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For information on the Lab Certification program, contact:
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Ultimate BOD header graphic

BOD sample pre-treatment requirements

Because the BOD test is a bioassay, it is critical to maintain optimal conditions for the "bugs" (bacteria and other microorganisms) to remain viable. Temperature, pH, oxygen levels and the presence of disinfectants all can all influence the outcome of the BOD test. Before proceeding with the BOD test, the following items must be checked. In many cases pretreatment maybe required.

  • Temperature
  • pH
  • residual chlorine or other disinfectants
  • Dissolved oxygen (DO) super-saturation

Preliminary testing - pH

pH extremes kill (or at least severely injure) the "bugs" (microorganisms). Consequently, you must test each sample to confirm that the pH is in the appropriate range before proceeding with the BOD test.

Test for proper pH ("pH extremes" kill bugs)
pH extremes are defined as less than pH 6 or greater than pH 8.5
pH extremes were undefined in previous editions of Standard Methods NOTE: 21st ed. Standard Methods states that if samples exceed a pH range of 6 to 8, then adjust to pH 7.0 to 7.2 (to match international standards).
Adjust pH as needed. ( as below)
If the pH of the undiluted sample is less than 6 or greater than 8.5, adjust the pH to 6.5 -7.5 with 1N H2SO4 or 1N NaOH.
Do not dilute sample by >0.5% (1.5 ml in a 300 ml bottle). If more acid or base is needed, use more concentrated solutions (i.e., 5N)
Phosphate buffer in the dilution water will often adjust the pH into the acceptable range, particularly in sample volume samples. However, this must be confirmed and recorded.
Diluted sample pH must be between 6.5 & 7.5.
Always seed samples that have been pH-adjusted.

Preliminary testing - disinfection and disinfection residuals

Check wastewater samples for residual chlorine unless it can be demonstrated that the sample was collected prior to where chlorine is added. A plan diagram with the sampling point shown in relation to the chlorine contact tank is usually satisfactory. However, as a general rule, commercial laboratories should check all treated wastewater for the presence of chlorine and document this on some type of bench record.

Chlorine (Remember: chlorine kills bugs!)
If any chlorination process is employed:
  1. Test for residual chlorine
  2. If detected, quench the chlorine residual as per Standard Methods and,
  3. SEED the sample(s)
Other disinfection (UV)
If ANY disinfection process is employed (UV, chlorine) SEED the sample(s).

Preliminary testing — oxygen super-saturation

Water only has a limited capacity to hold oxygen. This capacity, or saturation point, is driven by temperature and barometric pressure (see Calibration). Many people incorrectly believe that Standard Methods establishes the maximum dissolved oxygen concentration allowed in the BOD test is 9.0 mg/L. Actually, this is not correct. Actually the point of super-saturation could occur at a much lower oxygen concentration.

If the sample DO is greater than saturation point when the bottles are placed in the incubator, oxygen will physically come out of solution and appear to be an oxygen demand. The resulting BOD will be falsely high.

There must also be a minimal amount of oxygen present such that there will be enough present so the BOD dilutions will not go anaerobic during the 5 day test period. The following summarizes the preliminary oxygen testing requirements for the BOD test.

Warm samples THEN shake to saturate

Warm samples to room temperature (20 ± 3°C) and then shake them to prevent super-saturation problems.

Check for super-saturation of oxygen (results in high bias)

  • Know the saturation point at your facility/your conditions...
    • Definitely a problem if DOI > 9.0 mg/l at 20°C,
    • Can occur during winter months (the colder the water, the more oxygen it can hold!)
    • Can also be a problem in localities where algae are actively growing (e.g., lagoons)
  • Results in high bias (the 'extra' oxygen quickly comes out of solution during incubation, but is read as oxygen used during incubation)
  • Reduce excess DO by shaking sample(s) or aerate with filtered compressed air

Make sure there is enough oxygen!

Always start with an initial DO close to the saturation point for your facility (typically about 8.2 to 8.5 mg/L in Wisconsin)

If the DO is low, shake or aerate with filtered compressed air to increase the DO concentration. Remember, starting with a higher initial DO will allow you to cover a wide BOD range with each dilution.


Here's the bottom line on preliminary testing for the BOD test:

Residual chlorine
If sampled upstream of chlorination, have documentation to prove it (e.g., plant schematic)
If not using chlorine (e.g., UV) note such on the benchsheet and SEED all samples.
Otherwise, you must document that samples were checked for chlorine, how, and results.
Need documentation someplace that the pH of ANY sample analyzed is within requirements
If the whole sample meets pH requirements, so will any dilution
It's acceptable to use short range pH paper for this check
Need documentation someplace that temperature of ANY sample analyzed is within requirements (20 ± 3°C)
If the whole sample meets temperature requirements, so will any dilution.
It's acceptable to use DO meter thermometers that have been verified and corrected (if necessary)

Checklist: sample storage/pre-treatment

The Wisconsin Department of Natural Resources Laboratory Certification & Registration Program has developed checklists that are used by all auditors. The list in the image below identifies the sample storage, handling and pre-treatment items they will check during a laboratory on-site evaluation. Laboratories are expected to have records to show that each of these requirements have been meet. The list also provides guidance for laboratories so there should be no surprises during an audit.

Excerpts related to sample pre-treatment from WI DNR LabCert Progranm BOD Checklist

Critical BOD sample pre-treatment requirements excerpted from WI DNR Laboratory Certification Program BOD checklist.

Copyright 2006. University of Wisconsin Board of Regents.
Unauthorized use prohibited without the expressed written consent of the UW, State Laboratory of Hygiene and the DNR Laboratory Certification & Registration Program.

Last revised: Wednesday December 18 2013