SAMPLE COLLECTION : The present study was carried out during August 2010 to June 2012.

Surface water samples were collected between 7 am to 8 am from the pond using clean polythene bottles. Samples for Bacteriological parameters were collected using sterilized glass bottle. Samples for bacteriological analysis were brought to the laboratory as quick as possible in an ice box and analysed before 24 hours. The water sample for the estimation of dissolved oxygen was fixed immediately and for biological oxygen demand the water is collected in clean light and dark bottle and brought to the laboratory for further analysis. Phytoplankton samples were collected from the selected stations. Standard plankton net (No. 25) was used for the collection and sampling was done during 9.00 to 11.00 am. About 100 litres of water was filtered through the net and the filtrate was collected in the collecting vessel. The samples then concentrated to 25 ml and preserved in 1ml neutralized formaldehyde solution. Plankton count were done using Haemocytometer counting chamber. From the collected and concentrated filtrate, 1ml of the sample was taken after shaking the concentrate, in order to get an even distribution of the planktonic organisms for analysis. The analysis was repeated 5 times for each sample and the average number was computed and expressed in number per cubic meter. Standard literatures were used for identifying the phytoplankton (Fritsch, 1977; Prescott, 1978; Anand, 1980; Prasad and Srinivasan, 1992). PHYSICO-CHEMICAL PARAMETERS : Temperature: Air temperature and water temperature are analysised by using thermameter. Turbidity: Cloudiness of water is measured by secchi disc. Electrical Conductivity: The electrical conductivity is measured in terms of the resistance offered to the flow of current using a conductivity meter. pH: The pH meter is calibrated using suitable buffers. The pH meter is immersed in the sample of water in the beaker and the reading are taken directly. Hanna pen type was used for measuring pH of water samples Free carbon dioxide: Reagents 1. Sodium hydroxide solution (0.01 ) : Dissolve 2.5 g of sodium hydroxide in CO2 free (boiled and cooled) distilled water and make the volume 1 L . standardize the solution with 0.05 N potassium hydrogen phthalate using phenolphthalein indicator. 2. Phenolphthalein indicator: disolve 1 g of pHenolphthalein in 100 ml of ethyl alcohol and add 100 ml of distilled water. Add NaOH solution drop by drop until a faint pink colour appears. Method:

Free carbon dioxide is determined in water immediately after taking the sample.Take 50 ml of sample in a conical flash and add 2-3 drops of Phenolphthalein indicator. If the colour turns pink, free CO2 is absent in sample. If the sample remains colourless , titrate it against sodium hydroxide solution until pink colour appears. Calculation: free CO2 mg/L = Volume of titrant (mL) x 1000 x 44 x 0.05 Volume of sample(mL) where ,0.05 is the normality of NaOH Total carbon Dioxide: Total carbon dioxide is the sum of all three species of CO2 , CO3 - - , HCO3 - present in water. It may be used as an index of trophic status. Calculation: Tatal CO2 (mg/L) = free CO2 (mg/L) + 0.88 (b+c) Where b = bicarbonate content in mg/L and c =carbonate content in mg/L Total alkalinity: Reagents: 1. sulphuric acid(0.02N) 2. sulphuric acid(0.1N) 2.8 ml of conc. H2SO4 diluted to 1 l using distilled water. Take 200 ml from this stock solution and dilute to 1 l using distilled water. 3. Phenolphthalein indicator 1 g of phenolphthalein is dissolved in 100 ml of ethyl alcohol. After complete dissolution add 100 ml distilled water. Add NaOH reagent (0.2272N) in drops till a faint pink colour appears. 4. Methyl orange indicator 0.1 g of methyl orange is dissolved in 200 ml of distilled water. Procedure: 1.Estimation of alkalinity should be done immediately after the collection of the sample. 2.In 50 ml of sample ,add 2-3 drops of Phenolphthalein indicator. 3.If the solution shows pink colour titrate it against sulphuric acid. 4.Appearance of slight pink colour indicates the presence of hydroxides or carbonates whereas colourless sample conforms the presence of free CO2

5.

Note down the colourless end point as p

6. Now add 2-3 drops of methyl orange indicator to the same flask and proceed with the titration till the solution turns from yellow to orange. 7. Record the value as t and calculate the alkalinity using the formula p= px1000 ( as CaCO3 mg/l) v Total alkalinity Where p = ml of titrant used for Phenolphthalein alkalinity t = ml of titrant used for both titrations v = volume of sample S.NO Volume Burette of the reading sample (mL) (mL) Vol. Of std.H2SO4 used for half neatralization of carbonates Vol. Of std H2SO4 used for complete neatralization of carbonates 2x Vol. Of std H2SO4 used for complete neatralization of CO3 + HCO3 c -a =y Vol. Of std H2SO4 used for complete neatralization of HCO3 y-2x t = tx1000 ( as CaCO3 mg/l) v Calculation: Phenolphthalein alkalinity

a Calculations:

b c (b-a)= x

CO3 ---in milliequivalent per liter: CO3 ---in milliequivalent per liter = Vol. Of H2SO4 (2X) X N X 1000 mL of water sample CO3 ---in gram per liter: CO3 ---in gram per liter = Vol. Of H2SO4 (2X) X N X Eq.Wt. Of CO3 --- (30) mL of water sample HCO3 ---in milliequivalent per liter =Normality Of H2SO4X Vol. Of H2SO4 X 1000 mL of water sample HCO3 --- me / liter = (y-2x) x 0.1 x1000

mL of water sample HCO3 ---in gram per liter: HCO3 ---in gram per liter = Vol. Of H2SO4 (2X) X N X Eq.Wt. Of HCO3 --- (61) mL of water sample Total acidity: Reagents: 1. sodium hydroxide(0.1N Dissolve 4g of NaOH in little distilled water and make up to one litre. 2. sodium hydroxide(0.05N) Take 5 ml 0.1 N sodium hydroxide and make up to one litre 3. Phenolphthalein indicator 1 g of phenolphthalein is dissolved in 100 ml of ethyl alcohol. After complete dissolution add 100 ml distilled water. Add NaOH reagent (0.2272N) in drops till a faint pink colour appears. 5. Methyl orange indicator 0.1 g of methyl orange is dissolved in 200 ml of distilled water. Procedure: 1.Transfer 100 ml of sample in a volumetric flask,,add 2-3 drops of methyl orange indicator. 2.Observe for the colour change from orange to yellow. 3. 4. 5. 6. p. if the solution changes to pink colour titrate it with 0.05 N sodium hydroxide. Note the reading ,let it be m. Now contiue to titrate the sample after adding phenolphthaline indicator. Observe for the colour change from yellow to pink. Note the reading Let it be

Calculation: Methyl orange acidity = m x 0.05N of NaOH x50,000 Volume of sample phenolphthaline acidity = m x 0.05N of NaOH x50,000 Volume of sample Total acidity = m x 0.05N of NaOH x50,000 Volume of sample

Total hardness: Reagents: 1.Ammonia buffer solution(Dissolve 13.5 g of ammonium chloride in 114 ml of conc. Ammonium hydroxide. Make up to 200 ml using distilled water. 2.Erichrome black T indicator( Dissolve 0.5 g of v T Dye in 100 ml ethyl alcohol (80%) 3.EDTA solution (0.001M) (3.723 g of sodium salt of EDTA in little distilled water and make up to 1000ml. Use polyethylene nottle for storage. Procedure: 1.In 50 ml sample, add 1 ml of ammonium buffer solution and 4 drops of Erichrome black T indicator. 2.Now the solution turns wine red in colour. 3.Titarte it against EDTA solution till the colour changes from wine red to blue. 4.Record the end point, repeat the procedure to attain constant value 5.Calculate the total hardness using the formula. Calculation: Total hardness= E x1000 (as CaCO3 mg l-1) v where E = Volume of titrant in ml V = Volume of sample Calcium hardness : Reagents: 1. NaOHsolution(8%) 8 g of NaOH dissolved in 100 ml of distilled water. 2. Murexide indicator Add 0.2 g of ammonium purpurate to100 g of sodium chloride.Grind it well in a mortar and pestle. 3. EDTA solution (0.01M) 3.723 g of disodium salt of EDTA dissolved in distiied water. Make up to

1000ml. Procedure: 1. In 50 ml sample, add 1 ml of sodium hydroxide solution and a pinch of murexide indicator. 2. Swirl the flask to ensure complete mixing. 3. The colour of the solution turns pink. 4. Now titrate it against EDTA solution ,till the colour changes from pink to purple. 5. Note down the end point. Calculation: Calcium hardness (Ca mg/ l)= E x400.5 x 1.05 v Calcium hardness( CaCO3 mg/ l) = E x400.5 x 1.05 v where E = Volume of EDTA titrant (E) in ml V = Volume of sample Total dissolved solids: ( Gravimetric method) Pipette out 100 ml of water into a clean dry potash basin, the accurate weight of which has been taken by drying in a steam ovenfor an hour, cooling in a desiccator and weighing in a chemical balance. Evaporate the water in the basin to dryness over a water bath. Wipe the outside of the basin, dry in air oven at 105o C for an hour to remove moisture, cool and weigh. The difference between the weights is the weight of total soluble salts which is expressed as ppm. Calculation Volume of water taken = 100 ml Weight of empty basin = A gm Weight of basin + residue = B gm Therefore, weight of total soluble salts = B-A This is present in 100 mlof water Therefore, total soluble salts content of water ( pp m )

= (B-A) X 10 6 100 sulphate : Reagents: 1.Mehyl red indicator(Dissolve 10 mg of barium chloride in 100ml of distilled water. Filter it through Whatsman No1 filter paper and store for use. 2.Silver nitrate solution( Dissolve 8.5 g of silver nitrate powder in little nitric acid solution. Make up to 500 ml using distilled water.) 3.Hydrochloric acid(50%) (Add distilled water and conc. Hydrochloric acid in the ratio 1:1 procedure: 1. Transfer 100 ml of sample in to a 250 ml volumetric flask. 2.To this, add 2-3 drops of methyl red indicator. 3. Slowly add hydrochloric acid to the flash content and observe for the colour change from red to orange.(This indicates the acidic pH of the solution) 4.Add still more hydrochloric acid to the flash and boil it. 5.Now add warm barium chloride solution so as to favour the precipitation process. 6. Continue to add it till the precipitation is completed. 7.Again heat it in a hot- water bath for about 2 hours. Do not cool it. Immediately filter it through ash less filter paper. 8.Flash the filter paper with distilled water many times. 9.This should be repeated till the filtrate does not contain traces of chloride. 10.Every time after washing the filter paper, check the presence of chloride in the filtrate by filtrating it against silver nitrate solution. Absence of turbidity indicates nil chloride content and at this stage transfer the filter paper to a silica crucible. 11.Ignite it in a muffle furnace at 80o C for 1 hour. After one hour take the weight of the precipitate.

Calculation: Sulphate (mg /l) =W x 411.5 V Where , W = Weight of the precipitate V = Volume of the sample

Chloride: The chloride present in the water is precipitated as silver chloride by titration with standard silver nitrate solution using potassium chloride as the indicator. After all the chloride is precipitated, the excess of silver nitrate combines with potassium chlromate indicator to form flesh red precipitate of silver chromate. Procedure: Pipette out 50 ml of the water in to a porcelain dish. Add a few drops of pottasium chrpmate indicator and titrate against 0.1 N AgNO3 consumed, calculated the chloride constant. Calculation: Volume of water taken =50 ml Volume of 0.1 N AgNO3 used = A ml 1ml of 0.1N AgNO3 =0.00355gm of Cl Therefore, the amount of chloride in water sample(ppm)=0.00355 xA x 106 50 In terms of m.e. /liter = 0.00355 x A x1000 x 1000 50 35.5 Dissolved Oxygen: Dissolved oxygen content of the surface water sample was measured by using Winklers titration method (APHA, 1985). Principle A divalent manganese solution followed by strong alkali are added to the sample.The precipitated manganese hydroxide is dispersed evenly through the water sample, which completely fills the stoppered glass bottle. Any dissolved oxygen rapidly oxidizes an equivalent amount of divalent manganese to basic hydroxide of higher valancy states. When the solution is acidified in the presence of iodine equivalent to the original dissolved oxygen content of the water is liberated. This iodine is titrated with standardized thiosulphate solution.

Mn ++ + 2 OH 2 Mn(OH)2 + O2 MnO (OH)2 + 4 H+ + 2H I 3 + 2 S2O3 Reagents

Mn(OH)2 2 MnO (OH)2 Mn ++ + I 3- + 3 H 2O 2I - + S4O6

1. BOD bottles (100 300 mL ). 2. Manganous sulphate solution : Dissolve 100 g of manganous sulphate in 200 mL of reviously boiled distilled water and filter the solution . 3. Alkaline potassium iodide solution : Weigh 50 g of potassium iodide and 100 g of potassium hydroxide . Dissolve the chemicals in 200 mL of previously boiled distilled water . 4. Sodium thiosulphate (0.01N) : Take 6.205 g of sodium thiosulphate, dissolve it in previously boiled distilled water and make up the to 1 Litre. Add a pallet of sodium hydroxide as a preservative. Keep the solution in a coloured bottle. 5. Starch indicator : Dissolve 1 g of starch in 100ml of warm distilled water and add afew drops of toluene or formaldehyde as preservative. 6. Concentrated Sulphuric acid : Sp.gr. 1.84. Apparatus Narrow mouth glass stoppered darkened bottle, pipettes, burette, and conical flask. Sampling Rinse the dark bottle with the sample water and fill the bottle with water. Sample from the reversing bottle through a rubber tube, keeping the tube always at the bottom of the bottle. This minimized the turbulence and agitation. Allow water to overflow from the top of the bottle and withdraw the rubber tube. Stopper the bottle at once without entrapping any air bubble. Procedure 1. Take a glass stoppered BOD bottle of known volume and fill it with sample avoiding any bubbling. No air should be trapped in bottle after the stopper is placed. 2. Restopper the bottle and pour in it 1 mL of each mangraous sulphate and alkaline potassium oidide solutions using seperate pipettes. 3. A precipitate will appear. Place the stopper and shake the bottle thoroughly. Sample at this stage can be stored for few days. If required. 4. Add 2 mL . Of sulphuric acid and shake thoroughly to dissolve the precipitate. 5. Transfer gently (avoiding bubbling) whole content or a known part of it, in a conical flask. Put a few drops of starch indicator. Titrate against sodium thiosulphate solution and note the end point when initial blue colour turns to

colurless. Calculation 1. if the whole content is used for titration: DO mg/L = V1 N X 8 X 1000 V2 - V3 2. if the fraction of the content is used for titration: DO mg/L = V1 N X 8 X 1000 V4 (V2 _V3 /V2) V1= Volume of the titrant(mL) N =Normality of the titrant(0.025) V2 = Volume of the sample bottle after placing the stopper (mL) V3 = Volume of manganous sulphate+ Potassium iodide solution added(mL) V4= Volume of fraction of the contents used for titration(mL) The equivalent weight of oxygen is 8. To obtain the value of DO in mL/L divide the Doin mg/L by 1.43 Biological Oxygen Demand (BOD) BOD of water is the quantity of dissolved oxygen required to effect stabilisation by anaerobic bacterial action of that portion of DOM (Dissolved Organic Matter) which can be oxidized in 3 days at 20C. It is expressed as mg O2 / l. BOD is the good index of organic pollution useful in determining water use patterns. Procedure 1. 2. 3. 4. Fill 6 BOD bottles with samples Winklarise 3 bottles immediately to determine the dissolved oxygen Incubate the remaining 3 bottles in BOD incubator at 27C for 3 days. Determine dissolved oxygen in incubated bottles and subtract the mean value from the initial DO.

Calculation BOD 3 mg - 1 = D1 - D2 Where D1 = Initial DO in the sample D2 = DO after 3 days

Sediment analysis: pH: Weighed 20g of soil and transfered to 100ml glass beaker. Add 40ml of distilled water. Stirr well with glass rod and allow it to stand for 30 minutes with immediates stirring. Adjust the pH meter with the buffer solution, wash the electrode with distilled water and carefully wipe dry with a piece of filter paper. Immense the electrodes in the beaker containing soil water suspension (the suspension must be stirred well just before the electrode are immensed) and change the function. Switch to the particular pH range (0 - 7 or 7 - 14). The meter reading will directly indicate the pH value of the sample. Electrical conductivity: 20gm of the soil samples were weighted in an analytical balance and transfer it to a clean 100ml glass beaker. 40ml of distilled water is added to the sample. It is distilled continuously for 5 minutes and allowed to stand for 30 minutes, with constant stirring using a glass rod. After 30 minutes the sample is read in an prestabilished, precalibrated, checked ELICO conductivity fridge (EC metre). The electrical conductivity of the soil sample solution is read and displayed by the electronic display available in the meter. The result is in the 2 unit of mMhos/ cm . Nitrogen: Weight 20gm of soil in a I litre round bottom distillation flask (Preferably long necked). Add 20ml of distilled water followed by 100ml each of 0.32% KMnO4 and 2.5% NaOH. The frothing during boiling is prevented by adding 1ml liquid paraffin (or paraffin was peals 1gm) and bumbing is prevented by adding few glass beads. Distil the contents at steady state and collect the liberated ammonia in a 250ml conical flask or beaker containing 20ml boric acid with double indicator. Collect 30ml of distillate, with the absorption of ammonia the colour turns to green. After cooling it is titrated against N / 50 H 2SO4 to the original shade (Bluish purple). Calculation 1000ml of 1N H2SO4 1ml of 1N H2SO4 1ml of 0.1N H2SO4 1ml of 0.02N H2SO4 Hence Nitrogen Kg / ac = = 14gm of N. (eq.wt.of N = 14) 0.014gm of N 0.0014gm of N 0.00028 gm of N T.V. x 0.0028 x 1000 x 10 6 1000 20 N.T. x 14

N.B. This factor is applicable only when 20gm of soil is taken and N/50 acid is used. Estimation of available potash: Take 5gms of sample in a 100ml conical flask and add 25ml of neutral normal ammonium acetate. Shake the contents in a horizontal shaker for 5 minutes in medium and filter the entire solution through Whatman No. 1 filter paper into a 50ml (Plastic) beaker. Switch on the flame photometer and light the burner. Set up the flame photometer by atomising the 0 to 100ppm potassium solution, alternatively to 0 to 100 meter readings respectively till the instrument is steady. Feed the sample extract and note the meter reading. Check the meter with 20,50ppm. Check solution whose meter reading is known. Find the potash content of the sample from the standard curve drawn. Calculation ppm in solution corresponding to curve reading x Dilution factor 5 = Kg / ac of K Phytoplankton collection: The collections were made early in the morning by using a standard plankton net (No.25) with 30cm mouth diameter and length of 1m. 100 litre of surface water was filtered and the filtrate was put into a clean labelled plastic containers. The volume of the concentrate was adjusted to 25ml and it was preserved immediately with 4% formalin for further analysis. Counting From the collected and concentrated filtrate 1ml of the sample was taken, after shaking the concentrate inorder to get an even distribution of planktonic organisms for analysis. The analysis was repeated for 10 times and computed. The average number is expressed in per cubic / meter. Identification The collected microalgae were identified by using standard literatures (Desikachary, 1959; Prescott, 1978; Anand, 1998). An artificial key was prepared after observing the important character of all forms collected and their systematic position is given below following Fritsch (1935) classification. Species Diversity Shannon - Weaver Diversity index (Shannon and Weiner, 1949) was employed to determine species diversity index ( H ). K H = - Pi log Pi i=1 Where, Pi = Proportion of the observation of the found in the category K = Number of categories Dominance Index (DI) Dominance index (DI) of phytoplankton was calculated according to Odum (1971).

C = Where, Ni N = =

ni N

Importance value for each species (number of individual) biomass production and so forth. Total of important values

Species Richness Species richness index was calculated using the following formula (Gleason, 1922) SRI = S-1 ln N Where, S In

-Number of species - Natural logerithm of total number (H) of the individuals

BACTERIOLOGICAL PARAMETERS: I.Total coliforms (most probable number) MPN test is conducted in three steps. A.Presumptive coliform Test B.Confirmed coliform Test C.Complete coliform Test A.Presumptive coliform Test Presumptive coliform test is used to detect and estmate coliform population in water sample. The test is known as presumptive because of the development of a positive result in Mac Conkey both inoculated with water sample may vary occasionaly due to the coliform organisms. Procedure 1.Water samples collected as described was brought to the lab immediatly. 2.Three single strengh lactose tubes were labelled 0.1 ml another three as 1ml. Three double strength tubes were taken and labelled as 10ml. 3.Each 10ml tube was inoculated with 10ml samples aseptically using a sterile 10ml pipette. 4.Each 0.1ml and 1ml tubes were inaculated with 0.1ml and 1ml samples aseptically using respecvtive sterial pipeetes. 5.In to all the test tubes Durham`s tubes were added for checking gas production.

6.The inoculated tubes were inculated at 37o C for 24-48 hours. Positive result of the production of acid and gas after 24 hours of inoculation that indicates a positive presumptive test for coliform bacteria. The tubes showing positive presumptive test were retained and used for confirmatory test. The count of positive tubes was taken and compared with the MPN index to determine the most probable number of coliforms present in water sample.agar plate to perform gram staining. If there is production of acid and gas there gram negetive bacteria rode shaped bacteria, the presence of E.Coli in the sample is confirmed as complete. II. FAECAL COLIFORM (FC) : Faecal coliform are those among total colifirms that prefer to grow at high temperature. Growth at high temperature is indicative of their origin in the intestine of warm blooded animals. This temperature barrier is here specially made use of as an enumeration tool the faecal coliforms were estmated using M-FC Agar at an incubation temperature at 44.5o C for a period 24 hours. M - FC Agar INGREDIENTS(Gram/litre) Tryptose-10; peptone-5.0 ;Yeast extract-3.0;Sodium Chloride-5.0; Lactose-0.2; Procedure 22 grams of the dehydrated medium (M/S Hi-Media lab. Ltd.,Bombay,India.) was suspended in 990 ml distilled water, heated to dissolve completely, 10 ml of rosolic acid was added. Heated for one minute . Cooled to < 45 o C and 12-15 ml of the cooled medium was poured over sterile petriplates.Colonies having blue colour was counted as faecal coliforms. III. FAECAL STREPTOCOCCI(FS): Faecal streptococci were enumerated on M-Enterococcus Agar medium at incubation temperature of 35o C for 24-48 hours.The FS colonies were identified as Maroon colour. M-Enterococcus agar Ingredients (grams/ Litre) Casein enzyme hydrolysate - 15.0; Papaic digest of soyameal -5.0; Yeast extract -5.0; Dextrose -2.0; Dipotassium phosphate -4.0; Sodium azide -0.4; 2,3,5 tri phenyl tetrazolium chloride -0.1; Agar -15;

final pH -7.2+0.2 41.5 grams of the dehydrated medium (M/ s Hi- Media Lab . Ltd., Bombay, India.) was suspended in 1000 ml of distilled water and boiled to dissolve completly. Cooled to < 45o C, 0.5ml of Tween 80 and 2.0ml of sodium carbonate was added, and 12-15 ml of the cooled medium was poured over sterile petriplates. IV) Salmonella like Organisms(SLO): Salmonella like organisms were screened using XLD (Xylose Lysine Deoxycholate)Agar medium for 24-48 hrs at 37o C and identified as red colonies with black centre. V) Shigella like Organisms (SHLO): Shigella like organisms was screened using XLD (Xylose Lysine Deoxycholate)Agar medium for 24-48 hrs at 37o C and the red colonies were identified as SHLO.
VI)

Proteus and Klebsiella like organisms(PKLO):

Proteus and Klebsiella like organisms were screened using XLD (Xylose Lysine Deoxycholate)Agar medium for 24-48 hrs at 37o C and the yellow colonies were identified as (PKLO) XLD (Xylose Lysine Deoxycholate) Ingredients (Gram/ Litre) Yeast extract-3.0; L-Lysine-5.0; Sucrose-7.5; Xylose -3.5; Sodium chloride -5.0; Sodium Deoxychlolate -2.5; Sodium thiosulphate -6.8; Ferric ammonium citrate -0.8 Phenol red -0.08; Agar -15; Final pH 7+0.2 56.68 grams of the dehydrated medium was suspended in 1000ml distilled water and boiled to dissolve completly. Cooled to <45o C and 12-15 ml of the cooled medium was poured over sterile petriplates. Vibrio cholera like organisms (VCLO) Vibrio cholera like organism was screened using TCBS (Thiosulphate citrate Bile Salt Sucrose Agar) medium at 37o C for 24 hours.VCLO looks yellowish and were counted.