2.9. Determination of BOD5 by the standard dilution method
BOD5 was determined by the dilution method as indicated in [2].
Dissolved oxygen concentrations in a sample were measured before
and after the incubation period and adjusted by the sample corresponding dilution factor. To ensure that all other conditions were
equal, a very small amount of microorganism seed was added to
each sample tested. This seed was generated by diluting organisms
with buffered dilution water. The BOD test was carried out by diluting the sample with oxygen saturated dilution water, inoculating it
with a fixed aliquot of seed, measuring the dissolved oxygen (DO)
and then sealing the sample to prevent further oxygen dissolving
in. The sample was kept at 20 ◦C in the dark to prevent photosynthesis (and thereby the addition of oxygen) for five days, and the
dissolved oxygen was measured again. The difference between the
final DO and initial DO was the required BOD value.