— SSeS ee RPVVUHVVeuveececcEeccceccee 1. To estimate dissolved oxygen content of a given water sample. In order to maintain metabolic processes, grow and reproduce, all living organisms must have oxygen in one form or another. This oxygen is in the form of Dissolved Oxygen (DO) in the fluid (air or water) in which the organisms live. The presence of DO is the factor which determines whether the biological changes in a wastewater will be accomplished by aerobic or anaerobic organisms. Oxygen interchange among air, water, and soils is limited by biological, chemical, and physical factors related to oxygen availability. Oxygen content of water is limited to about 11.3 milligrams dissolved oxygen per liter at 10° C, Oxygen concentration of less than 16% causes blackoutsand concentrations above 40% causes the formation of toxic oxygen radicals thatdamage cell structure and function. This process is known as oxygen toxicity andcan lead to death. Thus, life is is greatly affected by the concentration ofoxygen. The analysis for DO is a key test in water pollution control activities and waste treatment process control. The DO test provides information about the condition of the wastewater for the operator tomake process control decisions. Two methods for DO analysis are: the Winkler Method, or iodometric, and the electrometric method. The Winkler is a wet chemistry, titrimetric procedure based on the oxidizing property of dissolved oxygen. The electrode method measures the rate of molecular oxygen diffusion across a membrane. Procedure selection considerations include: interferences present, desired accuracy, time or convenience. Winkler method The Winkler test is used to determine the level of dissolved oxygen in water samples and to estimate the biological activity in the water sample. An excess of Manganese({II) salt, iodide (1) and hydroxide (OH-) ions are added to a water sample causing a white precipitate of Man(OH)2 to form. This precipitate is then oxidized by the dissolved oxygen in the water sample into a brown Manganese precipitate. In the next step, a strong acid (either hydrochloric acid or sulphuric acid) is added to acidify the solution. The brown precipitates then convert the iodide ion (I-) to Iodine. The amount of dissolved oxygen is directly proportional to the titration of Iodine with a thiosulphate solution. The generalized principle is that iodine will be released in proportion to the amount of dissolved oxygen present in the sample. By using sodium thiosulfate with starch as the indicator, titrate the sample and determine the amount of dissolved oxygen.SOVOVI FF FUG CVCVKVCeC CCC ECC ECL ES MnSO,+2KOH = [=] Mn(OH): + K2SOu 4Mn(OH); + 02 + 2H20 (=| 4Mn(OH)s 2Mn(OH)3 + 3H2S04 Mn2(S04)3 +6H20 NazS,O¢ + 2Nal 2Na;S203 + br Reagents 0.0250 M Sodium thiosulfate: Dissolve 6.206 grams sodium thiosulfate crystals in distilled water and make up water to a volume of I liter. For preservation, add 0.4 gm or 1 pellet of sodium hydroxide. Solutions of thiosulfate should be used within two weeks to avoid loss of accuracy because of decomposition of the solution. Alkaline odine solution: 50 g sodium hydroxide (KOH and 15 g potassium iodide (KD) are dissolved in reagent water and diluted to 100 ml. Starch solution: 2 g starch is dissolved in 100 ml boiling water, cool. ‘Manganous Sulfate solution: Dissolve 40 g manganous sulfate dihydrate (MnSOx. 2H20) in reagent water, filter and dilute to 100 ml. Tan mono SEU gma hM nSOy Sulfuric acid. Use concentrated reagent grade acid (H2S0,). Handle carefully, since this ‘material will bum skin and clothes. Procedure 1. Collect sample in 300 ml BOD bottle taking special care to avoid aeration of the liquid being collected. Fill bottle completely (no air under cap). Insert stopper in bottle. ‘Add, under the surface of the sample, 1.00 ml manganous sulfate solution. Immediately add 1.00 ml alkaline iodide solution below the surface of the liquid. Insert stopper in bottle, avoid trapping air bubbles, and invert 5 times to mix well Repeat this shaking after the floc has settled halfway. Allow floc to settle to half the volume of the bottle a second time, then open and add 1.0 ml concentrated Sulfuric acid by allowing the acid to run down the neck of the bottle above the surface of the liquid. Insert stopper and invert to mix until the floc dissolves. Using a graduated cylinder, transfer 200 ml of the solution to an Erlenmeyer flask for the titration procedure. Titrate with the 0.0250 M sodium thiosulfate solution until the solution is pale yellow. ‘Add 2 ml starch and continue titration until the color is permanently discharged. Record the volume of sodium thiosulfate used. Each 1.00 ml of 0.025 M sodium thiosulfate used is equivalent to 1.00 mg/L DO. Record the answer.SSSuIIT SF TRVVHUUUDTUKUYY SS” SS” 2. Estimation of phenolic compounds in the drinking water and effluent sample Phenolic materials react with 4-aminoantipyrine in the presence of potassium ferricyanide at a pH of 10 to forma stable reddish-brown colored antipyrine dye. The amount of color produced is a function of the concentration of phenolic material. This method is applicable to the analysis of drinking, surface and salinewaters, domestic and industrial wastes. The method is capable of measuring phenolic materials at the 3 ng/L levelwhen the colored end product is extracted and concentrated in a solvent phaseusing phenol as a standard. The method is capable of measuring phenolic materials that contain more thanSO Hg/L in the aqueous phase (without solvent extraction) using phenol as astandard. It is not possible to use this method to differentiate between different kinds ofphenols.« oxidation of the NH, group cH HN. cH HN, CHy ls = \ i + aut wae + aN (a Wor Nets + IFe(CN),* +H electrophilic attack ro (PQ— «formation ofthe quinone imine dye fox i ; opt 3 3 ° Cen, — UN-CH, + 2Fe(CN),* + 2H* 3 0 1. Buffer solution: Dissolve 2 gm of NaOH in 50 ml of distilled water. Now add 4.2 gm of NaHCO3.Make the final volume to 100 ml. 2. Aminoantipyrine solution: Dissolve 2 g of 4AAP in distilled water and diluteto 100 ml. 3. Potassium ferricyanide solution: Dissolve 8 g of KsFe(CN)s in distilled water and dilute to 100 ml. 4, Standard phenol solution: Dissolve 100mg phenol in distilled water and dilute to 100ml. 1pL = 1 ug phenol. ReagentsCEFVVOIIDPIAIVUVVVUVUUVUKCCKCGE CC CUSGS 5. Use phenol in the range of 10 to 1501g/10 ml for making the standards Procedure 1. To 10ml of distillate/sample or standards, add 2 ml of buffer solution and mix. The pH of the sample and standards should be 10 + 0.2. Incubate at room temperature for 5 min. 2. Adazo nl aminoantipyrine solution and mix. Incubate at room temperature for 5 min 3. Add2oml potassium ferricyanide solution . Incubate at room temperature for 5 min, 4. After 15 minutes read absorbance at 510 nm, Calculation Prepare a standard curve by plotting the absorbance value of standards versus the corresponding phenol concentrations. Obtain concentration value of sample directly from standard curve.3. Quantification of total detergents /surfactants content in drinking water. Principle ‘The method is based on the fact that the cationic dye methylene blue forms blue salts with anionic surfactants (MBAS), which can be extracted with chloroform. To eliminate interference, the extraction is first effected from alkaline solution and the extract is then shaken with acidic methylene blue solution. The absorbency of the separated organic phase is measured photometrically at the wavelength of maximum absorption of 650 nm. Reagents 1. Buffer solution pH 10 - Dissolve 24 g sodium bicarbonate, NaHCO; and 27 g anhydrous sodium carbonate (Na,COs) in distilled water and dilute to 1000 ml. 2. Neutral methylene blue solution-Dissolve 0.35 g methylene blue in distilled water and dilute to 1 000 mi. Prepare the solution at least twenty-four hours before use. 3. Acidic methylene blue solution- Dissolve 0.35 g methylene blue in 500 ml distilled water and mix with 6.5 ml H)SO, (d= 1.84 g/ml). Dilute to 1 000 ml with distilled water. Prepare the solution at least twenty-four hours before use. 4. Chloroform (trichloromethane) 5. SDS (Stock solution)- dissolve 100mg of SDS in 50 ml of distilled water.Make the volume to 100 ml 6. Working solution — 10 mg/100 ml Procedure 1. Place 100 ml of the sample into a 250 ml separating funnel. For standards use SDS in the range of 20to 150 wg/100 ml. 2. Add to the sample 10 ml of buffer solution, 5 ml of neutral methylene blue solution and 15 ml of chloroform 3. Shake the mixture uniformly and not too vigorously for one minute. ~ 4, After phase separation, run the chloroform layer into a second separating funnel, containing 110 ml of distilled water and 5 ml of acidic methylene blue solution 5. Shake the mixture for one minute. 6. Pass the chloroform layer into a graduated flask Calculation of results Prepare a standard curve from sodium dodecyl sulphate( SDS). The amount of anionic surfactant (MBAS) in the sample is read from the calibration curve——_E_ TTT ne ae SS _ UlUmUttt”~—” eeccevv ORCeOVISV®IFVGVHUUKCHUSEGESCES 4. Assay of soil urease activity by colorimetric estimation of ammonia release. ‘The rapidly increasing importance of urea in agriculture has drawn attention to the need for methods to estimate its hydrolysis in soil. Urease activity in soil can be analysed by berthelot method. Urease is an enzyme that catalyzes the hydrolysis of urea into carbon dioxide and ammonia. CO(NH,), + H,0—=92NH, + CO, The indophenol method for determining ammonia in soil samples is based on the formation of an indophenol blue pigment during the reaction of phenol and hypochlorite in the presence of ammonia. In alkaline medium (pH 8-11.5) a chloramine is first produced. This reacts with the surplus hypochlorite and with phenol forms quinone chloramine in the presence of catalytic quantities of sodium nitroprusside. Quinone chloramines further reacts with surplus pheno! to produce indophenol. Reagents- Sodium hypochlorite Toluene Potassium citrate - 10g/100 ml Urea - 102/100 ml Phenolic solution: i. Solution A- 6.2gm phenol+ Sml methanol 1.8ml acetone+ Sml ethanol ii Solution B- 2.7gm NaOH/ 10 ml distilled water Mix solution A and B. Make the final volume to 50m! 7. Ammonium sulphate standard solution- 100mg/100ml. For standards use ammonium sulphate in the range of 20 to 300 yg /10 ml vaeyer Procedure Take 10gms of soil sample in a flask. ‘Add Smi toluene. Incubate at room temperature for 10 min. Add 20m! of potassium citrate. Incubate at room temperature for 10 min. Add 10m! of uréa. Incubate at 37°C for 10 min. Make the final volume to 100ml and filter it. Take 10 ml of filterate Add Smi of phenolic reagent and 3ml of sodium hypochlorite. Incubate at room tempefature for 5 min. _ saya eNeeeseeev rT PT fr Pr UL vPrFoVvHvHWOHeUVEBEWYY 992) 8 ae ee ~ov»v Calculation Prepare a standard curve by plotting the absorbance value of standards versus the corresponding ammonium sulphate concentrations. Obtain concentration value of sample directly from standard curve.e&vv VOWIDIIAIVVHGH Vso vesesesseesevse 5. Estimation of chemical oxygen demand of a given water sample. Principle water consumed for chemical oxidation COD is the measurement of the amount of oxygen in f COD in ground and surface waters, of pollutants. This method covers the determination o domestic and industrial wastewaters. The applicable range is 3-900 mg/L. ‘The organic matter present in sample gets oxidized completely by potassium dichromate sulphate (AgSOs) and mercury (KsCha0r) in the presence of sulphuric acid (H2S0%), silver 5 sulphate (HgS04) to produce CO2 and H20. The sample is refluxed with a known amount of potassium dichromate (K2Cn07) in the sulphuric acid ‘medium and the excess potassium ichromate (KaCr207) is determined by titration against ferrous ammonium sulphate, using ferroin as an indicator. The dichromate consumed by the sample is equivalent to the amount of O2 required to oxidize the organic matter. Reagents Potassium dichromate (K2Cr207) 0.25N Sulphuric acid (HiSO.) / silver sulphate (Ag:SOs) solution Mercuric sulphate (HgSOs) crystals Ferrous ammonium sulphate (FAS) [Fe(NHs)a(SOs)2}, 0.1N Ferroin indicator (I, 10-phenanthroline and ferrous ammonium sulphate) wane Chemical Preparation 1. Dissolve 12.25g of potassium dichromate in distilled water and dilute to 1 litre volume in a volumetric flask. 2, Add 2.2g of reagent grade silver sul (804) and mix until the silver sulphate goes into solution. 4, Use gpl°e of mercuric sulphate (H1gSO1) to complex 100 mg chloride (2,000 mg/l). 4. Dissolve 1.485g of 1,10-phenanthroline monohydrate and 0.695g of ferrous ammonium sulphate heptahydrate in distilled water and dilute to approximately 100ml. Dissolve 3.9g reagent grade ferrous ammonium sulphate hexahydrate in distilled water. ‘Add 2ml of concentrated sulphuric acid (HSO,). Cool and dilute to exactly 100ml in a volumetric flask using distilled water. phate to a 400ml of concentrated sulphuric acide©eeoeeee « SRVVOVVIIFXIF OH eovHostuesoss Procedure 1 9. Place a 25ml sample or an aliquot diluted to 25ml in a 500ml refluxing flask. The blank is prepared using 25m of distilled water. Add 5 to 7 glass boiling beads. ‘Add 0.5g of mercuric sulphate (HgSO»), 2.5ml of concentrated sulphuric acid / silver sulphate solution, and mix until the HgSOz is in solution. The function of the mercuric sulphate is to bind or complex chlorides. ‘Accurately add 12.5ml of 0.25 N potassium dichromate (K2Cr207) and mix. ‘Add while mixing, an additional 35ml of concentrated sulphuric acid-silver sulphate solution. ‘After thorough mixing, attach the flask to the reflux condenser, apply heat, and reflux for ation of organic 2 hours. Refluxing time can be decreased depending on the ease of oxidat materials. This time may be determined by refluxing for periods from 15 minutes to 2 hours and comparing the results. ‘A reagent blank containing 25ml of distilled water treated with the same reagent as the sample should be refluxed with each set of samples. Cool the apparatus to room temperature after the refluxing period. Wash down the interior of the condenser and flask twice with approximately 25ml portions of distilled water. Remove flask from the condenser and dilute to a final volume of approximately 175ml with distilled water ‘Add 4 to 5 drops of Ferroin indicator 10. Titrate with 0.1 N ferrous ammonium sulphate to the first red-brown endpoint. COD calculations COD (mg/l) = (a-b)(N) x 8,000 / sample size (ml)OVS HOVIIDIIVVHCVOHHHHETELSEEECSCEGES 6. To measure biochemical oxygen demand of a sample by titrimetric method. In the presence of free oxygen, aerobic bacteria use the organic matter found in wastewater as “food”. The BOD test is an estimate of the “food” available in the sample. The more “food” present in the waste, the more Dissolved Oxygen (DO) will be required. The BOD test measures the strength of the wastewater by measuring the amount of oxygen used by the bacteria as they stabilize the organic matter under controlled conditions of time and temperature. a. Principle: The method consists of filling with sample, to overflowing, an airtight bottle of the specified size and incubating it at the specified temperature for 5 days. Dissolved ‘oxygen is measured initially and after incubation, and the BOD is computed from the difference between initial and final DO. Because the initial DO is determined shortly afier the dilution is made, all oxygen uptake occurring after this measurement is included in the BOD measurement. Reagents 1. Phosphate buffer solution: Dissolve 8.5 g KH,POs, 21.75 g K.HPO., 334 g NasHPO,.7H20, and 1.7 g NH,Cl in about 500 mL distilled water and dilute to 1 L. The pH should be 7.2 without further adjustment. Alternatively, dissolve 42.5 g KH2PO, or 54.3 g KoHPOs in about 700 mL distilled water. Adjust pH to 7.2 with 30% NaOH and dilute to 1 L. 2. Magnesium sulfate solution: Dissolve 22.5 g MgSOx.7H20 in distilled water and dilute to IL. Calcium chloride solution: Dissolve 27.5 g CaCl in distilled water and dilute to 1 L. Ferric chloride solution: Dissolve 0.25 g FeCls.6H20 in distilled water and dilute to 1 L. ‘Acid and alkali solutions, IN, for neutralization of caustic or acidic waste samples. Acid—Slowly and while stirring, add 28 mL cone sulfuric acid to distilled water. Dilute tol L. 7. Alkali—Dissolve 40 g sodium hydroxide in distilled water. Dilute to 1 L. 8. Nitrification inhibitor, 2-chloro-6-(trichloromethyl) pyridine. 9. Glucose-glutamic acid solution: Add 150 mg glucose and 150 mg glutamic acid to distilled water and dilute to 1 L. Prepare fresh immediately before use. 10. Ammonium chloride solution: Dissolve 1.15 g NH4Cl in about 500 mL distilled water, adjust pH to 7.2 with NaOH solution, and dilute to 1 L. Solution contains 0.3 mg N/mL. 11. Dilution water: Use distilled, tap, or natural water for making sample dilutions. aya Preparation of dilution water: Place desired volume of water in a glass bottleand add 1 mL each of phosphate buffer, MgSO4, CaCI2, and FeCI3 solutions/L of water. Seeddilution water, if desired. ,