Judging Biodegradability: Five Test Methods and What Each One Misses

The question behind the question

When an engineer asks whether a wastewater is biodegradable, the useful question is narrower: can micro-organisms remove enough of the organic load, fast enough, to make a biological plant smaller than the alternative? Wastewater contains both easily metabolised compounds and fractions that resist attack or actively inhibit growth, and the relative abundance of the two decides whether aerobic treatment is feasible and how much tank volume it will need.

Rate matters as much as extent. A substance that is ultimately degradable but takes three weeks is, for design purposes, not degradable. Every method below is an attempt to answer the same two questions at once, and each answers them with a different bias.

Ratio methods: BOD5/COD and its relatives

The five-day biochemical oxygen demand divided by the dichromate chemical oxygen demand is the classic index. BOD measures the oxygen aerobic organisms consume while metabolising the pollutants; COD measures the oxygen a chemical oxidant consumes while oxidising them completely. The ratio is read as the biodegradable share of the total, and in general a higher value means a more treatable water.

Instrumental alternatives exist. Total organic carbon (TOC) and total oxygen demand (TOD) can be measured far faster and more reproducibly than COD, and reflect organic content more accurately. On that basis BOD/TOD and BOD/TOC ratios have been adopted abroad as indices in their own right, each with its own acceptance criteria. Whatever the denominator, the logic is identical: biodegradable carbon over total carbon.

The strength of the approach is that everybody understands it, the methods are standardised and the glassware is cheap. The weaknesses are less widely taught, and they matter more.

Three ways the ratio misleads

First, reproducibility. BOD is an empirical number whose comparability depends on identical dilution, seeding and analyst practice. There is no standard seed against which the error introduced by a particular inoculum can be checked, so no one can say which of two results is closer to the truth. Inter-laboratory agreement on the same sample is poor.

Second, the ratio is not the biodegradable fraction. Studies of industrial and municipal wastewaters fit relationships of the form COD = a + b multiplied by BOD, where a is the COD of the non-biodegradable fraction and b is the ratio of the COD of the biodegradable fraction to its BOD. The BOD/COD ratio is constant only when a is zero, which is almost never.

Third, specific water characteristics distort the result or invert it. Suspended organic solids are readily oxidised by dichromate and so appear in COD, but their physical form limits them in a BOD bottle, which depresses BOD5 and therefore the ratio; in a real plant those solids are flocculated, hydrolysed by extracellular enzymes and oxidised inside the cells, so a low ratio does not prove poor treatability. COD also counts oxygen consumed by inorganic reductants such as sulphides, sulphites, nitrites and ferrous iron, and BOD counts some of the same species, but the two methods leave them in different end states, so the oxygen they account for differs between methods. And a few compounds resist dichromate altogether: pyridine is not oxidised chemically but is metabolised biologically, so it contributes nothing to COD while appearing as BOD5 and pushing the ratio up.

The practical conclusion is that the ratio is a screening tool, not a verdict. A defensible number comes from a biological test.

Respiration curves: three shapes that answer the question directly

Plot oxygen uptake against time and the shape of the curve tells you what the biomass is doing. Oxygen uptake rates are measured with a Warburg respirometer or an electrode dissolved-oxygen meter. The reference is endogenous respiration: once the organisms reach the endogenous phase their uptake rate is constant, so the plot is a straight line through the origin whose slope is the endogenous rate.

• A curve above the endogenous line means the pollutants are biodegradable and the uptake rate exceeds the endogenous rate; when the curve later becomes parallel to the endogenous line, substrate degradation is essentially finished.
• A curve coinciding with the endogenous line means the pollutants are not biodegradable but do not inhibit the biomass.
• A curve below the line means the pollutants are not biodegradable and are inhibitory or toxic; if it reaches the horizontal axis, respiration has stopped and the biomass is dead.

Replacing time on the horizontal axis with substrate concentration converts the same experiment into the oxygen-uptake method, which is the same principle with a different presentation.

Carbon dioxide evolution

Degrading a pollutant consumes oxygen and releases a corresponding quantity of CO2, so measuring the gas produced gives an alternative index. The Sturm test, run over 28 days, compares actual with theoretical CO2 yield; the CO2/DOC ratio works on the same basis. Both need dedicated apparatus and careful operation, which is why they remain laboratory tools with little production-scale use.

Physiological indicators: dehydrogenase and ATP

Micro-organisms use wastewater organics as carbon and energy source, so the biochemical state of the biomass during that process is itself a measurement. Dehydrogenase activity is the most used: dehydrogenases catalyse the transfer of hydrogen from the substance being oxidised to an acceptor, they are extremely sensitive to toxins, and their activity falls when toxins are present. Activity rising in a culture grown on a given substrate means the substrate is degradable.

Adenosine triphosphate (ATP) works on the same logic from the energy side. Oxidative degradation is energy metabolism, and ATP is the cell energy-storage compound, so ATP level tracks microbial activity and therefore degradative capacity. Other physiological indicators include standard plate counts, DNA measurement, INT reduction and luminescent-bacteria light intensity. All of these need demanding instrumentation and reagents, so in practice they are used to judge single compounds and ecotoxicity rather than whole effluents.

Simulation: the closest thing to an answer

Shake-flask tests charge conical flasks with the pollutant or wastewater as carbon source, add nitrogen and phosphorus, adjust pH, inoculate with a strain or acclimatised sludge, and shake. Over a period the medium appearance, the population and indicators such as pH and COD are followed. Model bioreactor tests go further, reproducing the conditions of a real aeration tank, including MLSS, temperature, dissolved oxygen and F/M ratio, to predict removal and the influence of individual factors. Because the same organisms and the same wastewater are used under near-real conditions, this amounts to a pilot study and avoids the structural errors of the other methods.

Structure-based models

When no sample exists yet, biodegradability has to be predicted from molecular structure. Integrated modelling correlates structure with the known behaviour of large numbers of substances, using inventories such as the EU EINECS list; the main models are BIODEG and PLS. The approach needs a very large database and is expensive to run, so it is reserved for new compounds and for predicting what happens to them once released.

Other routes continue to appear: particle size and COD distribution from multistage filtration and ultrafiltration used as predictors, joint assessment combining oxygen consumption with reaction end products and activity values, and empirical flow charts. None replaces the pilot test, but each narrows the number of waters that need one.