Materials and Methods

Transcription

1 3.1 SAMPLING METHOD The study was carried out for 13 different lakes of Urban Ahmedabad. Among all these lake some of the lakes were from eastern Ahmedabad and some were from western Ahmedabad. Therefore proper sampling method was necessary to get accurate result. During the study of all the 13 lakes following points were kept in mind SELECTION OF SAMPLING SITES In the present study the sampling was done during morning hour. The water samples were collected from five different points of lake. Among these (1) The inlet point points where the feeder opens in the lake, (2) Center point - the point which represent the general water quality of the lake and (3) The outlets point - the place where the overflows occurs, whereas remaining two point were selected randomly COLLECTION OF SAMPLES The water samples were collected in the polyethylene bottles or in a glass bottle. Initially the prewashed bottles were rinsed with the sample water. The closed bottle was dipped in the lake at the depth of 0.5 to 0.7 m, and then a bottle was opened inside and was closed again to bring it out at the surface. The samples were collected from five different points and were mixed together to prepare an integrated sample SAMPLE HANDLING AND PRESERVATION From the time of sample collection to the time of actually analyses, many physical, chemical and biochemical reactions would change the quality of the water sample, therefore to minimize this change the sample were preserved soon after the collection. The water samples were preserved by adding chemical preservatives, lowering the temperature or by the combination of both the method. The water temperature, ph, dissolved oxygen, electrical conductivity and total dissolve solids Page 52

2 were analyzed immediately at a time of collection, whereas the analyses of remaining parameters were done in the laboratory. The study was carried for a period of 1 year. (March 2009 to February 2010). The collected water samples were brought to the laboratory and relevant analysis was performed. ph was determined electrometrically using digital ph meter, electrical conductivity was measured by conductivity meter, dissolved oxygen is measured by DO meter, total dissolve solid was measured by using TDS meter and similarly turbidity is measured by Nepthalo turbidity meter. Alkalinity, chloride, TDS, calcium, magnesium, total hardness, nitrate and phosphate were determined by method suggested by APHA (1985); Kumar and Ravindranath (1998); Trivedy and Goel (1984). Estimation of sodium was done by Flame Photometric method. 3.2 PHYSICAL AND CHEMICAL METHOD Physico chemical analysis of water means analysis of water for physical and chemical parameters. The methods used here to check the status of the lakes are as follows METHODS FOR PHYSICAL PARAMETERS Temperature Measurement The temperature of water was measured by using centigradee thermometer Procedure Bulb of thermometer was immersed in the water About 6" below the surface water It was kept steady for a period of sufficient time Temperature was noted Page 53

3 Electricall Conductivity Materials and Methods EC was determined by using Conductivity meter, Model: EQ 660. The unit of conductivity measurement is m.mho/cm. Principle Water usually consists of various types of ions, these ions help in passing the electric current through water. Hence, conductivity may be definedd as the measure of ability of aqueous medium to carry on electric current. Conductivity is totally dependent upon concentration of ions. Conductivity is generally measured by conductivity meter which consists of conductance cells having electrode of platinum, these electrode are placed parallel at a fixed distance. As the ionizations of solutes are totally dependent on temperature, therefore all the result is measured at 25 0 C. Procedure Conductivity cell was rinsed by using 0.01 M KCL solution Temperaturee was adjusted to 25 0 C Then cell constant was computed. Conductivity cell was then rinsed with sample Temperature was then adusted to 25 0 C Reading was noted in mho/cm Page 54

4 Method of calibration 1. Approx. method (accuracy 2% to 3%) for cell const. K = 1 only. The following method was proceed The instrument was allowed to warm up for 15 minutes. The range switch was thrown in 2mM position (indicated by arrow). Bring in standard conductance of 1.00mM by throwing the switch down in EQ-660. Now with the help of screwdriver turn the standardize shaft was turned till the Digital display reads Throw the standard cond. Switch up The instrument is now ready for use. 2. Accurate Method (0.5% to 1% accuracy) KCl Sol was prepared and was allowed to attain the room/ desired temperature. The solution so prepared is temperature dependant The cell K = 1 was dipped in the solution The range switches in appropriate position so that full range of display is read. The std. cond. was checked and the switch is in upward position. The standardize shaft was turned with screwdriver till the display reads the correct conductance of the solution. The temperature effect has to be considered. The instrument thus calibrated also accounts for deviation in cell constant to the extent of 10% K = 0.5 can also be used in this method. Page 55

5 Turbidity Turbidity of water sample was measured by using Digital Nephlo Turbidity meter, Model: 132 (Systronics) Principle In this method, when light is passed through a turbid sample, the particles present in sample scatter some of the light and the light scattered is directly proportional to the turbidity i.e. higher the turbidity, higher will be the light scattered, the amount of light scattered by the sample is compared with the intensity of light scattered by a standard suspension. Turbidity can be determined for any kind of water sample, but it should be debris and rapidly settable particles. Apparatus and reagent preparation (a) Nepthelometer (b) Sample tubes: Should be free from any scratch (c) Stock turbidity suspension I. 1.0 gm of hydrazine sulphate (NH 2 ) 2, H 2 SO 4 was dissolve in distilled water to prepare 100 ml of solution using volumetric flask II gm hexamethyline tetramine (CH 2 ) 6 N 4, was dissolve in distilled water to prepare 100 ml of solution in volumetric flask III. 5 ml of solution (I) and 5 ml of solution (II) were mixed and was allow to stand for one day at 25 0 C, then it was diluted to 100 ml. Therefore standard suspension of 400 NTU (Nephelometric turbidity unit) is prepared. This solution is stable up to one year. (d) Standard turbidity suspension: 40 NTU solutions were prepared by diluting 10 ml of stock solution to 100 ml. Page 56

6 Procedure Instrument was set to 100 by using standard suspension of 40 NTU. Sample was added in Sample tube Outer surface of the sample tube was cleaned by using tissue paper. Sample tube was then put inside the nepthelometer Reading was noted in NTU. Calculation Turbidity (NTU) = Nephelometer reading x 0.4 x dilution factor Total Dissolved Solid Total dissolvedd solid s are the solids present in water in the dissolved state and is determined as the residue left after evaporation of filtered water at C. Total suspended solids are the solids present in a suspended state. Page 57

7 Procedure After calibrating the instrument. Water Sample was collected in the Glass beaker. TDS meter was then immeresed into this water sample. It was allowed to stand until it achieves stable reading. Reading was noted in ppm. Calibration Method Preparation of solution having 1382 ppm TDS: The 20 ml sachet of HI70032 is dissolved in 1000 ml of distilled water to prepare solution having TDS value 1382 ppm. The TDS meter was immersed in the solution. Then it was allowed to stand until it achieves stable reading. Then with the help of small screw driver the calibration trimmer was adjusted, till the display shows the reading of 1382 ppm. Now the instrument is ready for use. Page 58

8 3.2.2 METHODS FOR CHEMICAL PARAMETERS ph Measurement The ph of the sample was determined by digital ph meter Model: 511 (Testronics). Instrument Calibration The combination electrode was cleaned with distilled water observing proper safety precautions The combination electrode was connected to the meter The temperature pot was kept on the temperature of the solution under test and switch (mv/ph) on mv position. The unit was switched on and was checked weather the led glows. The electrode was dipped in the reference standard solution of 7 ph (pure water) The STAD pot was set and was adjusted so that digital indicator show zero. Now mv/ph switch position was changed to ph, the digital indicator will read 7 ph. The electrode was cleaned with distilled water. The electrode was kept in 4 ph (reference standard solution) solutions. The digital indicator read 4 ph. If not the slope and pot was adjusted so that the digital indicators read 4 ph. Corresponding mv readout was checked for reference. Now the digital ph meter is ready to measure any solution Between 0-14 ph at any temperature between 0 to 100 o C Page 59

9 The electrode was cleaned with distilled water. The temperature adjust pot was kept to the temperature of the solution under test. And no change was made STAD adjust pot and slope adjusts pot. Procedure ph meter was calibrated by following manufacturer manual. Electrode was made dry by using tissue paper. Half of the electrode was then dipped into sample water. ph was noted Total Alkalinity Total alkalinity is the measure of the capacity of the water to neutralize a strong acid. The alkalinity in the water is generally imparted by the salt of the carbonates, bicarbonates, phosphates, nitrates, borates, silicates, etc. together with the hydroxyl ions in the Free State. However, most of the ware is rich in carbonates and bicarbonates with little concentration of other alkalinity imparting ions Total alkalinity, carbonates and bicarbonates can be estimated by titrating the sample with a strong acid (HCl or H 2 2SO 4 ), first to ph 8.3 using phenolphthalein as an indicator and then further to ph between 4.2 and 5.4 with methyl orange or mixed indicator. In first case, the value is called as phenolphthalein alkalinity (PA) and in second case; it is Page 60

10 total alkalinity (TA). Values of carbonates, bicarbonates and hydroxyl ion can be computed from these two types of alkalinities. Reagents Hydrochloric acid, 0.1 N: 12N concentrated HCl was diluted to 12 times ( ml) to prepare 1.0N HCl. It was further diluted to make 0.1N HCl (100 to 1000ml). It was Standardize against sodium carbonate solution. Methyl orange indicator, 0.05%: 0.5 g of methyl orange was dissolved in 100ml of distilled water. Phenolphthalein indicator: 0.5g of phenolphthalein was dissolved in 50ml of 95% ethanol and 50ml of distilled water was added. 0.05N CO 2 free NaOH solution was added drop wise, until the solution turns fainty pink. Sodium carbonate, 0.1N 5.300g of Na 2 CO 3, previously dried at 250 C for about 4 Hours was dissolved in distilled water to prepare 1Liter of solution. Calculation for alkalinity PA as CaCO 3, mg/l = (A X Normality) of HCl X 1000 X 50 ml of sample TA as CaCO 3, mg/l = (B x Normality) of HCl x 1000 x 50 ml of sample Page 61

11 Procedure for Alkalinity 100ml of sample was taken in a conical flask 2 drops of phenolphthalein indicator. was added The solution remain colourless Colour changes to pink Phenophthalein Alkalinity = 0 Titrated with 0.1N HCl 2-3 drops of methyl orange was added Colour disappears at end point Titrated with 0.1N HCl The reading was noted as A Colour changes from yellow to pink at end point The reading was noted as B Page 62

12 Where A = ml of HCl used with only phenopthalein B = ml of total HCl used with phenopthalein and methyl orange, PA = phenopthalein alkalinity TA = total alkalinity TOTAL HARDNESS Ethylene diamine tetra acetic acid (EDTA) and its sodium salt form a chelated soluble complex when added to a solution of certain metal cations. Additional of small amount of a dye such as Erichrome Black T to an aqueous solution of calcium and magnesium ions of ph 10.0, turns the solution wine red. When EDTA is added as titrant, the calcium and magnesium get complexed and the colour of the solution turns from wine red to blue, marking the end point of the titration. This on calculating using the formula gives the total hardness. Metal + Indicator Metal-Indicator (complex). Metal-Indicator + EDTA Metal-EDTA (Complex) + Indicator. Reagent Preparation Buffer Solution: 16.9 gm of ammonium chloride was dissolved in 143ml ammonium hydroxide, 1.25 gm of magnesium EDTA was added and diluted to 250 ml by using distill water. Inhibitor: 4.5 gm of hydroxyl amine hydrochloride was dissolved in 100 ml of 95% ethanol or isoprophyl alchol. Eriochrome Black T indicator: 0.5 gm of the dye was mixed with 100 gm of sodium chloride, to prepare a dry powder. Murexide Indicator: 200 mg of murexide and 100 gm of solid sodium chloride were grinded and mixed. 2 N Sodium hydroxide: 80 gm of sodium hydroxide was dissolved in distilled water and was diluted to 1000 ml. Page 63

13 0.01 M Standard EDTA solution: gm of EDTA sodium salt was dissolved and was diluted to 1000 ml in a volumetric flask. Procedure 50 ml of water sample 2 ml of buffer solution. 1 ml of inhibitor. Pinch of Erichrome Black T as indicator. Titrated against Standard EDTA (0.01 M) solution. Colour changes from wine red to blue. Volume of EDTA Used was Noted as A ml. Calculation Total Hardness mg/l as CaCO 3 = A X 1000 Volume of sample taken (in ml) Page 64

14 Calcium When the ph is adjusted to 12 or 13 by addition of sodium hydroxide, magnesium is precipiated as hydroxide. The Murexide indicator gives a colour change from pink to purple when all the calcium has been complexed. Reagent preparation (Same as in Total Hardness) Procedure 50 ml of water sample. 1 ml of 2 N NaOH. Pinch of murexide indicator Titrated against EDTA. Colour changes from pink to purple. Note down the volume of EDTA required (A ml). Calculation Calcium hardness = mg/l as CaCO 3 A x 1000 ml Volume of water sample taken in ml Calcium mg/l = A x Volume of water sample taken in ml Page 65

15 Magnesium Like calcium magnesium is also found in all natural waters and its sources are from rocks. Its generally occurs in concentrations lower than those of calcium. Magnesium is a necessary constituent of chlorophyll. Its high content reduces the utility of water for domestic use. A concentration above 500 mg/l imparts an unpleasant taste and renders the water unfit for drinking purposes. Procedure Total hardness and calcium hardness of water were determined by following the above method. From these values the magnesium content is calculated as follows Calculation Magnesium mg/l = (T C) x Where T = Total hardness (mg/l, as CaCO 3 ) C = Calcium hardness (mg/l, as CaCO 3 ) Dissolved Oxygen Dissolved Oxygen was analyzed by using Dissolved Oxygen meter, Model: VSI 14N (VSI Electronics). Principle The measurement of DO is based on Volta metric method. The diffusion of oxygen through membrane produces current which is proportional to concentration of dissolved oxygen. Calibration The main lead was connected to 230V 10% mains supply. Page 66

16 Procedure A 200 ma fused was provided at the rear panel for the safety of a circuit. The display was adjusted to zero with ZERO knob. The DO probe was connected in the DO sockets provide in the rear side of the instrument. The probe was put in 1-2% Sodium Sulphite Na 2 S0 3 solution and allows about 2 minutes to attain equilibrium. The instrument read zero or up to 0.2ppm. The probe is good enough. If it does not become zero, it was adjusted to zero by using ZERO knob. The DO probe was dipped in double distilled water after washing it thoroughly with distilled water. Referring to the value of DO in distilled and with the help of CALIB knob. The reading was adjusted to the proper value when the sample is being stirred at continues speed. Now the instrument was calibrated. After completion of calibration. Water sample was taken in a BOD bottle. The temperature of water sample was noted. Now DO proble was dipped in the sample water stirred by magnetic stirrer. The display showing reading was noted. This was the amount of DO recorded. Page 67

17 Biochemical Oxygen Demand (BOD) Principle Biochemical Oxygen Demand (BOD) is the measure of degradable organic material present in the water sample, and can be defined as the amount of oxygen required by the microorganisms in stabilizing the biological degradable organic matter under aerobic conditions. The principle of the method involves, measuring the difference of the oxygen concentration between the sample and after incubating it for 5 days at 20 C Reagents Preparation BOD-free water: Double distill water was used BOD Incubators: Having a temperature control at 20 0 C Phosphate buffer: 8.5 gm KH 2 PO 4, gm K 2 HPO 4, 33.4 gm Na 2 HPO 4.7H 2 O and 1.7gm NH 4 Cl was dissolved in distilled water to prepare 1 liter of solution. ph was adjusted to 7.2. Magnesium Sulphate: 22.5 gm MgSO 4.7H 2 O was dissolved in distilled water to prepare 1 liter of solution Calcium Chloride: 27.5 g of anhydrous CaCl 2 was dissolved in distilled water to prepare 1 liter of solution. Ferric chloride: 0.25 g FeCl 3 6H 2 O distilled water was dissolved to prepare 1 liter of solution. Allylthiourea solution: 100 mg of allylthiourea was dissolved in distill water and was diluted to 100 ml. Sodium Sulphite Solution, N: g Na 2 SO 4 was dissolved and was diluted to 1000 ml. Solution was freshly prepared. Preparation of dilution water: Dilution water was used to dilute water sample, containing large amount of organic matter and do not contain dissolved oxygen. Initially the BOD free double distilled water was aerated for about half an hour using an aerator. Then 1 ml of phosphate buffer, magnesium Page 68

18 sulphate, calcium chloride and ferric chloride solution was added to 1000 ml aerated distilled water This is dilution water. Preparation of Percentage Dilution of Polluted Water Dilution mixture Percentage Sample Water sample Dilution water Total dilution ml ml ml ml 500 ml 1000 ml Polluted ml 750 ml 1000 ml waters ml 800 ml 1000 ml ml 900 ml 1000 ml Procedure Water sample was diluted by dilution water if required. The ph of water sample was adjusted to 7.0 by using acid or base. Two BOD bottle were filled by this sample water. 1 ml of allylthiourea solution was added to each bottle. Dissolved oxygen of one bottle was analysed. It was noted as D1. Second bottle was kept in BOD incubator at 20 0 C for 5 days. Other two BOD bottles were filled with dilution water. Dissolved oxygen of one bottle of dilution water was noted immediately. Second bootle was also incubated for 5 days at 20 0 C. Dissolved oxygen for sample water was noted as D3. Dissolved oxygen for dilution water was also noted. Page 69

19 Calculation BOD (mg/l) = (D1 D3 BC) x 100 Percentage dilution of sample Where D1 = Initial dissolved oxygen in sample (mg/l). D3 = Dissolved oxygen left out in the sample after 5 days of incubation (mg/l). BC (Blank Correction) = Difference between the DO content of the blank on initial day and after 5 th day of incubation Chloride Principle Silver nitrate reacts with chloride to form very slightly soluble white precipitate of AgCl. At the end point when all the chlorides get precipitated, free silver ion reacts with chromate of reddish brown colour. Reagent preparation Potassium chromate indicator: 25 gm of potassium chromate was dissolved in 100 ml distilled water. Then silver nitrate solution was added to it until a definite red precipitated get formed. Then it was kept for 12 hours. After that it was diluted to 500 ml by using distill water N Silver nitrate solution: gm of silver nitrate was dissolved in distilled water and was diluted to 1000 ml. Page 70

20 n Sodium chloride: mg sodium chloride was dissolved in distilled water and was diluted to 100 ml. 1.0 ml of this solution contain 1.0 mg of chloride. Procedure 100 ml of water sample was taken. ph was adjusted between 7.0 and 8.0. Add 2 ml K 2 CrO 4 as indicator Titrated against silver nitrate. Brick red precipitate formed. Volume of silver nitrate used was noted as A (ml). Calculation (A B) x N x x 1000 Chloride mg/l = Volume of sample (ml) Where A= ml of AgNO 3 required for titration N= Normality of AgNO 3 used = Equivalent weight of chloride and the factor of 1000 is for conversion to one liter Page 71

21 Sodium Flame Photometric Method Principle Sodium can be determined by using flame photometer at 589nm. When sample is produced in flame it comes to excitation condition and produce characteristic light. The light intensity at 589nm is proportional to concentration of sodium which can be read by using light dispersion devices. Reagents Stock sodium solution (100 mg/l Na): g NaCl dried at C was dissolved in distilled water to make one liter of a solution (1 ml = 1.00 mg Na). Intermediate sodium solution: 10 ml of stock solution was dissolved in distilled water and make volume to 100 ml. (1 ml. = 0.10 mg Na) Nitric acid: HNO 3 concentrated. Hydrochloric acid: HCl concentrated. Hydrogen peroxide: H 2 O 2, 30% Ammonium hydroxide: NH 4 OH concentrated. Preparation of calibration curve 1. The following procedure was used for pretreatment of the sample The sample of suitable size in a 250 ml conical flask was taken and was acidify it with nitric acid. It was evaporated to dryness on a water bath. Again 25ml of concentrated HNO 3 was added to boil until the acid was evaporated to small volume. The presence of brown fumes indicates the un oxidized organic matter. Now some more conc. HNO 3 and small quantities of H 2 O 2 was added for complete ashing of organic matter. The final residue is colourless on drying. The presence of more iron and copper salts may impart a colour to it. The residue in small amount of HCl and distilled water was warmed. The Page 72

22 content was filtered and was neutralize with conc. NH 4 OH. It was diluted to suitable volume. 2. For non polluted samples or the sample where only dissolved sodium is to be estimated, the sample was through a filter paper to remove any suspended matter which otherwise may clog the capillary of the instrument. 3. Calibration curve was prepared in the range of 0 1, 0 10 or mg/l, by using standard sodium solution. Procedure After calibrating the instrument. Suitable amount of sample was filtered to remove suspended matter. By using flame photometer concentration of sodium was recorded. Calibration curve was prepared by following the instruction provided by manufacturer Sodium was determined by using calibration curve. Calculation Sodium mg/l = (mg Na/l in sample) x dilution ratio Page 73

23 Nitrates Principal Nitrate when reacts with brucine produces yellow colour is presence of H 2 SO 4 this can be measured spectrophotometrically at 410nm. Apparatus Spectrophotometer, nessler tubes, test tubes, 100 ml beakers, water bath measuring cylinder, physical balance and pipettes. Preparation of Reagents Nitrate Stock solution : 722 mg anhydrous potassium nitrate was dissolved in 100 ml distilled water and final volume was made 100 ml in a volumetric flask 1 ml of this solution will contain 0.1 mg nitrate nitrogen Standard nitrate solution: 100 ml. nitrate stock solution was prepared in a 1000ml volumetric flask and the level was made up to 1000 ml. 1 ml of this solution will contain 0.01 mg nitrate nitrogen. Brucine-sulfanilic acid solution: 1 gm of brucine sulphate and 100 mg. sulfanilic acid was dissolved in 70 ml. hot distilled water. 3 ml. concentrated HCl was added to this solution. The solution was cooled and was dilute to 100 ml with distilled water. This solution was stable for several months. Sulphuric acid solution: 500 ml of conc. sulphuric acid was added to 75 ml distilled water carefully. Then it is cooled at room temperature. Toxic, avoid ingestion Sodium Arsenite solution: 1.83 gm of sodium arsenite (NaAsO 2 ) was dissolved in 100 ml distilled water. Sodium Chloride solution: 300 gm NaCl was dissolved in distilled water and was diluted to 1000 ml. Page 74

24 Procedure 2ml of sample was taken in 50 ml nessler tube. 1 ml of Brucine sulfanilic acid solution was added and was mixed nicely. 10 ml of sulphuric acid was added and was mixed nicely. Then it was kept in dark for 10 minutes 10 ml of distill water was added. Then it was kept in dark for 20 to 30 minute Absorbance was measured at 410 nm. By using standard curve concentration of nitrate nitrogen in sample was calculated. Preparation of Standard Curve Into a series of 50 ml. nessler tubes, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 ml of nitrate standard solution was pipetted.the volume was made to 5 ml in each tube by adding the appropriate volume of distilled water. The standards were labeled. A beaker containing 5 ml of distilled water was taken as a blank. Page 75

25 1 ml of brucine sulfanilic acid solution was added to the blank and standard, then they were mixed nicely. 10 ml of sulphuric acid was carefully added to each nessler tube and was nicely mixed..the nessler tubes were kept in the dark for 10 minutes. 10 ml of distilled water was added to each of the standards and blank. Then all the nessler tubes were kept in the dark for 20 to 30 minutes. The blank was used to set the spectrophotometer or colorimeter at 100 % transmittance at a wave length of 410 nm. The absorbances of the standards were measured. After that standard curve was prepared to find out the concentration of nitrate nitrogen in sample. Calculation mg/l nitrate nitrogen = mg nitrate nitrogen x 1000 Volume of sample taken for estimation (in ml) Nitrate in mg/l = mg/l nitrate nitrogen x TOTAL PHOSPHATE Principal Organic phosphorus is converted to orthophosphates by heating or by persulphate digestion while inorganic phosphates are converted to orthophosphates by H 2 SO 4 digestion. The phosphates thus released can be determined calorometrically Page 76

26 Chemical Potassium dihydrogen phosphate (KH 2 PO 4 ), ammonium molybdate, concentrated sulphuric acid, stannous chloride (SnCl 2.2H 2 O), glycerol, concentrated nitric acid and distilled water, phenolphthalein, Sodium hydroxide and phenolphthalein indicator Apparatus Spectrophotometer or colorimeter, volumetric flasks, beakers, test tubes, nessler tubes, glass rod, measuring cylinder, physical balance, kjeldahl flask, burette, bunsen burner, tripod stand and wire gauze. Preparation of reagents Stock phosphate solution: 439 mg of potassium hydrogen phosphate was dissolved in distilled water and the volume was made to 1000ml. 1 ml = 100 microgram of phosphate or 0.1 mg of phosphate. Phosphate working solution: 10 ml stock solution was diluted to 1000 ml using distilled water. This solution was freshly prepared. 1 ml = 1 microgram of phosphate or mg of phosphate. Ammonium molybdate solution: 25 gm of ammonium molybdatewas dissolved in about 200 ml distilled water. 280 ml of concentrated sulphuric acid was added to 400 ml distilled water. To the dilute acid, molybdate solution was added to make final volume 1000 ml. Strong acid reagent: Carefully add 75 ml concentrated sulphuric acid to 150 ml distilled water. 2 ml of concentrated nitric acid, was added, allowed to cool and 250 ml was diluted with distilled water. Stannous chloride: 2.5 gm of stannous chloride was dissolved in 100 ml of glycerol. This was heated on a water bath to insure complete dissolution. It was mixed well by stirring with a glass rod. 1 N sodium hydroxide: 40 gm of sodium hydroxide pellets was dissolved in about 200 ml of distilled water. And total volume was made to 1000 ml in a volumetric flask. Page 77

27 Phenolphthalein: It was prepared as it was prepared in the section of the alkalinity measurement. Procedure 100 ml of the sample was taken in a kjeldahl flask. 1 ml of conc. sulphuric acid and 5 ml of conc. nitric acid was added. The sample was heated until the solution become colourless. It was Cooled. 20 ml of distilled water and 2 drops of phenolphthalein indicator was added. Titrated against sodium hydroxide until the appearance of pale pink colour. The solution was transfer to the 100 ml of volumetric flask and was dilute up to the mark. 4 ml of ammonium molybdate solution was added and was mixed well. 0.5 ml of stannous chloride was added. After 10 to 12 minutes colour developed. OD was measured at 690nm using spectrophotometer. Comparing the value with standard curve. phosphorus content can be found Preparation of Standard Curve 1. Appropriate amounts of phosphate working solution were pippeted to cover the range of 0.3 to 1.5 mg/l into the series of 100 ml nessler tubes. These tubes serve as standards. A nessler tube containing 100 ml distilled water was included as the blank. Page 78

28 2. To the standards and blank 4 ml of ammonium molybdate solution was added and was mixed well ml of stannous chloride was added to all the tubes and was mixed well 4. It takes minutes for the development of colour. 5. The spectrophotometer or colorimeter was calibrated using the blank solution and distilled water. 6. The intensity of blue coloured complex at 690 nm using a spectrophotometer was measured. 7. A standard curve was prepared by plotting the phosphate concentration of the standard solution on the x axis and the optical density on the y axis. 8. The phosphorous content of the sample was found by matching its absorbance (S) with the standard curve. 9. The result was expressed as mg phosphate as phosphorous. If it has to be expressed in term of Phosphates multiply by a factor of Calculation Phosphate mg/l = phosphorus mg/l x Page 79

29 3.3 BOTANICAL METHOD The physical and chemical characteristics of water affect the abundance, species composition, stability and productivity of the indigenous populations of aquatic organisms. The botanical methods used for assessing water quality include collection, counting and identification of phytoplankton. The work involving phytoplankton analysis would help in: Explaining the cause of colour and turbidity and the presence of objectionable odour, tastes and visible particles in waters. The interpretation of chemical analyses. Identifying the nature, extent and biological effects of pollution Phytoplankton net The phytoplankton net is a field-equipment used to trap phytoplankton. It has a polyethylene filter of a defined mesh size and a graduated measuring jar attached to the other end. A handle holds the net. The mesh size of the net determines the size range of the plankton trapped Sampling Procedure Plankton net number 25 of mesh size 20 μm was used for collecting samples. 100 liters of water was measured in a graduated bucket and filtered through the net and concentrated in a 100 ml bottle. Samples were collected as close to the water surface as possible in the morning hours and preserved for further analysis Labelling The samples were labeled with the date, time of sampling, study area-lake name and the volume measured and pasted on the containers Preservation of the sample Between the time that a sample is collected in the field and until its analysis in the laboratory, physical, chemical and biochemical changes may take place altering the intrinsic quality of the sample. It is therefore necessary to preserve the samples Page 80

30 before shipping, to prevent or minimize changes. This is done by various procedures such as keeping the samples in the dark, adding chemical preservatives, lowering the temperature to retard reactions by freezing or by a combination of these methods. For a phytoplankton sample to be analyzed for an extended period, commonly two preservatives are used: Lugol s iodine using acetic acid which will stain cells brownish yellow and will maintain cell morphology and of 4% formaldehyde. Preparation of Lugol Solution.: 100 gm of KI and 5 gm Iodide crystals were dissolved in 20 ml of distilled water. then 5 gm of anhydrous sodium acetate was dissolved in 50 ml distilled water now both of this were mixed and Lugol solution get prepared. 0.5 ml of lugol should be added to preserve 100 ml of sample Prparation of 4% Formaline: 4 ml of concentrated formalin was diluted to 100 ml with distill water Concentration technique The phytoplankton nets were used to collect samples for the quantitative estimation, by filtering a known volume of water (100 litres) through the net. The sample was allowed to settle for hours and was further concentrated to approximately 30 ml by decanting. The concentration factor is used during the calculations Qualitative and quantitative analysis of phytoplankton Detailed analyses of phytoplanktonic populations are done by estimating the numbers in each species. The phytoplankton consisting of individual cells, filaments and colonies are counted as individual cells. When colonies of species are counted, the average number of cells per colony is counted, and in filamentous algae, the average length of the filament has to be determined. Device used for this analysis is Sedgwick Rafter counting cell. Sedgwick Rafter cell is approximately 50 mm long, 20 mm wide and 1 mm deep. The total volume of the cell is 1 ml. Page 81

31 3.3.7 Mounting the slides Concentrated samples in a bottle are mixed uniformly by gentle inversion. Then by using bore pipette 1 ml of sample was transfer on Sedgwick Rafter count cell. Now it was covered by using cover slip, avoiding any kind of air bubble. Then it was kept for minutes so that all plankton may settle down. Now the Sedgwick rafter counting cell is placed under microscope and then plankton was counted by moving the cell horizontally and vertically. The process was repeated twice Microscope A binocular compound microscope is used in the counting of plankton with different eyepieces such as 10X and 40X. The microscope is calibrated using an ocular micrometer Procedure for Plankton Measurement 100 liter of water was filtered through plankton net. Suitable amount of preservative was added in the collected samples. The sample was allowed to settle for hours. Further concentrated to approximately 30 ml by decanting. 1 ml of concentrated sample was added to sedgwick Rafter counting cell. The cell was counted by moving the cell horizontally and vertically. The observed number of plankton was then applied to the formula. Page 82

32 Formula = n = (a x 1000) c l where, n = Number of plankton / liter of water. a = Average no of plankton in one small counting chamber of S-R cell. c = ml of plankton concentrate. l = Volume of original water filtered in litre Phytoplankton Identification From the concentrated sample, the slides for the plankton were prepared. Then these slides were placed under microscope, and the phytoplanktons were observed in 100 X in binocular microscope. The images of the phytoplankton were captured by using digital camera. Later on the phytoplankton were identified by using the book The Fresh Water Algae by Prescott (1970). The status of the water can be evaluated by performing all the above methodsphysical, chemical and botanical. The result are then compared with the WHO and BIS standard and on the basis of this the quality of water can be judged. And presence of some algal blooms also shows the richness of the nutrient in the water. Page 83

33 3.4 Correlation It is essential to understand the relationship between different water quality parameter when the study is completed Relationship between the parameters (Correlation) Any relationship between the two variable is known as correlation. If one variable increases or decreases with a corresponding increase or decrease of the other variable, a direct positive correlation exists between the two variables. If one variable decrease with an increase in the other variable, then there is a negative or inverse correlation. There are two different methods to study correlation Graphic method It is the simplest method of showing the relationship between two variable. in this one variable is represented on X-axis and other variable on Y-axis on graph paper. Data corresponding to X and Y axis were plotted in form of dots. And then estimated lines joining first and last points was drawn on the graph paper to find out correlation Correlation coefficient The graphic method indicates the existence of a correlation. But it is not possible to calculate the extent or degree of relationship using these graph. This was calculated by using following formula r = (dx. dy) (dx) 2. (dy) 2 Where, r is the correlation coefficient, x and y are the two variable dx is the deviation from the x-mean of the x variable, dy is the deviation from the y mean of the y variable, (dx. dy) is the sum of the products of the deviations, (dx) 2 is the sum of the squares of the deviations of the x variable, (dy) 2 is the sum of the squares of the deviations of the y variable, Page 84

34 The variable in table were written in short form, its full form were as follows TEMP = TEMPERATURE EC = ELCTRICAL CONDUCTIVITY TUR = TURBIDITY TDS = TOTAL DISSOVED SOLIDS ALK = ALKALINITY TH = TOTAL HARDNESS CAL = CALCIUM MAG = MAGNESIUM DO = DISSOLVED OXYGEN BOD = BIOCHEMICAL OXYGEN DEMAND Cl = CHLORIDE Na = SODIUM NO3 = NITRATE PO4 = PHOSPHATE CYN = CYANOPHYCEAE CHL = CHLOROPHYCEAE BAC = BACCILARIOPHYCEAE EUG =EUGLENOPHYCEAE The correlations were done seasonally for 13 different lakes. All the 19 parameters were correlated with each other to check there relationship. Page 85