1. PRINCIPLE
1.1. The biochemical oxygen demand (BOD) test is based in mainly bio-assay procedure which measures the dissolved oxygen consumed by micro-organism while assimilating and oxidizing the organic matter under aerobic conditions.
1.2. The standard test condition includes incubating the sample in an air tight bottle, in dark at a specified temperature for specific time.
2. APPARATUS
2.1 Incubation Bottles: 300 ml capacity narrow neck special BOD bottles with planed mouth with ground glass stoppers. New bottles should be cleaned with 5 N hydrochloric acid or sulphuric acid followed by rinsing with distilled water. In normal use, bottles once used for winklers procedure should only be rinsed with tap water followed by distilled water.
2.1.1. During incubation ( if incubator is used ) to ensure proper sealing, time to time add water to the flared mouth of the bottles.
2.2 Water Bath or Air Incubator : Air incubation with thermostatically controlled 27 Deg.C + 1 Deg. C. Avoid light to prevent possibility of photosynthetic production of oxygen.
NOTE - Thermostatically controlled at 27 Deg. C + 1 Deg C water bath with continuous stirring may be preferred.
3. REAGENTS
3.1 Phosphate Buffer Solution : Dissolve 8.5 gm potassium dihydrogen phosphate ( KH2PO4), 21.75 g potassium hydrogen phosphate |(K2HPO4) , 33.4 g dosodium hydrogen phosphate (Na2HPO4,7H2O) and 1.7 g ammonium chloride (NH4Cl) in about 500 ml distilled water and dilute to 1 litre, pH of the solution should be aroubd 7.2 without any further adjustment.
3.2 Magnesium Sulphate Solution : Dissolve 22.5 g magnesium sulphate ( MgSO4, 7 H2O) in distilled water and dilute to 1 litre.
3.4 Calcium Chloride Solution : Dissolve 27.5gm calcium chloride in distilled water and dilute to 1 litre.
3.5 Ferric Chloride Solution: Dissolve 0.25 gm hydrated ferric chloride (FeCl3, 6H2O) in distilled water and dilute to 1 litre.Acid and Alkali Solution 1 N Sodium hydroxide and 1 N sulphuric acid for neutralization of samples.
NOTE : Any of the above solutions showing any sign of biological growth may be discarded.
4. OTHER REAGENTS FOR DISSOLVED OXYGEN MEASUREMENT.
4.1 Manganous Sulphate Solution : Dissolve manganese sulphate ( 480 g of MnSO4, 4H2O or 400 g of MnSO4 . 2H2O or 364 g of MnSO4,H2O) in freshly boiled and cooled water, filter and make up to 1,000 ml . The solution should not give blue colour by addition of acidified potassium iodide solution and starch.
4.2 Alkaline Iodide Sodium azide Solution : Dissolve 500 g of sodium hydroxide ( or 700 g of potassium hydroxide ) and 135 g of sodium iodide ( or 150 g potassium iodide) in freshly boiled and cooled 950 ml water slowly add solution of 10 gm NaN3 in 40 ml H2O and dilute to 1 litre.
4.3 Sulphuric Acid, Concentrated : Starch Indicator
Disoolve 2 g of starch and 0.2 g of salicylic acid as preservative, in 100 ml of hot distilled water.
4.3 Sodium Thiosulphate Stock Solution : Dissolve approximately 25 g of sodium thiosulphate (Na2S2O3,5H2O) in boiled distilled water and make up to 1000 ml . Add 1 g of sodium hydroxide to preserve it.
4.4 Standard Sodium Thiosulphate Solution: Dissolve 250 ml of stock solution in boiled distilled water and make up to 1 litre and standardize sodium thiosulphate against known standard before use.
4.4 Preparation of dilution water : Aerate the required volume of distilled water in a container by bubbling compressed air for 8 to 12 hours to attain dissolved oxygen saturation. Let it stabilize for 4 h at room temperature( around 27 Deg. C ) At the time of use, add 1 ml each of phosphate buffer, magnesium sulphate , calcium chloride and ferric chloride for each litre of dilution water.
Add 2 ml to 5 ml of treated sewage per litre of dilution water for seeding purpose. Dilution of Sample and Incubation
4.5 Neutralization: Neutralis the sample to pH around 7.0 using alkali or acid of such strength that the quantity of reagent does not dilute the sample by more than 0.5 percent.
5. PROCEDURE
5.1 Determination of Initial Dissolved Oxygen ( DO) : Determine initial DO for one bottle and keep two bottles for incubation at 27 Deg.C + 1 Deg.C for 3 days. Prepare six blanks by siphoning out dilution water directly into the bottles. Determine initial DO in two bottles and incubate remaining four bottles at 27 Deg.C + 1 Deg. C for 3 days.
5.2 Determination of final DO : After 3 days incubation at 27 Deg C + 1 Deg. C , determine final DO in incubated bottles.
6. PROCEDURE TO ESTIMATE THE DISSOLVED OXYGEN
6.1 Add 2.0 ml MnSO4,Soln and 2.0 ml alkaline - Sodium azide , solution to sample in 250 or 300 ml BOD bottle, replace stoper , excluding air bubbles , and invert several times to mix,Let floc settle and repeat mixing. After floc has settled , leaving 100 ml clear supernate, remove stopper and add 2.0 ml H2SO4 , dawn neck of bottle.Restopper and mix by inversion untilI is uniformily distributed. Immediately titrate, 203 ml ( 3 ml is allowance for added reagents ) with 0.025 n Na2S2O3 to pale straw yellow. Add 1-2 ml starch indicator and titrate,to disappearance of blue . Disregard reappearance of blue.
6.2 Calculation of Dissolved Oxygen = (0.025 N Na2S2O3 used x 0.2N/200) x 1000.
7. CALCULATION OF BOD
BOD , mg
/I =
D1-D2-(B1-B2) f x
1000
P
Where
D1 = Initial Do of
sample in mg/Litre
D2 = Do of sample
after incubation in mg/Litre
B1 = DO of seed
control before incubation in mg/Litre
B2 = DO of seed
control after incubation in mg/Litre
f =
ratio of seed in diluted sample to seed in control ; (
percent seed in diluted sample ) : ( percent seed in
seed control ).
p = Percentage dilution of
sample ( sample volume in ml/10).
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