1.

0 INTRODUCTION
Biochemical oxygen demand (BOD) is used to measure the amount of dissolved
oxygen needed by microorganism (ex: aerobic biological organisms) in order to
break down organic matter present in water sample at certain temperature over a
specific time period.
Organic waste normally is decomposed by microorganism. Exist of the organic
matter will caused the aerobic bacteria decomposed the waste by consuming the
dissolved oxygen in water. Therefore, the demand of oxygen will be increased so
that the BOD level also will be increased. The higher the BOD level, the lower the
dissolved oxygen (DO) level in water. This is because the available oxygen in the
water is being used by bacteria to decompose the waste. As a result, BOD test
commonly used as the indicator to show the cleanliness of the waste water. The
higher BOD value is an indication of poor water quality.
Normally, when carried out the BOD test, the water sample is incubation at 20C
over a period of 5 days and it is called as BOD5. However, in this experiment BOD3
is used instead of BOD5. Thus, the water sample is incubation at 30C over a period
of 3 days. By doing so, the time used to conduct the experiment can be shorten.

2.0 OBJECTIVE
To measure the strength of the water sample ( water, wastewater, etc) based on the
amount of oxygen needed to stabilize the organic matter in the sample.
3.0 LEARNING OUTCOME
3.1 Student able to describe the importance of BOD in the environmental studies.
3.2 Students able to measure the BOD of samples with the right sample size.
4.0 THEORY
Biochemical Oxygen Demand is a common, environmental procedure for
determining the extent to which oxygen within a sample can support microbial life.
This method is popular in many environmental laboratories analyzing waste water,
compost, sludge, and soil samples.

BOD directly affects the amount of DO in water bodies. The greater the BOD, the
more rapidly oxygen is depleted in the water body, leaving less oxygen available to
higher forms of aquatic life. The following formula can be used to calculate the
BOD,

DO
sample

( initialDO final)
BOD=

300

Once a general range for the BOD of a sample has been determined, the dilution
can be established which will ensure that at least once dilution will meet the criteria
for valid BOD results. The following formulas are used to calculate the minimum
and maximum estimated dilution:
Min. mL sample added to BOD bottle =
min allowable depletion Volume of BOD bottle
Estimated BOD
Max. mL sample added to BOD bottle =
maz allowable depletion V olimeof BOD bottle
Estimated BOD

When a measurement is made of all oxygen consuming materials in a sample, the

result is termed Total Biochemical Oxygen Demand ( TBOD ) , or often just
simply Biochemical Oxygen Demand (BOD). Because the test is performed over
a five day period, it is often referred to as a five Day BOD, or a BOD5.
In addition, this procedure is only suitable for samples void of serious matrix
interferences. To gain a broader appreciation of oxygen demand, additional avenues
of interest may be explored including CBOD (carbonaceous oxygen demand), COD
(chemical oxygen demand), and TOC (total organic carbon).

Because of complications measuring this ultimate BOD (BODu), BODu is usually

extrapolated from laboratory 5-day BOD bottle tests
BODt = BODu ( 1 e-kt ).
In many biological treatment plants, the facility effluent large numbers of nitrifying
organisms which are developed during the treatment process. These organisms can
exert an oxygen demand as they convert nitrogenous compounds (ammonia and
organic nitrogen) to more stable forms (nitrites and nitrates). At least part of this
oxygen demand is normally measured in a five day BOD.
Sometimes it is advantageous to measure just the oxygen demand exerted by
organic (carbonaceous) compounds, excluding the oxygen demand exeried by the
nitrogenous compounds. To accomplish this, the nitrifying organisms can be
inhibited from using oxygen by the addition of a nitrification inhibitor to the
samples.The result is termed Carbonaceous Biochemical Oxygen Demand or
BOD.

5.0 EQUIPMENTS AND REAGENTS

5.1 3 units 300 mL BOD Bottles
5.2 Incubator , capable of maintaining 30 1 C
5.3 2 units 100 mL beaker
5.4 100 mL graduated cylinder
5.5 2 units 25 ml meaning pipettes
5.6 DO meter
5.7 pH meter
5.8 Phosphate Buffer
Dissolve 8.5g KH2PO4, 21.7g K2HPO4, 33.4g Na2HPO4, and 1.7g NH4Cl in
deionized water. Adjust pH to 7.2, if necessary, with either 1 N H2SO4 or
NaOH. Dilute to one liter.
Magnesium Sulfate
Dissolve 22.5g MgSO4.7H2O and dilute to one liter.
5.10 Calsium Chloride
Dissolve 27.5g CaCl2 and dilute to one liter.
5.11 Ferric Choloride
Dissolve 0.25g FeCl3.6H2O and dilute to one liter.
5.9

6.0 PROCEDURE

1. The waste water was collected from specified source.

2. Three units of 300ml BOD bottles were labelled separately; one BOD bottle for
blank of dilution water, another two BOD bottles for minimum and maximum
volume of water sample respectively.

3. The minimum and maximum volumes of water samples were calculated by

using formula.
4. The pH value and temperature of the water sample were determined. The
sample must be neutralize before test is performed.
5. Each BOD bottle was first filled with the proper volume of sample size by using
pipettes and then followed by dilution water that completely filled up the 300 ml
bottle. For the blank BOD bottle, the bottle only filled with dilution water.

6. The initial DO for each bottle was determined by DO meter and the data was
recorded on the table.

7. After that, the bottles stoppers were inserted to the bottles and small amount of
dilution water was pour on top of the stopper.
8. The bottles were placed in the incubator at 30C and incubated for 5 days.

9. The final DO was recorded after 5 days of the experiment.

10. Finally, the BOD for each dilution was calculated.

7.0 RESULT AND CALCULATION

BIOCHEMICAL OXYGEN DEMAND ( BOD )

Data : 29 March 2015
Analyst: Group5 members
Time: 2.00 pm 4.00 pm
Sample Details:
Waste water from water plant behind Kolej Kediaman Tun Fatimah was used as
sample for this BOD3 test. No pretreatment is done as the water sample is obeys the
criteria and exists as a neutral water sample.
pH
6.41
Temperature, C
22.35C

Sample Sample
Type

ID

Volume

Dilution

Initial

Final

DO

Sample
(mL)

Factor

DO
(mg/L)

DO
(mg/L)

Depletion
(mg/L)

0
0.01
0.03

8.30
8.33
8.29

7.29
6.30
5.87

1.01
2.03
2.42

BOD3
Blank
0
BOD3
1
4
BOD3
2
10
Average BOD3 = 141.83 mg/L

BOD
(mg/L)
203.00
80.67

8.0 DATA ANALYSIS

8.1 Calculate for the average BOD.
Sample 1+ Sample 2
Average BOD=
2

203+ 80.67
2

= 141.83 mg/L
8.2 Show all the calculation and state if any of the data needs to thrown out.
Sample Blank
Volume of sample ,V s
Dilution
factor
,
P=

Total volume , V T

0
300

=0
DO Depletion = DOi - DOf
= 8.30 7.29
= 1.01 mg/L
BOD = None
Sample 1

Dilution factor , P=

Volume of sample ,V s
Total volume , V T

4
300

= 0.01
DO Depletion = DOi - DOf
= 8.33 6.30
= 2.03 mg/L

BOD=

DO iDO f
Dilution factor , P

2.03
0.01

= 203..00 mg/L
Sample 2

Dilution factor , P=

Volume of sample ,V s
Total volume , V T

10
300

= 0.03
DO Depletion = DOi - DOf
= 8.29 5.87
= 2.42 mg/L
DO iDO f
BOD= Dilution factor , P
2.42

= 0.03
= 80.67 mg/L
8.3 The dillution water blank cannot deplete more than 0.2 mg/L. Was this
criteria met?
From the above calculation of the dilution water blank, it is shown that the
criterion is not valid. This is because the DO depletion of dilution water blank
obtained from the experiment is 1.01 mg/L. This value is more than 0.2 mg/L
may due to poor quality control of dilution water when preparing the dilution
water. If the dilution water is kept in poor environment this also will caused
growth of microorganism in the dilution water. In the end, the DO depletion
value sure will deviate from 0.2 mg/L.

8.4 Does you sample showsa toxic effect?

Toxicity in BOD testing means the characteristic of a sample that causes it to
interfere with biochemical oxidation of organic materials during incubation.
Toxic materials present in the sample might spoil the biochemical process.

Thus, proper test to identify whether the waste water sample contains toxicity
is needed in order to take proper action to eliminate those toxicants.
According to Standard Methods 5210B, if the average of all BOD bottles that
meet the criteria of 2.0 mg/L or more of dissolved oxygen (DO) depleted with
at least 1 mg/L DO retained, toxicity will not be occurred. Since the sample
(Sample 1 + Sample 2) has average value of DO depleted more than 2, thus
the toxic effect does not occur.
8.5 Could you rely on your BOD results? Why?
The BOD result in this experiment cannot rely on because of some errors were
occurred. Although the sample is free from toxicity, the dilution water blank
does not meet the requirement. The dilution water was prepared and stored for
some times without proper quality control before the experiment. This caused
growth of some biological that cannot be seen by naked eyes. Existence of
biological in the dilution water caused inaccuracy of the result. Moreover, the
waste water used in this experiment does not undergo pretreatment process. It
may contain dissolved heavy metal. The presence of heavy metal make the
result obtained may be different.

8.6 By referring to Sewage/Effluent Standard (DOE, Malaysia ) could your

sample be discharge to river untreated? If not, suggest the associated
treatment for BOD removal.
According to Third Schedule of Environmental Quality Act, 1974 under the
Environmental Quality (Sewerage and Industrial Effluent) Regulation, 1979,
regulation 8(1), 8(2) and 8(3), the parameter limits of effluent of Standard A
and B are as follow:
Parameter
(i) Temperature

Unit
C

Standard A
40

Standard B
40

(ii) pH Value
(iii) BOD5 at 20C
(Almost the same with BOD3)
(iv) COD

6.0 - 9.0

5.5 - 9.0

mg/L

20

50

mg/L

50

100

Although the temperature = 22.35C and pH value = 6.41 of the waste water
are satisfy the requirement, however, the average BOD for the waste water is
141.43 mg/L and it is exceed the BOD requirement for both Standard A and
Standard B. Thus, the waste water sample cannot be discharged to river
untreated. The process which can used to treat waste water is aeration.
Aeration is the process by which air is circulated through, mixed with or
dissolved in a liquid or substance. Normally, this treatment used in the
secondary treatment of wastewater through aerating mixers. By doing so, high
dissolved oxygen content of waste water will be produced. In the end, BOD
level of wastewater will reduced.

9.0 DISCUSSION
Q1. a) Calculate the min / max volume of sample to be added to prepare as
estimated BOD 150mg/L.
Estimated BOD=

depletion x total volume

( min . max . allowable
)
sample water

Suppose that estimated BOD of an influent sample is 150 mg/L.

Water sample ( min ) = (2 mg/L X 300 mL) / 150 mg/L
= 4 m/L
Water sample ( max ) = (5 mg/L X 300 mL) / 150 mg/L
= 10 m/L
b) Why must samples containing cautic alkalinity or acidity be adjusted
before preparing BOD dilution?

This is because if the sample too alkalinity or acidic will prevent the growth
of bacteria. Thus, in order to conduct BOD experiment, the sample must be
neutral which is in the range of 6.0 to 8.0. In this pH range, the microbes are
most active.
c) The completed composite sample arrive in the lab at eg:,___) What is the
latest day and time the sample can be started for BOD.
The sample must be tested in time less than 24 hours. If it is begin within 2
hours of collection, cold storage is unnecessary. However, if the test will
begin within more than 2 hours, the sample must keep at or below 4C.
d) Describe the function of BOD bottles cap and seal water.
BOD bottles cap and seal water are used to ensure that no air bubble is
trapped in the bottle that will affect the characteristics of sample. Moreover,
it is also to prevent further oxygen dissolving in the sample.
Q2. a) Why must samples containing residual chlorine be dechlorinated before
preparation of BOD dilutions?
This is because chlorine can kill the microbes and bacteria in samples. If the
sample is destroyed, the result of BOD test will be affected.
b) What reagents are required to chemically dechlorinate a BOD sample?
The reagents are sulphuric acid solution, acetic acid solution, potassium
iodide solution or sodium sulfite ( Na2SO3 ).
c) What must be done to samples which have been dechlorinated or adjusted
for pH variations?
The sample must be seeded. In the seeding process, the healthy active
organism is added. Lastly, a seed correction is used in the BOD calculation.
Q3. a) State the formula to calculate
i. Seed correction
Seed correction =
BOD of seed material x volume of seed material
300

ii.

BOD5 (seeded)
BOD5 (mg/L) =

( D 1D 2 )( B1B 2)f
P

where;
D1 = DO of diluted sample immediately after preparation
D2 = DO of diluted sample after 5 days incubation at 20C
B1 = initial seed DO
B2 = final seed DO
f = seed fraction
P = decimal volumetric fraction of sample used
b) Calculate the seed correction and BOD5 (seeded) for the data given as
below

Dilution #1

BOD5 of Seed Material

mL of seed material
mL of sample
Start D.O.
Final D.O.

95 mg/L
2 mL
100 mL
7.8 mg/L
2.9 mg/L

Seed correction = (95 x 2 ) / 300

= 0.633 mg/L
BOD5 (seeded) = [(7.8 2.9) - 0.633] x 300 / 100
= 12.801 mg/L
Q4. a) 30 mL of wastewater are placed in a 300 mL BOD bottle. The sample is
diluted to fill the bottle. The DO concentrations at the beginning and the
end of 5-day incubation period are 7.3 mg/L and 1.8 mg/L respectively.
What is the BOD?
DO iDO f
BOD 5= Dilution factor , P

( 7.31.8 ) x 300
30

= 55 mg/L

b) The BOD5 of a wastewater was determined to be 250 mg/L. If the reaction

coefficient was 0.23 l/d, calculate
i) ultimate BOD,
kt
BODt = Lo(1e )
Lo =

250
1e(0.23 )(5)

Lo = 365.84 mg/L

ii) BOD3
kt
BODt = Lo ( 1e )
(0.23)(3 )
BOD3 = 365.84 ( 1- e
)

= 182.34 mg/L
iii) BOD remaining at 3 days
BOD remaining at 3 days = BODu BOD3
= 365.84 182.34
= 183.50 mg/L
Q5. a) What is seeding process in BOD measurement?
Seeding is a process of adding live bacteria and microorganism to a sample.
If the samples tested contain materials which could kill or injure the
microorganisms, the condition must be corrected and healthy active
organisms added.
b) Explain preparation of seed material.
Select a material to be used for seeding which will have a BOD of at
least 180 mg/L. This will help ensure that the seed correction meets the

0.6 mg/L minimum specified in StandardMethods.

Place the material in a suitable container and incubate at 20C for 3
days. Usually, settle draw domestic sewage prepared in the manner

above will have sufficient BOD for use as a seed material.

As an alternative, commercially available seed material may be used.
The seed correction should not exceed 1.0 mg/L BOD; therefore, care
should be taken not to use too strong a seed material for the test.

Make sure that seed material is a relatively stable which produces a good
seed correction in every test situation.

c) What materials can be used to seed a BOD sample?

Settled raw sewage or commercially prepared seed material are the most
common sources. However, any source of water which can provide a
suitable population of organisms can be used.

Q6. What is the significance of dissolved oxygen?

Dissolved oxygen allows animals to breathe in water and it provides a suitable
habitat for the other animals. Bacteria in the water also use this oxygen to
break down animals and plants. If the oxygen level is reduced, the animals
begin to die. It is also to maintaining the aquatic life and aesthetic quality of
streams and lakes.

Q7. a) With regard to precision, ten percent duplicate or replicate samples should
be run. This would result in one duplicate sample or one replicate sample
being run every ten samples. Differentiate replicate and duplicate sample.

Replicates are two or more separate water samples collected in the field
from the same site and depth. It is used to determine the errors involved
in sample collection. If there are no errors in the collection and analysis,
and then the difference between two replicate analysis indicates the

natural variability in the water at that location.

Duplicates are two or more lab analyses on the same water sample. It is
used to determine the percentage difference between two samples in
order to estimate the error involved in the analysis.

b) When are DO levels at their highest and their lowest and why?

DO level is highest if the water is free from polluted materials like toxic
and bacteria. When the numbers of bacteria is decreased, the oxygen
demand also decreased and resulted in highest DO level. On the other hand,
water will have lowest DO level if water is highly polluted.
Microorganisms in water will used up the oxygen so will cause DO level
decreases. Moreover, DO level is often highest on the daytime as aquatic
plant will undergoes photosynthesis to produce oxygen. The DO level will
decreases during night as photosynthesis cannot occur without sunlight.

10.0 CONCLUSION
In conclusion, the objective of the experiment is achieved. The experiment was
carried out to measure the strength of the waste water sample based on the amount
of oxygen needed to stabilize the organic matter in the sample.
Normally, BOD5 will be used to determine the BOD level in water. However, in this
experiment, BOD3 is used instead of BOD5. By using BOD3, the time of experiment
can be shorten and the effect of experiment almost the same with BOD5.
Since the average BOD3 obtained from experiment is 141.83 mg/L so that it can be
concluded that the waste water from water plant behind Kolej Kediaman Tun
Fatimah has been polluted. The higher BOD3 reading will result in lower DO level.
When the DO level is lower, this indicated that the water is unsuitable for living
organisms. Besides, this waste water cannot simply discharge to the river untreated.
It must be treating first by any possible treatment to make sure that water is safe and
will not to be polluted to the river.