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OECD 302 Inherent Biodegradation Test


As the second tier of tests in OECD standard methods for biodegradability evaluation, OECD 302A-C (D is a proposed method) are designed to determine whether a material is inherently biodegradable. Prolonged exposure of the test substance to microorganisms and a low ratio of test substance to biomass are allowed, which offers a better chance to obtain a positive result compared to tests for ready biodegradability.

Biodegradation percentages above 20% may be regarded as evidence of inherent, primary biodegradability, whereas biodegradation percentages above 70% may be regarded as evidence of inherent, ultimate biodegradability. This indicates that the material is not likely to persist indefinitely in the environment.

If a sample does not pass the 70% threshold indicating inherent primary biodegradability, it may lead to a preliminary conclusion of environmental persistency. The higher tier of tests OECD 303 may be performed to evaluate the degradation under environmentally realistic conditions, or an evaluation of the environmental effects of the degradation products may be performed.

Please refer to the page "Test Method Overview" for more concepts or overview about OECD 302.

OECD 302B Zahn-Wellens/EMPA Test

The OECD 302B Zahn-Wellens/EMPA test measures the removal of dissolved organic carbon (DOC) or chemical oxygen demand (COD) during biodegradation and the degradation percentage is calculated as the ratio of DOC removal to original DOC loaded (or COD removal to original COD dosed).

Compared to other methods in the OECD 302 series, OECD 302B has the top levels of simplicity and applicability.

The carbon content of the test substance and its purity may be known to calculate the DOC, or otherwise the COD may be measured. Information on the toxicity of the test substance is helpful in determining the appropriate dosage so that the material does not inhibit bacteria at the concentration tested.

This method is capable of testing samples that are highly soluble (> 50 mg DOC/L), non-volatile, and non-adsorbing.

Key summaries of 302B

  1. Measurement: DOC or COD;
  2. Temperature: 20-25°C;
  3. pH: 6.5-8.0;
  4. Reference: ethylene glycol, diethylene glycol, lauryl sulfonate or aniline;
  5. Test substance dosage: 50-400 mg DOC/L (100-1000 COD/L);
  6. Inoculum dosage: 0.2-1.0 g/L of TSS by activated sludge;
  7. Make sure: DOC/L : TSS/L = 2.5:1 – 4:1;
  8. Duration: up to 28 days; prolong the test beyond 28 if adaptation occurs in the final days;
  9. Reactors: 1-5 L cylindrical glass vessels;
  10. Number of reactors: 1-2 for test substance; 1-2 for blank control; 1 for reference control;
  11. Sampling (by filtration or centrifugation) (replace the loss due to evaporation before sampling): a. 3h ± 30 mins after addition of the test substance to estimate any adsorption of it by the AS; b. 4 times between days 1 and 27; c. Twice on days 27 and 28 or the last two days if the test is terminated earlier.
  12. Test validity: a. Reference compound reaches 70% of degradation within 14 days; b. The removal of test substance takes place gradually (as an indicator of biodegradation instead of physico-chemical processes, which are generally faster).
  13. A pass level is not specified (but normally > 20% is regarded as inherently biodegradable, and > 70% ultimately biodegradable).


TestAnalytical methodSample info requiredPoorly solubleVolatileAdsorbing
OECD 302B (Zahn-Wellens/EMPA Test)DOC or CODCarbon content or COD---