Biochemical Oxygen Demand is a sum parameter and the amount of oxygen required to oxidise organic matter present in the water biochemically. So BOD is an indirect measure of the concentration of organic contamination in water. BOD analysis does not oxidise all of the organic matter present in the waste; only the organics that are biochemically degradable during n days time period at 20°C are oxidised. The day period is given as index in BOD_n. The standard for usual measurements is a 5-day period.

BOD₅ is the most widely used parameter of organic pollution applied to wastewater and is used:

• to determine the approximate quantity of oxygen that will be required to biologically stabilise the organic matter present,

• to determine the size of wastewater treatment facilities,

• to measure the efficiency of some treatment processes;

• to determine compliance with wastewater discharge permits.

For the measurement of BOD, different volumes of wastewater are mixed in special BOD bottles with a liquid called "dilution water". This may be final effluent of a wastewater treatment plant which still contains some microorganisms or primary clarifier effluent diluted with tap water; it has to be supplemented with nitrogen, e.g. urea, and phosphate, aerated for a period of 3 to 10 days prior to use for BOD analysis, which had been saturated with oxygen prior to BOD analysis by bubbling in air. Moreover, a nitrification inhibitor (e.g. allylthiourea) is added, because only the oxygen consumption due to biochemical oxdation of organic wastewater constituents - and not of ammonia - is desired to be determined. Also blanks are prepared (bottles containing only dilution water and nitrification inhibitor).

The BOD bottles are completely filled and sealed with a glass stopper in such a way that no more air bubbles are contained in the bottles. With every mixture a duplicate of bottles is prepared. In one bottle of each pair, the concentration of dissolved oxygen is determined (e.g. by means of an oxygen probe) immediately after mixing. The other bottle is stored for n days at 20°C in the dark (to prevent photochemical reactions). At the end of this period, the concentration of dissolved oxygen is measured also in this bottle. The difference of oxygen concentration in the two bottles of a pair is the oxygen consumption (OC) (mg O₂/l). From the oxygen consumption of a particularly diluted wastewater sample and the oxygen consumption of the blanks (OC_DW), the BOD_n is calculated as follows:

with DF being the dilution factor (V_{(diluted sample)}/V_{(sample before dilution)}). BOD values determined for different dilutions should give a straight line when drawn as a function of the term V_{(sample before dilution)}. When points corresponding to low dilution factors (i.e. to high V_{(sample before dilution)}) in this graph are lying below the extrapolated line, this is a hint, that inhibition of microorganisms occurred in samples with low dilution. These values must not be applied for BOD determination!