BOD5 at 20 Degrees C: What the Five-Day Number Measures, Where It Misleads, and How to Run It Properly

A test that measures behaviour, not mass

Biochemical Oxygen Demand (BOD) is the amount of dissolved oxygen aerobic microorganisms need to decompose organic matter into inorganic substances within a specific time at a certain temperature. It is not a precise quantitative test, because no single compound is being counted, but because it indirectly reflects the relative content of organic matter it has stayed in service for over a century as an environmental monitoring indicator. In water-environment modelling the same logic applies: tracking every compound individually is impractical, so BOD stands in for organic matter as it changes in the water.

BOD is also called biochemical oxygen consumption. It is the total dissolved oxygen consumed when organic matter is oxidised and decomposed into inorganic or gaseous form by the biochemical action of microorganisms. The higher the value, the more organic pollution and the more serious the pollution. Hydrocarbons, proteins, oils and lignin, suspended or dissolved in domestic sewage and in industrial wastewater from sugar, food, paper and fibre operations, are all oxygen-demanding pollutants. Discharge them in excess and dissolved oxygen is depleted, anaerobic bacteria take over, and methane, hydrogen sulfide, mercaptans and ammonia make the water putrid and malodorous.

Why five days, and why 20°C

Complete oxidation of all organic matter in sewage takes about 100 days. Waiting that long is useless for operations, so the convention, established by the British Royal Commission on Sewage Disposal, is to incubate at 20°C for 5 days and report the oxygen consumption in mg/L. That figure is the 5-day BOD, abbreviated BOD5; for domestic sewage it represents about 70% of the oxygen that complete oxidation would consume. Extending the incubation gives total BOD, but in practice it is only estimated from BOD5 using assumed biochemical oxygen-consumption patterns.

Reading the ratio

BOD on its own tells you how much oxygen demand exists. BOD against Chemical Oxygen Demand (COD) tells you what fraction of the organic load microbes can actually attack. COD is measured chemically: it is the amount of oxidant consumed by reducing substances in the sample under defined conditions, converted to the milligrams of oxygen needed to fully oxidise one litre, expressed in mg/L. It reflects pollution by reducing substances and is one of the comprehensive indicators of relative organic content. A BOD/COD ratio above 0.3 is generally taken as the point where biological treatment becomes the sensible choice; below it, the fraction that microorganisms cannot decompose is doing the environmental damage.

Benchmarks worth memorising

• A clean river typically sits at or below 2 mg/L for 5-day BOD; above 10 mg/L it starts to give off a foul odour.
• Industrial, agricultural and aquacultural water should stay below 5 mg/L.
• Drinking water should be below 1 mg/L.
• Municipal sewage runs around 200 mg/L, while high-concentration organic industrial wastewater can reach thousands or even millions of mg/L.
• Chinese municipal discharge grades for BOD: 10 mg/L for Grade 1A, 20 mg/L for Grade 1B, 30 mg/L for Grade 2 and 60 mg/L for Grade 3. The Integrated Wastewater Discharge Standard caps BOD at the factory outfall for Grade 2 at 60 mg/L and requires surface water BOD not to exceed 4 mg/L.

What the sensor method actually does

The rapid microbial-sensor approach pairs an oxygen electrode with a microbial membrane. Sample saturated with dissolved oxygen enters the flow cell and contacts the sensor; the strains in the membrane act on the dissolved biodegradable organic matter, consuming oxygen and reducing the amount diffusing to the electrode surface. Once the diffusion rate of biodegradable matter to the membrane becomes constant, the oxygen flux and therefore the current become constant too, and the constant-current difference quantifies BOD. It suits surface water, domestic sewage and industrial wastewater that does not contain substances markedly toxic to microorganisms.

Interference ceilings are explicit: CO2+ 5 mg/L, Mn2+ 5 mg/L, Zn2+ 4 mg/L, Fe2+ 5 mg/L, Cu2+ 2 mg/L, Hg2+ 5 mg/L, Pb2+ 5 mg/L, Cd2+ 5 mg/L, Cr6+ 0.5 mg/L, CN- 0.05 mg/L and suspended solids 250 mg/L. Samples carrying free or combined chlorine are dosed with sodium sulfite solution at 1.575 g/L to deactivate it, avoiding excess. Streams loaded with bactericides or pesticides toxic to the membrane strains are out of scope for the method.

Reagents and preparation, in the order they bite you

Use analytical-grade reagents and distilled water, and boil the distilled water for about 2–5 minutes before cooling it to room temperature. Glassware and plastic containers must be cleaned carefully; no toxic or biodegradable compound may remain on the walls, and contamination must be avoided during handling. The phosphate buffer stock at 0.5 mol/L dissolves 68 g potassium dihydrogen phosphate (KH2PO4) and 134 g disodium hydrogen phosphate (Na2HPO4·7H2O) and makes up to 1000 mL, giving pH around 7; the working solution used as cleaning solution is 0.005 mol/L. Hydrochloric acid (HCl) is 0.5 mol/L, sodium hydroxide (NaOH) is 20 g/L, and sodium sulfite (Na2SO3) at 1.575 g/L must be prepared fresh because it is unstable.

The glucose-glutamic acid standard weighs 1.705 g each of anhydrous glucose (C6H12O6) and glutamic acid (HOOC–CH2–CH2–CHNH2–COOH), dried at 103°C for 1 h and cooled, dissolved and diluted to give a 250 mg/L BOD standard solution. Taking 10.00 mL of that into a 250 mL volumetric flask and making up with 0.005 mol/L phosphate buffer working solution gives 100 mg/L. The cleaning buffer, prepared from potassium dihydrogen phosphate and disodium hydrogen phosphate, adjusts sample pH, cleans and maintains the sensor, and settles heavy-metal ions.

The microbial membrane itself has to be uniform and consistent between batches, stored wet or dried at room temperature, with a continuous service life above 30 days. Activation means soaking it in 0.005 mol/L phosphate buffer working solution for over 48 h before mounting. Temperature stability is not optional, because electrode response depends on a stable temperature field; that is what the constant-temperature control device in the instrument is for. A peristaltic pump feeds sample or cleaning solution into the measurement cell at a fixed ratio per unit time.

Sampling and arithmetic

If a sample cannot be analysed within 2 hours of collection, hold it at 0°C–4°C and analyse within 6 days; if analysis cannot start within 6 hours, report storage time and temperature alongside the result. Under no condition may storage exceed 24 hours.

The calculation is BOD (mg/L) = (D1 − D2) / P, where D1 is the initial dissolved oxygen of the diluted sample, D2 is the dissolved oxygen after 5-day incubation at 20°C, and P is the volume of water sample divided by the final volume of the diluted sample. Both incubation time and temperature affect oxygen consumption, which is why every country settled on the same 5 days at 20°C, written BOD5 at 20°C, or simply BOD5, or sometimes just BOD.

Where BOD5 misleads

BOD testing is not easy to do accurately. Sample storage, dilution and inoculation all have to follow standard methods, and toxic industrial wastewater often needs special equipment or cannot be measured at all. The five-day window also truncates what slow-degrading compounds would eventually demand, so total BOD is only ever an estimate. Treat the number as a comparative signal about biodegradable load and treatability, not as an inventory of what is in the water.