Laboratory Module

EXPERIMENT 1
BIOCHEMICAL OXYGEN DEMAND (BOD)
OBJECTIVES
To understand the nature of oxygen demand in water by determining
the degree of water pollution caused by microorganisms through
biodegradation.
To determine the biochemical oxygen demand (BOD) of the given
wastewater samples.

2.0 INTRODUCTION
BIOCHEMICAL OXYGEN DEMAND (BOD)
BOD is the amount of oxygen required by bacteria to stabilize organic
matter under aerobic conditions. The BOD test involves the
determination of oxygen uptake by bacteria under standard conditions
(5 day incubation at 20 C). Strong wastewater samples must be
diluted in keeping with the solubility of oxygen to ensure that DO will
be present throughout the test period.

The following guide may be used for preparing dilutions:

Typical Amount of Resulting Source

Sample

BOD5 range

Clear creek water

Clear creek water
Clear creek water
Weak sewage, polluted creek water
Weak sewage, polluted creek water
Weak sewage, polluted creek water
Weak sewage, polluted creek water
Weak sewage, polluted creek water
Strong sewage, industrial waste
Strong sewage, industrial waste
Strong sewage, industrial waste
Strong sewage, industrial waste
Strong sewage, industrial waste
Strong sewage, industrial waste

0 to 5
4 to 10
8 to 20
10 to 25
13 to 33
20 to 50
25 to 62.5
40 to 100
50 to 125
67 to 167
150 to 250
200 to 500
400 to 1000
667 to 1667
2000 to
5000
4000 to
10000

Strong sewage, industrial waste

Strong sewage, industrial waste

mg/L
sample
to add
(mL)
300
150
75
60
45
30
24
15
12
9
6
3
1.5
0.9

3.0 MATERIALS AND APPARATUS

3.1 Reagents
a) Phosphate buffer solution
b) Magnesium sulphate solution
c) Calcium chloride solution
d) Ferric chloride solution
3.2 Apparatus
a)
b)
c)
d)
e)

BOD sample bottle with stopper

50 mL pipettes
1 mL pipettes
10 L water container
DO meter

4.0 PROCEDURE

Concentratio
n
(%)
100
50
25
20
15
10
8
5
4
3
2
1
0.5
0.3

0.3

0.1

0.15

0.05

4.1

Set out ten cleaned 300 mL BOD bottles for the test and log the
bottle numbers on the labsheet. The sample will be prepared as
a duplicate as the Winkler DO method will be used. Six of the
bottles will be used for the three different dilutions of the sample
and two for the blanks.

4.2

Add some dilution water to each BOD bottle, and then add the
necessary amount of well-mixed sample using a pipette. Record
the amount on the labsheet. Prepare each dilution in the
following manner; 1 in 1 (100% sample concentration),1 in 2
(50% sample concentration), 1 in 4 (25% sample concentration)
and 1 in 5 (20% sample concentration). For blanks, fill the BOD
bottles full of only dilution water.
* The dilution water is readily available for use. The dilution
water is prepared by adding 1 mL of the following per litre
of distilled water and then aerate the mixture to oxygen
saturation (approximately 2 h) at room temperature.
- 1 mL phosphate buffer solution

- 1 mL magnesium sulphate solution

- 1 mL calcium chloride solution
- 1 mL ferric chloride solution
4.3

Fill each bottle to the top with dilution water. Stopper each BOD
bottle so that no bubbles are visible in the sample. Place a water
seal on each bottle and a aluminium foil cap over the stopper.

4.4

Determine and record the DO on the duplicate set bottles with

the Winkler titration. Place the duplicate set of bottles in the
incubator in the dark for 5 days at 201C. Record the
temperature, date and time of the beginning of the incubation on
the labsheet.

4.5

At the end of the incubation period, remove the BOD bottles from
the incubator and record the temperature, date and time on the
labsheet.

4.6

Determine the DO on each bottle with the DO meter. Record the

results on the labsheet.

4.7

Calculate the BOD for the samples.

When dilution water is seeded,

BOD (mg/l) = [(D1 D2) (B1 B2) x f] P

(1)
When dilution water is not seeded,
BOD

(mg/l)

[(D1

D2)]

(2)
where,
D1 = DO of diluted sample immediately after
preparation, mg/l
D2 = DO of diluted sample after 5-day incubation,
mg/l
B1 = DO of seed control before incubation, mg/l
B2 = DO of seed control after incubation, mg/l
f
= fraction of seeded dilution water volume in
sample to volume of seeded dilution water in
seed control (volume of seed in sample/volume
of seed control)
P
= fraction of wastewater sample volume to total
combined volume (volume of sample/volume of
mixture)

5.0 RESULTS
Record your sample details. Fill in your DO data to the table below.

Table 1: DO concentration
Dissolved oxygen (DO)
(mg/L)

Bottle *
Day
Day
Day
Day

Blank
Seed control

0
5
0
5

Sample dilution
Day
Day
Day
Day
Day
Day
Day
Day

100% sample concentration

50% sample concentration
25% sample concentration
20% sample concentration

0
5
0
5
0
5
0
5

BOD can be calculated using the formula given below:

BOD (mg/L) = [ (D1 D2) (B1 B2) x f ] / P
Table 2: BOD calculation
Blan
k

Seed
Ctrl

(A) Sample vol. in vol. of bottle

(mL)
(B) Seed vol. in vol. of bottle (mL)
(C) Initial DO, Day 0 (mg/L)
(D) Final DO, Day 5 (mg/L)
(E) DO depletion (mg/L), (C) - (D)
(F) f, ((E) x [(B) / vol. of bottle]
(G) P, (A) / vol. of bottle
(H) BOD5 (mg/L), [(E) - (F)] / (G)

6.0 DISCUSSION
Briefly discuss your experiments and results.

100%

Sample dilution
50%
25%

20%

7.0 CONCLUSION
Conclude your experimental results.

8.0 REFERENCES
8.1

Dunnivat, F.M. (2004). Environmental Laboratory Exercise for Instrumental

Analysis and Environmental Chemistry, Wiley, Interscience.

8.2

APHA., AWWA., & WPCF. (1992). Standard Methods for Examination of Water and
Wastewater. 16th Edition. American Public Health Association, Washington.

9.0 QUESTIONS
9.1 What is the need for using seeded dilution water in the BOD test?
9.2 What are the main sources of error in a BOD test?
9.3 Why is a nitrification inhibitor used in the test?
9.4 What is the significance of BOD?
9.5 A 20 mL wastewater sample is placed in a standard 300 mL BOD
bottle and the bottle is filled with dilution water. The bottle had an
initial dissolved oxygen concentration of 10 mg/L and a final
concentration of 3 mg/L. A blank (a BOD bottle filled with dilution
water) run in parallel showed no changes in dissolved oxygen over
the 5 day incubation period. Calculate the BOD5.
9.6 In a test for determination of BOD of a sample of water from a
polluted stretch of river, three dilutions were used. The
observations are given below. Calculate the BOD 5 of the sample.
Then, from the result discuss what is the discharge limit for BOD
according to Environmental Quality Act (EQA) ?
Sample
Dilution, %
Initial DO,
Final DO,
No
mixture
mg/L
mg/L
1
50
7.5
2.5
2
20
7.5
5.2
3
10
7.7
6.9