A Report on

Physico-chemical & Microbiological study of Drinking Water in Gwalior

Bachelor of Science In Biotechnology Submitted to Department of Biotechnology

I.A.S.C.A College, Gwalior

Affiliated to Jiwaji University, Gwalior

Under Guidance of:

Mr. Surendra Singh Parihar.

Microbiologist Department of Life Science, ITM University, Gwalior

Submitted By: RAUSHANI RAJ B.Sc. Biotechnology, 5th Semester I.A.S.C.A College, Gwalior
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School of Science, ITM University Gwalior

Certificate
This is to certify that RAUSHANI RAJ Student of B.Sc. Biotechnology 5th Semester, I.A.S.C.A College, Gwalior has undergone training program entitled Physico-chemical & Microbiological study of Water In our University from 15th November to 5th December During training his/ her conduct and progress was good. We wish her all success in her future endeavors.

Project guide:Mr.Surendra singh parihar Department of life sciences ITM UNIVERSITY

Dean School of science ITM UNIVERSITY

ITM UNIVERSITY CAMPUS: SITHOULI, N.H. 75, JHANSI ROAD, GWALIOR (M.P.) 474001 INDIA TEL. 91-751-2440060, FAX: 91-751-2432988 E.MAIL: conference.itmu2012@gmail.com, tims@itmuniversity.ac.in, iasca@itmuniverse.in, www.itmuniversity.ac.in 2

Declaration
I Adya Pushp, student of B.Sc. Biotechnology, 5th semester, hereby declared that the project work entitled Physico-chemical &

Microbiological study of Water

Which is being submitted to the department of Biotechnology, IASCA College, Gwalior, is my own work carried out at 15th November to 5th December 2011, under the guidance of Mr. Surendra Singh Parihar. I declare that the work has not been submitted to any other university or institute for the award of any degree of diploma.

Place: Date:

RAUSHANI RAJ

Acknowledgement
It is a great pleasure for me to put on record my appreciation and gratitude towards Dr. J.L. Bhat for his valuable support and suggestion for the improvement and editing of minor project report. I would like to specially thanks to Dr. Manoj Pathak, Dr. R.N. Gupta, Dr. Trapti Pathak, Dr. Sonia Johari, Dr. Rekha Vashishtha, Ms.Nidhi Agrawal, Mr. Himanshu Bhatnagar, Mr. Surendra Singh Parihar, Mr. Sukhveer Singh & Mr. Arbindra Kumar sagar Faculty of Department of Life Science ITM University Gwalior (M.P.) for their guidance and moral support and providing insight to this training time and helped me completing my project report. Last but not the least this opportunity, I would like to thanks Department of Life Science, ITM University, Gwalior for giving me this opportunity.

RAUSHANI RAJ B.Sc. 5th Semester I.A.S.C.A College, Gwalior

CONTENTS

Introduction Aims & Objectives Review of Literature Materials & Methods Results & Discussions References

Introduction
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We live on a Planet that is dominated by water. More than 70% of the Earths surface is covered with this simple Molecule. .Scientists estimates that the hydrosphere contains about 1.36 billion cubic Kilometers of this substance mostly in the form of a liquid (water) that occupies topographic depressions on the earth. Water is also essential for life. Water is the major constituent of almost all life forms. Most Animals and plants contain more than 60% water by volume. Without Water Life would probably never have developed on our planet. Water has a very simple atomic structure. This structure consists of two hydrogen atoms bonded to one oxygen atom. The nature of the atomic structure of water causes its molecules to have unique electrochemical properties. The hydrogen side of water molecules has a slight positive charge. On the other side of the molecule a negative charge exists. The molecular polarity causes water to be a powerful solvent and is responsible for its strong surface tension. Ground water is the major Source of drinking water in both urban and rural areas. The importance of ground water for the existence of human society cannot be over emphasized. Besides, it is an important source of water for the agricultural and industrial sector. Till recently it had been considered a dependable source of uncontaminated water. Groundwater crisis is not the result of natural factors. It has been caused by human actions. Much of ill health which effects humanity, especially in the developing countries can be traced to lack of safe and whole some water supply. Water plays a vital role in human life. The consequence of urbanization and industrialization leads to spoil the water. For agricultural purposes ground water is explored in rural areas especially in those areas where other sources of water like
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dam and river or a canal is not available. During last decade, this is observed that the ground water get polluted drastically because of increased human activities. Consequently number of cases of water borne diseases has been seen which a cause of health hazards. So, basic monitoring on water quality has been necessitated to observe the demand and pollution level of ground water. A good number of water analysis experiments are regularly conducted by different groups of chemists and biologists across the country. It is needless to emphasize the importance of water in our life. We need water for different purposes; we need water for drinking, for industries, for irrigation, for swimming, for fishing etc. Thus water for different purposes has its own requirements for the composition and purity and each body of water has to be analyzed on a regular basis to confirm to suitability. The types of analysis could vary from simple field testing for a single analyze to laboratory based multi component instrumental analysis. The analytical process involves sampling and sample storage since changes in composition of water do not stop once the sampling has been taken. Precaution has to be taken to make sure that the water reaching the laboratory has the same composition as it did when the sampling was done. The Key to increase human productivity and long life is good quality water. The provision of good quality household drinking water is often regarded as an important means of improving health. According to World health Organization (WHO), there were estimated 4 billion cases of diarrhea and 2.2 million deaths annually. The consumption of unsafe water has been implicated as one of the major causes of this disease. Most gradual deterioration of water quality was resulted by
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the increase in human populations and urbanization. As water pollution is getting serious, houses especially in the urban area started to equip with a water filter system. People are concern with the presence of pollutants such as heavy metals and toxic chemical in their daily drinking water. Filtered water is main source of safe and reliable drinking water. However there is still a debate on the effieciency of filtration system to comply with the regulations as water that physically looks colourless, odourless and even tasteless is not sufficient to determine that the water is safe for consumption. In fact, the drinking water should be examined on microbiological and physicochemical quality. The WHO in its 2002 report, recommended that increased emphasis be placed on home water treatment and storage that more research should conducted to assess the health benefits of such interventions.

 Aims & Objectives

Ground water samples collected from different area of Gwalior for laboratory analysis. Collected Sample stored in standard conditions at 40c. 1. To assess physical parameters viz., Color, pH, Taste, Temperature, TDS (Total Dissolved Solid), and Hardness of water samples collected from different regions of Gwalior. 2. To estimate chemical parameters like DO and BOD of water samples collected from different regions of Gwalior.
3.

To assess qualitative and quantitative microbial analysis, MPN (Most Probable Number), Total Plate Count for microbial assay will be carried out in the collected water samples of the study area.

 Review of the work already done

In developing countries, availability of water has become a critical and urgent problem and is a matter of great concern to families and communities depending on non public water supply. Confirmation of microbiological standard is of special great concern because water has great potential to spread diseases within a large population. Although the standards vary from place to place, the objective is to reduce the possibility of spreading water born diseases to the barest minimum, which implies that it must be wholesome and palatable in all respect (Edema et al., 2001). The Principal objectives of municipal water are production and the distribution of the safe water that is fit for human consumption (Lamikanra, 1999). A good knowledge of the chemical qualities of raw water is necessary so as to guide its suitability for use. Thus, regular physico-chemical analysis of water at source must be carried out to determine or check the effectiveness of treatment process. Water of good drinking quality is of basic importance to human physiology and mans continued existence depends very much on its availability (Lamikanra, 1999, FAO, 1997). The provision of portable water to rural and urban population is necessary to prevent health hazards (Nikoladze and Akastal, 1989; Lemo, 2002). Before water can be described as portable, it has to comply with certain physical, chemical and microbiological standards, which are designed to ensure that the water is palatable and safe for drinking (Tebutt, 1983). Potable water is defined as
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water that is free from diseases producing microorganisms and chemical substances deleterious to microorganisms and chemical substances deleterious to health (Ihekoronye and Ngoddy, 1985). Water can be obtained from a number of sources, among which are streams, lakes, rivers, ponds, rain, springs and wells (Linsely and Frazini, 1979; Kolade, 1982). Unfortunately, clean, pure and safe water only exists briefly in nature and is immediately polluted by prevailing environmental factors and human activities. Water from most sources is therefore unfit for immediate consumption without some sort of treatment (Raymond, 1992). The consequences of waterborne bacteria and virus infection; polio, hepatitis, cholera, typhoid, diarrhea, stomach cramps, etc, have been well established but nitrate contamination is just deadly. Consequence to the realization of the potential health hazards that may result from contaminated drinking water from any source is therefore of primary importance because of the danger and risk of water borne disease (Edema et al., 2001; Fapetu, 2000). The provision of good quality household drinking water is often regarded as an important means of improving health (Urrbansky, et. al 2002). The drinking water should be examined for microbiological and physico-chemical quality. The original source of any drinking water is rich in aquatic microbes, some of which could be dangerous if they enter the human body accordingly, the treatment of water for drinking involves stages where microbes are removed or destroyed before the water gets into homes. After purification the water is subjected to test by bacteriologist to ensure the safety for human consumption. A long series of dilution is not necessary by some sample because most water supplied are fatty low in

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bacteria content, while others require long series of dilutions (Fawole and Oso, 2001). According to WHO (WHO, 1996), the physical parameters that are likely to give rise to complaints from consumers are color, taste, odour, and turbidity while low pH causes corrosion and high pH results in taste complaints. Therefore the objective of this study is to determine the microbiological and physicochemical quality of water samples collected before and after the filtration treatment given.

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 Proposed Methodology of the research work

Collected ground water samples from Gwalior for analysis of following physicochemical & microbiological Water Quality parameters1. Determination of color: Visual comparison Method, BIS.IS:3025(part-4)-1983.

2. Measurement of Temperature by Laboratory and Fields Method; APHA 3. 4. 5. Determination of pH by Electrometric Method, BIS.IS:3025(part-2)-1983. Determination of Taste by Bureau of Indian standards. IS: 3025 (Part-16)-1984. Determination of Total Dissolved Solid by Bureau of Indian standards. IS: 3025(Part-16)-

1984. 6. Determination of Hardness of Water: EDTA Trimetric method by APHA, Gannon University Sim. 7. 8. 9. 10. Measurement of DO by Azide Modification; APHA. Measurement of Biological oxygen demand: 5-Day BOD Test from APHA. MPN for Portability testing of water sample.APHA. Total Plate Count (TPC). By Bureau of Indian Standards: IS 5402: 2002, ISO

4833:1991.

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METHODS:

Collection of sample:
Drinking water sample was collected in plastic container from Govindpuri, Gole ka mandir and Anupam nagar Gwalior M.P India. Immediately after collection of the water sample was bought to the laboratory and analyzed for the physico chemical & microbiological properties.

Sample No.

Locations

Type

S1 S2 S3

Govindpuri Gole ka Mandir Anupam Nagar

Bore well Municipal supply Bore well

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Media & Reagent used:-

Nutrient agar:Peptone Beef extract Sodium chloride Agar agar 5.0gm 3.0gm 5.0gm 20gm

2.8gm of nutrient agar was dissolved in 100ml of distilled water and pH was adjusted to 7.6.

Plate count agar(PCA)

Tryptone Yeast extracts Dextrose Agar agar 5.0gm 2.5gm 10.0gm 15.0gm

2.35gm of PCA was dissolved in 100ml of distilled water and Ph was adjusted to 7.6

Lactose Broth:
Beef extract 3.0gm
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Peptone Lactose

5.0gm 5.0gm

2.8gm of lactose broth was dissolved in 100ml distilled water and pH adjusted to 6.9.

MacConkey broth
Proteose peptone or polypeptone 3.0gm Peptone Lactose Bile salts no. NaCl Neutral red Crystal violet 17.0gm 10.0gm 3 1.5gm 5.0gm 0.03gm 0.001gm

2.8 gm of MacConkey broth was dissolved in 100 ml distilled water & pH was adjusted to 7.1

Staining Reagents: Grams Iodine Iodine 1.0gm Potassium iodide 2.0gm

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Distilled water Ethyl Alcohol Ethyl alcohol (100%) Distilled water Safranin

300 ml

1.0gm 5.0 ml

Safranin (2.5% solution in 95% ethyl alcohol) 10.0 ml Distilled water 100 ml

1.Physico-chemical analysis
All the collected drinking water samples were immediately examined and the physico-chemical properties of samples were recorded. Following methods were adopted for the various tests to examine the physico-chemical properties of drinking water as described by BIS(1986),APHA.

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1. Colour: The colour of the water sample was observed on the basis of its
appearance/transparency or clarity.

2. Temperature: The temperature of the water sample measured by glass

thermometer.

3. pH (Hydrogen- ion concentration):

.PH was recorded by Digital pH meter( EI, Model iiiE) set at room temperature. .To measure pH, Electrode was removed from the water and rinsed with distilled water. .Electrode was dried by gentle wiping with a soft tissue paper. . Instrument was standardized with a buffer solution of pH 7.0. . After setting the pH meter electrode was removed from the buffer solution, rinsed and dried and placed in the sample to be tested. . pH of the sample was recorded.

4. Taste: All the collected drinking water samples were tasted and observe the
taste of the water samples.

5. Total dissolved solids (TDS):

.100 ml of filtered sample was taken in the previously weighed evaporating dish on a water bath (980c).
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.Residue was heated at 103-1050c in an oven for one hour. . Final weight of dishes was noted. The final weight was taken after cooling in desiccators. Total dissolved solids were calculated by following formula: Total dissolved solids mg/L= (A-B10001000)/V
A= final weight of the dish in gm. B= initial weight of the dish in gm. V= Volume of sample taken in ml.

6. Total Hardness:
.Take 50 m l 0f samples, 1.0 ml of buffer solution and 200 mg of Eriochrome Black TIndicator were added. .The solution turned wine red. It was titrated against 0.01M-EDTA solution. At the end point colour changed from wine red to blue. .Total hardness was calculated by following formula: Hardness as mg/L caco3 =(ml EDTA1000)/ml sample .

7) Dissolve Oxygen (DO)

1) Collect 300 ml. water samples in BOD bottle. 2) 1ml. MnSo4 added, followed by 1ml. Alkali Azide reagent. 3) Stopper carefully to exclude air bubbles & mix by inverting bottle a few times. 4) When ppt. has settled sufficiently to leave clear supernatant above the manganese hydroxide.Add1 ml. conc. H2So4.
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5) Restopper and mix by inverting several times dissolution is complete. 6) Titrate 200 ml. original samples. After corrections for sample loss by displacement with reagent. Thus for a total of 2ml ( 1ml each ) of MnSo4 & Alkali Iodine Azide reagents in 300 ml bottle, Titrate 200*300/(300-2)= 201ml =198. 7) Titrate with 0.025 Mn2S2O3 solution to pale straw colour. Add few drops of starch solution. Blue colour appears titration to first disappearance of Blue colour.

8) Biological Oxygen Demand (BOD)

1) Collect 300 ml. water sample in BOD bottle, than added 1 ml dilution water, Incubate it 200c at BOD incubator for 5 days. After incubate add. 2) 1ml. MnSo4 added, followed by 1ml. Alkali Azide reagent. 3) Stopper carefully to exclude air bubbles & mix by inverting bottle a few times. 4) When ppt. has settled sufficiently to leave clear supernatant above the manganese hydroxide.Add1 ml. conc.H2So4. 5) Re-stopper and mix by inverting several times dissolution is complete. 6) Titrate 200 ml. original samples.After corrections for sample loss by displacement with reagent. Thus for a total of 2ml ( 1ml each ) of MnSo4 & Alkali Iodine Azide reagents in 300 ml bottle, Titrate 200*300/(300-2)= 201ml =198. 7) Titrate with 0.025 Mn2S2O3 solution to pale straw colour. Add few drops of starch solution. Blue colour appears titration to first
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disappearance of Blue colour.

9) Presumptive Coli form Count (Multiple tube technique)\MPN Test : 50 ml & 10 ml of double strength macConkey broth medium ; 5 ml of single strength MacConkey broth medium was sterilized in screw capped bottle containing Durhams tube for indicating gas production.

With sterile pipettes the following amount of water sample was added to each tube: 50 ml of water to one 50 ml tube containing double strength medium.

10 ml water to each five 10 ml tubes containing double strength medium.

1ml of water each to five 5 ml tube containing single strength medium.

0.1 ml of water each tube five 5 ml tube containing single strength medium.

All the screw capped bottles were incubated at 37C

Isolation of pathogenic organisms in drinking water:

For pathogenic organisms following media and methods were applied as described by Cruickshank et al. (1970).
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Isolation of Coli forms:

a)

Presumptive test for coli form bacteria:

Aseptically 10 ml of sample was shaken into sterile flask. 9.0 ml diluents (normal saline) was added & shakes vigorously to obtain 10-1 dilution. From this dilution 1 ml was transferred in 9.0 ml of diluents (normal saline 10-2). All decimal dilutions (10-2, 10-4, 10-5 and 10-6) were prepared by this method. From each dilution 1 ml was transferred to 3 tubes containing Lauryl tryptose broth (containing a Durhams tube for indicating gas production). Tubes were incubated for 48 hours at 35C. Tubes were examined after 48 hours. A confirmation test was performed on all presumptive positive (gassing) tube

b)

Confirmation Test for Coli forms

Each gassing tube containing lauryl tryptose broth was gentally agitated and loopful of suspension was transferred to tube containing brilliant green lactose Bile (BGLB) broth.

The tube containing brilliant green lactose bile (BGLB) broth was incubated for 58 hours at 350 +_ 2C.

Results were recorded after 48 hours. Confirmation test for E. Coli Each gassing tubes containing Lauryl tryptose broth was gentally agitated and loopful of each suspension was transferred to tube containing EC broth.

EC broth containing tubes were incubated at 48hrs at 350 Gas production was examined after 24 Hrs.

+_

2C.

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 Loopful of suspension from each gassing tube was strikked to Levines Eosin-methylene blue (L-EMB) agar. Plates were examined after 24 hours.

Colonies were picked from each L-EMB plate and transferred xylose lysine desoxycholate (XLD) agar, motility, salmonella shigella agar and MacConkey agar for morphological and biochemical tests. Plates were incubated for 18-24 hrs at 350C.

Cultural and microbiological characteristics of the colonies were recorded. Gram staining and biochemical test of cultures were performed.

Gram staining:
Thin smear of bacteria was prepared on a slide and air dried. The smear was heated on flame for a second. The smear was heated on flame for a second. The smear was covered with crystal violet for 30 seconds. The slide was washed with distilled water for few seconds. The smear was covered with Iodine solution for 30 seconds. The slide rinsed with 95% ethyl alcohol. The slide was rinsed with distilled water for few seconds. Saffranin was applied to smear for 30 seconds. The slide was rinsed with distilled water and air dried.
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10) Total (aerobic) Plate Count:

Aseptically 10 ml sample was taken into sterile flask.

9.0 ml diluents (normal saline) were added and shake vigorously to obtain 10_1 dilution. From this dilution 1 ml was transferred to 9 ml diluents (normal saline 10-2 ).All the decimal dilutions (10-2, 10-4, 10-5, 10-6) were prepared by this method.

1 ml of each dilution was separately pipetted into approximately marked Petri dishes in triplicates. Immediately within 15 min. 15ml plate count agar (cooled) was added to each plate and mixed thoroughly and uniformly.

Plates were incubated for 48 hrs at 350c 20C

Colonies were counted by colony counter.

Results & Discussion

The examined physico-chemical and microbiological parameters showed considerable variations from sample to sample. The observations of the study are depicted in Table given below.

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Water Sample
Sr. Sample pH No From .

Experiment
Temp Colou Tast eratu r e re TDS Ha rd ne ss DO BO TPC D MP N

S1. Govindp 7.24 27.50C Colou uri rless

Taste 0.07 15 less gm 2

4.6 4.0 1650

1.6

S2. Anupam 7.46 26.50C Colou Nagar rless

Taste 0.03 23 less gm 8

5.2 4.6 950

0.91

S3. Gole Ka 7.10 23.30C Colou Madir rless

Taste 0.11 33 less gm 2

5.6 4.0 128

0.30

Temperature
The temperature was found in the range of 23-250 C in month of December. The variation in the water temperature may be due to different timing of collection and influence of season. Water temperature varies with changing climatic condition. Hutchinson stated that temperature is important in controlling both the quality and quantity of plankton flora.
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pH
The pH is affected not only by the reaction of carbon dioxide but also by organic and inorganic solutes present in water. Any alteration in water pH is accompanied by the change in other physicochemical parameters. pH maintenance (buffering capacity) is one of the most important attributes of any aquatic system since all the biochemical activities depend on pH of the surrounding water. It was concluded that the pH of water were slightly alkaline (7.10 to 7.46) and were within the maximum limit set for domestic use as per APHA. The high pH in this case may be attributed to sewage discharge by surrounding human population.

Hardness
Hardness is an important parameter in decreasing the toxic effect of poisonous element. The hardness was found to be in the range of 152- 332 mg/lit. It is within desirable limit. The hardness of water increases in the polluted waters by the deposition of calcium and magnesium salts.

TDS
The most remarkable observation of investigation was the alarmingly high level of total dissolved solids (TDS). The TDS of all the samples were in range of 200- 1100 mg / lit. while the maximum permissible limiting value of TDS for potable water is 500 mg/ lit., according to WHO. High level of TDS in water used for drinking purposes leads to many diseases which are not water-borne but due to excess salts. The present investigation has provided a good platform for further study to analyze the types and amount of cationic/ anionic salts.

Dissolved Oxygen
DO is a very important parameter of water quality and an index of physical and biological process going on in water. In the present study, the maximum concentration of dissolved oxygen was observed in the month of July after heavy rainfall, which favours solubility of oxygen among the study
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sites. The highest concentration (5.6 mg/l) was recorded on S- 2 but the range was not narrow for other sites. DO is of great importance to all living organisms. It may be present in water due to direct diffusion from air and photosynthetic activity of autotrophs. Concentration of DO is one of the most important parameters to indicate water purity and to determine the distribution and abundance of various algal groups.

BOD (Biological Oxygen Demand)

BOD is the amount of oxygen required by the living organisms engaged in the utilization and ultimate destruction or stabilization of organic water (Hawkes, 1963). It is a very important indicator of the pollution status of a water body. Highest BOD found in sample-2 and lowest value were found sample -3.

MPN and total plate count

The microbiological analysis of the water is also showed in the table-2. The total plate count (TPC) indicate that the highest microbial load 1420 cfu/ml in S-1 after 24 & 48 h incubation and minimum load 128 cfu/ml in S2. MPN indexing of analyzed water samples showed wide variation and were in range of 0.30, 0.91 to 1.6. Gram negative rod shaped bacilli are observed in gram staining. The coliform bacterium is the primary bacterial indicator for faecal pollution in water.

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28

Fig. Showing Presumtive test for Coliform.

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Fig. Showing positive tube of presumptive test (Acid & Gas Production)

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 Conclusion:
The present investigation is essentially a primary work and needs to be further investigated to arrive at specified conclusion with respect to clinical implications. The observation of study strongly suggest that water of Gwalior region is of very high TDS and needs to be lowered down within prescribed limits before using it for drinking purposes. Also, the water samples were showing microbial content beyond the portability Range,which needs to be disinfected before consumption to avoid water-borne disease.

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