Biological oxygen demand

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Biological Oxygen Demand is the measure of uptake rate of oxygen by microorganisms in oxidation state. BOD is one of the most common measurement methods of measuring pollutant organic material in water. The dissolved oxygen (DO) level of a water sample is measured for five days which is stated as BOD5. The DO level of the sample is measured on the same day of collection of sample and the sample is incubated for five days in dark at a temperature of 20-oc and DO level is measured again. BOD5 is the difference between the DO levels of initial and final value on 5th day of incubation.

In 1908 The Royal Commission on River Pollution and The Royal Commission of Pollution Disposal has selected BOD5 as the definitive method to measure organic pollution of rivers. The test period five days appropriate as it the time taken for the river water to travel from source to destination.

The amount of Nitrates and Phosphates which are plant nutrients in water can contribute the increase in BOD levels. The plants and algae grow quickly which in turn decomposed by bacteria contributing the organic waste in water resulting increased BOD levels. The temperature of the water has also effect on BOD levels. The BOD levels increases with increase in temperature of water.

Dissolved Oxygen (DO) is determined by Winkler's Method developed by Lajos Winkler in 1988. The amount of dissolved oxygen is considered as measure of the biological activity of the water.

Materials Required:

Apparatus and Lab ware:

  1. Series of 250-300 ml BOD incubation bottles with ground glass stoppers, and caps
  2. Incubator set at 20 degrees Celsius and holding no light.
  3. Two large carboys (capacity depending on sample amount)
  4. Series of class A pipets (0.2 ml-10 ml)
  5. Aeration device
  6. pH meter
  7. Erlenmeyer flasks for preparation of reagents and titration.
  8. Seed
  9. 50 ml class A burette
  10. Series of volumetric flasks
  11. Series of beakers
  12. Double distilled and De-ionised water


  1. Phosphate buffer solution
  2. Dissolve 8.5g potassium dihydrogen phosphate (KH2PO4), 21.75g dipotassium hydrogen phosphate (K2HPO4), 33.4g disodium hydrogen phosphate (NA2HPO4.7H2O), 1.7g ammonium chloride (NH4Cl) in water (distilled) to makeup 1 litre of solution.

  3. Magnesium sulphate solution
  4. Dissolve 22.5g MgSO4.7H2O in distilled water to give 1 litre of solution.

  5. Calcium chloride solution
  6. Dissolve 27.5g anhydrous CaCl2 in distilled water to give 1 litre of solution.

  7. Ferric chloride solution
  8. Dissolve 0.25g FeCl3 6H2O in distilled water to give 1 litre of solution.

Reagents for Winkler Method:

  1. 0.125M Sodium thiosulphate solution:
  2. Dissolve 6.25g of Na2S2O3.5H2O in 1litre distilled water.

  3. Alkali-Iodide - azide solution:
  4. Dissolve 500g NaOH and 140g of NaI to 1 litre of distilled water with dissolved10g of NaN3 in 40ml distilled water.

  5. Concentrated sulphuric acid
  6. Manganese sulphate solution:
  7. Dissolve 480g MnSO4 .--------4H2O in 1litre of distilled water.

  8. Starch Indicator:

Dissolve 2g of soluble potato starch in 100ml of distilled water heated and stir to mix it well.


  1. The sample is aerated initially such that the initial DO level is a satured level.
  2. The sample is diluted with respective dilution factor based on the potential BOD level.
  3. Fill the incubation bottles with sample and dilution water to making up the volume and insert the stoppers.
  4. Precaution is taken such that no air is trapped inside the incubation bottles.
  5. Mix the samples in the bottles by inverting the them several times.
  6. The Dissolved oxygen is determined by the Winkler method.
  7. The bottles are incubated at 200C for 5 days in dark and are used for measuring DO level by Winkler Method.

Winkler Method:

  1. Add 2ml of Manganese sulphate solution and 2ml of Alkali Iodide - Azide solution to water sample.
  2. Care should be taken while adding; the pipette tip should be below the surface level of the sample in the bottle.
  3. Replace the stopper carefully without air bubbles by inclining the bottle .
  4. Invert the bottle several times such that the contents of bottle are thoroughly mixed.
  5. Thus leave the sample to settle down the precipitate. Mix and allow settling until a clear supernatant is formed.
  6. Add 2ml of concentrated Sulphuric acid along the walls of the bottle.
  7. Precaution is taken in handling the concentrated acid by wearing protective gloves.
  8. Allow the precipitate to dissolve by inverting mix the contents thoroughly. The sample turns into bright gold color.
  9. Transfer 200ml of sample to Erlenmeyer flask for titration with 0.0125M sodium thiosulphate solution.
  10. Titration is carried out with sodium thiosulphate solution until a pale straw color appears.
  11. Few drops of starch solution is added to produce blue color as indication of presence of iodine.
  12. The titration is continued until blue color disappears. The amount of sodium thiosulphate used for titration is recorded.


Calculation of Initial Dissolved oxygen (DO0) by Winkler method:

1 ml of 0.0125M thiosulphate solution = 0.0125x10-3/4 mole of dissolved oxygen

= (0.0125x10-3/4) X 32 g of dissolved oxygen

= 0.1 mg dissolved oxygen.

Amount of sample used for titration = 272ml

Amount of Sodium thiosulphate solution used for titration = 26.8ml

Initial Dissolved oxygen (DO0) = (2.68 x 1000)/272

DO0 = 9.853 mg/L

... (Since 1ml = 0.1mg DO, 26.8ml = 2.68mg DO)

Calculation of Final Dissolved oxygen (DO1) by Winkler method:

Amount of sample used for titration = 200ml

Amount of Sodium thiosulphate solution used for titration = 10.2ml

Final Dissolved oxygen (DO1) = (1.02 x 1000)/200

DO1 = 5.1 mg/L

.... (Since 1ml = 0.1mg DO, 10.2ml = 1.02mg DO)

Calculation of Biological Oxygen Demand (BOD)

Biological Oxygen Demand (BOD) = (DO0 - DO1)/F

Where, DO0= initial dissolved oxygen concentration (mg/L)

DO1= dissolved solution level after 7 days incubation (mg/L)

F= dilution factor

Therefore, BOD (mg/L) = (9.853- 5.1) / 1/5

= 4.753 X 5

= 23.765 mg/L

The BOD test was carried out for the primary and secondary wastewater samples. The tabular column shows the BOD values obtained.


Effluent samples were collected from primary treatment effluent plant and secondary treatment effluent plant. BOD values of these effluents are determined by Winkler method. The samples are diluted with respective dilution factor, for primary treated effluent sample a dilution factor of 1:20 is used and for secondary treated effluent sample a dilution factor of 1:5 is used.

The initial DO values are determined by Winkler method and the samples are incubated at 20oC in dark for 7days (practically for 5days). The samples are added with Manganese sulphate and Alkali- Iodide azide solution which produces white precipitate which turns into Orange-brown precipitate by the dissolved oxygen. By the addition of concentrated sulphuric acid to solution the iodine from potassium iodide is liberated by manganese hydroxide and the solution appears in brown color. On titration with sodium thiosulphate the brown color turns into pale straw color. The amount of iodine is found by addition of starch to it which results in blue coloration. The titration is continued until the blue color disappears. The amount of sodium thiosulphate drained to titrate is recorded and the value of Dissolved Oxygen (DO) is found. 1ml of sodium thiosulphate (0.0125M) is equivalent to 0.1mg dissolved oxygen.

BOD values of primary effluent are higher than the secondary effluent as the organic matter in the primary effluent is more either in suspended solids form or dissolved form. The BOD levels of primary effluent are found to be ranging from 128-157mg/L, but the acceptable range for primary effluent treatment is 80-120mg/L(Table:1) hence the primary effluent is to be treated further to release into environment. The BOD levels of secondary effluent is found to be ranging from 17-23mg/L (Table: 2), the acceptable range for release of secondary effluent is less than 20mg/L. The initial DO value is found to be 9.853 mg/L and final DO value for secondary effluent is 5.1mg/L. The BOD level of the secondary effluent is found to be 23.8. The overall


  • BOD values of primary effluent and secondary effluent are determined.
  • Winkler method is used to determine the DO values.
  • The BOD values of primary effluent are higher than the secondary effluent.
  • The organic matter in the primary samples is more than the secondary effluents.
  • The BOD levels of secondary effluent


  • Montgomery, H.A.C., Thorn, N.S. and Cockburn, A. Determination of dissolved oxygen by the Winkler method and the solubility of oxygen in pure water and sea water. J. Applied Chemistry 1964, 14, 280.
  • "Standard Methods for the Examination of Water and Wastewater," American Public Health Association, 18th Edition, 1992.
  • Warren Viessman, Jr, Mark J. Hammer. 1993. Water Supply and Pollution Control. Harper Collins College Publishers. 5th ed.