EXPERIMENT NO.

10 : BIOLOGICAL
OXYGEN DEMAND
Structure
10.1 Introduction
Objective

10.2 Apparatus
10.3 Reagents

i
b
10.4 Procedure
10.5 Calculations

L
10.1 INTRODUCTION
The Biochemical Oxygen Demand (BOD) determination is an empirical test in which
standardized laboratory procedures are used to determine the relative oxygen requirement
of waste water, effluents and polluted waters. The test has its widest application in
measuring waste loadings to treatment plants and in evaluating the BOD-removal
efficiency of such treatment systems.
The test measures the oxygen utilized during a specified incubation period for the
biochemical degradation of organic material. It also may measure the oxygen used to
oxidize reduced forms of nitrogen unless their oxidation is prevented by an inhibitor.
The method consists of filling with sample to overflowing an airtight bottle of the
specified size and incubating it at the specified temperature (20°C) for 5 days. Dissolved
oxygen (D.O.) is measured initially and after incubation, and-the BOD is computed from
the difference between initial and the final D.O. determined immediately after the
dilution is made.
It is important to ensure that sufficient amount of oxygen exists in the water sample. Even
at the expiry of 5 days incubation period, a minimum level of 1 mg 1 L of dissolved
oxygen should be available in the water sample.
Sometimes the bacterial content existing in the water sample may not be sufficient for the
stabilization of decomposable organic matter. In such a case, water is required to be
seeded and after the analysis, its result needs to be corrected accordingly, taking into
account the effect of settled sewage (added to the dilution water).

Objective
To detemine 5-day BOD of the given sample of waste water.

(a) Incubation bottle (300 ml)

(b) BOD incubator
(c) Air compressor

10.3 REAGENTS
(a) Magnesium Sulphate (MgS04. 7H20) Solution
Take 22.5 g of mangesium sulphate, mix and dissolve it in the distilled water and
dilute it to 1.0 litre.
(b) Calcium Chloride (CaC12) Solution
Take 27.5 g of anhydrous calcium chloride, mix and dissolve it in distilled water and
dilute it to 1.0 litre.
Laboratory - II (c) Ferric Chloride (FeCt . 6H20) Solution
Take 0.25 g of Ferric chloride, mix and disslove it in distilled water and diluteit i o
I-Olkre. .
(d) Phosphate Buffer Solution
To prepare the phosphate buffer solution, take the following chemicals in the quantity
as given against each of it below :
Potassium dihydric phosphate (KH2P04) 8.5 g
Dipotassium hydrogen phosphate (K2HP04) 21.75 g
Disodium hydrogen phosphate (Na2HP04 . 7H20) 33.40 g
Ammonium chloride (NHACI) 1 7 ~

Figure 10.1 :BOD Incubator

Mix and dissolve the above chemicals in distilled water and dilute to 1.0 litre. Adjust
the pH value of the solution to 7.2 by adding IN, NaOH solution (which is prepared
by mixing 40 g of NaOH per litre of solution using distilled water). This is done to
provide favourable conditions for the proper growth and propagation of bacteria.
Sodium and Potassium phosphates help in maintaining the proper osmatic conditions.
(e) Alkali-Iodide-Azide reagent
(0 Sulphuric Acid (Concentrated)
(g) Starch Indicator
(h) Standard (0.025N)Sodium Thiosulphate titrant
(i) Dilution Water
Saturate thedietilled water with Dissolved Oxygen by aerating it With compressed air,
maintaining a temperature of about 20°C, till water attains saturation. Then add and
mix to it, 1 ml of Phosphate buffer solution + 1 rnl of Magnesium Sulphate solution +
1 ml of Calcium Chloride solution + 1 ml of Ferris Chloride solution. This solution
which serves as nutrients fur micro-organisms and provide favourable conditions for
their growth and propagation, is used for diluting the water sample.
 Seeded dilution water is used when the sample to be tested for BOD does not possess Environmental Engineering
sufficient amount of micro-organisms to stabilize the organic matter in it. For this
purpose 2 ml of settled sewage is added to the dilution water which eventually
increases the bacterial content in the water sample, when diluted which such type of
dilution water.
In such a case, the BOD result needs to be corrected in regard to the amount of BOD
existing in the settled sewage (initially and after 5 days).

10.4 PROCEDURE
(i) Water sample, collected in the BOD bottle is required to be diluted with dilution water
in such a manner that the dissolved oxygen in the resulting solution becomes half of .
the level of dissolved oxygen existing in the dilution water. Sometimes the raw water
sample may contain excessive amount of Dissolved Oxyge? because of the presence
of algae, causing supersaturation. In such a case, it needs to be reduced by aeration and
agitation of water sample before the test. For raw water sample (from lake, river or
reservoir) at least 4 to 5 dilutions, in the range of 25% to 100% are prepared for BOD
test, by using the dilution water and the dilution in which 50% reduction in the
Dissolved Oxygen content occurs, is finally chosen for testing.
(ii) After fixing up the percentage dilution, calculate the volume of dilution water and
water sample needed for preparing the total volume of diluted sample. Syphon half of
the calculated volume of dilution water into a graduated cylinder and add the desired
amount (as calculated earlier) of well mixed water sample and then add the remaining
amount of dilution water. .
(iii) Syphon the diluted water sample into 4 BOD bottles, each of the capacity of 300 ml
and put the stoppers. Out of these four bottles, two will be used for determination of
initial Dissolved Oxygen content and the remaining two bottles will be incubated for
5 days at 20°C.
(iv) Similarly prepare two BOD bottles with blank dilution water. Out of these two, one
bottle will be used for determination of initial Dissolved Oxygen content and another
bottle will be incubated for 5 days at 2 0 ' ~ .
(v) For determination of Dissolved Oxygen, take 300 ml of sample and add 1 ml
Magnesium Sulphate solution, shake the bottle, again add 1 ml Alkali-Iodide-Azide
reagent, stopper the bottle carefully to exclude air bubbles and mix the content
thoroughly by inverting the bottle repeatedly.
If no oxygen is present, the manganous ion (Mnct) reacts only with the hydroxide ion
to form a pure white precipitate of Mn(OH)2. If oxygen is present, some of the ~ n *is
oxidized to a higher valance (Mnte*) and precipitates as a brown coloured
i
oxide (Mn02).

After the bottle has been shaken and sufficient time has been allowed for all the
oxygen to react, the chemical precipitates are allowed to settle leaving'clear liquid in -
the upper portion. Now add 1.0 ml of concentrated Sulphuric Acid. Restopper the
bottle and mix by inverting until the suspension is completely dissolved and the yellow
colour is uniform throughout the bottle.
The Equation (10.3) shows the reaction that takes place; the manganic oxide is reduced
to manganous manganese while an equivalent amount of iodide ion is converted to
s
free iodine. The quality of is equivalent to the dissolved oxygen in the original
sample.
 -
Laboratory I1 Now pour 201 ml of sample (it is equivalent to 200 ml of the original sample after
correction for the loss of sample displaced by reagent additions) from the BOD
bottle into a container. Titrate this ~olutionagainst standardized 0.025 N Sodium
Thiosulphate solution till pale straw colour develops and then add a few drops of
starch indicator solution and continue the titration to the first disappearance of blue
colour. If 0.025 N Sodium Thiosulphate is used to measure the dissolve oxygen in
a volume equal to 200 ml of original sample, 1.0 mi of tirant is equivalent to
l . O m g / L D.O.
Find the initial D.O. and D.O. after 5-days incubation of the water sample and the
blank, as per the procedure stated above.

10.5 CALCULATIONS
(DOo - Do5) - (DOB - DOB5) X 100
BOD5, mg 1 L =
percent dilution

where, DO0 = DO of diluted sample immediately after preparation, (in mg / L),

DOB = initial DO of blank (in mg / L),
DO5 = DO of diluted sample after 5 day incubation (in mg / L), and
DOB5= DO of blank after 5 day incubation (in mg / L).