of microbial communities (Kelly et al., 2005; Siripong et al., 2006), so it has been
shown to be effective, but this approach is time consuming and technically challenging. For
example, effective discrimination of single base mismatches depends on mismatch position
(Urakawa et al., 2003; Wick et al., 2006), which places constraints on probe design. In
addition, the characterization and analysis of large numbers of melting profiles is difficult.
However, several recent publications have developed software tools to address this challenge
(Urakawa et al., 2002; Pozhitkov et al., 2005).
Another approach to optimizing specificity is the use of mismatch probes that are
designed to differ in sequence relative to a perfect-match probe. Bavykin et al. (2008) used
this strategy on a microarray that contained probes targeting 16S rRNA and 23S rRNA gene
sequences in Bacillus anthracis and closely related microorganisms. This microarray was
designed to include perfect match / mismatch probe pairs for each target, and Bavykin et al.
found that analysis of perfect / mismatch signal ratios enabled specific identification of
targets both singly and in mixtures (Bavykin et al., 2008). Wilson et al. (2002) used a similar
approach with a SSU rRNA-based microarray and demonstrated specific detection of bacteria
in pure cultures and simple mixtures.
Specificity can also be addressed by designing microarrays in which single base
mismatch discrimination is not a necessity. For example, Zhou (2003) built a microarray
which included 50-mer probes targeting specific variants of genes involved in nitrogen
cycling (nirS, nirK, nifH, amoA and pmoA) and sulfur reduction (dsrA and dsrB). Zhou found
that genes with less than 8690% sequence identity were clearly differentiated using
hybridization conditions of 50C and 50% formamide. Although this does not reflect a single
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John J. Kelly
base mismatch level of specificity, Zhou suggested that this level of specificity should
provide species level resolution, since the average similarity of these functional genes at the
species level ranged from 74 to 84% (Zhou 2003).
Figure 2.Example of non-equilibrium dissociation curves (signal versus temperature) obtained with a
DNA microarray containing oligonucleotide probes. S0 represents a perfectly matched probe-target
duplex, S30 represents a probe-target duplex with a single mismatch, and S59 represents a probe-target
duplex with two mismatches. Data reprinted from Urakawa et al. (2003).
CHALLENGES FOR MICROARRAY TECHNOLOGY: SENSITIVITY

Sensitivity is another critical parameter for microarray applications. Planar microarrays
have been shown to be 105-fold less sensitive than membrane hybridization (Cho and Tiedje,
2002), due to significant differences in the amount of nucleic acid immobilized: a membrane
immobilizes > 1ug per dot, whereas a planar microarray immobilizes 10 to 20 pg per spot
(Cho and Tiedje, 2002). As mentioned above, gel-pad microarrays have a higher probe
binding capacity than planar arrays due to their three-dimensional nature. Gel-pad arrays have
been used to immobilize 3 pmol of oligonucleotide probe per gel pad (Urakawa et al., 2002),
which corresponds to approximately 18 ng for a typical 20 base pair probe. Therefore, gel-pad
microarrays should result in an increase in sensitivity over planar arrays, but no direct
comparisons of the sensitivity of these two formats are available in the literature.
Several design and methodological approaches can also be used to increase sensitivity,
such as increasing probe length and reducing the stringency of the hybridization and/or wash
conditions. Several studies have demonstrated that increasing probe length results in
increased sensitivity of microarray hybridization (Zhou , 2003). In addition, a number of
studies have demonstrated higher hybridization signals at lower stringency: Guschin et al.
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(1997) showed higher hybridization signals at lower hybridization temperatures, and several
studies have reported higher hybridization signals at lower denaturant (formamide)
concentrations (Urakawa et al., 2002; Zhang et al., 2007). However, increasing probe length
and reducing stringency also have the drawback of decreasing hybridization specificity, so it
is necessary in any microarray application to find an appropriate balance between specificity
and sensitivity. Increasing probe concentration is another approach that has been shown to
increase sensitivity (Guschin et al., 1997), and this approach has the advantage of not
decreasing specificity.
Several groups have demonstrated that microarrays can be designed with adequate
sensitivity to detect nucleic acids directly from environmental samples. As mentioned above,
ribosomal RNA is a good target for hybridization because active cells contain thousands to
tens of thousands of copies of ribosomal RNA per cell (Amann and Ludwig, 2000). Several
groups have demonstrated successful microarray-based detection of ribosomal RNAs from a
variety of environmental samples: Small et al. (2001) detected Geobacter chapellei 16S
rRNA directly from a total-RNA soil extract; El Fantroussi et al. (2003) successfully detected
Acidobacteria as well as Alpha, Beta, and Gamma Proteobacteria by hybridizing RNA
extracted directly from estuarine sediments to a microarray containing 16S rRNA targeted
oligonucleotide probes; and Kelly et al. (2005) detected nitrifying bacteria by hybridizing
RNA extracted directly from a wastewater treatment plant aeration tank to a microarray
containing 16S rRNA-targeted oligonucleotide probes.
Detection of bacterial DNA in environmental samples via direct hybridization is more
challenging since most bacterial cells contain only a single chromosome which generally
includes one or perhaps a few copies of each gene. Nevertheless, Wu et al. (2001)
successfully hybridized DNA isolated from soil and sediment samples to a microarray using
large PCR products (0.76 kb) as hybridization probes. These very large probes enabled high
sensitivity (1 ng of target DNA), but low specificity (genes had to be at least 15% to 20%
different in sequence in order to be discriminated). Zhou (2003) used much smaller
oligonucleotide probes (50 mer) and successfully hybridized DNA isolated from soil. These
shorter probes resulted in improved specificity (genes that were at least 10% to 14% different
in sequence were discriminated), but lower sensitivity (8ng of target DNA). However, Zhou
(2003) suggested that this level of sensitivity should be sufficient for many studies in
microbial ecology since DNA yields from soil and sediment samples typically range between
10 and 400 g of DNA per gram of soil dry weight.
AMPLIFICATION OF TARGETS PRIOR

TO MICROARRAY HYBRIDIZATION
Although the studies cited above have shown some success in detecting bacteria in
environmental samples by direct hybridization of either RNA or DNA, the sensitivity of these
approaches may not be adequate to detect nucleic acids in low biomass systems or to detect
nucleic acids that represent a small fraction of the total nucleic acid pool in a sample. For
example, microarrays may not be sensitive enough to directly detect organisms whose rRNA
makes up a small fraction of the total community rRNA pool either because the organisms are
present in low numbers or because their cellular rRNA content is low due to a low level of
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metabolic activity. In addition, functional genes present in low concentrations may not be
detectable by direct hybridization. For this reason, several groups have used PCR
amplification prior to microarray hybridization to increase target concentration.
Because highly conserved universal primers for amplifying rRNA genes are available,
PCR amplification can be run with universal 16S primers to simply increase the overall 16S
rRNA gene concentration prior to hybridization with a microarray containing specific 16S
rRNA probes. This approach was used by Loy et al. (2002) to detect sulfate-reducing
prokaryotes in a hypersaline cyanobacterial mat, by Wang et al (2002) to detect a variety of
human intestinal bacteria in fecal samples, and by Loy et al. (2005) to detect members of the
betaproteobacterial order Rhodocyclales in activated sludge from an industrial wastewater
treatment plant. This approach has the advantage of increasing sensitivity, and general
amplification of all 16S rRNA genes requires only a single PCR reaction.
PCR can also be run with group-specific primers to selectively amplify the 16S rRNA
genes from specific phylogenetic groups. For example, Siripong et al. (2006) improved their
detection of ammonia oxidizing bacteria (AOB) in wastewater treatment plant samples by
selectively amplifying the 16S rRNA genes of beta-proteobacterial AOB with specific PCR
primers prior to microarray hybridization, and Loy et al. (2005) found that the selective
amplification of Rhodocyclales 16S rRNA genes prior to microarray hybridization allowed
the detection of rare Rhodocyclales groups in activated sludge. Group-specific PCR
amplification has the advantage of improving detection of the targeted group, but it does
require separate PCR reactions for each group, which can limit the number of groups detected
in a particular experiment.
Several groups have also used PCR amplification prior to microarray hybridization to
detect functional genes in environmental samples. For example, Taroncher-Oldenberg et al.
(2003) amplified nirS genes from sediment samples using general nirS primers and detected
specific nirS variants by hybridization of amplicons to a microarray containing a set of 64
nirS-specific probes. Zhang et al. (2007) used a similar approach to detect nifH variants in
roots of wild rice. One limitation of PCR amplification of functional genes prior to
microarray hybridization is that each functional gene targeted by the array must be amplified
via a separate PCR reaction. This was not a problem for the Zhang et al. (2007) study, as their
array targeted only one functional gene, nifH, so universal nifH primers were used to amplify
all nifH sequences in the samples followed by hybridization of amplicons to a set of 56
specific oligonucleotide probes on the microarray. However, the need for separate PCR
reactions for each targeted gene could be problematic for microarrays targeting a large
number of different functional genes. For example, the microarray used by TaroncherOldenberg et al (2003) included probes targeting four distinct functional genes (amoA, nifH,
nirK, and nirS), each of which would have required a separate PCR reaction prior to
microarray hybridization. Running multiple PCR reactions prior to microarray hybridization
is not desirable for a number of reasons: 1) it will significantly increase the time and labor
involved in microarray analysis, 2) it could introduce experimental error due to sample
splitting, and 3) it limits the main advantage of DNA microarrays, namely the potential for
highly parallel analysis of a high number of targets.
One way to avoid multiple PCR reactions is via Multiplex PCR, in which multiple primer
sets are included in a single PCR reaction. Several groups have utilized multiplex PCR
amplification prior to microarray hybridization. Panicker et al. (2004) demonstrated detection
of Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of four gene
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targets followed by microarray hybridization. Gonzalez et al. (2004) detected five marine fish
pathogens by multiplexing nine primer sets and subsequently hybridizing PCR products to a
microarray that included nine specific oligonucleotide probes. However, multiplex PCR is
restricted in the number of targets that can be amplified simultaneously due to primer-primer
interactions (Edwards and Gibbs, 1994). For example, Panicker et al. (2004) were able to
multiplex four primer pairs targeting Vibrio parahaemolyticus, but were unable to multiplex
ten primer pairs.
Pemov et al. (2005) developed a unique approach to multiplex PCR called multiplex
microarray-enhanced PCR (MME-PCR) that avoids some of the issues associated with
standard multiplex PCR. MME-PCR enables PCR amplification of multiple targets via
specific primers immobilized within a gel-pad based microarray. MME-PCR prevents primerprimer interactions because each primer pair is immobilized within a separate gel-pad, so
each primer pair is physically separated, and the specific amplification occurs within the gel
pad. Amplification within each gel pad is detected by subsequent hybridization of
fluorescently labeled, gene-specific probes. Pemov et al. (2005) demonstrated successful,
specific on-chip amplification via MME-PCR of six genes from Bacillus subtilis using six
different, gene-specific primer pairs. This approach could also be useful for many microbial
ecology applications. For example, our lab is currently designing a MME-PCR chip for the
detection of specific variants of nitrogen cycling functional genes.
Another way to avoid multiple PCR reactions prior to microarray hybridization is through
a process known as multiple displacement amplification (MDA). The MDA reaction uses
DNA polymerase and random primers to amplify entire genomes (Raghunathan, 2005). MDA
has been used to amplify whole genomes from pure cell cultures (Raghunathan, 2005), and
Wu et al. (2006) recently developed an MDA method for the amplification of entire genomes
from mixed microbial communities, which they termed whole-community genome
amplification (WCGA). Wu et al. (2006) used WCGA to amplify all of the genes in a
microbial community prior to hybridization to an oligonucleotide (50 mer) functional gene
array in order to improve sensitivity. They compared direct hybridization of genomic DNA
isolated from groundwater to WCGA amplification of dilutions of this DNA prior to
hybridization, and they found that WCGA-assisted microarray hybridization with as little as 1
ng of community DNA produced results that were highly similar to direct DNA
hybridization. These results suggest that WCGA may be a useful method for improving the
sensitivity of microarray-based assays for low biomass samples.
As demonstrated above, the addition of an amplification step to a microarray detection
protocol can increase sensitivity and enable the detection of low abundance targets, but the
incorporation of an amplification step does create some challenges as well. For example,
double stranded nucleic acids such as PCR amplicons do not hybridize as effectively as single
stranded nucleic acids (i.e. ssDNA or ssRNA) due to the potential for double stranded DNAs
to reanneal instead of hybridizing to the microarray immobilized probes (Zhang et al., 2007).
Several studies have avoided this problem by removing one of the DNA strands prior to
hybridization via amplification with a 5-biotin-labeled reverse primer and subsequent
removal of this strand via streptavidin-coated paramagnetic beads (Peplies et al., 2003; Zhang
et al., 2007). Another potential challenge related to amplification of targets prior to
microarray hybridization is the fact that amplification may introduce biases, as was discussed
above for PCR-based assays. Since an amplification step may not amplify all targets in an
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unbiased manner, amplification prior to microarray hybridization may interfere with the
ability to extract quantitative information from microarray data.
CHALLENGES FOR MICROARRAY TECHNOLOGY: QUANTIFICATION

The value of microarrays as a tool for microbial ecologists would be greatly enhanced if
microarrays could not only detect target nucleic acids but also provide quantitative
information on their abundance. In theory, it should be possible to extract quantitative
information from microarray hybridization if the amount of hybridization to a probe (i.e. the
fluorescent intensity) correlates to the amount of target present in the sample. Several studies
have confirmed linear relationships between target concentrations and signal intensities for
specific probes: Wu et al (2001) found a log-linear relationship between the concentration of
target genomic DNA and the hybridization signal for a 760 bp probe, and Rhee et al. (2004)
found strong log-linear relationships between signal intensity and DNA concentrations for a
large set of 50-mer oligonucleotide probes. Subsequently, Cho and Tiedje (2002) developed a
method that involved co-immobilization of functional gene probes (500-900 bp) with a
control probe (500 bp) and simultaneous hybridization of differentially labeled target genes
and control DNA, and they achieved good log-linearity between signal ratio and DNA
concentration ratio for three gene targets. These data confirmed that a correlation exists
between the hybridization intensity for a microarray immobilized probe and the concentration
of that probes target.
However, the challenge for microbial ecologists is that quantification of distinct bacterial
populations in a complex community would require the comparison of hybridization signals
from multiple probes with a microarray. This is a challenge because different probes can vary
significantly in hybridization potential due to differences in probe length (Wu et al., 2001)
and G+C content (Siripong et al., 2006) among other factors. These differences in
hybridization potential have significant implications. Several studies have shown that
redundant probes targeting the same organism often do not produce identical signal intensities
when hybridized with RNA from the target organism (e.g. Peplies et al., 2003; Guschin et al.,
2007). For example, Loy et al. (2005) demonstrated that probes targeting different regions of
the 16S rRNA gene of the same organism can vary by a factor of 240 in signal intensity when
hybridized with DNA from that organism. In addition, different probes can yield dramatically
different hybridization signals even when hybridized to equal amounts of their respective
targets (Ward et al., 2007). These situations make it extremely challenging to extract
quantitative information on relative target abundance from multiple probes within a
microarray. Since linear relationships have been shown to exist between target concentration
and hybridization signal (Wu et al., 2001; Rhee et al., 2004), a standard curve could be
developed for each probe on an array (Cho and Tiedje, 2002), as is routinely done for dot-blot
membrane hybridizations. However, having to develop a standard curve for every probe on a
microarray would be extremely labor intensive, and would limit the main advantage of
microarrays, their high probe capacity. Therefore, attempting to determine the relative
abundance of distinct bacterial populations in a complex community based on microarray
hybridization is a very difficult task, and further work is needed to determine if microarrays
can provide this type of information.
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MICROARRAY-BASED MONITORING OF GENE EXPRESSION IN

MIXED MICROBIAL COMMUNITIES
Functional gene arrays in which the presence of specific functional genes is detected via
either direct hybridization of DNA extracted from an environmental sample (e.g. Zhou 2003)
or via hybridization of functional genes amplified by PCR (e.g. Taroncher-Oldenberg et al.,
2003; Zhang et al., 2007) can provide valuable information on the distribution of specific
functional guilds in the environment as well as insight into the functional potential of
microbial communities. However, the presence of a functional gene in an environmental
sample does not necessarily mean that the gene is being expressed. One way to determine
expression is by detection of gene transcripts (mRNAs). As discussed above, microarrays
have been used extensively to investigate gene expression patterns in eukaryotic cells, such as
human cell lines (DeRisi et al., 1996) and yeast (Lashkari et al., 1997), via analysis of
mRNAs. Microarray analysis of mRNAs has been less extensively applied to prokaryotic
cells, due to difficulties in priming cDNA synthesis from bacterial mRNA (Dennis et al.,
2003). In addition, most microarray studies examining prokaryotic mRNA have been
conducted under controlled laboratory conditions on single cell lines or pure bacterial cultures
(e.g. de Saizieu et al., 1998; Meth et al., 2005) due to the difficulties associated with
obtaining sufficient quantities of high quality mRNA from environmental samples (Parro et
al., 2007).
One of the first studies to demonstrate the detection of bacterial mRNAs from mixed
communities used a microarray containing near-full length amplicons from 25 catabolic genes
involved in the degradation of chlorinated aromatic compounds (Dennis et al., 2003). The
steps used in this study were extraction of total RNA, synthesis of cDNAs via reverse
transcription, labeling of cDNAs, and hybridization of labeled cDNAs to the microarray. This
approach demonstrated the induction of catabolic genes in response to substrate additions in
pure cultures, in an artificial six member microbial community, and in sludge-fed pulp mill
effluent (Dennis et al., 2003). Zhang et al. (2007) used a similar approach to detect expression
of nifH gene variants in roots of wild rice samples with a microarray containing short (15-25
mer) oligonucleotide probes. Recently Parro et al. (2007) took a slightly different approach
and built a DNA microarray containing a gene library from a single target organism,
Leptospirillum ferrooxidans. They used this microarray to assess the relative expression of L.
ferrooxidans genes in two habitats with low bacterial diversity that differed in salt and oxygen
contents. The results demonstrated increased expression of genes required for adaptation to
each of the two habitats: for example, genes related to halotolerance were preferentially
expressed in the high salinity environment (Parro et al., 2007).
One of the challenges associated with microarray detection of bacterial mRNAs in
environmental samples such as soil and sediments is the isolation of sufficient quantities of
high quality mRNA for analysis (Parro et al., 2007). Several recent studies have addressed
this challenge by developing strategies for amplification of low quantity bacterial mRNAs
prior to microarray hybridization. Moreno-Paz and Parro (2006) used random-primed reverse
transcription coupled to in vitro transcription as a method for total bacterial RNA
amplification, and they demonstrated their ability to amplify as little as 250 ng of total
bacterial RNA from a pure culture and successfully hybridize the product to a microarray.
Gao et al. (2007) utilized a similar approach, which they termed whole-community RNA
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amplification (WCRA), to amplify RNA from mixed bacterial communities. The WCRA
approach used fusion primers (six to nine random nucleotides with an attached T7 promoter)
for the first-strand synthesis, followed by second strand synthesis and in vitro transcription.
This approach resulted in representative microarray detection from as little as 50 to 100 ng
total RNA, and Gao et al. (2007) demonstrated that bacterial mRNAs could be detected in
groundwater samples via WCRA amplification followed by hybridization to an
oligonucleotide (50-mer) functional gene array.
The publications described above illustrate the potential for microarrays to monitor
bacterial gene expression in environmental samples, which would provide extremely valuable
insights into bacterial community function. The success of these pioneering studies should
lead to further applications of this approach.
PARALLEL ANALYSES OF MICROBIAL COMMUNITY COMPOSITION

AND FUNCTION WITH MICROARRAYS
One of the key goals in microbial ecology research is the elucidation of the relationship
between the composition of a microbial community (i.e. the species present) and the function
of that community, but assessing this relationship for complex microbial communities in
environmental samples has always been extremely challenging. As described above,
numerous studies have demonstrated the ability of microarray technology to assess microbial
community composition, via hybridization of either 16S rRNAs, 16S rRNA genes, or
functional genes, and a few studies have demonstrated the ability of microarrays to assess
microbial community function through the detection of mRNAs (Dennis et al., 2003; Gao et
al., 2007; Parro et al., 2007). Several recent studies have expanded upon this work and
developed highly innovative, microarray-based approaches to examine both microbial
community composition and function in parallel.
Adamczyk et al. (2003) developed an isotope array approach. In this approach, a
microbial community is incubated with a 14C-labelled substrate, and after an incubation
period, total RNA is extracted from this community, fluorescently labeled, and hybridized
with a microarray containing 16S targeted oligonucleotide probes. The array can then be
scanned for fluorescence as well as for radioactivity. For each probe on the array,
fluorescence indicates the presence of the target organism in the community, and radioactivity
indicates that the target organism has incorporated 14C into RNA, indicating active growth
and utilization of the labeled substrate. Adamczyk et al. (2003) demonstrated this approach
using a microarray containing oligonucleotide probes targeting 16S rRNA genes of ammoniaoxidizing bacteria. In this study, activated-sludge samples were incubated with 14C-labelled
bicarbonate, and detection of radioactivity for specific probes on the array was taken as an
indication of CO2 fixation by the targeted organisms. This study demonstrated the potential
use of an isotope array for the simultaneous assessment of community composition and
function.
Another novel approach to examining community composition and function was
developed by Zhang et al. (2007). They assessed both the presence and expression of different
variants of nifH in roots of wild rice using a microarray containing 56 oligonucleotide probes
(15-25 mers) targeting nifH variants. In this study, DNA and RNA were coextracted from
375
plant roots, and separate aliquots were treated with either DNase I or RNase A to yield pure
RNA and DNA samples, respectively. DNA was amplified prior to microarray hybridization
via PCR with universal nifH primers. RT-PCR with a universal nifH primer was used to
convert mRNA to cDNA, and PCR with universal nifH primers was then used to amplify the
RT-PCR products prior to microarray hybridization. In this creative approach, hybridization
of amplified DNA indicated the presence of specific nifH variants, and hybridization of
amplified mRNA indicated the expression of specific nifH variants. The results of this study
demonstrated that only a small subset of the nifH-containing organisms found in the roots
were expressing the nifH gene (Zhang et al., 2007). This approach has tremendous potential
for simultaneous exploration of microbial community composition and function, which could
provide fascinating insights into the functioning of microbial communities in the
environment.
APPLICATIONS OF DNA MICROARRAYS TO MICROBIAL ECOLOGY

Since the pioneering work of Guschin et al. (1997) the use of microarray technology in
microbial ecology research has increased rapidly (Figure 3). The vast majority of microarray
studies published to date in the field of microbial ecology have focused on demonstrating the
capabilities of microarrays and developing the technology, but recently a growing number of
studies have been applying microarrays as tools in studies asking ecological questions.
Figure 3.Number of publications per year that include application of microarray technology to
microbial ecology. Numbers are based on a search of the Web of Science database on February 1, 2008
using the following search string: (TS=microarray* OR TS=microchip*) AND (TS=microbial ecology
OR TS=microbial community OR TS=microbial detection OR TS=microbial identification). This
approach was based on Wagner et al.(2007).
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John J. Kelly
Some examples include the following: Taroncher-Oldenberg et al. (2003) used a

microarray to reveal differences in the distribution of nirS variants in sediment samples from
two stations in the Choptank River that differed in salinity, inorganic nitrogen, and dissolved
organic carbon. Loy et al. (2002) used a 16S rRNA gene-based oligonucleotide microarray to
detect sulfate-reducing prokaryotes in an unusual habitat, a low-sulfate acidic fen, and they
found differences in the distribution of these sulfate-reducers in fen samples from different
locations. Rhee et al. (2004) demonstrated that the community composition of naphthalenedegraders in soil microcosms differed depending on incubation conditions based on analysis
with a functional-gene microarray containing oligonucleotide probes. Sanguin, Remenant, et
al. (2006) used a 16S rRNA-based microarray to reveal a significant maize rhizosphere effect
on soil bacterial community composition. Ward et al. (2007) demonstrated variations in the
composition of ammonia oxidizing communities across a freshwater/marine transect
extending from the Choptank River through the Chesapeake Bay and out into the Sargasso
Sea using an oligonucleotide (70 mer) functional gene microarray targeting amoA genes. This
study also revealed correlations between amoA guilds and environmental parameters,
suggesting that different amoA-containing organisms occupy different ecological niches
within the estuarine/marine environment (Ward et al., 2007).
All of the studies cited above are excellent illustrations of the effective use of microarrays
in microbial ecology, and based on these and other successes it is likely that the uses of
microarrays in this field will continue to expand rapidly.
CONCLUSIONS
Microarrays have the potential to revolutionize the field of microbial ecology via high
throughput analysis of microbial community structure, function, and population dynamics.
There are significant challenges to the use of microarrays in microbial ecology studies,
including optimization of specificity and sensitivity and quantification of targets. However, as
reviewed above, the last decade has seen tremendous progress in addressing these challenges,
and this progress suggests that microbial ecologists are now on the verge of finally being able
to realize the tremendous potential of microarray technology.
ACKNOWLEDGMENTS
JJKs efforts in preparing this review were supported by a Research Support Grant from
Loyola University Chicago and by USDA Grant 2005-35107-16098.
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ISSN: 2158-5717
Chapter 14
FOOD SAFETY IN INDIA:

CHALLENGES AND OPPORTUNITIES
Wasim Aktar*
Pesticide Residue Laboratory, Department of Agricultural Chemicals, Bidhan Chandra
Krishi Viswavidyalaya, Mohanpur-741252, Nadia, West Bengal, India
1. INTRODUCTION
Rising incomes and urbanization, an expanding domestic consumer base concerned about
food quality and safety, and rapidly growing agricultural exports have been important drivers
for the increased attention to food safety in India. But the development of effective food
safety systems is hampered by a number of factors, including: restrictive government
marketing regulations, weak policy and regulatory framework for food safety, inadequate
enforcement of existing standards, a multiplicity of government agencies involved, weak
market infrastructure and agricultural support services. The small farm structure further limits
farmer capacity to meet increasing domestic and export food safety and SPS requirements.
Addressing food safety concerns in India will require adoption of appropriate legislation,
strengthening capacity to enforce rules, promoting adoption of good agricultural,
manufacturing and hygiene practices, greater collective action, and some targeted
investments. Implementing these actions will require joint efforts by the government and the
private sector.
Developing countries are paying increased attention to food safety, because of growing
recognition of its potential impact on public health, food security, and trade competitiveness.
Increasing scientific understanding of the public health consequences of unsafe food,
amplified by the rapid global transmission of information regarding the public health threats
associated with food-borne and zoonotic diseases (e.g. E. coli and salmonella, bovinespongiform encephalopathy (BSE), severe acute respiratory syndrome (SARs) and H5N1
avian flu) through various forms of media and the internet has heightened consumer
awareness about food safety risks to new levels globally (Lindsay 1997, Unnevehr 2003,
*
Correspondence to: wasim04101981@yahoo.co.in
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Wasim Aktar
Buzby and Unnevehr 2003, Kafersteing 2003, Ewen et al. 2006, Bramhmbatt 2005).
Increased understanding of the impact of mycotoxins, which can contaminate dietary staples
such wheat, maize, barley and peanuts, has further raised food security and public health
concerns in many developing countries (Dohlman 2003, Bhat and Vasanthi 2003, Unnevehr
2003).
As developing countries seek to expand agricultural exports especially to OECD
countries, many are receiving a wake-up call on the challenges of meeting both government
and private sanitary and phyto-sanitary (SPS) standards in export markets (Otsuki et al. 2001,
Henson 2003, Unnevehr 2003, World Bank 2005a). Private standards or supplier protocols
have grown in prominence over the past decade as a means to further ensure compliance with
official regulations, to fill perceived gaps in such regulations, and/or to facilitate the
differentiation of company or industry products from those of competitors. Trends in private
standards increasingly tend to blend food safety and quality management concerns (i.e. the
recent creation of ISO 22000), or to have protocols which combine food safety,
environmental, and social (child labor, labor conditions, animal welfare) parameters (Willems
et al. 2005, World Bank 2005). At the same time, increasing globalization of trade introduces
greater risks of cross-border transfer of food-borne illnesses. Recent cases of disease episodes
in the United States resulting from imported food produce, such as cyclospora from
raspberries, hepatitis A from strawberries and salmonella from cantaloupe (Calvin 2003),
illustrate to developing countries the potential food safety challenges that can arise in a more
globalized market.
Weaknesses in food safety systems can have a high cost to society and the global
economy. The World Health Organization (WHO) estimates that 2.2 million people
worldwide die from diarrheal diseases caused by a host of bacterial, viral and parasitic
organisms, which are spread by contaminated water (WHO 2006a). In India, it is estimated
that 20% of deaths among children under five are caused by diarrheal disease (WHO 2006b).
The SARs outbreak in 2003 in East Asia is estimated to have caused an immediate economic
loss of about 2% of the Regions GDP in the second quarter of that year, even though only
800 people died from the disease (Brahmbatt 2005).1 The Lowy Institute for International
Policy (2006) estimates that a mild global outbreak of the avian flu can cost the world 1.4
million lives and close to 0.8% of GDP (US$330 billion) in lost economic output. At the
same time, country reactions to protect its citizens from food safety risks can also have large
consequences for exporting countries. Otsuki et al (2001) examined the projected impact of
the EUs new harmonized aflatoxin standard on the value of trade flows to 15 European
countries from 9 African countries and found that it could decrease African exports by 64%
(US$670 million).
Food safety concerns are getting widespread attention in India. The countrys rural
development strategy, for which a key element is the promotion of increased agricultural
exports as a means to foster rural growth and poverty reduction, is coming up against
tightening food safety and SPS standards in prospective markets (World Bank 2006a, 2006b).
From a domestic perspective, the large national market of 1.2 billion people is undergoing
rapid change. Increasing incomes, a growing middle class, increased urbanization and
1
The large economic impact resulted primarily from uncoordinated efforts of individuals to avoid becoming
infected, contributing to a contraction in services sectors (tourism, mass transportation, retail, hotel and restaurant
sales) and workplace absentiism (Brahmbatt 2005).
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literacy, and a population highly tuned to international trends fueled by the information
technology boom are creating a large consumer base giving increasing value to food quality
and safety. Improving food safety systems, to meet domestic and export requirements,
however, face a number of policy, regulatory, infrastructural and institutional obstacles.
2. OBJECTIVES
(i) To review the main drivers for the increased priority to addressing food safety risks in
India in both the export and domestic markets, (ii)To examine the nature and effectiveness of
government and private responses to the food safety challenges, with special focus on high
value agriculture; (iii)To identify the constraints to more effective responses; (iv) To examine
the implications for policy; v) To review food safety with special relation to Pesticides; and
vi) To discuss briefly about the food safety from consumer point of view.
3. TYPES OF FOOD SAFETY RISKS
Figure 1. Food Supply Chain: Potential Sources of Food Safety Hazards
Food safety risks, as they relate to human health, arise from of a number of factors. These
include: (i) microbial pathogens (bacteria, viruses, parasites, fungi and their toxins); (ii)
pesticide residues, food additives, livestock drugs and growth hormones; (iii) environmental
toxins such as heavy metals (e.g. lead and mercury); (iv) persistent organic pollutants (e.g.
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dioxins); and (v) zoonotic diseases (e.g.Avian flu, Japanese encephalitis, tuberculosis) (Buzby
and Unnevehr 2003, Ewen et al. 2004).2 The health risks associated with these agents impact
the whole food supply chain, starting from input supply to the farm to the consumer table
(Figure 1).
Common use of pesticides in modern farming inevitably leaves some residues on food
crops.
Potential food safety hazards at HOME can be divided into three categories:
There remains considerable debate regarding the food safety risks associated with genetically modified organisms
(GMOs). The paper will not be covering issues relating to GMOs.
1. Biological
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2. Chemical
3. Physical
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While all the above type of hazards are important from viewpoint of prevention, the focus
here will be on the microbiological hazards and in that on foodborne bacteria, which can lead
to illness if the food is mishandled, particularly for those more at risk -- the very young, the
elderly and the immuno-compromised.
Certain processes or handling practices by consumers in the home have been identified as
being essential or critical in preventing foodborne illness. These practices, which prevent or
control the "meals" microbial contamination associated with foodborne illness, are under the
direct control of the consumer, from food acquisition through disposal.
They are purchasing, storing, pre-preparation, cooking, serving, and handling leftovers.
Failure to take appropriate action at these critical points could result in foodborne illness.
4. PESTICIDES AND FOOD SAFETY

Fruits, vegetables and cereal crops treated with pesticides are perceived by some as a
health risk, and this belief along with affordability, and time pressures may all play a role in
limiting consumption of plant foods, such as cereal grains, fruit and vegetable consumption of
consumers in Asia. The World Health Organisation (WHO), the World Cancer Research Fund
(WCRF) and many other national and inter-governmental agencies recommend that adults
consume at least 400g of fruit and vegetables per day and 25-30 grammes of dietary fibre per
day, but analysis of current dietary patterns around the world indicate that many consumer are
not achieving these dietary goals, particularly those who are less affluent. AFICs Short
Briefing on Pesticides, Food Safety and Health is intended to provide a science-based factual
overview of the issue, to enable consumers to make better informed choice about their diet, in
particular fruit, vegetables and grains consumption, and allay unwarranted anxieties and
concerns.
Definition of Pesticide
The Food and Agriculture Organisation (FAO) defines a pesticide as any substance or
mixture of substances intended for preventing, destroying, attracting, repelling, or controlling
any pest including unwanted species of plants or animals during the production, storage,
transport, distribution, and processing of food, agricultural commodities, or animal feeds or
which may be administered to animals for the control of ectoparasites
Natural Toxins
Substances that are capable of causing cancer are virtually everywhere, even in natural
compounds. The FDA estimates that the intake of carcinogens from man-made pesticide
residues is extremely small compared to carcinogenic residues that plants produce naturally.
According to Bruce Ames, a professor of molecular biology and biochemistry at the
University of California, more than 99.99 percent of the pesticides Americans ingest are
"nature's pesticides" or "natural toxins" (Hotchkiss, 1992; Moore, 1989).
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Natural toxins are present in all plants and such food products as beans, lettuce, apple
juice, wine, black pepper, spinach, peanut butter and many others. Of the known natural
toxins, which concentrate in parts per thousand versus parts per billion in synthetic pesticides,
none has been shown to cause cancer (Hotchkiss, 1992; Moore, 1989).
Reasons of Pesticide Residues in Food

Pesticide residues may be present in food because of the following reasons:
1) Direct use of pesticides on food crops;
2) Animal feeding on pesticide treated feed;
3) Environmental contamination
Pesticide Use on the Farm

Many of today's food producers are taking an Integrated Pest Management (IPM)
approach to preventing, reducing or eliminating pest problems. Growers and processors must
make complicated decisions prior to planting, during the growing season, and during
postharvest handling. Scientific IPM strategies give the grower economic incentives for
sustaining long-term crop protection with minimal disruption to the environment. The
agricultural community typically will use pesticides judiciously as part of the IPM strategy
whenever proven alternatives are not available for pest control. Growers are hiring
professional crop consultants with increasing frequency for advice on maintaining or
increasing production through the utilization of IPM programs structured toward their specific
agronomic situations.
Integrated Pest Management

It is an ecological approach to pest management in which all available control techniques
are consolidated into a unified program so that pest populations can be managed in such a
manner that economic damage is avoided and adverse side effects are minimized. Practices
used as a part of this management philosophy include the following: 1) destruction of crop
debris, 2) having pests feed and concentrate on trap crops, 3) crop rotation, 4) selectivity of
planting and harvest dates, 5) soil test analysis for crop nutrient needs, 6) planting crop
species adapted for local conditions, 7) using genetically improved crop varieties with
resistance to specific pests, 8) using biological control, 9) predicting pest outbreaks with
computers, 10) pheromones for trapping pests, 11) scouting and monitoring for pests, 12)
economic thresholds as guides to pest control, 13) better timing and application of pesticides,
14) use of biological insecticides, 15) improved pesticide application efficiency, 16) adapting
promising technology, including the use of infrared scanners, satellite photos, gene-splicing
biotechnology, and new pesticide delivery systems that incorporate farm-specific information
on tractor mounted computers.
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Pesticide Limits and Regulation

Approval for use of any pesticide in a country is subject to its safety evaluation. Safety
levels for any pesticide are calculated over a number of formal assessments. The Codex
Alimentarius Commission is an international body which sets international guidelines on
many elements of food safety, including pesticides residues on food. These guidelines are not
mandatory, but many countries in Asia use these guidelines, sometimes with additional
scientific data determined by their national regulatory agencies to establish limits on use and
also acceptable residue levels at point of sale.
Acceptable Daily Intake

One of the most important tools in the safety evaluation of pesticide use on food crops is
the calculation of what is an Acceptable Daily Intake (ADI). The ADI for any given pesticide
is a measure of the quantity of a particular chemical in food that can be consumed daily over a
lifetime without any known risk to health. It is expressed in relation to bodyweight.
ADI is derived by first conducting diet trials on laboratory animals and observing the
maximum level of pesticide that can be consumed by the animal with no observable adverse
effect on health. This level expressed as percentage of body weight is known as the No
Observable Adverse Effect Level (NOAEL or NOEL), The investigations include checks for
birth defects, cancer, reproductive changes, damage to the nervous system, harm to organs
such as the kidney or liver, and many other measurable health indicators.
A safe level for human consumption is estimated by dividing the NOAEL on humans by
an uncertainty factor (usually 100) to allow for the possibility that humans may more
sensitive than the animals used for testing and also to account for possible variation in
sensitivity to the pesticide between human individuals, for example adults and children. These
results in an ADI for humans which is 100 times lower than the NOAEL consumption rate
established from trials on laboratory animals.
Acute Reference Dose

Safety evaluation of all pesticides also requires an estimate of the acute refrence dose
(ARfD). The ARfD is an estimate of the amount of a substance in food or drinking water
expressed as percentage of body weight, that can be consumed over a short period of time,
usually one meal or one day, without any known effect on health. This figure is also
expressed as a percentage of body weight.
Maximum Residue Levels

A maximum reside levels (MRL) is the maximum permissible quantity of pesticide that
may still be present on the crop at point of sale. It is derived from an assessment of the
residues found when the crop is treated according to good agricultural practices. The MRL is
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the maximum concentration of a pesticide residue that is legally permitted in, or on, a food
commodity, and is set by national governments if the approval is given for the use of the
pesticide on specified crops. MRLs are set to determine legal trading limit, and are not an
indicator of risk to health. MRLs are set at levels which would result in consumption of any
residue at a level substantially lower than the ADI or the ARfD for the pesticide, and any
pesticide whose MRL could result in dietary intake which might exceed the ADI or ARfD
would not receive approval.
Pesticide Residue Monitoring

Under FFDCA, the Food and Drug Administration (FDA) and USDA share responsibility
for monitoring levels of pesticide residues on foods. FDA enforces pesticide tolerances for all
domestically produced food shipped in interstate commerce and in imported foods, except for
meat, poultry and some egg products, which are monitored by USDA. Many agriculturallyintensive states such as California and Florida also conduct extensive pesticide residue
monitoring programs. FDA uses three approaches for pesticide residue monitoring: 1)
incidence/level monitoring, 2) regulatory monitoring, and 3) Total Diet Study (FDA, 1994).
Total Diet Studies

To assess potential health problems from contaminants, both natural and man-made in the
food supply, the WHO recommends total diet studies (TDS) as the one of the most costeffective means for assuring that people are not exposed to unsafe levels of toxic chemicals
through food. TDS provides an additional tool to assess whether or not any pesticides may be
present in the diet at levels which might pose a risk to health. A TDS is conducted by
purchasing through standard retail outlets a typical selection of foods commonly consumed in
the country or region. The basket of foods is processed and prepared as if for normal
consumption and then analysed in the laboratory to measure total levels of the substances of
interest, for example pesticides. Drinking water and water used in cooking are also included
in the assessments. The TDS provides a measure of the average amount of the pesticide
consumed by different age/sex groups living in a country. See box for an example of an actual
TDS and results for estimate of pesticide consumption.
Risk Calculation
Risk = exposure x toxicity. Risk of harm from a chemical depends on both the level of
exposure to the chemical and on the toxicity of the chemical (Chaisson et al., 1991).
Therefore, to quantify potential risks from consuming minute quantities of a particular
chemical residue in food, scientists consider the toxicity of the chemical, the residue content
of foods and the amounts of these foods eaten by population subgroups. Population subgroups
such as infants, children, women, women of child-bearing age and ethnic subgroups may be
considered in risk assessments in addition to the total population. The groups considered
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depend on the toxicologic characteristics of a particular chemical. Risk assessments that

consider regional and seasonal variations also are performed.
Exposure = residue concentration in food x amount of food consumed. Potential exposure

to a chemical in a specific food is assessed by multiplying the residue concentrations in food
times the amount of food consumed by each person in the population. This exposure is
expressed as milligrams of residue per kilogram of body weight per day (mg/kg BW/day).
Potential dietary exposure to a chemical is assessed by adding together residue intakes from
all foods. Different assumptions regarding residue concentrations in food may be used to
assess exposure. A worst-case exposure scenario may be calculated using tolerance levels for
pesticides in food. This exposure assessment is the theoretical maximum residue contribution.
Exposure may also be calculated using anticipated residue levels (Chaisson et al., 1991;
California Agriculture,1994).
5. FOOD SAFETY AND THE INDIAN DOMESTIC MARKET

Increasing incomes, urbanization, and literacy, improved infrastructure and closer ties to
global trends, especially during the last decade, are driving changes in consumer demand and
preferences in India. Sustained economic growth (6.0% per year in real terms from 1990/91 to
2003/04) resulted in GDP per capita increasing by about 70%, from about US$315 in 1990 to
US$538 in 2004 (constant 2000 dollars). National poverty rates (headcount) declined from
38.9% (Central Statistical Organization 2002) in 1987/88 to 28.5% in 1999/00 (Deaton and
Dreze 2002).3 The middle class, which now accounts for about 15% of the 1.2 billion people
in India, is the fastest growing income group and is a major force shaping the diet revolution
that is occurring (Landes and Gulati 2003).
3
There continues to be a debate on the headcount poverty rate in 1999/00, arising from the adjustment in the design
of the 1999/00 National Sample Survey. Depending on the methodology used, the poverty estimates range from
26.1% (Planning Commission) to 28.9% (Sundaram and Tendulkar) (Virmani 2006).
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Figure 2. Diversification on Food Consumption Expenditures
These structural changes are reshaping consumer demand. The Indian food consumption
basket is diversifying away from cereals towards higher value and more perishable products,
such as fruits and vegetables, dairy, meat and fish (Figure 2). Increasing female participation
in the work force and higher disposable incomes to spend on non-home cooked foods are
driving growth in demand for prepared and semi-prepared foods, and thus the growth of the
processed food industries (Pingali and Khwaja 2004). These trends bring increased attention
to safety concerns in the handling, processing and marketing of foods.
In addition, growing consumer preference for shopping convenience, increased exposure
to the media (TV, cable and the internet) and ownership of durables such as refrigerators and
cars are fostering the growth of modern retailing (i.e. supermarkets and hypermarkets), which
in turn demand greater efficiency and food quality and safety standards in the supply chain
Mukherjee and Patel 2005, Chenggapa, et al 2005).
Increased vigilance by NGOs, consumer groups, and local research institutes is also
raising awareness and spurring action among consumers and policy makers to address food
safety risks. Findings of high levels of pesticides in bottled water and soft drinks in 2003 by
the Centre for Science and Environment (CSE), an NGO, shook the country and forced the
Government of India (GOI) to take swift action (Mathur et al 2003, CSE 2004). The CSE
tested 30 bottled water brands from the major cities of Delhi and Mumbai in Maharashtra and
found that all except one contained pesticide residues.
The Delhi brands on average contained pesticide residues 36.4 times the maximum
pesticide residues stipulated by the European Union standards for bottled water (CSE 2004).
Shortly thereafter, Mathur et al. (2003) tested 12 brands of soft drinks sold in Delhi for 16
organochlorine and 12 organophosphorus pesticides and 4 synthetic pyrethroids commonly
used in agricultural fields and homes in India. Their analysis found that all brands exceeded
the EU maximum pesticide residue limit of 0.0005 ppm (Figure 3).
397
Figure 3. Pesticide Residues in Soft Drinks in India, 2003
To deal with the back-to-back crises, the GOI established a special Joint Parliamentary
Committee on Pesticide Residues in and Safety Standards for Soft Drinks, Fruit Juice and
Other Beverages in August 2003 to investigate the allegations. Two GOI Laboratories were
instructed to conduct tests on the 12 brands (but using different samples) and their findings
showed that 9 of the 12 samples exceeded the EU limits (Hindu Business Line 2003).
Weak regulations and inadequate standards were major causes of these high profile food
safety crises. In the case of bottled water, while the existing norm set out by the Bureau of
Indian Standard (BIS) required that no pesticides should be detectable, the prescribed
methodology could only detect pesticides at extremely high levels. Consequently, GOI issued
a notification revising the standards for pesticide residues on bottled water, adopting the EU
single residue limit of 0.0001 ppm and multiple residue limit of 0.0005 ppm (CSE 2004). In
the case of soft drinks, the BIS only had voluntary standards, not mandatory standards for
pesticide residues. To address the problem, BIS constituted a 39 member committee,
consisting of representatives from the soft drinks industry, government scientists, NGOs and
consumer groups to formulate the new BIS standards. The outcome was the Indian Ready to
Serve Non-Alcoholic Beverages Specifications, which established the limits for 16 pesticides
in the finished product (0.0001 mg/l for individual pesticides and total pesticide residue limit
of 0.0005 mg/l) (CSE 2004).
Even the government-sponsored Mid-day Meals program encountered serious food safety
incidents. The National Program for Nutritional Support to Primary Education (NPNSPE),
more popularly known as the Mid-Day Meals Scheme, aims to improve child enrollment in
primary school and encourage regular attendance by providing supplementary feeding, while
improving their nutritional status. It covers children enrolled in classes I to IV in government
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398
and government-aided schools in the whole country (Jha and Umali-Deininger 2003). In June
2006, 85 students from a Chennai primary school were admitted to the hospital because of
food poisoning after consuming food prepared under mid-day meal scheme.4 In February
2004, 281 children attending municipal schools in Delhi fell ill and were admitted to the
hospital after consuming their mid-day meal.5 There have been many other cases, despite
quality norms being established for the mid-day meal program.
While issues related to pesticides in bottle water and carbonated drinks, and out-breaks of
food-borne illnesses received wide media attention, there are other serious domestic food
safety concerns that have been identified including heavy metal contamination in foods.
Marshall, et al. (2003), tested fresh cauliflower, okra, and spinach common vegetables in
the Indian diet in 5 production sites around the Delhi region and in Delhis Azadpur
wholesale market from May 2001 to June 2003. They found that 72% of the 222 spinach
samples exceeded the Indian MRLs for lead of 2.5 mg/kg, and 100% exceeded the Codex
MRL of 0.3 mg/kg. They attributed the high lead content to a number of possible causes,
including contamination of the irrigation water by sewage and industrial effluent and
industrial pollution.6 Contamination was exacerbated by their locationsthe production sites
and market were in peri-urban and urban areas. When tested for zinc, 21% of samples
exceeded both the Indian and international standards. Currently, however, no regular testing
for heavy metals in vegetables is undertaken by government agencies in India. Tests
undertaken by the Indian Council for Agricultural Research found pesticide residues above
the MRL in 5.3% of 666 samples of vegetables in 2003 and 15% of 468 samples of milk
tested in 2001 (Directorate of Plant Protection and Quarantine 2006).
The long term use of pesticides in agriculture and for disease control (e.g. DDT for
malaria control) is manifesting itself in the blood, human milk and fatty tissue in the
population in many states. Table 1 presents the results of micro-research studies in selected
states in India from 1980 to 2005.
Table 1. Level of DDT and HCH Content in Human Blood Samples in Selected
States in India.
Location
Year
Number of Samples
Total DDT
(ppm)
Total HCH
(ppm)
Lucknow, Uttar Pradesh

Delhi
Lucknow, Uttar Pradesh
Delhi
Ahmedabad, Gujarat (rural)
Ahmedabad, Gujarat (urban)
Punjab (rural)
1980
1982
1983
1985
1992
1997
2005
25
340
48
50
31
14
20
0.020
0.710
0.028
0.301
0.048
0.032
0.0652
00.022
0.049
0.075
0.148
0.039
0.057
Note: HCH - Hexachlorocyclohexane

Source: ICMR 2001, Mathur et al. 2005.
4
http://www.newkerala.com/news3.php?action=fullnews&id=11595
http://www.hindu.com/2004/02/27/stories/2004022713760300.htm
6
Potential sources of industrial pollution include emissions from vehicles, industrial plants, coal power generation
plants, and diesel generator sets and re-suspended road dust. Marshall et al. found that washing the spinach twice
reduced the lead contamination by 50% indicating that a large proportion of the lead was air-borne.
5
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6. FOOD SAFETY CONCERNS IN INDIAN EXPORTS

Increased globalization and liberalization of markets, facilitated by the World Trade
Organization (WTO), are opening new export markets for Indian agricultural products, both
fresh and processed. Indian agricultural exports grew at an average annual rate of 7.2% from
1990/91 to 2003/04. In response to these new opportunities, Indias agriculture exports
diversified from traditional exports of tea, spices, and coffee to include horticultural, fish and
livestock products. Between the triennium ending (TE) 1991/92 and TE 2003/04, the value of
fresh and processed fruit and vegetable exports rose from US$84 million to US$394 million
in real terms (1993/94 dollars) while marine product exports rose from US$516 million to
US$1.5 billion during the same period (Figure 4).
As Indian agricultural exports diversified, and the value of exports to high income
countries increased, India has had to confront new food safety challenges. Concerns over
numerous rejections of Indian agro-food exports on food safety grounds have spilled over
domestically, generating greater domestic attention to pervasive food safety problems in
the supply chain including high levels of pesticide residues, presence of heavy metals in food,
and micro-biological contamination. The following section describes recent food safety
challenges in Indian horticultural, spice and fisheries exports.
Figure 4. Trend in Agricultural Exports, Triennium Ending (TE) 1990/91 to TE 2003/04
Horticultural Exports
In 2004, India exported US$575 million of fresh and processed fruits, vegetables and
flowers. Traditionally Indias fresh fruit and vegetables exports were targeted to markets in
neighboring South Asian countries, to the Middle East and to East Asia. Since the early 1990s
India achieved some success in exporting fresh horticultural produce to Western Europe.
India has been quite proud of its penetration into the U.K, Netherlands and German fresh
400
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grape markets. Grapes are a highly seasonal crop and Indian exporters have been targeting a
crucial March to April window in the European market, which falls at the end of the main
southern hemisphere production season (in South Africa and Chile) and before Egypt and
Turkey enter the market. Virtually all of Indias grape exports are of the Thompson Seedless
variety.
The Indian grape export crisis in May 2003 was a pivotal wake-up call to Indian
exporters concerning the costs of failing to meet food safety standards. In the midst of a
commercial dispute with an Indian grape exporter, a Dutch importer had samples of the
Indian grapes tested by a private laboratory. On finding that the grapes contained residues of
the insecticide methomyl in excess of the EU maximum residue limit (0.05 microgram/kg.),
the importer placed an advertisement in the local paper warning that grapes from this Indian
supplier contained poison (World Bank, 2006b). Dutch authorities, who were alerted about
the finding, tested samples from the 28 containers of Indian grapes then in Rotterdam port and
found that about 75% of the samples exceed the MRLs for methomyl and/or acephate.7 The
problem was reported on the EU Rapid Alert system, causing not only significant short term
economic losses, but also considerable longer term reputation damage. The price of Indian
grapes dropped sharply, and the Indian grape shippers incurred losses, either in Dutch sales or
by diverting the shipments to other markets.
Spice Exports
India is the worlds largest consumer and producer of spices and is also a significant
exporter of spices (Jaffee, 2005). In 2004/05, Indias spice exports totaled US$399 million.
India, however, has encountered a number of food safety problems in its spice exports
including high pesticide residues, aflatoxin contamination and the use of prohibited food
colorants. In the mid-nineties, Indian dry chili exports faced several rejections including
rejections in Spain due to pesticide residue in excess of permissible MRLs, and in the United
States because residues of quinalphos, a pesticide not registered in the United States (Jaffee,
2005). Between 1998 and 2000, Indian dry chili exports also faced rejection in Germany,
Italy, Spain and the U.K. due to the presence of aflatoxin.8 More recently, exports of chili and
curry powder faced problems due to the use of the prohibited red dye Sudan 1 (Jaffee, 2005).
In February 2005, a massive recall of some 600 food products took place in the UK because
of the detection of Sudan 1 in Worcester sauce. This was the largest ever food recall in the
U.K. and it affected all major retailers as well as large numbers of food manufacturers and
food service companies, as the Worcester Sauces had been used in the preparation of a large
number of different products. It is estimated that this recall, and associated expenses, cost the
U.K. and other European food manufacturers some 200 million Euros (Jaffee, 2005). The
source of the Sudan 1 dye in the Worcester sauce was traced to chili powder imported from
India in 2002.
Of the twenty Indian samples with violative levels of methomyl, six exceeded the MRL by ten times, but most of
the others were also far in excess of the MRL (Schee 2004).
8
Aflatoxin may emerge in dried chilies as a result of improper dying (Jaffee, 2005).
401
Fish and Fish Product Exports

Fish and fish products are one of Indias largest agricultural export earners, totaling
US$1.3 billion in 2004/05. Over the years, India has encountered several food safety
problems with its fish and fish product exports. Most prominent, in 1997, the European
Commission found the industry to be non-compliant in maintaining hygiene standards in fish
processing plants. In May 1997 the European Commission banned Indian exports of fresh
crustaceans and cephalopods and imposed border testing for Salmonella and Vibrio spp. for
frozen products (Henson, Saqib and Rajasena, 2005). Because of continued detection of
salmonella, all exports of fish and fishery products to the EU from India were banned in 1997.
While India has for the most part been able to address the hygiene-related problems plaguing
its export of fishery products in the late nineties, Indian exports are now under scrutiny
because of problems related to antibiotic residues and bacterial inhibitors (antibiotics,
preservatives and chlorine) (Henson, Saqib and Rajasena, 2005). It is widely acknowledged
that in the future, heavy metals and other contaminants could be an emerging issue
particularly because of the increased attention to heavy metals in the EU. Surveillance of
fisheries products for heavy metals has already begun in the U.K.
Although India has been able to broadly comply with food safety requirements for each
of the export commodities mentioned above, it continues to face problems across a range of
agro-food exports. Evidence of continuing trouble is clearly apparent from Import Refusal
Reports issued each month by the USFDA for food and drug imports into the United States.
Most recently, in both April and May 2006, India had one of the highest rejections among all
countries exporting to the USA; India faced 176 rejections in May, 2006 and 211 rejections in
April, 2006.9 While a significant number of the 176 rejections were issued for drugs and
cosmetics, the grounds for rejection among the various food items included salmonella and/or
filth in raw peeled shrimp, prepared Indian breads (paratha, roti), basmati rice, sesame seeds,
pepper, coriander and chili powder; pesticide residues in lentils; failure to declare the color
additive FD & C Yellow No. 5 in banana chips; and unsafe coloring in cream biscuits. The
number of rejections and the range of problems reveal extensive safety problems in Indian
food products. It is also reasonable to assume that the extent of the problems faced by
domestic consumers is far more serious as there many more micro, small and medium
enterprises that cater to domestic consumers and generally pay less attention to food safety
issues. By contrast, exporters are likely to be more well-established and larger firms with
better technology and relatively more cognizant about food safety concerns.
7. CHALLENGES TO IMPROVE FOOD SAFETY IN INDIA

Improving food safety in India, whether for the domestic market or for export trade, is
hampered by a number of structural, policy, institutional, technical and cultural barriers.
India had the most rejections of any country in May and the second highest number of rejections, behind Mexico,
in April, 2006. http://www.fda.gov/ora/oasis/5/ora_oasis_cntry_lst.html.
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Policy and Regulatory Environment

A number of policies and regulations governing agricultural marketing and food
processing complicate the implementation of food safety measures by the government and by
the private sector. Two critical marketing regulations are the State level Agricultural Produce
Marketing (Development and Regulation) Acts and the Small Scale Industry Reservation
Policy. Almost all states in India have an Agricultural Produce Marketing (APM) Act, which
gives state governments the sole authority to establish and manage wholesale markets.10 The
Act, adopted by most states in the 1960s and 1970s, prescribes the setting up of a network of
state controlled regulated markets or mandis and the establishment of Market Committees
to operate each. All notified agricultural commodities grown in areas surrounding the
market are required by law to be sold only through these markets, with the number of notified
commodities varying by state and market. Implementation of the Act and its enforcement
vary considerably by state. In 2005, there were nearly 8,000 regulated markets in the whole
country.11 The requirement that all agricultural commodities be channeled through the
regulated markets not only increases transactions costs, but is also a major obstacle to
preserving produce quality and traceability. In 2003, the GOI formulated a model Agricultural
Produce Market Act for state governments to adopt, which removes the restrictions on farmer
direct sales and permits entities outside of government to establish and operate wholesale
markets. To date only 10 of the 28 states and Union Territories have adopted the model Act.12
The Small Scale Industry (SSI) Reservation restricts the processing of certain
commodities to the small scale sector. Although the list of commodities subject to this
restriction has been reduced significantly during the last decade, several processed
agricultural products are still subject to SSI reservation, such as rapeseed, mustard and ground
nut oil,13 bread, pastry, pickles and chutneys, and hard boiled sugar candy (Department of
Small Scale Industries 2006). The SSI reservation imposes constraints on enterprises ability
to undertake the necessary investments (e.g. HACCP) and certifications required to meet the
domestic and international food safety and SPS requirements.14
There is a complex web of laws governing the processed food sector which complicate
implementation of food safety measures. These laws are enforced by 8 different ministries.
Some of the most critical are: Prevention of Food Adulteration Act 1954 implemented by the
Ministry of Health and Family Welfare; Milk and Milk Products Order 1992 and Agricultural
Produce Grading and Marking Act 1937 implemented by the Ministry of Agriculture; the
Essential Commodities Act 1955, Standards of Weights and Measures Act 1976, Consumer
Protection Act 1986, and Bureau of Indian Standards Act 1986 implemented by the Ministry
of Food, Consumer Affairs and Public Distribution; the Fruit Products Order 1955
10
The states of Kerala, Jammu and Kashmir, Manipur, Andaman and Nicobar Islands, Dadra and Nagar Haveli,
and Lakshadweep do not have the regulation.
11
In 2003, there were 7,383 wholesale markets in the country of which 7,360 were regulated markets. In addition,
there were 27,294 rural periodic markets (Ministry of Agriculture as cited in www.indiastat.com).
12
The states that adopted the model Act include : Punjab, Madhya Pradesh, Andhra Pradesh, Orissa, Maharashtra,
Rajasthan, Chhattisgarh, Himachal Pradesh, Sikkim and Nagaland.
13
Exceptions are rapeseed, mustard, and ground oil through solvent extraction and those processed by growers
cooperatives and state agro-cooperatives (Ministry of Small Scale Industries 2005)
14
This issue is more serious for domestic consumers since food processing units exporting more than 50% of
production are not subject to the SSI reservation.
403
implemented by the Ministry of Food Processing Industries; import and export regulations
implemented by the Ministry of Commerce; Trade in Endangered Species Act implemented
by the Ministry of Forest and Environment; Atomic Energy Act 1962/Control of Irradiation
of Food Rule 1991 implemented by the Ministry of Science and Technology; and Infant Milk
Substitutes, Feed Bottles and Infant Foods (Regulation of Production, Supply and
Distribution) Act 1992 implemented by the Ministry of Human Resource Development
(Patnaik 2005).
These laws also authorize several agencies to lay down standards for food products: (i)
Bureau of Indian Standards (BIS) of the Ministry of Food, Consumer Affairs and Public
distribution under the BIS Act, (ii) Ministry of Food Processing Industry under the Fruit
Products Order, (iii) Ministry of Agriculture under Ag Mark and the FPO, (iv) Ministry of
Health and Family Welfare (MOHFW) under the PFA Act; (v) Export Inspection Council
under the Export-Import Policy, and (vi) the Defense Ministry for their own purchases.
These laws and associated regulations in some cases prescribe contradictory or differing
standards. For example, while the Fruit Products Order (FPO) allows the use of artificial
sweeteners in fruit products, the Prevention of Food Adulteration (PFA) Act bans it.
Mandatory declaration labels required by the PFA differ from those of the Packaged
Commodity Regulation Rules (1977) under the Standard Weights and Measures Act. The
emulsifier and stabilizers permitted for use in jams and chutneys under the PFA differ from
those allowed under the FPO.
In 1998, the GOI began the process of rationalizing the legal and regulatory framework
for food and food processing. The Prime Ministers Council on Trade and Industry
established a Task Force on Food and Agro-Industries Management Policy to recommend
options for rationalizing the various policies and regulations. The outcome was a new Food
Safety and Standards Bill, which was submitted to Parliament in August 2005 and is awaiting
approval. The Bill aims to consolidate the laws relating to food. The key provisions of Bill
include: (i) the repeal of a number of Acts and Orders;15 (ii) the establishment of a Food
Safety and Standards Authority of India; (iii) definition of the standards for food additives,
contaminants, genetically modified and organic foods, packaging and labeling, and food
imports; (iii) accreditation of laboratories, research institutions and food safety auditors; (iv)
licensing and registration of food business and setting penalties for offenses; and (v)
establishment of a Food Safety Adjudication Tribunal (Ministry of Food Processing
Industries 2005). Approval of the Bill will be an important milestone in strengthening food
safety systems in India.
There are a large number of government agencies involved in agricultural marketing
activities, more broadly or with respect to specific commodities, which complicates effective
implementation of a coherent food safety strategy for the country. As in the case of the soft
drink contamination, the multiple laws and agencies added to the confusion. The BIS was
charged with setting the standards for pesticides in soft drinks, while the MOHFW is charged
with setting the pesticide standards for bottled water.
15
The laws and orders repealed are the: Prevention of Food Adulteration Act 1954 (37 of 1954), Fruit Products
Order 1955, Meat Food Products Order 1973, Vegetable Oil Products (Control) Order 1947, The Edible Oils
Packaging (Regulation Order) 1998, Solvent Extracted Oil, De-oiled Meal and Edible Flour (Control) Order
1967, Milk and Milk Products Order 1992, and other orders under the Essential Commodities Act 1955 (10 pf
1955) relating to food.
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Smallholder Agriculture
The current structure of the farm sector in India constrains farmer capacity to meet
domestic and international food safety standards. Farming in India is dominated by small
farmers the average farm size in 1990/00 was 1.8 ha (NABARD 2002). Most farmers face
credit constraints (World Bank 2004), and literacy rates are low.16 These constraints impose
limits on the number of farmers capable to adopt more sophisticated farm practices and
undertake the necessary investments (e.g. land improvements, obtaining necessary
certifications, cold storage) to meet more stringent food quality and safety requirements. They
increase the cost of transacting business and monitoring compliance with food safety
standards. Stringent land policies, e.g. land ceilings and restrictions on land rental, limit
possibilities for greater land amalgamation (World Bank 2006c). International experience
indicates, however, that farm size constraints may be overcome through innovative
interventions such as organizing farmers into producer groups, establishing collection centers
(by supermarkets and exporters), using contract farming arrangements, and by creating
public-private partnerships to assist farmers in a variety of ways, including help in obtaining
the capital required to make on-farm improvements and other investments (e.g. grading or
cooling facilities), developing and improving farming skills through joint extension provision,
and assistance in acquiring the required national and international certifications (Berdegu et
al. 2003, Boselie et al. 2003, Dries et al 2004, Reardon and Swinnen 2004, Reardon and
Timmer 2005a, 2005b).
In order to address various food safety concerns in both the spices and fresh and
processed fruit and vegetable sectors, some exporters initiated contract farming operations or
vendor screening programs. One industry that has been especially successful in establishing
contract farming arrangements and meeting stringent food safety and quality standards is the
pickled gherkin industry. The industry, consisting of some 42 companies and nearly 50,000
smallholder outgrowers, is concentrated in Karnataka, Andhra Pradesh, and Tamil Nadu. The
leading gherkin exporting companies each have several thousand farmers under contract. The
companies provide intensive oversight and maintain extensive records of farmer practices,
especially related to pesticide use. At least one company began the process of getting
outgrowers certified under EurepGAP (World Bank 2006b). Contract farming has worked
relatively well in the case of gherkins as almost the entire production from India is exported
and there is no local market. Hence contract enforcement has not been a major challenge as in
the case of other commodities where the export intensity is much lower and the majority of
production is consumed domestically.
Until recently, contract farming was illegal in India as per the provisions of the APM Act.
The only way entrepreneurs can legally enter into contract farming with farmers is to obtain a
special waiver from the APM Act from the State Government. The new model APM Act
provides the legal framework and guidelines for contract farming. The provisions in the
model Act allow contract buyers to directly purchase commodities from farmers under
individual contracts or from farmers markets. It also allows the direct sale of farm produce at
the farmers fields without having them routed through regulated markets. Adoption of the
16
The rural literacy rate in 1999/2000 was 50% http://www.indiastat.com/india/ShowData.asp?secid=16611

&ptid=367635&level=5
405
model Act by state governments will therefore facilitate not only more efficient marketing,
but also improved food safety and the adoption of improved agricultural practices.
Weak Extension Systems

The public agricultural extension systems at the state level are very weak and have not
effectively caught up to the changing needs of farmers and the market (World Bank 2005b).
In view of the GOIs earlier concentration on food self-sufficiency, the state-level Department
of Agriculture (DoA) extension systems generally focused on cereals, particularly rice and
wheat, with an emphasis on the transfer of improved varieties and management practices. The
weak coordination between the state DoAs and the other line departments (e.g. Departments
of Irrigation, Horticulture, Livestock, Marketing, etc) and the limited staff capacity beyond
the Department of Agriculture also often translated to limited extension activities beyond
cereals, limiting its impact on agricultural and market diversification trends. The weak
coordination with research at the central level further increased the difficulty of ensuring
effective research-extension-farmer linkages at the state level. In many states, tight fiscal
constraints contributed to the breakdown of the state extension machinery (Hanumantha Rao
2003). Private extension provision (fee for service) is emerging. There are an increasing
number of input suppliers, traders, contract buyers, supermarkets, and exporters which
provide extension services to farmers as an integral part of their trading arrangements (World
Bank 2005b). However in the national context, private extension remains limited.
Table 2. Farmer Sources of Information.
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The findings of a World Bank agricultural marketing survey, covering 1,579 farmers
producing high value crops (tomatoes, potatoes, mangoes, maize and tumeric) in four states in
India (Orissa, Tamil Nadu, Uttar Pradesh, and Maharashtra) conducted during February to
May 2005, confirm the limited effectiveness of the national extension system. Farmers
primarily depended on personal observation or on other farmers for information about crop
prices, post harvest practices, irrigation, fertilizer and pesticide use (Table 2).
Although food safety concerns have not been a major focus in the extension program, it is
partly addressed through the increased Ministry of Agriculture (MoA) priority to integrated
pest management (IPM). MoA established the National Center for Integrated Pest
Management in1988 to develop and promote IPM technologies. Notably there has been a
decline in total pesticide consumption in India from 75,000 mt in 1990/91 to 48,400 mt in
2003/03 (Directorate of Plant Protection and Quarantine 2006).
Poor Infrastructure and Services in the Marketing System

Reducing food safety risks from the farm to domestic and export markets is constrained
by inadequate infrastructure and facilities, particularly at the wholesale markets. The World
Bank Agricultural Marketing Survey also collected information on the operations of 78
wholesale markets in the four states. The survey found that the infrastructure and facilities in
these markets are limited and rudimentary. Overall, Maharashtra and UP had slightly better
infrastructure than the other two states. About 83% of markets had covered shops, but only
18% had paved roads within the market and 51% had public toilets. Access to warehouses is
limited, except in Maharashtra (85%). Less than 40% of markets had a drying area and no
markets in Orissa or Uttar Pradesh had cold storage facilities (compared to 5% in Tamil Nadu
and 20% in Maharashtra).
Waste management and pest control in the markets are very weak. Officials working in
the wholesale markets were asked how the spoiled produce and waste products were disposed
off. Fifty-four percent responded that market employees or contracted firms handled garbage
disposal and waste management; 29% reported that they were just left to rot in the market,
while 13% reported that they were left for the animals to eat. Market officials were also asked
about the pest control measures they undertake. Fifty-nine percent indicated that no particular
control measure for rats and insects are implemented in their market, 32% indicated it was up
to the individual shop owners to take care of their rat problems. Only 8% reported the market
management or association or a subcontracted firm took care of rat problems. Reducing food
safety risks will require significant public and private investments to upgrade the market
infrastructure and services. For regulated markets, this will also require improving the
operational and fiduciary management to ensure that more resources are re-invested back into
the markets.
Cultural Issues
Religious beliefs further constrain the kinds of food safety measures that could be
adopted in India. The sacred value attached to cattle imposes limits on disease control
407
measures to address food safety and public health (BSE, foot and mouth disease), such as
culling to limit disease spread or to create disease free zones.
Inadequate Grades and Standards for the Domestic

Market and Poor Enforcement
The Directorate of Marketing & Inspection under the Department of Agriculture and
Cooperation is responsible for enforcing and implementing the Agricultural Produce (Grading
and Marking) Act. Its mandate includes promoting standardization and grading of agricultural
products. Grades and standards have been prescribed for 164 commodities under the APM
Act for domestic trade, for export trade and for grading at the producers level. The
AGMARK grades are primarily voluntary grades covering aspects such as size, variety,
weight, color, and moisture levels. For certain items they also cover parameters such
acceptable levels of organic and inorganic foreign matter (in pulses, for example) and other
chemical properties such as specific gravity for essential oils. Different grades and standards
are laid out under AGMARK for domestic consumption versus exports.
The Directorate provides third party certification under the AGMARK quality
certification scheme. The AGMARK seal is supposed to ensure quality and safety. Any
consumer, trader or manufacturer can have products tested at one of the 23 regional
AGMARK laboratories for designated commodities. Typically, testing is only carried out for
adulteration prone commodities such as oils, ghee, whole and ground spices, honey, and
whole and milled food grains. Blended edible vegetable oils and fat spreads are compulsorily
required to be certified under AGMARK. The Prevention of Food Adulteration Act also sets
standards for food products including aspects such as permissible food colorings,
preservatives, pesticide residues, packaging and labeling. As illustrated by the bottled water
and soft drink pesticide residue incidents, inadequate standards and weak enforcement remain
a problem.
The grades specified under AGMARK and standards as laid out in the PFA are designed
to facilitate trade as well as ensure food safety. The food safety standards under the PFA in
general need to be aligned with international standards. However there are many commodities
that are not grown or consumed outside of India. For these commodities it may not be
possible to align domestic standards with international standards because there are no
established international standards. In these instances it is important for research to be
conducted in India to set appropriate standards for the domestic market.
Lack of Pro-Activity in Addressing Food-Safety Issues

Domestic food safety scares and the more notable food-safety problems faced by Indian
agro-exports, reveal the overall absence of any pro-activity in addressing food safety concerns
in India. Several factors contribute to this. In the case of exports, many if not most of the
emerging SPS and international standards are widely viewed as not scientifically based and as
representing unfair barriers to trade (World Bank, 2006b). These measures are viewed as
efforts to protect foreign farmers or processors from competition, or are being fueled by
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unreasonable consumer fears in high income countries and improved technologies for
detecting hazards. Consequently, the approach of the government and private sector has been
to try to negotiate away the problems with trading partners and, failing that, addressing the
various measures in international standard-setting or dispute flora. As a consequence,
insufficient attention is devoted to monitoring the requirements of official and private
standards, interpreting their implications for Indian agriculture and using current and
anticipated requirements as catalysts to upgrade existing operations and strengthen supply
chain management (World Bank 2006b).
This absence of pro-activity has meant that India has either had to adopt a defensive
strategy avoiding markets with more stringent food safety and agricultural health standards or
launch into a fire-fighting mode when it faces potential disruption or loss of trade due to
noncompliance with standards.17 The absence of pro-activity is well illustrated through
examples of problems faced with exports of fishery products in the late nineties and the more
recent troubles with grape exports to Europe. In both cases, although there were signs of
potential problems for a considerable period of time, the food safety problems were not given
serious attention until India was faced with a crisis.
In the case of exports of fish and fishery products, necessary monitoring and enforcement
measures for ensuring that exports complied with food safety concerns were not put in place
until the loss of EU markets in 1997 (Henson, Saqib and Rajasena, 2005). This was despite
the fact that India had continually faced rejections because of failure to meet hygiene
standards and other food safety requirements since the 80s, and in spite of regulatory reforms
to provide safety assurance for fish and fishery products undertaken in 1995 (Henson, Saqib
and Rajasena, 2005).
Similarly, in the case of grape exports to the EU, pesticide residue problems had surfaced
since the late nineties. During this period, some limited testing was done for pesticide
residues in export-oriented grapes. Testing was made mandatory in 2000, but most of the
available testing equipment was not up to date, could not test to the same level of detection as
was common in Europe and was unable to detect certain heat-sensitive chemicals such as
acephate and methomyl (World Bank, 2006b).18 Only after EU Rapid Alerts were issued in
2003 did the Government and industry step into action to address the problem. In general
India has not viewed complying with food safety and agricultural health standards as a means
to both improve its competitive position and to enhance the effectiveness of its negotiations
on particular technical and commercial matters, which is in stark contrast to the approach of
leading agro-food exporting countries (World Bank, 2006b).
A consequence of the lack of pro-activity and the crisis management mode of operation
has been the adoption of very rigorous and strict controls for commodities threatened with the
loss or disruption of trade. This has led to extremely high costs of compliance in some cases
(e.g. grapes) (World Bank, 2006b) or rather onerous requirements (e.g. requirements for
processing facilities exporting fishery products) (Henson, Saqib and Rajasena, 2005). In the
case of grapes, the Government of India (GOI) Agricultural and Processed Food Products
Export Development Authority (APEDA), formulated an integrated system of intensive grape
supply chain oversight that included:
17
An example of a defensive strategy is the existing trend where many of Indias mango pulp exporters are forced
to sell to less remunerative markets because they are not HACCP compliant.
18
As reported in Buurma et al 2001.
409
A requirement that all farms growing grapes for export to Europe have to register
with the Department of Agriculture. About 6200 growers registered for the 03/04
season;
Three field inspections (for registered exporters) during the crop cycle by a newly
constituted cadre of horticultural field inspectors. Some 244 such officers were
initially appointed and trained. There are now 291 such officers;
The inspection and registration of all grape export packinghouses by APEDA.
Mandatory pesticide residue testing from each registered field of export grapes.
Testing would be done prior to harvest and only if the tests were passed would
authorization be given for harvesting for export. Grapes from fields with failed
results would need to be sold in other markets or re-tested.
Every consignment would be checked by AGMARK to ensure conformity with EU
quality specifications for grapes. AGMARK would issue certificates.
Obtaining a phytosanitary certificate issued by Plant Protection, Quarantine and
Storage for every consignment; and
Later, in 2005, another procedure was added whereby National Research Center for
Grapes would take a 5% sample of ex-packhouse grape consignments to re-test for
pesticide residues.
The extensive system of checks and controls primarily focused on end-of-the-pipeline

solutions. In addition to the protocols that potential exporters to the EU have to follow, the
government also invested heavily in upgrading laboratory testing equipment, training field
inspectors, subsidizing packhouse upgrades, and strengthening the National Research Centre
for Grapes. Overall, it is estimated that the cost of this control system for pesticide residues
(to government and the private sector) is about US$1.2 million, equivalent to 7.9% of the
FOB value of Indias grape trade to Europe in 2005 (Table 3). If all other costs associated
with the oversight of the grape supply chain are added to the costs of pesticide residue testing,
SPS compliance costs are estimated to account for 13% of this FOB value.
Table 3. Estimated Annual Cost of Meeting EU SPS Standards2005 US $
While it is arguable that there are many spillovers and important lessons that have been
learned from the handling of the pesticide residue problem with grape exports, and that these
measures have been successful in that they have not resulted in further alerts or rejections,
the heavy handed approach with which the problems were addressed, and the costs involved,
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clearly suggest that it is not a strategy that should be replicated. Although India has not faced
further rejections of exports to the EU, routine laboratory testing still reveals violative
residues, indicative of the continuing need to focus on improving overall agricultural
practices to assure food safety.
Lack of Good Agricultural, Manufacturing and Hygiene Practices

In addition to constraints that arise due to small farm sizes, the lack of good agricultural,
manufacturing and hygiene practices remain a major challenge for improving food safety both
for the domestic and export market. It is only recently that efforts are being made to promote
good practices. For example, Marine Products Export Development Authority (MPEDA)
promoted codes of good practice, particularly with regards to addressing antibiotic use. To
this extent the organization was involved in monitoring antibiotic usage levels, providing
training and disseminating information (Henson, Saqib and Rajasena, 2005). In the spices
sector, the Spices Board (SB) undertook measures to address problems with regards to
pesticide residues and aflatoxin. The SB, in conjunction with State Departments of
Agriculture and various NGOs, supported measures to promote integrated pest management
(IPM) and the production of organic spices (Jaffee, 2005). They helped address the aflatoxin
concern by promoting better drying practices. The Ministry of Food Processing Industries and
APEDA have both been promoting adoption of HACCP and ISO certification among
processed food manufacturers through a range of training initiatives and private sector
investment grant for upgrading processing plants to obtain HACCP/ISO certification.
However, the adoption of good practices remains limited. Much remains to be done in
improving practices with regards to the manufacture and use of pesticides and improving post
harvest techniques. Although there have been some limited spillovers from the export sector
into the domestic market, in terms of improving production practices, for most commodities,
including spices and fresh fruit and vegetables, farmers do not necessarily see any advantages
or necessity for altering their production practices since the vast majority of production is
consumed in the domestic market. Until domestic consumer awareness and willingness to pay
for improved food safety becomes more widespread, it is unlikely that addressing food safety
concerns will become standard practice nationally. Similarly, significant measures are needed
to improve the safety of processed foods. In the food processing sector there are a growing
number of firms with modern factories and good quality assurance systems, but this segment
co-exists with large numbers of small and older firms that would need to make significant
upgrades to implement HACCP and other quality assurance systems.19
In the short term, developments in the food retail sector in India are likely to bring about
improvements in food safety. International experience shows that modernization of the food
retail sector is an important driver for change not only in the structure of production and
wholesale marketing of produce, but also in fostering adoption of improved grades and food
safety standards (Berdegu et al 2003, Reardon and Timmer 2005a, 2005b). Despite the ban
on foreign direct investments in food retailing, the supermarket industry is growing rapidly,
19
For instance, in recent work on the mango pulp sector in India one company reported costs of $35,000 to put it in
a position to implement a proper HACCP system (World Bank, 2006b).
411
driven by investments from the Indian corporate sector.20 Many of the modern retail outlets
are beginning to undertake direct procurement from individual farmers or farmers
associations. In some cases farmers or associations supplying these outlets are required to
follow a code of practice to meet quality and safety requirements of their buyers. The retail
outlets are also involved in disseminating new agricultural techniques and information to their
suppliers as well as providing training on quality control of produce handling, grading and
packaging.
There are also efforts by the public sector to promote good agricultural practices among
producer groups and to help establish linkages with the organized food retail sector.21 The
Government of India and State governments are working closely with the supermarket
industry (with support from USAID) to develop an India Good Agricultural Practice standard
for agricultural produce (INDIA-GAP), which will in turn also provide the framework for
government extension support to farmers.
Need for More Collective Action

International experience highlights the importance of collective action within the private
sector to promote awareness of SPS matters, find solutions to emerging challenges, promote
good agricultural and manufacturing practices, and otherwise provide a degree of selfregulation, which in turn reduces the need for government agencies to play enforcement roles.
While there are some examples of successful collective action in both the spice and fishery
export industries in India, it has been lacking in many other sectors, notably in horticulture
(World Bank, 2006b). For example, the Seafood Exporters Association of India (SEAI) has
developed a model to provide a number of pre-processing units with common water,
ice and effluent infrastructure. SEAI in collaboration with MPEDA has also been
involved in developing a system to ensure traceability for shrimp from aquaculture in order to
address quality problems (Henson, Saqib and Rajasenan, 2005). In the Spices sector, the All
India Spice Exporters Forum has been an important player in trying to influence standards for
pesticides in spices grown under tropical conditions and in finding solutions to address food
safety concerns in its export markets (Jaffee, 2005).
20
Corporate manufacturers such as Hindustan Lever Ltd, International Tobacco Company, Godrej, Bharti,Reliance,
DCM Sriram Conolidated, RPG Group, Pantaloon Group are setting up or have set hypermarkets, supermarkets
and retail outlets in rural areas, recognizing the huge untapped potential (World Bank 2006a). Gasstation stores
are also another growing retail outlet. Petroleum companies like Hindustan Petroleum Corporation Limited,
Indian Oil, and Bharat Petroleum have introduced branded outlets like Speedmart (around 60-65 in number),
ConveniOs (around 150), and In&Out Stores (around 100) which sell food items (Singh 2004).
21
The Marashtra Agricultural Agricultural Marketing Board in collaboration with USAID is trying to promote
good agricultural practices among mango farmers in the state and link these farmers with various supermarkets
and other retail outlets that are interested in procuring better quality and safer fruit.
Wasim Aktar
412
8. CONSUMER CONTROL POINTS

Minimizing Possible Consumption of Pesticide Residues
For those consumers who wish to take additional measures to reduce any possible
pesticide residues on their foods, here are some tips from AFIC.
1) Raw foods should be washed thoroughly before cooking and/or consumption.
Washing in dilute vinegar solution, or solution of sodium bicarbonate, then rinsing
with clean water will help to remove any chemical residues and also any soil or other
foreign matter on the produce.
2) Many chemicals applied to crops to protect from insects and disease are sprayed onto
external surfaces, so peeling outer layer or skin when preparing fresh fruit and
vegetables will remove any surface residues.
3) Look out for the many of the quality assurance schemes, which guarantee chemical
treatment of produce has strictly followed manufacturers recommendations and
residues levels at point of harvest are either zero or very low. There are also an
increasing number of retailers and growers offering organic produce, however, be
aware that organic farming often uses some pest control substances, approved by
the various associations established to promote this form of cultivation.
4) Do not consumer berries, leaves or other edible plant material picked from roadsides
or other public areas, as these plants as it is not possible to know if these plants have
been sprayed intentionally or unintentionally contaminated with pesticides or other
substances and will not be subject to safety restrictions of designated food crops.
Certain processes or handling practices by consumers in the home have been identified as
being essential or critical in preventing foodborne illness. These practices, which prevent or
control the "meals" microbial contamination associated with foodborne illness, are under the
direct control of the consumer, from food acquisition through disposal.
They are purchasing, storing, pre-preparation, cooking, serving, and handling leftovers.
Failure to take appropriate action at these critical points could result in foodborne illness.
Critical Point 1: Purchasing
Purchase as far as possible perishables on a daily basis.

Procure processed food items only after checking properly the `Best Before date
validity and prefer processed branded food items with product certification marks
viz. ISI, FPO & AGMARK.
Procure milk and milk products, fish, seafood, poultry, meat and meat products, eggs
and other perishables last when out on purchasing and keep packages of raw meat
and meat products separate from other foods, particularly foods that will be eaten
without further cooking. Consider using plastic bags to enclose individual packages
of raw meat and meat products.
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Make sure milk and milk products, fish, seafood, poultry, meat and meat products,
eggs and other perishables are refrigerated as soon as possible after purchase. Plan to
drive directly home after purchases from the grocery store.
Canned food items, if purchased, should be free of leaks, dents, cracks or bulging
lids.
Critical Point 2: Home Storage
Verify the temperature of your refrigerator and freezer with an appliance

thermometer - refrigerators should run at 5C or below; freezers at -18C. Most
foodborne bacteria grow slowly at 5C, a safe refrigerator temperature. However,
freezer temperatures of -18C stops bacterial growth.
Keep High risk products such as fish, seafood, poultry, meat and meat products in the
freezer cabinet of refrigerator immediately on reaching home. Keep the milk and
milk products in the designated racks in the refrigerator or on the first topmost rack
just under the freezer cabinet.
To prevent raw juices drippings & pieces of meat and meat products from falling on
cooked to other foods kept in the refrigerator. Keep them in shelves below the stand
cooked food. Use plastic bags or place meat and poultry on a plate.
Wash hands with soap and water for 20 seconds before and after handling any raw
fish, seafood, poultry, eggs, meat and meat products.
Store canned foods in a cool, clean dry place and as per the labeling directions of the
manufacturer. Avoid extreme heat or cold which can be harmful to canned food
items.
Never store any foods directly under a sink and always keep foods off the floor, on a
rack or pallet and completely separate from cleaning supplies.
Critical Point 3: Pre-Preparation
The importance of hand washing cannot be overemphasized. This simple practice is

the most economical, yet often forgotten way to prevent contamination or crosscontamination.
Wash hands with soap and water thoroughly (for 20 seconds): before beginning
preparation; after handling raw meat, poultry, seafood or eggs; after touching
animals; after using the toilet/bathroom; after changing diapers; or after blowing the
nose.
Don't let juices from raw meat, poultry or seafood come in contact with cooked foods
or foods that will be eaten raw, such as fruits or salads.
Wash hands, kitchen slabs / tables, equipment, utensils, and cutting boards with soap
and water immediately after use. Kitchen slabs / tables, equipment, utensils and
cutting boards can be sanitized with a chlorine solution of 1 teaspoon liquid
household bleach per quart of water. Let the solution stand on the board after
washing, or follow the instructions on sanitizing products.
Before processing frozen fish, seafood, poultry, meat and meat products, thaw the
same properly in the refrigerator but NEVER ON THE KITCHEN SLAB / TABLE.
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414
It is also safe to thaw in cold water in an airtight plastic wrapper or bag, changing the
water every 30 minutes till thawed. Thawing may also be carried out in the
microwave and followed immediately by cooking.
Marinate foods in the refrigerator and NEVER ON THE KITCHEN SLAB / TABLE.
Critical Point 4: Cooking
Make sure to cook food thoroughly, particularly the meat products, Depending upon
the type of dish made, thoroughly cooking would be signified by colour, texture and
taste. For example boiled/steamed rice shall have grains double the size of original
rice grains, with each grain separate; Fried item viz cutlets, pokoras etc should be
brought to golden brown coloir at medium flame, which ensures thorough cooking
upto the core of the fried item.
If harmful bacteria are present, only thorough cooking will destroy them (core
temperature of product to be higher than 75 C) ; remember freezing or rinsing the
foods in cold water is not sufficient to destroy bacteria.
Avoid interrupted cooking. Never refrigerate partially cooked products to later finish
cooking on the grill or in the oven. Fish, seafood, poultry, meat and meat products
must be cooked thoroughly the first time and then they may be refrigerated and
safely reheated later.
When microwaving foods, carefully follow manufacturers instructions. Use
microwave-safe containers, cover, rotate, and allow for the standing time, which
contributes to thorough cooking.
Critical Point 5: Serving:
Wash hands with soap and water before serving or eating food.
Serve cooked products on clean plates with clean utensils and clean hands. Never put
cooked foods on a dish that has held raw products unless the dish is washed with
soap and hot water.
Hold hot foods above 60C and cold foods below 5C.
Never leave foods, raw or cooked, at room temperature longer than 2 hours.
Critical Point 6: Handling Leftovers
Wash hands before and after handling leftovers. Use clean utensils and surfaces.
Divide leftovers into small units and store in shallow containers for quick cooling.
Refrigerate within 2 hours of cooking.
Discard anything left out too long.
Never taste a food to determine if it is safe.
When reheating leftovers, reheat thoroughly (temperature of 75 C) until the dish is
hot and steamy. Bring soups, sauces and gravies to a rolling boil.
If in doubt, throw it out.
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Ten Steps to a Safe Kitchen:

Step 1 Keep your refrigerator at 4 C or less.A temperature of 4 C or less is important
because it slows the growth of most bacteria. The fewer bacteria there are, the less likely you
are to get sick from them.
Step 2 Refrigerate cooked, perishable food as soon as possible but within two hours after
cooking. A temperature of 4 C or less is important because it slows the growth of most
bacteria. The fewer bacteria there are, the less likely you are to get sick from them. Date
leftovers so that they can be used within one day. If in doubt, throw it out!
Step 3 Sanitize your kitchen dishcloths and sponges regularly. Wash with a solution of
one teaspoon (5 ml) chlorine bleach to one litre of water, or use a commercial sanitizing
agent, following product directions.
Step 4 Wash your cutting board with soap and hot water after each use to prevent any
subsequent contamination in food during preparation. Never allow raw meat, poultry, and fish
to come in contact with each other as they have generally high bacteria count including
pathogens which cause food poisoining .Washing with only a damp cloth will not remove
bacteria. Periodically washing in a bleach solution is the best way to prevent bacteria from
remaining on your cutting board.
Step 5 Cook meats , seafood and poultry products thoroughly so as to ensure that cooked
food is free from harmful bacteria. Cooking meat products on a law/medium flame such that
the core temperature reaches at least 75 C usually protects against foodborne illness. Welldone meats reach that temperature.
Step 6 Don't consume raw or lightly cooked eggs as they may contain the harmful
Salmonella bacteria. Always cook the eggs thoroughly before eating them.
Step 7 Clean kitchen counters and other surfaces that come in contact with food with hot
water and detergent or a solution of bleach and water. Bleach and commercial cleaning agents
are best for getting rid of pathogens. Hot water and detergent are good, but may not kill all
strains of bacteria. Keep sponges and dishcloths clean because, when wet, these materials
harbor bacteria and may encourage their growth.
Step 8 When washing dishes by hand, its best to wash them with warm water and
detergents all within two hours--before bacteria can begin to form. Allow dishes and utensils
to air-dry in order to eliminate re-contamination from hands or towels.
Step 9 Wash hands with soap and warm water immediately after handling raw meat,
poultry, or seafood. Wash for at least 20 seconds before and after handling food, especially
raw meat. If you have an infection or cut on your hands, wear rubber or plastic gloves.
Step 10 Defrost frozen meat, poultry and fish products in the refrigerator, microwave
oven, or cold water that is changed every 30 minutes. Cook microwave-defrosted food
immediately after thawing. Changing water every 30 minutes when thawing foods in cold
water ensures that the food is kept cold, an important factor for slowing bacterial growth on
the outside while inner areas are still thawing.
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CONCLUSION
The Indian experience illustrates the many challenges faced by developing countries in
addressing food safety concerns in domestic and export markets. Despite a large number of
food safety incidents in the past, it is only in the past five years or so that food safety issues
have begun to receive greater attention. As elaborated in this paper, this has partly been due to
greater consumer awareness arising from campaigns led by NGOs, increased coverage of
food-safety incidents in the media, wider access to media and the internet, and the problems
encountered with agro-food exports in high income markets. Despite this, considerable efforts
are still needed to give the issue of food safety the attention it warrants.
Because of low consumer awareness, the private sector engaged in agriculture, food
processing and the food retail industry in India, for the most part, has not taken the necessary
steps to improve the quality and safety of food products. In most cases, the responsibility of
ensuring food safety fell into the hands of government through enacting and enforcing
legislation and setting standards. While government has taken actions in instances where
there have been immediate public health scares or disease outbreaks, less attention has been
given to food safety concerns whose impact is only apparent in the medium to long-term. One
of the positive results of globalization and the emergence of modern food retailing in India is
the increased attention to quality and safety issues. As incomes are increasing, consumers are
also more willing and able to pay for better quality and safer food.
Addressing food safety issues in India will require the adoption of more appropriate
legislation and their enforcement (Table 4). Parliamentary approval of the Food Safety and
Standards Bill will be critical to removing the uncertainty arising from, and the associated
additional cost of dealing with, overlapping and conflicting food safety regulations. Broad
based adoption of the model APM Act and the removal of the remaining agricultural
commodities from the SSI reservation will foster both increased market efficiency and
facilitate adoption by firms of appropriate food safety measures.
Joint efforts by the government and the private sector will be needed in a number of
areas. These include better risk management, the promotion and adoption of good
agricultural, manufacturing and hygiene practices, greater collective action and some targeted
public investments. Responsibilities for these functions need to be shared between the private
and public sectors. While there are many critical regulatory, research and management
functions that are normally carried out by governments, the private sector also has an
important role in the actual compliance with food safety requirements.
Quality grades should be voluntary for fruits, vegetables and for most other fresh
produce, since they are set primarily to facilitate trade and are not a regulatory instrument.
Yet, for matters of food safety, standards should be mandatory rather than voluntary. These
standards would apply for pesticide residues, heavy metal and other forms of environmental
contamination, and especially for microbiological contaminations for which there could be
acute health risks. A coordinated program of food safety product surveillance can be used to
highlight the nature and scope of pertinent problems and also be used as a basis for
developing consumer and supply chain awareness and good practice promotion. Overall there
is a large role for extension service providers to promote good practices in order to ensure that
farmers follow recommended dosages for agro-chemicals and observe appropriate pre-harvest
417
intervals. Soil and water testing should also be routinely conducted through the extension
apparatus (World Bank, 2006a).
Table 4. Role of Public and Private Sector in Enhancing Food Safety Capacity.
Role of Public Sector
Policy and Regulatory Environment
Adopt domestic food safety legislation and
standards suited to local risk conditions and
preferences and consistent with Indias WTO and
other treaty obligations.
Risk Assessment and Management:
Strengthen national or state-level systems of pest
and animal disease surveillance and market
surveillance programs to gauge the incidence of
various food safety hazards in the domestic
agrofood system.
Find solutions to animal health constraints that
limit domestic (for imports) and foreign (for
exports) market access. This might entail, product
inspection, agreed development of disease-free
areas. etc
Awareness building and promoting good
practices:
Consumer awareness campaigns about food safety
risk and improve hygiene in the home
Raise stakeholder awareness about and promote
good agricultural, hygiene, and manufacturing
practices and quality management. Incorporate
these areas into curricula of public agricultural/
technical institutes and universities.
Incorporate food-safety focused practices in
extension program, including through public-private
partnerships
Accredit private laboratories and conduct
reference/consistency testing.
Facilitate technical, administrative and
institutional change and innovation within the
private sector for example through publicprivate
partnerships
Public Expenditures
Investments in water supply and sanitation,
marketing facilities, to reduce food safety hazards
Support research to address food safety and
agricultural health concerns
International Trade Diplomacy:
Undertake continuous dialogue and periodic
negotiations to address emerging constraints or
opportunities.
Source: Adapted from World Bank 2006a.
Role of Private Sector

Good Management Practices:
Implement appropriate management practices to
minimize food safety risks. Examples include
good agricultural, hygiene, and manufacturing
practices and HACCP principles.
Where commercially valuable, gain formal
certification for such adopted systems.
Develop incentives, advisory services and
oversight systems to induce the similar adoption of
the above good practices by supply chain
partners.
Traceability:
Develop systems and procedures to enable the
traceability of raw materials and intermediate and
final products in order to identify sources of
hazards, manage product recalls or other
emergencies, etc.
Develop Training, Advisory, and Conformity
Assessment Services:
On a commercial basis provide support services
to agriculture, industry, and government related to
quality and food safety management. Invest in the
needed human capital, physical infrastructure and
management systems to competitively supply such
services.
Collective Action and Self-Regulation:
Work through industry, farmer, and other
organizations to share the costs of awarenessraising and systems improvement, alert
government to emerging issues, advocate for
effective government
services, and provide a measure of self-regulation
through the adoption and oversight of industry
codes of practice
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Wasim Aktar
The use of agricultural chemicals in relation to food safety will continue to be a complex
subject. Some studies have shown that pesticides may affect ground-water, wildlife and
occupational workers if the chemicals are not used in accordance with the law. But the future
looks promising as food scientists, research-ers, government officials and manufacturers
search for methods to improve agricultural techniques while reducing pesticide-related risks.
Today's consumers can feel confident that they can choose from an abundant and safe food
supply for themselves and their families.
There is also a need for regular inspection of health and sanitary conditions at certain
types of food premises that may be associated with more severe consumer health risks,
(abattoirs, for example). Inspection should not be random, but should be targeted based upon
risk assessments that government may do on different types of food establishments to help
pinpoint areas requiring particular attention, not only in the form of inspection, but also
including awareness-raising, training, periodic licensing, etc. The challenges for ensuring
food safety in the domestic market and in its food exports remain large. India has made some
progress in the last decade to strengthen food safety measures at home and in meeting food
safety and SPS standards abroad. The challenge for the future will be to adopt a more
strategic, rather than crisis management approach. This will be essential to ensuring the
sustainability and cost effectiveness of these efforts.
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ISSN: 2158-5717
Chapter 15
IMPACT OF PESTICIDE USE IN INDIAN AGRICULTURE

THEIR BENEFITS AND HAZARDS
Wasim Aktar*
Pesticide Residue Laboratory, Department of Agricultural Chemicals, Bidhan Chandra
Krishi Viswavidyalaya, Mohanpur-741252, Nadia, West Bengal, India
1. INTRODUCTION
The term pesticide covers a wide range of compounds including insecticides, fungicides,
herbicides, rodenticides, molluscicides, nematicides, plant growth regulators and others.
Among these, organochlorine (OC) insecticides, used successfully in controlling a number of
diseases, such as malaria and typhus, were banned or restricted after the 1960s in most of the
technologically advanced countries. The introduction of other synthetic insecticides
organophosphate (OP) insecticides in the 1960s, carbamates in 1970s and pyrethroids in
1980s and the introduction of herbicides and fungicides in 1970s - 1980s contributed greatly
in pest control and agricultural output. Ideally a pesticide must be lethal to the targetted pests,
but not to non-target species, including man. Unfortunately, this is not, so the controversy of
use and abuse of pesticides has surfaced. The rampant use of these chemicals, under the
adage, if little is good, a lot more will be better has played havoc with human and other life
forms.
1.1. Production and Usage of Pesticide in India

The production of pesticides started in India in 1952 with the establishment of a plant for
the production of BHC near Calcutta, and India is now the second largest manufacturer of
pesticides in Asia after China and ranks twelfth globally[9]. There has been a steady growth
in the production of technical grade pesticides in India, from 5,000 metric tonnes in 1958 to
102,240 metric tonnes in 1998. In 1996-97 the demand for pesticides in terms of value was
*
Correspondence to: wasim04101981@yahoo.co.in
Wasim Aktar
424
estimated to be around Rs. 22 billion (USD 0.5 billion), which is about 2% of the total world
market.
The pattern of pesticide usage in India is different from that for the world in general. As
can be seen from Figure 1, in India 76% of the pesticide used is insecticide, as against 44%
globally.[9] The use of herbicides and fungicides is correspondingly less heavy. The main use
of pesticides in India is for cotton crops (45%), followed by paddy and wheat.
Figure 1. Consumption pattern of pesticides
2. BENEFITS OF PESTICIDES
2.1. Improving Productivity
Tremendous benefits have been derived from the use of pesticides in forestry, public
health and the domestic sphere - and, of course, in agriculture, a sector upon which the Indian
economy is largely dependent. Food grain production, which stood at a mere 50 million
tonnes in 1948-49, had increased almost fourfold to 198 million tonnes by the end of 1996-97
from an estimated 169 million hectares of permanently cropped land. This result has been
Impact of Pesticide Use in Indian Agriculture: Their Benefits and Hazards
425
achieved by the use of high-yield varieties of seeds, advanced irrigation technologies and
agricultural chemicals.[1]
Similarly outputs and productivity have increased dramatically in most countries, for
example, wheat yields in the United Kingdom, corn yields in the USA. Increases in
productivity have been due to several factors including use of fertiliser, better varieties and
use of machinery. Pesticides have been an integral part of the process by reducing losses from
the weeds, diseases and insect pests that can markedly reduce the amount of harvestable
produce. Warren (1998) also drew attention to the spectacular increases in crop yields in the
United States in the twentieth century. Webster et al. (1999) stated that "considerable
economic losses" would be suffered without pesticide use and quantified the significant
increases in yield and economic margin that result from pesticide use. Besides this, most of
the pesticides, in environment, undergo photochemical transformation to produce metabolites
which are relatively non-toxic to the human beings as well as environment.[47]
2.2. Protect Crop Losses/Yield Reduction

In medium land rice even under puddle conditions during the critical period warranted an
effective and economic weed control practice to prevent a reduction in rice yield due to weeds
that ranged from 28 to 48% based on comparisons that included control (weedy)
plots[43].Weeds reduce yield of dry land crops[43] by 37-79%. Severe infestation of weeds
particularly in early stage of crop establishment ultimately accounts for a yield reduction of
40%. Herbicides provided an economic and labour benefit too.
2.3. Vector Disease Control

Vector-borne diseases are most effectively tackled by killing the vectors. Insecticides are
often the only practical way to control the insects that spread deadly diseases such as malaria
that results in an estimated 5000 deaths each day (Ross, 2005). In 2004, Bhatia wrote that
malaria is one of the leading causes of morbidity and mortality in the developing world and a
major public health problem in India.
2.4. Quality of Food

In the countries of first world, it is now observed that a diet containing fresh fruit and
vegetables far outweigh potential risks from eating very low residues of pesticides in crops
[27]. Increasing evidence (Dietary Guidelines, 2005) shows that eating fruit and vegetables
regularly reduces the risk of many cancers, high blood pressure, heart disease, diabetes,
stroke, and other chronic diseases.
Lewis et al (2005) discussed the nutritional properties of apples and blueberries in the US
diet and concluded that their high concentrations of antioxidants act as protectants against
cancer, heart disease. Lewis attributed doubling in wild blueberry production and subsequent
increases in consumption chiefly to herbicide use that improved weed control.
426
Wasim Aktar
2.5. Other Area-Transport, Sport Complex, Building

The transport sector makes extensive use of pesticides, particularly herbicides. Herbicides
and insecticides are used to maintain the turf on sports pitches, cricket grounds and golf
courses. Insecticides protect buildings and other wooden structures from damage by termites
and wood boring insects.
3. HAZARDS OF PESTICIDES
3.1. Direct Impact on Human Being
If the credits of pesticides include enhanced economic potential in terms of increased
production of food and fibre, and amelioration of vector-borne diseases, then their debits have
resulted in serious health implications to man and his environment. There is now
overwhelming evidence that some of these chemicals do pose potential risk to humans and
other life forms and unwanted side effects to the environment [17-19]. No segment of the
population is completely protected against exposure to pesticides and the potentially serious
health effects, though a disproportionate burden is shouldered by the people of developing
countries and by high risk groups in each country.[20] The world-wide deaths and chronic
illnesses due to pesticide poisoning number about 1 million per year[21].
The high risk groups exposed to pesticides include the production workers, formulators,
sprayers, mixers, loaders and agricultural farm workers. During manufacture and formulation,
the possibility of hazards may be more because the processes involved are not risk free. In
industrial settings, the workers are at increased risk since they handle various toxic chemicals
including pesticides, raw materials, toxic solvents and inert carriers.
In India, the first report of poisoning due to pesticides was from Kerala in 1958, where
over 100 people died after consuming wheat flour contaminated with parathion.[2] This
prompted the Special Committee on Harmful Effects of Pesticides constituted by the ICAR to
focus attention on the problem[3]. Further, Carlson in 1962 warned that OC compounds could
pollute the tissues of virtually every life form on the earth, the air, the lakes and the oceans,
the fishes that live in them and the birds that feed on the fishes.[4] Later, the US National
Academy of Sciences stated that the DDT metabolite, DDE causes eggshell thinning and that
the bald eagle population in the United States declined primarily because of exposure to DDT
and its metabolites[5]. Certain environmental chemicals including pesticides termed as
endocrine disruptors are known to elicit their adverse effects by mimicking or antagonising
natural hormones in the body and it has been postulated that their long-term, low-dose
exposure are increasingly linked to human health effects such as immunosuppression,
hormone disruption, diminished intelligence, reproductive abnormalities and cancer[6-8].
A study on workers (N=356) in four units manufacturing HCH revealed neurological
symptoms (21%) which were related to the intensity of exposure[22]. The magnitude of the
toxicity risk involved in the spraying of methomyl, a carbamate insecticide, in field
conditions was assessed by the National Institute of Occupational Health (NIOH) [24].
Significant changes were noticed in the ECG and the levels of serum LDH and ChE activities
in the spraymen indicating the cardiotoxic effects of methomyl.
427
Observations confined to health surveillance in male formulators engaged in production

of dust and liquid formulations of various pesticides (malathion, methyl parathion, DDT and
lindane) in industrial settings of the unorganised sector revealed a high occurrence of
generalized symptoms (headache, nausea, vomiting, fatigue, irritation of skin and eyes)
besides psychological, neurological, cardiorespiratory and gastrointestinal symptoms coupled
with low plasma cholinesterase (ChE) activity [23].
Data on reproductive toxicity were collected from 1,106 couples when the males were
associated with the spraying of pesticides (OC, OP and carbamates) in cotton fields.[25]
A study in malaria spraymen was initiated to evaluate the effects of a short term (16
week) exposure in workers (N=216) spraying HCH in field conditions.[26]
3.2. Impact through Food Commodities

The UK Pesticide Residue Committee annual report (2002) showed that over 70% of the
food in the UK contained no pesticide residues at all and only 1.09% contained residues
above the statutory maximum residue levels (MRLs). It concluded that none of these
residues caused concern for people's health. Yet these very small quantities of chemicals in
our food, detected at ever lower levels due to increasingly sensitive laboratory equipment, are
now easy targets for the media. In India, a study revealed that 50% of the vegetable samples
taken from farm gate were found contaminated with various pesticides (0.01-2.23 ppm) of
which 16% were above MRL.[48]
3.3. Impact on Environment

Pesticides can contaminate soil, water, turf, and other vegetation. In addition to killing
insects or weeds, pesticides can be toxic to a host of other organisms including birds, fish,
beneficial insects, and non-target plants. Insecticides are generally the most acutely toxic
class of pesticides, but herbicides can also pose risks to non-target organisms.
3.3.1. Surface Water Contamination

Pesticides can reach surface water through runoff from treated plants and soil.
Contamination of water by pesticides is widespread. The results of a comprehensive set of
studies done by the U.S. Geological Survey (USGS) on major river basins across the country
in the early to mid- 90s yielded startling results. More than 90 percent of water and fish
samples from all streams contained one, or more often, several pesticides.45 Pesticides were
found in all samples from major rivers with mixed agricultural and urban land use influences,
and 99 percent of samples of urban streams.[28] The USGS also found that concentrations of
insecticides in urban streams commonly exceeded guidelines for protection of aquatic life 41.
Twenty-three pesticides were detected in waterways in the Puget Sound Basin, including 17
herbicides. According to USGS, more pesticides were detected in urban streams than in
agricultural streams. [29]
428
Wasim Aktar
3.3.2. Ground Water Contamination

Pesticides, including herbicides, can and do leach to contaminate ground water.
According to the USGS, at least 143 different pesticides and 21 transformation products have
been found in the ground water, including pesticides from every major chemical class. Over
the past two decades, detections have been found in the ground water of more than 43
states[30]. Contamination of ground water is of concern because ground water supplies 50
percent of the U.S. population with Drinkingwater.[31] During one survey in India it has been
found that 58% of drinking water samples drawn from various hand pumps and wells around
Bhopal are contaminated with Organ Chlorine pesticides above the EPA standards.[46] Once
ground water is polluted with toxic chemicals, it may take many years for the contamination
to dissipate or be cleaned up. Cleanup may also be very costly and complex, if not impossible
[32-34].
3.3.3. Soil Contamination
Pesticides have various characteristics that determine how they act once in soil. Mobility
refers to how much a pesticide will move around in the soil. The half life of a pesticide refers
to the length of time it takes for half of the pesticide to degrade. Persistence refers to the
length of time until all measurable residues of a pesticide are gone.
3.3.4, Effect on Soil Fertility (Beneficial Soil Microorganisms)
One spoonful of healthy soil has millions of tiny organisms including fungi, bacteria, and
a host of others. These microorganisms play a key role in helping plants utilize soil nutrients
needed to grow and thrive. Microorganisms also help soil store water and nutrients, regulate
water flow, and filter pollutants.[38] The heavy treatment of soil with pesticides can cause
populations of beneficial soil microorganisms to decline. Sometimes pesticides have a
negative impact on the available NPK from soil.[49] According to soil scientist Dr. Elaine
Ingham, If we lose both bacteria and fungi, then the soil degrades. Overuse of chemical
fertilizers and pesticides have effects on the soil organisms that are similar to human overuse
of antibiotics. Indiscriminate use of chemicals might work for a few years, but after awhile,
there arent enough beneficial soil organisms to hold onto the nutrients. [40].
3.3.5. Contamination of Air, Soil, and Non-Target Vegetation
Pesticide sprays can directly hit non-target vegetation, or can drift or volatilize from the
treated area and contaminate air, soil, and non-target plants. Some pesticide drift occurs
during every application, even from ground equipment.[35] Drift can account for a loss of 2
to 25% of the chemical being applied, which can spread over a distance of a few yards to
several hundred miles. There are thousands of reported complaints of off target spray drift
each year in the U.S. [36]. Many pesticides can volatilize (that is, they can evaporate from
soil and foliage, move away from the application, and contaminate the environment.)[38]. As
much as 80-90 percent of an applied pesticide can be volatilized within a few days of
application[39]. Despite the fact that only limited research has been done on the topic, studies
consistently find pesticide residues in air. According to the USGS, pesticides have been
detected in the atmosphere in all areas of the USA sampled.[40] Nearly every pesticide
investigated has been detected in rain, air, fog, or snow across the nation at different times of
429
the year.[41] Many pesticides have been detected in air at more than half the sites sampled
nationwide.
3.3.6, Non-Target Organisms:

Pesticides are found as common contaminants in soil, air, water and on non-target
organisms in our urban landscapes. Once there, they can harm plants and animals ranging
from beneficial soil microorganisms and insects, non-target plants, fish, birds, and other
wildlife.[37]
CONCLUSION
Pesticides are often considered a quick, easy, and inexpensive solution for controlling
weeds and insect pests in urban landscapes. However, pesticide use comes at a significant
cost. Pesticides have contaminated almost every part of our environment. Pesticide residues
are found in soil and air, and in surface and ground water across the nation, and urban
pesticide uses contribute to the problem. Pesticide contamination poses significant risks to the
environment and non-target organisms ranging from beneficial soil microorganisms, to
insects, plants, fish, and birds. Contrary to common misconceptions, even herbicides can
cause harm to the environment. In fact, weed killers can be especially problematic because
they are used in relatively large volumes. The best way to reduce pesticide contamination
(and the harm it causes) in our environment is for all of us to do our part to use safer, nonchemical pest control (including weed control) methods.
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State Univ. Cooperative Extension (August). http://hermes.ecn.purdue.edu:8001/cgi/.
[31] ONeil, W. and Raucher, R. (1998). Groundwater public policy leaflet series #4: The
costs of groundwater contamination. Wayzata, MN:Groundwater Policy Education
Project. http:// www.dnr.state.wi.us/org/water/dwg/gw/costofgw.htm (Aug).
[32] US EPA. (2001). Managing small-scale application of pesticides to prevent
contamination of drinking water. Water protection practices bulletin, Washington, DC:
Office of Water (July). EPA 816-F-01-031.
[33] Johnson, J. and Ware, W.G. (1991). Pesticide litigation manual 1992 edition. Clark
Boardman Callaghan Environmental Law Series, New York, NY. 65. US EPA. 1999.
Spray drift of pesticides. Washington, DC: Office of Pesticide Programs (December).
http:// www.epa.gov/pesticides/citizens/spraydrift.htm#1.
[34] US EPA. (1999). Spray drift of pesticides. Washington, DC:Office of Pesticide
Programs (December). http://www.epa.gov/pesticides/citizens/spraydrift.htm#1.
[35] Glotfelty and Schomburg. (1989). Volatilization of pesticides from soil in Reactions
and Movements of organic chemicals in soil. Eds. BL Sawhney and K. Brown.
Madison, WI: Soil Science Society of America Special Pub.
[36] Que, S. et al. (1975). Factors effecting the volatility of DDT, dieldrin, and
dimethylamine salt of (2,4-dichlorophenoxy) acetic acid (2,4-D) from leaf and glass
surfaces. Bull. Environ. Contam. Toxicol. 13(3):284-290.
[37] USGS. (1995). Pesticides in the atmosphere: current understanding of distribution and
major influences. Fact Sheet FS- 152-95. http://water.wr.usgs.gov/pnsp/atmos/
[38] Marx, J et al. (1999). The relationship between soil and water, how soil amendments
and compost can aid in salmon recovery. Soils for Salmon 1-18.
[39] Majewski, M. and P. Capel. (1995). Pesticides in the atmosphere: distribution, trends,
and governing factors. Volume one, Pesticides in the Hydrologic System. Ann Arbor
Press Inc. pg. 118.
[40] Savonen, C. (1997). Soil microorganisms object of new OSU service. Good Fruit
Grower. http://www.goodfruit.com/archive/ 1995/6other.html.
[41] U.S. Geological Survey. (1999). The quality of our nations waters nutrients and
pesticides. Circular 1225. Reston VA: USGS. http://water.usgs.gov/pubs/circ/circ1225/
[42] Bhatia, Mrigesh R., Fox-Rushby, J and Mills, M. (2004) Cost-effectiveness of malaria
control interventions when malaria mortality is low: insecticide-treated nets versus inhouse residual spraying in India. Soil Science and Medicine, Vol. 59, p-525.
[43] Behera, Basudev, Gauri Shankar Singh. (1999) Studies on Weed Management in
Monsoon Season Crop of Tomato. Indian Journal of Weed Science, Vol. 31, No.1+2, p67.
432
Wasim Aktar
[44] Porwal, M.K. (2002) Relative Economics of Weed Management Systems in Winter
Sweet Potato (Ipomoea batatus L.) in Command Area of Southern Rajasthan. Indian
Journal of Weed Science, Vol. 34, No.1+2, P.88.
[45] Kole R.K., Banerjee H. and Bhattacharyya A. (2001) Monitoring of market fish
samples for Endosulfan and Hexachlorocyclohexane residues in and around Calcutta.
Bull. Envir. Contam. Toxicol 67: 554-559.
[46] Kole R.K. and Bagchi M.M. (1995) Pesticide residues in the aquatic environment and
their possible ecological hazards. J. Inland Fish. Soc. India. 27(2): 79-89.
[47] Kole R.K., Banerjee H., Bhattacharyya A., Chowdhury A. and AdityaChaudhury N.
(1999) Photo transformation of some pesticides. J. Indian Chem. Soc. 76:595-600.
[48] Kole R.K., Banerjee H. and Bhattacharyya A. (2002) Monitoring of pesticide residues
in farm gate vegetable samples in west Bengal. Pest. Res. J. 14(1): 77-82.
[49] Sardar D. and Kole R.K. (2005) Metabolism of Chlorpyriphos in relation to its effect on
the availability of some plant nutrients in soil. Chemosphere 61: 1273-1280.

ISSN: 2158-5717
Chapter 16
OZONE DECOMPOSITION BY CATALYSTS AND ITS

APPLICATION IN WATER TREATMENT:
AN OVERVIEW
J. Rivera-Utrilla, M. Snchez-Polo, J. D. Mndez-Daz
Inorganic Chemistry Department, Faculty of Science,
University of Granada, Granada, Spain
ABSTRACT
Ozone has recently received much attention in water treatment technology for its
high oxidation and disinfection potential. The use of ozone brings several benefits but has
a few disadvantages that limit its application in water treatment, including: i) low
solubility and stability in water, ii) low reactivity with some organic compounds and iii)
failure to produce a complete transformation of organic compounds into CO2, generating
degradation by-products that sometimes have higher toxicity than the raw micropollutant.
To improve the effectiveness of ozonation process efficiency, advanced oxidation
processes (AOPs) have recently been developed (O3/H2O2, O3/UV, O3/catalysts). AOPs
are based on ozone decomposition into hydroxyl radicals (HO), which are high powerful
oxidants. This chapter offers an overview of AOPs, focusing on the role of solid catalysts
in enhancing ozone transformation into HO radicals. Catalytic ozonation is a new way to
remove organic micropollutants from drinking water and wastewater. The application of
several homo- and heterogeneous ozonation catalysts is reviewed, describing their
activity and identifying the parameters that influence the effectiveness of catalytic
systems. Although catalytic ozonation has largely been limited to laboratory applications,
the good results obtained have led to investigations now under way by researchers
worldwide. It is therefore timely to provide a summary of achievements to date in the use
of solid materials to enhance ozone transformation into HO radicals.
434
1. INTRODUCTION
Demographic development and an exponential increase in industrial activity have largely
been responsible for the large rise in the demand for water for domestic, public, and industrial
use, and for the high volume of effluents discharged into waters. Legislation has been
introduced to reduce permissible contamination levels. However, conventional treatment
systems cannot completely remove a large amount of organic and inorganic contaminants
present in waters, because most cannot be metabolized by microorganisms as carbon source
and can even inhibit the activity of these microorganisms, leading to their bioaccumulation in
the food chain. Hence, there is an increasing demand for more effective treatments to reduce
the potential environmental impact of effluents and to comply with increasingly strict
legislations. Successful water treatment requires the use of more sophisticated methods,
including:
Biological systems for nitrogen removal: Ammonium can be transformed into nitrate
by using nitrifier microorganisms in aerobic medium. Nitrate can be removed in a
subsequent stage under anaerobic conditions, when it is transformed by denitrifying
bacteria into molecular nitrogen.
Advanced oxidation processes for removal of toxic organic compounds,
chromophore compounds, and other non-biodegradable organic compounds. These
are based on the use of highly oxidizing agents, e.g., ozone or hydrogen peroxide. A
greater effectiveness is observed when these oxidizing agents are used in the
presence of UV radiation.
Ionic exchange for ion removal. This is highly effective for removing cations and
anions from the aqueous phase but transfers the problem to the solid phase by
concentrating the pollutant in the adsorbent medium.
Adsorption on activated carbon for removal of metals, organic compounds, etc. This
has the same drawback as described above for ionic exchange.
Chemical precipitation for phosphorus removal: Chemical agents (Al2(SO4)3,
Fe2(SO4)3 or FeCl3) are used to precipitate phosphorus.
Distillation for removal of volatile organic compounds. This is only appropriate
when there are high concentrations of the contaminant and its recovery brings
economic benefits.
Liquid-liquid extraction. This is also only useful under the above conditions.
Chemical oxidation processes currently play a very important role in water treatment.
Table 1 lists the most common chemical oxidants used in water treatments and their
corresponding reduction potentials [1]. Oxidants can be used to remove both inorganic
pollutants and toxic organic compounds (pesticides, hydrocarbons, toxins, etc.) [2,3]addition,
oxidants are widely used to degrade compounds responsible for odor, color or taste [4,5,6]
Ozone, due to its high oxidation and disinfection potential, has recently received much
attention in water treatment technology. Despite several advantages of using ozone, it has a
few disadvantages that limit its application in water treatment, including: i) low solubility and
stability in water, ii) low reactivity with some organic compounds, and iii) failure to produce
a complete transformation of organic compounds into CO2, generating degradation by-
Ozone Decomposition by Catalysts and its Application in Water Treatment:
435
products that sometimes have a higher toxicity than the raw micropollutant. To improve the
effectiveness of ozonation, advanced oxidation processes (AOPs) have recently been
developed (O3/H2O2, O3/UV, O3/catalysts). AOPs are based on the decomposition of ozone
into hydroxyl radicals (HO), a very powerful oxidant.
This chapter offers an overview of the role of different solid catalysts in enhancing ozone
transformation into HO radicals. Catalytic ozonation is a new way to remove organic
micropollutants from drinking water and wastewater. The application of several homo- and
heterogeneous ozonation catalysts is reviewed, describing their activity and identifying the
parameters that influence the effectiveness of catalytic systems. Although catalytic ozonation
has largely been limited to laboratory applications, the good results obtained have led to
investigations now under way by researchers worldwide. It is therefore timely to provide a
summary of achievements to date in the use of solid materials to enhance ozone
transformation into HO radicals.
Table 1. Reduction potentials of different oxidants used in water treatments.
Oxidant
Ozone
Reduction semireaction
O3 (aq) + 2H+ + 2e- O2 (aq) + H2O
E0 (V)
2.08
Permanganate
2MnO4- + 8H+ + 6e- 2MnO2 (s) + 4H2O
1.68
Chlorine dioxide
ClO2 + e ClO2
Hypochlorous acid
HOCl + H+ + 2e- Cl- + H2O

-
0.95
1.48
Hypochlorite ion
Dichloramine
ClO + 2H + 2e Cl + H2O
NHCl2 + 3 H+ + 4e- 2Cl- + NH4+
1.64
1.34
Oxygen
Hydroxyl radical
O2 (aq) + 4H+ + 4e- 2H2O

HO + H+ + e- H2O
1.23
2.85
Hydrogen peroxide
H2O2 + 2H+ + 2e- 2H2O
1.78
2. OZONE
Ozone, O3, discovered by Schnbein in 1840 [7], is an allotrope of oxygen consisting of
three atoms. It is a diamagnetic compound that is an unstable gas at room temperature with a
characteristic sharp odor. Experimental results show a bond angle of 116.8 0.5 and an
interatomic distance of 127.8 pm between central oxygen and each terminal [8]. Fig 1 depicts
the two resonant forms of the ozone molecule according to the valence bond theory:
+
O
:O :
O+
:O :
Figure 1. Chemical structure of ozone.
:O :
O
: :
436
Due to its configuration, ozone is a highly oxidant compound (E = +2.08 V), and its
natural tendency is to transfer an oxygen atom and release O2. Applications of ozone
applications in water treatment can be grouped into three categories: i) as disinfectant or
biocide, ii) as oxidant for the removal of organic pollutants and, iii) as pre- or post-treatment
in another procedure (coagulation, flocculation, sedimentation, biological oxidation,
adsorption on activated carbon, etc.).
Ozone began to be used as bactericide agent to treat drinking waters in Nice (France) at
the beginning of the 20th century. As a result of the large amount of resources invested in the
study of ozone, other advantages of its use in treatment plants [9] are now widely known,
including: i) removal of compounds that produce odor, taste or color in water, ii) oxidation of
inorganic chemical compounds, e.g., iron and manganese, iii) removal of algae and other
aquatic microorganisms, iv) oxidation of organic micropollutants, v) absence of the increase
in the presence of organochlorinated compounds found when chlorine is used for the
treatment, and vi) enhanced performance of adsorption and coagulation processes.
Nevertheless, its large-scale application to treat industrial liquid waste did not become
widespread due to the high economic costs involved and the chemical complexity of
industrial effluents. However, ozone has now become an attractive option for the treatment of
these effluents because conventional systems are inadequate to reduce the toxic organic
compounds they contain to levels required by new environmental legislation.
O3 + OH-
O2- + HO2
Start
2HO2
O3 + H2O
HO2
H+ + O2-
O2- + O3
O2 + O3-
(2)
(3)
(4)
HO + O2
(5)
O3-
O- + O2
(6)
HO
O- + H+
(7)
O3- + H+
Propagation
HO + O3
End
(1)
HO2 + O2
(8)
(9)
HO2 + HO2
H2O2 + O2
HO2 + O2-
HO2- + O2
(10)
2HO- + 2O2
(11)
O3- + O2- + H2O
Figure 2. Mechanism of ozone decomposition in aqueous medium.
Over the past two decades, there has been a notable increase in research into the reaction
between ozone and numerous organic and inorganic compounds, especially aromatic
compounds [10,11,12,13,14]. Because it is highly reactive, ozone can interact with a large
number of organic and inorganic substances by direct oxidation/reduction reaction,
cycloaddition-substitution, or nucleophilic addition [15,16,17]. The direct reaction between
437
ozone and a given compound is not the only pathway by which ozone can act to degrade
pollutants, since ozone is very unstable in aqueous solution and spontaneously decomposes
by a complex chain mechanism (Fig 2) in which different radical species participate [18]. The
radicals generated are of great interest for water treatment because some of them, e.g., the
hydroxyl radical HO, are even more reactive than ozone and play an essential role in
removing pollutants in solution [19]. These systems will be described in greater depth in next
section, which is devoted to AOPs.
Despite its high efficacy in some systems, ozone has inadequate capacity to degrade
surfactants and tensioactive substances, among other pollutants. Narkis et al. [20] observed
that ozone treatment favored the biodegradation of non-ionic surfactants but was unable to
remove them. Other authors also reported the lack of reactivity of saturated cationic
surfactants [21]. Regarding anionic surfactants, although some researchers achieved a high
degradation of alkylbenzene-sulphonates with ozone [22], the reaction rate constant values
were subsequently determined to be low [23], suggesting that an indirect mechanism was
largely responsible for the degradation.
3. ADVANCED OXIDATION PROCESSES BASED ON OZONE

Polluted waters can generally be effectively treated by biological, adsorbent or
conventional chemical approaches (chlorination, ozonation or oxidation with permanganate).
However, as stated earlier, these methods are sometimes inadequate to degrade pollutants to
levels required by law or necessary for subsequent utilization of the effluent. AOPs have
proven highly effective in the oxidation of numerous organic and inorganic compounds and
are based on the generation of free radicals, notably hydroxyl radical HO. These free radicals
are highly reactive species that can successfully attack most organic and inorganic
compounds with very high reaction rate constants (106-109 M-1s-1). The numerous systems
that can be produced by these radicals (Table 2) account for the high versatility of AOPs.
Advanced oxidation processes based on the use of ozone are briefly described below,
highlighting catalytic ozonation (also see section 4).
Table 2. Water treatment technologies based on advanced oxidation processes.
Non-photochemical processes
Oxidation in sub/supercritical water
Fentons reagent (Fe2+/H2O2)
Photochemical processes
Photolysis of water with vacuum UV
(VUV)
UV/hydrogen peroxide
Electrochemical oxidation
UV/ozone
Radiolysis
Non-thermal plasma
Ultrasound
Ozonation in alkaline medium (O3/OH-)
Ozonation in the presence of hydrogen
peroxide (O3/H2O2)
Catalytic ozonation
Photo-Fenton
Heterogeneous photocatalysis
438
3.1. Ozonation in Alkaline Medium

Based on studies by Hoign et al.[10,11,12], Aieta et al. [24] published an illustrative
diagram in 1988 describing ozone in aqueous solution. Figure 3 summarizes the two reaction
pathways of molecular ozone: i) direct reaction of the substrate with molecular ozone, which
is selective but slow or null with some species, and ii) decomposition and generation of HO
hydroxyl radicals, which attack rapidly but not selectively.
Direct oxidation of the substrate
O3
Products
Slow / Selective
OHRadical formation
HO
Radical oxidation
Fast / Non-selective
Products
Figure 3. Reaction pathways of ozone.
Moreover, hydroxyl radicals react rapidly with molecular ozone, contributing towards
autocatalytic decomposition of the ozone. Several researchers have studied the mechanism
and reaction kinetics involved in ozone decomposition in aqueous phase
[18,25,26,27,28,29,30,31,32,33]. Ozone stability largely depends on the aqueous matrix,
especially the pH, type of organic matter present, and alkalinity [34]. The water pH is
especially important, because hydroxyl ions considerably increase the ozone decomposition
rate [18]. Thus, according to equations 12 and 13, ozone decomposition spontaneously
accelerates with an increase in the solution pH, leading to an AOP. However, it must be taken
into account that a high pH increase can have a negative effect on the degradation, depending
on the composition of the water, because of the inhibiting action of HO radical scavengers,
e.g., bicarbonate or carbonate ions [35].
O3 + OH- HO2- + O2 k = 70 M-1s-1 (13)
O3 + HO2- HO + O2- + O2 k = 2.8 106 M-1 s-1
(12)
3.2. Ozonation in the Presence of Hydrogen Peroxide

The addition of hydrogen peroxide favors the ozonation of organic compounds in the
medium. The combination of ozone and hydrogen peroxide is largely used to oxidize
pollutants that require high ozone consumption. Hydrogen peroxide is a weak acid (pKa =
11.6), a powerful oxidant (See Table 1), and an unstable compound that easily dismutes
(equations 14-16).
H2O2 HO2- + H+ (14)
H2O2 + 2e- +2H+ 2 H2O
(15)

H2O2 + HO2- H2O +O2 +HO-
439
(16)
The mechanism by which H2O2 favors free radical generation was described by Forni et
al. [25], who demonstrated that the conjugated base of hydrogen peroxide initiates ozone
decomposition in aqueous phase via an electronic transference reaction.
HO2- + O3 HO2 + O317)
Taking advantage of the capacity of H2O2 to initiate ozone decomposition in aqueous
phase (Figure 4), numerous researchers have used this process for a faster and more effective
oxidation of organic matter [36,37,38,39,40,41,42,43. The presence of H2O2 in the system
favors oxidation, although the possibility that added H2O2 is consumed in reactions with other
contaminants has hampered application of this method to treat industrial effluents. Fernndez
et al. [44] compared the efficacy of the O3/H2O2 system and photolysis to remove linear
alkylbenzene-sulphonates (LAS), observing the complete degradation of the mixture of
surfactants after 20 min of ozonation.
HO2- + H+
H2O2
O3
O3O2- *
HO2*
HR+
HO3
RHO2*
Degradacin
HO*
HR*
O2
HRH
Figure 4. Mechanism of oxidation of an organic compound (HRH) by means of O3/H2O2.
3.3 Ozonation in the Presence of UV Radiation

Ozonation coupled with UV radiation is one of the most effective chemical oxidation
techniques to treat polluted waters. This process is capable of oxidizing organic substances at
room temperature and generates products that are innocuous to the environment. As in the
O3/H2O2 system, the UV radiation of O3 generates hydroxyl radicals in solution [45]. The
reactions involved in this process are:
O3 + h (<310 nm) O2 + O (18)
O + H2O 2HO
(19)
440
Prengle et al. [46] of Houston Research Inc. were the first researchers to describe the
application of an O3/UV-based system for water treatment. Glaze et al. [47,48] subsequently
analyzed the effectiveness of the O3/UV system to remove microcontaminants and
determined the reactions involved in the mechanisms of this process [49]. They demonstrated
that compounds not degraded by UV light behave very similarly in response to O3/UV and
O3/H2O2 systems [50].
The O3/UV process has been mainly applied in the oxidation of aromatic derivatives
[51,52] but it has also shown high efficacy to remove surfactants [53,54,55], pesticides [56],
and other organic compounds [57]. Taking advantage of the high photocatalytic capacity of
TiO2, a new treatment process has been developed based on the simultaneous use of
O3/UV/TiO2. Table 3 displays information on the type of pollutant, experimental conditions
and the main conclusions of publications on the application of the O3/UV system to remove
organic pollutants from waters; in the majority of these studies, TiO2 was used as catalyst.
Table 3. Application of systems based on O3, UV radiation, and catalysts for removing
pollutants from waters.
System
Catalyst
O3, H2O2, UV,

3/UV, O3/H2O2,
UV/H2O2,
O3/UV/H2O2
Pollutants
Experimental conditions Observations
Semi-continuous; pH 8;
17C; flow 1-1.5 L/min;
Phenolic compounds
low-pressure lamps
(254nm)
Ref.
Treatments without ozone are

less effective in pollutant
58
removal
UV, V/TiO2,
V/Fenton
Different
TiO2 (Degussa P25)
wastewaters
Better performance of
Semi-continuous; 25C;
catalyzed experiments; Fenton
medium-pressure Hg lamp
59
process more cost-effective
400 W; 2 g/L of catalyst;
than UV
O2/UV/cat
Ag/ZnO
Semi-continuous; pH 3-7; Total removal of toxicity of

medium-pressure Hg lamp certain effluents, but little
125 W; flow 10 mL/min reduction in TOC
O2/UV/cat
TiO2 (Degussa P25),

Colorants
ZnO
20C; medium-pressure Hg
lamp 125 W; gas flow10
mL/min; 0.8 g/L catalyst
UV/TiO2
TiO2
Formic acid
Discontinuous with
Better performance in
ecirculation; pH 3.8; 20C; combined use; determination 62
Lamp 6W
of reaction constants
UV/H2O2,
2-methyl
benzimidazole
carbamate
Discontinuous; lowpressure Hg lamp
O3/UV
Oxalic acid
Semi-continuous; pH 7; 1.5 Kinetic model proposed; study

L/min; UV lamps 3-910-6 of effect of operational
64
E/L-s
variables
O2/UV/TiO2
O3, H2O2, UV,

O3/UV,
O3/H2O2,
UV/H2O2,
Textile effluent
TiO2 (Degussa P25) Tetracyclines
Diazinon
Semi-continuous; flow
0.2L/min; 3 lamps of
different power; 0.5-1 g/L
catalyst
60
High mineralization; better

catalytic activity of ZnO;
61
synergic effect between TiO2
and ZnO
Determination of radical
reaction rate constants
(hydroxyl and carbonate);
identification of degradation
products
63
Degradation is not observed in

the absence of TiO2;
65
significant mineralization with
use of catalyst
Kinetic study and comparison

Discontinuous; pH 2-9; 20- between systems;
40C; low-pressure Hg
determination of quantum
66
lamp 15W
performances and reaction
constants
441
Table 3. (Continued)
System
UV, O3/UV,
O3/TiO2,
O3/UV/TiO2
Catalyst
Pollutants
TiO2
Acetic acid,
monochloroacetic
acid, phenol,
dimethyl-2,2,2trichlorine-1hydroxyethylphosphate
Semi-continuous; lowpressure Hg lamp 6 W
O3, UV/TiO2,
TiO2
O3/UV/TiO2
Ref.
Proposal of the reaction

mechanism; detection of
formic, acetic, glyoxylic, and 67
glycolic acids as reaction
intermediates
Study of kinetics and

Semi-continuous; pH 3-11; operational variables; O3/UV
2,4-dichlorine
18-22C; flow 1-220 mg improves treatment efficiency; 68
phenoxyacetic acid
O3/L; lamp 20W
identification of reaction
intermediates
O3/UV/TiO2
TiO2
25C; flow 510-4 mol
O3/min; medium-pressure
Hg lamp 125 W; 2 g/L of
catalyst
Proposal of reaction
mechanism; simultaneous use
of O3/UV/TiO2 is more
69
effective versus O3 and
UV/TiO2 in parallel
O3, O3/UV,
UV/TiO2,
O3/UV/TiO2
Alkylamines,
Semi-continuous; pH 6.5;
alkanolamines,
20C; flow 35 L/h; Xe
TiO2 (Degussa P25) nitrogenated
Lamp 450 W; 0-3 g/L of
heterocyclic and
catalyst
aromatic compounds
mechanism; identification of
intermediate reaction
70
products; study of effect of
structure and concentrations
on reaction rate
UV/TiO2,
O3/UV,
O3/UV/TiO2
Pyridine;
TiO2 (Degussa P25) monochloroacetic
acid
20C; flow 20 L/h; UV
Lamp; 0.5g/L catalyst
mechanism; much higher
71
performance of system based
on O3/UV/TiO2
, UV/TiO2,
O3/UV/TiO2
TiO2 (Degussa P25) Textile effluent
flow 6 L/h; high-pressure
Hg lamp 125 W; 0.1 g/L
catalyst
Determination of toxicity and

TOC and color of effluent as a 72
function of treatment time
O3/UV/TiO2
TiO2
Aniline
Higher efficiency of the
4flow 1 L/min; low-pressure
combined system; detection of 73
chlorobenzaldehyde Hg lamp 10 W; 1 mg/mL
degradation by-products
of catalyst
UV/TiO2,
2-dimethyl-pyrazine,
Semi-continuous; highO3/UV/TiO2, TiO2(Degussa P25) monochloroacetic
pressure Hg lamp 125W
UV/Fenton
acid
O3/UV/TiO2
TiO2 (Degussa P25

Cyanides
y BDH)
Proposal of disjunctive
mechanism: UV promotes
electrons in TiO2 or radical
reaction generation
74
Kinetic study and mechanism;

formation of carbonate,
20C; medium-pressure Hg
75
cyanate, and nitrate; strong
lamp 400 W
O3/TiO2 interaction
O3, O3/H2O2,
O3/H2O2/UV
O3/UV/TiO2, TiO2
O3/H2O2/UV/T
iO2
Wastewater
O3/H2O2/UV/TiO2 is the only

one able to appreciably reduce
flow 500 L/h; Hg Lamp
TOC; after treatment,
76
(200-280 nm); 2 g/L of
anaerobic digestion improves
catalyst
CH4 production
UV/TiO2, O3,
TiO2
O3/UV/TiO2
Formic acid
Semi-continuous; pH 2-12;
Diffusion effects worsen
10-50C; gas flow 510-3
performance
3
m /min; 6W Lamp
O3/UV/TiO2
Cyanides
Mechanisms is proposed and

Continuous; pH 10; gas
products identified; effluent is
flow 4 L/min; low-pressure
78
reutilized to obtain deionized
lamp 40 W
water
TiO2
77
442
System
O3/UV/TiO2
Catalyst
TiO2 (Degussa P25 ,

Phenol
SG)
O2, O3, O3/UV,

UV/TiO2,
Carbon-TiO2
O3/UV/TiO
O3/UV/TiO2
Pollutants
Catechol
TiO2 (Degussa P25) Humic acid
O3, O3/UV,
UV/TiO2,
Acetic acid, formic
TiO2 (Degussa P25)
O2/UV/TiO2,
acid, propionic acid
O3/UV/TiO2
O3/Foto
fenton,
O3/UV/TiO2
Ref.
Semi-ontinuous; 25C;
Detectionof intermediates;
flow 2.1 mg O3/L; Hg
improvement of
Lamp (220-380nm); 15 g/L mineralization with use of
of catalyst
combined system
79
Semi-continuous; lowpressure lamp 15 W
Determination of rate constant

and reaction order; higher
80
efficacy of O3/UV/TiO2
system to remove TOC
Kinetic study; UV
Semi-continuous; flow 4.8 compensates for low capacity
81,82,
mg O3/min; 125 W Lamp; of O3 to remove organic
83
0.25-1 g/L of catalyst
matter; adsorption study of
pollutant on catalyst
25C; flow 0.9 L/min;
medium-pressure Hg lamp
6 W; 1 g/L catalyst
Identifies intermediates; better

performance of combined
84
process versus ozone and
photocatalysis separately
Semi-continuous; pH 3, 7;
Alachlor, atrazine,
Photo-fenton system (order 1)
25C; flow 1.6 g O3/h;
TiO2 (Degussa P25) diuron, isoproturon,
shows better results than
85
UVA Lamp 6W; 0.25 g/L
pentachlorophenol
O3/UV/TiO2 (order 0)
of catalyst
O3/UV,
O3/TiO2, O3/V- TiO2, V-O/TiO2
O/TiO2
Higher removal efficacy of

Semi-continuous; pH 4, 7,
O /V-O/TiO2 system;
Sulphosalicylic acid 9; 20C; Lamp (254 nm) 14 3
86
maintains activity even in the
W; 1.6 g/L of catalyst
presence of carbonates
O3, O3/TiO2,
Phenol, pO3/UV/TiO2,
TiO2 (Degussa P25) chlorophenol, pO3/UV, UV,
nitrophenol
UV/TiO2
O3, UV/TiO2,
TiO2
O3/UV/TiO2
flow 51 L/h; high-pressure
Hg lamp 700 W; 1.5 g/L
catalyst
Semi-continuous; UV
Water with fungicide
Lamp 6W
Kinetic study and

identification of intermediates;
87
determination of reaction
constants
Synergic effect; O3/UV/TiO2
system removes organic
88
compounds and inhibits
germination of fungi
4. CATALYTIC OZONATION
Research into novel alternatives to conventional advanced oxidation processes has
significantly increased over the past few decades. These new treatments are based on the
addition of reagents to the system, generally heavy metals that increase the effectiveness of
ozone as oxidizing agent. These substances, called catalysts, participate in the reaction
mechanism but are not consumed in the process. Depending on the state of the species that
acts as catalyst, two types can be distinguished: homogeneous catalysis (catalyst dissolved in
aqueous phase) and heterogeneous catalysis (solid or supported catalyst). Each of these
catalytic processes is studied in depth below.
443
4.1. Homogeneous Catalytic Ozonation

One of the first investigations in this field was by Hewes and Davison [89], who observed
that addition of certain salts during ozonation of phenolic compounds increased the
mineralization of organic matter. Subsequently, other researchers [90,91] found that these
catalysts could improve the decoloration of effluents from textile industries and accelerate the
oxidation of compounds at acid pH values.
Unlike for other AOPs, a general reaction mechanism cannot be established for
homogeneous catalytic ozonation because the system can behave very differently according
to the compounds involved. Whereas some researchers indicated that the presence of
transition metals does not promote the generation of free radicals [92], explaining that the
higher oxidation of the substrate is due to the formation of a complex that is highly reactive to
ozone, others explained the enhancement of substrate degradation by the generation of
hydroxyl radicals [93]. A review of the literature shows that metals (either in cation or anion
form) that are susceptible to oxidization in the ozonation process can act as
initiators/promoters of the transformation of ozone into HO radicals. Despite the
improvement in performance, the need to add metal species to the system increases the
difficulties in applying homogeneous catalytic ozonation in liquid waste treatments, because
it introduces highly contaminant species into the system. Table 4 summarizes data from the
main published studies on the use of homogeneous catalytic ozonation to remove organic
microcontaminants from waters.
Table 4. Most relevant data from publications on homogeneous catalyzed ozonation.
System
Catalyst
ContaminantsExperimental Conditions
Observations
Ref.
Ozone/H2O2
Fe2+, Co2+, Ni2+,

Cu2+ Sulfates
Discontinuous; acid pH
Study of the catalytic effect of

several cations; reaction
mechanism is proposed
94
Ozone
Co2+
Discontinuous; acid pH; 0C
Study of mechanism and kinetics;

95
acid pH inhibits the process
Ozone
Co2+
Acetic acid
Discontinuous; acid pH; 0C
Study of mechanism and kinetics 96
Ozone
Co2+, Ti2+, Mn2+
Secondary
effluent
Discontinuous and
Improvement of DQO removal
recirculation; pH 5-10, 30-60C
Discontinuous; acid pH; [O3]

310-4M
2+
Determination of reaction
stoichiometry; mechanism
proposal; influence of several
radicals
89
Ozone
Fe
97
Ozone
Cu2+, Zn2+, Cr2O3
Azoic
colorants
Semi-continuous; flow 100 L/h Improvement in discoloration rate 90
Ozone
Fe2+, Mn2+
Discontinuous, pH 5-8, 25C,

1mg/L O3
Determination of reaction
stoichiometry and rate constant
98
Ozone
NOM
Mn2+
Discontinuous, 0.5-2 mg/L
Significant removal of Mn2+;

influence of O3 dose
99
Ozone
Mn2+
Oxalic Acid
Discontinuous and semiKinetic study and determination of

continuous (flow 36 L/h); acid rate constant; proposal of reaction 91
pH; 20C
mechanism
444
System
Catalyst
ContaminantsExperimental Conditions
Observations
Ozone
Fe2+
Kinetic study and determination of

Reactor with flow stop; pH 0-2; rate constant at the beginning of
100
25C; 110-4-610-5 M O3
the process; mechanism is
proposed
Ozone
NOM
Mn2+, DQO
Continuous; diffusion plate
Efficacy is tested in different

waters
Ref.
101
-5
Ozone
Mn2+
Discontinuous; pH 2-8; 4-510

Reaction mechanism is proposed 102
M O3
Ozone
Mn2+
Pyrazine,
oxalic acid
Semi-continuous; pH 4; flow 36
Oxalic- Mn complex as catalyst
L/h
2+
103
3+
Ozone
Ag , Fe , Fe ,
Co2+, Cu2+, Mn2+, TOC
Zn2+, Cd2+
Discontinuous; pH 7; flow 1.5g Intermediates are identified; TOC

104
O3/h
reduction improves with catalyst
Ozone
Mn2+
Atrazine
Semi-continuous and
recirculation; pH; 20C
Considerably increases atrazine

removal with Mn2+
105
Ozone
Mn2+
TOC
Semi-continuous; pH 8; flow
1.6g/ O3/h
TOC removal increases with the

catalyst; intermediates are
identified
106
Ozone
Mn2+
Pyruvic acid
Semi-continuous; pH 2-4; 25C; Only removes pyruvic acid if

flow 36 L/h
catalyzed; negative effect of pH
107
Ozone
Mn2+
Atrazine,
DQO
Semi-continuous and
recirculation; pH 7; 20C; 1
mg/L Mn2+
108
Ozone
Fe2+, Fe3+, Mn2+
Chlorobenzene
Semi-continuous; pH 7
s
Ozone
Mn2+
Glyoxylic acid
Semi-continuous; pH 2-5.4;
flow 36 L/h
Ozone
Mn2+
Pyruvic acid
Semi-continuous; pH 1-3; flow Kinetic study; similar behavior of

111
36 L/h
Mn2+ and Mn4+
Ozone
Mn2+
p-Nitrotoluene
Determination of the kinetic
Semi-continuous; acid pH; flow
in acetic
constant and reaction
-2
10 L/s
anhydride
intermediates
112
Ozone/H2O2
Fe2+
Acids and
colorants
113
2+
Humic acids improve atrazine

removal; reaction mechanism is
proposed
Presence of the catalyst improves

109
contaminant removal
Reaction intermediates are
110
identified; mechanism is proposed
Semi-continuous; 4-10.5; flow

Kinetic study
29mg O3/min
2+
Ozone
Pb , Cu , Zn ,
Fe2+, Mn2+
Nitrates
OrthoSemi-continuous; pH 3; flow
chlorophenol mg O3/min
Ozone
Co2+
Oxalic acid
Semi-continuous; acid pH;

20C; flow 24 L/h
Ozone
Co2+
Discontinuous; pH 1.6-8.4; 10- Influence of pH, O3 dose and

5
-210-4 M
catalyst; mechanism is proposed
116
Ozone
Fe3+
Oxalic acid
Improvement in oxalic acid

Semi-continuous; pH 2.5; 20C,
removal; reaction mechanism is
flow 24 L/h
proposed
117
Ozone
Mn2+, Cu2+
Phenol
Semi-continuous; pH 3 and 10
*Publications on ozone decomposition in the absence of pollutants
Mechanism is proposed; order of

metal catalyst activity is
114
established
Kinetic study and reaction
stoichiometry determination
Phenol and TOC removal

improved with catalyst
115
118
445
4.2. Heterogeneous Catalytic Ozonation

Heterogeneous catalysis takes place in systems with two or more phases, most frequently
with catalyst in solid phase and reagents in gas or liquid phase. Most publications in this field
have centered on the study of reactions in gas phase, although increasing numbers of
researchers have studied reactions in liquid phase in recent years. Since Chen et al. [119]
published results on heterogeneous catalytic ozonation of some compounds using Fe2O3 as
catalyst, numerous catalysts have been tested, including metal oxides [120,121], supported
metal oxides [122,123], supported metals [124], mesoporous materials [125], and activated
carbons [126]. In general, the catalytic activity of these catalysts is mainly based on the
generation of radical species such as hydroxyl; therefore, the efficacy of heterogeneous
catalysis in ozone decomposition will be largely influenced by solution characteristics (pH,
temperature, ionic strength, etc.) and by chemical and textural properties of the catalyst.
Among the numerous materials that have been used as heterogeneous catalysts, there are four
major types:
[1]
[2]
[3]
[4]
Supported metals.
Metal oxides.
Supported metal oxides.
Activated carbons.
4.2.1. Supported Metals

After the first results published by Heinig et al. [127], the use of catalysts based on metals
deposited on different supports began to be studied in the late 1990s. At present there is
considerable uncertainty about the mechanisms involved and the influence of different
operational variables on this process. Results obtained by Karpel Vel Leitner et al. [128]
indicated that adsorption of dissolved humic matter on the surface of the catalyst reduces the
catalytic activity of metal catalysts supported on alumina, whereas catalysts supported on
TiO2, where there is little such adsorption, show a better performance. Although the value of
solution pH and the adsorption process are important, they do not always determine the
activity of the catalyst in the ozonation of organic contaminants. Thus, the catalyst synthesis
method and consequently its structure are critical for its capacity to transform ozone into HO
radicals[129,130]. Lin et al.[131] recently studied the catalytic activity of more than 20
catalysts (alumina, silica, activated carbons impregnated with metals, etc.) in the ozonation of
formic acid, concluding that the transformation of ozone into HO radicals is mainly
influenced by its diffusion rate on the catalyst surface. Table 5 lists the catalysts tested in
some of the most important research studies in this field, including data on the pollutants
studied, the experimental conditions, and some observations.
446
Table 5. Ozonation catalyzed by supported metals.

System
Catalyst
Contaminants
Experimental
Conditions
Observations
O3, O2
Ag/Al2O3
Microorganisms
Fixed bed; 500 g of

catalyst
Bacteria and virus removal; strong

catalytic interaction with dissolved 127
O2
O3
Cu supported on
Organic compounds
alumina, anatase, and
(TOC), succinic acid
attapulgite, Ru/CeO2
Discontinuous; and
semi-continuous
(flow 1.25 g O3/h);
0.2-1.2 g/L catalyst
Demonstrates the catalytic activity

of supported metals; proposes
132
mechanism by adsorption and/or
hydroxyl radical attack
O3
Continuous (6.5 mg TOC removal increases with certain

Metals supported on Salicylic, peptide acid,
O3/L) and semicontaminants; low adsorption on
128
Al2O3, TiO2 and clay humic substances
continuous; acid pH catalyst surface
O3
Ru/CeO2
Succinic acid
Semi-continuous; pH
High TOC mineralization in
3.4; flow 2.7510-4
catalysts prepared by impregnation 129
mol O3/h; 0.8 g/L of
and reduction
catalyst
O3
Ru/CeO2
Succinic acid
Semi-continuous; pH Effect of the catalyst structure on its

3.4; flow 1.25 g O3/h; efficacy; organic molecules produce 130
0.8-3.2 g/L of catalyst metal sintering
O3
Ru/CeO2-TiO2
Chloroacetic acid,
chlorosuccinic acid
Semi-continuous; pH
2.6-3.6; flow 15.6
High contaminant removal rates
L/h; 0.8 g/L of
catalyst
O3
Cu/Al2O3
Alachlor
Semi-continuous;
Use of the catalyst increases TOC,
134
flow 0.5 mg O3/min chloride and nitrate formation
O3
Mg and Al on Fe, Co,

Phenol, oxalic acid
Ni and Cu oxides
Ref.
133
Semi-continuous; pH
Identifies oxalic acid as product of
2; 20C; flow 200
phenol ozonation; oxalic acid
135
mL/min; 1 g/L of
appears to cause losses of Cu and Ni
catalyst
4.2.2. Metal Oxides

Besides its chemical stability, the most important parameter determining the ability to use
a metal oxide as catalyst of ozone decomposition is its acidity or basicity. Thus, active centers
of the surface of a catalyst are located in groups that act as Brnsted or Lewis acids, which is
largely conditioned by the medium pH. Adsorption of organic compounds on the catalyst
surface plays an essential role in the catalytic activity of the oxide. This is because: i) it is the
first step in the catalytic mechanism of ozone decomposition, and ii) the affinity of the metal
oxide for certain inorganic species in solution (carbonates, phosphates, etc) can block active
sites and reduce the efficacy of the catalyst. Researchers have used numerous metal oxides to
study ozone decomposition and/or the ozonation of organic contaminants in solution. Thus,
different groups successfully degraded oxalic acid by using catalysts such as TiO2, MnO2 and
Al2O3[120,136,137] Several authors have studied the optimization of experimental conditions
and have investigated reaction mechanisms (Table 6), proposing catalysts of different origin
to achieve or improve the degradation of organic contaminants and mineralize dissolved
organic matter in waters. Table 6 shows the most recent investigations into the simultaneous
use of ozone and metal oxides to remove pollutants from waters, including the main study
conclusions.
447
Table 6. Most recent research on catalyzed ozonation with metal oxides.

System
Catalyst
Contaminants
Experimental
conditions
Observations
Ozone
Fe2O3
Phenol,
TOC
Continuous; pH 4.3-6.3
High efficacy to remove phenol and

119
DQO; mechanisms is proposed
Ozone
CuO, Fe2O3, NiO,

Aniline, DQO
Cr2O3, Co2O3
Good catalytic
12 L/h; 55 g/L of
leaching
catalyst
Ozone
Mo, Zn, Sn, V, Ni,

Co, Cu, Pb, Mn,
*
Fe, Ce, Ag and Bi
oxides
Continuous; 25C; flowDeactivation of certain oxides due to

0.15 L/min; 0.5 g ofpossible adsorption of H2O on active139
catalyst
sites
Ozone
-FeOOH
Chlorobenzene,
bicarbonates
Increased chlorobenzene removal in
0.2 L/min; 0.05-0.2 g/L
140
the presence of catalyst
of catalyst
Ozone
MnO2
Oxalic acid
Semi-continuous; pH 3Improved performance with lower pH;

7, flow 36 L/h; 0.25 g/L
120
reaction on surface
of catalyst
Ozone
-Al2O3
2-chlorophenol
Semi-continuous; pH 3Kinetic study; toxicity and TOC

9; flow 18 mg O3/h; 2
141
behaviors
g/L of catalyst
Ozone
TiO2
Oxalic acid
Semi-continuous;
pH
Total removal of TOC; zero-order
2.5, 5; 30C; 5 g/L
136
reaction mechanism is proposed
catalyst
Ozone
Tierra, Fe(O), *
FeOOH
Continuous;
catalyst
Ozone
FeOOH
Semi-continuous, pH 7,
Proposal of radical
flow 0.5 L/min; 10 g/L
improved TOC removal
catalyst
Ozone
Mn2+, -MnO2, -Sulfosalicylic andSemi-continuous, pH 1-Study of effects of pH and state of Mn

144
MnO2, MnSO4
propionic acid
8.5
on catalytic activity
Ozone
Mixed metal
oxides, metal
oxides
p-chlorobenzoic
acid
Discontinuous (pH 7 and

deionized water); semi- No catalysis in deionized water; Co
continuous (natural
and Cu-Zn-Al oxides catalyze in145
water); 43 mg/L of
natural water
catalyst
Ozone
CuO-Al2O3
Phenol,
chlorophenol,
nitrophenol
Reactor tubular; pH 3-Intermediates are identified; the

11; 21C; flow 20 L/h;catalyst reduces ozone consumption146
0.25-2 g/L catalyst
and increases rate
Ozone
Perfluorinated
alumina
Methyl tert-butyl
Semi-continuous
with
ether,
isopropyl
Alumina does not catalyze; kinetic
and without recirculaether, ethyl tertconstant is determined and reaction by-147
tion; pH 5; 10-60 g/L of
butyl ether, tertproducts are identified
catalyst
amyl methyl ether
Ozone
Al2O3
Discontinuous and semiOxalic,

acetic,
continuous (flow 100Removal increase depends on type of
salicylic, and suc137
L/h); pH 5.5; 20-50 g/Lcontaminant
cinic acids
of catalyst
Ozone
Al2O3
NOM (DQO)
Semi-continuous; pH 7-Increased
NOM
removal
and
8; flow 0.4 mg O3/L; 75decreased formation of reaction by-148
g/L of catalyst
products
Ozone
TOCCATA
Wastewaters
(DQO)
Semi-continuous; pH 7-Improvement in DQO removal by

149
8; flow 40 mg O3/L
oxidation and precipitation
Ozone
Sludge with metals

*
and metal oxides
DQO,
NOM (TOC)
5-50
Ref.
activity;
catalyst
138
Study of operational variables; effect

g/L
of metal oxides and NOM on the142
process
mechanism;
143
Semi-continuous; pH 3;Feasibility study on the use of sludge

150
25C
as catalyst of ozone decomposition
*Publications on ozone decomposition in absence of contaminants
448
4.2.3. Supported Metal Oxides

Attempts have been made to enhance the catalytic activity of metal oxides in the
transformation of ozone into HO radicals by increasing their surface area, supporting them on
alumina, clays, silica, or zeolites. However; results obtained were not completely satisfactory
because of leaching. According to Copper et al. [151] the presence of a heterogeneous surface
facilitates the transference of ozone in solution and its decomposition into radical species. For
this reason, many organic compounds were more effectively degraded when ozonation was
carried out in the presence of these supported catalysts. Table 7 summarizes the main
conclusions of different research groups, and describes the experimental conditions for the
simultaneous use of ozone and supported metal oxides to remove organic contaminants from
waters.
Table 7. Removal of organic contaminants by systems based on the simultaneous use of
ozone and supported metal oxides.
System
Catalyst
Ozone
CuO, MnO2, Pd,

supported on
Phenol
Al2O3
Contaminants
Ref.
Without catalyst activity
20C; flow 1.43 L/min
152
Catalyst activity removes more

Semi-continuous; acid pH TOC; it does not oxidize
hydroquinone and phenol
153,154
Continuous; 25C; flow

0.15 L/min; 0.5 g of
catalyst
Ozone
Fe2O3/Al2O3
Phenol,
hydroquinone,
carboxylic acids,
aldehydes
Ozone
Ag y Ni
supported on
zeolite
Ozone
High removal of TOC and

TiO2, TiO2/Al2O3, Fulvic acid, proteins, Semi-continuous; pH 8; 30
pollutants in the presence of the 155
TiO2/arcilla
disaccharides
g of catalyst
catalyst
High efficacy of Ag to
decompose O3; it does not appear 139
to affect adsorbed H2O
Ozone
MnO2/Al2O3
Semi-continuous
Decomposition of O3, in the

presence of the catalyst is zero
order with respect to water;
mechanism proposed with
superoxide and peroxide
intermediates
Ozone
TiO2/Al2O3
Fulvic acid
Discontinuous; pH 7.5;
20C; 10 g/L de catalyst
Use of the catalyst increases

mineralization
Ozone
Al2O3,
Fe2O3/Al2O3,
TiO2/Al2O3
Use of the catalyst increases

Chlorophenol, oxalic Semi-continuous; flow 24
solubility and decomposition of 151
acid, chloroethanol mg O3/(Lh)
O3,improving pollutant removal
Ozone
TiO2/Al2O3
Humic acid, natural

river water (TOC)
Semi-continuous; pH 7.2; By-products are identified; TOC

123,
flow 0.4 g O3/h; 2.5-10 g/L removal increases at low humic
158
catalyst
acid concentration
Ozone
V-O/SiO2, VO/TiO2, MnO2
Sulfosalicylic acid
Semi-continuous, pH 3,
flow 0.67 L/min
Ozone
Metal oxides on
Al2O3, SiO2, TiO2 *
y SiO2Al2O3
Fixed-bed; flow 0.175

Proposes a mechanism of ozone
L/min of aqueous solution decomposition; catalytic efficacy 160
with O3; 1.5 g of catalyst of noble metals; catalyst effect
Ozone
MnOx/Al2O3,
MnOx/SiO2,
MnOx/TiO2,
MnOx/ZrO
Fixed-bed; gas flow of

Catalyst deactivation and
0.25-1L/min; 20-75 mg/L subsequent regeneration at high 161
of catalyst
temperatures
Benzene
Formation of oxalic acid;

improved TOC removal and
reaction rate with catalysts;
MnO2 does not catalyze
156
157
159
449
Ozone
TiO2/Al2O3
Oxalic acid
The catalyst notably improves

pollutant removal; Eley-Rideal
10-40C; flow 12-36 L/h;
mechanism; reaction order and
1.25-3.75 g/L of catalyst
activation energy determined
Ozone
MnOx/activated
carbon
Nitrobenzene
Higher catalytic activity at acid

Continuous (flow 0.075
pH; pollutant oxidation and
L/h) and semi-continuous;
163
adsorption is detected on catalyst
pH 2-9
surface
Ozone
MnOx/Al2O3
Cyclohexane
Indicates formation of
Semi-continuous; flow 0.5
intermediate compounds on
L/min; 0.1 g/L of catalyst
catalyst surface
Ozone
Co oxides
supported on
alumina and
hydrotalcite
Naphthol blue black
Co leaching; dispersion is studied

Semi-continuous; 25C; 30
in catalysts and reutilization in 3 165
L/h; 0.3 g/L of catalyst
cycles
162
164
*Publications on ozone decomposition in the absence of contaminants
4.2.4. Activated Carbons

The porous texture and chemical nature of activated carbons makes them ideal materials
for use as adsorbents and also as catalysts or catalyst supports [166]. Although there has been
far less study on the use of activated carbons as catalyst or metal catalyst support for reactions
in aqueous solution than on their role as adsorbent, research interest has been increasing over
the past 10 years. In the 1990s, it was observed that the presence of activated carbon catalyses
ligand substitution between complex compounds [167] and can produce oxidation of sulfide
into sulfate [168]. These studies found a relationship between the catalytic activity of
activated carbon and its porous structure, although they did not consider the influence of the
surface chemistry of the carbon, which was subsequently addressed by other authors[169,170]
Table 8. Relevant publications on catalyzed ozonation with activated carbons.
System Catalyst
Contaminants
Ref.
Ozone
Granular activated
carbon
Phenols
15-35C
Lower consumption of O3 with AC;

AC improves selectivity but not the 179
reaction constant
Ozone
Activated carbon
Chlorinated
hydrocarbons,
COD
Fixed bed
ECCOCLEAR
Combines catalyzed ozonation with

biological treatment and nano
187
filtration; low influence of
carbonates as scavengers
Ozone
Activated carbon
Subterranean
waters, domestic
and industrial
wastewaters
Fixed bed
ECCOCLEAR
Results of industrial plants;

catalyzed ozonation requires less
ozone and is effective over a wide
pH range
Ozone
Activated carbon,
carbon black
Radical pathway process; Inhibitor

p-Chlorobenzoate, Discontinuous; 20 mg/L
and promoter effects of ozone
178
acetate, methanol of catalyst
decomposition
Ozone
Activated carbon
Textile
wastewaters
Ozone
Granular activated
carbon
Fixed bed; pH 2, 8; 25C; Solid-gas catalytic reaction;

1,2
flow 5 L/min; 4 g/L of
increases % of oxidized groups after 190
dihydroxybenzene
catalyst
activated carbon ozonation
Ozone
Granular activated
carbon
Kinetic and stability study and
60-360 L/h; 200 g of
catalyst activity; removes color
activated carbon column
188
189
Proposes mechanism; heterogeneous

Discontinuous; pH 2-9; 5- and homogeneous processes
191
30C; 2 g/L de CA
simultaneously; influence of
variables and kinetic study
450
System Catalyst
Contaminants
Ref.
Higher catalytic activity of basic

activated carbon with/without
metals in mineral matter
180
181
Ozone
Granular activated
carbon
1,3,6 naphthaleneDiscontinuous; pH 2.3

trisulfonic acid
Ozone
Granular activated
carbon
(mono,di,tri)
naphthalenetrisulfonic acids
Semi-continuous; pH 2Study of adsorption capacity and

12; flow 76 mg O3 /min;
surface chemical modification of
0.5-2 g/L of activated
ozonated activated carbon
carbon
Ozone
Granular activated
carbon
(mono,di,tri)
naphthalenetrisulfonic acids
flow 76 mg O3/min
Ozone
Granular activated
carbon
Identification of intermediates;
1,3,6 naphthalene- Discontinuous; pH 2.3; 2
reduction in catalytic activity of
trisulfonic acid
g/L of catalyst
activated carbon with ozonation
Ozone
Activated carbon
Pyruvic acid
Catalytic and non-catalytic reaction

Semi-continuous; pH 7.5; rate constants determined; transfer
194
20C; 2.5 g/L CA
of O3 mass is limiting step at high
carbon doses
Ozone
Granular activated
carbon
Discontinuous; 22C; 1
g/L of catalyst
Ozone
Granular activated
carbon, pellets
Succinic acid
Use of activated carbon permits

Semi-continuous; pH 7; 5,
almost complete mineralization;
15, 25C; flow 20-60 L/h;
195
reaction via 2 pathways: water and
10-20 g/L CA
activated carbon surface
Ozone
Activated carbon (0.1*

0.3 mm)
Because of activated carbon

Semi-continuous; pH 3-9; oxidation, the surface chemistry is
25C; 4-15 mg/L O3; 0.2 only important in the first cycles, 196
g/L CA
after which the texture is more
influential
Ozone
Activated carbon
Columns in line; 100 g

CA; 3 mg/L O3
Rainwater (DQO)
Reduces genotoxicity of ozonation

products when catalyzed with
192
activated carbon
193
Higher catalytic activity of basic

activated carbon and larger surface
183
area; greater ozone decomposition
with higher concentration
Optimization of operational
variables; synergic effect in the
O3/AC /CAB system
197
Effect of operational variables

Ozone,
Discontinuous; pH 7;
Granular and powdered Sodium dodecyl(activated carbon size and dose, O3
ozone/H2
25C; 210-5M; 0.1 g/L of
198
activated carbon
benzenesulfonate
dose, etc.); activated carbon reduces
O2
activated carbon
effect of radical scavengers
Ozone,
Granular activated
ozone/H2
carbon
O2
Ozone
Carbon aerogels with

Mn2+, Co2+y Ti4+
Ozone
Granular activated
carbon
Removal of TOC and

Organic and
Discontinuous; pH 7, 9; microcontaminants; similar
inorganic
-5
25C; 210 M O3; 0.5 g/L performance of O3/GAC and
compounds in
CA
O3/H2O2 in generation of HO
natural lake water
radicals
199
Metals susceptible to oxidation with

Discontinuous; pH 7;
Para-chlorobenzoic
O improve the generation of HO
-5
25C; 210 M O3; 2.5-10 3
200
acid
radicals; activated (more basic)
mg/L aerogel
samples are more effective
Benzothiazole
Proposes mechanism and

Semi-continuous; pH 2,7,
determines kinetic constants; high
11; 15C; flow 54 L/h; 1
201
radical contribution to contaminant
g/L CA
removal
Notable catalytic activity in phenol
removal; in situ regeneration of
activated carbon fiber during
202
ozonation, although its chemical and
textural properties are modified
Ozone
Activated carbon fiber Phenol
Semi-continuous; 25C;
flow 1.5 L/min; 4 g/L
activated carbon fiber
Ozone
Granular activated
Semi-continuous; pH 2.5, Identification of reaction by-
Polyphenols
203

carbon
Ozone
Activated carbon (0.1Oxalic acid,

0.3 mm), oxidized with
oxamic acid
HNO3
451
5; 25C; flow 30 L/h; 10 products; improves TOC removal;

g/L CA
generation of H2O2
Study of the effect of pH, presence
Semi-continuous; pH 3, 7; of radical scavengers and surface
25C; flow 0.15 L/min; chemistry; proposes mechanism;
204
0.5 g/L CA
better result with basic activated
carbons
The catalytic activity of activated carbon in aqueous solution has been studied in the
synthesis[171,172]and
degradation
of
ammonic
nitrate
[173],
phenolic
compounds[174,175]or chlorinated compounds [176,177] among others. The main interest in
this behavior of carbon is related to the oxidation of organic matter. Thus, it has been
observed that activated carbon can decompose ozone in aqueous solution into radicals with a
high oxidizing power, increasing the extent of ozonation [178]. Therefore, the combined use
of ozone and activated carbon in a single process was proposed as an attractive option to
destroy toxic organic compounds.
Until recently, the major advantage of adding activated carbon to the treatment system
was considered its high adsorption capacity [179]. However, Jans and Hoign [178] showed
that both carbon black and activated carbon catalyze the decomposition of ozone in aqueous
phase. These authors indicated that both types of carbon initiate the radical-type chain
reaction of ozone, which continues in aqueous phase and accelerates the transformation of
ozone into free hydroxyl radicals. Zaror et al.[179] reported that the presence of activated
carbon drastically reduced the stability of ozone in aqueous solution, which they attributed to
a combination of the decomposition catalyzed by the surface and the participation of activated
carbon surface groups. Subsequently, in-depth research into ozone/activated carbon systems
by Rivera-Utrilla et al.[180,181,182,183] demonstrated that the surface chemistry and textural
characteristics of activated carbon play a major role in its behavior as catalyst of ozone
decomposition into hydroxyl radicals. These authors ascribed this effect to basic carbon
groups, e.g., pyrone and chromene [180]. Zaror et al. found that addition of activated carbon
to the system favored the oxidation rate of 1,2-dihydroxybenzene with ozone[179]. Hu et
al.[184] reported a more effective removal of organic matter from textile industry effluents by
using reduced Cu supported on activated carbon. Table 8 summarizes the results of some of
the most relevant studies on the simultaneous use of ozone and activated carbon in water
treatments.
It is necessary to study the mechanisms underlying this catalytic process in order to
achieve its economic and effective large-scale optimization and implementation. Various
researchers have investigated the mechanisms involved in heterogeneous catalytic
ozonation[185,186], but these have not yet been fully elucidated because of the complexity of
these simultaneous pollutant oxidation and adsorption processes.
CONCLUSIONS
There is now a considerable body of knowledge on advanced oxidation processes
(O3/H2O2, O3/UV) in the literature, including data on the mechanisms and reactions involved
and on the influence of operational variables in the transformation of ozone into HO radicals.
452
This information has allowed the large-scale application of these processes to remove a wide
variety of organic microcontaminants.
The use of dissolved or supported metals to transform ozone into HO radicals is not very
effective. Studies show that i) the simultaneous use of ozone and dissolved metals must be
approached in a more systematic manner; and ii) there is considerably controversy about the
reactions involved in the process and the metal characteristics that promote the transformation
of ozone into HO radicals. Research is required into the mechanisms that underlie this
catalytic process in order to achieve its economical and effective optimization and
implementation on a large scale. Because of the complexity of simultaneous pollutant
oxidation and adsorption processes, the mechanisms involved in heterogeneous catalytic
ozonation have not yet been fully elucidated.
Finally, activated carbon appears to be the most appropriate solid catalyst to use for
enhancing micropollutant ozonation. Besides considerably accelerating the process of ozone
transformation into HO radicals, thereby increasing the rate of organic contaminant removal,
activated carbon also has a high adsorbent capacity, further contributing towards the removal
of microcontaminants from waters.
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ISSN: 2158-5717
Chapter 17
USE OF MICROARRAYS TO STUDY

ENVIRONMENTALLY RELEVANT ORGANISMS:
A UK PERSPECTIVE
Michael J. Allena, Andrew R. Cossinsb, Neil Hallb, Mark Blaxterc,
Terry Burked and Dawn Fielde
a
Plymouth Marine Laboratory, Prospect Place, Plymouth, UK.

NERC Molecular Genetics Facility, School of Biological Sciences,
University of Liverpool, Liverpool, UK
c
NERC Molecular Genetics Facility, Institute of Evolutionary Biology,
University of Edinburgh, Edinburgh, UK
d
NERC Molecular Genetics Facility, Dept of Animal & Plant Sciences,
University of Sheffield, Sheffield, UK
e
NERC Environmental Bioinformatics Centre, NERC Centre for Ecology
and Hydrology, Mansfield Road, Oxford, UK;
b
ABSTRACT
Historically, the majority of microarray work has been restricted to well-defined
model organisms. This was primarily due to the limited availability of genomic or
transcriptomic sequence data and the then high cost involved in developing microarrays.
However, recent technological developments have greatly enhanced the speed of
generating the underpinning sequence data for non-model species, and have opened up
more cost-effective approaches for microarray production to make them far more
affordable for researchers at the lower end of the budget range. These developments have
been seized upon by the environmental genomics community within the UK. The
creation of a network of closely integrated facilities for sequencing, microarray printing
and bioinformatics has opened the gateway for the study of environmentally relevant
organisms. Here, we describe the infrastructure for microarray development within the
UK, and the diverse applications for which they are currently being used.
466
Michael J. Allen, Andrew R. Cossins, Neil Hall et al.
INTRODUCTION
The advent of the genomic era has heralded a new dawn in the study of biological
systems. The intensive study of model species such as E. coli, yeast, Drosophila and the
mouse has provided a wealth of information on the molecular workings inside the cell. These
advances were largely dependent on large-scale sequencing projects which determined the
entire genome sequence of several well studied model strains [1-4]. The development of
microarray technology for massively parallel detection of sequence targets defined from
large-scale genome and EST sequencing projects, has allowed transcriptional profiles from
cells, tissues and organisms at various life stages to be analysed and mapped on a global scale
with increasing precision [5].
It cannot be denied that the intensive study of model organisms is of great benefit.
However, the study of ecologically important, non-model species in their natural environment
is arguably of greater relevance when analysing the response of species to real-world
environmental conditions. Model organisms are often chosen for reasons of experimental
power and convenience, rather than for ecological interest or evolutionary significance [6]. In
an era of increasing awareness of the environmental changes occurring through global
warming, habitat destruction, deforestation, globalisation and ocean acidification there has
never been a more important time to study the impact on the Earths ecosystem, and this
means adapting advanced genomic tools for this purpose.
Historically, tools such as microarrays have been restricted to model species due to
limitations in the genomic sequence available and the high cost involved in developing
microarrays. However, technical advances have led to a significant decrease in the costs
involved, opening the field to researchers working on non-model organisms. The uptake of
microarray technology by environmental researchers, in particular the marine sciences, has
been relatively slow partly due to a lack of both focused funding and technical expertise in the
community. In the UK this has been addressed by the predominantly NERC-funded
environmental genomics community which has created a dedicated network of facilities and
resources to aid the use and development of microarrays.
FUNDING
The United Kingdom Natural Environment Research Council (NERC) has invested over
26 million in two directed science programmes; (i) Environmental Genomics (EG) and (ii)
Post-Genomics & Proteomics (PGP). To complement these initiatives, NERC has also funded
the creation of dedicated technical support centres covering DNA sequencing, genotyping,
microarray design and fabrication (through NERCs Services and Facilities branch), and
bioinformatics (through the thematic programmes themselves). Each centre offers a strong
technical capability built on the existing expertise in established laboratories, and also
consultation, training, and support not only to undertake the lab work but also in the
subsequent data analysis and management. Access is open to all UK-based scientists, but
priority is given to NERC grant holders based on an appraisal of science quality. Applicants
for NERC-funded research are advised to take advantage of these facilities whenever
possible.
467
In addition, to promote the use of these advanced technologies in environmental science,

an annual small projects competition has allowed researchers to conduct pilot studies in
support of full-scale future project applications. National and international collaboration
between environmental scientists often working in vastly different areas is promoted through
this system. The diverse range of subjects for which new microarrays have been developed
include: marine phytoplankton viruses (see Case Study 1); adaptive osmoregulatory
physiology of euryhaline teleosts; ecotoxicological responses of terrestrial annelids; genetic
control of avian plumage traits; sexual differentiation and endocrine disruption in fish; and
responses to cold and hypoxia in carp (see Case Study 2).
INFRASTRUCTURE
Every microarray experiment is dependent on a number of steps, all of which are
essential to produce results that are scientifically competent and of publishable quality. These
steps are listed in Table 1, and can be grouped into 3 main categories: the acquisition of
genomic sequence data, the design and construction of the array and the experimental use of
the array. Of course, this is only the beginning of a researchers challenges: correct
experimental design with the appropriate number and type of replicates, in combination with
the large volume of data produced, demands the use of complicated statistical analysis to
provide meaningful results. An environmental scientist working alone on a microarray project
will need to be expert in a range of disciplines, such as molecular biology, bioinformatics and
statistics. In addition, ready access to sequencing, array printing and hybridisation facilities is
essential.
It is important that the researcher has an understanding of the processes involved at each
stage, and also that the correct expertise is available at each stage in line with current opinion
in a rapidly developing field. Thus the use of microarrays is a daunting prospect to any
researcher who lacks significant experience or expertise in the technologies involved. In
particular this includes environmental researchers whose field has been historically dominated
by non-molecular scientists (i.e. large numbers of field biologists). It is perhaps for these
reasons that environmental scientists studying non-model organisms have been relatively
slow to embrace microarray technology.
Fortunately for the UK-based environmental science community, NERC foresaw this
problem and put in place the infrastructure required to facilitate the technical development of
microarray platforms and their application to environmental science. The new provision was
based on the pattern established by an existing NERC genotyping centre based in Sheffield
(MGF-Sheffield) and consists of three additional Molecular Genetics Facilities (MGF) nodes:
two facilities are involved with sequencing (MGF-Edinburgh and MGF-Liverpool through its
Advanced Genomics Facility, AGF-Liverpool), and a third facility with microarray
construction (MGF-Liverpool through its Liverpool Microarray Facility, LMF-Liverpool). A
fourth but separate facility, The NERC Environmental Bioinformatics Centre (NEBC)
provides the necessary bioinformatic expertise required at all levels of the process. Combined
these facilities provide a comprehensive suite of technical platforms and expertise, and they
interact seamlessly with one another to satisfy the needs of the researcher.
468
GENOMIC SEQUENCING
There are three currently applicable methods for generating sequence data for
environmentally relevant organisms: capillary Sanger sequencing, massively parallel
pyrosequencing, and massively parallel sequencing-by-synthesis. Each method has its
benefits and drawbacks, and any particular environmental genomics project will choose the
route(s) most suited to its needs. Both MGF-Edinburgh and AGF-Liverpool are dedicated
sequencing facilities and thus generally not directly involved in physical sample acquisition:
it is assumed that a researcher working on a particular environmental organism has the
necessary expertise for culture and/or sample extraction to provide suitable genomic material
(quantity and quality) for sequence analysis.
Capillary Sanger sequencing, the workhorse of DNA sequencing for over 30 years, is
offered through the MGF-Edinburgh. This generates 500700 bases per read, and is suited to
both survey sequencing (such as expressed sequence tag sequencing, or ESTs) and also
whole-genome sequencing. The MGF-Edinburgh offers medium-throughput (~1000 samples
per day) Sanger sequencing using ABI3730 instruments. This suits projects from ~100 to
50,000 sequencing reads from targets such as PCR products, cloned inserts in plasmids and
other vectors provided by the user. It also carries out the complete sequencing of large DNA
fragments (>30 kb; for example from fosmid and BAC clones) using shotgun strategies.
Bioinformatic support is offered by MGF- Edinburgh, including preliminary sequence trace
processing, assembly and annotation. For EST projects, a suite of in-house developed
sequence annotation tools can also be applied to sequence datasets using programs such as
PartiGene (databasing tool for EST datasets) [7], trace2dbEST (for processing EST
sequencing traces) [7], prot4EST (for prediction of peptides from neglected genome sequence
datasets) [8], and annot8r (annotation of peptide or nucleotide sequences with GO, KEGG
and EC tags based on sequence similarity).
The Advanced Genomic Facility (AGF) based at the University of Liverpool offers
access to the ultra-high throughput pyrosequencing Genome Sequencer FLX system (GS
FLX, Roche), which at present can generate in excess of 100 Mb of genome sequence in a
single run with a mean length of 250 bases. The FLX pyrosequencing platform incorporates
single-molecule amplification and parallel sequencing of up to ~400,000 individual molecules
for 250 bases, making large-scale scientific projects feasible and more affordable. By mid2008 this instrument is expected to yield up to 1 Gb with read lengths approaching 500 bp.
FLX pyrosequencing is suited to deep surveying of transcriptomes, complete sequencing of
large-insert clones and smaller genomes, resequencing of larger genomes, and metagenomic
analyses. AGF-Liverpool also has the latest computational tools and trained staff to deliver
bioinformatic services for data handling and analysis, in particular for assembling sequence
reads (Newbler assembly) and sequence alignment (FlowMapper).
Sequencing by synthesis is also massively parallel, but generates ~30 million reads of
~35 bases per run, generating 1 Gb of sequence. This technology is ideal for resequencing of
complex genomes, metagenomic surveys of mesocosms, digital transcriptomics and de novo
sequencing of smaller genomes. MGF-Edinburgh also offers a sequencing-by-synthesis
service using an Illumina Genome Analyser instrument.
The production of raw sequence data is only the start of the process: the genomic or
transcriptomic sequence must be correctly assembled and annotated in order to provide
469
meaningful information that can be used at the next stage for microarray design. As shown
above, both facilities have their own particular expertise in certain areas. However, in
addition to the bioinformatic facilities offered by MGF-Edinburgh and AGF-Liverpool,
additional bioinformatic support for genomic annotation is offered by the NERC
Environmental Bioinformatics Centre (NEBC). The bioinformatic support provided by MGFSheffield is focused on population genetic data analyses, including genomic mapping.
MICROARRAY DESIGN AND FABRICATION

The key requirement for generating a DNA array is access to suitable genomic resources.
For arrays printed in-house using amplified DNA, this comprises cloned cDNAs or largeinsert genomic fragments isolated from libraries [9]. Alternatively, arrays can be printed from
oligonucleotides synthesised in bulk with defined sequences as probes against known DNA
targets of interest. Both techniques are expensive in time (for cloning and amplification) and
costs (for oligonucleotide purchase). However, for situations where sequence data already
exist it is now preferable to design probes in silico against all known targets for a particular
species using one of several design algorithms, followed by on-chip synthesis by one of
several manufacturers, notably Agilent. Advancing fabrication techniques now makes this a
relatively inexpensive yet highly effective approach to array provision.
The design of oligonucleotide probes from a known target DNA sequence can be
achieved using any one of several open source or commercial packages [10]. The MGFLiverpool uses a commercial package (ArrayDesigner, Premier Biosoft) as an alternative to
the widely used open source package OligoArray2 [11]. Design algorithms continue to
advance, and one algorithm might suggest different probe designs to another. Commercial
providers of oligonucleotides or arrays offer a design service (e.g. Agilents eArray system),
but the precise algorithms employed remain confidential to the company and so one cannot be
totally confident of design quality or the criteria used in its execution. It is also worth noting
that the outputs of any design algorithm do not necessarily provide for optimal function as a
hybridisation probe, since they are based on thermodynamic criteria that are surrogates of the
hybridisation process. This is why the MGF-Liverpool uses an optimisation approach,
comparing the properties of several competing probe designs generated from a single
algorithm against stringent performance criteria [12] in order to define the best performing
design.
All of these options are available at the MGF-Liverpool which has access to both contact
and non-contact (piezo) printing robots for array fabrication, has service centre status for both
Affymetrix and Agilent platforms, and has access to the Illumina BeadStation, this being the
third major commercial platform. As a central array facility, it has negotiated special rates for
purchasing large numbers of synthesised oligos and all of the other necessary consumables,
including catalogue arrays. It is thus well able to provide support to users with all aspects of
microarray design, construction and commissioning. In the past few years MGF-Liverpool
has constructed cDNA arrays for a wide range of species (e.g., common carp, flounder,
rainbow trout, ground squirrel and eel) all of which were preceded by large-scale cDNA
sequencing projects. It has also been involved in a series of microbial metagenomics projects
printing commercially synthesised oligoarrays directed against rRNA targets defined by the
470
user. MGF-Liverpool has also some experience in constructing oligoarrays for environmental
model species directly from public EST data, taking advantage of the rapidly increasing
capacity of new platforms. Thus, an oligoarray directed against 22,000 BLAST-identified
genes in the rainbow trout was fabricated by Oxford Gene Technology to a MGF-Liverpool
design, and this has been expanded to 44,000 probes [12]. This design was optimised in the
sense that multiple predicted probes were quantitatively compared using several different
tests and the best performing probe identified. The resulting arrays displayed a dynamic range
and sensitivity that far exceeded that of conventional cDNA arrays [12] .
ANALYSING THE RESULTS OF MICROARRAY EXPERIMENTS

Once a microarray has been designed and fabricated the actual experimental biology can
begin. The first important issue is the statistical design of the array experiment. since the shift
from the early reference-based designs to more complex ANOVA-based loop designs [13],
and the need to account for multiple sampling corrections in such large-scale statistical
comparisons, makes this the realm of specialist statistical advice. Only rarely do biologists
who develop skills in laboratory technique have the numerical and statistical skills for highlevel data analysis and pattern searching. The staff of the MGF-Liverpool and NERC
Environmental Bioinformatics Centre (NEBC) provide specialist advice to define the most
appropriate and cost-effective experimental plan to success. The researcher also needs to
understand the very fine definition offered by array experiments to ensure that the experiment
is adequately controlled and that sufficient independent biological replicates are generated for
detailed statistical analysis. Dedicated microarray bioinformaticians at both the MGFLiverpool and at NEBC keep abreast of the latest developments and issues with microarray
analysis and are best positioned to advise on correct experimental design. The MGFLiverpool also supports all of the stages of array usage, including sample preparation,
hybridisation protocols, imaging/data acquisition and interpretation of the resulting data. This
includes the training of research students and staff from NERC-funded laboratories.
The fundamental responsibility for sample acquisition and for extraction of DNA or RNA
lies with the client. Indeed, sample processing, hybridisation and data acquisition can be
performed at the researchers own laboratory if facilities are available. Alternatively the entire
programme can be performed as a service by MGF-Liverpool staff or the latter can provide
the necessary training for clients to undertake the work. Emphasis is placed on the researcher
remaining in control of the experiment, but with the full benefit of state-of-the-art protocols
and equipment.
MGF-Liverpool offers several scanning platforms for image acquisition including
Agilent, Perkin-Elmer and Axon. It also advises on the critical issue of interpreting the image
file in order to quantify the distribution of fluorescence intensities across the population of
probes. Once microarray images have been processed and quantified, it is strongly
recommended that that the researcher interacts further with the MGF-Liverpool or the NEBC
if they require help progressing the statistical analysis. NEBC offers access to the commercial
GeneSpring (Agilent) and the open-source maxD analysis platforms, and both the NEBC and
the MGF-Liverpool routinely use algorithms from within the Bioconductor package written in
the R statistical language. This combined provision has three main advantages. Firstly, the
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researcher is directed to well established statistical and data visualisation algorithms. Second,
he/she can be trained in analytical technique through interactions with the facilities. Third,
he/she can be exposed to objective approaches for drawing inferences both in terms of which
genes are responding to experimental treatment, but also how this relates to the biological
pathways and processes, and to the questions being addressed. This avoids biasing the
analysis to favoured genes or to genes that fit with particular hypotheses. Bioinformaticians at
any NERC facility can explain the data output, its strengths and its limitations. However, it is
entirely up to the researcher to interpret and publish the data. This involves the use of gene
ontology profiling tools such as GOMatrix, GoMiner, Reactome etc, as well as the newer
gene set analysis techniques originated in the Broad Institute, MIT.
At the final stage of publication, it is necessary for all microarray data to be submitted to
a public repository such as GEO or ArrayExpress [14, 15]. This is aided by the development
of the Biolinux based maxdload2 data submission program, developed by NEBC partners at
the University of Manchester, which ensures it is MIAME-compliant [16]. Indeed, in
response to the issues associated with environmental microarrays, the NEBC has developed
its own guidelines for MIAME for environmental projects, designated MIAME/Env [17].
Researchers funded by NERC are also required to submit their data to the NERC-supported
database EnvBase (http://nebc.nox.ac.uk/).
DATA ANALYSIS AND LONG-TERM MANAGEMENT

While each of the sequencing facilities have specialist informatics services focused on
specific sequencing platforms, the NEBC is focused on delivering multi-omic approaches to
analysis and data management. NEBC was formed in 2002 to specifically provide support for
the NERC Environmental Genomics Programme, and subsequently for the sister PostGenomics and Proteomics thematic programme. NEBCs primary remit is to archive all
NERC-funded data generated under these programs subscribing to the NERC data policy of
spending up to 15% of the programme budget on data management and archiving activities.
This was one of the earliest data policies operated by a major UK funding agency and its
wider implementation is mediated by a set of seven designated data centres that receive core
funding from NERC for long-term data management.
To guide the data management of the programme, and the stewardship of omic data in
NERC as a whole, NEBC has also produced an extension of the NERC data policy to cover
omic data. This policy includes very specific mechanisms for submission of data. All data
are catalogued in the EnvBase portal and must be submitted to an appropriate public
repository where one exists. EnvBase contains discovery-level information about all listed
projects, and for each data-holding includes a list of relevant hyperlinked accession numbers
leading to databases such as ArrayExpress [14, 15]. EnvBase also provides a mechanism to
house all data for which a recognized database does not exist.
NEBC was designed to build informatic capacity within the newly forming NERC
environmental genomic community. NEBC is therefore staffed by bioinformaticians and
computer scientists, in addition to data managers, who provide direct consultation to the
wider community. NEBC was established in part to realize the vision of providing the entire
Environmental Genomics community with a powerful and uniform computing environment
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based on Linux. This system, called Bio-Linux, is one of a growing suite of highly
customized Linux distributions [18]. It contains over 60 software packages and is distributed
free of charge to all labs supported by the program. The Bio-Linux system has been used to
build teaching labs and computer clusters in several locations across the UK and is used by
the core facilities and researchers to coordinate research.
The primary focus of NEBC efforts under these programs has been support for research
that is generating microarray and EST data. Microarrays were a focus because of their widespread use among funded researchers, the expense and effort of tooling up to produce and
interpret them for new users, and the added complexity that several NERC-funded groups
aimed to build custom arrays for novel species and sets of genes. In order uphold its remit to
provide stewardship over the collective data holdings of these programmes; the centre has
become progressively more involved in standardization activities. While at first, this simply
meant monitoring emerging standards and compliance activities, over time this led to active
participation and leadership in standardization activities that expressly benefit the
environmental omics community and that have made submission of data to public
repositories possible.
The NEBC spearheaded a standardization project in response to the difficulties NERC
researchers were facing in complying to the MIAME standard, a requirement for submission
to ArrayExpress [19]. The MIAME/Env project emerged out of work aimed at simplifying
this process for researchers across the board. This initiative extended the core MIAME
specification to capture additional information about the environmental context of samples
(for example geographical location, biological interactions, and environmental conditions)
[17]. Work on MIAME/Env has since been extended into the metabolomics and genomics
domains [20]. Because of participation in such projects, and its focus on multi-omic data sets,
NEBC is also a founding member of the Minimum Information about a Biological and
Biomedical Investigation (MIBBI) community [21].
CONCLUSION
The UK has developed a powerful network of specialist facilities to promote the use of
high-density microarrays that help researchers overcome the myriad challenges of working
with non-model but environmentally-focussed organisms. Depending on the resources
available to them and their level of expertise, environmental researchers can selectively
choose the facilities while having as much or as little control in the development, construction
and processing of the microarrays as they are comfortable with. The nature of the organisms
involved can vary dramatically, as can the type of experiments undertaken, but help is
available at every stage of the process. This has had a strong beneficial effect on the UKbased environmental community by promoting the use of microarrays and driving
environmental sciences into the 21st century. To illustrate this, the following are two case
studies, typical of the type of work that has been undertaken, by researchers in the
environmental genomics community.
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NON-MODEL ORGANISM CASE STUDY 1:

THE COCCOLITHOVIRUS MICROARRAY
The Science
Emiliania huxleyi is a marine coccolithophorid with worldwide distribution capable of
forming vast blooms that can cover up to 10 000 km2 [22, 23]. Its production of calcium
carbonate coccoliths and its role in CO2 cycling and dimethyl sulphide (DMS) production
makes E. huxleyi an important species with respect to past, present and future marine primary
productivity.
Viruses have been shown to be a major cause of bloom termination [24-26]. Emiliania
huxleyi virus 86, EhV-86, is a giant virus that was originally isolated from a bloom in the
English Channel off the south coast of England in 1999 [27]. EhV-86 is the type species of
the genus Coccolithovirus within the family Phycodnaviridae and is currently its largest fully
sequenced member [28]. The 407,339 bp genome is the second largest virus genome
sequenced and is predicted to encode around 472 protein coding sequences (CDSs) [29]. The
majority of EhV-86 CDSs exhibit no similarity with proteins in the public databases; a mere
21% of the CDSs contain proteinprotein basic local alignment search tool (BLAST) results
that matched an E value lower than 0.01 [29]. The large size (and availability) of the EhV-86
genome and its highly unknown nature easily justified the construction of a microarray to aid
in the molecular characterisation of this unique virus family.
The Array
The EhV-86-based coccolithovirus microarray has been through 2 stages of development.
Version 1 was dominated by probes for the 472 predicted CDSs of EhV-86 and contained
only a handful of E. huxleyi probes, and consisted of around 1,600 features. As the work has
progressed and developed, and the nature of the questions asked of the array changed, so the
latest version 2 of the array (printed at MGF-Liverpool) has around 25,000 features and
contains probes for multiple coccolithovirus strains and for ESTs from the host species E.
huxleyi (developed with the aid of MGF-Edinburgh).
The Research
The majority of the EhV-86 genome is composed of genes of which the function is totally
unknown [29]. Indeed, unusually among sequenced genomes, a number of predicted genes
fail to find any matches whatsoever in the public databases [30]. This novelty of the genome
makes the task of annotation difficult: without functional information it is difficult to assess
whether what is predicted to be a gene is actually a gene. Thus, the first use to which the
coccolithovirus array was put was simply to detect the presence of viral transcripts. The
likelihood of an open reading frame being a functional coding sequence and gene is vastly
increased by detecting the presence of a mRNA transcript. This approach was used effectively
474
during the annotation of the EhV-86 genome, where message for 65% of the 472 predicted
genes of EhV-86 was confirmed from a lytic phase total RNA extraction [29].
Following the lytic phase transcriptional work the microarray was used to outline the
transcriptional profile generated by EhV-86 during the early stages of infection. Using a
highly sensitive amplification method, the precise expression profile of the EhV-86 infection
of E. huxleyi was determined over the first 4 h of infection [31]. This strategy allowed the
designation of EhV-86 CDSs into three groups on the basis of when their transcripts are first
detected (1, 2 or 4 h post infection). The results obtained suggested two distinct phases to the
infection process: a primary phase dominated by a group of CDSs localized to a sub-region of
the genome, which have no database homologues and are associated with a unique promoter
element; and a secondary phase during which CDSs are transcribed from the remainder of the
genome [30-32]. Since the majority of EhV-86 CDSs are unknown, the designation of CDSs
into transcriptional categories is a significant step forward in determining their function. The
distinctive transcriptional profile has answered and raised many interesting questions about
the infection strategy and life style of the virus, in particular the role and function of the
virally encoded RNA polymerase.
It is important to note that a microarray based on sequence data for a particular strain is
not restricted for sole use with that strain. The Plymouth Virus Collection (PVC) contains 12
virus strains all capable of infecting E. huxleyi [33]. These strains were collected over the 4year period between 1999 and 2003 from sampling sites in the English Channel and in a
Norwegian Fjord [25, 27, 34]. The construction of the coccolithovirus microarray allowed the
genetic diversity of the viruses in the PVC to be assessed. A major disadvantage of using a
specific strain/species microarray for this purpose is that the microarray can only tell you
what is highly variable or not present in a particular genome. Genomic additions go
undetected since there is no probe on the array to detect it. However, the use of the array for
screening for genomic content can provide a wealth of information on genetic diversity within
a family of closely related species. At least 70 genes found in EhV-86 are absent or highly
variable from one or more of the genomes of the coccolithoviruses found in the PVC [33].
The distinctive pattern of genetic content displayed by each of the strains (essentially a
genomic fingerprint) suggested a complicated series of gene loss/gain events. In particular, a
high frequency of deleted or variable genes was found in a 100 kb region of unknown
function suggesting this section of the genome may be under a high selection pressure [30,
33]. Thus the microarray method is a relatively cheap, efficient and quick way to glean an
insight into the genetic content of a family of closely related species. Genes of interest that
are conserved among all strains or are lacking and/or variable in some strains can be
identified rapidly for further downstream analysis.
In summary, over a relatively short period of time, a predominantly uncharacterized virus
has had its genome annotated, the kinetic profile of its transcripts determined and the
diversity within its family assessed through the use of microarrays [29, 31, 33, 35]. The
results have indicated a plethora of new and novel genes, a distinctive biphasic transcriptional
pattern during the infection process and an unexpectedly large degree of genomic content
variability among the coccolithoviruses. The construction of the microarray rapidly increased
the understanding of the virus and its infection strategy and serves an excellent model for
other researchers to follow.
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CASE STUDY 2: GENE DISCOVERY AND SYSTEM-WIDE OVERVIEWS

OF ENVIRONMENTAL STRESS RESPONSES IN THE COMMON CARP
The Science
Abiotic environmental stress, such as heat and cold, hypoxia, UV and pollution pose
major problems for all forms of life, and organisms respond to stress by both protecting
themselves from the damaging effects of extreme stress, but also by adjuting themselves to
allow life processes continue unaffected by the change in environmental conditions. Animals
from temperate climatic zones and which inhabit small freshwater ponds are routinely
exposed to seasonal fluctuations of temperature and daily fluctuations of environmental
oxygen concentrations, and survival depends upon the existence of powerful stress-coping
mechanisms [36-38]. Understanding the underpinning mechanisms of stress responses and
adaptation is important not only academically but practically in agriculture and in human
society [39]. Cold presents special problems both in the form of reduced biochemical activity
but also from the damaging effects of freezing [36]. Our understanding of response patterns to
chronic cold in complex animals has been transformed by microarray analyses, from one
composed of a few key or candidate genes to one where the system-wide effects on largescale networks of genes and proteins can be appreciated [40, 41]. Since 2000 NERC has
funded a large-scale analysis of response patterns at the transcript level in the tissues of the
common carp, Cyprinus carpio L..
The Array
Given that there were no genomic resources for the carp, it was first necessary to generate
a collection of cDNA clones, representing genes, all of which was accomplished by the MGFLiverpool. Fish were exposed to the full range of environmental treatments under
investigation, including hypoxia, and RNA isolated, normalised to rebalance representation
the representation of genes, and then cloned directionally into a suitable vector. The inserts
were sequenced from the 5 end, clustered clones into groups containing overlapping
sequences and then their identity established by homology searching. The inserts of 13,500
clones were amplified by PCR and the amplicons were used to print a microarray on glass
slides using a contact-printing robot. More recently, another 20,000 clones have been added
to the collection which were cherry-picked to reduce redundancy. The new, more
representative collection consists of 26,000 clones, which have been printed using a noncontact, piezo-deposition robot. The evolution of the carp array has been detailed in Williams
et al (2008) [42]
The Research
Our first experiment [9] followed changes in the expression of genes in 7 different tissues
of the carp during long-term transition from warm (30C) to cold conditions (10C). The fish
476
were cooled over a 3-day period and then held at 3 cooled temperatures (23, 17 and 10C) for
up to 21 days. On each sampling interval 6-8 animals were killed and the tissues dissected
into a plastic (sushi) tray for long-term freezing at -80C. Samples of these tissues were used
to generate target cDNA, which were hybridised to over 400 high-density cDNA arrays.
The data was subjected to refined statistical analysis to reveal a very complex picture of
gene responses [9]. Over 3000 probes were found to be significantly different between warm
and cooled specimens in at least one tissue. The first focus was on genes that were
differentially expressed (DE) in all 7 tissues, which generated a list of 260 genes of which
180 were identified. This list was composed of genes that were largely involved in cell
homeostasis, with the greatest and most consistent responses being for the 9-desaturase and
CIRBP. The former gene is widely invoked in cold responses as being the key gene in
transforming lipids from saturated to more unsaturated, thereby fluidising the cold-ordered
lipids in cell membranes [43, 44]. The latter gene is an RNA-binding protein of unknown
function, but its prominent responses indicate that it is important to all tissues during cold
adaptation. A large number of genes involved in protein turnover were found, indicating
strong up-regulation in all tissues.
The second focus was on the remaining ~1,700 identified genes that were regulated in
one or more tissues. Clustering of this data using unsupervised techniques generated 23
groups of genes with characteristic tissue-specific responses. Each cluster was profiled by
statistical analysis of the Gene Ontology annotations [45] and this pointed directly at the
nature of gene responses in relation to tissue function. For example, cluster 5 was composed
of genes that were up-regulated exclusively in the intestinal mucosa. This cluster included an
unusually large number of transport-associated genes, which is entirely consistent with the
role of the intestinal epithelium in nutrient absorption and ion/pH regulation. This gene
profiling technique allows the broader gene expression properties across treatment space to be
interpreted within the context of tissues function.
Comparing between clusters allowed the identification of major effects in the pathways
of intermediary metabolism [9] [46]. Thus, it was found that genes of glycolysis were
generally up-regulated in heart and brain, but down-regulated in the other tissues, indicating a
shift between tissues in the patterns of energy provision and storage. Consistent responses
were identified in all of the major tissues, despite them being mediated by different genes or
isoforms. Thus brain, liver, heart and liver all showed a very high representation of genes
involved in energy pathways and carbohydrate metabolism in the responding gene clusters,
which is consistent with a cold-induced modification of the substrates and sources of energy
metabolism, and of the pathways involved.
A second major experiment involved the exposure of carp to chronic hypoxia [47]. Carp
is unusually tolerant to hypoxia which allows it to prosper in natural ponds. This experiment
involved over 700 microarrays, comprising responses to hypoxia in 7 tissues but also at two
environmental temperatures, 17C and 30C. Again this revealed strong patterns within and
between tissues which were surprisingly different between the two exposure temperatures.
Curiously, a transcript corresponding to myoglobin was substantially up-regulated by hypoxia
in carp liver. Myoglobin is an oxygen-binding protein which was famous for its role in
protecting oxidative muscle cells including cardiac myocytes. Expression of this gene in nonmuscle tissues is linked to the presence of myoglobin protein in the liver of hypoxia-treated
fish [48] and that the amount of protein increases in carp exposed to chronic hypoxia. It was
also found that carp possess a second isoform of this important gene, whose expression was
477
limited to the brain, this being the only known vertebrate species possessing two myoglobin
isoforms. This fact is likely to be linked to a whole genome duplication event some 15MYA
in a clade of fish that includes the goldfish and crucian carp [49].
This discovery of non-muscle expression of this gene was an entirely serendipitous and
unexpected finding, arising directly from an open microarray screen that was directed at the
discovery of both genes and their environmental responses. It is notable because it flies in the
face of conventional respiratory physiology of vertebrates animals. Similar myoglobin
expression has been found in zebrafish and the mouse system is now being explored. This
phenomenon is likely to have major implications for understanding hypoxia responses in
vertebrate animals generally but particularly in humans where hypoxia injury caused by
ischaemia and infarct are major clinical conditions in the developed world.
Table 1. Integration of different UK research facilities in the microarray research
What?
DNA sequencing
Sample acquisition
Sequencing
Annotation
Microarray Construction
Oligo design
Oligo synthesis
cDNA clones and EST
sequencing
Microarray printing or
submission of in silico
oligoprobes to 3rd part
fabrication
Microarray experiment
Experimental design
Sample acquisition
Array hybridisation
Image acquisition and
quantification
Data analysis
Interpretation
Publication
Researcher
Who?
MGF- MGF-Liverpool MGF-Liverpool NEBC
Edinburgh
(AGF)
(LMF)
The combination of data from both cold and hypoxia treated carp constitutes a very large
data set which can be used to explore the correlation of gene expression properties between
pairs of genes. This allows the regulatory relationships between genes to be explored, as
whether the correlated profiles between genes are conserved between species. More than 400
gene pairs have been identified whose close expression relationships are conserved between
carp and human (Li, W and AR Cossins, to be published), and thus by extension across all
vertebrate animals. Finally, the expression data collected on such a large scale and across
several different tissues and stress modalities, constitutes another resource with which to
differentiate the identities of closely-related members of gene families, which completes the
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more usual sequence-based approaches. This is a particular problem in recently duplicated

genomes, such as the common carp [49] and the salmonid fish. Indeed, genes given a
common identity by homology alignment proved to have distinguishable expression
properties. On further investigation these expression forms have been found to constitute
separate genes.
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Reviewed by William H. Wilson of Bigelow Laboratory for Ocean Science Research,

Maine, USA.
INDEX
A
abatement, 126, 458
absorption, x, 43, 64, 77, 173, 175, 176, 179, 192,
212, 320, 322, 476
abstraction, 326, 335, 353
abuse, xv, 423
accessibility, 237, 238, 242, 244, 245, 249, 254, 255
acclimatization, 235
accounting, 21, 212
accreditation, 403
accuracy, 27, 290, 292, 293, 351, 352, 353
acetic acid, 431
acid, 17, 19, 27, 33, 120, 122, 141, 144, 153, 155,
156, 192, 222, 226, 227, 228, 233, 235, 236, 362,
368, 369, 377, 379, 431, 435, 438, 440, 441, 442,
443, 444, 445, 446, 447, 448, 449, 450, 451, 455,
456, 457, 458, 459, 460, 461, 462, 463
acidity, 446
activated carbon, 130, 179, 180, 434, 436, 445, 449,
450, 451, 452, 461, 462, 463
activation energy, 449, 457
active centers, 446
active site, 212, 446, 447
adaptability, 96
adaptation, 118, 373, 475, 476
additives, 31, 150, 154, 387, 403
adjudication, 403
adjustment, 97, 265, 395
adsorption, x, 18, 65, 68, 70, 97, 173, 175, 176, 177,
179, 182, 436, 442, 445, 446, 447, 449, 450, 451,
452, 459, 461, 462
advantages, xiii, 25, 31, 61, 62, 64, 81, 221, 357,
364, 378, 410, 434, 436, 470
advocacy, 120
aerobic bacteria, 48
aerogels, 450, 463

aflatoxin, xv, 386, 400, 410
AFM, 183
Africa, 5, 17, 20, 35, 116, 400
agar, 25, 26, 34, 222
agencies, xiv, 385, 391, 393, 398, 403, 411
aggregation, 221
aging process, 259, 260, 265, 266, 267
agricultural exports, xiv, xv, 385, 386, 399
agricultural market, 402, 403, 406
agricultural sector, 309, 310, 312
agriculture, 54, 75, 84, 100, 127, 141, 153, 162, 220,
307, 308, 309, 310, 311, 312, 315, 319, 387, 398,
399, 408, 416, 417, 424, 475
AIDS, 24, 25
air emissions, 155
air pollutants, 112
alanine, 227, 233
aldehydes, 448
algae, 24, 36, 436
algorithm, 469
allele, 27
alters, 379
aluminum oxide, 460
amino acid, 227, 235
amino acids, x, 63, 85, 141, 219, 224, 227, 228, 233
ammonia, 55, 82, 123, 163, 227, 358, 359, 365, 366,
370, 374, 376, 377, 378, 380, 382
ammonium, 63, 82, 377, 462
amphibians, 480
amplitude, 259
amylase, 82, 163
anaerobic bacteria, 60
anaerobic sludge, 46, 146
anatase, 446
anger, 468
482
Index
animal disease, 358, 417

animal welfare, xiv, 386
anionic surfactants, 437, 455
annealing, 383
annotation, 468, 469, 473, 479
annual rate, 399
ANOVA, 470
anoxia, 478
antagonism, 85
anthrax, 358, 380
antibiotic, 401, 410
antibody, 28
antioxidant, 280
anus, 45
apples, 425
aquaculture, 411
aquatic habitats, 320
aquatic life, 427
aqueous solutions, 174, 175, 182, 224, 453, 454,
457, 458, 459, 460, 461, 463
aquifers, xii, 16, 325, 326, 327, 328, 331, 333, 334,
335, 337, 338, 339, 350, 352, 353, 354
architecture, 278
Argentina, 100, 107, 174
Aristotle, 43
aromatic compounds, 373, 436, 441, 454
aromatic hydrocarbons, 43, 61, 76, 454
aromatics, 147
arrest, 45, 139, 152
arrests, 75
ARs, xiv, 385
arsenic, ix, x, xiii, 130, 156, 173, 174, 175, 325, 326,
327, 328, 338, 353
arthritis, 20, 38
arthropods, 84
asbestos, 131, 152, 154
aseptic, 16
Asia, xv, 17, 20, 76, 105, 114, 116, 120, 168, 287,
289, 386, 391, 393, 399, 419, 420, 421, 423
asian countries, 114, 399
assessment, xii, 5, 6, 31, 164, 191, 213, 254, 285,
289, 299, 304, 318, 324, 366, 374, 382, 383, 393,
395, 429
assimilation, 319
atmosphere, vii, 3, 130, 428
atmospheric pressure, 130
atomic force, 183
atomic force microscope, 183
atoms, 435
atypical pneumonia, 24
Austria, 37, 114, 157, 164, 168

authorities, 100, 116, 309, 310, 314, 400
automation, xiii, 29, 357
automobiles, 122, 123, 136, 141, 148, 156
avoidance, 134
awareness, xii, xiv, 31, 119, 132, 133, 135, 158, 305,
306, 310, 319, 320, 385, 396, 410, 411, 416, 417,
466
B
BAC, 463, 468
Bacillus subtilis, 222, 371
bacteria, viii, xiii, 12, 13, 19, 20, 23, 24, 25, 29, 32,
36, 39, 43, 48, 55, 60, 64, 75, 78, 82, 83, 85, 97,
99, 107, 122, 126, 127, 145, 146, 147, 162, 222,
357, 358, 359, 361, 362, 363, 365, 367, 369, 370,
374, 377, 379, 381, 382, 383, 387, 391, 413, 414,
415, 428, 434
bacterial infection, 24
bacterium, 19, 25, 26, 98, 358, 382
Bangladesh, vi, xiii, 174, 325, 326, 327, 328, 353,
354, 355
banks, 240, 316, 323
barium, 119, 156
barometric pressure, 338
barriers, 30, 156, 401, 407
base pair, 367, 368, 382
basicity, 446
baths, 120, 192
batteries, 121, 122, 123, 124, 136, 141, 149, 155
bauxite, 149, 153
beef, 18, 222
behaviors, 255, 447
Beijing, 323
Belgium, 116, 139, 155
beneficial effect, 472
benefits, xv, 122, 142, 162, 242, 252, 255, 424, 430,
433, 468
benzene, 123, 129, 156, 461, 462
benzo(a)pyrene, 98
beta particles, 125
beverages, 150
bias, 29, 360, 377, 381
bicarbonate, 374, 412, 438
bile, 25
bioaccumulation, 77, 80, 188, 434
biochemistry, 391
bioconversion, 147, 161
biodegradability, 122, 127
Index
biodegradable wastes, 47, 126
biodegradation, viii, x, 42, 43, 49, 51, 58, 59, 64, 65,
78, 82, 97, 98, 99, 108, 124, 126, 127, 141, 144,
145, 159, 169, 219, 220, 221, 222, 223, 224, 225,
227, 233, 234, 235, 382, 437
biodiversity, 286, 289, 306, 320, 322, 323, 360
biofuel, 146, 159
biogas, 146, 159, 160, 161, 162
bioindicators, 188, 216
bioinformatics, xvi, 465, 466, 467
biological control, 392
biological stability, 13
biological systems, xi, 257, 258, 264, 265, 466
biomarkers, 268
biomass, vii, 3, 7, 43, 45, 47, 48, 58, 59, 60, 62, 63,
65, 67, 76, 97, 105, 126, 127, 143, 145, 146, 147,
162, 163, 226, 235, 236, 369, 371
biomonitoring, xi, 271, 272
bioremediation, 81
biosensors, 29, 36
biosphere, 100, 258, 479
biosynthesis, 282
biotechnology, viii, 42, 82, 122, 143, 144, 392
biotic, 63, 290
biotin, 371
birds, xi, 154, 160, 163, 217, 271, 272, 273, 274, 275,
277, 279, 281, 282, 323, 324, 426, 427, 429
bisphenol, 75
blood pressure, 425
body fat, x, 187
body fluid, 121
body size, 192, 207
body weight, 43, 192, 193, 206, 208, 393, 395
boilers, 149, 150, 156
bonds, 453
bone, 189, 191, 205, 212
bones, 44, 52, 118, 220
boundary conditions, 352
brain, 16, 476, 477
Brazil, 45, 145, 147
breakdown, 99, 145, 163, 213, 405
breeding, xi, 271, 274, 275, 279
brevis, 30
bridges, 148
Britain, 242, 255
Brno, 215
buffalo, 49
buildings, 131, 152, 241, 307, 426
Bulgaria, 257, 271
Burma, 289
483
burn, 264
bursa, 191
butyl ether, 447
buyers, 132, 138, 139, 165, 404
by-products, xv, 70, 122, 154, 433, 435, 441, 447,
450, 460
C
cabbage, 84
cabinets, 121
cadmium, x, 54, 57, 64, 76, 77, 119, 123, 130, 155,
156, 187, 191, 193, 196, 198, 201, 205, 207, 208,
209, 210, 211, 212, 213, 214, 215, 216, 217
calcium, 56, 59, 77, 85, 153, 473
calcium carbonate, 59, 473
campaigns, 416, 417
cancer, 17, 378, 391, 392, 393, 425, 426
candidates, 307
capillary, 468
capital intensive, 131
capitalism, 252
capsule, 127, 189
carbohydrate, 233, 476
carbohydrate metabolism, 476
carbohydrates, 44, 49
carbon, vii, 3, 4, 7, 11, 33, 35, 61, 62, 63, 64, 123,
125, 126, 128, 130, 143, 145, 146, 147, 153, 156,
159, 179, 180, 220, 234, 376, 434, 436, 449, 450,
451, 452, 453, 454, 457, 459, 461, 462, 463
carbon dioxide, 61, 62, 125, 128, 145, 159, 220
carbon materials, 461
carbon monoxide, 64, 128, 147
carboxylic acids, 448, 459
carcinogenicity, 122
Caribbean, 242
carotenoids, 279, 282
carp, 467, 469, 475, 476, 477, 480
cartilage, 189
case study, 251, 255, 323, 377
cash, 140
casting, 141
catalysis, 442, 445, 447, 458, 461
catalyst, 440, 441, 442, 444, 445, 446, 447, 448, 449,
450, 451, 452, 456, 458, 459, 460, 461, 462
catalytic activity, 440, 445, 446, 447, 448, 449, 450,
451, 463
catalytic effect, 443
catalytic reaction, 449, 450
catalytic system, xvi, 433, 435
484
Index
category a, 121
cation, 224, 443
cattle, 21, 45, 48, 50, 56, 59, 61, 63, 85, 86, 87, 89,
90, 117, 143, 150, 235, 293, 361, 406
CBS, 280
cDNA, 373, 375, 378, 469, 475, 476, 477
cell culture, 27, 371
cell line, 25, 362, 373
cell membranes, 476
cellulose, 46, 48, 49, 145, 222
cement, 21, 112, 115, 141, 146, 150, 151, 153, 154,
156
cement plants, 141, 154
census, xi, 237, 238, 239, 240, 241, 242, 247, 255
ceramic, 138, 150, 151, 192, 222
certificate, 409
certification, 407, 410, 412, 417
cesium, 125
cestodes, 188, 216
charitable organizations, 155
cheese, 143
chemical, viii, 17, 19, 21, 42, 43, 44, 46, 48, 54, 55,
56, 58, 62, 67, 70, 76, 77, 78, 81, 82, 85, 87, 89,
90, 91, 92, 95, 96, 99, 100, 119, 122, 123, 124,
125, 126, 130, 135, 137, 138, 142, 144, 145, 147,
150, 153, 154, 156, 174, 182, 183, 222, 235, 236,
259, 260, 378, 393, 394, 395, 407, 412, 428, 429,
434, 436, 437, 439, 445, 446, 449, 450, 463
chemical kinetics, 259
chemical properties, 236, 378, 407
chemical reactions, 58
chemical reactivity, 122
chemical stability, 446
chemicals, viii, ix, 42, 43, 44, 54, 55, 61, 66, 75, 76,
77, 78, 82, 84, 90, 98, 118, 119, 121, 122, 123,
129, 130, 133, 138, 139, 141, 158, 394, 408, 412,
416, 418, 426, 427, 428, 431
chemiluminescence, 25
chemoreceptors, 44
Chicago, 302, 376
chicken, 53, 281
child labor, xiv, 386
childhood, 34
Chile, 174, 400
China, 45, 67, 114, 120, 139, 145, 289, 322, 323, 423
chitin, 21, 84
chlorinated hydrocarbons, 130
chlorination, viii, 12, 13, 19, 30, 36, 437
chlorine, viii, 12, 21, 30, 401, 413, 415, 436, 452
chlorobenzene, 447, 460
chloroform, 274
cholera, 20, 23, 35
cholinesterase, 427
chromatography, 454
chromium, 77, 119, 123, 156
chromosome, 369
chronic diseases, 260, 267, 425
chronic hypoxia, 476
chronic illness, 426
chronology, 377
circulation, vii, 3, 209, 210, 212, 214
cities, 12, 124, 131, 145, 396
citizens, xv, 133, 386
city, 11, 55, 112, 140, 161, 166, 168, 240, 421
civilization, viii, 42, 97
class, 114, 121, 211, 290, 293, 427, 428
classes, 153, 290, 397
classification, 5, 6, 158, 252
cleaning, 46, 68, 76, 136, 143, 154, 156, 413, 415,
456
cleanup, 152
clients, 470
climate, vii, 3, 4, 5, 6, 7, 50, 165, 307, 308, 313
climate change, 5, 7, 50, 165
clitellum, 44
clone, 358, 359, 360, 366
cloning, 282, 359, 360, 378, 469
closure, 160
clusters, 472, 476
coagulation process, 436
coal, 78, 120, 145, 148, 149, 153, 156, 159, 398
coastal management, 322
cobalt, 457, 458
cocoon, 45
coding, 381, 473
coffee, 399
coke, 148, 149
colonization, 24, 31, 39, 68
color, 56, 150, 222, 282, 328, 401, 407, 434, 436,
441, 449
colour patches, 272
combined effect, x, 187
combustion, vii, 3, 123, 130, 146, 147, 153, 164, 175
commercial crop, 221
commodity, 113, 165, 394
communication, 26
community, xvi, 13, 24, 29, 32, 38, 39, 40, 61, 108,
135, 161, 164, 166, 169, 252, 286, 293, 335, 359,
360, 361, 364, 369, 371, 372, 373, 374, 375, 376,
Index
377, 378, 379, 380, 381, 382, 383, 384, 392, 452,
465, 466, 467, 471, 472, 479
competition, 203, 279, 364, 407, 467
competitive advantage, 165
competitiveness, xiv, 385
competitors, xiv, 386
complaints, 428
complement, 466
complementary DNA, 382
complexity, ix, 112, 131, 258, 259, 436, 451, 452,
472
compliance, xiv, 386, 404, 408, 409, 416, 472
complications, 19
composites, 121
composition, 46, 54, 118, 144, 163, 221, 222, 224,
227, 228, 233, 242, 243, 247, 290, 312, 313, 355,
360, 364, 366, 374, 376, 377, 379, 382, 384, 438
compost, 43, 46, 47, 54, 55, 59, 60, 61, 62, 63, 78,
79, 80, 82, 85, 86, 87, 89, 90, 93, 94, 95, 97, 99,
102, 105, 126, 136, 140, 141, 143, 144, 145, 146,
160, 161, 162, 163, 222, 227, 235, 236, 431
composting, viii, ix, 42, 43, 44, 45, 47, 49, 50, 55, 57,
58, 59, 60, 61, 62, 63, 87, 96, 97, 98, 99, 105, 106,
107, 108, 112, 125, 126, 127, 137, 143, 144, 145,
159, 161, 162, 163, 164, 222, 234, 236
compounds, xv, 17, 62, 63, 76, 78, 79, 80, 82, 98,
119, 123, 128, 129, 130, 150, 156, 177, 179, 181,
220, 221, 226, 227, 231, 367, 373, 391, 423, 426,
433, 434, 436, 437, 438, 440, 441, 442, 443, 445,
446, 448, 449, 450, 451, 452, 453, 454, 455, 456,
460, 462
compression, 220
computing, 471
conceptual model, 253
conditioning, 151, 319
conductance, 25, 34
conduction, 333
conductivity, 65, 221, 258, 327, 331, 340, 341, 342,
352, 355
conference, 104
configuration, 159, 436
conflict, 309, 313
conformity, 409
connective tissue, 210
connectivity, xiii, 287, 307, 321, 325, 338, 353
consciousness, 132
consensus, 131
consent, 312
conservation, 126, 138, 164, 165, 286, 287, 289,
290, 291, 292, 301, 302, 304, 305, 307, 308, 309,
485
310, 313, 314, 315, 316, 317, 318, 320, 321, 322,
323, 324
constant rate, 332, 350
constitution, 286
construction, 30, 113, 128, 131, 140, 144, 145, 148,
152, 153, 165, 307, 308, 311, 316, 319, 351, 353,
467, 469, 472, 473, 474
consumer choice, 254
consumer demand, 395, 396
consumer education, 132, 164
consumer electronics, 121, 130
consumer goods, 122, 132, 137, 138, 164
consumers, 13, 30, 114, 119, 120, 132, 133, 137,
138, 140, 157, 164, 165, 310, 391, 396, 401, 402,
412, 416, 418
consumption, vii, 11, 16, 17, 20, 22, 33, 38, 64, 112,
113, 114, 132, 133, 135, 136, 139, 147, 148, 157,
162, 164, 189, 213, 224, 391, 393, 394, 396, 406,
407, 412, 425, 438, 447, 449
contact time, 17
contaminant, 31, 99, 188, 191, 434, 443, 444, 446,
447, 450, 452
contaminated sites, 76
contaminated soil, 78, 79, 80, 98
contaminated soils, ix, 42, 43, 76, 78, 99
contaminated water, xiv, 13, 24, 30, 327, 386
contamination, viii, xiii, 12, 13, 16, 17, 25, 31, 35,
123, 162, 174, 211, 263, 272, 325, 326, 354, 391,
392, 398, 399, 400, 403, 412, 413, 415, 416, 428,
429, 431, 434
content analysis, 306
contract enforcement, 404
control measures, 406, 407
controversies, 314
convention, 126
cooking, 48, 119, 143, 146, 150, 391, 394, 412, 414,
415
cooling, 48, 64, 404, 414
coordination, 174, 175, 176, 179, 260, 365, 379, 405
copper, 54, 77, 85, 144, 148, 149, 174
copulation, 44
corporate sector, 411
correlation, 208, 279, 372, 477
correlation coefficient, 208
correlations, 268, 376
corrosion, 148
corrosivity, 122
cosmetics, 123, 401
cost, viii, ix, xiii, xiv, xvi, 12, 13, 22, 25, 29, 30, 32, 40,
54, 76, 96, 97, 98, 99, 112, 113, 114, 120, 128,
Index
486
131, 138, 139, 140, 141, 145, 147, 148, 149, 152,
153, 154, 161, 162, 165, 357, 359, 386, 394, 400,
404, 409, 416, 418, 429, 440, 465, 466, 470
cost effectiveness, 418
costs of compliance, 408
cotton, 127, 136, 138, 149, 169, 424, 427, 430
covering, 47, 159, 286, 287, 293, 300, 306, 312, 388,
406, 407, 466
coxsackievirus, 17
cracks, 413
crew, 221
crisis management, 408, 418
critical period, 425
critical value, 248
criticism, 311
crop production, 221
crops, 18, 39, 84, 85, 87, 89, 90, 95, 96, 100, 150,
221, 235, 388, 391, 392, 393, 394, 406, 412, 424,
425
cross-fertilization, 44
crude oil, 43
crust, 125
cryptosporidium, 37
crystalline, 175
cues, 279
cultivation, 221, 231, 232, 308, 311, 377, 412
cultural barriers, 401
culture, 25, 26, 27, 28, 36, 37, 65, 113, 119, 132,
162, 163, 219, 221, 222, 223, 229, 230, 231, 232,
233, 234, 235, 236, 358, 359, 373, 468
culture media, 25, 37
cumulative frequency, 247, 248
cumulative percentage, 248
current limit, 380
curricula, 417
customers, 140
cuticle, 44, 212
cyanide, 130, 456
cycles, 7, 19, 21, 38, 449, 450
cycling, 7, 366, 367, 371, 383, 473
cyst, x, 24, 39, 187, 189, 190, 191, 193, 194, 196,
197, 198, 199, 201, 202, 203, 204, 208, 209, 210,
211
cystathionine, 280, 283
cysteine, xii, 227, 233, 272, 280, 283
D
damages, 260, 264
danger, 30, 121
Darwin, Charles, 43, 96, 100

data analysis, 29, 351, 353, 355, 466, 470
data collection, 32, 338
data set, 344, 345, 472, 477
database, 5, 239, 240, 241, 375, 378, 471, 474, 478
Dead Sea, 308
deaths, xv, 18, 121, 386, 425, 426
decay, 75, 244, 245
decentralization, 161
decision makers, 310, 318, 319, 320
decomposition, vii, xv, 3, 5, 6, 7, 43, 48, 50, 59, 61,
63, 64, 96, 126, 127, 159, 160, 162, 163, 235, 236,
433, 435, 436, 438, 439, 444, 445, 446, 447, 448,
449, 450, 451, 453, 454, 456, 457, 458, 460, 461,
463
defects, 393
deficiencies, 306, 312, 317, 319, 320
deficiency, 221, 231, 252, 279, 283
deforestation, 137, 148, 164, 466
degradation, viii, xv, 17, 42, 46, 49, 50, 52, 55, 58,
59, 65, 86, 98, 99, 125, 145, 338, 360, 373, 377,
378, 433, 434, 437, 438, 439, 440, 441, 443, 446,
451, 455, 456, 457, 459, 460, 461, 463
degradation process, 46
Degussa, 440, 441, 442
dehydration, 22, 54, 191
dematerialization, 136
demographic data, 302
demography, 126
denitrification, 365, 366
denitrifying, 236, 377, 382, 434
Denmark, 20, 116, 131, 139, 155, 164, 479
Department of Agriculture, 405, 407, 409, 421, 430
Department of Energy, 83
Department of Health and Human Services, 33, 430
Department of the Interior (DOI), 355, 431
dependent variable, 208, 289
deposition, 6, 68, 198, 209, 212, 213, 214, 279, 323,
327, 475
deposits, x, 40, 187, 191, 206, 207, 212, 214
depression, 331, 333, 346
deprivation, xi, 237, 238, 242, 243, 247, 249, 250,
251, 252, 255
derivatives, 440
desorption, x, 173, 174, 175
destination, 244, 245
destiny, 182
destruction, viii, 42, 59, 63, 130, 213, 293, 392, 458,
459, 460, 466
Index
detection, xiii, 17, 25, 26, 27, 28, 29, 31, 32, 33, 34,
35, 36, 37, 39, 40, 272, 273, 357, 362, 363, 364,
365, 366, 367, 369, 370, 371, 373, 374, 375, 377,
378, 379, 380, 381, 382, 383, 384, 400, 401, 408,
441, 466
detection system, 28
detergents, 122, 415
detoxification, 130, 209, 210
developed countries, vii, 11, 12, 17, 21, 22, 30, 31,
32, 124, 163
developed nations, 115, 117, 120, 139, 154, 155
developing countries, vii, xiv, 11, 17, 19, 20, 21, 116,
155, 158, 165, 174, 386, 416, 426, 430
developing nations, 76, 120, 126
deviation, 245
diabetes, 425
diagnosis, 26, 268, 430
diamonds, 177
diaphragm, 210
diarrhea, 18, 38, 421
diesel engines, 146
diet, xii, 189, 209, 212, 272, 273, 279, 281, 391, 393,
394, 395, 398, 425, 480
dietary intake, 209, 210, 394
diffraction, 175, 176
diffusion, 258, 365, 444, 445
digestion, 23, 136, 161, 192, 441, 456
dimorphism, 211, 281
dioxin, 43, 120
dioxins, 119, 130, 131, 146, 151, 154, 388
direct investment, 410
directives, 157, 164, 318, 320
dirt, 127, 152
disability, 242
disadvantages, xv, 433, 434
discharges, 330
discriminant analysis, 274
discrimination, 366, 367, 383
disinfection, viii, xv, 12, 13, 21, 24, 30, 34, 36, 37, 42,
433, 434, 458
dispersion, 82, 449
displacement, 29, 248, 371
disposable income, 396
dissipative structure, 258
dissociation, 367, 368, 379, 380, 384
dissolved oxygen, 222, 224
distillation, 156
distilled water, 192
disturbances, 259, 273, 286, 293, 303
diversification, 405
487
diversity, xiii, 13, 252, 286, 306, 323, 357, 358, 359,
360, 373, 377, 379, 380, 381, 382, 383, 384, 474,
479
DNA, vi, xiii, 28, 29, 32, 37, 273, 277, 357, 359, 360,
361, 362, 365, 368, 369, 370, 371, 372, 373, 374,
375, 377, 378, 379, 380, 381, 382, 383, 384, 466,
468, 469, 470, 477, 478
DNA extraction, 360, 381
DNA polymerase, 371
DNA sequencing, 466, 468, 477
DNase, 375
DOC, 454
dogs, 21, 116, 117
domestic markets, 387
DOP, 317
dosage, 283
double bonds, 453
draft, 255
drainage, 62, 82, 151, 306, 307, 310, 311, 312, 316,
319, 324
drawing, ix, 112, 471
drinking water, viii, ix, xv, 12, 13, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37,
38, 40, 120, 151, 173, 174, 393, 428, 431, 433,
435, 436, 452, 453, 454, 460
drosophila, 466, 478
drug resistance, 20
drugs, 387, 401
drying, 192, 308, 406, 410
dumping, 99, 116, 120, 165, 220
duodenum, 210
dyeing, 122
dyes, 27, 123, 130, 362, 455, 457, 458
E
E.coli, 18, 60, 61
early warning, 38
earthworms, viii, ix, 41, 42, 43, 44, 45, 46, 47, 48, 50,
51, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, 100, 101, 102, 104, 107, 108, 126,
127, 143, 145, 146, 169
East Asia, xv, 386, 399
Eastern Europe, 419
ecology, xiii, 13, 31, 32, 303, 322, 323, 324, 357,
359, 360, 369, 371, 374, 375, 376, 377, 379, 380,
381, 383
economic consequences, 13
488
Index
economic development, 317

economic growth, 395
economic incentives, 392
economic independence, 315
economic losses, 22, 400, 425
economic problem, 140, 155
economy, 97, 116, 136, 138, 140, 141, 309, 424
ecosystem, xii, 122, 126, 136, 138, 141, 148, 278,
285, 286, 289, 302, 313, 321, 322, 466
editors, 38, 282
education, 31, 119, 132, 166
educators, 133
efficiency, 50, 72, 105, 107, 136, 462
effluent, 18, 67, 220, 373, 379, 398, 411, 437, 440,
441, 443, 455, 456
effluents, xii, 36, 46, 67, 150, 161, 220, 305, 306,
307, 308, 378, 434, 436, 439, 440, 443, 451, 455,
460, 462
egg, 21, 52, 139, 149, 394
Egypt, 400
electric charge, 260
electric conductivity, 327
electric current, 130, 148, 258
electricity, 131, 146, 150, 159, 160, 161, 162, 165
electrolysis, 155
electromagnetic, 125, 259
electromagnetism, 258
electron, 175, 176, 183
electronic structure, 174
electrons, 441
electrophoresis, 26, 27, 29, 360, 378, 380, 381
elongation, 83, 227, 229
elucidation, 374
emission, ix, 48, 61, 64, 96, 112, 119, 124, 125, 129,
131, 142, 144, 146, 147, 148, 149, 156, 159, 160,
163, 165, 175, 177, 178, 179, 180
emitters, 125
employment, 242
encephalitis, 16, 388
encephalopathy, xiv, 385
encoding, 358, 380, 480
endangered species, 289, 292
endocrine, 75, 98, 426, 429, 430, 467
endonuclease, 26
energy, viii, xi, 42, 62, 74, 75, 124, 125, 126, 131,
134, 135, 136, 137, 142, 143, 146, 148, 149, 150,
151, 154, 156, 164, 165, 177, 178, 179, 180, 181,
209, 213, 235, 257, 259, 260, 261, 262, 268, 358,
449, 457, 476
energy consumption, 136
energy recovery, 131, 146

energy supply, 165
energy transfer, 260
enforcement, xiv, 309, 314, 385, 402, 404, 407, 408,
411, 416
engineering, 126, 127, 153
England, vi, xi, 37, 39, 45, 145, 237, 238, 239, 252,
254, 473
enlargement, 283
enrollment, 397
enterovirus, 16, 33
entrepreneurs, 404
entropy, 258, 264, 265
environmental awareness, 319, 320
environmental change, 273, 466
environmental conditions, xi, 21, 144, 252, 268, 271,
280, 466, 472, 475
environmental contamination, 31, 211, 416
environmental degradation, 17
environmental effects, 158
environmental factors, 292, 379
environmental impact, 133, 135, 434
environmental influences, xi, 257
environmental issues, 120, 133, 319
environmental management, viii, 41, 320
environmental policy, 305, 324
environmental protection, 137
Environmental Protection Agency (EPA), 13, 22, 109,
166, 428, 431
environmental quality, 252, 313
environmental regulations, 120
environmental temperatures, 34, 476
enzymatic activity, 106
enzymes, 25, 48, 49, 59, 81, 82, 84, 99, 122, 145,
162, 163, 365, 382
eosinophils, 210
epidemic, 17
epidemiology, 22, 26, 32, 40
epithelial cells, 280
epithelium, 476
EPR, 157
equilibrium, 176, 179, 269, 332, 335, 340, 342, 367,
368, 380, 384
equilibrium sorption, 179
equipment, 29, 123, 131, 139, 141, 148, 155, 156,
224, 364, 408, 409, 413, 427, 428, 470
equity, xi, 237, 238, 252, 253, 254, 255, 256
erosion, 6, 139, 145, 156, 221, 327
esophagus, 55, 56
EST, 466, 468, 470, 472, 477
Index
ethanol, 143, 146, 147, 175, 176, 177
ethics, 139, 155
ethnic groups, xi, 237, 242, 247, 248, 249, 252, 255
ethnicity, 237, 238, 242, 252
ethyl alcohol, 150
etiology, 22, 23, 37, 380
eukaryotic cell, 373
European Commission, 401
European Community, 115
European Union (EU), xv, 113, 131, 139, 151, 155,
157, 166, 386, 396, 397, 400, 401, 408, 409, 410,
418
evaporation, 150, 160
EXAFS, 175, 176, 184
excretion, 77, 99
execution, 469
exercise, 253
expenditures, 126, 255
experiences, ix, 112
experimental condition, 440, 445, 446, 448
experimental design, 467, 470
expertise, 466, 467, 468, 469, 472
exploitation, xii, 305, 306
exploration, 255, 257, 274, 375
exporter, 400
exports, xiv, xv, 385, 386, 399, 400, 401, 407, 408,
409, 416, 417, 418
exposure, 12, 17, 209, 211, 213, 394, 395, 396, 426,
427, 430, 476
expressed sequence tag, 468, 478
external environment, 21
extinction, 287
extraction, 47, 107, 125, 151, 212, 360, 373, 381,
402, 434, 468, 470, 474
F
fabrication, 466, 469, 477
factor analysis, 22
factories, 220, 410
false negative, 302
false positive, 302
famine, 479
farm size, 404, 410
farmers, 43, 45, 82, 85, 96, 100, 144, 145, 404, 405,
406, 407, 410, 411, 416
farmland, 76, 82, 100
farms, 43, 54, 97, 140, 144, 162, 235, 241, 409
fat, x, 187, 191, 192, 205, 206, 207, 212, 214, 407
fatty acids, 150
489
faults, 312, 317

fauna, 43, 293
FDA, 391, 394
fears, 408
feces, 18, 20, 47, 48
feedback, 253, 260, 261, 262
femur, 191, 192, 203, 205, 212
fencing, 151, 241
fermentation, 146, 147, 220, 224, 236
ferrous ion, 457, 458
fertility, viii, 42, 45, 82, 83, 96, 100, 145, 220, 227,
233, 307
fertilization, 44, 221, 222, 231, 235
fertilizers, x, 81, 85, 86, 87, 89, 90, 91, 92, 95, 96, 99,
100, 143, 146, 219, 221, 223, 224, 226, 227, 228,
229, 230, 231, 232, 233, 307, 428
fever, 16, 18
fiber, 152, 450, 463
fibers, 121, 127, 150
fibrin, 210
fibrosis, 378
field trials, 84
films, 122, 151, 457
filters, 40
filtration, viii, 18, 25, 30, 42, 65, 66, 68, 74, 75, 76,
97, 98, 449
financial resources, 155
financial support, xii, 285
Finland, 18, 40, 116
fish, x, 53, 63, 127, 174, 188, 212, 215, 216, 219,
220, 221, 222, 233, 234, 235, 240, 279, 371, 379,
396, 399, 401, 408, 412, 413, 415, 427, 429, 432,
467, 475, 476, 478, 480
fisheries, 220, 235, 399, 401
fishing, 114
fission, 124, 125
fitness, 191
fixation, 99, 361, 366, 374, 381
flame, 130, 192, 414, 415
flammability, 122
flatness, 332
flexibility, 123, 309
flocculation, 30, 436
flooding, 306, 313, 335
flora, 48, 80, 408
flotation, 150
flour, 426
fluctuations, xiii, 308, 325, 335, 338, 353, 475
flue gas, 153
fluid, 20, 47, 64, 75, 136, 463
Index
490
fluorescence, 25, 28, 175, 177, 178, 179, 180, 181,

361, 362, 374, 470
fluorophores, 29
foams, 222
food additives, 387, 403
food industry, 46, 145
food poisoning, 398, 429
food production, 46, 122
food products, 20, 54, 97, 135, 392, 400, 401, 403,
407, 416
food safety, xiv, xv, 385, 386, 387, 388, 393, 396,
397, 398, 399, 400, 401, 402, 403, 404, 405, 406,
407, 408, 410, 411, 416, 417, 418, 419
food security, xiv, 386
foodborne illness, 18, 391, 412, 415
foreign direct investment (FDI), 410, 420
formaldehyde, 123
formamide, 367, 369
formula, 179, 181, 192, 223, 245, 332, 338, 346
fouling, viii, 12, 13
foundations, 152
fragments, 378, 468, 469
framing, 152
France, 45, 46, 115, 125, 139, 145, 146, 164, 165,
436
free radicals, 261, 437, 443, 454
free trade, 120
freezing, 414, 475, 476
frequencies, xi, 209, 259, 271, 278
frequency distribution, 247
freshwater, xiii, 38, 162, 216, 358, 359, 376, 377,
475
fruits, 84, 85, 96, 108, 136, 221, 396, 399, 413, 416
funding, 31, 151, 256, 302, 466, 471
fungi, 24, 43, 60, 78, 82, 83, 84, 97, 122, 126, 127,
145, 235, 387, 428, 442
fungus, 52, 55, 60, 75, 81, 84
fusion, 24, 25, 374
G
gamma rays, 125
garbage, ix, 61, 96, 112, 115, 116, 141, 147, 162,
163, 308, 406
gastroenteritis, 16, 18, 20, 22, 32, 37
gastrointestinal tract, 20, 24, 211
GDP, xv, 157, 386, 395
GDP per capita, 395
gel, 26, 27, 360, 364, 365, 367, 368, 371, 378, 380,
381, 382
gender differences, 203

gene arrays, 28, 373, 384
gene expression, xiii, 280, 357, 362, 373, 374, 378,
380, 382, 476, 477, 478, 479, 480
genes, 24, 27, 28, 272, 358, 359, 360, 362, 363, 365,
366, 367, 369, 370, 371, 372, 373, 374, 376, 377,
378, 379, 380, 381, 382, 383, 384, 470, 471, 472,
473, 474, 475, 476, 477, 478, 480
genetic diversity, 474
genetic information, 260, 378
genetic mutations, 265
genetics, 289, 302
genome, 27, 28, 40, 261, 371, 378, 379, 380, 383,
384, 466, 468, 473, 474, 477, 478, 479, 480
genomics, xvi, 465, 466, 468, 472, 480
genotype, 479
geography, 126
geology, 355
Georgia, 168, 255
Germany, 62, 115, 117, 139, 224, 400
germination, 83, 87, 95, 223, 226, 229, 231, 232,
235, 442
gizzard, 49, 56, 81, 97, 99
glasses, 116, 136, 138, 142, 147, 150, 151, 161
global climate change, 5
global consequences, 165
global economy, xiv, 386
globalization, xiv, 386, 399, 416
glucose, 33
glutamate, 280
glutathione, 280, 281, 283
glycine, 227, 233, 280
glycol, 123
glycolysis, 476
gonads, 191
Gore, Al, 174
governance, 309
government intervention, 132
government policy, 164
governments, 31, 132, 137, 155, 166, 394, 402, 405,
411
GPS, 289
grades, 149, 407, 410, 416
gradient formation, 234
grading, 290, 404, 407, 411
granules, 280
graph, 347, 348, 351, 353
grass, 48, 59
grasses, 47, 48, 147
grassroots, 120
Index
gravity, 21, 69, 75, 129, 156, 160, 407
grazing, 114
Greece, 38
green revolution, 82, 118
greenhouse gases, vii, ix, 3, 61, 112, 124, 125, 128,
139, 149, 156, 160
greening, 323
groundwater, xiii, 22, 29, 32, 40, 119, 129, 130, 162,
325, 326, 328, 329, 331, 333, 334, 335, 338, 342,
352, 353, 354, 355, 371, 374, 431, 453, 454
group activities, xi, 237
grouping, 244
growth factor, 234
growth hormone, 48, 85, 99, 387
growth rate, 361
Guangdong, 120
guidelines, 4, 121, 314, 317, 393, 404, 427, 430, 471
H
habitats, xii, 24, 32, 285, 287, 289, 290, 291, 293,
294, 295, 296, 297, 298, 299, 300, 301, 305, 306,
307, 310, 314, 316, 320, 358, 360, 361, 373
hair, 141
half-life, 125
hammer, 155
hardener, 150
hardness, 280, 281
harvesting, 35, 95, 96, 409
hazardous materials, 156, 157, 454
hazardous waste, 120, 122, 154, 157, 158, 160, 256
hazardous wastes, 99, 116, 118, 120, 121, 122, 123,
124, 128, 131, 133, 139, 141, 142, 145, 154, 156,
158, 165
hazards, 30, 35, 131, 388, 391, 408, 417, 426, 430,
432
HDPE, 56, 78, 143, 151, 152
headache, 427
Health and Human Services, 430
health care, 13, 24
health effects, 23, 38, 426, 430
health problems, 394
health status, xi, 257, 259, 272, 279
heart disease, 425
heavy metals, 43, 47, 54, 55, 56, 57, 58, 59, 61, 64,
74, 75, 76, 77, 78, 96, 98, 106, 119, 123, 129, 130,
157, 216, 217, 234, 261, 265, 387, 398, 399, 401,
442
height, 86, 224, 330, 331
hematite, 183
491
hemisphere, 5, 6, 400
hepatitis, xiv, 17, 386
herbicide, 425
heterogeneous catalysis, 442, 445
high blood pressure, 425
highlands, 287
HIV, 20
homeostasis, xi, 257, 259, 260, 476
homocysteine, 280
homogeneity, 192
Hong Kong, 139, 167, 170, 236
host, x, xiv, 20, 24, 28, 31, 33, 37, 39, 187, 188, 189,
208, 209, 210, 211, 212, 213, 214, 215, 216, 217,
386, 427, 428, 473
hotel, 386
hotels, 162
house, 281
housing, ix, 111, 242, 252, 317, 323
hub, 216
human activity, 174
human behavior, 164
human capital, 417
human genome, 28
human health, ix, 112, 121, 122, 151, 154, 174, 387,
426, 430
human immunodeficiency virus, 33
human milk, 398
humoral immunity, 261
humus, 49, 81, 82, 100, 222
Hunter, 19, 35, 272, 281
hunting, 273, 279, 283
hyaline, 189
hybrid, 161, 175
hybridization, xiii, 28, 29, 357, 361, 362, 363, 364,
365, 366, 367, 368, 369, 370, 371, 372, 373, 374,
375, 378, 380, 381, 382, 383
hydrocarbons, 76, 78, 80, 98, 99, 130, 147, 434, 449,
454
hydrogen, 56, 59, 64, 70, 128, 154, 163, 358, 434,
437, 438, 439, 453, 454, 455, 457, 460, 461, 462
hydrogen chloride, 154
hydrogen peroxide, 434, 437, 438, 439, 453, 454,
455, 457, 460, 461, 462
hydrolysis, 222, 462
hydroponics, 221, 235
hydroquinone, 448
hydroxide, ix, x, 155, 173, 174, 175, 182, 453
hydroxyl, xv, 433, 435, 437, 438, 439, 440, 443, 445,
446, 451, 453
hygiene, xiv, 156, 385, 401, 408, 410, 416, 417
Index
492
hypertrophy, 196, 198, 213
hypoxia, 467, 475, 476, 477
I
ice, 151, 411
ideal, 65, 68, 76, 97, 131, 238, 449, 468
identity, 367, 475, 478
ideology, 322
image, 183, 287, 308, 470
image interpretation, 287
images, 27, 183, 470
immigration, 316, 319, 320, 321, 322
immobilization, xiii, 77, 78, 357, 372
immune memory, 261
immune reaction, 261
immune response, 260, 261
immune system, 260
immunity, 261
immunocompromised, 13
immunodeficiency, 33
immunosuppression, 426
impacts, xii, 61, 87, 89, 90, 93, 95, 214, 305, 306,
307, 308, 317, 319, 323
imports, 401, 403, 417
impregnation, 446
impurities, 68
incidence, viii, 12, 17, 20, 36, 37, 84, 90, 259, 394,
417
income support, 242
incomplete combustion, vii, 3
incubation period, 374
independence, 315, 319
independent variable, 208, 289
India, vi, ix, xiv, xv, 41, 44, 46, 49, 50, 83, 85, 87, 89,
100, 101, 102, 104, 105, 107, 108, 109, 112, 114,
116, 120, 132, 139, 162, 167, 168, 169, 289, 302,
303, 355, 385, 386, 387, 395, 396, 397, 398, 399,
400, 401, 402, 403, 404, 406, 407, 408, 409, 410,
411, 416, 417, 418, 419, 420, 421, 423, 424, 425,
426, 427, 428, 430, 431, 432
Indians, xi, 114, 237
individual differences, 211
Indonesia, 116
induction, 373
industrial processing, 137
industrial revolution, 118
industrial wastes, ix, 112, 141, 142, 144, 147, 152,
154, 164, 165
inequality, 248, 255, 265
inequity, 249
infants, 124, 394
inferences, 471
inflammation, 17
information exchange, 258
information technology, xv, 387
infrastructure, xiv, xvi, 139, 307, 311, 312, 313, 324,
385, 395, 406, 417, 465, 467
ingest, 49, 54, 55, 63, 70, 75, 77, 81, 98, 391
ingestion, 12, 13, 17, 43, 64
initiation, 319, 453
inoculum, 159
insecticide, 400, 424, 426, 431
insects, 84, 123, 406, 412, 425, 426, 427, 429
inspections, 409
inspectors, 409
institutional change, 417
institutions, ix, 112, 155, 310, 312, 313, 314
insulation, 123, 152
integration, 30
intelligence, 426
intercepts, 347
interface, 38, 306
interference, x, 173
International Atomic Energy Agency, 168
international law, 120
international standards, 398, 407
intervention, xii, 31, 32, 291, 305
intervention strategies, 31
intestinal malabsorption, 21
intestine, 43, 48, 49, 56, 60, 81, 107
intoxication, 260
invertebrates, 22
investments, 31, 402, 404, 406, 411
ion-exchange, 181
ionic strength, 445
ions, 70, 176, 365, 438, 453, 456, 458
Iowa, 215
Ireland, 11, 40, 44, 61, 76, 77, 104, 164
iron, 77, 85, 148, 149, 326, 338, 436, 457, 460
irradiation, 30, 455
isolation, 25, 28, 373
isotherms, 176, 177
isotope, 7, 374, 376
Israel, vi, xii, 45, 145, 166, 305, 306, 307, 308, 309,
312, 313, 314, 315, 316, 317, 319, 320, 321, 322,
323, 324
Italy, 43, 45, 46, 102, 109, 115, 145, 146, 155, 164,
400
Index
J
Jammu and Kashmir, 402
Japan, 45, 46, 115, 121, 125, 131, 139, 141, 145,
155, 165, 173, 174, 175, 282, 456
Jordan, 308
Jordan River, 308
jurisdiction, 310, 311, 312, 313, 315, 316
juveniles, 209, 277, 279
K
Kenya, 166, 170
keratin, 21, 280, 281
keratinocytes, 282
kidney, x, 187, 188, 189, 190, 191, 192, 193, 194,
196, 197, 198, 199, 201, 202, 203, 206, 208, 209,
210, 211, 212, 213, 214, 215, 393
kidneys, 18, 190, 191, 193, 196, 201, 208, 209, 210
kill, 21, 45, 84, 114, 124, 415
kinetic constants, 450
kinetic model, 454, 455
kinetics, 258, 259, 360, 367, 438, 441, 443, 452, 455,
457, 460
Korea, 45, 139, 145, 155, 219, 220, 223, 224, 233
L
labeling, 365, 373, 376, 380, 382, 403, 407, 413
lack of confidence, 13
lakes, viii, 12, 22, 38, 379, 426
landfills, ix, 46, 48, 54, 55, 62, 76, 96, 99, 112, 113,
114, 119, 120, 121, 124, 125, 126, 127, 128, 129,
130, 131, 132, 133, 134, 135, 139, 140, 142, 144,
145, 151, 152, 154, 159, 160, 164, 165, 316
landscape, xii, 6, 285, 286, 287, 289, 290, 299, 300,
301, 306, 307, 308, 310, 311, 313, 314, 315, 316,
318, 319, 321, 322, 323, 324
landscapes, xii, 300, 305, 306, 307, 308, 309, 310,
311, 312, 313, 314, 315, 316, 317, 318, 319, 320,
321, 429
Latin America, 17
leaching, 119, 129, 447, 448, 449
lead, ix, x, 18, 44, 57, 63, 76, 77, 85, 119, 120, 123,
130, 144, 145, 149, 154, 155, 173, 174, 179, 181,
182, 187, 198, 201, 203, 205, 207, 208, 209, 210,
211, 212, 213, 214, 215, 216, 217, 280, 309, 313,
374, 387, 391, 398
lead content, 398
493
leadership, 472
leakage, xiii, 130, 325, 326, 332, 335, 338, 340, 341,
353
leaks, 413
legislation, xiv, 156, 306, 309, 311, 312, 315, 319,
320, 385, 416, 417, 436
leisure, vii, 11, 12, 240, 254, 307
lens, 280, 282, 342
lesions, 210
Lewis acids, 446
liberalization, 399
liberation, 17
life cycle, 188
lifetime, 115, 177, 179, 393
ligand, 449, 461
lignin, 69, 126, 127, 145, 150, 455
limestone, 59, 153, 154
limitations, 360
lipases, 49
lipids, 21, 126, 145, 476
liquid chromatography, 454
liquid phase, 224, 445
liquids, 123
listeria monocytogenes, 23
literacy, xv, 387, 395, 404
literacy rates, 404
litigation, 431
liver, x, 17, 187, 188, 189, 191, 192, 193, 196, 198,
201, 203, 206, 209, 210, 211, 214, 215, 393, 476,
480
liver cancer, 17
liver damage, 214
livestock, 12, 18, 19, 20, 22, 31, 34, 46, 47, 67, 387,
399
living environment, 242
local authorities, 314
local government, ix, 48, 112, 140
localization, 274
logging, 47
low temperatures, vii, 11, 130
lubricants, 156
lubricating oil, 156
lupus, 188
lymphocytes, 210
lysine, 44, 63, 227, 233, 362
lysis, 23
lysosome, 24
Index
494
M
machinery, 141, 148, 405, 425
macromolecules, 258, 260, 261, 265
macronutrients, 221
macrophages, 25, 210
magazines, 115, 123, 138
magnesium, 62, 82, 85
magnetic resonance, 176
magnets, 148
magnitude, 359, 426
Maine, 480
majority, xiii, xvi, 5, 12, 21, 30, 134, 198, 240, 308,
357, 375, 404, 410, 440, 465, 473, 474
malabsorption, 21
malaria, xv, 398, 423, 425, 427, 429, 430, 431
Malaysia, 120, 235, 302
management, viii, ix, xii, xiv, 30, 35, 41, 43, 45, 46,
61, 67, 96, 101, 112, 126, 130, 131, 132, 133, 134,
135, 139, 145, 156, 158, 161, 164, 165, 166, 285,
286, 303, 308, 309, 312, 313, 314, 320, 321, 322,
324, 326, 386, 392, 405, 406, 408, 410, 416, 417,
418, 431, 466, 471, 479
mandarin, 421
mandible, xi, 271, 272, 273, 274, 275, 276, 277
manganese, 77, 326, 436, 457, 458, 459, 461
manpower, 139
manufacture, 113, 122, 132, 135, 138, 149, 150,
151, 152, 153, 154, 155, 157, 410, 426, 430
manufacturing, xiv, 47, 122, 132, 136, 141, 220, 221,
365, 385, 410, 411, 416, 417, 426
manure, 18, 19, 46, 55, 56, 59, 61, 107, 146, 220,
234, 235
mapping, 256, 469, 478
marine environment, 376
markers, 361, 377
market access, 417
marketing, xiv, 385, 396, 402, 403, 405, 406, 410,
417
marketplace, 165
marsh, 382
Marx, 431
Maryland, 324
mastitis, 20
mathematical methods, 339
matrix, xii, 28, 285, 300, 438
meat, 20, 52, 151, 394, 396, 412, 413, 414, 415
mechanical engineering, 126
media, xiv, 23, 25, 34, 37, 69, 132, 133, 166, 355,
385, 396, 398, 416, 427, 454
median, 243, 247, 249, 287

medicines, 122
Mediterranean, 307, 308, 316, 324
Mediterranean climate, 308
MEK, 129
melanin, xii, 272, 280, 282
melt, 130, 151
melting, 151, 190, 211, 214, 215, 366, 367, 382
melting temperature, 366
membrane separation processes, 234
membranes, 24, 476
memory, 261
meningitis, 16
mercury, 54, 76, 77, 119, 120, 123, 124, 130, 144,
155, 174, 212, 387
mesoporous materials, 175, 176, 183, 445
messages, 132
meta-analysis, 303
metabolic disturbances, 269
metabolic pathways, 226, 279
metabolism, 59, 61, 112, 174, 214, 224, 236, 261,
281, 476
metabolites, 227, 425, 426
metal oxides, 445, 446, 447, 448, 459
metals, 43, 44, 47, 54, 55, 56, 57, 58, 59, 61, 64, 74,
75, 76, 77, 96, 98, 106, 112, 116, 117, 119, 120,
123, 129, 130, 136, 138, 140, 142, 148, 152, 154,
155, 156, 157, 161, 163, 188, 190, 191, 192, 202,
203, 205, 208, 209, 210, 212, 214, 215, 216, 217,
234, 261, 265, 387, 398, 399, 401, 434, 442, 443,
445, 446, 447, 448, 450, 452, 457
meter, 65, 68, 75, 97, 98, 151, 160, 163, 326
methanol, 449
methodology, 252, 255, 306, 395, 397
metropolitan areas, 12
Mexico, 102, 123, 401
mice, 25, 214, 215, 269
microarray detection, 371, 373, 379, 382
microarray technology, xiii, 37, 358, 364, 366, 374,
375, 376, 380, 382, 466, 467, 478, 480
microbial cells, 145, 377
microbial communities, xiii, 24, 28, 29, 30, 34, 357,
359, 360, 361, 363, 365, 366, 367, 371, 373, 374,
375, 377, 378, 379, 382, 383
microbial community, 13, 29, 40, 61, 359, 360, 361,
364, 371, 373, 374, 375, 376, 377, 379, 381, 383,
384, 479
microbial metagenomics, 469
microcosms, 376
micrometer, 28
Index
micronutrients, 81, 82, 90, 95, 144, 145
microorganism, 17
microscope, 175, 176, 183, 361, 362
microscopy, 183, 362, 363
microspheres, 29
middle class, xv, 386, 395
Middle East, 131, 399
migration, 128, 210
military, 131
milligrams, 395
mineral water, 34
mineralogy, 6, 67
miniaturization, 362
mining, 76, 124, 125, 148, 149, 165, 190, 191, 211,
214, 215
Ministry of Education, 184
minorities, 238, 253
misconceptions, 429
missions, vii, 3, 5, 139, 147, 152
mixing, 46, 64, 79, 80, 86, 120, 131, 174, 175
modeling, 290, 301, 302, 458
modelling, 244, 455
models, vii, 3, 6, 24, 119, 155, 254, 287, 290, 292,
293, 301, 480
modern society, 128, 164
modernization, ix, 111, 410
modification, 25, 34, 267, 274, 346, 450, 476
moisture, 43, 45, 55, 57, 58, 76, 78, 85, 95, 97, 152,
159, 163, 407
moisture content, 55, 78, 85, 159
molecular biology, 26, 257, 391, 467
molecular weight, 209, 227, 455
molecules, 126, 145, 152, 259, 269, 365, 446, 454,
468
monitoring, viii, 12, 28, 29, 30, 31, 35, 37, 128, 174,
183, 225, 272, 278, 313, 328, 329, 335, 342, 361,
366, 380, 382, 392, 394, 404, 408, 410, 472
monoclonal antibody, 28
morbidity, vii, 11, 20, 22, 425
morphology, 361
morphometric, 191, 206
mortality rate, 20
mosquitoes, 163
mRNA, 362, 373, 375, 473
mRNAs, 362, 373, 374
mucoid, 19
mucosa, 476
mucous membrane, 24
mucus, 49, 81
multiple regression, xii, 285, 289, 290, 292, 301
495
multiplication, 23, 48, 78, 83

muskrat, 213, 215
mutation, 287, 378
mutations, 265, 362, 379
Myanmar, 287, 300, 301
mycobacteria, 13, 19, 23, 24, 38
mycorrhiza, 235
mycotoxins, xiv, 386
myocarditis, 16
myoglobin, 476, 477, 480
N
NaCl, 83, 222
NAD, 355
nanoparticles, 174, 175, 176, 179
nanotechnology, 31, 175, 183
naphthalene, 156, 376, 450
National Bureau of Standards, 192
national parks, xii, 285, 287, 308, 315, 319
national policy, 313
National Research Council, 11
national security, 319
natural disasters, 152
natural habitats, xii, 305
natural resources, 113, 137, 165, 286
nausea, 427
necrosis, 213
needy, 155
negative relation, 213
neglect, 308, 311, 346
nematode, 188, 211
nervous system, 393
Netherlands, 103, 105, 106, 116, 124, 131, 139, 155,
167, 354, 355, 399
neural network, 382, 383
New England, 37, 39
New South Wales, 109
New Zealand, 46, 48, 76, 116, 430
next generation, 252
NGOs, 133, 396, 397, 410, 416
nickel, x, 156, 187, 191, 196, 197, 198, 201, 203,
205, 207, 208, 210, 211, 212, 213, 214
Nigeria, 116
nitrate, 174, 434, 441, 446, 451, 462
nitrates, 49, 63, 81, 82
nitrification, 366
nitrifying bacteria, 358, 363, 369, 379, 381, 382
nitrobenzene, 461
Index
496
nitrogen, 44, 48, 54, 59, 62, 63, 64, 67, 78, 81, 82,
83, 85, 87, 90, 95, 99, 128, 143, 144, 145, 146,
221, 222, 234, 235, 236, 358, 366, 367, 371, 376,
377, 381, 383, 384, 434, 453, 456
nitrogen fixation, 99, 366, 381
nitrogenase, 384
nitrogen-fixing bacteria, 82, 99
nitrous oxide, ix, 61, 62, 112, 125
NMR, 176
noble metals, 448
nodes, 467
nodules, 78, 358
nonequilibrium, 258
nonequilibrium systems, 258
non-renewable resources, 138
normal aging, 267
normal distribution, 192
North America, 21, 117, 161, 252, 323
North Sea, 479
Northern Ireland, 11, 40
Norway, 116, 133, 139, 155
nuclear energy, 124
nuclear power, 125, 131, 455
nucleic acid, xiii, 17, 27, 28, 33, 357, 362, 364, 365,
366, 368, 369, 371, 372, 377, 380
nucleotides, 361, 365, 374
nuisance, 252, 253
null, 438
nutrients, 20, 23, 48, 54, 55, 62, 63, 64, 65, 67, 78,
82, 83, 85, 86, 99, 100, 136, 144, 146, 162, 209,
220, 221, 226, 227, 228, 231, 428, 431, 432
nutrition, 83, 221, 267
O
obstacles, xv, 387
oceans, 384, 426
offenders, 314
oil, 43, 47, 76, 85, 107, 123, 131, 141, 143, 147, 150,
151, 152, 153, 156, 165, 220, 363, 379, 402
oligonucleotide arrays, 378, 379, 381
omentum, 210
omission, 302
open spaces, xi, 237, 240, 253, 305, 314, 316
operating costs, 75
operations, 29, 190, 191, 214, 314, 404, 406, 408
operon, 358
opportunities, 32, 307, 399, 417
optical fiber, 121
optimization, xiii, 225, 357, 376, 381, 446, 451, 452
oral cavity, 383

oral health, 383
ores, 125, 140, 148, 149
organ, 30, 91, 192, 193, 196, 197, 206, 208
organic chemicals, 122, 141, 431
organic compounds, xv, 63, 123, 128, 130, 156, 433,
434, 436, 438, 440, 442, 446, 448, 451, 452, 454,
455, 456, 462
organic food, 403
organic materials, 43, 46, 75, 126, 130, 143, 159,
161
organic matter, vii, 3, 6, 43, 48, 49, 55, 59, 63, 64,
66, 69, 70, 77, 81, 82, 85, 96, 99, 100, 127, 144,
146, 163, 174, 220, 226, 227, 233, 234, 236, 378,
438, 439, 442, 443, 446, 451, 460, 461
organic solvents, 123
organism, xi, 26, 27, 29, 30, 118, 121, 257, 260, 263,
271, 358, 363, 372, 373, 374, 468
organizing, 183, 404
organochlorine compounds, 123
osmosis, 98
overgrazing, 293
overlap, 289, 311, 313, 318, 320
overlay, 247
oversight, 404, 408, 409, 417
ownership, 396
oxidation, xv, 179, 222, 225, 358, 365, 433, 434, 436,
437, 439, 440, 442, 443, 447, 449, 450, 451, 452,
453, 454, 455, 456, 457, 458, 459, 460, 461, 462,
463
oxidation rate, 451
oxidative stress, 280, 282
oxygen, 58, 59, 61, 64, 67, 69, 70, 97, 125, 128, 160,
222, 224, 235, 280, 373, 381, 435, 436, 459, 475,
476, 480
oxygen consumption, 64, 224
oyster, 192
oysters, 40
ozonation, xv, 433, 435, 437, 438, 439, 443, 445,
446, 447, 448, 449, 450, 451, 452, 453, 454, 455,
456, 457, 458, 459, 460, 461, 462, 463
ozone, xii, xv, 21, 30, 272, 279, 280, 433, 434, 435,
436, 437, 438, 439, 440, 442, 443, 444, 445, 446,
447, 448, 449, 450, 451, 452, 453, 454, 455, 456,
457, 458, 459, 460, 461, 462, 463
ozone reaction, 462
ozonization, 458, 460
ozonolysis, 456
Index
P
Pacific, 16, 40, 76, 105, 114, 116, 117, 304, 377
pain, 400
paints, 123, 141
pairing, 273, 279
Pakistan, 116, 120, 355
palladium, 461
palpation, 210
parallel, 29, 335, 362, 370, 374, 380, 441, 455, 466,
468
paralysis, 16
parasite, viii, x, 12, 30, 34, 187, 188, 189, 190, 191,
208, 210, 211, 212, 213, 214, 215, 216, 272, 273
parasites, 21, 188, 203, 212, 213, 215, 216, 217, 281
parasitic infection, x, 187, 209, 211, 213, 214
parliament, 403
partition, 153
pathogens, viii, 11, 12, 13, 16, 18, 19, 20, 21, 22, 23,
24, 26, 28, 29, 30, 31, 32, 34, 36, 37, 38, 40, 45,
47, 48, 54, 55, 56, 57, 59, 60, 61, 75, 84, 96, 97,
98, 145, 163, 371, 379, 387, 415
pathology, 32
pathophysiology, 281
pathways, 226, 279, 438, 450, 471, 476
peat, 459
penalties, 403
peptides, 17, 459, 468
percentile, 248
percolation, 66, 129, 333, 337
performance, 35, 87, 308, 330, 430, 436, 440, 441,
442, 443, 445, 447, 450, 469
peri-urban, 398
permeability, 335, 340
permit, 324
peroxide, 434, 435, 437, 438, 439, 448, 453, 454,
455, 457, 460, 461, 462
person-to-person contact, 19
Perth, 101, 161, 244
pest populations, 392
pesticide, xv, 61, 76, 387, 391, 392, 393, 394, 396,
397, 398, 399, 400, 401, 403, 404, 406, 407, 408,
409, 410, 412, 416, 418, 423, 424, 425, 426, 427,
428, 429, 430, 432
pesticides, xv, 43, 76, 85, 100, 122, 123, 307, 388,
391, 392, 393, 394, 395, 396, 397, 398, 403, 410,
411, 412, 418, 423, 424, 425, 426, 427, 428, 429,
430, 431, 432, 434, 440, 457
pests, xv, 84, 85, 90, 123, 221, 392, 423, 425, 429
PET, 136, 143, 151
497
petroleum, 122, 141

phagocytosis, 24
PHB, 236
phenol, 274, 441, 446, 447, 448, 450, 456, 459, 460,
461, 462
phenotype, 479
Philippines, 45, 120, 145, 421
phosphates, 49, 81, 83, 236, 446
phosphorus, 48, 54, 63, 64, 67, 78, 82, 83, 90, 95,
143, 144, 221, 231, 236, 434
phosphorylation, 282
photocatalysis, 437, 442, 456, 457
photodegradation, 454, 455
photoemission, 183
photolithography, 362
photolysis, 78, 439, 454
phylogenetic tree, 365
phylum, 363
physical activity, 253, 254
physical environment, 254
physical exercise, 253
physical fields, 269
physical fitness, 191
physical properties, 220, 234
physics, 258, 259, 263, 268
physiology, 76, 221, 358, 467, 477, 478, 480
phytoplankton, 467
pigs, 116, 117
pilot study, 67
pioneer species, 293
pitch, 241
placenta, 121
plants, ix, xiii, 21, 30, 45, 54, 55, 63, 83, 84, 85, 90,
91, 92, 94, 95, 96, 97, 99, 112, 123, 131, 141, 145,
150, 153, 154, 156, 159, 160, 220, 221, 222, 224,
226, 227, 228, 234, 235, 306, 316, 357, 362, 391,
392, 398, 401, 410, 412, 427, 428, 429, 430, 436,
449, 455
plasmid, 378
plastic products, 139
plasticity, 480
plastics, 112, 113, 116, 117, 119, 122, 123, 126, 130,
135, 136, 138, 144, 147, 150, 151, 152, 157, 161,
163
platform, 68, 317, 367, 468, 469
platinum, 183, 462
PLS, 5
plutonium, 125
pneumonia, 24
point mutation, 379
498
Index
poison, 120, 400

policy makers, 31, 133, 396
polio, 16, 33
pollutants, 76, 130, 150, 151, 154, 188, 387, 434,
436, 437, 438, 440, 445, 446, 448, 454, 455, 458,
463
polluters, 314
pollution, x, xii, 25, 46, 113, 135, 137, 139, 142, 144,
148, 149, 161, 164, 165, 184, 187, 190, 208, 211,
216, 217, 221, 234, 252, 272, 305, 306, 308, 313,
318, 398, 475
polychlorinated biphenyls (PCBs), 48, 76, 123, 130,
430
polycyclic aromatic hydrocarbon, 43, 61, 76
polymerase, 27, 36, 40, 371, 381, 474
polymerase chain reaction (PCR), xiii, 26, 27, 28, 29,
33, 35, 36, 39, 274, 281, 282, 357, 359, 360, 361,
366, 369, 370, 371, 373, 375, 377, 378, 379, 380,
381, 382, 383, 468, 475
polymerization, 280
polymers, 365
polymorphism, 26, 38, 360, 377, 380, 381, 382, 383
polymorphisms, 362, 379, 380
polypeptide, 366
polypropylene, 68, 151, 192
polystyrene, 151
pools, vii, 3, 21, 22, 137
population density, 65, 67
population group, xi, 237, 248, 249
population size, 242, 286, 291, 300, 303
porosity, 62, 66, 82, 220, 342, 353, 365
porous media, 355
portability, 29
Portland cement, 153
Portugal, 139, 155
positive relationship, 205, 212
potassium, 54, 62, 63, 82, 85, 90, 95, 143, 221
potato, 39, 47, 52, 53, 234, 235
poultry, 18, 31, 34, 394, 412, 413, 414, 415
poverty, xv, 46, 120, 386, 395
poverty reduction, xv, 386
power plants, 145, 153, 316, 455
pozzolana, 153
precipitation, 129, 150, 434, 447
predation, 17
predators, 24
prejudice, 252
preparation, 29, 69, 317, 359, 400
prevention, 22, 27, 165, 235, 293, 391
primary school, 397
priming, 210, 373

prioritizing, 308
private investment, 406
private sector investment, 410
probability, 63, 248, 250, 251, 274, 278, 290, 291,
294
probability distribution, 248, 250, 251
probe, 175, 176, 183, 361, 363, 364, 366, 367, 368,
372, 374, 378, 469, 470, 474
process control, 34, 225
process gas, 130
procurement, 119, 140, 411
producers, 134, 139, 157, 392
productivity, viii, 5, 6, 22, 42, 82, 83, 96, 99, 213,
425, 473
profit, 141
project, xii, 151, 159, 162, 239, 254, 285, 287, 300,
301, 314, 378, 467, 468, 472
prokaryotes, 370, 376, 380
prokaryotic cell, 373
proliferation, 33, 60, 99, 209
promoter, viii, 42, 87, 95, 99, 374, 449, 474
propagation, 479
propane, 175
prophylaxis, 25
proteases, 49
protected areas, 286, 287, 292, 293, 300
proteins, 21, 49, 63, 82, 126, 141, 145, 163, 234,
265, 279, 383, 448, 473, 475
prototype, 382
Pseudomonas aeruginosa, 23
public awareness, 132, 158
public health, xiv, 12, 27, 34, 38, 40, 129, 131, 253,
310, 385, 407, 416, 424, 425
public interest, 289
public investment, 416
public parks, xi, 237, 238, 244, 252, 255
public policy, 323, 431
public sector, 411, 416
public-private partnerships, 404, 417
pulp, 46, 47, 63, 144, 145, 149, 150, 156, 373, 408,
410, 460
pumps, 335, 428
pure water, 112
purification, viii, 16, 29, 42, 65, 67, 183, 381, 458
PVC, 68, 69, 113, 121, 123, 143, 151, 160, 474
pyrolysis, 131
Index
Q
quality assurance, 410, 412
quality control, 378, 411
quality of life, 134, 136
quality standards, 404
quantum mechanics, 258, 263
quartile, 249
Queensland, 55
quinone, 280
R
race, 125, 255
racism, 255
radial distance, 326, 346, 347
radiation, vii, xii, 3, 125, 128, 272, 279, 280, 434,
439, 440, 454, 455
radical mechanism, 447
radical reactions, 452, 453
radicals, xv, 261, 433, 435, 437, 438, 439, 443, 445,
448, 450, 451, 452, 453, 454, 462
radioactive waste, 125, 165
radium, 125
radius, 330, 333, 338, 344, 346, 354
radon, 125
rain forest, 289
rainfall, 6, 309, 335
rash, 16
raw materials, 119, 137, 138, 139, 140, 151, 164,
165, 417, 426
RDP, 378
REA, xii, 26, 285, 289
reaction mechanism, 441, 442, 443, 444, 446, 447,
460
reaction order, 442, 449
reaction rate, 437, 440, 441, 448, 450
reaction rate constants, 437, 440, 450
reactions, xv, 27, 29, 58, 59, 128, 130, 182, 220, 224,
280, 370, 371, 386, 439, 440, 445, 449, 451, 452,
453, 457, 461, 462
reactive arthritis, 20, 38
reactive oxygen, 280
reactivity, vii, xv, 3, 122, 262, 433, 434, 437
reading, 289, 473
reagents, 29, 442, 445
real terms, 395, 399
reality, 252, 253, 331
recall, 400
499
recognition, xiv, 17, 26, 385

recommendations, 273, 412
recovery plan, 131
recovery processes, 259, 260, 262, 267
recreation, ix, xii, 111, 240, 253, 305, 307, 308, 310,
313, 314, 316
recurrence, 310, 312
recycling, 116, 120, 126, 132, 133, 134, 137, 138,
139, 140, 141, 142, 143, 145, 147, 148, 149, 150,
151, 152, 154, 155, 156, 157, 158, 159, 160, 164,
165, 166, 169, 220
red blood cells, 18, 210, 213
Red List, 302
redistribution, 210
redundancy, 475
reflectivity, 273
reforms, 408
regeneration, 448, 450
regression, xii, 285, 289, 290, 292, 301
regression method, 301
regression model, 289, 290, 292
regulatory framework, xiv, 385, 403
rehabilitation, xii, 305, 306, 310, 314
rehydration, 20
rejection, 134, 400, 401
relevance, 466
reliability, 192, 281, 352
relief, 6, 163
remediation, viii, 42, 81, 83, 98, 99, 160
repair, 134, 155, 260
reparation, 460
replacement, 153, 189
replication, 25, 39
reporters, 28
reprocessing, 138, 156
reproduction, 45, 58
reproductive organs, 92, 94, 95
reputation, 400
requirements, 34, 78, 209, 401, 408, 411
research facilities, 477
research institutions, 403
researchers, xvi, 95, 363, 433, 435, 437, 438, 439,
440, 443, 445, 451, 465, 466, 467, 470, 472, 474
reserves, 191, 241, 303, 308, 319
residuals, 13
residues, 59, 61, 76, 96, 123, 144, 220, 236, 387,
388, 391, 392, 393, 394, 396, 397, 398, 399, 400,
401, 407, 408, 409, 410, 412, 416, 425, 427, 428,
429, 430, 432
resins, 121, 150
Index
500
resistance, viii, 12, 13, 20, 25, 84, 99, 221, 261, 262,
265, 272, 326, 392
resolution, 175, 177, 178, 179, 180, 183, 242, 243,
290, 363, 368
resources, 84, 96, 112, 113, 133, 134, 135, 136, 137,
138, 141, 153, 155, 165, 220, 253, 286, 308, 309,
310, 316, 355, 406, 436, 466, 469, 472, 475, 480
respiration, 63, 221, 236, 358
restaurants, ix, 51, 112, 127, 147, 162
restriction enzyme, 382
restriction fragment length polymorphis, 26, 360,
377, 380, 381, 382
retail, 386, 394, 410, 411, 416
retardation, 22
revenue, 55, 140
ribosomal RNA, 358, 359, 361, 369, 378, 379
rights, 133, 324
rings, xv, 53, 433
risk assessment, 394, 418
risk factors, 39
risk management, 416
risks, xiv, xv, 32, 115, 131, 385, 386, 387, 388, 394,
406, 425, 427, 429
river basins, 427
river systems, 327
RNA, 28, 358, 359, 361, 363, 369, 372, 373, 374,
377, 378, 379, 380, 381, 382, 470, 474, 475, 476
robotics, 362
rodents, 429
room temperature, ix, 112, 192, 414, 435, 439
root hair, 98
roundworms, 188
rowing, 409
rubber, 118, 121, 138, 144, 415
rubbers, 147
rules, 139, 141
runoff, 129, 427
rural areas, 97, 411
rural development, xv, 386
rural women, 46
Russia, 100
S
salinity, viii, 42, 83, 306, 326, 327, 373, 376
saliva, 363
salmon, 431
salmonella, xiv, 385, 386, 401
salts, 150, 220, 443
sanctuaries, xii, 285, 287
Saudi Arabia, 148, 149

sawdust, 46, 60, 145
scarcity, 308, 309
scattering, 176
scavengers, 438, 449, 450, 451
scientific knowledge, 144
scientific understanding, xiv, 385
screening, 47, 404, 474
sea level, xiii, 325, 327, 338
seafood, 220, 234, 412, 413, 414, 415
sea-level, 328, 334
seasonal changes, 360, 378
seasonality, 30, 242
secrete, 49, 82, 112
secretion, 24, 99
sediment, 7, 66, 156, 352, 359, 369, 370, 376, 377,
381, 382
sedimentation, 30, 436
sediments, viii, xiii, 5, 6, 7, 12, 64, 307, 327, 328,
352, 358, 363, 369, 373, 377
seed, 83, 86, 87, 95, 223, 229, 231, 232
seedlings, 83, 95
segregation, 162
selectivity, 183, 392, 449
selenium, 130
self-organization, 258
self-regulation, 411, 417
semiconductors, 121
sensitivity, xiii, 7, 28, 29, 30, 183, 188, 357, 368, 369,
370, 371, 376, 393, 470
sequencing, xvi, 28, 39, 359, 360, 362, 366, 378,
383, 465, 466, 467, 468, 469, 471, 477, 478
serum, 426, 430
service provider, 416
settlements, 300, 315, 319
severe acute respiratory syndrome, xiv, 385
sewage, 18, 36, 43, 45, 46, 54, 55, 56, 60, 61, 62, 63,
64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 76, 96, 97,
98, 101, 112, 141, 145, 146, 151, 153, 154, 159,
161, 162, 234, 306, 307, 308, 398
sex, xi, 204, 271, 273, 278, 281, 394
sexual dimorphism, 211, 281
shade, 78
shape, 189, 242, 331
shear, 13, 36
sheep, 21
shellfish, 22, 370, 381
shelter, 13, 292
shingles, 144, 152
ships, 148
Index
shoot, 87, 264
shortage, 231, 308
shrimp, 401, 411
side effects, 392, 426
signals, 175, 272, 279, 365, 368, 372
signs, 16, 289, 408
silica, 141, 151, 153, 445, 448
Silicon Valley, 120, 154, 168
silver, 96, 140, 141, 144, 149, 156, 460
Singapore, 105, 115, 120, 121
single-nucleotide polymorphism, 379, 383
sintering, 446
skin, 24, 58, 412, 427
slag, 131
Slovakia, 217
sludge, 43, 45, 46, 47, 54, 55, 56, 57, 59, 60, 62, 63,
64, 68, 74, 75, 77, 97, 98, 101, 102, 105, 112, 141,
145, 146, 154, 159, 162, 234, 236, 370, 373, 374,
447, 460
social activities, 132
social change, 309
social conflicts, 319
social group, 238, 252, 253
social justice, 238
social problems, viii, 42
socioeconomic status, 254
sociology, 126
sodium, 35, 124, 150, 155, 174, 175, 412, 455, 463
sodium hydroxide, 155, 174
software, 367, 472, 479
soil erosion, 6, 139, 145
soil particles, 81, 307, 360
soil pollution, 135
solid phase, 434, 445
solid surfaces, 29
solid waste, viii, ix, 18, 42, 43, 48, 96, 98, 107, 112,
113, 115, 116, 117, 118, 124, 129, 130, 131, 132,
133, 135, 142, 144, 146, 148, 154, 159, 161, 162,
163, 164, 166
solubility, xv, 224, 433, 434, 448
solvents, 123, 154, 156, 426
sorption, 174, 175, 179, 180, 183
South Africa, 153, 400
South Asia, 399, 419, 420, 421
South Korea, 139, 155
Southeast Asia, 287, 289
sovereignty, 309
SPA, 378, 381
Spain, 116, 400, 433
specialists, 289
501
speciation, 234
species richness, 323
specific gravity, 21, 407
specific surface, 65, 175, 176
specifications, 27, 409
spectrophotometry, 192
spectroscopic techniques, 175
spectroscopy, x, 173, 174, 175, 177, 183
speculation, 210, 213
spicule, x, 187, 189, 190, 191, 192, 193, 194, 196,
197, 199, 201, 202, 203, 205, 208, 209, 211, 212,
214, 215
spiders, 123
spillovers, 409, 410
spleen, x, 187, 191, 206, 207, 208, 213, 214
splenomegaly, 213
spreadsheets, 354
Spring, 282, 429
Sri Lanka, 121
SSI, 402, 416
stabilization, viii, 42, 54, 55, 63, 65, 153, 227, 233,
236, 316
stabilizers, 403
stakeholders, xii, 285, 286, 300, 301
standard deviation, 245
standard error, 194, 196, 199, 204, 205, 206
standardization, 407, 472
starch, 122, 147, 222
state control, 402
State Department, 410, 431
statehood, 315
states, 132, 310, 311, 313, 394, 398, 402, 406
statistics, 13, 238, 240, 242, 243, 248, 249, 250, 267,
282, 467
steel, 113, 115, 138, 139, 148, 149, 152, 153, 156
sterile, 47, 60, 192, 223
stimulus, 210
STM, 183
stoichiometry, 443, 444, 457
stomach, 189
storage, xiii, 29, 54, 61, 68, 76, 126, 146, 156, 158,
209, 210, 212, 234, 326, 330, 331, 332, 333, 335,
340, 342, 343, 345, 352, 353, 354, 355, 391, 404,
406, 476
stormwater, 159
stoves, 121
stratification, 234, 331
streams, viii, 12, 47, 220, 235, 293, 307, 308, 309,
340, 427, 431
streptococci, 108
502
Index
stroke, 425
strontium, 125
structural changes, 396
structural gene, 382
style, ix, 111, 122, 309, 474
subgroups, 248, 394
subjective judgments, 351, 353
submarines, 221
substitution, 436, 449, 461
substitution reaction, 461
substrates, 25, 163, 476
Sudan, 400
sugar beet, 220
sugarcane, 47, 83, 147
sulfate, 150, 225, 235, 358, 366, 376, 449
sulfur, 62, 82, 147, 153, 227, 233, 366, 367
sulfuric acid, 155
Sun, 109, 170, 283, 379, 456
supermarkets, 135, 152, 164, 396, 404, 405, 411
supplier, xiv, 386, 400
suppliers, 411
supply chain, 388, 396, 399, 408, 409, 416, 417
support services, xiv, 385, 417
suppression, 84
surface area, 65, 160, 175, 176, 364, 448, 450
surface chemistry, 449, 450, 451
surfactant, 453, 463
surplus, 65, 335
surrogates, 32, 469
surveillance, 16, 30, 416, 417, 427
survey, 124, 133, 182, 239, 242, 243, 244, 245, 255,
288, 289, 301, 406, 428, 468
survival, vii, 11, 13, 24, 35, 58, 164, 475
susceptibility, 208, 221
suspensions, 456
sustainability, 164, 314, 326, 418
sustainable development, viii, 41, 169, 326
Sweden, 36, 37, 116, 123, 131, 139, 155, 166
Switzerland, 116, 117, 139, 155, 158, 224, 235, 302,
303
symmetry, 175
symptoms, 16, 17, 19, 426, 427
synchronization, 260
syndrome, xiv, 18, 385
synergistic effect, 456
synthesis, xii, 176, 260, 261, 272, 280, 283, 373, 374,
445, 451, 468, 469, 477
T
tags, 361, 468, 478
Taiwan, 120, 139, 155, 235
tanks, 46, 146
tannins, 150
tapeworm, 215, 217
tar, 152
target, xv, 27, 28, 29, 157, 289, 290, 291, 296, 297,
298, 299, 300, 301, 361, 363, 364, 365, 366, 367,
368, 369, 370, 372, 373, 374, 380, 423, 427, 428,
429, 469, 476
taxation, 165
taxonomy, 40
technical support, 466
technological developments, xvi, 119, 465
technological revolution, 118, 121
technologies, viii, xiii, 29, 42, 59, 74, 97, 142, 145,
147, 154, 220, 259, 357, 408, 425, 437, 467
technology, xiii, xv, 37, 64, 76, 96, 97, 98, 99, 119,
121, 130, 131, 138, 140, 142, 143, 144, 146, 147,
158, 159, 162, 164, 165, 357, 362, 364, 366, 374,
375, 380, 382, 387, 401, 433, 459, 462, 466, 468,
478, 480
TEM, 175, 176, 183
temperature, vii, ix, 3, 4, 16, 43, 45, 47, 48, 50, 58,
59, 60, 67, 76, 96, 97, 112, 124, 130, 147, 151,
160, 163, 170, 192, 222, 223, 258, 259, 272, 306,
366, 367, 368, 413, 414, 415, 435, 439, 445, 462,
475
territory, 327
test data, 278, 331, 341, 351, 352, 354, 355
testing, 26, 393, 398, 401, 407, 408, 409, 410, 417
tetrachlorodibenzo-p-dioxin, 43
textiles, 123, 144, 151
textural character, 451
texture, 5, 47, 56, 67, 414, 449, 450
Thailand, vi, xii, 33, 45, 121, 145, 285, 286, 287, 288,
289, 292, 302, 303, 304
therapy, 268
thermal analysis, 175
thermal energy, 146
thermodynamics, 258, 259, 263, 265, 269
thermometer, 413
thin films, 457
thinning, 426
Third World, 139
threats, xiv, 116, 293, 385
threonine, 234
thyroid, 429
Index
time periods, 264
time pressure, 391
time use, 113
tin, 148, 183
tissue, x, 149, 150, 187, 188, 192, 193, 196, 198,
199, 201, 203, 205, 208, 209, 210, 211, 212, 213,
214, 215, 398, 476
titanium, 456, 459
TLR, 238
toluene, 380
tones, ix, 83, 112, 115, 116, 125, 140, 145, 149, 153,
154, 157, 159, 160, 162, 163, 165
total energy, 260
tourism, 286, 314, 316, 386
toxic effect, 227, 229
toxic gases, 48, 146, 152
toxic metals, 188, 190, 191, 209, 212, 215
toxic waste, 76, 97
toxicity, xv, 215, 379, 394, 426, 427, 430, 433, 435,
440, 441, 447, 456
toxin, 18, 20
toys, 123, 136, 174
trace elements, 54, 99, 145, 188, 216
tracks, 289
trading partner, 408
training, 37, 242, 260, 302, 409, 410, 411, 418, 466,
470
traits, xi, 271, 273, 467
transactions, 402
transcription, 27, 362, 373
transcriptomics, 468, 479
transcripts, 373, 473, 474
transference, 439, 448
transformation, xv, 20, 55, 175, 420, 425, 428, 432,
433, 434, 435, 443, 445, 448, 451, 452, 454, 462
transformation processes, 55
transformation product, 428
transformations, 192
transition metal, 443, 457
translocation, 20
transmission, xiv, 16, 17, 19, 21, 22, 28, 30, 31, 35,
36, 38, 156, 175, 178, 181, 189, 215, 385
transport, 6, 55, 126, 141, 158, 165, 209, 238, 391,
426, 476
transportation, ix, 31, 112, 131, 161, 315, 317, 386
trauma, 260
treatment methods, 98
trial, 161
trickle down, 67
triggers, 319
503
tropical forests, 302

trucks, 148
tsunami, 120
tuberculosis, 20, 38, 358, 388
tumors, 121
tumours, 429
tunneling, 183
Turkey, 400
Turks, 308
turnover, vii, 3, 4, 476
typhoid, 18
typhoid fever, 18
typhus, xv, 423
typology, xi, 237, 240, 252, 253
tyrosine, 228, 280
U
U.S. Geological Survey, 354, 355, 427, 431
Ukraine, 271, 282
UNESCO, 289, 303
uniform, 63, 68, 85, 160, 193, 211, 252, 330, 331,
471
united, xiv, 11, 14, 16, 18, 24, 33, 38, 112, 114, 115,
133, 158, 166, 167, 170, 322, 380, 386, 400, 401,
421, 425, 426, 430, 466
United Kingdom (UK), vi, xi, xvi, 11, 21, 45, 49, 50,
101, 103, 113, 114, 128, 132, 135, 145, 151, 153,
157, 164, 167, 168, 237, 238, 239, 254, 255, 400,
420, 425, 427, 430, 431, 465, 466, 467, 471, 472,
477, 479
United Nations (UN), 112, 115, 158, 354
universities, 46, 166, 417
uranium, 124, 125
urban area, xi, 237, 251, 252, 253, 320, 398
urban population, 252
urbanization, xiv, xv, 385, 386, 395
urea, 82, 85, 87
ureter, 188
ureters, 210
urinary bladder, 188
urine, 20, 64, 188, 279, 283
USDA, 376, 394, 418, 419
USSR, 316
UV irradiation, 30, 455
UV light, 279, 440
UV radiation, 280, 434, 439, 440, 455
Index
504
V
vacuum, 129, 156, 437
valence, ix, 173, 175, 177, 435
validation, 377, 382, 478
valuation, 321
vanadium, 183
vapor, 156
variables, 35, 208, 289, 445, 449, 450, 451
variations, 211, 254, 273, 274, 276, 317, 338, 353,
366, 376, 395
varieties, 392, 405, 425
vector, 426, 475
vegetable oil, 47, 143, 147, 407
vegetables, 18, 20, 136, 221, 391, 396, 398, 399,
410, 412, 416, 425
vegetation, xii, 285, 289, 290, 291, 293, 300, 304,
306, 335, 427, 428
vehicles, 17, 126, 162, 398
vein, 274
velocity, 180, 182, 342, 353
ventilation, 223
versatility, 437
vertebrates, 22, 215, 477
vertical transmission, 31
video, 151
Vietnam, 120, 121, 174
vinasse, 456
vinyl chloride, 129
violence, 132
viral meningitis, 16
virus infection, 33, 37
viruses, 13, 16, 17, 25, 27, 32, 60, 387, 467, 474, 479
viscosity, 156, 331
vision, 273, 279, 281, 283, 312, 314, 317, 320, 471
visual impression, 249
vitamins, 85
VOCs, 123, 128, 129, 159
volatility, 431
volatilization, 78
vomiting, 427
vulnerability, 307
W
waiver, 404
Wales, 45, 109, 145
walking, 254
Washington, 109, 115, 133, 149, 166, 170, 302, 303,

304, 323, 377, 418, 419, 420, 421, 431, 455
waste disposal, 131, 132, 133, 139, 160, 165
waste incineration, 131
waste incinerator, 131
waste management, ix, 61, 96, 112, 126, 131, 132,
133, 134, 135, 156, 161, 164, 165, 166, 406
waste treatment, 131, 149, 161, 443, 452
wastewater, viii, x, xv, 13, 28, 34, 36, 42, 43, 44, 47,
54, 64, 65, 66, 67, 68, 69, 70, 74, 75, 76, 96, 97,
98, 101, 104, 109, 153, 219, 220, 225, 233, 234,
235, 236, 363, 369, 370, 378, 379, 382, 433, 435,
453, 455, 456, 460, 462, 463
water policy, 308, 309, 315
water quality, viii, 12, 13, 17, 25, 28, 33, 38, 40, 309,
313, 316, 326, 330
water resources, 220, 308, 309, 310
water supplies, 12, 17, 18, 22, 24, 29, 326, 428, 452
watershed, 30, 311, 313, 318, 320
waterways, 114, 427
weakness, 151
wealth, 46, 252, 466, 474
wear, 115, 415
web, 402, 418
websites, 170
weight changes, 208, 215
weight loss, 22
welfare, xiv, 386
wells, xiii, 29, 153, 174, 325, 328, 329, 330, 331, 332,
333, 334, 335, 337, 338, 339, 342, 343, 348, 350,
352, 353, 354, 355, 428
Western Europe, 309, 399
wetlands, 323
wholesale, 398, 402, 406, 410
wild animals, viii, 12, 13, 17
wildlife, xii, 12, 18, 20, 31, 38, 190, 240, 285, 287,
289, 290, 291, 293, 301, 303, 418, 429
windows, 148
withdrawal, xiii, 325, 326, 328, 333, 335, 338, 340
wood, 114, 117, 127, 138, 144, 147, 150, 152, 426
workers, 120, 121, 163, 418, 426, 427, 430
working hours, 32
workplace, 136, 386
World Bank, xiv, xv, 166, 386, 400, 404, 405, 406,
407, 408, 410, 411, 417, 419, 420, 421
World Health Organisation, 40, 391
World Trade Organization (WTO), 399, 417
worldwide, ix, xiv, xvi, 6, 12, 13, 16, 21, 36, 129, 131,
155, 156, 173, 386, 433, 435, 473
Index
worms, x, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54,
56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 75, 76, 78,
79, 81, 82, 83, 84, 85, 87, 90, 91, 92, 94, 95, 97,
98, 99, 100, 106, 145, 146, 187, 188, 189, 190,
191, 192, 193, 194, 196, 197, 198, 199, 201, 202,
203, 204, 205, 206, 208, 209, 210, 211, 212, 214,
216
X
x-axis, 267
XPS, 183
x-ray, x, 173, 175, 176, 183, 184
x-ray diffraction (XRD), 175, 176, 183
505
Y
Yale University, 269
yeast, xiii, 147, 150, 222, 357, 362, 373, 466, 478
yield, 83, 87, 89, 90, 146, 220, 228, 234, 245, 326,
330, 351, 353, 372, 375, 425, 468
Z
zeolites, 448
Zimbabwe, 104
zinc, 54, 76, 77, 85, 123, 144, 156, 398
ZnO, 440, 455

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ADVANCES IN ENVIRONMENTAL RESEARCH

ADVANCES IN ENVIRONMENTAL RESEARCH

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ADVANCES IN ENVIRONMENTAL RESEARCH

Nova Science Publishers, Inc.

Copyright 2011 by Nova Science Publishers, Inc.

ISBN: 978-1-61209-049-8 (eBook)

Published by Nova Science Publishers, Inc. New York

Research and Review Studies

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A New Trait of Gentoo Penguin: Possible Relation to Antarctica

Assessing Population Viability of Focal Species Targets in the

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Hydraulic Characterization of Aquifer(s) and Pump Test Data

Application of DNA Microarrays to Microbial Ecology Research:

Food Safety in India: Challenges and Opportunities

Impact of Pesticide Use in Indian Agriculture Their Benefits and

Ozone Decomposition by Catalysts and its Application in Water

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