A 527

OULU 2009

U N I V E R S I T Y O F O U L U P. O. B . 7 5 0 0 F I - 9 0 0 1 4 U N I V E R S I T Y O F O U L U F I N L A N D

U N I V E R S I TAT I S

S E R I E S

SCIENTIAE RERUM NATURALIUM

Professor Mikko Siponen

HUMANIORA
University Lecturer Elise Krkkinen

TECHNICA
Professor Hannu Heusala

ACTA

ENVIRONMENTAL
APPLICATIONS OF
MANOMETRIC
RESPIROMETRIC METHODS

MEDICA
Professor Olli Vuolteenaho

SCIENTIAE RERUM SOCIALIUM

Senior Researcher Eila Estola

SCRIPTA ACADEMICA
Information officer Tiina Pistokoski

OECONOMICA
University Lecturer Seppo Eriksson

EDITOR IN CHIEF
Professor Olli Vuolteenaho
PUBLICATIONS EDITOR
Publications Editor Kirsti Nurkkala
ISBN 978-951-42-9078-7 (Paperback)
ISBN 978-951-42-9079-4 (PDF)
ISSN 0355-3191 (Print)
ISSN 1796-220X (Online)

UN
NIIVVEERRSSIITTAT
ATIISS O
OU
ULLU
UEEN
NSSIISS
U

Katri Roppola

E D I T O R S

Katri Roppola

A
B
C
D
E
F
G

O U L U E N S I S

ACTA

A C TA

A 527

FACULTY OF SCIENCE,
DEPARTMENT OF CHEMISTRY,
UNIVERSITY OF OULU

SCIENTIAE RERUM
RERUM
SCIENTIAE
NATURALIUM
NATURALIUM

ACTA UNIVERSITATIS OULUENSIS

A Scientiae Rerum Naturalium 527

KATRI ROPPOLA

ENVIRONMENTAL APPLICATIONS
OF MANOMETRIC RESPIROMETRIC
METHODS

Academic dissertation to be presented with the assent of

the Faculty of Science of the University of Oulu for public
defence in Auditorium TS101, Linnanmaa, on 2 May 2009,
at 12 noon

O U L U N Y L I O P I S TO, O U L U 2 0 0 9

Copyright 2009
Acta Univ. Oul. A 527, 2009

Supervised by
Docent Toivo Kuokkanen

Reviewed by
Associate Professor Yen Peng Ting
Professor Tuula Tuhkanen

ISBN 978-951-42-9078-7 (Paperback)

ISBN 978-951-42-9079-4 (PDF)
http://herkules.oulu.fi/isbn9789514290794/
ISSN 0355-3191 (Printed)
ISSN 1796-220X (Online)
http://herkules.oulu.fi/issn03553191/

Cover design
Raimo Ahonen

OULU UNIVERSITY PRESS

OULU 2009

Roppola, Katri, Environmental applications of manometric respirometric methods

Faculty of Science, Department of Chemistry, University of Oulu, P.O.Box 3000, FI-90014
University of Oulu, Finland
Acta Univ. Oul. A 527, 2009
Oulu, Finland

Abstract
In this work a manometric respirometric measuring system was applied to practical environmental
cases related to wastewater management and biodegradation studies of oil-contaminated soils and
materials used in landfill structures. Pollution of groundwater, surface water and soils is a
worldwide problem. Therefore, tests simulating the biodegradation behaviour of organic
compounds in water media and soils have become increasingly important. Respirometric methods
provide direct measurement of the oxygen consumed by micro-organisms in biodegradation
processes from an air or oxygen-enriched environment in a closed vessel.
Biochemical oxygen demand (BOD) is a crucial environmental parameter used to measure the
quality of water and treatment results in wastewater. Generally, BOD is measured with
standardised methods, which are usually time-consuming as well as laborious. In this work the
manometric respirometric test was compared with conventional BOD tests by determining the
BOD of pulp and paper mills as well as domestic wastewater samples. The effect of different
factors such as type, amount and pre-treatment of inoculum and the effect of dilution of a sample
on the BOD values were also tested. A right dilution was noticed to be the most significant factor
affecting the BOD values of the industrial wastewater samples. The mathematic estimation of the
BOD7 values from the respirometric data was proved to work reliably after a 23 day incubation
period. Characterisation of organic fractions of the pulp and paper mill wastewater was carried out
with methods including filtration, long term BOD measurements and COD analyses. The most
significant observation in characterisation analyses was that a remarkable part of the detected
oxygen demand was consumed for the biotransformation of biodegradable fractions into new inert
decomposition products, not only for mineralisation of the biodegradable COD fraction.
Biodegradation behaviour of the peat samples with different decomposition rates was studied
in order to evaluate the applicable peat types that can be used in landfill structures. Only minor
(BOD/ThOD < 0.4%) biodegradation was observed with compaction peat samples, and the stable
state, in which biodegradation stopped, was achieved during a two month period. The manometric
respirometric method was also applied for the biodegradation studies in which the effect of the
modification of soil properties on biodegradation rates of bio-oils was tested. Modified properties
were the nutrient content and the pH of the soil. Fertiliser addition and pH adjustment increased
both the BOD/ThOD% values of the model substances and the precision of the measurement. The
manometric respirometric method was proved to be an advanced method for simulating
biodegradation processes in soil and water media.

Keywords: biochemical oxygen demand, biodegradation, BOD test, organic compound,

respirometric method, wastewater

Acknowledgements
This work was carried out at the Department of Chemistry, Laboratory of
Physical Chemistry, University of Oulu. This research was financially supported
by the University of Oulu, Stora Enso Veitsiluoto Mills, North Ostrobothnia
Regional Environment Centre (NOREC), Oulu Waterworks, Vapo Oy and Kemira
Oyj. I was fortunate in that I received personal grants from the Oulu University
Scholarship Foundation, Maa- ja Vesitekniikan Tuki ry, Multidisciplinary
Environmental Graduate Net School (EnviroNet), University of Oulu; Faculty of
Science and the Tauno Tnning Foundation. All of these founding bodies are
greatly acknowledged. In addition, I would like to thank the following people
who have helped and encouraged me during this work:
For a start, I would like to express my deepest gratitude to my supervisor
Docent Toivo Kuokkanen for providing me with the opportunity to work with this
interesting subject. Toivo has always instructed me patiently and given me very
important advice in relation to my research. Special thanks go to Hanna Prokkola
(M.Sc.) for her valuable contributions with the experimental work, and to Jaakko
Rm (D.Sc., Tech.) for his momentous comments during this project. Also other
co-authors of my articles Hannu Nurmesniemi (Ph.D., Docent), Risto Pyki
(Ph,D., Docent), Pekka Vhoja (Ph.D.), Ilkka Vlimki (M.Sc.), Juhani
Kaakinen (M.Sc.), Kauko Kujala (D.Sc.,Tech., Docent), Matti Kuokkanen (M.Sc.,
Tech.), Eeva Heiska (M.Sc.) and Jussi Ruotsalainen (M.Sc., Tech.) are warmly
acknowledged. In addition, I would like to thank Suomen Ympristpalvelu Oy
for the nutrient analyses of the soil and peat samples.
I would like to thank Professor Tuula Tuhkanen (Tampere University of
Technology) and Associate Professor Yen Peng Ting (National University of
Singapore) for reviewing this thesis. A special thank you goes to Ilari Sohlo
(M.Sc.) for proof-reading this text.
I want to thank all of my fellow students and friends, especially my best
friend Anu. I am grateful that I have a friend like you! My warmest gratitude goes
to my parents Leena and Mauno, and to my sister Hanna-Leena and her family.
They all have encouraged and supported me during my long-lasting studies.
Lastly, I want to express my loving thanks to my husband Ari for his
encouragement and for believing in this project. I personally did not always
believe it was possible. And of course our son, Arttu, you are my sunshine...
Kempele, 2009

Katri Roppola
5

Abbreviations and nomenclature

Bunsen absorption coefficient

AS
activated sludge
ASM
activated sludge model
ATP
adenosine triphosphate
ATU
allylthiourea
BCODsol
soluble biodegradable COD [mg/l]
BCODtot
total biodegradable COD [mg/l]
BOD
biochemical oxygen demand [mg/l]
BODn
biochemical oxygen demand in n days [mg/l]
BODst
short term biochemical oxygen demand [mg/l]
BOD/ThOD biochemical/theoretical oxygen demand ratio (degree of
biodegradation)
Cfilt
final total COD of filtered wastewater sample (after BOD
measurement) [mg/l]
Craw
final total COD of raw wastewater sample (after BOD measurement)
[mg/l]
COD
chemical oxygen demand [mg/l]
CODinf,tot
influent total COD [mg/l]
CODinf,sol influent soluble COD [mg/l]
CODinf,part influent particulate COD [mg/l]
CODeff,sol effluent soluble COD [mg/l]
DF
dilution factor
DO
dissolved oxygen [mg/l]
DOC
dissolved organic carbon [mg/l]
ECn
concentration of toxicant yielding n% inhibition in BOD or OUR.
EU
European Union
HBOD
headspace biochemical oxygen demand
IAWQ
International Association on Water Quality
ISO
International Organization for Standardization
kBOD
kinetic rate constant for BOD [d1]
OECD
Organisation for Economic Co-operation and Development
OUR
oxygen uptake rate [mg/l h]
R
gas constant
RAS
return activated sludge inoculum
REACH
Registration, Evaluation and Authorisation of Chemicals
7

RSD
Sfilt
SFS
SI
SI
SP
Sraw
SS
SS,min
STOWA
ThCO2
ThOD
TOC
TOFA
UV
VSS
WW
WTP
WWT
XI
XP
XS
XS,min

relative standard deviation

final soluble COD of filtered wastewater sample (after BOD
measurement) [mg/l]
Finnish Standards Association
sewage inoculum
soluble inert COD [mg/l]
soluble residual microbial product (inert) [mg/l]
final soluble COD of raw wastewater sample (after BOD
measurement) [mg/l]
soluble biodegradable COD [mg/l]
mineralised part of the SS fraction [mg/L]
Dutch acronym for the Foundation for Applied Water Research
theoretical carbon dioxide
theoretical oxygen demand
total organic carbon
tall oil fatty acid
ultraviolet
volatile suspended solids
wastewater
wastewater treatment plant
wastewater treatment
particulate inert COD [mg/l]
particulate residual microbial product COD (inert) [mg/l]
particulate biodegradable COD [mg/l]
mineralised part of the XS fraction [mg/L]

List of original publications

This thesis is based on the following articles, which are referred to in the thesis by
their Roman numerals.
I

Roppola K, Kuokkanen T, Nurmesniemi H, Rm J, Pyki R & Prokkola H (2006)

Comparison Study of Manometric Respirometric Test and Common Chemical
Methods in the Determination of BOD7 in a Pulp and Paper Mills Wastewaters.
J Autom Meth and Manage Chem ID 90384: 15. DOI: 10.1155/JAMMC/2006/90384.
II Roppola K, Kuokkanen T, Rm J, Prokkola H & Heiska E (2007) Comparison Study
of Different BOD Tests in the Determination of BOD7 Evaluated in a Model Domestic
Sewage. J Autom Meth and Manage Chem ID 39761, 14. DOI: 10.1155/2007/39761.
III Roppola K, Kuokkanen T, Rm J, Prokkola H & Ruotsalainen J (2009)
Characterisation of Organic Fractions of Pulp and Paper Mill Wastewater with a
Manometric Respirometric BOD Method and Automatic COD Analyses. Chem Spec
Bioavail. In press.
IV Roppola K, Kuokkanen T, Kujala K & Kuokkanen M (2008) Utilization Potential of
Peats a Study on Peat Biodegradability Determined by Respirometric Method.
Water Air Soil Pollut 192: 5966. DOI 10.1007/s11270-008-9634-y.
V Kaakinen J, Vhoja P, Kuokkanen T & Roppola K (2007) Studies on the Effects of
Certain Soil Properties on the Biodegradation of Oils Determined by the Manometric
Respirometric Method. J Autom Meth and Manage Chem ID 34601: 17. DOI:
10.1155/2007/34601

The present author was the primary author of Articles IIV, and co-author of
Article V. The designing of the analyses, the bulk of the experimental work as
well as the analysis of the results related to Articles I, II and IV were carried out
by the present author. The designing of the research and the analysing of the
results related to Article III were the work of the present author. The Articles IIV
were written by the present author and modifications were carried out in coordination with the other authors. The designing of the experimental work as well
as the analysing of the results described in Article V were carried out together
with the first author. Experimental work related to Article V was the work of the
present author.
In addition, the results of the article Vhoja P, Roppola K, Vlimki I &
Kuokkanen T (2005) Studies of Biodegradability of Certain Oils in Forest Soil as
Determined by the Respirometric BOD OxiTop Method. Int J Environ Anal Chem
85: 10651073 are discussed in this thesis.

10

Contents
Abstract
Acknowledgements
5
Abbreviations and nomenclature
7
List of original publications
9
Contents
11
1 Introduction
13
2 Review of the literature
15
2.1 Biochemical oxygen demand and biodegradation................................... 15
2.2 Respirometric techniques ........................................................................ 16
2.3 Present day respirometers ....................................................................... 17
2.4 Applications of the respirometric methods ............................................. 19
2.4.1 Biodegradation measurements...................................................... 20
2.4.2 Applications in wastewater management ..................................... 21
3 The aims of this dissertation
29
4 Experimental work
31
4.1 Theory of biochemical oxygen demand measurements (I, II and
III) ........................................................................................................... 31
4.2 Theory of biodegradation measurements in soil (IV, V) ......................... 32
4.3 Other analysis methods ........................................................................... 35
4.4 Samples ................................................................................................... 35
4.5 Measurements ......................................................................................... 36
4.5.1 Biochemical oxygen demand measurement (I, II, III).................. 37
4.5.2 Characterisation of wastewater (III) ............................................. 38
4.5.3 Biodegradation measurements (IV, V) ......................................... 40
5 Results and discussion
43
5.1 Biochemical oxygen demand measurement ............................................ 43
5.1.1 Comparison between the manometric respirometric test
and conventional chemical BOD methods (I, II).......................... 43
5.1.2 Estimation of BOD7 value (I, II) .................................................. 46
5.1.3 Effect of different variables on BOD value (III) .......................... 49
5.1.4 Characterisation of wastewater fractions (III) .............................. 52
5.2 Biodegradation measurements ................................................................ 55
5.2.1 Biodegradation of peat samples (IV)............................................ 55
5.2.2 Effect of different variables of soils on biodegradation
behaviour of oil samples (V) ........................................................ 59
11

5.3 Precision of the manometric respirometric test ....................................... 64

6 Conclusion
67
7 Future work
71
References
73
Original publications
81

12

Introduction

Chemicals bring about benefits on which modern society is entirely dependent,

but then certain chemicals have caused serious damage to the environment and to
human health resulting in suffering and premature death [1]. Pollution of
groundwater and soil is a worldwide problem that can result in uptake and
accumulation of toxic chemicals in food chains and harm the flora and fauna of
affected habitats [2]. Many activities are known to cause contamination of soil,
surface water and groundwater, but in most cases, contamination is the results of
careless use of chemicals, or is simply a consequence of discharge or ignorance.
In Finland, typical activities or establishments that have caused contamination of
soils have been the distribution and storage of fuels, sawmills, impregnation
plants, various industries, depots and garages, greenhouses and shooting ranges.[3]
The European Union chemicals policy must ensure a high level of protection of
human health and the environment as enshrined in the treaty both for the present
generation and future generations while also ensuring the efficient functioning of
the internal market and the competitiveness of the chemical industry.
Fundamental to achieving these objectives is the Precautionary Principle. New
and existing chemicals should be properly tested in order to protect human health
and the environment. [1] It is very important to possess knowledge of the ecotoxicological properties of chemicals such as biodegradability for example in the
case of accidents.
Wastewater treatment plants are falling under increasing pressure to increase
the efficiency of their operations. Plants have to control the cost of wastewater
treatment but at the same time the quality of effluents that are discharged must be
improved. [4] The Water Framework Directive (Dir 2000/60/EC) points out the
necessity of a progressive reduction of pollutants in water effluents. The Directive
sets the goal of achieving a good status for all of Europe's surface waters and
groundwater by 2015. [5] Biochemical oxygen demand (BOD) is an important
parameter in water resource management and it is used to measure the quality of
water and treatment results in wastewater. BOD is a measure for the quantity of
oxygen required to decompose the organic matter by aerobic micro-organisms in
a sample of water. Certain factors such as toxic compounds and insufficient
nutrient content of wastewater can affect bacterial respiration and biodegradation
of organic compounds. Tests simulating the biodegradation behaviour of organic
compounds in surface water, groundwater and soils have become increasingly
important. Respirometric methods provide direct measurement of the oxygen
13

consumed by micro-organisms from an air or oxygen-enriched environment in a

closed vessel under conditions of constant temperature and agitation. [4]

14

Review of the literature

2.1

Biochemical oxygen demand and biodegradation

Biochemical oxygen demand (BOD) is a measure for the quantity of oxygen

required for the biodegradation of organic matter (carbonaceous demand) in water
and the oxygen used to oxidise inorganic material such as sulphides and ferrous
iron. It can also indicate the amount of oxygen used to oxidise reduced forms of
nitrogen (nitrogenous demand), unless their oxidation is prevented by an inhibitor.
[6] The five-day BOD test was developed over a hundred years ago in England
and the length of the test period was chosen based on the maximum time required
for any British river to flow from its source to the sea. [7] BOD is a crucial
environmental index for monitoring organic pollutants in wastewater. It measures
the rate of uptake of oxygen by micro-organisms in a water sample at a fixed
temperature and over a given period of time (usually 5 or 7 days). Probably the
most sensitive indicator of toxicity of wastewater is inhibition of the respiration
rate of the activated sludge. When respiration is inhibited, biochemical oxygen
demand and the rate of biodegradation of wastewater is reduced. [4]
Conventionally, BOD is measured with universal methods, such as iodometric
titration or the oxygen electrode, which are usually time-consuming as well as
laborious, due to the necessary oxygen content measurement of the sample before
and after the incubation period. A poor choice of dilution can produce a lack of a
part of the BOD data thus causing the test to be imprecise. [8, 9] Therefore, it is
necessary to develop new potential methods to measure this important parameter.
There are two types of bacteria in activated sludge: chemoautotrophs (mainly
nitrifiers) and heterotrophs (which consume the organic carbon of the BOD). The
chemoautotrophs oxidise ammonia to nitrite (Nitrosomonas) and then to nitrate
(Nitrobacter), and this process is not actually respiration. True respiration is only
displayed by the heterotrophic bacteria. Respiration involves the breakdown of
simple organic carbon molecules to the end products carbon dioxide (CO2) and
water (H2O). The major benefit of the energy (adenosine triphosphate, ATP) made
available in respiration is that it can be used to synthesise the various molecular
components required for cell growth and reproduction. In addition, growth
requires a supply of organic carbon molecules. Thus, bacteria need carbon and
other nutrients for both respiration and growth. [4, 10]

15

Biodegradation is the process whereby organic matter is decomposed by the

action of micro-organisms present in the environment which utilise carbon as
food. Depending on the nature of the micro-organisms, they might assimilate only
a part of the organic molecule, destroying the intact parent substance in a process
known as primary biodegradation, or they may assimilate it completely in a
process known as ultimate biodegradation. In either situation, some of the carbon
is burned to provide the energy necessary to sustain life and the rest is used to
generate more micro-organisms in the process of cell growth. The energyproducing part of the metabolic activity consumes oxygen, resulting in the
immediate formation of carbon dioxide, water and mineral salts in a process
known as mineralisation. [11, 12]
In heterotrophic bacteria, toxic compounds in wastewater can inhibit either
growth or respiration. The inhibitory compounds can act in a number of ways.
They may be inhibitory to their own biodegradation or influence the rate of
biodegradation of other compounds. [13] If growth is inhibited, the energy
requirements will decrease and the respiration rate will also be reduced. Since less
carbon will thus be required for respiration, biodegradation decreases. Thus,
wherever the toxicity acts, there is inhibition of both the respiration rate and the
rate of biodegradation. [4]
2.2

Respirometric techniques

Respirometry is a measurement and interpretation of the biological oxygen

consumption rate (aerobic conditions) or biogas generation rate (anaerobic
conditions) under well-defined experimental conditions. In general, the term
respirometry is used to describe the measurement of oxygen consumption in
wastewater samples or activated sludge. Under aerobic conditions the microorganisms consume oxygen in proportion to the organic matter and biomass in the
sample. The respiration rate is the amount of oxygen per unit of volume and time
[mg/l d] that is consumed by the micro-organisms. [13, 14] The first application
based on a respirometric technique for measuring the oxidation of wastewater was
reported in 1924 by Otto Heinrich Warburg (18831970). This respirometric
technique was manometry, which is based on the analysis of changes in the
pressure of the gas over an enclosed measuring system. Most of the respirometers
available today are based on Warburgs respirometer. [13, 15] After the invention
of the Warburg respirometer several researchers have developed different kinds of
respirometric measuring techniques based on manometeric, electrolytic or direct
16

measurement of oxygen consumption. Development of instrumentation and

automation has widened the application possibilities of respirometers in terms of
the optimisation and operation of wastewater treatment systems and waste
management and biodegradation studies. [1632]
2.3

Present day respirometers

Present day respirometers range from simple, manually operated bottle equipment
with dissolved oxygen (DO) sensors to complicated instruments that
automatically perform the sampling, calibration and calculation of the respiration
rate. Because DO concentration can be measured relative easily and reliably, it
has often been used in controlling activated sludge processes. However, DO
concentration-based control is not similar to respiration rate-based control.
Respirometers, monitoring of the rate of oxygen utilisation by metabolising
micro-organisms, can be categorised as the manometric, electrolytic or direct
measurement of DO. All respirometers include certain technologies that enable
assessing the rate at which the biomass takes up DO from the liquid. The
operation of all existing respirometers can be explained in terms of two criteria: 1.
the phase where oxygen concentration is measured and 2. whether or not there is
input and output of liquid and gas (flowing or static). [14, 33]
There are two types of respirometers: on-line versions and traditional
versions involving a set of laboratory instruments. Ideally, on-line respirometers
should operate on a continuous basis, taking measurements in real time.
Laboratory respirometers have an additional role to play in process control and
management. Decrease in oxygen concentration can be sensed by a manometric
device in the air space above the liquid, or by an oxygen electrode inserted into
the liquid. Respirometers that are used for long-term biotreatability studies
require that respired oxygen is replenished or that there is sufficient oxygen space
(gas space). Oxygen replenishment may be achieved either by electrolytic
generation of oxygen or by bleeding in small quantities of the compressed gas. [4]
Manometric respirometers relate oxygen uptake to the change in pressure
caused by oxygen consumption while maintaining a constant volume. As the
micro-organisms respire they use oxygen converting the organic carbon in the
solution to CO2, which is absorbed to alkali (usually KOH or NaOH). Pressure
drops in the system, which are directly proportional to BOD values, are measured
by a pressure sensor. The BOD values are stored in the sensor memory and can be
called up on the large-format display at any time without the need for time17

consuming conversion using factors. [3436] Before measurement, it is essential

to estimate the measuring range (BOD in the unit mg/l) of the sample to that is to
be analysed. That determines the volume of the gas space and the amount of
wastewater sample necessary. [37]
Since the development of the first electrolytic respirometer in 1965,
electrolytic respirometers have been widely used to optimise the wastewater
treatment processes as well as to determine the biodegradability of compounds.
[2728, 31, 33, 3841] The measuring principle for all electrolytic respirometers
is quite similar. As micro-organisms respire they use oxygen converting the
organic carbon in the solution to CO2 gas, which is absorbed to alkali. This causes
a reduction in the gas pressure, which can be sensed with various sensors or
membranes. A small current is created in electrolysis cell and this generates
oxidation/reduction reactions in the electrolysis cell and oxygen is formed at the
anode. The amount of oxygen produced by the electrolysis correlates with the
amount of oxygen consumed by bacteria. [33, 40]
Headspace biochemical oxygen demand test (HBOD) is a batch respirometric
method based on a mass balance of oxygen in a sealed tube using a measurement
of the oxygen concentration in the headspace of the tube. In the first HBOD test,
oxygen concentration was evaluated by measuring the DO of a sample by pouring
it into a small sample holder. [15] The method has since been developed and the
today oxygen demand can be calculated from the decrease in oxygen
concentration in the gas phase with the HBOD probe. [9, 4244]
Biosensors are defined as devices built up by a biological recognition element
combined with a suitable transducer, e.g. electrodes. The biological component
produces a biochemical signal which is converted into an electrical signal by the
transducer element. Biological sensing components such as enzymes, antibodies
or whole microbial cells are responsible for the high selectivity and sensitivity
and also for the wide range of substances to be analysed by the biosensor. [45]
The BOD sensors can be generally classified as being either of the biofilm and
respirometer -type. Both types are based on the respirometric principle. Biofilm
type sensors have generated a great interest among scientists due to their various
advantages including rapid analysis, simplicity, compact design, low cost and
possibility of instrumentation for on-line applications. A common BOD -sensor
consists of a Clark type probe (Pt-cathode, Ag-anode and KCl electrolyte solution)
and a synthetic microbial membrane. [6, 8, 4647]

18

2.4

Applications of the respirometric methods

Within the last decade, biological treatment of wastewater, wastes and

contaminated soils has become one of the major treatment technologies used in
environmental engineering. The primary objective in wastewater treatment
systems usually involves the removal of carbonaceous material from the waste
stream through the growth of heterotrophic bacteria. Carbonaceous material
removal and related processes are not necessarily the only sinks for oxygen in
activated sludge systems. Nitrification, which involves oxidation of ammonia to
nitrate by autotrophic organisms, will often account for approximately 40 percent
of the total oxygen demand. In an activated sludge system, respiration rate and
oxygen demand are related to all of the aerobic organism activities, and vary in
space and time. [14]
Many respirometry-based control strategies have been proposed in the
literature but very few implementations are reported. Respirometry is a powerful
tool for assessing the condition of a system. [14] The importance of respiration in
this context is that it is quickly and easily measurable. Using new technologically
advanced respirometers it is possible to utilise respiration measurements for
biodegradation measurements in water media and soils, for kinetic analysis and
biokinetic constant determination, for modelling the wastewater treatment process,
for managing toxicity, for measuring short-term BOD, nitrification capacity,
activity of activated sludge, readily degradable BOD treatment capacity and
aeration requirements. [4, 13, 36] Even the biodegradability of poorly soluble
substances such as oils and organic compounds can be analysed with
respirometric techniques. [4853]
There are requirements and limitations for the test methods, and new methods
(non-animal) must be constantly developed and validated. [1] European Union
Directive 67/548/EEC (Annex V) lists the testing requirements for new chemicals.
The accepted methods for the determination of eco-toxicity and biodegradation
are listed in Annex V, Part C. [54] A number of guidelines have been developed to
standardise evaluations of the ultimate or ready biodegradability of specific
compounds or formulations [11, 5556]. These guidelines usually involve longterm (14 to 28 day) respirometric testing to evaluate the extent to which a
compound is oxidised relative to its theoretical oxygen demand (ThOD) or
measured chemical oxygen demand (COD).

19

2.4.1 Biodegradation measurements

Biodegradability is an important parameter due to the simple fact that an easily
degradable substance will cause no long-term risk to the environment. Knowing
the ecological behaviour of substances and products is one of the responsibilities
of the manufacturing chemical industry. [57] The global annual production of
chemicals has increased from 1 million tonnes in 1930 to 400 million tonnes
today. The White Paper formulated by the European Commission presents
proposals for a strategy on a future chemicals policy in the EU with the overriding
goal of sustainable development. The present system for general industrial
chemicals distinguishes between existing substances, i.e. all chemicals declared to
be on the market in September 1981, as well as new substances i.e. that have
entered the market since that date. Risks presented by new chemicals to human
health and the environment must be tested and assessed according to Directive
67/548/EEC. Existing substances are not subject to the same testing requirements,
although there is not enough knowledge concerning the properties and uses of
existing substances. The Commission proposes that existing and new substances
should in the future, following the phasing in of existing substances by 2012, be
subject to the same procedure under a single system. The current new substances
system should be revised and made more effective and efficient, and the revised
obligations should be extended to existing substances. The proposed system is
called REACH (Registration, Evaluation and Authorisation of Chemicals). [1, 54]
Information on the degradability of chemicals may be used for hazard
assessment or for risk assessment. Hazard and risk assessment as well as aquatic
hazard classification are generally based on data obtained in standardised tests for
ready biodegradability, but results of tests simulating biodegradation in water,
aquatic sediment and soils may also be used for these purposes. Other types of
test data that may be considered in an assessment of the potential environmental
hazard or risk include sewage treatment plant simulation data, inherent
biodegradability, anaerobic biodegradability, biodegradability in seawater and
abiotic transformation. [58] Readily biodegradable is an arbitrary classification
for chemicals that have passed certain specified screening tests for ultimate
biodegradability. These tests are so stringent that it is assumed that such
compounds will rapidly and completely biodegrade in aquatic environments
under aerobic conditions. In OECD 301 guidelines the pass levels for ready
biodegradability are a 70% removal of dissolved oxygen carbon (DOC), and a
60% production of theoretical oxygen demand (ThOD) or theoretical carbon
20

dioxide (ThCO2) for respirometric methods. Some of the carbon from the test
chemical is incorporated into new cells, thus the percentage of CO2 produced is
lower than the percentage of carbon being used (ultimate biodegradation). In
practice, a substance showing 60% biodegradability in a mineralisation test would
be expected to be consumed in excess of 90% when allowance is made for
incorporation of organic carbon into the growing bacterial population. These pass
values have to be reached in a 10-day window within the 28-d period of the test,
except where mentioned below. The 10-d window begins when the degree of
biodegradation has reached 10% DOC, ThOD or ThCO2 and must end before day
28 of the test. Chemicals that reach the pass levels after the 28 day period are not
deemed to be readily biodegradable. [11, 58]
Degradation processes are constantly taking place on a large scale in the
natural environment, in water and soils. Under favourable conditions, microorganisms can oxidatively degrade organic contaminants completely into nontoxic by-products such as carbon dioxide and water or organic acids and methane.
[2] Development of respirometric techniques enables simulations of the natural
biodegradation processes under optimised and controlled conditions. [38, 40, 49
51, 57, 5960] In the course of growing environmental consciousness, tests
simulating biodegradation in soils have become increasingly important. In general,
the methods are designed for the evaluation of the mineralisation rate of a
14
C-labelled compound in soil. [61] At present, it is possible to measure soil
respiration and biodegradation of chemicals in soils without 14C labelling by
respirometric tests. [6263]
2.4.2 Applications in wastewater management
The aim of wastewater treatment is to remove contaminants from wastewater
using physical, chemical and biological processes. Wastewater can contain solid
wastes (organic and inorganic), organic material which consumes oxygen (BOD),
nutrients, greases and oils, metals, different kinds of chemicals, and microorganisms. Wastewater treatment usually involves three different stages called the
primary, secondary and tertiary treatment. In primary treatment, the influent
wastewater is strained to remove all large objects that are deposited in the sewer
system. Secondary treatment is designed to substantially degrade the biological
content of the wastewater (biological treatment), whereas tertiary treatment
provides a final stage to raise the effluent quality before it is discharged to the
receiving environment. [64]
21

A general principle in biological wastewater treatment is to accelerate the

oxidation process of the organic matter, which occurs naturally in the receiving
water. This is usually achieved by using an activated sludge technique. A common
principle of biological wastewater treatments is to use the right micro-organisms
(bacteria) that both biodegrade organic material and transform undesirable
substrate into biomass that is no longer in solution and can be separated from the
treated water through settling techniques. This results in BOD reduction, COD
reduction as well as wastewater odour control. Micro-organisms provide not only
organic material as food but also oxygen to breathe. Thus, adequate supply of
oxygen is required. [64] In addition, micro-organisms require nutrients (mainly
nitrogen, N and phosphorus, P) for growth and reproduction. It is usually stated
that the ratio of BOD:N:P in the wastewater to be treated should be approximately
100:5:1 for aerobic treatment, but for example pulp and paper mill wastewater
can be treated with a BOD:N:P ratio of 100:2,53,5:0,10,5. [65] Municipal
wastewater contains a sufficient amount of nutrients, whereas industrial
wastewaters can suffer from lack of nitrogen, phosphorus and other traceelements. Therefore nitrogen and phosphorus must be added to the biological
treatment process in order to attain an adequate BOD:N:P ratio. Temperature and
pH of the wastewater also affect wastewater treatment efficiency. [64]
Respiration rate is used as the most sensitive variable in detailed modelling of
an activated sludge process theory. Thus, a respirometer can give crucial
information for the control of a treatment plant. Each test produces a
characteristic pattern of the way in which the oxygen uptake rate (OUR) changes
as the treatment proceeds. Every change during the treatment process has an
effect on the respiration rate and developing respirograph. [66] In general, oxygen
uptake curves consist of a brief lag period (adaptation period), an extended linear
region of rapid oxygen uptake (exogeneous phase) and an endogeneous
respiration period of very low OUR (so-called plateau BOD). When the substrate
is completely removed by the bacteria a plateau occurs. [67]
Modelling and optimising the treatment process
A wastewater treatment plant is, however, never at steady state, but is
continuously subject to disturbances. In process control, oxygen uptake rates
(respiration rates) can be used to model the aerobic treatment process. Different
kinds of sources, which cause problems in the treatment plant, can be detected
with respirometric measurements. In addition, every process changes and
22

operations can be monitored closely. All the variables that influence the process
are called inputs. Some of them can be manipulated and they are hence called
manipulated variables. Other inputs affect the process and are generated
externally. These inputs cannot be manipulated and they are defined as
disturbances. [14] The effect of the temperature and pH of wastewater on
biochemical oxygen demand can be controlled and studied with respirometric
tests. [20, 34] In addition, the effect of different bacterial inoculums as well as the
effect of volume, concentration and pre-conditioning of bacterial seed on BOD
and wastewater treatment efficiency have been studied by several researchers. [23,
3334, 39, 6873] Also the nutrient content has effect on biochemical oxygen
demand. If there are no sufficient nutrient salts present in the sample (industrial
wastewater), accurate results can be only achieved by diluting the sample with
dilution water. The nature of the carbon source also has an influence on the
biodegradation and BOD value. Some carbon sources (for example glucose) have
higher and more variable oxidation rates than others (for example glycerol). That
influences the repeatability of the results. [34, 74]
Toxicity management
Until early 2000s, the effects of toxicity on process control has not received a
great deal of attention. That is probably because of the unavailability of suitable
instrumentation for measuring toxicity. Pressure from the regulators to ensure that
toxic effluents are not discharged in treatment plants provides an opportunity for
managers to introduce formal toxicity management plans within the framework of
operational procedures concerning sewage treatment. There are four stages to any
such toxicity management plan: prevention, toxicity monitoring, identification of
toxic streams and toxicity reduction. Prevention has an important role in toxicity
management, particularly for plants that receive discharges by sewer from
multiple industrial sources. In that case, early-warning devices such as on-line
respirometers can be used to control receiving wastewaters. [4] It is noted that
standard domestic wastewater has generally a constant composition and rarely
contains compounds or factors that would inhibit biodegradation. In contrast to
domestic sewage, industrial effluents are characterised as complex mixtures with
varying concentrations of pollutants and inhibitory substances, such as heavy
metals, volatile organic compounds, and poly-aromatic and polychlorinated
organic substances, which may affect system performance. [75] Respirometry is
an advantageous mechanism in identifying compounds that might potentially
23

have an adverse effect on a wastewater treatment system. [10, 3435, 74, 76] A
respiration inhibition test is important for managers of treatment facilities in order
to check the toxicity of the accepted wastes. EC50, EC20 and EC10 values are used
in respiration inhibition and nitrification inhibition tests to represent the
concentration of wastewater (%) which produces 50%, 20% and 10% inhibition
of the oxygen uptake rate in comparison to the control sample. [4]
Nitrification monitoring
Nitrification, which involves oxidation of ammonia nitrogen to nitrate nitrogen by
autotrophic organisms, is one of the most significant factors contributing to
problems in BOD measurements. In spite of that, it is difficult to monitor and
adjust nitrification in a plant operation. The decrease of the substrate (NH4+) and
increase of the end-product (NO3) gives information but does not reflect the
actual activity of the nitrifiers. Selective inhibitors of NH4+ and NO2 oxidation
have been tested to determine the nitrifying activity of sludge. NaClO3 is an
inhibitor of NO2- oxidation by Nitrobacter, but has no immediate negative effects
on NH4+ oxidation by Nitrosomonas bacteria. Nitrosomonas becomes detectable
only after 30 minutes due to the slow conversion of NaClO3 to NaClO2, which
inhibits both populations. Allyl-thiourea (ATU, C4H8N2S) is a selective inhibitor
of NH4+ oxidation. [77] Respirometers make it possible to rapidly measure the
nitrification capacity of wastewater and activated sludge. Fig. 1 presents a
schematic representation of a typical oxygen uptake profile recorded with
respirometry.

24

Fig. 1. A typical oxygen uptake rate profile recorded with the developed nitrification
activity determination method. [77].

Evaluation of BODn value

Measures of respiration rate may also be used as an indicator of short term BOD
(BODst). Traditional measurement values such as BOD5 or BOD7 involve a 5 or
7-day incubation period of wastewater in the presence of bacteria. However, BOD
measurements over such a long time frame are not useful in assessing the
biodegradability of organic matter passing through a wastewater treatment plant.
The extended measuring time causes problems in controlling a wastewater
treatment plant because the wastewater in the process keeps changing during such
a long test period. The microbial oxygen consumption measured by the
conventional method is the sum of the oxygen used to oxidise both the easily
assimilable compounds and biodegradable polymers (e.g. starch and proteins),
whereas BOD biosensors can normally only give responses to the fast and easily
assimilable compounds in the samples. [78] BODst uses activated sludge biomass
of the treatment plant and its purpose is to measure readily biodegradable BOD. It
is carried out by measuring the increase in oxygen consumption when a small
amount of influent wastewater is added to activated sludge in the endogenous
state in a closed chamber. Usually BODst measurements are made with microbial
BOD sensors based on the respirometric principle. The BOD5 and BOD7 values of
wastewater samples are calculated from the calibration curve of the calibration
25

solutions such as OECD synthetic wastewater with known BOD5 values. The
proper selection of the calibration solution is a key issue for obtaining good
agreement between results of the sensor BOD measurements and conventional
BOD analysis. [6]
BODst can also be determined by measuring the OUR [mg/l h] over a period
ranging from one to four hours after adding wastewater to the mixed liquor.
However, correlating BODst and BOD5 are futile, since the discrepancy is simply
an indication of the relative amount of non-readily degradable material in the
wastewater. BODst should, therefore, be viewed as a tool that is useful in process
control, rather than as a cost-saving predictor of BOD5. [4, 6]
Characterisation of wastewater fractions
Wastewater contains various organic components with different physical and
biochemical characteristics. Characterisation of wastewater has been an important
aspect in terms of the design, optimisation and operation of wastewater treatment
systems. Advanced mathematical models summarise recent knowledge on
activated sludge wastewater treatment processes. The models are useful in
designing and in the operational assistance of activated sludge processes. In 1987,
the International Association on Water Quality (IAWQ) introduced the Activated
Sludge Model No1 (ASM No 1), in which a simple mathematical model having
the capability of realistically predicting the performance of single-sludge systems
carrying out carbon oxidation, nitrification and denitrification was presented. This
model is probably still the most widely used for describing wastewater treatment
(WWT) processes around the world. The development of ASMs led to a much
better understanding of different treatment processes but it also necessitated more
intensive wastewater characterisation. Generally, determination of biokinetic
parameters and wastewater components by mathematical models is quite timeconsuming (at least by conventional BOD tests) and usually some modifications
and assumptions have to be made on the models. Even then, some of the
parameters are usually unidentifiable. [7981]
Many researchers have applied and modelled the ASM, and several
characterisation methods for organic fractions of wastewater have been proposed
[39, 73, 7996]. Some of these methods focus on the determination of both
biodegradable and inert COD fractions [73, 80, 82, 86, 89, 9293, 95] while the
other focus on the characterisation of either biodegradable [39, 79, 8385, 88, 91,
96] or inert COD fractions [87, 90, 94]. Typically, the carbonaceous material is
26

divided into biodegradable COD, non-biodegradable COD (inert material) and

biomass (Fig. 2). The biodegradable COD is further divided into readily
biodegradable substrate (SS) and slowly biodegradable, particulate substrate (XS).
It is hypothesised that the readily biodegradable substrate consists of simple
soluble molecules that can be readily absorbed by organisms and metabolised for
energy and synthesis, whereas the slowly biodegradable substrate is assumed to
be made up of particulate/colloidal/complex organic molecules that require
enzymatic breakdown prior to absorption and utilisation. The non-biodegradable
COD is divided into soluble (SI) and particulate (XI) material. Both are
considered to be unaffected by the biological action in the system. The inert
soluble material leaves the system by the secondary clarifier effluent, whereas the
inert particulate material is enmeshed in the sludge mass and accumulates as inert
volatile suspended solids (VSS). [8081]

Fig. 2. Chemical oxygen demand (COD) components in ASM No1. [81]

The total biodegradable COD (BCODtot) of influent is the sum of the readily
biodegradable soluble (SS) and the slowly biodegradable particulate (XS) COD
fractions. SS is taken up by activated sludge in a matter of minutes and
metabolised, giving rise to a high unit rate of oxygen demand for synthesis. XS
must first be sorbed onto the micro-organisms, and broken down to simple
chemical units by extracellular enzymes before finally being metabolised by the
27

micro-organisms [8081]. Generally, determination of BCODtot is based on a

long-term respirometric BOD test applying an unfiltered influent combined with a
curve-fit. A reaction rate of BOD consumption can be modelled as first order
kinetics and the BODtot and the kinetic rate constant kBOD can be evaluated from
the fitted BOD-curve according to Eq. (1). When kBOD is determined for a certain
wastewater, BODtot can be determined from the BOD5 or BOD7 value. [93, 95]
BODtot = BODt / (1 e kBOD t)

(1)

Heterotrophic biomass is both generated by growth on readily biodegradable

material and lost by decay, which incorporates a large number of mechanisms
including endogenous metabolism, death, predation and lysis. The decay acts to
convert biomass to a combination of slowly biodegradable substance XS (f), and
new inert products (1 f). [80] The new inert metabolic products (Sp and Xp) can
be determined by COD measurements of unfiltered and filtered samples after the
biodegradation measurements of raw (Craw and Sraw) and filtered (Cfilt and Sfilt)
influent. [87, 88, 92, 96]. If the values of the new metabolic products are not
determined, a value between 0.10.2 (1020%) can be assumed and applied to the
Eq. (2) for the municipal wastewater. [93]
BCOD = BODtot / (1 f)

(2)

In most of the methods the soluble influent COD (CODinf,sol) is subdivided into
biodegradable (SS) and inert (SI) fractions by assuming that inert COD can be
found from the effluent COD of low loaded activated sludge plants. Subsequently,
the inert fraction SI is subtracted from the CODinf,sol to give the fraction SS. Since
BCOD is determined, the particulate biodegradable fraction (XS) can then be
calculated by subtracting the fraction SS from the BCOD. Finally, the particulate
inert fraction (XI) can be determined by subtracting the determined fractions (SS,
SI and XS) from CODinf,tot. [93, 95]
Generally, the characterisation methods are developed for municipal
wastewater, in which COD levels are quite low. Hence, these methods are not
suitable for industrial wastewaters with high organic content. Therefore, it is
important to both model these existing characteristic methods as well as develop
new methods suitable for industrial wastewater.

28

The aims of this dissertation

This dissertation consists of five articles (IV) in which the manometric

respirometric method is applied for biodegradation and biochemical oxygen
demand studies. In addition, results of our former research [63], in which the
biodegradation of different oils in soils was studied, is also discussed. That
research was closely connected to this doctoral thesis.
BOD is a crucial environmental parameter for monitoring organic pollutants
in wastewater. However, conventional BOD tests are usually time-consuming and
laborious due to the oxygen content measurements of the sample before and after
the incubation period. It is not possible to detect intervening values during the
incubation period in conventional BOD tests. Managing a wastewater treatment
plant can be very difficult when using these tests, because the conditions at a
plant change during test periods. Conventional tests are also affected by factors
such as dilution and sample preparation and possible inhibition. On the other hand,
conventional BOD tests have some benefits as compared to new, rapid, automated
and analytical techniques. The classical methods are used universally and the
results are therefore comparable. However, various microbial biosensors and
respirometric techniques have been developed to make BOD measurement faster
and easier. The aim of this work was to investigate the applicability of the
manometric respirometric test in the BOD determination of pulp and paper mills
as well as municipal wastewaters (Article I and II). The respirometric test was
compared with conventional chemical BOD measuring methods and the seventhday BOD values were estimated after a short-term measuring period (13 days).
Conventional chemical methods give only one BOD value after a seven-day
incubation period. Predictability of the BOD7 value could reduce the
measurement time and for example improve the chemical adjustment of
wastewater treatment processes.
Characterisation of wastewater is an important aspect for the design,
optimisation and operation of wastewater treatment systems. Hence, research
related to BOD measurements was broadened in this study to include the
characterisation of organic material of pulp and paper mill wastewater. To begin
with, preliminary tests including experiments with additional microbial seed as
well as dilution series were carried out before the characterisation analyses in
order to optimise the method. After the optimisation, different organic fractions of
pulp and paper mills wastewater were characterised with three physical-chemical
methods, which include long term BOD measurements and COD analyses of
29

unfiltered and filtered wastewater samples. The characterisation of organic

fractions of wastewater is discussed in Article III.
Finnish legislation concerning waste management is guided by several EU
directives, Council decisions and decrees, which have been transferred into the
national legislative framework by a set of decisions and decrees issued by
government bodies [97100]. Requirements concerning the structure and ground
conditions of the landfills are defined in the EU landfill directive (Council
Directive 1999/31/EC) [101]. The purpose is to prevent contamination of soil,
groundwater and surface water. Peat has numerous properties that make it an
excellent material for use in the barrier layers of landfills. As an organic material
it can absorb and store several organic and inorganic elements and compounds.
The manometric respirometric test was used to evaluate the applicable peat types
that can be used in landfill structures for the base structures of new landfill areas
as well as to improve the surface of sites that are no longer in active use. The
biodegradation rates of different peat types were analysed and the effect of the pH
of peat samples on biodegradation behaviour was evaluated (Article IV).
Certain man-made chemicals may cause serious problems when they are
spilled in soil or in water. The most common harmful substances causing
contamination of soils are heavy metals such as cadmium, lead and arsenic, oil
products, polyaromatic hydrocarbons, polychlorinated biphenyls, chlorophenols,
and pesticides. [3] The characteristics of these chemicals affect biodegradation
ability and toxicity, as well as their aptitude to bioaccumulate or move in nature.
It was shown earlier that a manometric respirometric test is a suitable method for
the biodegradation studies of oils in water media [4951]. In addition, this test is
also applicable to biodegradation studies of oils in forest soils [63]. In this project
the aim was to apply the manometric respirometric method for biodegradation
studies in which the effect of the modification of soil properties on biodegradation
rates was tested. The same substances as in the previous researches [4951, 63]
were used as model substances. The properties modified included nutrient content
and the pH of the soil. The results of this study are presented in Article V.

30

Experimental work

A manometric respirometric measuring system was applied in practical

environmental cases such as that of controlling wastewater treatment processes
(Articles IIII) and studies of biodegradation in soil media (Articles IVV).
4.1

Theory of biochemical oxygen demand measurements (I, II and

III)

The manometric respirometric equipment used in biochemical oxygen demand

analyses was the OxiTop Control 6 instrumentation presented in Fig. 3. (WTW
Weilheim, Germany)

Fig. 3. Oxitop Control 6 instrumentation (WTW, Weilheim).

The test is based on very accurate automated pressure measurements via

piezoresistive electronic pressure sensors in a closed bottle under constant
temperature (20.0 0.2 oC). A certain amount of oxygen is consumed during the
31

biodegradation process of the organic matter. At the same time the CO2 gas that is
formed is removed from the gas space by means of an absorber (NaOH), so that
the resulting pressure decline is a measure of the biochemical oxygen demand. In
the BOD measurements of water samples the instrument calculates BOD in the
unit mg/l using the ideal gas law that is modified for closed space conditions, as
described in Eq. (3):
BOD [mg/l] = M(O2)/RTm [(Vtot Vl)/Vl + Tm/T0] p(O2)

(3)

M(O2) is the molar mass of oxygen (32.00 g/mol), R is the gas constant (83.144 l
hPa mol1 K1), Tm is the measuring temperature (K), T0 is 273.15 K, Vtot is the
bottle volume (ml), Vl is the liquid phase volume (ml), is the Bunsen absorption
coefficient (0.03103) and p(O2) is the difference in partial oxygen pressure (hPa).
The chemical BOD measuring method used (Article I, II) was SFS-EN
1899-1 [102]. Residual oxygen after a seven-day incubation was determined
according to the standards SFS-EN 25813 and SFS-EN 25814 [103104]. The
analyses with electrochemical probe method were carried out with the Stirrox G,
WP3-ST, WTW oxygen sensor. The iodometric method is a titrimetric procedure
based on the oxidising property of dissolved oxygen, while the membrane
electrode procedure is based on the rate of diffusion of molecular oxygen across a
membrane.[105106] The titration end point was detected visually. A UV
absorption meter (Endress-Hauser, Stamosens CSM 750 / CSS 70) measurement
was used to measure the biologically treated wastewater (Article II).
Measurement is based on the spectral absorption of organic substances in the UV
range (the wavelength of measurement l = 254 nm). Measured spectral absorption
coefficient [1/m] was set in correlation to BOD7 value [mg/l]. The UV absorption
measurement is direct measurement of wastewater and does not require chemical
addition or sample preparation. [107]
4.2

Theory of biodegradation measurements in soil (IV, V)

The biodegradation measurements were carried out with the OxiTop Control
B6M instrumentation specified for soil respiration analyses (Fig. 4).

32

Fig. 4. Oxitop Control B6M instrumentation (WTW, Weilheim).

The principle of the measurement is the same as with BOD measurements, but the
calculations concerning the biodegradation degree of the sample are different. The
degree of biodegradation can be calculated when the volume of soil, the free gas
volume of the measuring bottle, the amount of oxygen consumed and the
theoretical oxygen demand are known. When the weight of the soil (msoil (g)) and
its bulk density (dsoil (g/l)) are known, the volume of the soil (Vsoil (l)) can be
calculated according to Eq. (4). [108109] Bulk densities of soil samples were
measured using equipment with a measuring cylinder of 1 l and a weight of 650 g
(surface pressure 8 g cm2). [110]
Vsoil = msoil /dsoil

(4)

The free gas volume (Vfr (l)) of the measurement bottle can be calculated using
Eq. (5), where Vbottle is the volume of the bottle (0.96 l), Vvessel is the characteristic
volume of the absorption vessel (0.01 l) and VNaOH is the characteristic volume of
the absorption agent (0.05 l).
Vfr = Vbottle Vvessel VNaOH Vsoil

(5)

In biodegradation measurements of soils the instrument gives the pressure decline

(p) in hectopascal (hPa) which must be converted to the amount of oxygen
consumed (m (g)) according to Eq. (6).
m = p Vfr MO2 / (R T)

(6)

33

In which MO2 is the molar mass of oxygen (32.00 g/mol), R is the specific gas
constant (8.301 Jmol1K1) and T is the measuring temperature (293.15 K). To be
able to monitor only the biodegradation of the chemical, soil respiration is
excluded by subtracting the m value of the soil (i.e. blank value) from the m
value of the soil/chemical mixture. The biochemical oxygen demand of the
chemical (e.g. oil) is calculated from the m value of the chemical (mchem (g))
and given in Eq. (7), where msample is the amount of the chemical weighed in the
experiment.
BOD (g/g) = mchem / msample

(7)

Theoretical oxygen demand (ThOD [g/g]) can be calculated when the carbon
content of the sample is known.
ThOD (g/g) = [mC (MO2 / MC)] / msample

(8)

MC is the molar mass of carbon and mC is the mass of carbon (measured with
CHNS/O analyzer, see chapter 4.3). The degree of biodegradation as a percentage
can be calculated using Eq. (9).
Degree of biodegradation = (BOD/ThOD) 100%

(9)

Other elements besides carbon also have an effect on theoretical oxygen demand.
Hydrogen and phosphorus atoms for example increase oxygen consumption,
whereas the oxygen atom in a molecule diminishes the ThOD value. The
hydrogen content could easily be determined with elemental analysis, but the
oxygen determination is not possible using our equipment. Since, the influence of
these atoms on ThOD value is not very significant when compared to carbon,
calculation of the ThOD values were carried out in this work as in our earlier
BOD studies using only the carbon content.
Generally, theoretical oxygen demand (ThOD) may be calculated if the
elemental composition is determined or known. For the compound
CcHhClclNnNanaOoPpSs the ThOD, without nitrification, would be
ThOD = 16 [2c + (h cl 3n) + 3s + 5/2p + 1/2na o] / M

(10)

M is the molar mass of the compound. Since the actual theoretical oxygen
demand values of the studied samples could be slightly larger than values
calculated using only the carbon content, the biodegradation results presented
herein can be a little bit larger than actual biodegradation degrees. [11]

34

4.3

Other analysis methods

The chemical oxygen demand measurements used in characterisation studies of

wastewater were determined according to standard SFS 5504 [111]. The CODCr
measurements were carried out with Hach Lange HT 200 S (sample digestion)
and Hach Lange DR 2800 (spectrometric analysis) analysers. The carbon contents
of the oils, soil samples and peat samples used in biodegradation measurements
were determined with a Perkin Elmer 2400 Series II CHNS/O analyser based on
combustion of the samples and determination of carbon in carbon dioxide. The
soil type, pH, conductivity, the amounts of major nutrients in the soil and peat
samples were determined before the biodegradation studies at Suomen
Ympristpalvelu Plc, which is a laboratory specialised in soil fertility studies of
agricultural land. Conductivity and pH were measured from a 1:2.5 (v/v) soilwater suspension according to standard ISO 10390:2005 [112] with a Consort C
831 pH/EC analyser. Concentrations of exchangeable calcium, potassium,
magnesium and easily soluble phosphorus were determined from a 1:10 (v/v)
extract of a mixture of 0.5 M acetic acid 0.5 M ammonium acetate. Phosphorus
was determined with a spectrophotometric molybdenum blue method using a
Foss-Tecator 5000 FIA Star Flow Injection Analyser. The concentrations of
calcium, potassium and magnesium were measured with a Perkin Elmer AAnalyst
700 FAAS instrument. The moisture content of the soil samples were determined
by drying the soil in a heating chamber (105 oC) for 2 hours.
4.4

Samples

Biochemical oxygen demand measurements were carried out with pulp and paper
mill wastewater samples and domestic sewage samples. Pulp and paper mill
wastewater samples were collected at the Stora Enso Veitsiluoto Mills in the City
of Kemi in Northern Finland (Article I) and at the Mets-Botnia Mill in the same
city Kemi (Article III). The Veitsiluoto Mill in Kemi is an integrated production
facility manufacturing office papers, coated mechanical papers and sawn products,
whereas Mets-Botnia Mill manufactures bleached (elemental chlorine free) and
unbleached sulphate pulp and is specialised in the production of pulp suited for
paper and linerboard manufacture. At the Stora Enso Mill the samples were
collected from sewer of the chemical pulp mill after the sampled wastewater had
been subject to biological treatment (henceforth referred to as pulp mill sample)
and from the sewer of the paper mill after chemical poly-aluminium treatment
35

(henceforth referred to as paper mill sample). At the Mets-Botnia Mill the

influent samples (from here on referred to as Bio in samples) were collected
from the primary equalisation basin before biological treatment, and effluent
samples (from now on referred to as Bio out samples) were collected from the
sewer after the secondary sedimentation basin. Municipal wastewater samples
were collected at the Oulu Waterworks (Taskila WTP) separately from three
different stages of the wastewater treatment process: after pre-clarification (preclarified), the post-aeration basin (biologically treated) and after final
clarification (outflowing water). The sewage inoculum (SI) used in the
experiments was collected from the Taskila WTP after the SI had passed through
the grizzly. Return activated sludge inoculum (RAS) was collected at the MetsBotnia Mill while one inoculum was a commercial product (Polyseed). The
samples were taken by the personnel separately at each of the treatment plants.
Biodegradation measurements were carried out with the peat samples
collected from different peat bogs in Finland by the company Vapo Inc. The peat
samples consisted of different types of peat with decomposition rates varying
from H 1 to H 8 on the von Post decomposition scale [113]. The peat samples
with the decomposition rates H 1 to H 4 are classified herein as sphagnum peat
and the samples with decomposition rates H 5 to H 8 are classified as compaction
peat.
The soil that was used as a measurement medium (Article V) was collected
from a forest area in a small town called Lumijoki (Snow River in English),
which is located in Northern Ostrobothnia, Finland. The model substances used in
the studies were sodium benzoate, a rapeseed oil-based multipurpose lubricating
oil, and chain oil. Sodium benzoate is a typical, quickly biodegrading model
substance, and its use is recommended in OECD guidelines. The research was a
continuation of our former studies and therefore, the selected model substances
were the same substances as used in previous studies [4951, 63].
4.5

Measurements

The manometric respirometric test was applied to studies of biochemical oxygen

demand of domestic and pulp and paper mill wastewater as well as in studies
related to biodegradation measurements.

36

4.5.1 Biochemical oxygen demand measurement (I, II, III)

It is essential to estimate the measuring range and the volume of the sample to be
analysed before respirometric BOD measurements. If the selected measuring
range is too low, the oxygen in the bottle will ultimately run out, whereas a
measuring range that is too large will lead to inaccurate results. The choices made
concerning the measuring ranges of the respirometric measurements were based
on preliminary COD and BOD measurements.
Some wastewaters contain a sufficient amount of micro-organisms and
nutrients, whereas industrial wastewaters can contain unsuitable micro-organisms,
toxic and inhibitory substances and have a too low nutrient content. On this
account, these kinds of samples must be diluted with dilution water that contains a
sufficient amount of nutrients in order to attain an adequate BOD:N:P ratio, and to
eliminate the inhibitory effect of toxic substances. Furthermore, an extra microbe
source (inoculum) is needed often. The dilution solution contains a phosphate
buffer (pH 7.2), magnesium sulphate heptahydrate (22.5 g/l), calcium chloride
(27.5 g/l) and iron(III)chloride hexahydrate (0.25 g/l). The nitrification inhibitor
(e.g. allylthiourea, ATU) can be used in the cases where the purpose is to
eliminate the effect of nitrification. The bottles must be sealed with a rubber
sleeve containing CO2 absorber (NaOH pellets) immediately after the bottles are
filled with the sample and the nitrification inhibitor. The bottles are then sealed
with the measuring heads and the samples are stabilised in the incubation cabinet
(20.0 0.2 oC) for a few hours before measurements are started.
The effect of dilution on the BOD7 values of both the pulp and paper mill
samples were tested in order to detect possible inhibition or too low nutrient
content of the samples. Both samples were analysed using diluting factors (DF) 1
and 2, and in addition, a few measurements on the paper mill samples were
carried out using DF 20. A measurement range of 040 mg/l and 080 mg/l was
decided upon for the chemical pulp mill and paper mill samples, respectively. An
extra microbe source used in these measurements was sewage inoculum (SI) and
to follow common mill practice, a nitrification inhibitor was not used in the most
of the measurements. Only a few tests with ATU were carried out, the purpose of
which was to ascertain that a major nitrification process does not take place in the
wastewaters of the pulp and paper mills. All measurements by the chemical
reference methods [102104] were carried out with diluted samples using dilution
factors 5 and 20 for the pulp and the paper mill samples, respectively.

37

All the respirometric measurements with domestic wastewater were carried

out with undiluted samples, because municipal wastewater does not generally
contain toxic or retarding substances, and there are usually sufficient nutrient salts
and suitable micro-organisms present in the wastewater. The measurement range
of 0200 mg/l was chosen for pre-clarified water and the range 040 mg/l was
chosen for the other two sample types. The measurements by the chemical
methods were carried out with diluted samples with a dilution factor from 2 to 50.
A UV absorption meter was continuously measured the biologically treated
wastewater.
BOD curves developed during the biodegradation process of organic matter
are in general regular in shape for particular wastewater samples. Hence, the
seventh day BOD value can already be evaluated at an early stage of the
respirometric test (e.g. after 13 days). The evaluation of the BOD7 values of the
pulp and paper mill and the domestic wastewater samples were calculated with
the TBL-curve and Microsoft Excel programmes.
The measurements of Bio In and Bio Out samples collected from the MetsBotnia mills were carried out with unfiltered and filtered samples (filter pore size
0.1 m) and the extra microbial seed was always filtered through the 7 m pore
size filter. The efficiency of three different inocula (SI, RAS and Polyseed) to
biodegrade the organic material of Bio In and Bio Out samples were compared
and the effect of the volume of the extra seed was tested as well. In addition, the
influence of the pre-treatment of SI and RAS inoculum by aeration was studied.
The indication of inhibition of the samples was tested with dilution series by
diluting the samples with nutrient solution. Additionally, the effect of nutrient
content in the wastewater sample on BOD value was tested with two dilution
solutions with different nutrient contents. Detailed descriptions of the analyses are
presented in Article III.
4.5.2 Characterisation of wastewater (III)
Characterisation of pulp and paper mill wastewater was carried out with three
different methods. In these combined filtration, COD and BOD measuring
methods, the wastewater influent COD (CODinf,tot) is divided into a soluble
(CODinf,sol = SS + SI) and a particulate (CODinf,part = XS + XI) fraction by filtration
over a membrane filter with a pore size of 0.1 m. In the first method (Method 1),
which is originally developed for municipal sewage by STOWA (Dutch acronym
for the foundation for applied water research), it is assumed that the soluble inert
38

fraction (SI) can be found from the effluent COD of a low loaded activated sludge
plant [93]. Thereafter, the inert fraction SI is subtracted from the CODinf,sol to give
the fraction SS. In Method 2, the SS fraction is determined by a long term BOD
measurement of the filtered influent (BCODsol). In the third method, along with
the influent fractions, the new metabolic products generated during the
biodegradation process as well as an estimate of the mineralised part of the
influent biodegradable fraction were determined without any hypothetical
correction factors. All the equations and determinations related to the
characterisation methods 1, 2 and 3 are given in Tables 1 and 2. Specific
descriptions of the characterisation methods are described in Article III.
Table 1. Equations and determinations for calculating the different COD fractions with
Method 1 and 2.
Component

Definition

Determination / Measurement

CODinf, tot

SS + SI + X S + X I

COD of unfiltered influent

CODinf, sol

SS + SI

COD of filtered influent

CODinf,part

XS + XI

CODinf, tot CODinf, sol

CODeff,sol

COD of filtered effluent

BODtot

long-term BOD of unfiltered influent

BODsol

long-term BOD of filtered influent

BCODtot

SS + XS(1 and 2)

BODtot/(1 f), f = 0.15

BCODsol

SS(2)

BODsol/(1 f), f = 0.15

SI

CODeff,sol 0.9 (low loaded wtp)(1) or (CODinf,sol SS)(2)

SS

CODinf,sol SI(1)

XS

BCODtot SS(1) or
BCODtot BCODsol(2)

XI

CODinf,tot SS SI XS(1 and 2)

Method 1 (STOWA method)

Method 2 (method modelled on STOWA and ASM No 1)

39

Table 2. Equations and determinations for calculating the different COD fractions with
Method 3.
Component

Definition

Determination / Measurement

CODinf, tot

SS + SI + X S + X I

COD of unfiltered influent

CODinf, sol

SS + SI

COD of filtered influent

CODinf,part

XS + XI

CODinf, tot CODinf, sol

BODtot

SS + X S

long-term BOD of unfiltered influent

BODsol

SS

long-term BOD of filtered influent

BCODtot

SS + XS =SS,min + XS,min+ XP + SP

BCODsol

SS = SS,min + XP + SP

Craw

SI + X I + S P + X P

Sraw

SI + S P

COD of unfiltered raw influent after initial state of the

BOD measurement
COD of filtered raw influent after initial state of the BOD
measurement

Cfilt

SI + S P + X P

COD of unfiltered soluble influent after initial state of

Sfilt

SI + S P

COD of filtered soluble influent after initial state of the

the BOD measurement

BOD measurement
SS,min + XS,min

CODinf, tot Craw

XI + X P

Craw Sraw

XP

Cfilt Sfilt

SSmin

CODinf, sol Cfilt

XS,min

(CODinf, tot Craw) SSmin

XI

(Craw Sraw ) XP

XS

CODinf,part XI

SI

CODinf, tot SS - XI XS

SP

Sraw SI or Sfilt SI

4.5.3 Biodegradation measurements (IV, V)

The biodegradation studies of peats were carried out with a 50100 gramme peat
sample with normal moisture content, which is typically 4060%. A sodium
hydroxide solution (1 M), which was used as an absorption agent for CO2 gas,
was placed on the holder and the bottles were sealed tightly, and measurement
heads were screwed onto the bottles. The bottles were held in an incubation
cabinet at a temperature of 20.0 0.2 C for a period of 80210 days. The effect
of the pH of the peat on biodegradation behaviour was tested for 40 days. The pH
of two different peat samples was adjusted to the pH values of 7.0 and 9.0 with a
solution of calcium hydroxide (Ca(OH)2). The base solution was sprinkled onto
the peat and the pH of the sample was then allowed to stabilise for about two
40

weeks. The pH value was then continuously checked. When the desired pH was
achieved, analyses on the properties of the peat and biodegradation measurements
were started. The final base consumption was determined with soil property
analyses in which the amount of calcium of the original as well as pH adjusted
peat samples was measured.
The effect of the nutrients (trace elements) and nitrogen content of the soil on
the biodegradation behaviour was tested with sodium benzoate and two oil
samples. The nutrients and nitrogen content of the soil were increased with
complete fertiliser (Kemira multipurpose fertiliser). Before fertiliser processing
the soil was bisected. The fertiliser was dissolved in distilled water and then
sprinkled onto one of the soil portions. The other soil portion was sprinkled with
the distilled water in order to maintain the same moisture content. After the
addition of fertiliser the soil was allowed to stabilise for about two weeks.
Subsequently the pH, conductivity, nutrient and nitrogen contents were
determined. Furthermore, the effect of the pH of the soil on the biodegradation
process was tested. The pH of the soil was adjusted with calcium carbonate
(CaCO3) solution. The base solution was sprinkled onto both soil portions in the
same way as the fertiliser. The pH values were allowed to stabilise for about two
weeks before commencing soil property analyses. Oil samples were added to soils
in concentrations of about 1000 mg/kg. The bottles were held in an incubation
cabinet at a temperature of 20.0 0.2 C for 30 days without opening during the
biodegradation measurements.

41

42

Results and discussion

This section summarises the results and discussions of Articles IV. In addition,
results of our previous article [63] concerning biodegradation studies of oils in
soil media are discussed.
5.1

Biochemical oxygen demand measurement

Comparison studies concerning manometric respirometric test and conventional

BOD methods, as well as on BOD7 value estimates after short-term measurements
of pulp and paper mill and domestic wastewater samples are discussed in Articles
I and II. The effect of different variables on the BOD7 value as well as the
characterisation of organic fractions of pulp and paper mill wastewater are
discussed in Article III.
5.1.1 Comparison between the manometric respirometric test and
conventional chemical BOD methods (I, II)
The manometric respirometric test was compared with conventional chemical
methods in order to study the applicability of the respirometric test in BOD
analyses of pulp and paper mill wastewater as well as domestic wastewater
samples.
Table 3 lists the BOD7 results of pulp and paper mill wastewater samples
measured by the respirometric test and the conventional methods. Most of the
paper mill samples were analysed with a respirometric test using diluted samples,
whereas the bulk of the pulp mill samples were analysed without dilution. The
results revealed that dilution of the paper mill samples increased their BOD7
values. A possible explanation to this is the lack of nutrients or the presence of
inhibitory substances in chemically treated wastewater. Adequate dilution was
achieved with dilution factor 2, since the use of dilution factor 20 did not increase
BOD7 values significantly. Incorrect dilution choices can have a major influence
on the accuracy and precision of conventional BOD tests. Experienced analysts
can maintain a precision of 50 g/l with visual end-point detection with the
iodometric titration method [105]. With most commercially available membrane
electrode systems the accuracy of 0.1 mg DO/l and the precision of 0.05 mg
DO/l can be obtained. [106] Dilution factor 20 was used for paper mill samples in
conventional BOD measurements in order to ensure adequate oxygen content at
43

the bottle. After correct dilutions, the BOD7 values measured by the conventional
methods and the respirometric test were very similar (relative standard deviation
for three different tests ranged between 3 and 13%). However, the BOD7 values
of undiluted paper mill samples represent the real situation at the mill. The
dilution of the pulp mill samples in respirometric measurements has no significant
effect on BOD values probably because these samples naturally contain a
sufficient amount of nutrients required for the biodegradation process. However,
all the BOD values measured with the respirometric test were higher than those
obtained with the iodometric method. Dilution of the samples with relatively low
BOD values can have a major influence on the precision of the iodometric test. In
addition, more oxygen is available for use in respirometric test bottles than in
Winkler bottles (dissolved oxygen only). Therefore, the biodegradation of organic
compounds can be quicker and BOD values higher with a respirometric test.
Table 3. The BOD7 results of pulp mill and paper mill wastewater samples determined
by the respirometric method and conventional BOD tests.
Sample

Dilution factor used

Respirometry

Iodometric method*

Oxygen sensor*

in respirometric test

BOD7 [mg/l]

BOD7 [mg/l]

BOD7 [mg/l]

Chem.pulp. (I)

22

22

16

Paper (I)

50

81

73

Chem.pulp. (II)

33

19

15

Paper (II)

30

79

58

Chem.pulp. (III)

38

20

19

Paper (III)

58

110

91

Paper (III)

110

110

91

Chem.pulp (IV)

30

17

19

Chem.pulp (IV)

28

17

19

Chem.pulp. (IV)

26

17

19

Paper (IV)

67

100

95

Paper (IV)

100

100

95

Paper (IV)

20

110

100

95

Chem.pulp (V)

37

19

26

Chem.pulp (V)

39

19

26

Paper (V)

70

120

130

Paper (V)

120

120

130

Paper (V)

20

130

120

130

Chem.pulp (VI)

19

13

11

Paper (VI)

63

130

110

Paper (VI)

100

130

110

*Measurements with the iodometric method and oxygen sensor were carried out using dilution factors 5
and 20 for pulp mill and paper mill samples, respectively.

44

The precision of the respirometric test was determined by measuring the BOD
values of the wastewater samples two times. The precision of the manometric
respirometric test was good; the relative standard deviation (RSD) values varied
between 0.00 and 4.32% for pulp mill samples and between 1.45 and 9.08% for paper
mill samples.
A few experiments were carried out with the respirometric test using a
nitrification inhibitor. The measured values for the pulp mill sample were 33.4
mg/l with ATU and 36.5 mg/l without ATU. Corresponding values for the paper
mill samples were 115.6 mg/l and 121.2 mg/l, respectively. The values were thus
within a 10% range and confirmed the mill experience that no significant
nitrification process took place in treatment processes of the wastewater of the
pulp and paper mills.
Pre-clarified, biologically treated and outflowing domestic wastewater
samples were analysed with the respirometric and iodometric method as well as
with a UV absorption meter. Table 4 lists the BOD7 results of four different BOD
tests. The UV absorption meter gave the absorption value (BOD7 value) at every
hour (24 measuring data per day) and the presented values are the mean values of
one days continuous measurements.
Table 4. The BOD7 results of domestic wastewater samples determined by the
respirometric test, iodometric method, oxygen sensor and UV absorption test.
Sample

Respirometry

Iodometric method

Oxygen sensor

UV meter

BOD7 [mg/l]

BOD7 [mg/l]

BOD7 [mg/l]

BOD7 [mg/l]

Pre-clarified (I)

65

56

Biol.leaving (I)

4.5

4.8

5.7 (4.86.7)*

Leaving (I)

5.6

5.6

5.0

Pre-clarified (II)

106

108

4.9 (4.95.0)*

Biol.leaving (II)

4.0

6.7

Leaving (II)

4.2

5.8

4.0

Biol.leaving (III)

6.2

4.5

5.0

10.6 (7.420.2)*

Leaving (III)

7.0

6.0

8.0

Pre-clarified (IV)

86

66

2.1 (0.93.7)*

Biol.leaving (IV)

5.3

6.0

4.0

Leaving (IV)

6.0

7.3

6.0

*mean value of one days measurement

According to Table 4 there were no considerable differences between the results

of the three separate BOD tests: the respirometric, the iodometric, and the oxygen
sensor test. RSD between the three tests were between 1 and 35%. These
45

deviations can partly be explained to be due to the heterogeneity of the samples.

Considering the results in which the UV-technique was applied, the composition
of wastewater must remain similar, as varying compositions in the organic matrix
will have an effect on BOD results.
5.1.2 Estimation of BOD7 value (I, II)
The long duration of conventional BOD tests causes problems in controlling a
wastewater treatment plant because wastewater has sufficient time to change
during the test period. Accordingly, the results are ordinarily out of date for
process control or monitoring purposes. Thus, the evaluation of BOD7 values after
a short-term measuring period is an important aspect in terms of the optimisation
of wastewater treatment processes. The BOD curves generated during the
respirometric measurements of chemical pulp mill samples, as well as
biologically treated and outflowing domestic wastewater samples were linear in
shape, whereas the shape of the BOD curves of paper mill samples and primary
clarified domestic wastewaters were clearly non-linear (Figs. 5 and 6).
120
100

BOD [mg/l]

80

Chem. pulp mill, rep 1

Chem. pulp mill, rep 2

60

Paper mill, rep 1

Paper mill, rep 2

40
20
0
0

tim e [d]

Fig. 5. Respirometric BOD7 curves of the pulp and paper mill wastewater samples
collected from Stora Enso Plc Veitsiluoto Mills.

46

120

10

Pre-clarified water,
BO D [m g/l]

80

60

6
4

40

3
2

20

0
0

Pre-clarif ied rep 1

Biol. treated and Outflowing

water, BOD [mg/l]

100

Pre-clarif ied rep 2

Bio l. treated rep 1
Bio l. treated rep 2
Outf lo w ing rep 1
Outf lo w ing rep 2

tim e [d]

Fig. 6. Respirometric BOD7 curves of pre-clarified, biologically treated and outflowing

wastewaters collected from the Oulu Waterworks, Taskila WTP.

The TBL curve programme calculates dozens of equations from which the most
suitable ones were selected. The evaluated BOD values for pulp and paper mill
samples and domestic wastewater samples are represented in Table 5 and 6,
respectively.
Table 5. Evaluated BOD7 values of pulp and paper mill wastewater samples.
Sample

Equation

Evaluated BOD7 Evaluated BOD7 Evaluated BOD7 Measured BOD7

number*

value after 2 day value after 3 day value after 4 day

[mg/l]

[mg/l]

value [mg/l]

[mg/l]

Chem.pulp. (II)

32.7

33.5

33.1

32.5

Chem.pulp (III)

37.4

37.6

38.8

37.9

Chem.pulp (V)

32.1

34.8

35.9

36.5

Chem.pulp (VI)

20.4

20.2

19.8

19.2

Paper (IV)

114

95

92

102

Paper (V)

129

120

119

121

Paper (VI)

96

97

96

103

*Equation number 1: y = ax + b
*Equation number 2: y = [(a + cx)/(1 + bx)]2

Estimated BOD7 values for the chemical pulp mill samples using the Equation
number 1(y = ax + b) were very accurate. Estimation of the BOD7 values of paper
47

mill samples also worked well. The estimated BOD7 values for both samples were
reliable as early as after a two-day incubation period. The difference between the
evaluated and measured BOD7 values ranged only from 1 to 12 percent in both
cases.
Table 6. Evaluated BOD7 values of domestic wastewater samples.
Sample

Equation

Evaluated BOD7 Evaluated BOD7 Evaluated BOD7 Measured BOD7

number

value after 2 day value after 3 day value after 4 day

[mg/l]

[mg/l]

[mg/l]

value
[mg/l]

Pre-clarified (I)

67

64

64

65

Pre-clarified (II)

98

106

104

108
100

Pre-clarified (III)

92

93

93

Pre-clarified (IV)

113

94

88

86

Biol.leaving (I)

4.5

4.5

4.1

4.5

Biol.leaving (I)

4.3

4.1

4.4

4.5

Biol.leaving (II)

3.7

4.1

4.0

4.0

Biol.leaving (II)

3.3

3.4

4.0

Biol.leaving (III)

8.3

8.0

7.2

6.2

Biol.leaving (III)

8.3

7.1

6.3

6.1

Biol.leaving (IV)

7.2

6.0

5.6

5.3

Leaving (I)

6.2

8.5

5.7

5.6

Leaving (I)

5.2

5.6

5.3

5.6

Leaving (II)

3.8

4.8

4.7

4.2

Leaving (II)

2.2

4.4

4.6

4.2

Leaving (III)

9.7

9.2

8.8

7.0

Leaving (III)

8.4

8.4

8.0

7.0

Leaving (IV)

8.1

6.0

6.1

5.9

Equation number 1: y = ax + b
Equation number 2: y = [(a + cx)/(1 + bx)]2
Equation number 3: y = [(a + cx) / (1 + bx)]

The estimation of BOD7 values of domestic wastewater samples worked well and
the estimated values were very accurate. Eventually, the two best fitting equations
for outflowing and biologically treated wastewater were tested; Equation number
2 (y = [(a + cx)/(1 + bx)]2) was noticed to work better after three days incubation.
The average deviation after two days incubation using the Equation number 2
was 27%, while after three days incubation it was only 10%. However, for the
linear equation (Equation 1) the average deviations were 29% and 18%,
respectively. Although the precisions of the evaluated BOD7 values were better by
using Equation 2, it would be simpler and more practical to use Equation 1 at the
48

wastewater treatment plant. It was discovered that Equation 3 (y = [(a + cx) /

(1 + bx)]) worked best for pre-clarified sewage. The average deviation was 13%
after two days and only 5% after three days incubation. Thus, the accuracy of the
estimated BOD values can be deemed good.
It is very important to know the BOD7 values of influent wastewaters
beforehand in order to adjust the chemical feed (in other words to reach the
correct BOD:N:P ratio) and aeration in an exact manner. This is a significant
advantage when treating industrial wastewater in which nutrient contents can be
very low. The factors have a major influence in maintaining a suitable microbial
population, affecting for example BOD-reduction, nitrification, precipitation and
flotation. Therefore, a respirometer could be a useful tool for process control
purposes.
5.1.3 Effect of different variables on BOD value (III)
The manometric respirometric measuring procedure was optimised especially to
correspond with the wastewater qualities of the pulp and paper mills in order to
characterise different organic fractions of the wastewater reliably.
The functionality and sufficiency of the own bacterial strain of the Bio Out
samples was tested with adding additional seed inoculants. The results revealed
that bacterial population presented in the Bio Out samples did not biodegrade the
organic material sufficiently. The adaptation period of the BOD curves of the
seeded samples was much shorter and hence, the readily biodegradable organic
material biodegraded more quickly than non-seeded samples.
Aeration of the SI or RAS inoculums had no remarkable effect on the final
BOD7 values of the samples. Aeration slightly increased the oxygen consumption
of sewage inoculum and it also caused variation between the duplicated
measurements. A similar influence was not detected with RAS inoculum. The
RSD of BOD7 values for the Bio In and Bio Out samples seeded with SI
inoculum were 6.2% and 18%, respectively. The RSD of BOD7 values for Bio In
and Bio Out samples seeded with RAS inoculum were 4.0% and 7.7%,
respectively.
The comparison studies of the inoculums SI, RAS and Polyseed were
carried out with Bio In and Bio Out samples as duplicated measurements. The
BOD curves of unfiltered Bio In samples seeded with three additional inoculums
are presented in Fig. 7. The figure reveals that the precision of duplicated
measurements was very good, but that the biodegradation efficiency between the
49

SI and two other seeds diverged. Sewage inoculum worked most efficiently for
Bio In samples. Bacterial population present in the sewage inoculum (SI) can be
more heterogeneous and diverse, in comparison to the two other seeds. However,
for Bio Out samples such a clear difference in biodegradation efficiency between
three seeds was not detected. The RSD of comparison BOD measurements
concerning the three seeds was 10% for unfiltered Bio In samples, whereas the
RSD for the Bio In samples seeded with RAS and Polyseed was only 3%. The
RSD for the Bio Out samples seeded with the three different inoculums was 9%
(mean value of BOD7 7.4 mg/l). To simulate actual plant conditions, the rest of
the preliminary tests as well as the characterisation analyses of the biodegradable
organic fraction of the influent were carried out with RAS inoculum collected
from the Mets-Botnia mill (results presented further). However, if the
respirometric test is used along with conventional BOD methods for example, for
controlling a treatment plant, the same additional inoculums must be used in both
tests. In that case, sewage inoculum is usually preferred.
300
SI, rep. 1
SI, rep. 2

250

RAS, rep. 1
RAS, rep. 2
Polyseed, rep. 1

BOD [mg/l]

200

Polyseed, rep. 2.

150

100

50

0
0

time [d]

Fig. 7. The BOD curves of unfiltered Bio In samples seeded with three different
inoculums.

50

The volume of extra inoculum given in BOD standards and applications is usually
5 to 20 ml of sewage per litre of sample. This wide-ranging scale was tested with
Bio In and Bio Out samples with the inoculum volumes 5, 10 and 20 ml per litre
of sample. The volume of the additional seed (RAS) did not change the BOD7
value or the shape of the respirogram of the Bio In or the Bio Out samples. The
RSD of the BOD7 results of the three different seed amounts were 13% (average
value of BOD7 172 mg/l) and 3% (average value of BOD7 137 mg/l) for
unfiltered and filtered Bio In samples, respectively and 8% for filtered Bio Out
samples (average value of BOD7 6.0 mg/l). The variation of the BOD7 results of
unfiltered samples can be explained by the heterogeneity of the samples.
The clear inhibition was noticed in dilution experiments of Bio In samples
(Fig. 8). Undiluted samples as well as samples diluted with the dilution factor 2
(DF 2) produced almost 30% lower BOD7 results than samples diluted with DF
10, 20 or 50. However, the repeatability and precision of the BOD results
concerning the highly diluted samples (DF 20 or 50) were much worse than with
samples with DF from 1 to 10. The BOD curve attained the stable state with
samples diluted with DF 10, which reveals that the biodegradation of organic
material was completed. Additional nutrients had no effect on the biodegradation
behaviour of wastewater samples. Consequently, the characterisation analyses of
the influent samples were carried out with diluted samples (DF 10) in order to
eliminate the effect of inhibition. The inhibition was not detected with Bio Out
samples.

51

500
Bio In, undilluted

450

Bio In, DF 2
Bio In, DF 10

400

Bio In, DF 50

BOD [mg/l]

350
300
250
200
150
100
50
0
0

time [d]

Fig. 8. Dilution series of the unfiltered Bio In sample.

5.1.4 Characterisation of wastewater fractions (III)

Characterisation of the wastewater of the pulp and paper mills was carried out
with diluted Bio In samples (DF 10) and undiluted Bio Out samples. The organic
fractions of the pulp and paper mill samples 1 and 2 (WW 1 and WW 2)
determined according to the Methods 1, 2 and 3 are presented in Table 7.

52

Table 7. Organic fractions of samples 1 and 2 (WW 1 and WW 2) determined by

Methods 1, 2 and 3.
Method 1

Method 2

Method 3

Method 1

Method 2

Method 3

WW 1

WW 1

WW 1

WW 2

WW 2

WW 2

[mg/l]

[mg/l]

[mg/l]

[mg/l]

[mg/l]

[mg/l]

CODinf, tot

840

840

840

787

787

787

CODinf, sol

670

670

670

639

639

639

Fraction

CODeff,sol

197

175

BODtot

585

585

585

350

350

350
310

BODsol

492

492

310

BCODtot

688

688

412

412

BCODsol

579

365

Ss

493

579

492*

481

365

310*

XS

196

109

143**

47

148**
329

SI

177

91

178

158

274

XI

60

27

218

101

XP

19

65

SP

243

80

Ss, min

230

165

XS, min

142

135

*Influent SS fraction (BODsol) transforms to SS,min and new inert products.

**Influent XS fraction is almost totally mineralised to XS,min.

In Method 1 the influent soluble fraction SI was determined from the effluent
residual soluble COD (CODeff,sol), and the new inert soluble products (SP)
generated during the biological treatment are reduced from the effluent COD by
multiplying it with 0.9 in order to give the fraction SI. However, as can be seen
from the results of Method 3, the SP fraction generated during the biodegradation
process composes a remarkable part of the residual organic matter of the
wastewater. A significant part of the detected oxygen demand is consumed in the
biotransformation of biodegradable fractions into new inert decomposition
products, and not only in the mineralisation. Pulp and paper mill effluents have
been shown to contain biotransformation products such as oxidation products of
resin acids and sterols which can be more persistent and toxic than the original
substances [114116]. Therefore, it is very important to determine the amounts of
the decomposition products of organic fractions.
As it was noticed in preliminary tests, pulp and paper mill wastewaters
usually contain inhibitory substances which can have a major effect on
biodegradation processes. On this account, the biodegradation measurements
53

must be carried out with the right dilutions in order to obtain accurate information
of the biodegradable part of the wastewater. Therefore, the evaluations of the SI
and SS fractions made on the basis of the CODeff,sol can be incorrect if the
biological treatment process does not work sufficiently well. However, as can be
seen from the CODeff,sol values in Table 7, the real activated sludge treatment plant
can oxidise organic matter more effectively, as compared to a respirometric
device.
In Method 3 the influent inert organic fractions and new inert products were
determined by the COD analyses and the biodegradable fractions were
determined with the BOD measurements. The aim was to eliminate the factors
used in Methods 1 and 2 for evaluating the residual inert part of the wastewater.
The BOD curves of unfiltered and filtered Bio In samples are presented in Fig. 9.
700
WW 1, unfilt.

WW 1, unfiltered

600

WW 1, filtered
WW 1, filt.

WW 2, unfiltered

500

WW 2, filtered

BOD [m g/l]

400

WW 2, unfilt.

300
WW 2, filt
200
100
0
0

10

15

20

25

30

-100
time [d]

Fig. 9. BOD curves of unfiltered and filtered Bio In samples 1 and 2.

The BOD curves attained the stable state within 28 days, which reveals that the
biodegradation of organic material was completed. The proportion of inert
fractions that remained in the solution after the biodegradation measurement was
significant. However, the CODeff,sol values were much smaller than Sraw values,
although these values should in fact have been similar. The reason to this can be
found in the volatile organic compounds (VOCs) present in the pulp mill
54

wastewater such as methanol, mercaptans, aldehydes and ketones.[117] VOCs can

be liberated during the biological treatment process which is not possible with the
respirometric device, as it is closed system. In consequence, the concentration of
chemically oxidable compounds present in the BOD measuring bottle can be
higher compared to the effluent. The composition of the wastewater samples
collected at different times can vary considerably, although there was only a
minor difference between the measured CODinf,tot values (840 mg/l and 787 mg/l).
Influent sample 1 contained 58% of soluble biodegradable organic material (SS),
21% of soluble inert material (SI), 17% of particulate biodegradable material (XS)
and 3% of particulate inert organic material (XI). For influent sample 2 the
composition was the following: SS 39%, SI 42%, XS 19% and XI 0%.
The value of the first order rate constant kBOD was determined for both
filtered and unfiltered influent samples from the fitted BOD-curve according to
Eq. (1): BODtot = BODt / (1 e kBOD t) (detailed description in Section 2.4.2). Four
different wastewater samples collected during spring 2008 were analysed. The
average kBOD for the filtered influent sample was 0.184 d1 (0.182; 0.189; 0.183
and 0.183 d1) and 0.191 d1 (0.175; 0.178; 0.185 and 0.227 d1) for the unfiltered
influent sample. Hence, the half-lives were 3.8 and 3.6 days.
5.2

Biodegradation measurements

Biodegradation studies, carried out with the respirometric test were a continuation
of our previous study [63]. The biodegradation of different peat types is discussed
in Article IV, whereas the effect of different variables of soils on the
biodegradation behaviour of oil samples is discussed in Article V.
5.2.1 Biodegradation of peat samples (IV)
The biodegradation rates of different peat types were studied with the manometric
respirometric method in order to evaluate the potential to use them as hydraulic
barrier material in landfill layers. In addition, the effect of the pH in some of the
peat samples on biodegradation behaviour was analysed. Certain properties of the
peat samples used in the research (peat samples 18) are given in detail in Article
IV. Biodegradation behaviour was tested with the peat samples 16, whereas the
peat samples 7 and 8 were used in the pH tests.
The results of the biodegradation measurements (Fig. 10 and 11) were
calculated on a dry weight basis, although the measurements were carried out
55

with samples with normal moisture contents, which are typically 4060%. Peat
samples 4, 5 and 6 degraded within the first two months and thereafter the
biodegradation attained a stable state (a state in which biodegradation stopped).
The attainment of the stable state took longer with the other peat samples (peat 1,
2 and 3). It was noticed, that the decomposition rates and biodegradation
behaviour of the peat samples were linked to each other. The peat samples with a
low decomposition rate (sphagnum peat) required a longer time period to
biodegrade whereas the peat samples with higher decomposition rates
(compaction peat) attained the endogeneous state faster. Moreover, the degree of
biodegradation was higher with sphagnum peat samples. This kind of behaviour
can be understood, knowing that well humified peat is almost completely
degraded.

0,5
0,45

Peat 4, H 6-7

0,4

Peat 5, H 6-7

0,35
BOD/ThOD%

peat 5

Peat 6, H 8

0,3

peat 4

0,25
0,2
0,15
0,1

peat 6

0,05
0
0

10

20

30

40

50

60

70

80

90

tim e [d]

Fig. 10. Biodegradation rates of the peat samples 4, 5 and 6 (compaction peat
samples).

56

1,2

peat 3

Peat 1, H 1-2
Peat 2, H 2-3

Peat 3, H 4

BOD/ThOD %

0,8

peat 1
0,6

peat 2
0,4

0,2

0
0

50

100

150

200

t ime [d ]

Fig. 11. Biodegradation rates of the peat samples 1, 2 and 3 (sphagnum peat samples).

The biodegradation rates of the different peat types were quite small in spite of
the decomposition rate of the sample. Peat sample 3 (sphagnum peat) has the
largest BOD/ThOD % value of 1.1%. However, only compaction peat is used as a
barrier material in landfills. If the thickness of the compaction peat layer is 1000
mm, the moisture content of the layer 55% (real thickness of the peat is only 450
mm) and the biodegradation of the peat 0.36% (Peat 5, Fig. 10), the peat layer
will only become 1.62 mm thinner. Such a minor reduction of the peat layer does
not have to be taken into account when designing landfills and the figure is much
lower than the compaction of the material during the same time period. In
addition, the air and oxygen contents in landfill layers are substantially lower than
in our measuring bottles. Therefore, the biodegradation of peat in the hydraulic
barrier layer of a landfill is probably even lower than shown in our results.
The pH of peat samples 7 and 8 were adjusted to the pH values of 7 and 9
with Ca(OH)2 solution. After the pH adjustment, the calcium contents were
determined in order to find out how large the final base consumption was. The pH
values as well as the calcium and carbon contents of the peat samples measured
both before and after the pH adjustment are presented in Article IV. The amount
of calcium hydroxide consumed in the pH adjustment process was considerable.
57

Since the bulk densities were determined, the amount of consumed base per
kilogramme of peat could be calculated. For example, when the pH of one tonne
of peat 7 is adjusted from the value 4.7 to the values 7.1 and 8.8, the amount of
Ca(OH)2 consumed is 30 kg and 70 kg, respectively. The biodegradation studies
of base treated peat samples lasted for 40 days. The biodegradation behaviour of
the pH adjusted and original peat sample 8 is presented in Fig. 12.

BOD/ThOD%

1
0,9

Peat 8 or iginal, H 6

0,8

Peat 8 pH ~7

0,7

Peat 8 pH ~9

0,6
0,5
0,4
0,3
0,2
0,1
0
0

10

20

30

40

tim e [d]

Fig. 12. The effect of the pH of peat 8 on the biodegradation behaviour.

The results revealed that pH has an influence on the biodegradation rate. The
original peat sample achieved a stable state in which all the biodegradable organic
material was consumed later than in the case of pH adjusted samples. The peat
sample, the pH of which was about 9 achieved this stage 10 days earlier than the
original peat sample. A same kind of behaviour was also found in the
biodegradation studies of peat sample 7. Hence, if alkaline waste overtakes the
peat layer in a landfill, it does not increase the degree of biodegradation. However,
in this case the stable state is attained more quickly. Despite the pH of the peat
samples, the degrees of biodegradation were very low (BOD/ThOD % < 0.5%).

58

5.2.2 Effect of different variables of soils on biodegradation

behaviour of oil samples (V)
The effect of soil mineral concentration and pH on the biodegradation behaviour
of oil samples was analysed with the manometric respirometric method. The soil
properties were modified in order to make the biodegradation of oil samples more
efficient. Certain mineral concentrations and other properties of the soil used
(marked as soil 1 hereafter) were measured before and after processing with
mineral solution and CaCO3 (Table 8).
Table 8. Mineral concentrations and other properties of a sandy soil sample measured
before and after soil processing.
Sample

pH

Conductivity

Ca

Mg

[cS/cm]

[mg/l]

[mg/l]

[mg/l]

[mg/l]

Ca/Mg

Soil 1

6.1

0.2

150

2.4

36

56

2.70

Soil 1 + ms*

5.7

4.3

139

11.6

163

66

2.10

Soil 1 + CaCO3

8.0

0.9

1245

2.9

29

29

42.9

Soil 1 + CaCO3 + ms*

7.7

5.3

822

19.7

186

52

15.8

*ms = mineral solution (multipurpose fertiliser)

The soil 1 used in the experiments was sandy soil. Its conductivity and
concentrations of basic nutrients were low and the pH value was almost neutral.
The phosphorus and potassium as well as the conductivity of the soil were
increased by fertiliser addition. Addition of CaCO3 increased the pH value to 8.0
and clearly multiplied the calcium concentration at the same time. When mineral
solution was added after pH adjustment, a soil sample with high amounts of basic
nutrients was obtained. The influence of the pH adjustment and the mineral
addition on the concentrations of different types of nitrogen is given in Table 9.
Table 9. Influence of soil processing on nitrogen concentrations of sandy soil.
NH4+

NO3

Exchangeable nitrogen

[mg/l]

[mg/l]

[mg/l]

Soil 1 + CaCO3

0.50

8.40

8.90

Soil 1 + CaCO3 + ms*

0.30

75.9

76.1

Sample

*ms = mineral solution

Fertiliser addition increased the nitrate concentration, whereas the ammonium

concentration remained almost the same. However, the concentration of
59

exchangeable nitrogen (the sum of ammonium and nitrate) increased over 8-fold.
Considering the ability of the processed soil 1 to biodegrade chemicals, the
options are quite good. There should be no limitations in terms of mineral, pH, or
moisture concerning biodegradation, because those levels were adjusted to more
optimal values. The temperature in the measurements is also high (20 oC),
corresponding to a typical mid-summer temperature in Northern Finland. The
only potential limitation could be the capacity of the soil microbes, i.e. whether
the soil will be able to adapt in order to be able to biodegrade the studied
chemicals, in this case oils.
The biodegradation of sodium benzoate, chain oil (chain oil, replication 1/2) and
lubricating oil in soil 1 was followed up by either including or omitting mineral
additions. The moisture of the soil was always adjusted to a steady level. A precision
test for the biodegradability of chain oil was carried out exactly after one month
had elapsed from the first measurement (chain oil, replication 2/2). Results are
presented in Fig. 13.
90
Sodium benzo at e (1)
Chain o il 1/2 (2)

80

Chain o il 2/2 (3)

5.

Lubricat ing o il (4)

Sodium benzo at e +mineral so lut io n (5)

70

Chain o il + mineral so lution 1/2 (6)

Chain o il + mineral so lution 2/2 (7)

BOD/ThOD%

60

Lubricat ing o il + mineral so lutio n (8)

6.

50

1.
40

7.
8.

30

20

10

4.
2. and 3.

0
0

10

15

tim e [d]

20

25

30

35

Fig. 13. BOD/ThOD values of sodium benzoate, chain oil and lubricating oil in fertilised
o

and non-processed soil 1 at 20 C.

60

Sodium benzoate biodegraded moderately without mineral addition; the observed

BOD/ThOD value was 45.1% after thirty days. However, in our previous study
[62] sodium benzoate biodegraded in a fertile forest soil (marked as soil 3s
hereafter) significantly better, since the BOD/ThOD was 85% in 14 days.
Especially the starting phase of biodegradation (adaptation period) seems to go on for
a relatively long period in soil 1. The addition of minerals enhanced the
biodegradability potential of this soil significantly. The observed BOD/ThOD value
of sodium benzoate was 76% after thirty days, and the stable phase was reached in 15
days. However, even with fertiliser addition in soil 1, sodium benzoate biodegraded
better in fine sandy till type of soil (marked as soil 3s), which was used in our former
study [63].
The chain oil examined was also used in our former study [63]. The
biodegradation degree of chain oil was on average 43% after 14 days in soil 3s,
and the biodegradation was still effective after 14 days. The non-processed soil 1
was not at all suitable to biodegrade this type of chain oil and no biodegradation
was observed during the measurement period of 30 days. However, addition of
fertiliser to the soil made biodegradation possible. The BOD/ThOD value in this
case was 51% after 30 days and the 14-day value was 22%, which was about 50%
less than the values previously obtained for soil 3s [63]. Lubricating oil
biodegraded weakly in soil 1 without nutrient addition; the BOD/ThOD value was
only 5.9% after 30 days. The nutrient addition raised the degree of biodegradation
to a moderate value of 32.9% after 30 days.
Precision of the biodegradability measurement for chain oil was not good in soil
1 whether there was a nutrient addition or not. Without nutrient addition chain oil 1
reached the BOD/ThOD value of 1.8% after 30 days in this test, whereas the
corresponding value in the previous test was 0%. However, the observed value of
1.8% is almost insignificant in terms of biodegradability. When fertilisation was used,
the degree of biodegradation was 36% in the precision test, whereas the
corresponding value in Fig. 13 was 51%. It seems that soil 1 is not as
homogeneous as soil 3s, in which replication tests of both this chain oil and tall
oil fatty acid (TOFA) produced significantly more precise results in a 14-day
measurement period [63] than was observed in this case. Perhaps, the microbial
activity of the sandy soil 1 depending on the used batch varies considerably, thus
causing variations in the aptitude of the soil to biodegrade oils, because
temperature, moisture, and mineral concentrations were adjusted to the same level.
The pH value of the soil was adjusted to approximately 8.0 using calcium
carbonate. Experiments with and without nutrient addition were carried out. Moisture
61

was always adjusted and kept at the same steady level. The results of pH adjustment
experiments are presented in Fig. 14.

Fig. 14. The influence of pH adjustment of soil 1 on the biodegradability of lubricating oil
o

and chain oil at 20 C.

When the pH of the soil was adjusted to 8.0, the lubricating oil reached a
BOD/ThOD value of 9.5% in 30 days. This value is slightly higher than the one
observed in the non-processed soil (5.9%), but it is only less than one third of the
corresponding BOD/ThOD value in the soil with mineral addition (33%). The
small enhancing influence on the BOD/ThOD value is probably due to the
increased calcium concentration in the soil, but the measurement accuracy also
plays a significant role. When both the pH of the soil was adjusted and extra
minerals were added, the BOD/ThOD value of the lubricating oil increased
significantly, being 52% after 30 days.
A similar behaviour was also observed when chain oil was studied. The lowest
BOD/ThOD values (on average 0.9% in 30 days) were observed in the original soil,
the pH adjustment increased the BOD/ThOD value slightly to 7.4%, and mineral
addition produced the average BOD/ThOD values of 43%. Again when both pH
62

adjustment and mineral addition were implemented, the BOD/ThOD values observed
were significantly enhanced, up to 76% after 30 days. One important compound
influence of the pH adjustment and mineral addition on the biodegradation is that
biodegradation reactions seem to start at an earlier stage than when soil is not
processed.
Precision of the biodegradability measurements of the lubricating oil was
examined in soil 1, when extra minerals were added, and the pH was adjusted to
approximately 8.0 (the exact pH value was 7.7). The time period between the two
experiments was about two months. Results are presented in Fig. 15.
60
Lubricating o il with pH
adjustment + mineral
additio n 1/ 2
Lubricating o il with pH
adjustment + mineral
additio n 2/ 2

50

BOD/ThOD%

40

30

20

10

0
0

10

15

20

25

30

35

tim e [d]

Fig. 15. Precision test of the biodegradability measurement of lubricating oil in soil 1 at
o

20 C.

When the precision test was carried out for chain oil in soil 1 with mineral
addition, the results were not very promising. However, when lubricating oil was
tested in soil with an adjusted pH and added extra minerals, the results were
completely different. The degrees of biodegradation observed were 52 and 51%
after 30 days, and the behaviour of both samples seems to be quite similar during
the measurement period. Thus the precision of the OxiTop test in soil
measurements seems to be accurate.
63

5.3

Precision of the manometric respirometric test

Accuracy is a measure of how close a number is to the actual value, as in for

example how far from the actual value a particular measurement falls. In many
experiments, as in this case, accuracy cannot be determined, and only an estimate
of the accuracy can be found, due to the fact that the actual value is not known.
Precision, on the other hand, refers to how close repeated measurements are to
each other (a measure of the variation present in a set of readings). [118]
The precision of the Oxitop measuring system has been tested with hydraulic oil
and chain oils in ground water in the previous studies of our research group [4950].
The relative standard deviation (RSD) of the degrees of biodegradation of three
replicates of hydraulic oil was 1.1%, whereas the RSD for different chain oils varied
between 0.5 and 4.7% [4950]. In this research, the precision of the BOD
measurements was tested by measuring two or three replicate wastewater samples
with the Oxitop system. The RSD values varied between 0.0 and 4.3% for pulp mill
samples and between 1.4 and 9.1% for paper mill samples. The RSD of BOD for
domestic wastewater samples varied between 0.0 and 9.4%. Bio In and Bio out
samples were measured with and without filtration. The RSD values for filtered Bio
In samples varied between 0.0 and 10.5%, whereas for unfiltered Bio In samples the
RSD values ranged between 0.0 and 5.7%. The Bio Out samples gave the highest
variations for the BOD values between two or three replicated samples. For unfiltered
and filtered Bio Out samples the RSD values varied between 0.0 and 18.4%, and
between 2.7 and 14.6%, respectively. This could be due to the low BOD values of the
Bio Out samples. Heterogeneity of the wastewater samples also has an effect on the
precision of the tests. However, the precision of the Oxitop test as a whole was quite
good.
Precision of the biodegradation measurements in soil was tested with tall oil fatty
acid (TOFA) in our previous study [63]. Replicate tests on the biodegradation of
TOFA in three different soils revealed that the precision of the Oxitop method was
rather good. In this research the precision of the method was tested with chain oil and
lubricating oil as duplicate measurements. Precision of the biodegradability
measurement for chain oil was not too good in soil 1 whether there was a nutrient
addition or not. The BOD/ThOD values of the replicate measurements were 1.8% and
0.0% after 30 days. With fertilisation the degrees of biodegradation were 35.6% and
50.8%. Lubricating oil was tested in a soil with an adjusted pH and added extra
minerals. The degrees of biodegradation observed were 52.3 and 51.4% after 30
days, and the behaviour of both samples seemed to be quite similar during the
64

measurement period. This supports the results observed in our previous study
[63]. The soil simply has to be homogeneous enough in order for precise results
to be achieved.

65

66

Conclusion

This work discusses the application possibilities of the manometric respirometric

method in the environmental field.
In the first part of this work the manomeric respirometric method was applied
to cases related to wastewater management. Biochemical oxygen demand is an
important parameter in water resource management. BOD is commonly measured
with conventional BOD tests such as iodometric titration or the oxygen electrode
test. Nowadays, BOD can be measured quickly and easily using advanced
respirometers. Large differences in BOD results were not detected in a
comparison study involving a respirometric test and conventional methods, when
the right dilutions were used in both tests. Incorrect dilution choices can have
major influence on the accuracy and precision of conventional BOD testa. It was
observed that a right dilution was the most significant factor affecting the BOD
values of industrial wastewater, which can contain inhibitors, toxic substances
and have an insufficient nutrient content. However, domestic wastewater, which
naturally contains sufficient nutrient salts and suitable micro-organisms, can be
analysed without dilution using a respirometric test. This reduces the potential
source of errors resulting from sample preparation. It was observed that the type
of inoculum has an effect on BOD value, due to a bacterial population present in
the additional seed. Some seeds can contain more heterogeneous bacterial
populations than others and biodegrade the organic material more effectively. The
volume of the additional seed (520 ml/litre of sample) did not have remarkable
effect on BOD7 values. The long measuring period used in conventional BOD
tests can cause problems in controlling a wastewater treatment plant. BOD7 value
estimates based on the respirometric data were proved to be reliable after 23
days incubation period for both pulp and paper mill and domestic wastewater
samples. The analysis of BOD7 values after a short-term measurement period is
an important aspect in terms of the optimisation of wastewater treatment
processes, for example in terms of adjusting the correct BOD:N:P ratio and
aeration exactly.
Characterisation of wastewater is an important aspect for the design,
optimisation and operation of wastewater treatment systems. Characterisation can
be carried out by focusing on the determination of both biodegradable and inert
COD fractions or by characterising either the biodegradable or inert COD
fractions. In this work both biodegradable and inert fractions were specified. The
most significant observation concerning the characterisation analyses of pulp and
67

paper mill samples was that a remarkable part of the detected oxygen demand was
consumed for the biotransformation of biodegradable fractions into new inert
decomposition products, not only for mineralisation of the BCOD fraction. Hence,
the amount of new metabolic products (SP) generated during the biodegradation
process can be significant. These biotransformation products can be more
persistent and toxic than original substances. Therefore, it is very important to
determine the amounts of the decomposition products of organic fractions.
In the second part of this thesis the applications of the manometric
respirometric method in terms of biodegradation measurements in soil media is
discussed. The biodegradation behaviour of different peat samples was tested in
order to evaluate the applicable peat types to be used in landfill structures both for
the base structures of new landfill areas and in order to improve the surface of
sites that are no longer in active use. The results showed that the degrees of
biodegradation of the compaction peat samples were very low (0.040.35%) and
the stable state, in which biodegradation stopped, was achieved during the twomonth period. The degradation of sphagnum peat samples took longer and the
biodegradation degrees were larger as compared with compaction peat samples.
However, the BOD/ThOD % values were still very low. Alkaline pH accelerated
the biodegradation rate and the achievement of the stable state. It should be noted
that the air and oxygen content in landfill layers is substantially lower than in our
measuring bottles. Therefore, the aerobic biodegradation of peat in a hydraulic
barrier layer of a landfill is probably even lower than our results showed.
The possibilities of enhancing the biodegradation of bio-oils by mineral
addition in the soil and soil pH adjustment were also studied. The combined effect
of a suitable mineral addition and pH adjustment enhanced the biodegradation of
these oils significantly. Mineral addition alone increased the BOD/ThOD values
of oils in the soil relatively considerably, whereas pH adjustment alone did not
improve biodegradation considerably. One important benefit in applying mineral
addition and pH adjustment besides improved biodegradation is that soil can
be made more homogeneous and the measurements will be significantly more
precise.
In conclusion, the manometric respirometric test was shown to be a useful
method for various kinds of environmental studies. The equipment is simple to
use and the application possibilities are wide-ranging. Therefore, only a few
different target applications were studied in this work. The manometric
respirometric method is an advanced method for simulating biodegradation
processes in soil and water media. Therefore, the results can be applied in real-life
68

environmental cases, such as for designing a purification strategy for

contaminated soils.

69

70

Future work

As the results of this doctoral dissertation attested, the manometric respirometric

test is an advanced and useful method for various kinds of environmental studies.
Only a few different target applications were studied in this work and therefore,
the studies will be continued and expanded. The effect of temperature on
biodegradation behaviour of chemicals in water media and soils will be studied in
the near future. In addition, the intention is to study the decomposition products
generated during biodegradation processes. It is very important to specify these
so-called biotransformation products because these compounds can be more
persistent and toxic than the original substances. In this work the respirometric
test was applied to kinetic rate constant (kBOD) determinations of pulp and paper
mill wastewaters. These kinetic studies will be continued and extended to concern
municipal wastewaters as well as industrial wastewaters from different sources.
The aim is also to apply the manometric respirometric test for biodegradation
studies in anaerobic conditions where degradation processes take place in the
absence of oxygen (N2 atmosphere). The use of special measuring bottles with a
septum-sealed nozzle allows conducting anaerobic biodegradation studies and
biogas (CH4 and CO2) generation measurements. The interference-free addition of
substrates and solutions can be made with these special measuring bottles. These
septum-sealed nozzles allow selective sampling, which facilitate for example the
specification of biotransformation products.
Biodegradation behavior of all new chemicals (chemicals placed on the
market since 1981) has to be evaluated by 2012. The European Commission
proposes that also existing substances should in the future be subjected to the
same testing requirements. [1] Testing and assessing the properties of the
chemicals is a challenging task, hence the development and validation of testing
methods is pivotal.

71

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80

Original publications
I

Roppola K, Kuokkanen T, Nurmesniemi H, Rm J, Pyki R & Prokkola H (2006)

Comparison Study of Manometric Respirometric Test and Common Chemical
Methods in the Determination of BOD7 in a Pulp and Paper Mills Wastewaters.
J Autom Meth and Manage Chem ID 90384: 15. DOI: 10.1155/JAMMC/2006/90384.
II Roppola K, Kuokkanen T, Rm J, Prokkola H & Heiska E (2007) Comparison Study
of Different BOD Tests in the Determination of BOD7 Evaluated in a Model Domestic
Sewage. J Autom Meth and Manage Chem ID 39761, 14. DOI: 10.1155/2007/39761.
III Roppola K, Kuokkanen T, Rm J, Prokkola H & Ruotsalainen J (2009)
Characterisation of Organic Fractions of Pulp and Paper Mill Wastewater with a
Manometric Respirometric BOD Method and Automatic COD Analyses. Chem Spec
Bioavail. In press.
IV Roppola K, Kuokkanen T, Kujala K & Kuokkanen M (2008) Utilization Potential of
Peats a Study on Peat Biodegradability Determined by Respirometric Method.
Water Air Soil Pollut 192: 5966. DOI 10.1007/s11270-008-9634-y.
V Kaakinen J, Vhoja P, Kuokkanen T & Roppola K (2007) Studies on the Effects of
Certain Soil Properties on the Biodegradation of Oils Determined by the Manometric
Respirometric Method. J Autom Meth and Manage Chem ID 34601: 17. DOI:
10.1155/2007/34601

Reprinted with permission from Science Reviews 2000 Ltd (III) and Springer
Science and Business Media (IV).
Original publications are not included in the electronic version of the dissertation.

81

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SERIES A SCIENTIAE RERUM NATURALIUM

511.

Risteli, Maija (2008) Substrate specificity of lysyl hydroxylase isoforms and

multifunctionality of lysyl hydroxylase 3

512.

Korsu, Kai (2008) Ecology and impacts of nonnative salmonids with special
reference to brook trout (Salvelinus fontinalis Mitchill) in North Europe

513.

Laitinen, Jarmo (2008) Vegetational and landscape level responses to water level
fluctuations in Finnish, mid-boreal aapa mire aro wetland environments

514.

Viljakainen, Lumi (2008) Evolutionary genetics of immunity and infection in social

insects

515.

Hurme, Eija (2008) Ecological knowledge towards sustainable forest management.

Habitat requirements of the Siberian flying squirrel in Finland

516.

Kaukonen, Risto (2008) Sulfide-poor platinum-group element deposits. A

mineralogical approach with case studies and examples from the literature

517.

Rokka, Aare (2008) Solute traffic across the mammalian peroxisomal

membranethe role of Pxmp2

518.

Sharma, Satyan (2008) Computational Studies on Prostatic Acid Phosphatase

519.

Chen, Zhijun (2008) Characterization of the 2-enoyl thioester reductase of

mitochondrial fatty acid synthesis type II in mammals

520.

Hilli, Anu (2009) The effect of crop quality and pre-treatment on germination in
Scots pine and Norway spruce seeds

521.

Kreivi, Marjut (2009) Conservation genetics and phylogeography of endangered

boreoarctic seashore plant species

522.

Riihijrvi, Jorma (2009) Tietojenksittelytieteiden koulutuksen tyelmvastaavuus. Esimerkkitapauksena Oulun yliopiston tietojenksittelytieteiden
laitoksen koulutusohjelma

523.

Ilmonen, Jari (2009) Benthic macroinvertebrate and bryophyte assemblages in

boreal springs: diversity, spatial patterns and conservation

524.

Pujol, Franois (2009) Experiments on fatty acids chain elongation and glycan
flipping in the ER membrane

525.

Lampila, Satu (2009) The causes and consequences of population declines of two
boreal forest species. The case of the willow tit (Parus montanus) and the Siberian
flying squirrel (Pteromys volans)

526.

Anttila, Katja (2009) Swimming muscles of wild, trained and reared fish. Aspects
of contraction machinery and energy metabolism

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