BIOCHEMICAL OXYGEN DEMAND (BOD5)
Being able to measure the “strength” of wastewater is important for controlling treatment systems and for measuring the effectiveness or treatment. The Biochemical Oxygen Demand (BOD) is a test that measures the biological and chemical oxygen demand of wastewater. In the BOD test, wastewater samples are incubated at 20° C for five days. During the incubation, microorganisms metabolize nutrients in the sample. In doing so, they use oxygen. If a lot of nutrients are present, the organisms with reproduce actively, creating a larger population and thus, using a lot of oxygen. Furthermore, chemical substances in the wastewater sample (such as hydrogen sulfide or sulfur dioxide) will react with oxygen, which also causes an oxygen demand. Because both types of oxygen demand are measured, the test is called the Biochemical Oxygen Demand.
Samples for BOD analysis may degrade significantly during storage between collection and analysis, resulting in low BOD values. For this reason, analyze samples promptly or cool samples to 4º C for storage. Warm stored samples to 20ºC before analysis. For grab samples, analysis should be performed within two hours or the sample should be cooled to 4º C at time of sample collection. Standard Methods states that every effort should be made to begin analysis within 6 hours, but under no circumstances start analysis of grab samples more than 24 hours after sample collection. For composite samples, keep sample aliquots at 4º C during compositing and limit the compositing period to 24 hours. Use the same holding time criteria as for grab samples, starting the measurement of holding time from the end of the compositing period. Under the BOD methodology detailed in the Federal Register under the Code of Federal Regulations, (40 CFR 136), a maximum sample holding time of 48 hours following the last composite sample aliquot is allowable. However, be aware that this longer holding time should only be used out of clear necessity and that 40 CFR 136 should be cited as the sampling protocol. For all BOD samples, state the storage time, temperature and conditions as part of the result.
The BOD test is conducted in special 300 mL glass bottles, known as “BOD bottles”. To begin setting up the test, a measured volume of sample is added to a BOD bottle. The amount of sample that is used depends upon how strong the lab analyst suspects the wastewater to be. For example, if the sample is raw influent, the analyst may only use a small portion, say 20 mL, whereas if the sample is very clear effluent, the analyst may use 250 mL. If the sample was disinfected, (effluent), there may not be enough live microorganisms in it to conduct the test, and so extra microorganisms must be added. These extra microorganisms are known as “seed”. Usually, 1 – 3 mL of settled influent is used as a seed, which is added to the BOD bottles containing sample. After the addition of seed, the BOD bottles are filled up the rest of the way with buffered dilution water that contains all of the things (other than food) that the microorganisms need to reproduce. Using a dissolved oxygen meter equipped with a special stirring probe, the dissolved oxygen in the BOD bottles containing sample, seed and buffered dilution water is then measured. This beginning dissolved oxygen level is known as the initial D.O.
After the initial D.O. has been measured in each of the BOD bottles, the bottles are sealed so that no oxygen can get in or out. Then, the BOD bottles are placed in an incubator that is specially designed for the BOD test. The samples are incubated at 20º +/- 1º C for 5 days. It is critical that the incubation temperature stay as close to 20º C as possible if the test is to be accurate. When samples are placed into the incubator in the morning, they should be read in the morning 5 days later. When placed into the incubator in the afternoon, they should be read 5 days later, in the afternoon. If the incubator allows the temperature to exceed 21 ºC or go below 19 ºC, all samples in the incubator become invalidated and cannot be used for reporting purposes.