Experiment No.

10
Determination of Biochemical Oxygen Demand

Introduction
When biodegradable organic matter/waste (the most common category of pollutant: affecting
surface water) is released into a water body, microorganisms (especially bacteria) feed on the
wastes, breaking it down to simpler organic and inorganic substances. When this decomposition
takes place in an aerobic environment (i.e., in the presence of oxygen; Eq. -1), it produces non-
objectionable, stable end products (e.g., CO2, SO4, PO4, and NO3) and in the process draws down
the dissolved oxygen (DO) content of water.

Organic matter + O2 = CO2 + H2O + New cells + Stable products (1)

(bacteria)
When insufficient oxygen is available or when oxygen is exhausted by the aerobic decomposition
of wastes, then different set of microorganisms carry out the decomposition anaerobically (Eq. -2)
producing highly objectionable products including H2S, NH3, and CH4.

Organic matter = CO2 + CH4 + New cells + Unstable products (2)

(bacteria)

The amount of oxygen required by micro-organisms to oxidize organic wastes aerobically is called
biochemical oxygen demand (BOD). BOD may have various units, but most often it is expressed
in mg of oxygen required per liter of water/wastewater (mg/L). The total amount of oxygen that
will be required for biodegradation is an important measure of the impact that a given waste stream
would have on the receiving water body.

Environmental Significance
Dissolved oxygen is the most commonly used indictor of the general health of a surface water
body. If DO in the water body goes below 4 to 5 mg/L, forms of aquatic life that can survive begin
to be reduced. When anaerobic condition exists, higher life forms are killed or driven off. Noxious
condition, including floating sludge, bubbling, odorous gases, and slimy fungus growth prevails.

According to Bangladesh Environment Conservation Rules (1997), drinking water standard for
Biochemical Oxygen Demand (BOD) is 0.2 mg/L (at 20°C). For wastewater effluent allowable
concentration of BOD varies from 50- 250 mg/L depending on discharge point of the effluent e.g.,
inland water, irrigation land, public sewer etc. respectively (see appendix for details).

Theory on Experimental Method

The biochemical oxygen demand (BOD) determination is an empirical test in which standardized
laboratory procedures are used to determine the oxygen requirements of wastewater, effluents and
polluted waters. It has become a standard practice to simply measure and report the oxygen demand

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over a 5-day period, realizing that the ultimate oxygen demand (for complete degradation of
organic matter) is considerably higher and would take a much longer time to determine in the
laboratory.
The 5-day BOD or BOD5 is the amount of oxygen consumed by micro-organisms during the first
5 days of biodegradation. In its simplest form, a BOD5 test would involve putting a sample of
waste/wastewater into a stoppered bottle, measuring the dissolved oxygen (DO) in DO would be
the BOD5 of the water/wastewater.

Light must be kept out of the bottle to keep algae from adding oxygen by photosynthesis and the
stopper is used to keep air from replenishing DO from outside. To standardize the procedure, the
test is run at a fixed temperature of 20°C. Since the oxygen demand of typical waste is sever
hundred milligrams per liter, and since the saturated value of DO for water at 20°C is only 9.1
mg/L, it is usually necessary to dilute the sample to keep final DO above zero, if during the five
days of experiment, the DO drops to zero, then the test is invalid since more oxygen would have
been removed had more been available.

The five-day BOD of a diluted sample is given by

BOD5 = [DOi, - DOf] x D.F. (3)

Volume of (waste water +dilution water)

Where, D.F. = Dilution Factor =
Volume of wastewater

In some cases, it becomes necessary to seed the dilution water with micro-organisms to ensure that
there is an adequate bacterial population to carry out the biodegradation. In such cases, two sets of
BOD bottles must be prepared, one for just the seeded dilution water (called the “blank”) and the
other for the mixture of wastewater and dilution water. The changes in DO in both bottles are
measured. The oxygen demand of the wastewater (BODw) is then determined from the following
relationship:

BODm Vm = BODw Vw + BODd Vd (4)

Where, BODm is the BOD of the mixture of wastewater and dilution water and BODd is the BOD
of the dilution water alone. Vm and Vd are the volumes of wastewater and dilution water,
respectively, in the mixture, and Vm = Vw + Vd.

Dilution water and “Seed”

A wide variety of dilution waters have been used for BOD work. Natural surface waters have been
used, but they suffer from a number of disadvantages including variable BOD, variable micro-
organism population, and variable mineral content. Tap water has also been used, but it also suffers
from many of the limitations of surface water plus the possibility of toxicity from chlorine
residuals. Synthetic dilution water prepared by adding different salts to distilled water has been

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found to be the most ideal for use as dilution water. The pH of the dilution water should be in
between 6.5 to 8.5. Potassium, sodium, and magnesium salts are added to distilled water to give
buffering capacity and proper osmotic pressure. Ferric chloride, magnesium sulphate, and
ammonium chloride are added to supply the requirements for iron, sulphur, and nitrogen. The
phosphate buffer provides phosphorus that may be needed. The dilution water is usually well
aerated (through aeration process) to raise its DO level before mixing with wastewater.

A wide variety of materials have been used for "seeding" purpose. Experience has shown that
domestic wastewater, particularly from combined sewer systems provides a well-balanced
population of mixed micro-organisms. Usually 2 mL of wastewater per liter of diluted water is
sufficient. Some river waters are also satisfactory, but care must be taken to avoid using waters
that contain algae or nitrifying bacteria in significant amount. The dilution water should always be
"seeded” to ensure a uniform population of micro-organisms.

Modeling of BOD
It is often assumed that the rate of decomposition of organic waste is proportional to the amount
of waste available. If Lt represents the amount of oxygen demand left after time t. then, assuming
a first order reaction, we can write,

dLt/dt = - K Lt (5)

Where, k = BOD reaction rate constant (time-1).

Integrating, we get, Lt = L0 e-kt (6)

Where, L0 = the ultimate carbonaceous oxygen demand, which is the total amount of oxygen
required by microorganisms to oxidize the carbonaceous portion of the waste.

Now, L0 = BODt + Lt (7)

and combining, we get, BODt = L0 (1 – e-kt) (8)

Fig. - 1(a) and 1(b) shows the graphical representations of Eq. (6) and Eq. (8), respectively.

Besides the oxidation of carbonaceous organic matter, a significant additional demand may be
caused by the oxidation of nitrogenous compounds. To distinguish this nitrogenous biochemical
oxygen demand from the carbonaceous biochemical oxygen demand, the two are sometimes
referred to as NBOD and CBOD, respectively. Fig. - 2 illustrates the two demands for a typical
municipal waste. Mote that the NBOD does not begin to exert itself for at least 5 to 8 days, so most
5-day BOD tests are not affected by the nitrification process.

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 (a) (b)
Fig.- 1: Idealized carbonaceous oxygen demand: (a) the BOD
remaining as a function of time, and (b) the oxygen consumed or the
biochemical oxygen demand as a function of time

Fig, - 2: Illustration of Carbonaceous and Nitrogenous

Biochemical Oxygen Demand

Theory of DO Determination
Dissolved oxygen (DO) determination is done by Winkler Method. The reactions involved in the
various steps are presented below:

Manganous sulphate reacts with potassium hydroxide in the alkaline potassium iodide solution to
produce a white precipitate of manganous hydroxide.

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 MnSO4 + 2KOH = Mn(OH)2 ↓+ K2SO4 (9)
white ppt

If the white precipitate is obtained, there was no dissolved oxygen in the sample and there is no
need to proceed further. A brown precipitate shows that oxygen is present and reacted with the
manganous hydroxide and forms manganic basic oxide.

2Mn(OH)2 + O2 = 2MnO(OH)2 ↓ (10)

brown ppt

Upon the addition of (sulphuric) acid, this precipitate is dissolved, forming manganic sulphate:

MnO(OH)2 + 2H2SO4 = Mn(SO4)2 + 3H2O (11)

This compound immediately reacts with potassium iodide, liberating iodine and resulting in the
typical iodine (blue) coloration of the water.

Mn(SO4) + 2KI = MnSO4 + K2SO4 + I2 (12)

The quantity of iodine liberated by these reactions is equivalent to the quantity of oxygen present
in the sample. The quantity of iodine is determined by titrating a portion of the solution with a
standard solution of sodium thiosulphate solution. The end point of titration is indicated by the
disappearance of blue color produced by the reaction between iodine and starch (which is added
as an indicator in the titration).

2Na2S2O3 + I2 = Na2S4O6 + 2Nal (13)

Reagents
- Manganous sulfate solution [57]
- Alkaline potassium iodide solution [58]
- 0.025N sodium thiosulfate [59]
- Starch solution (indicator) [60]
- Concentrated sulfuric acid

Apparatus
- BOD bottle : 3 nos
- Beaker (250 ml) : 1 no.
- Measuring cylinder : 1 no.
- Dropper : 1 no.
- Stirrer : 1 no.

Procedure
(For determination of DO)

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Fill two BOD bottles with sample (or diluted sample); the bottles should be completely filled.
Determine initial DO (DOi) in one bottle immediately after filling with sample (or diluted sample).
Keep the other bottle in dark at 20°C and after particular days (usually 5-days) determine DO (Dof)
in the sample (or diluted sample). Dissolved oxygen (DO) is determined according to the following
procedure:

1. Add 1 mL of manganous sulphate solution to the BOD bottle by means of pipette, dipping
in end of the pipette just below the surface of the water.
2. Add 1 mL of alkaline potassium iodide solution to the BOD bottle in a similar manner.
3. Insert the stopper and mix by inverting the bottle several times.
4. Allow the "precipitates” to settle halfway and mix again.
5. Again allow the “precipitates" to settle halfway.
6. Add 1 mL of concentrated sulphuric acid. Immediately insert the stopper and mix as before.
7. Allow the solution to stand at least 5 minutes.
8. Withdraw 100 mL of solution into an Erlenmeyer flask and immediately add 0.025N
sodium thiosulphate drop by drop from a burette until the yellow color almost; disappears.
9. Add about 1 mL of starch solution and continue the addition of the thiosulphate solution
until the blue color just disappears. Record the ml. of thiosulphate solution used (disregard
any return of the blue color).

Calculation
Dissolved oxygen, DO (mg/L) = mL of 0.025N sodium thiosulphate added x MF

normality of Na2S2O3 x equivalent wt. of oxygen x 1000

Where, MF =
mL of sample taken

Calculate BOD of the sample according to Eq. -4.

Assignment Questions
1. In a BOD test on a diluted wastewater sample (1:20 dilution, but not seeded), the initial
DO is 8.2 mg/L and final DO after 5 days is 3.2 mg/L. If the reaction rate constant is
0.2/day, calculate; (a) 5-day BOD of the wastewater, (b) Ultimate carbonaceous BOD of
the wastewater, (c) Remaining Oxygen demand after 5-days.
2. A test bottle containing just seeded dilution water has its DO level drop by 0.5 mg/L in a
5-day test. A 300 mL BOD bottle filled with 40 mL of wastewater and the rest with seeded
dilution water experiences a drop of 7.1 mg/L in the same period (5-day). Calculate the
BOD5 of the wastewater.
3. A sample of sewage is mixed with water (no seeding done) in the ratio of 1:30 (i.e., 1 mL
of sewage diluted to 30 mL by adding water) for BOD test. The initial DO is 8 mg/L and
final DO, after 5 days, is 2.1 mg/L. Calculate BOD5 of the sewage.

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