STP Determination of Biochemical Oxygen Demand (BOD)

Determination of Biochemical Oxygen Demand (BOD)


B.O.D bottles capacity 300 ml (or 250 ml)

B.O.D incubator maintained at 20C


0.025 N Sodium thiosulphate

1N Sodium hydroxide

1N Hydrochloric acid.

Phosphate buffer

Dissolve 8.5 gm of K2HPO4, 33.4 gm of Na2HPO4.7H2O and 1.7 gm of NH4Cl in about  500 ml of water and dilute to 1000 ml. The pH of this solution should be 7.2

Magnesium Sulphate Solution.

Dissolve 39.2 gm of magnesium sulphate (MgSO4.7H2O) in water and dilute to 1 litre.

Calcium Chloride Solution

Dissolve 27.5 gm of anhydrous calcium chloride in water and dilute to 1 litre.

Ferric Chloride Solution

Dissolve 0.25 gm od water ferric chloride (FeCl3.6H2O) in water and dilute to 1 litre.

Dilution Water

Distilled water of good, aerated and maintained at temperature of 200 C and containing dissolved oxygen not less than 8 mg/litre

Manganous Sulphate Solution

Dissolve 480 gm of manganous sulphate (MnSo4.4H2O) in distilled water and dilute to 1 litre. Filter it before use.

Iodide – Azide Reagent

Dissolve 700 gm of KOH (or 500 gm NaOH) and 150 gm of KI in distilled water dilute to 1 litre. Dissolve 10 gm of sodium azide in 40 ml of distilled water add to 950 ml of the first solution with constant stirring.

Starch Indicator Solution

Boil about 100 ml of water in a beaker. Add aqueous slurry of 0.5 gm starch, continue boiling for 2-3 minutes and cool to room temperature. Store in a refrigerator.


Neutralise the sample to pH about 7.0 with sodium hydroxide or hydrochloride acid. Add 1 ml each of phosphate buffer, magnesium sulphate , calcium chloride and ferric chloride solution for every litre of dilute water. Dilute the sample with dilution water such that oxygen depletion is between 5-6 mg/litre after incubation at 200 C for 5 days.

Fill up two BOD bottles with dilution water (blank) and two with diluted sample. Find out dissolved oxygen in one of the each bottles of blank and sample preparation as below, immediately.

To the blank and sample as collected in a 250-300 ml bottle, add 2 ml manganous sulphate, followde by 2 ml of iodideazide reagent well below the surface of the liquid. Stopper with care to exclude air bubbles and mix by inverting the bottle several times. When the precipitates settle leaving a clear supernatant liquid above manganous hydroxide fioc,

repeat the shaking second time. When the precipitate has settled, carefully remove the stopper and immediately add 2 ml of concentrated sulphuric acid allowing the acid to run down the neck of the bottle; restopper and mix by gentle inversion until solution is completely is completely clear. Now take out 203 ml of the solution in aconical flask and titrate with 0.025 N Sodium thiosulphate solution , using starch solution as indicatior.

   Dissolved oxygen  =  V x N

   (mg/ml)                       0.025

  Where :  

                 V = Volume of 0.025 N sodium thiosulphate used

                 N = Normality of sodium thiosuphate

     Important Instructions

     Dilution of the sample can be adjusted by the following formula.

i)   Untreated effluent

For untreated effluent.

Expected BOD = COD  x  2  =  a


       So Dilution can be adjusted as

      a  x  1   =  b


      a  x  1  =  c


  i.e, dilute 1 ml of sample in between b & c volume with water.

 e.g, if COD = 9000 ppm

Then expected BOD = 9000 x 2  i.e, 6000 ppm    

 So dilute can be adjusted as

6000  x  1   =  2000


6000  x  1   =  1200


Therefore, dilute 1 ml of sample to 1500 ml (i.e any valve between (1200 to 2000)

For more accuracy dilute can be adjusted  like.

10 ml  =  100 ml                    100  x  1500  =  1500 times

10 ml  =   1500 ml                  10         10


ii) Treated Effluent


For treated effluent, expected BOD can be calculated as:

   BOD = COD  x  1   =   d


  Rest of the calculation is as in untreated effluent.

 After determination of dissolved oxygen in blank and sample of zero day at 200 C, keep the other two bottle one each of blank and sample at 200 C in a BOD incubator for 5 days

After incubation at 200 C for 5 days, find out the dissolved oxygen in blank and sample.


     Biochemical oxygen demand = (D1- D2)-(C1-C2)  x  F


      (5 days at 200 C)

Where :

     D1, D2  = Dissolved oxygen in diluted sample on zero day and after 5 days incubation

                     at 200 C respectively.

    C1, C2  = Dissolved oxygen in blank on zero day and after 5 days incubation at 200 C


    F          = Dilution factor for sample.

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