FACULTY : CIVIL & ENVIRONMENTAL ENG.

EDITION:
DEPART : WATER & ENVIRONMENTAL
REVISION NO: 02
ENG.
EFFECTIVE 28/12/2015
LAB : ENVIRONMENTAL ENGINEERING
DATE:
EXPERIMENT : BIOCHEMICAL OXYGEN AMENDMENT
DEMAND (BOD) DATE:

1.0 OBJECTIVE
To measures the strength of the water sample ( water, wastewater, etc) based on the amount of
oxygen needed to stabilize the organic matter in the sample.

2.0 LEARNING OUTCOMES

i) Students should be able to describe the importance of BOD in the environmental studies.
ii) Students should be able to measure the BOD of samples with the right sample size.

3.0 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.

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.
 FACULTY : CIVIL & ENVIRONMENTAL ENG. EDITION:
DEPART : WATER & ENVIRONMENTAL
REVISION NO: 02
ENG.
EFFECTIVE 28/12/2015
LAB : ENVIRONMENTAL ENGINEERING
DATE:
EXPERIMENT : BIOCHEMICAL OXYGEN AMENDMENT
DEMAND (BOD) DATE:

Sometimes it is advantageous to measure just the oxygen demand exerted by organic

(carbonaceous) compounds, excluding the oxygen demand exerted 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 Demandor BOD.

Generally, high BOD indicates a high content of easily degradable organic material in sample,
and low BOD indicates a low volume of organic material substances which are difficult to break
down.

4.0 EQUIPMENT & REAGENTS

i. 6 units BOD bottles 300ml

ii. 100ml beaker
iii. 100ml graduated cyclinder
iv. 1unit pipettes (ml)
v. DO meter
vi. pH meter
vii. Air pump
viii. Incubator, capable of maintaining 20 1C

Nutrient Buffer solution

1. Phosphate Buffer
- Dissolve 8.5g potassium di hydrogen phosphate (KH2PO4), are dissolved in approximately
500ml distilled water, 21.75g di potassium hydrogen phosphate (K2HPO4) are added,
followed by 33.4g di sodium hydrogen phosphate (Na2HPO4) and 1.7g ammomium chloride
(NH4CI). Adjust pH to 7.2 if necessary with either 1 NH2SO4 or NaOH. Dilute to 1liter
2. Magnesium sulfate
- 22.5g magnesium sulphate (MgSO4.7H2O) is diddolved in distrilled water and the solution
made up to 1liter.
3. Calcium chloride
- 27.5g of anhydrous calcium chloride (CaCI2) are dissolved in distilled water and the solution
made up to 1liter.
4. Ferric chloride
- 0.25g FeCI3.6H2O is dissolved in distilled water and the solution made up to 1liter.
 FACULTY : CIVIL & ENVIRONMENTAL ENG. EDITION:
DEPART : WATER & ENVIRONMENTAL
REVISION NO: 02
ENG.
EFFECTIVE 28/12/2015
LAB : ENVIRONMENTAL ENGINEERING
DATE:
EXPERIMENT : BIOCHEMICAL OXYGEN AMENDMENT
DEMAND (BOD) DATE:

BOD Dilution water

Add 1ml of each of the 4 nutrient buffer solutions to 1liter of deionized water and aerate for
at least 1 hr before conducting BOD test to ensure the DO concentration in the dilution
water is at least 7.5 mg/L.

Chemical for pH adjustment

1N of sulfuric acid, H2SO4 or 1N sodium hydroxide, NaOH.

5.0 BOD5 MEASUREMENT PROCEDURES

5.1 DETERMINATION OF SAMPLE SIZE :

Once a general range for the BOD of a sample has been determined, the dilutions can be
established which will ensure that at least one dilution will meet the criteria for valid BOD
results. The following procedure can be used to calculate volumes for sample dilution from the
estimated BOD.

For example, suppose the estimated BOD of an influent sample is 400 mg/L and assume the DO
of saturated dilution water is 8.0 mg/L. since the criteria for most valid results states that the
DO depletion at the end of five days incubation should be at least 2.0 mg/L and the residual DO
at least 1.0 mg/L , the formulas to calculate the minimum and maximum estimated dilution are
as follows:

A. mL sample added to BOD bottle = ( minimum allowable depletion, mg/L x Volume of BOD
bottle, mL ) / estimated BOD , mg/L.

example:
minimum mL sample = [( 8 mg/L 6 mg/L ) x 300 mL ] / 400 mg/L

minimum mL sample = ( 2 x 300 ) / 400 = 600 / 400 = 1.5 mL

B. ml sample added to BOD bottle = ( maximum allowed depletion , mg/l x

Volume of BOD bottle, ml ) / estimated BOD, mg/l.

example:
maximum mL sample = [ ( 8 mg/L 1 mg/L ) x 300 mL ] / 400 mg/L

maximum mL sample = ( 7x 300 ) / 400 = 5.24 mL

 FACULTY : CIVIL & ENVIRONMENTAL ENG. EDITION:
DEPART : WATER & ENVIRONMENTAL
REVISION NO: 02
ENG.
EFFECTIVE 28/12/2015
LAB : ENVIRONMENTAL ENGINEERING
DATE:
EXPERIMENT : BIOCHEMICAL OXYGEN AMENDMENT
DEMAND (BOD) DATE:

NOTE: Those sample dilutions which deplete less 2 mg/L, or have a final DO of less than 1 mg/L
would not be use in the calculation of the average sample BOD.

Table 1: Volume of sample for dilution

Expected BOD Volume of sample to be diluted to
Range 300 ml in BOD bottle (ml)
(mg O2/L)
0-7 300
7 - 21 100
12 - 42 50
30- 105 20
60 - 210 10
120 - 420 5
300 - 1050 2
600 - 2100 1
1200 - 4200 0.5
3000 - 10500 0.2
6000 - 21000 0.1

To determine the value of the BOD in mg/l, use the following formula:

When dilution water is not seeded:

D1 D2
BOD5, mg/L =
P

D1 = DO of diluted sample immediately after preparation, mg/L,

D2 = DO of diluted sample after 5 d incubation at 20oC, mg/L,
P = reciprocal of dilution factor, D= volume of BOD bottle/volume of sample used
in mL

NOTE: The samples must undergo dilution if they have a BOD > 7, otherwise all the
dissolved oxygen will be used up before 5 days have elapsed. [Note: raw domestic
wastewater typically has a BOD of around 300 mgO2/l].
 FACULTY : CIVIL & ENVIRONMENTAL ENG. EDITION:
DEPART : WATER & ENVIRONMENTAL
REVISION NO: 02
ENG.
EFFECTIVE 28/12/2015
LAB : ENVIRONMENTAL ENGINEERING
DATE:
EXPERIMENT : BIOCHEMICAL OXYGEN AMENDMENT
DEMAND (BOD) DATE:

5.2 PRE-TREATMENT SAMPLES.

1) That contain caustic alkalinity or acidity

2) Caustic alkalinity or acidity can prevent bacteria from growing during the course of the
BOD test.
3) To prevent this, samples which have pH values higher than pH 8.0 or lower than pH 6.0
must be neutralized to pH 7.0 before test is performed.

NOTE : Neutralized samples must be seeded for the BOD test.

Procedure for neutralizing samples:

1. Pour 50 mL of samples into a 100 mL beaker.

2. Measure the pH of the sample using a pH meter. If the pH is out of the range of pH 6.0 to
pH 8.0 countinue with steps 3 6, otherwise perform the BOD test on the untreated
sample.
3. Add 1 N sulfiris acid if the sample is alkaline, or 1N sodium hydroxide if the sample is acidic,
intil the pH reaches 7.0.
4. Calculate the amount of sulfuric acid or sodium hydroxide needed to neutralize 1000ml of
the sample.
5. Add the calculated amount of acid or base to the sample.
6. Repeat steps 1-5 until the pH test shows pH 7.0.
7. Calculation the amount of 1 N sodium hydroxide or 1 N sulfuric acid needed to neutralized
the sample to pH 7.0 using the following formula:

mL needed = ( mL acid or base used x mL total test sample ) mL sample portion used for
neutralization.

For example, suppose 1.3 mL of 1 N NaOH are use to neutralize 50 mL of sample to pH 7.0
calculate the volume of NaOH to be added to neutralize the sample as follows:

mL 1 N NaOH needed = ( 1.3 mL x 1000 mL ) /50 mL = 1300 / 50 = 26ml

 FACULTY : CIVIL & ENVIRONMENTAL ENG. EDITION:
DEPART : WATER & ENVIRONMENTAL
REVISION NO: 02
ENG.
EFFECTIVE 28/12/2015
LAB : ENVIRONMENTAL ENGINEERING
DATE:
EXPERIMENT : BIOCHEMICAL OXYGEN AMENDMENT
DEMAND (BOD) DATE:

5.3 BOD5 PROCEDURES

1. Label separately 4 unit of 300ml BOD bottles (1 blank, 3 samples)

2. Pipette appropriate volumes into bottles. Fill all the bottles to the top with dilution water.
3. Measure the initial DO concentration using DO meter.
4. Place all the bottles into the BOD incubator, this is set at 20oC and is dark to prevent the
growth of algae. Leave for 5 days.
5. After 5 days, remove the samples from the incubator and measure the final DO content of
each.

NOTE: The dilution water blanks are used only to check the quality of the dilution water. If the quality
of the water is good and free from impurities, the depletion of DO should NOT be less than
0.2 mg/l. in any event, do not use the depletion obtained as blank correction.

6.0 RESULT AND CALCULATION

a) Type of sample: .
b) Initial pH sample: .
c) Adjustment pH sample: .
d) Volume acid used: .
e) Volume base used: .
f) Volume of sample used: .
 FACULTY : CIVIL & ENVIRONMENTAL ENG. EDITION:
DEPART : WATER & ENVIRONMENTAL
REVISION NO: 02
ENG.
EFFECTIVE 28/12/2015
LAB : ENVIRONMENTAL ENGINEERING
DATE:
EXPERIMENT : BIOCHEMICAL OXYGEN AMENDMENT
DEMAND (BOD) DATE:

Volume of Volume DO
Initial DO, Final DO, BODx,
Sample incubation of sample depletion,
mg/l mg/l mg/l
bottle, ml size, ml mg/l
BLANK
1
2
3
4
5

7.0 DATA ANALYSIS

i. Show all the calculation and state if any of the data needs to be discarded.

ii. Does your BOD dilution water show toxic effect?

iii. Could you rely on your BOD results? Why?

iv. Determine the pollution level of samples based on the BOD results.

v. Differentiate between seeded and unseeded BOD.

8.0 DISCUSSIONS

PREPARED BY: SIGNATURE:

POSITION:
DATE: