To Seed or Not To Seed - BOD Explained
Dilution, pH Adjustment, Seeding and BOD Calculations

Article written by Jessica Raney, Quality Control Group Leader, Analytical Products Group

Reprinted for Edition 13 originally published for the Edition 4 APG eNewsletter

Typically, 75% of the questions fielded by the APG technical staff around DMRQA time center around the Demand sample and BOD analysis. The test for BOD is time consuming, confusing, and there is little room for error.

What is Biochemical Oxygen Demand?
Biochemical Oxygen Demand or BOD is a measure of the amount of oxygen used by aquatic microorganisms to breakdown organic material found in the water. This organic material can be introduced into the water from natural sources such as plant growth and decay or by humans such as waste streams and agricultural runoff. These added organic materials are used as nutrients that promote the growth of the microorganisms in the water. There is a limited amount of dissolved oxygen available in the water. As the microorganisms metabolize the organic matter, they deplete the limited amount of dissolved oxygen. When the amount of dissolved oxygen in the water reaches a certain concentration, the water system will fail to support larger aquatic organisms. Dissolved oxygen levels below 3 ppm are very stressful to aquatic organisms and any level below 2 ppm will result in fish kills. It is important that the BOD levels be carefully monitored in order to keep the aquatic ecosystem in balance.

In the test for BOD, a sample is seeded for microorganisms and saturated with oxygen. Some samples, such as wastewater from a treatment facility already contain the population of bacteria necessary to oxidize the organic material, therefore wastewater treatment operators do not generally need to seed the routine samples that they analyze. The initial amount of dissolved oxygen (Do_i) is measured and the sample is then incubated for five days in a temperature controlled, dark environment. After the incubation period, the final amount of dissolved oxygen (DO_f) is measured. The difference between the DO_i and the DO_f are used to calculate the initial BOD of the sample.

APG's Demand sample can be tested for four parameters. In addition to BOD, Carbonaceous Biochemical Oxygen Demand (CBOD), Chemical Oxygen Demand (COD), and Total Organic Carbon can be measured (TOC). CBOD is simply a BOD analysis with a nitrification inhibitor added to prevent the oxidation of various forms of reduced nitrogen. The analysis of an APG PT sample for BOD may seem to differ slightly than the samples run on a routine basis. This is not the case. Once prepared according to the provided instructions, the PT sample should be treated in the same manner as any other unknown sample.

Initial Dilution
In order to ensure stability, the APG sample is a concentrate. The APG sample should be diluted 20 mL to one liter of laboratory grade water. There is approximately 23 mL of sample contained in the vial. Therefore, there is only enough of the sample for one dilution, and good pipetting technique is necessary in order to accurately prepare the sample. The diluted sample should be mixed for at least 15 minutes before it is tested.

pH Adjustment
One of the most critical steps in the preparation of the PT sample for BOD analysis is the pH adjustment. The pH of the resulting one-liter solution should be adjusted to 6.8-7.2. Hydrochloric acid and sodium hydroxide are acceptable for the adjustment. If the pH is not properly adjusted, it will result in the failure of any microorganisms to live in the solution no BOD will be measured.

Another important step in the process is to have properly prepared dilution water. Refer to Standard Method 5210 B. for the preparation procedure. Essentially, dilution water is water that has had nutrients added to it such as nitrogen, phosphorous, and trace metals that promote bacterial growth.

Do I need to seed the PT sample?
As was mentioned previously, most wastewater operators do not seed normal samples because the sample already contains the bacteria necessary to deplete the oxygen. In the case of the APG PT sample, specific steps have been taken to inhibit bacterial growth in order to provide a stable sample. This is why the PT sample must be seeded. Seeding kits can be purchased. One recommended source for seeding material is Polyseed produced by Interbio. You can find more information about their seeding kits at www.InterLabSupply.com. Effluent from the primary settling tank may also be used. How much seed is used depends on the instructions provided by the seed manufacturer.

How much of the PT sample should I put in the BOD bottle?
There is no easy answer to that question. The easiest answer is to treat the PT sample in the same manner that you would treat any unknown sample and do multiple dilutions. Dilutions of 5,10, 25,50, and 100 mL are recommended. You may use any dilution where the DO_f has depleted by two and is still greater than one.

Calculation of the BOD
The final step in the analysis is the calculation of the BOD from the data obtained. The formula for the calculation is as follows:

The Importance of Quality Control Samples
It is recommended that quality control samples be used to ensure that the procedure is under control. An APG quality control standard is essentially a PT sample that has been used for a round robin study. By analyzing the known sample and achieving acceptable results, you can be assured that your BOD analysis is operating correctly. Once you have verified that your analysis is under control, you can have more confidence in the data you generate about the PT sample. For more information about the BOD test, refer to Standard Method 5210 B: 5-Day BOD Test or EPA Method 405.1.