After the 5 days of incubation, the BOD bottles are removed from the incubator and the level of dissolved oxygen is again measured in the bottles. This is known as the Final D.O. The amount of dissolved oxygen that was consumed during the incubation is related to the strength of the sample and the volume of sample added to the bottle. Because the lab analyst knows the sample volume, a simple calculation can be performed to determine the concentration of BOD in milligrams per liter.
BOD, mg/L = [(Initial D.O. – Final D.O.) X 300] / (Volume of Sample used, mL)
As discussed earlier, when we are testing samples that have been disinfected (like effluent), there are not enough microorganisms alive in the sample to allow the test to run. Therefore, we add “seed” microorganisms to these types of samples in order to ensure there are live bugs to use the dissolved oxygen during the incubation time. Good seeding material is usually obtained by settling raw influent for at least 1 hour but less than 36 hours and then pipetting from 1 cm below the surface of the settled liquid. Primary effluent, non-disinfected
secondary effluent and commercially prepared seed can also be used.
Unfortunately, no matter what the origin of the seed, some oxygen-demanding material (BOD) will be carried along with the seed microorganisms. In order to obtain the true BOD of the sample, we must subtract out the amount of organic material that came along with the seed microorganisms. To do this, we determine how many mg/ L of D.O. are used per mL of seed by running seed controls along side the regular samples.
Seed Control =
(Initial D.O. Seed – Final D.O. Seed) / (mL of seed added to seed control bottle)
Knowing the BOD of the each seed control allows us to calculate the seed correction factor, which is the average of the seed controls multiplied by the mLs of seed added to each of the sample bottles.
Seed Correction Factor = Average of seed controls X mL of seed added to each sample bottle
Ideally, the Seed Correction Factor should be between 0.6 and 1.0 mg/L. If it is too high, too much seed has been used in the sample bottles. If it is too low, more seed should be used in the sample bottles next time. Often, lab analysts have to adjust the amount of seed (and even the source of the seed) in order to stay within this range.
BOD, mg/L =[(Initial D.O. – Final D.O. – Seed Correction Factor) X 300] / (Volume of sample used, mL)
To calculate the BOD for a seeded sample, we use the same equation shown above, except that we eliminate the oxygen demand caused by the seed by subtracting out the seed correction factor.
It is important to note that not all samples require seeding. Influent samples typically have a multitude of live microorganisms and therefore do not require seeding. If a lab analyst does not know if a sample has been disinfected or not, it is safest to seed the samples to ensure good test results.