Running the BOD Test: General Questions and Pointers

1. When performing the BOD test what are the acceptable ranges Standard Methods allows?

  • The Dilution Water Blank (i.e. water check) depletion must be <0.2 mg/L over a 5-day period
  • The Seed Control Factor (i.e. SCF, seeded blank, or seed check) must fall between 0.6-1.0 mg/L
  • The GGA standard (i.e. controls) must be 198 ± 30.5

2. Why is it important to have a watertight seal on my BOD bottles?

As a precaution against drawing air into the BOD bottle during the 5-day incubation, you must have a watertight seal.

Having a water tight seal will prevent the following:

  • Possible contamination
  • A change in the volume of solution in the bottle
  • The bottle stopper from becoming stuck

3. What type of BOD bottles should I use?

To ensure a watertight seal, InterLab Supply recommends using Wheaton’s 300-mL BOD bottles.  Wheaton’s BOD bottles have a flared mouth and use tapered ground-glass stoppers which are the bottle features recommended in The Standard Methods for Examination of Water and Wastewater.

4. Why is it important to run the BOD test?

Most importantly, the BOD test is required by law. The BOD test is used to determine how much oxygen is being used by aerobic microorganisms in the water to decompose organic matter. If these aerobic bacteria use too much of the dissolved oxygen in the water, there will not be enough left over for the fish, insects, and other organisms that rely on the oxygen to live. In this situation, the rich diversity of life in a healthy river is reduced to a low diversity (but sometimes high volume) of pollution-tolerant organisms.

5. Why is it important to read my 5-day DO±6 hours of incubation?

You want to keep a tight time frame when you read your final DO. The 21st edition of Standard methods now suggests that reading the final DO ± 6 hours will yield the most accurate test results.

6. What temperature should I store my BOD bottles during the 5-day incubation period?

Standard Methods states your bottles should be stored at 20 ± 1°C throughout the 5-day incubation period. Remember, temperature directly affects the metabolic rate of the bacteria. Incubating your bottles at 20 ± 1°C provides the perfect “controlled” environment for the bacteria to reproduce.

7. Why is it important to store my BOD bottles in the dark during the 5-day incubation period?

Excluding all light prevents the possibility of photosynthetic (algae) production of DO.

8. What dilutions should I use when performing the BOD test?

Below are the dilutions we recommend. Please note that these dilutions may be adjusted to achieve a SCF between 0.6-1.0mg/L and a GGA of 198 ± 30.5:

  • 2 dilution water blanks
  • 4 seeded controls-preferably; 15, 20, 25, and 30 ml of PolySeed solution
  • 2 Glucose-Glutamic Acid Controls-using 6 mls of GGA and 4 mls of PolySeed solution
  • Add 4 ml of PolySeed solution to each live BOD sample bottle, and Glucose-Glutamic Acid controls

9. Why is it important not to have any bubbles present in any the BOD bottles?

You do not want to add additional oxygen to your samples. Therefore, no visible bubbles should be present during preparation, or any time during the test.

10. Why should I use an air pump to aerate my seed solution instead of using the vortex method?

Aeration is very important when rehydrating PolySeed. The bacteria in PolySeed require a sufficient amount of air (i.e. oxygen) to reproduce correctly. Many labs aerate PolySeed by pulling a vortex (the tornado-like funnel that occurs when a liquid is being stirred fast). When this method is used, the vortex sucks outside air into the solution. We do not recommend this method due to the stress it puts on the bacteria.