Determination of Biological Oxygen Demand (BOD)

Chemicals Required:
1. Manganous Sulphate Solution (MnSO4.4H2O)
2. Alkali-iodine
Winkler’s method for
3. Conc. Sulphuric Acid (H2SO4) dissolved oxygen
4. Starch Solution
5. Std. Sodium Thiosulphate Solution (0.025 N)
Procedure:- Winkler Method

1. Take 300 ml of water sample in the bottle. Add 2 ml of manganous sulfate

solution and 2 ml of alkali-iodide solution to the sample.
2. Mix the solution obtained by inverting the bottle for at least 15 times.
3. Precipitate begin to form and allow it to settle ,leaving a clear supernatant liquid
above ,shake again.
4. After two minute of settling ,remove the stopper and add 3 ml of conc. H2SO4 by
allowing acid to run from the neck of the bottle.
5. Apply the stopper and mix again until the dissolution is complete.
6. Take about 203 ml of the solution from the bottle to flask.
7. Titrate with 0.025 N sodium thiosulfate solution to pale straw color.
8. Add 1.2 ml starch solution and continue the titration until blue color disappears
and note down the volume of sodium thiosulfate added which give DO value in
mg/L
Preparation of dilution water: To prepare dilution water, aerate the required volume of BOD
free distilled water in a container by bubbling compressed air for 1 to 2 days to attain
dissolved O2 saturation. Add per liter in this water 1 ml each of phosphate buffer solution,
MgSO4 solution, CaCl2 solution and ferric chloride solution.

Sample Preparation: Use dilution water to prepare BOD samples:

- Blank (only dilution water) in 300 mL BOD bottle. Minimum two sets for
each sample
- 20 mL sample in 300 mL BOD bottle and then fill up with dilution water.
- 50 mL sample in 300 mL BOD bottle and then fill up with dilution water.

Keep one set for DO analysis for day 0 (DO0)and keep another sample set in BOD incubator
for 5 days at 20°C. Take out the bottles after 5 days and determine immediately their
dissolved oxygen content (DO5)