Determination of Biochemical Oxygen Demand (BOD)



Biochemical oxygen demand or BOD is a chemical procedure for determining the uptake rate of dissolved oxygen by the biological organisms in a body of water. 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. In this experiment dissolved oxygen is measured by winkler’s method.


The Winkler test is used to determine the concentration of dissolved oxygen in water samples. . An excess of manganese (II) salt, iodide (I-) and hydroxide (HO-) ions are added to a water sample causing a white precipitate of Mn(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 sulfuric acid) is added to acidify the solution. The brown precipitate then converts the iodide ion (I-) to Iodine. The amount of dissolved oxygen is directly proportional to the titration of Iodine with a thiosulfate solution.


1)      Water sample to be tested.

2)      Phosphate buffer solution. 

3)      MgSO4 solution. 

4)      FeCl3 solution .

5)      CaCl2 solution. 

6)      Six BOD bottles. 

Reagents for Winkler’s test

1)      MnSO4 solution.

2)      Alkali iodide azide reagent.

3)      Concentrated H2SO4 solution.

4)      0.025 N Na2S2O3 solution. 

5)      Starch indicator (2 gm%). 

6)      Conical flask, burate and pipettes.


I.        Preparation of dilution water

1.     Take distilled water as per the requirement in a glass container and bubble compressed air for 24 hours.

2.     Add 1 ml of phosphate buffer, MgSO4 solution, FeCl3 solution and CaCl2 solution for each liter of distilled water.

3.     Preferably do not store the prepared dilution water for more than 24 hours, after adding minerals, nutrients and buffer unless dilution water blanks consistently meets quality control limits.

II.      Determination of BOD

1.     If the sample is not expected to contain sufficient bacterial population, add seed to dilution water (2 ml settled sewage for 1000ml of dilution water or activated sludge).

2.     Neutralize the sample to pH 7.0.

3.     The dilution of the sample is made such that residual dissolved oxygen after incubation will not be less than 1 mg/lit. And dissolved oxygen uptake of at least 2 mg/lit.

4.     Mix measured volume of a sample in BOD bottle with dilution water to get desired dilution.

5.     Take six BOD bottles and number them 1 to 6.

6.     Add the contents in BOD bottles as follow: 

BOD bottle No:

Contents to be added:


Dilution water


Dilution water + Measured volume of sample


Dilution water + Measured volume of sample


Dilution water + Measured volume of sample


Dilution water + Measured volume of sample


Dilution water

Bottle No: 1 & 6 are known as dilution water blanks. 

1.     As a precaution against drawing air into the BOD bottle, use a water seal by adding water to the flared mouth of the BOD bottle.  

2.     Determine initial dissolved oxygen (DO) in bottle no.1 by Winkler’s method known as DO of blank on ‘0’ day. Take average reading of DO in bottle no. 2 & 3 which is known as DO of sample on ‘0’ day.

Winkler’s method for determination of DO

o   Take the sample in 300ml BOD bottle. Add 2ml of MnSO4 solution, 2ml of alkali iodide azide reagent. The tip of the pipette should dip below the liquid surface.

o   Invert several times the BOD bottle to mix. Thereby allowing brown   precipitates to form. Allow the precipitates to settle down to at least half of the bottle

o   Add 2ml of concentrated H2SO4 solution with rubber bulb pipette. Restopper the BOD bottle and mix by inverting several times. This will dissolve the precipitates and turn the sample to golden yellow color.

o   Transfer 203ml of clear solution to 250ml flask and titrate it against 0.025 N Na2S2O3 till pale yellow color appears.

o   Add 2-3 drops of starch indicator. The color becomes deep blue. Continue the titration slowly until the color changes to colorless. Note down the burette reading.

o   The concentration of DO in the sample is equivalent to the number of ml of titrant used.    

1mg/ Lit Dissolve oxygen = 1ml of titrant

o   Keep bottle no: 4, 5 & 6 in BOD incubator for predetermined 3 days incubation at 27oC by covering with black paper to exclude light, to prevent possible photosynthetic production of DO.

o   After 3 days incubation, determine DO of bottle no. 4, 5 & 6 by Winkler’s method.

o   Average DO of bottle no.4 & 5 is known as DO of   sample on 3rd day. Whereas DO of bottle no. 6 is known as DO of blank on 3rd day. The DO uptake of dilution water blank should not be more than 0.2 mg/lit.                   

o   Calculate BOD of the sample. 


         Clesceri L.S., Greenberg A.E., Eaton A.D. (1998) Satandard methods for the examimnation of water and waste water. 23rd edition APHA.

         Kulkarni P.D. Laboratory manual in environmental engineering. Jaico Publishing House. Bombay. 


BOD5 Days            =     (DO 0th Day  - DO 5th Day) X Volume of BOD bottle /  Volume of sample per BOD bottle


=      _____________________ mg/Lit   

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