Subtleties of the Biochemical Oxygen Demand Test

BOD BottlesBiochemical Oxygen Demand (BOD) is a procedure used to measure the biodegradable organic material in a given water sample. This is one of the most commonly permitted parameters due to the drastic negative effects BOD laden water can have on natural streams. Bacteria rapidly degrade organic material through the process of aerobic respiration; which in turn depletes the dissolved oxygen of the water column. Oxygen depletion caused by untreated BOD discharge results in massive fish kills and poor water quality. Good environmental stewardship requires that BOD be removed from wastewater prior to discharge into natural waters.

For compliance sampling, the BOD test is run for 5 days. This 5-day incubation period was established, or so the rumor has it, to mimic the time it takes sewage water released in London, England to travel down the Thames River to the ocean. Samples are incubated in special refrigerators to hold the temperature at 20 °C +/- 1 °C over the 5-day test period. Holding time for the BOD5 test is 48 hours after collection.

The BOD5 test can be separated into total BOD5, which measures all biodegradable material in the sample, and soluble BOD5, which measures dissolved biodegradable material in the sample. In short, water samples are diluted with artificially aerated water that has been supplemented with nutri­ents and then seeded with a known volume of bacteria. The seed can be bought from commercial providers or pulled from working wastewater systems. Dilutions are determined by estimating the amount of BOD5 in the sample.

A common technique is using the Chemical Oxygen Demand (COD) as a surrogate to estimate the BOD5. Dissolved oxygen (DO) of the sample is measured initially after mixing the solution and again after five days. Initial DO should fall between 8.6 and 9.2mg/L at 20 °C. Final DO readings must change by 2.0 mg/L and have a residual above 1.0 mg/L. Final DO re­quirements are set to ensure the DO readings changed above background levels and that enough oxygen was available for the bacteria to fully degrade all BOD in the sample. The amount of BOD degraded in each sample is relative to the amount of oxygen removed from the water. Therefore, the sample dilution and amount of oxygen removal can be used to calculate the amount of BOD5 in the sample.

The BOD5 test can be affected by many different environmental factors. For example, nitrifying bacteria can oxidize ammonia in the BOD bottle from either the nutrient solution added or from the actual wastewater sample. Nitrification results in approximately 4 mg/l BOD impact per mg/l of ammonia nitrogen. This results in overestimating the amount of organic, or carbonaceous, BOD remaining in the effluent sample. To compensate, a nitrification inhibitor can be added to the nutrient water. When using a nitrification inhibitor, the test is called carbonaceous BOD or CBOD. These results can only be used for regulatory testing if the permit allows for CBOD. In addition, swings in pH below 6 or above 8 can affect bacterial growth. If the initial sample is outside this range, adjust the pH at room temperature to between 7.0 to 7.2 pH units prior to running the test. Sample volumes below 0.25 mL in a dilution can skew the data if a representative aliquot of sample is not used. To avoid this interference, dilute the sample in series to more uniformly distribute the sample’s biodegradable material prior to diluting into the BOD bottles. Multiply by this dilution factor when calculating the BOD5. Chlorine residuals in samples will kill the seed bacteria added during the BOD5 test. Allow the sample to sit in the light for 1 to 2 hours to remove the chlorine. If any chlorine is left after this time period, Na2SO3 can be added to scavenge the remaining ions. These interferences are common problems associated with BOD testing but are a far from complete list. Every plant has its own idiosyncratic combination of interferences and should be treated as a unique situation when troubleshooting.

Environmental Business Specialists provides laboratory audits and procedure development and/or revision for a variety of compliance tests, including BOD5. For more information contact us at

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