BIOCHEMICAL OXYGEN DEMAND (BOD)

DEFINITION
The biochemical oxygen demand is an empirical test to determine the relative oxygen requirements of wastewaters, polluted
waters and effluents.

APPARATUS

1. Incubation bottles 300-ml capacity, with ground-glass stoppers.

2. Incubator, thermostatically controlled at 201oC. Exclude Iight

REAGENTS

1. Distilled water, from an all-glass distiller.

2. Phosphate buffer solution, pH 7.2: Dissolve 1.70g ammonium chloride, NH4Cl; 21.75g dipotassium hydrogen phosphate,
K2HPO4; 33.4g disodium hydrogen phosphate heptahydrate, Na2HPO4.7H2O and 8.5g potassium dihydrogen phosphate,
KH2PO4, in about 500 ml distilled water and dilute to 1 liter. Discard the solution if presence of biological growth is seen.

3. Magnesium sulphate solution: Dissolve 22. 50g MgSO4.7H2O in distilled water and dilute to 1 liter.
4. Calcium chloride solution: Dissolve 27.50 anhydrous CaCl2 in distilled water and dilute to 1 litre.
5. Ferric chloride solution: Dissolve 0.25f FeCl3.6H2O in distilled water and dilute to .l Iitre.
6. Sulphuric acid, approximately 1M: Dilute 56ml concentrated H2SO4 to 1 liter. For neutralization of caustic waste
samples.

7. Sodium hydroxide solution, 1M: Dissolve 40g NaOH in 200ml distilled water and dilute to 1 litre. For neutralization of
acidic waste samples.

8. Sodium sulphite solution, 0.0125M: Dissolve 1.575g anhydrous Na2SO3 (or 3.152g Na2SO3.7H2O) in 1 liter distilled
water. The solution is not stable and should be prepared daily. The solution is for removal of residual chlorine
compounds.

PRELIMINARY STEPS
1. General
Clean B0D bottles with dilute hydrochloric acid and rinse with large amounts of water, followed by distilled water. Store
all samples at 4oC if analysis cannot be carried out immediately. Begin incubation not later than 24 hr after the sample
is collected. Carry out BOD determination without removing the algae for routine analysis.

2. Preparation Of Dilution Water

a. Aerate distilled water with a supply of clean compressed air. Plug the inlet of air with cotton wool to trap dust
particles. Use distilled water which has been stored at 20 1oC.

b. Place the dilution water in a suitable container. Add 1 ml each of calcium chloride, ferric chloride, magnesium
sulphate and phosphate buffer solutions to each litre of water.

c. If the dilution water is to be stored in the incubator, add the phosphate buffer solution just before using the water.

3. Pretreatment Of Samples
a. Samples containing acidity or alkalinity

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 Neutralize the samples with 1M NaOH or H2SO4 to about pH 7.0 as determined by a pH meter.

PROCEDURE
A. DILUTION TECHNIQUE
The most suitable dilution is one where 50-60% of the DO is consumed at the end of 5 days at 20oC.
a. Prepare standard dilution water in a vessel and add well-mixed samples to obtain the desired dilution.
b. Siphon this mixed dilution into 3 BOD bottles, 2 for incubation and the third for initial DO determination.
c. Stopper the B.O.D. bottles tightly and incubate for 5 days at 20oC.
d. Add water to the flared mouth of special water-seal bottle.
OR
a. Pipette suitable volume of sample into the BOD bottles. Fill with sufficient dilution water to permit insertion
of the stopper without leaving air bubbles.
b. For dilutions less than 1% (v/v), dilute the sample in volumetric flask.

B. Carry out a blank determination using dilution water alone following the procedure above (as for sample).
C. Determine the initial DO using the Azide modification of the iodometric method (see below)
D. Incubate the rest of bottles for 5 days in the dark at 201oC
E. Determine the DO after 5 days using the Azide modification of the iodometric method as given for Dissolved Oxygen.
F. For samples which are sterile (industrial waste), a suitable seed (settled sewage effluent) should be added to the
dilution water. Add 1-2 ml of seed solution per litre of dilution water. Carry out a blank determination of the seeded
dilution water.

CALCULATION
When dilution water is not seeded
BOD mg/l = D1 – D 2

When dilution water is seeded :

BOD mg/l = (D1 – D2)-(B1 –B2)f
P

Where :
D1 = DO of diluted sample immediately after preparation, mg/L
D2 = DO of diluted sample after incubation, mg/L
P = decimal volumetric fraction of sample used
B1 = DO of seed control before incubation
B2 = DO of seed control after incubation
f = ratio of seed in diluted sample to seed in seed control
= (% seed in diluted sample) / (%seed in seed control)

UNIT
mg per litre
EXPRSSION OF RESULTS
Results are to be expressed to the nearest whole number.

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METHOD TO DETERMINE DISSOLVED OXYGEN (DO)
AZIDE MODIFICATION METHOD

PRINCIPLE
The DO determination is based on the addition of manganese solution, followed by strong alkali, to the sample in a glass-
stoppered bottle. The DO rapidly oxidizes the manganous hydroxide to form higher hydroxides. On subsequent acidification
with presence of iodide, iodine is liberated and is equivalent to the original DO in the sample. The iodine is then titrated with
standard sodium thiosulphate.

APPARATUS

1. BOD bottles, 300-ml capacity, with ground glass stoppers and flared mouths.
2. Burette, 50 ml capacity, 0.05-ml

REAGENTS

1. Alkali iodide azide reagent: Dissolved. 500 g sodium hydroxide NaOH, and 135g sodium iodide, NaI, in distilled water
and, dilute to 1 liter. Add 10g sodium azide, NaN3, dissolve in 40 ml of of distilled water.

2. Manganese sulphate solution: Dissolve 480 g MnSO4.4H2O or 400g MnSO4.2H2O or 364g MnSO4. H2O in distilled water,
filter, and dilute to 1 Iitre.

3. Standard sodium thiosulphate solution, 0.0250M: Dissolve 6.205 g Na2S3O3.5H2O in freshly boiled and cooled distilled
water. Dilute to 1 litre. Preserve by adding 0.4g NaOH to 1 litre of solution. Standardize with 0.00208M standard
potassium hydrogen di-iodide.

4. Standard potassium hydrogen di-iodate 0.00208M: Dissolve 0.8124g KH(IO3)2 in distilled water and dilute to 1 litre.

5. Starch, aqueous solution: Add a cold water suspension of 5g soluble starch to about 800 ml boiling distilled water with
constant stirring. Dilute to I liter. Boil for a few minutes, allow to settle overnight. Use the clean supernatant which is
preserved by adding a few drops of toluene.

6. Sulphuric acid, H2SO4 concentrated

7. Sulphuric acid, H2SO4 10% (v/v)

PROCEDURE

1. To the sample in 300 ml-bottle, add 1ml- manganese sulphate solution, followed by 1ml alkali-iodide-azide reagent,
well below the surface of the sample.

2. Stopper carefully to exclude air bubbles. Mix well by inverting the bottles 10-15 times. Allow to settle.

3. After about 2 min, remove the stopper carefully. Add 1.0ml concentrates sulphuric acid down the neck of the bottle.
Iodine is liberated.

4. Stopper the bottle again carefully. Mix by gentle inversion until dissolution is complete.

5. Pipette 201ml flask. Titrate with 0.025M sodium thiosulphate solution to a pale straw color. Add 1.0 ml starch solution
and titrate until blue colouration disappears.

CALCULATION
Therefore , 1 ml 0.0205M sodium thiosulphate = 1 mg/L DO

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UNIT
mg per litre

EXRESSION OF RESULTS
Results are to be expressed to the nearest whole number

Reference
Standard Methods for the Examination of Water and Wastewater 20th Edition (APHA, AWWA, WEF)