BIOCHEMICAL OXYGEN DEMAND

OBJECTIVE
The purpose of this study is to determine the biochemical oxygen demand (BOD) of
domestic and industrial waste streams and surface waters.

INTRODUCTION
The BOD test is a common industrial procedure in which the strength of a waste stream is
measured. Dissolved oxygen (DO) in these waste streams is utilized by microorganisms as they
break down the waste within the stream. The stronger the waste stream, the more dissolved
oxygen will be used. The concentration of dissolved oxygen in the stream can be decreased to a
concentration where it can not support other aquatic life.
Determination of BOD is important as it measures the affect that a waste has on a receiving
body. Release of strong wastes into the natural environment can have negative effects on life
forms such as fish and other aquatic species. This, in turn, can affect other species as one
progresses up the natural food chain.
Complete stabilization of certain wastes may take long periods of time. Due to this fact, an
industry standard called the BOD5 has been established. The waste sample in question is initially
analyzed to determine the DO concentration (DO0). The sample is then allowed to stabilize for
five days under controlled conditions. After five days, the sample is again analyzed to determine
the final concentration of DO in the sample (DO5). Knowing the DO0 and DO5 concentrations,
one can calculate the BOD5. The BOD5 value can be compared to other waste samples to find
the relative strengths of wastes.
All DO concentrations are determined electronically with the use of a BOD probe connected
to a DO meter.
The BOD Probe
The probe uses a membrane covered polarographic sensor with a built-in thermistor for
measuring temperature. The special membrane, which allows only oxygen and certain other
gases to pass through, is stretched over the sensor, isolating the sensor elements from the
environment. When a polarizing voltage is applied across the sensor, oxygen that has passed
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through the membrane reacts at the cathode causing a current to flow. The membrane passes
oxygen at a rate proportional to the pressure difference across it If the oxygen pressure
increases, more current flows through the sensor.
Note: Do not touch the membrane on the probe, and be careful with the stir paddle.

REFERENCES
APHA, AWWA, & WPCF. 14th edition 1975. Standard Methods for the Examination of Water
and Wastewater. American Public Health Association, Washington DC.

EQUIPMENT
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.

YSI Model 50B Dissolved Oxygen Meter

YSI 5907 BOD Probe
Magnetic stirrer
Glass carboy
Air incubator
Siphon tube and cut-off valve
Vacuum pump with aeration hose and diffuser stone
300-mL incubation bottles with ground glass stoppers
Magnetic stir bar
10-mL and 25-mL pipets
Pipet bulb

CHEMICALS/MATERIALS
1.
2.
3.
4.
5.
6.

Deionized (DI) water

Gelatin seed capsule or settled waste water from sewage treatment plant
Phosphate buffer solution
Magnesium sulfate solution
Calcium chloride solution
Ferric chloride solution

EXPERIMENTAL PROCEDURE
Preparation of seed solution
1. Place the contents of one gelatin capsule (discard the gelatin capsule) in 500 mL of
dilution water.
2. Stir the seed solution while aerating for 60 minutes using a magnetic stirrer.
3. If the seed solution is settled waste water received from a sewage treatment plant,
aerate the sample when received until it is pipetted into the seed or BOD bottles.
Preparation of dilution water
1. Determine the total amount of dilution water needed for the test. Add 3 liters to this
amount.
2. Fill the glass carboy with the volume of deionized (DI) water calculated above.
3. Add the buffer and chemical solutions to the deionized water.
a. Add phosphate buffer solution, 1mL/L of DI water
b. Add magnesium sulfate solution, 1 mL/L of DI water
c. Add calcium chloride solution, 1 mL/L of DI water
d. Add ferric chloride solution, 1 mL/L of DI water
4. Place the aeration tubing and stone in dilution water.
5. Start the aeration pump and saturate the dilution water with oxygen. (The sample
should be aerated for a minimum of 30 minutes.)
Calibration of DO meter and BOD probe in air
Calibration should be done in the BOD bottle used for storage of the probe. Keeping a water
level in the bottle is crucial.
1. Turn the DO meter on by setting the function knob to the oC position. An audible tone
will sound. A second tone will sound in about seven seconds to signal the end of the
Power On Self Testing (POST) diagnosis.
2. Temperature will now be displayed, observe the reading for stability. Temperature
equilibration may take up to 5 minutes.
3. Set the meter function switch to the mg/L position and allow the meter to stabilize for
15 minutes.
4. After the meter has stabilized, set the function knob to the mg/L CAL position. Press
the CAL key once. The mg/L reading will automatically correspond to the amount of
oxygen in the air.
5. Turn the function switch to mg/L. The display will show CAL. In a few seconds one
or two audible tones will sound.
Next, the appropriate calibration value in mg/L (0.02 mg/L) will be displayed.
Observe the reading for stability for two or three minutes. Drift in the reading of more
than two digits may mean insufficient time was allowed for instrument stabilization.
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This completes the calibration in air in mg/L for fresh water measurements.
Preparation of BOD sample bottles
Note: * When filling bottles with dilution water, do so in a manner so that no entrained
air is allowed in the sample.
* Fill the bottles with enough dilution water so that NO air is left in the bottle when
the glass stopper is placed in the bottle.
* Shut off aeration system before siphoning dilution water
Dilution water control
1. Siphon dilution water into four 300-mL BOD bottles.
2. Use two for testing of DO0.
3. Use two for testing of DO5.
Seed correction
1. Pipet 10, 15, 20, 25, and 25 mL of seed solution into five BOD bottles.
2. Fill the bottles with dilution water.
3. Create a table similar to Table 1. Seed Correction
Table 1. Seed Correction
Bottle
Number

Concentratio
n
mg/L

Volume of
added
Seed
Solution

DO5 (10 mL)

10 mL

DO5 (15 mL)

15 mL

DO5 (20 mL)

20 mL

DO5 (25 mL)

25 mL

DO0 (25 mL)

25 mL

DO0
mg/L
Initial

DO5
mg/L
Final

After completing the seed correction step, add 6.67 mL/L (2mL/300 mL BOD bottle) of seed
solution to the dilution water remaining in the carboy

Waste Samples
Follow this procedure for each type of waste.
1. Pipet 1,3,6, and 6 mL of waste sample into four BOD bottles.
2. Fill the bottles with seeded dilution water.
3. Create a table similar to Table 2. Waste samples or add on to Table 1.
Table 2. Waste samples
Bottle
Number

Concentratio
n
mg/L

Volume
of
waste
sample

DO5 (1 mL)

1 mL

DO5 (3 mL)

3 mL

DO5 (6 mL)

6 mL

DO0 (6 mL)

6 mL

DO0
mg/L
Initia
l

DO5
mg/
L
Final

Determine the initial DO0 concentrations in the specified samples.

Place the remaining BOD5 bottles in the refrigerator set at 20oC. Add water to the flared mouth
of the bottle to provide a water seal. Water will have to be added each day to ensure there is a
water seal.
Note: When inserting the BOD probe into the sample, ensure that all air is evacuated from
the sample. Any air in the sample can attach to the membrane and cause false results.
Remember to turn on the stir paddle to allow for proper mixing of the sample.
After the five day incubation period, determine the DO5 levels in the samples.
If the samples are suspected to be low in BOD5 a modified procedure will have to be used.
These type of samples may include: tap water, creek/river water, lake water, pond water, etc.
1. Aerate the sample for 5 to10 minutes.
2. If the amount of sample collected is one liter or more, prepare six BOD bottles for
analysis. If the amount of sample collected is only 0.5 L prepare four BOD bottles for
analysis.
3. Pipet 2 ml of seed into each of one or two bottles depending on the amount of sample
available.
4. Fill the remainder of these bottles with your sample.
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5. If you had sufficient sample keep one bottle for determination of the initial DO
concentration of your sample. Place the other bottle in the incubator for a
determination of the DO after 5 days..
6. If you did not have sufficient sample, you will have to assume the initial DO
concentration in your sample is the same as the initial concentration of DO in the
dilution water.
7. Pipet into the other four BOD bottles 1 mL, 3 mL, 6 mL, and 6 mL of sample.
8. Fill these bottles with the dilution water. Keep one of the bottles that you pipetted 6
mL of sample into for a determination of the initial DO concentration of the dilution
water and place the other bottles into the incubator for determination of the DO after 5
days.
9. Create a table similar to Table 3.
Table 3. Waste samples
Bottle
Number

Concentration
mg/L

Volume
of
waste
sample

DO5 (298 mL plus 2 mL seed)

298 mL

DO0 (298 mL plus 2 mL seed)

If you have enough sample
available otherwise use DO
concentration from Bottle number
6 for the initial concentration

298 mL

DO5 (1 mL)

1 mL

DO5 (3 mL)

3 mL

DO5 (6 mL)

6 mL

DO0 (6 mL)

6 mL

DO0
mg/L
Initia
l

DO5
mg/
L
Final

CALCULATIONS
Biochemical oxygen demand

BOD5 calc =

( DO

DO5 )

(1)

where DO0 is the initial amount of dissolved oxygen, DO5 is the amount of dissolved oxygen
after five days of incubation, and P is the decimal fraction of the sample used.
Seed Correction

Seed Correction = ( DO0 seed DO5 seed ) f

(2)

where DO0-seed is the amount of initial DO in the seed correction solution, DO5-seed is the amount
of DO in the incubated seed corrected solution, and f is the seed solution ratio.
Seed Solution Ratio
f =

% Seed Waste Sample

% Seed Seed Correction Sample

(3)

Corrected BOD5

BOD5 corrected =

( D0

DO5 ) ( DO0 seed DO5 seed ) f

(4)

SAFETY NOTES
1. Personal protective equipment shall include goggles.
2. Latex gloves are optional depending on the wastes being tested.
3. Keep liquid away from ALL power supplies.

WASTE DISPOSAL PROCEDURES

Dispose of all BOD solutions down the drain with the tap water running for dilution purposes.
11/16/09
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