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.