1.0 Scope
This SOP is applicable for the determination of Biochemical Oxygen demand in waste water.
2.0 Responsibility – Chemist
3.0 Principle
The biochemical oxygen demand (BOD) test is based on mainly bio-assay procedure which Measures the dissolved oxygen consumed by micro-organisms while assimilating and oxidizing the organic matter under aerobic conditions. The standard test condition includes incubating the sample in an air tight bottle, in dark at a specified temperature for specific time.
4.0 Apparatus
- BOD Bottle 300 ml
- BOD incubator
- Burette
- Pipette
- Beaker
5.0 Reagents
5.1 Phosphate Buffer Solutions
Dissolve 8.5 g potassium dihydrogen phosphate (KH2 PO4), 21.75 g di-potassium hydrogen phosphate (K2HPO4), 33.4 g disodium 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 around 7.2 without any further adjustment.
5.2 Magnesium Sulphate Solution
Dissolve 22.5 g magnesium sulphate (MgSO4.7H2O) in distilled water and dilute to 1 litre.
5.3 Calcium Chloride Solution
Dissolve 27.5 g calcium chloride in distilled water and dilute to 1 litre.
5.4 Ferric Chloride Solution
Dissolve 0.25 g hydrated ferric chloride (FeCl3.6H2O) in distilled water and dilute to 1 litre.
5.5 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.
5.6 Glucose-Glutamic Acid Solution
Dry reagent grade glucose and reagent grade glutamic acid at 103°C for 1 hour. Add 150 mg of glucose and 150 mg of glutamic acid to distilled water and dilute to 1 litre. Prepare fresh immediately before use.
5.7 Other Reagent for Dissolve Oxygen Measurement
Refer SOP of Dissolve Oxygen
6.0 Procedure
6.1 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°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 to 5 ml of treated sewage per litre of dilution water for seeding purpose.
NOTE — Seeding is not required for domestic Sewage or surface water samples.
6.2 Dilution of Sample and Incubation
6.2.1 Neutralization
Neutralize the sample to pH around 7.0 using alkali and acid of such strength that the quantity of reagent does not dilute the sample by more than 0.5 Percent.
7.0 Procedure
7.1 Sample Volume and Dilution Technique
- On the basis of chemical oxygen demand (COD), determine expected BOD. Use the Following formula for calculating sample volume:
X
- Sample volume in ml, per litre dilution = —————————— X 1000
Expected BOD
- For Keeping 2 dilutions Take X=2.5 and 4.0
- For single dilution take X = 3.0 to 3.5
- Round off to nearest convenient volumes fraction.
- In case of high BOD samples, Prepare primary dilution with distilled water and then make the final dilution.
- Take requisite quantity of sample in one litre volumetric flask. Dilute to the mark with the dilution water by siphoning from the container (7.1). Mix well.
- Rinse three BOD bottles with the diluted sample and fill up these bottles with the diluted sample. Stopper the bottles immediately after removing the air bubbles.
Note: Samples of natural surface water bodies like river, lake and marine, generally do not require seeding and dilution due to naturally available microbiological population and low BOD values. For such samples which are likely to have BOD less than 5 mg/l, BOD determination may be carried out as such (100 percent) without any dilution.
7.2 Determination of Initial Dissolved Oxygen (DO)
- Determine initial DO for one bottle and keep two bottles for incubation at 27°C ± 1°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°C ± 1°C for 3 days.
Note: DO analysis As per SOP of Dissolved Oxygen
7.3 Determination of Final Dissolved Oxygen
After 3 days incubation at 27°C ± 1°C, determine final DO in incubated bottles.
Note: DO analysis As per SOP of Dissolved Oxygen
8.0 Calculations
8.1 When Sample is Undiluted
BOD, mg/l = DO before incubation – DO after incubation.
8.2 When Dilution Water is seeded
BOD, mg/l = (D1-D2)/P X 1000
8.3 When Dilution water is seeded
BOD, mg/l = D1-D2-(B1-B2)f/P X 1000
Where,
D1 = Initial DO of sample in mg/l,
D2 = DO of sample after incubation in mg/l,
B1 = DO of seed control before incubation in mg/1,
B2 = DO of seed control after incubation in mg/1,
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).
9.0 Glutamic Acid Check
Note: BOD being a bioassay test is greatly influenced by factors like toxicants, poor seeding, etc.
- For periodical checking of these factors, use a mixture of 150 mg glucose and 150 mg glutamic acid per litre as a standard check solution.
- Determine the 3 days 27°C BOD of 2 percent dilution of the glucose-glutamic acid standard check solution as in 8.0.
- If the BOD value of the check is outside the range of 200 ± 37 mg/l, reject any BOD determinations made with the seed and dilute water and seek the cause of the problem.
10.0 Safety Guidelines
Proper Safety and health precaution are taken while doing the analysis by using safety goggles, Apron, Safety shoes and Using Fuming Chamber for fume generating chemicals.
11.0 References
IS 3025 (P-44): 1993 RA-2019