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“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
School of Science, ITM University Gwalior
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: firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, www.itmuniversity.ac.in 2
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.
Mr. ITM University. Mr. Dr. RAUSHANI RAJ B. Dr. Ms.A College. Mr.C. Rekha Vashishtha. I would like to thanks Department of Life Science. I would like to specially thanks to Dr.Sc. Gupta. Sukhveer Singh & Mr.S. Last but not the least this opportunity.Nidhi Agrawal. Arbindra Kumar sagar Faculty of Department of Life Science ITM University Gwalior (M. Sonia Johari. Gwalior for giving me this opportunity. Dr.Acknowledgement It is a great pleasure for me to put on record my appreciation and gratitude towards Dr.) for their guidance and moral support and providing insight to this training time and helped me completing my project report. Himanshu Bhatnagar. 5th Semester I. Dr. Surendra Singh Parihar. Gwalior 4 .L. J.P. Manoj Pathak. R.N.A. Trapti Pathak. Bhat for his valuable support and suggestion for the improvement and editing of minor project report.
CONTENTS Introduction Aims & Objectives Review of Literature Materials & Methods Results & Discussions References • Introduction 5 .
The hydrogen side of water molecules has a slight positive charge.Scientists estimates that the hydrosphere contains about 1. Till recently it had been considered a dependable source of uncontaminated water. It has been caused by human actions. More than 70% of the Earth’s surface is covered with this simple Molecule. Besides. Groundwater crisis is not the result of natural factors. it is an important source of water for the agricultural and industrial sector. Water has a very simple atomic structure. Water is also essential for life. On the other side of the molecule a negative charge exists. especially in the developing countries can be traced to lack of safe and whole some water supply. The importance of ground water for the existence of human society cannot be over emphasized. Without Water Life would probably never have developed on our planet. . Water is the major constituent of almost all life forms.36 billion cubic Kilometers of this substance mostly in the form of a liquid (water) that occupies topographic depressions on the earth. This structure consists of two hydrogen atoms bonded to one oxygen atom. Much of ill health which effects humanity. Ground water is the major Source of drinking water in both urban and rural areas.We live on a Planet that is dominated by water. The molecular polarity causes water to be a powerful solvent and is responsible for its strong surface tension. The consequence of urbanization and industrialization leads to spoil the water. The nature of the atomic structure of water causes its molecules to have unique electrochemical properties. For agricultural purposes ground water is explored in rural areas especially in those areas where other sources of water like 6 . Most Animals and plants contain more than 60% water by volume. Water plays a vital role in human life.
2 million deaths annually. According to World health Organization (WHO). It is needless to emphasize the importance of water in our life. The types of analysis could vary from simple field testing for a single analyze to laboratory based multi component instrumental analysis. So. A good number of water analysis experiments are regularly conducted by different groups of chemists and biologists across the country. The consumption of unsafe water has been implicated as one of the major causes of this disease. for swimming. we need water for drinking. We need water for different purposes. 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 analytical process involves sampling and sample storage since changes in composition of water do not stop once the sampling has been taken. there were estimated 4 billion cases of diarrhea and 2. this is observed that the ground water get polluted drastically because of increased human activities. for irrigation. Consequently number of cases of water borne diseases has been seen which a cause of health hazards. 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. Most gradual deterioration of water quality was resulted by 7 . for fishing etc.dam and river or a canal is not available. for industries. The Key to increase human productivity and long life is good quality water. During last decade. The provision of good quality household drinking water is often regarded as an important means of improving health. basic monitoring on water quality has been necessitated to observe the demand and pollution level of ground water.
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. odourless and even tasteless is not sufficient to determine that the water is safe for consumption. houses especially in the urban area started to equip with a water filter system. the drinking water should be examined on microbiological and physicochemical quality. The WHO in its 2002 report. As water pollution is getting serious. However there is still a debate on the effieciency of filtration system to comply with the regulations as water that physically looks colourless. 8 .the increase in human populations and urbanization. People are concern with the presence of pollutants such as heavy metals and toxic chemical in their daily drinking water. In fact. Filtered water is main source of safe and reliable drinking water.
Temperature. 1. Color. TDS (Total Dissolved Solid). and Hardness of water samples collected from different regions of Gwalior. Collected Sample stored in standard conditions at 40c. Taste. 3. 9 . 2.. Total Plate Count for microbial assay will be carried out in the collected water samples of the study area.• Aims & Objectives Ground water samples collected from different area of Gwalior for laboratory analysis. pH. To estimate chemical parameters like DO and BOD of water samples collected from different regions of Gwalior. To assess physical parameters viz. MPN (Most Probable Number). To assess qualitative and quantitative microbial analysis.
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. it has to comply with certain physical. Although the standards vary from place to place. 2001). which implies that it must be wholesome and palatable in all respect (Edema et al. the objective is to reduce the possibility of spreading water born diseases to the barest minimum. Before water can be described as portable. 2002). 1999). The provision of portable water to rural and urban population is necessary to prevent health hazards (Nikoladze and Akastal.• Review of the work already done In developing countries. The Principal objectives of municipal water are production and the distribution of the safe water that is fit for human consumption (Lamikanra. 1999. FAO. which are designed to ensure that the water is palatable and safe for drinking (Tebutt. A good knowledge of the chemical qualities of raw water is necessary so as to guide its suitability for use. chemical and microbiological standards. Thus. Lemo. Potable water is defined as 10 . Confirmation of microbiological standard is of special great concern because water has great potential to spread diseases within a large population. Water of good drinking quality is of basic importance to human physiology and man’s continued existence depends very much on its availability (Lamikanra.. 1989. regular physico-chemical analysis of water at source must be carried out to determine or check the effectiveness of treatment process. 1997). 1983).
springs and wells (Linsely and Frazini. After purification the water is subjected to test by bacteriologist to ensure the safety for human consumption. rain. among which are streams.water that is free from diseases producing microorganisms and chemical substances deleterious to microorganisms and chemical substances deleterious to health (Ihekoronye and Ngoddy. A long series of dilution is not necessary by some sample because most water supplied are fatty low in 11 . 1985). al 2002).. ponds. 1992). Water can be obtained from a number of sources. 2001. 1979. The consequences of waterborne bacteria and virus infection. Unfortunately. 2000). Water from most sources is therefore unfit for immediate consumption without some sort of treatment (Raymond. 1982). The original source of any drinking water is rich in aquatic microbes. the treatment of water for drinking involves stages where microbes are removed or destroyed before the water gets into homes. typhoid. polio. The drinking water should be examined for microbiological and physico-chemical quality. etc. lakes. 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. pure and safe water only exists briefly in nature and is immediately polluted by prevailing environmental factors and human activities. Fapetu. et. hepatitis. clean. have been well established but nitrate contamination is just deadly. rivers. cholera. stomach cramps. The provision of good quality household drinking water is often regarded as an important means of improving health (Urrbansky. diarrhea. some of which could be dangerous if they enter the human body accordingly. Kolade.
odour.bacteria content. the physical parameters that are likely to give rise to complaints from consumers are color. 12 . and turbidity while low pH causes corrosion and high pH results in taste complaints. while others require long series of dilutions (Fawole and Oso. 2001). 1996). According to WHO (WHO. taste. 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.
ISO 4833:1991.• Proposed Methodology of the research work Collected ground water samples from Gwalior for analysis of following physicochemical & microbiological Water Quality parameters1. Determination of Taste by Bureau of Indian standards. Total Plate Count (TPC).IS:3025(part-4)-1983. By Bureau of Indian Standards: IS 5402: 2002. 9. Measurement of DO by Azide Modification. 4. 10. BIS. APHA. Determination of Hardness of Water: EDTA Trimetric method by APHA. MPN for Portability testing of water sample. BIS. 7. 5. 2. 13 . 8. Gannon University Sim. APHA 3. Measurement of Temperature by Laboratory and Fields Method.IS:3025(part-2)-1983.APHA. IS: 3025(Part-16)- 1984. Determination of Total Dissolved Solid by Bureau of Indian standards. Determination of pH by Electrometric Method. Determination of color: Visual comparison Method. IS: 3025 (Part-16)-1984. 6. Measurement of Biological oxygen demand: 5-Day BOD Test from APHA.
P India.• METHODS: Collection of sample: Drinking water sample was collected in plastic container from Govindpuri. Locations Type S1 S2 S3 Govindpuri Gole ka Mandir Anupam Nagar Bore well Municipal supply Bore well 14 . Immediately after collection of the water sample was bought to the laboratory and analyzed for the physico chemical & microbiological properties. Gole ka mandir and Anupam nagar Gwalior M. Sample No.
35gm of PCA was dissolved in 100ml of distilled water and Ph was adjusted to 7.8gm of nutrient agar was dissolved in 100ml of distilled water and pH was adjusted to 7.0gm 5. Media & Reagent used:- Nutrient agar:Peptone Beef extract Sodium chloride Agar agar 5.6.0gm 15 .0gm 20gm 2.6 Lactose Broth: Beef extract 3.0gm 15. Plate count agar(PCA) Tryptone Yeast extracts Dextrose Agar agar 5.0gm 2.0gm 3.5gm 10.0gm 2.
0gm Peptone Lactose Bile salts no.0gm Potassium iodide 2.Peptone Lactose 5.1 Staining Reagents: Grams Iodine Iodine 1.9.0gm 2.8 gm of MacConkey broth was dissolved in 100 ml distilled water & pH was adjusted to 7.0gm 10.8gm of lactose broth was dissolved in 100ml distilled water and pH adjusted to 6.5gm 5.001gm 2.0gm 16 .03gm 0.0gm 5.0gm 3 1.0gm 0. NaCl Neutral red Crystal violet 17. MacConkey broth Proteose peptone or polypeptone 3.
5% solution in 95% ethyl alcohol) 10. Following methods were adopted for the various tests to examine the physico-chemical properties of drinking water as described by BIS(1986).0gm 5.0 ml Distilled water 100 ml 1.0 ml Safranin (2. 17 .Physico-chemical analysis All the collected drinking water samples were immediately examined and the physico-chemical properties of samples were recorded.APHA.Distilled water Ethyl Alcohol Ethyl alcohol (100%) Distilled water Safranin 300 ml 1.
2. Total dissolved solids (TDS): . Temperature: The temperature of the water sample measured by glass thermometer.0. .Electrode was dried by gentle wiping with a soft tissue paper. . . 5. 3.ion concentration): . Taste: All the collected drinking water samples were tasted and observe the taste of the water samples. Model iiiE) set at room temperature. Colour: The colour of the water sample was observed on the basis of its appearance/transparency or clarity. pH of the sample was recorded. 18 . .PH was recorded by Digital pH meter( EI.100 ml of filtered sample was taken in the previously weighed evaporating dish on a water bath (980c). Electrode was removed from the water and rinsed with distilled water. Instrument was standardized with a buffer solution of pH 7. rinsed and dried and placed in the sample to be tested. 4.To measure pH. . pH (Hydrogen. After setting the pH meter electrode was removed from the buffer solution.1.
MnSo4 added.. B= initial weight of the dish in gm. V= Volume of sample taken in ml. 4) When ppt. conc.The solution turned wine red. H2So4. 3) Stopper carefully to exclude air bubbles & mix by inverting bottle a few times. Final weight of dishes was noted. .Add1 ml. 7) Dissolve Oxygen (DO) 1) Collect 300 ml. 6. It was titrated against 0. .01M-EDTA solution.Total hardness was calculated by following formula: Hardness as mg/L caco3 =(ml EDTA×1000)/ml sample . has settled sufficiently to leave clear supernatant above the manganese hydroxide. water samples in BOD bottle.0 ml of buffer solution and 200 mg of Eriochrome Black TIndicator were added. followed by 1ml.Residue was heated at 103-1050c in an oven for one hour. Total Hardness: . . 1. 19 . Alkali Azide reagent. 2) 1ml. At the end point colour changed from wine red to blue. Total dissolved solids were calculated by following formula: Total dissolved solids mg/L= (A-B×1000×1000)/V A= final weight of the dish in gm. The final weight was taken after cooling in desiccators.Take 50 m l 0f samples.
7) Titrate with 0. MnSo4 added.025 Mn2S2O3 solution to pale straw colour. original samples.After corrections for sample loss by displacement with reagent.Add1 ml. 7) Titrate with 0. 6) Titrate 200 ml. Add few drops of starch solution. Blue colour appears titration to first 20 . Alkali Azide reagent. 3) Stopper carefully to exclude air bubbles & mix by inverting bottle a few times. 4) When ppt.H2So4. Thus for a total of 2ml ( 1ml each ) of MnSo4 & Alkali Iodine Azide reagents in 300 ml bottle. than added 1 ml dilution water. water sample in BOD bottle. 6) Titrate 200 ml. Incubate it 200c at BOD incubator for 5 days. Titrate 200*300/(300-2)= 201ml =198. 5) Re-stopper and mix by inverting several times dissolution is complete. followed by 1ml. Thus for a total of 2ml ( 1ml each ) of MnSo4 & Alkali Iodine Azide reagents in 300 ml bottle.025 Mn2S2O3 solution to pale straw colour. Titrate 200*300/(300-2)= 201ml =198. 8) Biological Oxygen Demand (BOD) 1) Collect 300 ml. After corrections for sample loss by displacement with reagent. Blue colour appears titration to first disappearance of Blue colour. Add few drops of starch solution. 2) 1ml. original samples. conc.5) Restopper and mix by inverting several times dissolution is complete. After incubate add. has settled sufficiently to leave clear supernatant above the manganese hydroxide.
5 ml of single strength MacConkey broth medium was sterilized in screw capped bottle containing Durham’s tube for indicating gas production. • 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. 9) Presumptive Coli form Count (Multiple tube technique)\MPN Test : • 50 ml & 10 ml of double strength macConkey broth medium .disappearance of Blue colour. • All the screw capped bottles were incubated at 37°C Isolation of pathogenic organisms in drinking water: For pathogenic organisms following media and methods were applied as described by Cruickshank et al. • 0.1 ml of water each tube five 5 ml tube containing single strength medium. (1970). 21 . • 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.
+_ 2C. • The tube containing brilliant green lactose bile (BGLB) broth was incubated for 58 hours at 350 +_ 2C. Coli • Each gassing tubes containing Lauryl tryptose broth was gentally agitated and loopful of each suspension was transferred to tube containing EC broth. • 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.Isolation of Coli forms: a) • Presumptive test for coli form bacteria: Aseptically 10 ml of sample was shaken into sterile flask.0 ml diluents (normal saline) was added & shakes vigorously to obtain 10-1 dilution. • From this dilution 1 ml was transferred in 9. 10-4. 9. All decimal dilutions (10-2.0 ml of diluents (normal saline 10-2). • Results were recorded after 48 hours. • EC broth containing tubes were incubated at 48hrs at 350 Gas production was examined after 24 Hrs. From each dilution 1 ml was transferred to 3 tubes containing Lauryl tryptose broth (containing a Durham’s tube for indicating gas production). Confirmation test for E. Tubes were examined after 48 hours. • Tubes were incubated for 48 hours at 35°C. 10-5 and 10-6) were prepared by this method. 22 .
Gram staining: • Thin smear of bacteria was prepared on a slide and air dried. • The smear was covered with crystal violet for 30 seconds. • Saffranin was applied to smear for 30 seconds. 23 .• Loopful of suspension from each gassing tube was strikked to Levine’s Eosin-methylene blue (L-EMB) agar. • The slide was washed with distilled water for few seconds. motility. • The slide was rinsed with distilled water for few seconds. • Colonies were picked from each L-EMB plate and transferred xylose lysine desoxycholate (XLD) agar. • The slide rinsed with 95% ethyl alcohol. Plates were incubated for 18-24 hrs at 350C. • Gram staining and biochemical test of cultures were performed. • • Cultural and microbiological characteristics of the colonies were recorded. • The smear was covered with Iodine solution for 30 seconds. • The slide was rinsed with distilled water and air dried. • Plates were examined after 24 hours. • The smear was heated on flame for a second. salmonella shigella agar and MacConkey agar for morphological and biochemical tests. • The smear was heated on flame for a second.
10-5.10) Total (aerobic) Plate Count: • Aseptically 10 ml sample was taken into sterile flask. • 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. Immediately within 15 min. 15ml plate count agar (cooled) was added to each plate and mixed thoroughly and uniformly. From this dilution 1 ml was transferred to 9 ml diluents (normal saline 10-2 ).0 ml diluents (normal saline) were added and shake vigorously to obtain 10_1 dilution. • Plates were incubated for 48 hrs at 350c ±20C • Colonies were counted by colony counter. • • 1 ml of each dilution was separately pipetted into approximately marked Petri dishes in triplicates. 10-6) were prepared by this method. 10-4. • 9. 24 .All the decimal dilutions (10-2.
Hutchinson stated that temperature is important in controlling both the quality and quantity of plankton flora.11 33 less gm 2 5.6 4.Water Sample Sr.03 23 less gm 8 5. Experiment Temp Colou Tast eratu r e re TDS Ha rd ne ss DO BO TPC D MP N S1. Gole Ka 7.2 4.0 128 0. Water temperature varies with changing climatic condition.6 950 0.07 15 less gm 2 4.10 23.30 Temperature The temperature was found in the range of 23-250 C in month of December. Sample pH No From .0 1650 1. 25 .24 27.6 4.6 S2.50C Colou uri rless Taste 0.30C Colou Madir rless Taste 0.46 26.50C Colou Nagar rless Taste 0.91 S3. The variation in the water temperature may be due to different timing of collection and influence of season. Govindp 7. Anupam 7.
The TDS of all the samples were in range of 200. Hardness Hardness is an important parameter in decreasing the toxic effect of poisonous element. In the present study. which favours solubility of oxygen among the study 26 . the maximum concentration of dissolved oxygen was observed in the month of July after heavy rainfall. It is within desirable limit.10 to 7.46) and were within the maximum limit set for domestic use as per APHA.1100 mg / lit.pH The pH is affected not only by the reaction of carbon dioxide but also by organic and inorganic solutes present in water. The present investigation has provided a good platform for further study to analyze the types and amount of cationic/ anionic salts. while the maximum permissible limiting value of TDS for potable water is 500 mg/ lit.332 mg/lit. according to WHO. 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. The high pH in this case may be attributed to sewage discharge by surrounding human population. 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). High level of TDS in water used for drinking purposes leads to many diseases which are not water-borne but due to excess salts. Any alteration in water pH is accompanied by the change in other physicochemical parameters. Dissolved Oxygen DO is a very important parameter of water quality and an index of physical and biological process going on in water. The hardness was found to be in the range of 152.
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.91 to 1. Gram negative rod shaped bacilli are observed in gram staining.6.sites. 27 . DO is of great importance to all living organisms. MPN and total plate count The microbiological analysis of the water is also showed in the table-2. 1963). Highest BOD found in sample-2 and lowest value were found sample -3.6 mg/l) was recorded on S. MPN indexing of analyzed water samples showed wide variation and were in range of 0. 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. The highest concentration (5.30. The coliform bacterium is the primary bacterial indicator for faecal pollution in water. 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. It may be present in water due to direct diffusion from air and photosynthetic activity of autotrophs.2 but the range was not narrow for other sites. 0. It is a very important indicator of the pollution status of a water body.
Showing Presumtive test for Coliform. 29 .Fig.
Showing positive tube of presumptive test (Acid & Gas Production) 30 .Fig.
• 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. Also.which needs to be disinfected before consumption to avoid water-borne disease. the water samples were showing microbial content beyond the portability Range. 31 . 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.
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