Laboratory 2: Measurement of Biochemical Oxygen Demand (BOD)

Objectives:

1. To determine the amount of oxygen necessary for biological oxidation of wastewater

effluent.
2. To determine the amount of oxygen required by bacteria while stabilizing
decomposable organic matter.

Methodology:

In this experiment students will be diluting the wastewater with a known amount of
dilution water and measuring dissolved oxygen (DO) concentrations over the course of 5 days.
This requires an accurate DO determination. DO will be determined using DO meter and
iodometric titration method (Winkler method).

BOD5 values are acceptable only if the DO concentration after 5 days must be at least
1 mg/L and at least 2 mg/L lower in concentration than the initial DO. Therefore it is crucial to
determine the dilution(s) that will be needed. The table below provides general dilutions based
on anticipated ranges of BOD5.

When less than a 300-mL sample is to be analyzed, samples volumes are added to a
standard solution of dilution water. Dilution water is usually prepared by using distilled water,
added with various minerals such as potassium, sodium, calcium, ferrous chloride, magnesium
sulfate and phosphate buffer. The pH of the water is adjusted to 7.0 and the water is then
saturated with oxygen.

1. Make sure the BOD bottles are clean from organic contamination. They have to be
sealed properly by wetting the stoppers with a little bit of water to avoid any exchange
of air during incubation. Label the bottles appropriately: one with “initial” and the other
with “final”.
2. Add _____ mL of wastewater into both of the BOD bottles and fill them up to 300 mL
with provided dilution water. Take DO readings using DO probe for one of the bottle
which has been labeled as “initial”.
 3. Add 2 mL of manganese sulfate (MnSO4) just below surface of liquid in the “initial”
BOD bottle.
4. Add 2 mL of alkali-iodide-azide in the bottle.
5. Mix by inverting several times. Check for air bubbles.
6. Add 2 mL of sulfuric acid (H2SO4) just above surface of liquid. Invert sample to mix.
7. Put 200 mL of sample into a conical flask. Titrate with sodium thiosulfate (0.025N
Na2S2O3) to pale color. Stir and swirl continuously.
8. Add 2 mL of starch solution until blue color forms. Continue titration, stirring
continuously until sample is clear.
9. Calculate DO concentration. DO (mg/L) = ____ mL of titrant (mL) x 1 mg DO/1 mL.
10. Incubate the bottle labeled “final” for 5 days at 20C. After 5 days, take DO
measurements using DO probe and titration method (steps 3 – 9).

Results:

The BOD of the sample can be calculated using the equations provided in the following.

First equation is provided by the standard method:

𝑚𝑔 𝐷1 − 𝐷2
𝐵𝑂𝐷5 , =
𝐿 𝑃

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

D2 = DO of diluted sample after 5-day incubation at 20C, mg/L
P = fraction of wastewater sample volume to total combined volume

Discussion:

1. Obtain results from two other groups and compare the results. Calculate the average
of BOD of the wastewater.
2. Compare the BOD results using DO meter and Winkler method.
3. If DO of dilution water and D1 are known, estimate the initial DO of the wastewater.
4. Explain any changes on the control after 5 days.