CHAPTER - 4 MATERIALS AND METHODS

CHAPTER - 4 MATERIALS AND METHODS

CHAPTER - 4 MATERIALS AND METHODS (A) STUDY OF WASTE WATER QUALITY OF MILK PROCESSING UNIT: [1] Collection and preservation of waste water of milk processing unit: Waste water from milk processing unit is discharged through a underground pipe which terminates in to a pit of size 1.44 m deep and 0.49 m diameter. Waste water sample was collected from the surface of the pit, by using a glass bottle of one liter capacity. The bottle was sterilized and then rinsed with waste water before collection. Collections were made at monthly interval between 10:00 am to 12:00 noon, throughout the study period during the year 2008. The collected waste water sample was transported to laboratory from field. Ten liters of waste water sample was used for the analysis of physico chemical and biological parameters for the study of water quality, while 2000 2100 liters of diluted water sample in ratio 1:1 (waste water and tap water) was used for the cultivation of plants in the field during the period of investigation (2007 2009). Measurement of temperature and fixation of dissolved oxygen was done immediately at sampling site, while analyses of other parameters were carried out in preserved sample within forty eight hours of collection. The preservation of waste water sample was done as prescribed in APHA-AWWA-WPCF (1980). [2] Methods for physico-chemical and biological analysis of waste water of milk processing unit: (a) PHYSICAL PARAMETERS: (1) TEMPERATURE: Temperature of waste water was measured in C with a digital temperature recorder. (2) HYDROGEN ION CONCENTRATION (ph): ph is a value of negative log 10 of H + concentration in water sample, it was determined at room temperature by a Systronic digital ph meter. The standardization Materials and Methods 17

of instrument was done with a buffer solution of 7.4 and 9.2 ph. (3) TURBIDITY: Turbidity of waste water was measured in Nephelometric turbidity units (NTU) by Systronics turbidity meter. The standardization of the instrument was done against the reference turbidity suspension of 40 NTU. The reference suspension solution was prepared as per the method prescribed in APHA-AWWA-WPCF (1980). Standard 40 NTU suspensions: Step -1 Solution (A) : l gm of hydrazine sulfate was dissolved in 100 ml distilled water. Step -2 Solution (B) : 10 gm hexamethylene tetramine (Eurotropin) was dissolved in 100 ml distilled water. 5 ml each of the solution (A) and (B) were mixed together in 100 ml volumetric flask and was kept standing for 24 hours. It forms 400 NTU suspensions. Taking 10 ml of 400 NTU suspensions in volumetric flask, volume was made up to 100 ml that forms 40 NTU suspensions. (4) SALINITY: meter. Salinity was measured in parts per million (ppm) by elico digital salinity (5) ELECTRICAL CONDUCTIVITY: AND (6) TOTAL DISSOLVED SOLIDS: Electrical conductivity and total dissolved solids are related to each other because dissolved ionic compounds are responsible for conductance of electric current. Both the parameters were estimated by using elico EC/TDS - analyzer and values were expressed in µ mhos/cm and ppm respectively. Materials and Methods 18

(b) CHEMICAL PARAMETERS: (7) ALKALINITY: The alkalinity of water sample was determined by titrating the sample with standard solution of strong acid using phenolphthalein and methyl orange as an indicators, expressed as phenolphthalein, methyl orange, total hydroxyl, bicarbonate and carbonate alkalinities, as prescribed in APHA-AWWA-WPCF (1980). (i) Phenolphthalein alkalinity (PA): PA alkalinity (mg CaCO 3 /L) was determined by Titrimetric method as prescribed in APHA-AWWA-WPCF (1980). PA alkalinity expresses value for bicarbonate alkalinity in mg CaCO 3 /L at ph 8.3. M + H 2 CO 3 + H 2 O = M + + H 2 CO 3 + OH (a) Phenolphthalein indicator solution: 1 gm phenolphthalein indicator was dissolved in 100 ml 95% ethyl alcohol and diluted to 200 ml with distilled water. (b) Standard hydrochloric acid solution: 2.0 ml conc. HCl was dissolved in 200 ml of distilled water and normality of solution was calculated by acid base titration. (c) Standard sodium carbonate solution (0.045 N): 2.5 gm dried sodium carbonate was dissolved in distilled water and volume was made to 1L. 200 ml water sample was titrated against the standard HCl solution in the presence of phenolphthalein indicator. End point was noted at the point of disappearance of pink colour and the value was computed in the formula:- Phenolphthalein Alkalinity mg CaCO 3 /L = Where:- A = ml titrant A x B x 50,000 ml sample Materials and Methods 19

B = Normality of titrant (ii) Methyl orange alkalinity (MA): Methyl orange alkalinity (mg CaCO 3 /L) was determined by Titrimetric method as prescribed in APHA-AWWA-WPCF (1980). Methyl orange alkalinity expresses value for bicarbonate alkalinity in mg CaCO 3 /L at ph 4.5. (a) Methyl orange indicator solution: 50 mg Methyl orange powder was dissolved in 100 ml distilled water. (b) Standard Hydrochloric acid solution: 2.0 ml. conc. HCl was dissolved in 200 ml of distilled water and normality of solution was calculated by acid base titration. (c) Standard Sodium hydroxide solution (0.1 N): 400 mg sodium hydroxide was dissolved in 100 ml distilled water. 200 ml waste water sample was taken for titration against the standard HCl solution using methyl orange as an indicator and end point was noted when colour changed from yellow to orange. Values were computed in the following formula:- Methyl orange alkalinity mg CaCO 3 /L = Where:- A = ml titrant B = Normality of titrant A x B x 50,000 ml sample (iii) Total alkalinity (TA): The total alkalinity encompassing carbonate, hydroxyl and bicarbonate alkalinity, was determined by adding the values for phenolphthalein and methyl orange alkalinities. Total alkalinity mg CaCO 3 /L = PA mg CaCO 3 /L + Methyl orange alkalinity mg CaCO 3 /L Materials and Methods 20

(iv) Carbonate, Bicarbonate & Hydroxide alkalinities: Carbonate and Bicarbonate alkalinities were determined with the help of following table showing the alkalinity relationships APHA-AWWA-WPCF (1980). Result of Titration Hydroxide alkalinity as CaCO 3 Carbonate alkalinity as CaCO 3 Bicarbonate alkalinity as CaCO 3 P = 0 0 0 T P < ½ T 0 2P T-2P P = ½ T 0 2P 0 P > ½ T 2P-T 2 (T-P ) 0 P = T T 0 0 P = Phenolphthalein alkalinity, T = Total alkalinity (8) FREE CARBON DIOXIDE: Free CO 2 was determined in mg/l by Titrimetric method using phenolphthalein as an indicator at ph 8.3, as described in APHA-AWWA-WPCF (1980). (a) Phenolphthalein indicator solution: 500 mg Phenolphthalein indicator was dissolved in 50 ml of 95% ethyl alcohol and diluted to 100 ml by distilled water. (b) Sodium hydroxide solution (0.01 N): 40 mg NaOH was dissolved in 100 ml of distilled water and then standardization was done against standard oxalic acid. (c) Oxalic acid solution (0.01 N): 6.3 mg oxalic acid was dissolved in 100 ml of distilled water. 100 ml waste water was titrated against 0.01 NaOH solution using phenolphthalein as an indicator. The end point was marked by appearance of pink colour at ph 8.3. The quantity of free CO 2 was calculated by using the formula: Materials and Methods 21

Free CO 2 mg/l = A x B x 44,000 ml sample Where: A = ml titrant. B = Normality of titrant. (9) TOTAL CARBON DIOXIDE: Quantity of total CO 2 was determined indirectly by summing the values of carbonate and bicarbonate in formula as prescribed in APHA-AWWA-WPCF (1980). Total CO 2 mg/l = Free CO 2 mg/l + 0.88 (A + B) Where: A = mg/l bicarbonate alkalinity B = mg/l carbonate alkalinity (10) CHLORIDE: Chloride content was determined in mg/l by Argentometric titrimetric method following from APHA-AWWA-WPCF (1980). Chloride was precipitated as silver chloride and potassium chromate indicator marked the end point of the titration by colour change from yellow to pinkish yellow. (a) Potassium chromate indicator solution: 5 gm potassium chromate was dissolved in little amount of distilled water and silver nitrate solution was added till the formation of red precipitate. The solution was allowed to stand for 12 hours and then filtered. The volume was made to 100 ml by distilled water. (b) Standard silver nitrate titrant (0.0141N): 2.395 gm AgNO 3 was dissolved in distilled water and diluted to 1 liter. Solution was standardized against standard sodium chloride solution. (c) Standard sodium chloride solution (0.0141 N): 824.0 mg NaCl (dried at 140 C) was dissolved in chloride free water and diluted to 1 liter. (1 ml of standard solution = 500 µgm Cl ) Materials and Methods 22

100 ml waste water sample or sample diluted to 100 ml was titrated against the silver nitrate solution in the presence of potassium chromate indicator. End point of titration was indicated by appearance of pinkish yellow colour of silver chromate. A blank was also titrated simultaneously, and the obtained values were computed in the formula. Chloride mg/l = Where:- A = ml titrant used for sample B = ml titrant used for blank. N = Normality of titrant. (A - B) x N x 35,450 ml sample (11) DISSOLVED OXYGEN (DO): Dissolved oxygen content in waste water sample was determined in mg/l by using Azide modification method, as per the APHA-AWWA-WPCF (1980). Dissolved oxygen combines with manganous sulfate to form higher hydroxide, which on acidification liberates iodine equivalent to oxygen fixed. The liberated iodine was titrated against the hypo solution using starch as an indicator. (a) Manganous sulfate solution: 36.4 gm MnSO 4.H 2 O was dissolved in distilled water, filtered and diluted to 100 ml should not give any colour with starch. (b) Alkali iodide azide reagent: 1 gm. sodium azide was dissolved in 50 ml of distilled water to form solution A, while 48 gm Sodium hydroxide and 75 gm sodium iodide was dissolved separately in distilled water to form solution B, later both the solutions A and B were mixed together and volume was made to 100 ml. (c) Conc. H 2 SO 4 : (d) Starch indicator solution: 50 mg soluble starch powder was dissolved in 80 ml of hot distilled water and was diluted to 100 ml. Materials and Methods 23

(e) Standard sodium thio sulfate solution (0.025N): 1.241 gm Na 2 S 2 O 3.5H 2 O was dissolved in freshly boiled and cooled distilled water and 80 mg of solid NaOH was added, and then final volume was made to 200 ml. The standardization of hypo solution was done against the standard potassium biiodate solution. (f) Standard potassium bi-iodate solution (0.025N): 812.4 mg potassium bi-iodate was dissolved in distilled water and was diluted to 1000 ml. 300 ml waste water sample was taken in BOD Bottles. 1 ml each of manganous sulfate and alkali iodide azide solution were added by partly replacing the cap. Bottle was shaken for 10 times and then brown precipitate was allowed to settle down. Later 2 ml. of conc. H 2 SO 4 was added to the bottle to dissolve the precipitate, which resulted in yellow colour solution on complete dissolution. 203 ml of this yellow solution was titrated against standard hypo solution in presence of starch indicator. The end point was indicated by disappearance of blue colour. The amount of dissolved oxygen was calculated by computing the value in formula. 1 ml standard hypo solution of 0.025 N = 200 µg dissolved oxygen. (12) BIOCHEMICAL OXYGEN DEMAND (BOD): Biochemical oxygen demand in waste water sample was determined in mg/l as method prescribed in APHA-AWWA-WPCF (1980). The BOD measurement of waste water sample was the difference of oxygen concentration in the sample before and after incubation in dark. Incubation was made at 20ºC in BOD incubator for five days. The same as required for dissolved oxygen determination. Freshly collected waste water sample was diluted to about 100 times only to get measurable amount of oxygen after five days of incubation. This was filled into four BOD bottles. Initial oxygen concentration was determined immediately in two of the Materials and Methods 24

bottles. Remaining two bottles were incubated in BOD incubator at 20ºC for five days, and then oxygen concentration was again determined. BOD mg/l = D l - D 2 Where: D l = initial oxygen value. D 2 = oxygen value after 5 days incubation. (13) CHEMICAL OXYGEN DEMAND (COD): Chemical oxygen demand was determined by Dichromate reflux method as per APHA-AWWA-WPCF (1980). It is the measure of oxygen consumed by strong oxidizing agent (Potassium dichromate) during oxidation of organic matter. (a) Standard potassium dichromate solution (0.25 N): 2.451 gm of K 2 Cr 2 O 7 was dissolved in distilled water and diluted to 200 ml. (b) H 2 SO 4 reagent: 2.2 gm of silver sulfate was added to 400 ml of H 2 SO 4 and was allowed for 2 days to get dissolved. (c) Mercuric sulfate crystals: (d) Ferroin indicator solution: (e) Standard ferrous ammonium sulfate titrant (0.05N): 19.6 gm of ferrous ammonium sulfate was dissolved in distilled water then 20 ml. of conc. H 2 SO 4 was added to the solution. Later it was allowed to cool and diluted to 1L. Standardization was done by mixing 30 ml of H 2 SO 4 in 10 ml of the above solution and was titrated against K 2 Cr 2 O 7 solution using ferroin as an indicator. 10 ml waste water sample or sample diluted to 10ml distilled water was taken in 250 ml refluxing flask. 0.2 gm of HgSO 4 and 15 ml of H 2 SO 4 reagent were added. Solution was cooled and 5 ml of 0.25 N, K 2 Cr 2 O 7 solution was added. This solution was refluxed for two hours. After 2 hours of reflux, solution was cooled and diluted to final volume of 70 ml by distilled water. Excess dichromate of 25 ml refluxed solution was titrated against ferrous ammonium sulfate in presence of 3 drops of ferroin Materials and Methods 25

indicator. End point of titration was indicated by a colour change from blue green to reddish brown. Simultaneously a blank was also run and COD value was calculated by using the formula:- COD mg/l = (A - B) x N x 8,000 ml sample Where: A = ml. ferrous ammonium sulfate titrant used for blank. B = ml. ferrous ammonium sulfate titrant used for sample N = Normality of ferrous ammonium sulfate titrant. HARDNESS: It was estimated as total hardness and calcium hardness. (14) TOTAL HARDNESS: Total hardness was determined by EDTA titrimetric method, following APHA-AWWA-WPCF (1980). At a ph of 10.0 EDTA combines with both calcium and magnesium to form blue colour at the end point. (a) Buffer solution: 16.9 gm ammonium chloride was dissolved in 143 ml. of conc. NH 4 OH. This was added to l.25 gm. of Magnesium salt of EDTA dissolved in 50 ml of distilled water. The solution was further diluted to 250 ml. (b) Eriochrome Black T indicator mixture: Dry mixture of 0.40 gm of Eriochrome black T and 100 gm of anhydrous sodium chloride was prepared. Both the chemicals were grinded. (c) Standard EDTA titrant (0.01M): 3.723 gm of disodium salt of EDTA was dissolved in distilled water and diluted to1l; prepared titrant was standardized against the standard calcium solution. (d) Standard calcium solution: 1 gm anhydrous CaCO 3 was dissolved in 1+1 HCl then 200 ml distilled water was added and boiled to expel carbon dioxide. Solution was cooled and 2 drops of Materials and Methods 26

methyl red indicator was added. Intermediate orange colour was adjusted by adding 1+1 HCl solution. This solution was diluted to 1L with distilled water. The solution was equivalent to 1mg CaCO 3 /ml. Pretreatment of water sample 100 ml waste water sample was taken for pretreatment. It was acidified with dilute HNO 3 and H 2 SO 4. Evaporation of water sample was done till the formation of white residue. Dried white residue was dissolved in 1+1 HCl and was neutralized with conc. NH 4 OH. Finally volume was adjusted to 100 ml by distilled water. Titration: 50 ml of pretreated water sample was taken for titration. 4 ml of buffer solution was added to raise the ph of sample to 10 ph. This was titrated against the standard EDTA solution using a pinch of powdered eriochrome black T indicator. End point of titration was indicated by sharp colour change from wine red to blue. Total hardness as calcium carbonate mg/l was determined by using the formula: Total hardness mg CaCO 3 /L = A x B x 1,000 ml sample Where: A = ml EDTA titrant B = mg CaCO 3 equivalent to 1 ml of EDTA solution. (15) CALCIUM HARDNESS: Calcium hardness was determined by EDTA titrimetric method as prescribed in APHA-AWWA-WPCF (1980). In this method EDTA was added to water sample contained both Ca & Mg ions but in presence of muraxide indicator, it formed the pink coloured complex, and then was titrated with disodium salt of EDTA solution. At the end point of titration, colourless chelate complex leaved the solution purple. (a) NaOH (6N): 24 gm NaOH was dissolved in distilled water and volume was adjusted to 100 ml. Materials and Methods 27

(b) Murexide indicator (Ammonium perpurate): 200 mg murexide powder was grinded with 100 gm of anhydrous sodium chloride to 40 to 100 meshes. (c) Standard EDTA titrant (0.01M): (d) Standard Calcium solution: Both the reagents (c) and (d) were prepared as per the methodology described in Total hardness determination. (e) Conc. HNO 3 (f) Conc. HCl (g) Conc. H 2 SO 4 Pretreatment of sample was done similarly as was done in total hardness determination. Titration: 50 ml pretreated waste water sample was taken and 4 ml of 6 N NaOH solution was added to raise the ph up to 13, then 0.2 gm murexide indicator was added to this solution and was titrated against the standard EDTA solution. End point of titration was indicated by the colour change from pink to purple. Titration value was computed in formula given below Calcium hardness mg CaCO 3 /L = A x B x 1,000 ml sample Where: A = ml EDTA titrant B = mg CaCO 3 equivalent to 1 ml of EDTA solution. (16) CALCIUM: Calcium content was determined in mg/l by titrimetric method following APHA - AWWA - WPCF (1980). The value obtained from titration was computed in the formula: Calcium mg/l = Where: A x B x 400.8 ml sample Materials and Methods 28

A = ml EDTA titrant B = mg CaCO 3 equivalent to 1 ml of EDTA solution. (17) MAGNESIUM: Magnesium content was calculated in mg/l by deducting the value of calcium hardness in total hardness value and was multiplied by a conversion factor as given in APHA-AWWA-WPCF (1980). Magnesium mg/l = (Total hardness mg CaCO 3 /L - Calcium hardness mg/l CaCO 3 x 0.244). NITROGEN: Nitrogen content was determined in waste water in Ammonical, Nitrite and Nitrate form. (18) AMMONICAL NITROGEN: Ammonical nitrogen was determined in waste water by using Nesslerization method following APHA-AWWA-WPCF (1980). In this method Nessler reagent combines with ammonical nitrogen of water sample and formed yellow brown colour. (a) Zinc sulphate solution: 10 gm ZnSO 4.H 2 O was dissolved in distilled water and diluted to 100 ml. (b) EDTA reagent: 50 gm EDTA was dissolved in 60 ml of distilled water contained 10 gm of NaOH and was diluted to 100 ml after cooling. (c) Nessler reagent: 10 gm Mercuric iodide and 7 gm Potassium iodide were dissolved in small quantity of distilled water and was added to the cooled solution of 160 gm Sodium hydroxide in 500 ml of water, this solution was diluted to 100 ml. (d) Stock Ammonium solution: 3.819 gm anhydrous ammonium chloride was dissolved in distilled water and diluted to 1 liter by distilled water (1 ml = 1 mg NH 3 -N) Materials and Methods 29

(e) Standard Ammonium solution: 2 ml stock ammonium solution was diluted to 1000 ml with distilled water (1 ml = 0.02 mg NH 3 -N). Dechlorination: 100 ml waste water sample was taken for the removal of residual chlorine and an equivalent amount of sodium thiosulphate prepared in ammonia free water was added to the sample, later 1 ml of ZnSO 4.7H 2 O and 0.5 ml of 6N Sodium hydroxide solution was added to obtain a ph of 10.5. Sample was allowed to stand for few minutes and clarified by centrifugation. Volume was made to 100 ml. Colour development: 10 ml water sample was diluted to 50 ml with ammonia free water. Two drops of EDTA was added to prevent the turbidity caused by the precipitation of calcium and magnesium salt, then 2 ml of nessler reagent was added to the sample and % transmission of yellow colour was measured at 425 nm. Amount was determined with the help of standard curve. (19) NITRITIE NITROGEN: Nitrite nitrogen was estimated in mg/l by Diazotization method. In this determination reddish purple azo dye was formed at ph 2.5 by the coupling of diazotized sulfanilic acid with N l Naphthyl ethylene diamine dihydrochloride (NED) and formed pink colour. (a) Sulfanilamide solution: 1 gm sulfanilamide was dissolved in the mixture of 50 ml of conc. HCl, 300 ml. distilled water, and final volume was made to 100 ml. (b) N - 1 naphthyl ethylene diamine dihydro chloride solution (NED): 100 mg N-1 naphthyl ethylene diamine dihydro chloride was dissolved in 100 ml distilled water and was stored in dark brown bottle. Materials and Methods 30

(c) Stock Nitrite Solution: Stock nitrite solution was prepared by dissolving 1.232 gm sodium nitrite in nitrite free water (Distilled water).volume of this solution was made to 1 liter. (1 ml of this solution is equivalent to 250 microgram of NO 2 -N). (d) Standard Nitrite solution: 1 ml stock solution dissolved in distilled water and diluted to 100 ml. (1 ml = 2.5 µgm) 10 ml waste water sample was diluted to 50 ml by distilled water. 1 ml sulfanilamide solution was added to lower the ph 2 to 2.5. Sample was allowed to stand for 2 minutes to 8 minutes, after then 1 ml of N-1 naphthyl ethylene diamine dihydrochloride (NED) solution was added. Pink colour was developed and intensity of colour (% Transmission) was measured at 543 nm. Amount was determined with the help of standard curve and it was computed in formula:- mg/l Nitrite N = µg Nitrite N ml sample (20) NITRATE NITROGEN: Nitrate nitrogen was determined by Phenol disulphonic acid method by following Jackson (1973) and Trivedy and Goel (1984). Alkali nitrate - N reacts with 2, 4-phenol disulphonic acid and formed yellow colour. The % Transmission of yellow colour was measured on spectrophotometer at 410 nm. (a) Phenol disulphonic acid: 25 gm of crystal white phenol was dissolved in 150 ml of conc. H 2 SO 4 and was heated on water bath for 2 hours. (b) Ammonium hydroxide: (c) Stock Nnitrate solution: 721.8 mg anhydrous KNO 3 was dissolved in distilled water and diluted to 1 liter. (1 ml = 100 µg) (d) Standard nitrate solution: Materials and Methods 31

20 ml stock nitrate solution was diluted to 100 ml with distilled water. 1ml = 20 µg NO 3 -N. 50 ml waste water sample was taken and evaporated over a hot plate till the formation of residue, which was dissolved in 3 ml of phenol disulphonic acid. Reaction was allowed to stand for 10 minutes and then 15 ml of distilled water was added. Later 7 ml of ammonia solution was added and final volume was made to 50 ml. Intensity of yellow colour as percentage transmission was measured at 410 nm. Valuation of NO 3 -N as mg/l was obtained in reference of calibration curve and value was computed in the following formula:- mg/l Nitrate N = PHOSPHATE: µg Nitrate N ml sample Phosphorous was estimated as phosphate in waste water sample, in four forms viz. Total ortho, Acid hydrolyzable, Total & Organic phosphate, following APHA- AWWA-WPCF (1980). The determination was made by Vanadomolybdophosphoric acid method. In this method ammonium molybdate reacts under acidic condition in presence of vanadium and formed a yellow Vanadomolybdo phosphoric acid. The % Transmission of yellow colour was measured at 490 nm. Phosphate value was determined with the help of calibration curve, prepared from standard solution. Amount of phosphorus/l was calculated by using following formula :- Phosphate mg/l = mg P x 1000 ml sample (21) TOTAL ORTHO PHOSPHATE: Total ortho phosphate content was determined directly without making any pretreatment to the sample. (a) Phenolphthalein indicator: (b) Strong acid solution: Materials and Methods 32

300 ml conc. H 2 SO 4 was added to 600 ml of distilled water and cooled, later 4 ml of conc. HNO 3 was added and solution was diluted to l liter. (c )Vanadate molybdate reagent: 25 gm of ammonium molybdate was dissolved in 300 ml of distilled water. This solution was added to the solution of 1.25 gm of ammonium metavandate in 300 ml distilled water, then the solution was cooled and 330 ml conc. HCl was added to the solution and volume was made up to l liter by distilled water. (d) Standard Phosphate solution: 219.5 mg anhydrous potassium dihydrogen phosphate was dissolved in distilled water and diluted to 1 liter. (1 ml of the solution is equivalent to the 50 µg of phosphorous as phosphate). 35 ml filtered water was taken and 10 ml vanadomolybdate reagent was added. Finally the volume was made to 50 ml by distilled water. Percentage Transmission of yellow colour was measured at 490 nm. (22) ACID HYDROLYZABLE PHOSPHATE: Acid hydrolyzable phosphate was determined after mild acid hydrolysis of the waste water by following Vanadomolybdophosphoric acid method, as prescribed in APHA-AWWA-WPCF (1980). All the reagents used were same as orthophosphate determination. Pretreatment of water sample (Acid hydrolysis): 100 ml water sample was taken and 5 drops of phenolphthalein indicator was added. In some estimation solution was turned light pink. Colour was discharged by strong acid solution. This solution was boiled for 90 minutes and volume was maintained in between 25-50 ml by adding distilled water. Thereafter solution was cooled and neutralized to faint pink colour with 6N NaOH solution, and then the volume was adjusted to 100 ml with distilled water. This pretreated solution was used for the Materials and Methods 33

determination of acid hydrolyzable phosphate by Vanadomolybdophosphoric acid method as described in orthophosphate determination. (23) TOTAL PHOSPHATE: Total phosphate content includes both the soluble and insoluble forms of phosphate. It was determined by digesting the sample more rigorously with H 2 SO 4 and HNO 3, and then the estimation was done by following vanadomolybdophosphoric acid method following APHA-AWWA-WPCF (1980). (a) Conc.H 2 SO 4 : (b) Conc.HNO 3 : (c) Other reagents used are mentioned in the orthophosphate determination. 100 ml water sample was taken in conical flask. 5 drops of conc. HNO 3 and 1 ml of conc. H 2 SO 4 were added and digestion was done to a volume of about 1 ml. The digestion was continued till the formation of white residue in order to remove HNO 3. Then in cooled sample 20 ml distilled water and 5 drops of phenolphthalein indicator were added. This solution was neutralized by 6N NaOH till the formation of faint pink colour. Prepared solution was filtered and volume was made up to 100 ml by distilled water. In this digested sample total phosphate content was determined by vanadomolybdophosphoric acid method as mentioned in orthophosphate determination. (24) ORGANIC PHOSPHATE: Organic phosphate was determined by deducting the amount of inorganic phosphate estimated as ortho and acid hydrolyzable phosphate in total phosphate. Organic phosphate = Total phosphate - Total orthophosphate - Acid hydrolyzable phosphate. (25) PROTEIN: Protein was determined in waste water by using Folin Lowry method following Plummer (1971). In this method, protein reacts with the Folin Ciocalteau reagent to give a blue complex. Materials and Methods 34

(a) Alkaline sodium carbonate solution: 2 gm Sodium Carbonate dissolved in distilled water and diluted in 100 ml of distilled water contained 400 mg of NaOH. (b) Copper Sulfate Sodium Potassium Tartrate solution: (i) Copper sulfate solution : 500 mg Copper sulfate dissolved in distilled water, and diluted to 100 ml. (ii) Sodium potassium tartrate solution : 1 gm Sodium potassium tartrate dissolved in distilled water and diluted to 100 ml. (iii) Prepare fresh solution by mixing Stock solutions (i) and (ii) in ratio of 1 : 1. (c ) Alkaline solution: Prepared on day of use by mixing 50 ml of reagent (a) and 1 ml of reagent (b) (iii). (d) Folin Ciocalteau reagent: Dilute the commercial reagent with an equal volume of distilled water on the day of use. (d) Standard Proteins solution: 20 mg of albumin was dissolved in distilled water and diluted to 100 ml. (1 ml = 0.2 mg protein). Take 1 ml of waste water and add 5 ml of the alkaline solution. Mix thoroughly and allow standing at room temperature for 10 minutes or longer. Add 0.5 ml of diluted Folin Ciocalteau reagent rapidly and mixing simultaneously. After 30 minutes it gets transformed to blue colour. The % Transmission of blue colour was measured on spectrophotometer at 750 nm. Estimate the protein concentration with the help of standard curve. (26) CARBOHYDRATE: Carbohydrate was determined in waste water by using Anthrone method following Plummer (1971). The anthrone reaction is the basis of a rapid and convenient method for the determination of hexoses, aldopentose and hexuronic acids either free or present in polysaccharides. In this method carbohydrates reacts with the anthrone reagent and forms a coloured complex. Materials and Methods 35

(a) Anthrone reagent: 200 mg Anthrone was dissolved in conc. H 2 SO 4 and diluted to 100 ml. (b) Stock Glucose Solution: 10 mg of Glucose was dissolved in distilled water and diluted to 100 ml. (1 ml = 0.1 mg carbohydrate). (c) Standard Glucose Solution: 1 ml stock Glucose solution was dissolved in distilled water and diluted to 100 ml. (1 ml = 0.001 mg carbohydrate). (d) 2.5 N HCl Solution: 22.7 ml concentrated HCl was dissolved in distilled water and diluted to 100 ml. (e) Sodium Carbonate: (i) Pretreatment for obtaining Protein free Carbohydrates: 100 ml of waste water was taken in the 250 ml conical flask and 5 ml 2.5 N hydrochloric acid solutions was added, then it was heated to 100 C for 3 hours in a water bath. After cooling sodium carbonate was added till the formation of effervescences, and then this solution was centrifuged at 3000 rpm for 10 minutes. The collected solution was protein free carbohydrates. (ii) Analysis: 4 ml of the anthrone reagent was added in 1 ml of a protein free carbohydrate solution and was mixed rapidly, then it was placed in a boiling water in water bath for 10 minutes, with a marble on top to prevent loss of water by evaporation. Thereafter solution was cooled and found transformed in to blue-green colour. The % transmission of golden-green colour was measured on spectrophotometer at 620 nm. Carbohydrates concentration was determined with the help of standard curve. (27) FAT: Fat of waste water was measured in % by Electronic Milk Tester. This instrument is simple, economical and accurate milk fat testing instrument. It measures the Materials and Methods 36

fat content instantaneously on a digital readout. It is based on the photometric measurement of light scattered by the waste water sample. (28) SULFATE: Sulfate was determined in waste water by using Turbidimetric Method following APHA-AWWA-WPCF (1980). Sulfate ions are precipitated in HCl acid medium with barium chloride so as to form barium sulfate crystals of uniform size. Reagents and Apparatus: (a) Conditioning reagent: Mixed 50 ml glycerol with a solution containing 30 ml conc. HCl, 300 ml distilled water, 100 ml 95% ethyl alcohol and 75 gm NaCl. (b) Barium chloride (BaCl 2 ): Crystals of 20 30 meshes used. (c) Stock Sulfate solution: Dissolved 147.9 mg anhydrous Sodium sulfate (Na 2 SO 4 ) in distilled water and diluted to 1000 ml. (1 ml = 100 µg SO 4 ²). (d) Standard Sulfate solution: Dilute 1 ml of stock solution to 100 ml with distilled water. (1 ml = 1 µg SO 4 ²). (e) Measuring spoon: Capacity 0.2 to 0.3 ml. (f) Magnetic stirrer with stirring bars: (g) Stop watch: 5 ml waste water sample diluted to 100 ml distilled water was taken in an Erlenmeyer flask. 5 ml conditioning reagent was added and mixed in stirring apparatus. While stirring a spoon full of BaCl 2 crystals (0.5 gm) was added and stirred for exactly 1 minute at constant speed. Immediately after striving, solution was poured in spectrophotometer and turbidity was measured at 30 seconds intervals for four minutes. Maximum reading of Turbidity obtained in the four minutes interval was noted. The quantity of sulfate was calculated in mg/l by using calibration curve. Materials and Methods 37

Sulphate mg/l = mg SO 4 ² x l000 ml sample (29) SODIUM: Sodium was determined in waste water by using Flame emission photometric method by following APPHA-AWWA-WPCF (1980). Reagents & Auxiliaries: (a) Stock Sodium solution: Dissolved 2.542 gm sodium chloride dried at 140 ºC and diluted to 1000 ml de-ionized distilled water (1 ml = 1000 ppm Na). (b) Standard Sodium solution: 10 ml stock solution was dissolved in de-ionized distilled water & diluted to 100 ml (1 ml = 100 ppm Na). (c) Glasswares: Rinsed glassware s with 1 + 15 HNO 3 followed by several portions of deionized distilled water. (d) De-ionized distilled water: Used de-ionized distilled water to prepare all the reagents for calibration and standardization. (a) Pre treatment of waste water sample: Sample was containing particulates or organic materials and turbidity > 1 NTU, hence digestion was done before analysis:- (b) Digestion: 100 ml sample was mixed in a conical flask with 5 ml concentrated HNO 3 and few boiling chips and heated on a hot plate at 70-80ºC until a volume was reduced. Heating was continued and a small volume of concentrated HNO 3 was added until a clear solution was obtained. The digested solution was filtered and diluted with distilled water to the original volume. The sample was measured and sodium concentration was determined with the help of calibration curve. Materials and Methods 38

(30) POTASSIUM: Potassium was determined in waste water by using Flame emission photometric method as prescribed in APPHA-AWWA-WPCF (1980). Reagents & Auxiliaries: (a) Stock Potassium solution: 1.907 gm potassium chloride (dried at 110 C) dissolved in distilled water and diluted to 1000 ml.(1 ml =1000 ppm K). (b) Standard potassium solution: 10 ml stock potassium solution was diluted with de-ionized distilled water to 100 ml. (1 ml = 100 ppm). (c) Glassware: Glassware was rinsed with 1 + 15 HNO 3 and followed by de-ionized distilled water. (d) De-ionized distilled water: De-ionized distilled water was used to prepare all the reagents for calibration and standardization. (a) Pre treatment of waste water sample: Sample was containing particulates or organic materials and turbidity > 1 NTU, hence digestion was done before analysis:- (b) Digestion: 100 ml sample was mixed in a conical flask with 5 ml concentrated HNO 3 and few boiling chips and heated on a hot plate at 70-80ºC until volume was reduced. Heating was continued and a small volume of concentrated HNO 3 was added till a clear solution was obtained. Digested solution was filtered and diluted with distilled water to the original volume. (c) The sample was measured and potassium concentration was determined with the help of calibration curve. Materials and Methods 39

(C) BIOLOGICAL PARAMETER: (31) ENUMERATION OF MOST PROBABLE NUMBER (MPN): Multiple-Tube Fermentation Test or Most Probable Number (MPN) Test is the most often used technique for the analysis of water. The test was used to detect coli forms. Coli forms are defined as facultative anaerobic, gram-negative, non-sporing, rod shaped bacteria that ferment lactose with the production of acid and gas within 24 hours of incubation at 35ºC. A statistical method estimated the MPN of bacteria present in inoculums which was used to make a dilution series. Several series were made with different initial volumes of inoculums; the results were recorded as a series of positives, i.e. growth in the tube, can then be calculated to give an MPN. The result is the probable number of micro-organisms that would be expected to yield this result. This method was followed by Aneja (2001). Requirements: Media specification: Lactose fermentation broth (LB 1X and LB 2X). Peptone 1.25 gm Lactose 1.25 gm Beef extract 0.75 gm Brilliant green 0.003 gm Distilled water 250 ml Following media components were used to prepare Lactose Fermentation broth. ph of the media was maintained at 6.9 after sterilization. Collected water sample were labeled as 5 double strength lactose broth tubes 10 and 5 single strength broth tubes 1 another 5 tubes as 0.1. Waste water sample was mixed thoroughly by shaking. Three sets of 5 tubes each containing (LB 2X, LB 1X and LB 1X) were inoculated with 10 ml, 1ml and 0.1 ml of waste water Materials and Methods 40

sample using 10 ml,1ml and 0.1ml sterile pipettes. Later all the 15 inoculated tubes were incubated aerobically at 35 ºC for 48 hours. All the lactose fermentation tubes were examined for the production of acids (yellow colour) and gas after 24 & 48 hours of incubation. Production of acid and gas (appearance of a bubble large enough to fill the concavity at the top of the Durham tubes) after 24 hours incubation indicated a positive presumptive test for coli form bacteria. If there was no gas produced after 48 hours of incubation showed negative presumptive test (i.e. coli form absent). A count of number of positive tubes showed production of gas following the incubation period was taken in to consideration for calculation of MPN. STATISTICAL ANALYSIS: Pearson Correlation analysis was done for all the physico-chemical parameters investigated during the present study. The correlation was determined at P<0.05 & P<0.01 level of significance. P<0.05 is indicated in the table by single * and P<0.01 by double ** mark. (B) UTILIZATION OF WASTE WATER FROM MILK PROCESSING UNIT IN CULTIVATION OF PLANTS: To study the utilization of waste water from milk processing unit in cultivation of plants, an experiment was conducted, in which three selected plants were grown in field irrigated with waste water. Simultaneously control experiment was also set, which was irrigated with tap water. The net primary productivity of all the plants were determined in both the experiment to investigate the utility of waste water in promoting growth of plants. Determination of Net Primary Productivity (NPP) of cultivated plants: The Net Primary Productivity (NPP) of cultivated vegetables Trigonella foenum-graecum L., Spinacea oleracea L., and Coriandrum sativum L was determined by Harvest method. All the three plants were grown in home garden. An area of 1m x1m in triplicate was used each for experiment and the control. Similar kind of garden soil was used for growing plants. In each of the experimental and control field equal number of Materials and Methods 41

soaked, uncontaminated & healthy 200 seeds of Trigonella foenum-graecum L. was sown for the period of 30 days while Spinacea oleracea, 40 days and Coriandrum sativum for 45 days in winter. Experimental fields were irrigated with 18 liters of 1:1 waste water: tap water /day, while control fields were irrigated with same quantity of tap water/day. The 200 seeds of each plant was weighed before sowing and this weight was noted as initial biomass. After the 30 days, 40 days and 45 days interval, harvesting was done. Entire plant body was uprooted and washed with tap water. Later it was wrapped in a paper and dried in hot air oven at 105 ºC for 24 hours. Thereafter, it was weighed and recorded as final biomass (Bf). NPP was determined through obtained value of initial biomass, final biomass & number of days of the plant growth with the help of following formula:- Net Primary Productivity (gm m ² day ¹) = Bf - Bi dt Where :- Bf = Final biomass (gm m ²) Bi = Initial biomass (gm m ²) dt = Number of days of cultivation plants. Entire experiment was repeatedly conducted in winter season of year 2007-09 in order to investigate the utility of waste water in cultivation of plants. ----- ----- Materials and Methods 42