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OECD 302B - Inherent Biodegradability Test - Zahn-Wellens/EMPA Test

OECD 302B is an inherent aqueous aerobic biodegradation test that determines the inherent biodegradability of a material by measuring dissolved organic carbon or chemical oxygen demand. As one of the second-tier tests among OECD 301-303 standard methods, it is typically used for materials that are not readily biodegradable. The test normally lasts for 28 days, but the prolonged exposure of the test substance to microorganisms is allowed.

CONCEPT OF OECD 302 TESTS

As the second tier of tests in OECD standard methods for biodegradability evaluation, OECD 302A-C 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 biodegradation.

Biodegradation percentages above 20% may be regarded as evidence of inherent biodegradability, whereas biodegradation percentages above 70% may be regarded as evidence of 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, 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.

Learn More at Aropha Resource Center
OECD 302 test overview

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 Point Summary

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

Requirement and Applicability

Different testing methods are applicable for materials with different properties. Below is a summary of the applicability of OECD 302B. You can find the applicability for many other methods on Aropha Resource Center
.

Test Analytical method Sample info required * Poorly soluble Volatile Adsorbing
OECD 302B Zahn-Wellens/EMPA
DOC or COD Organic carbon content* or COD +/- - -

*"Sample info required" is the information needed to calculate the biodegradation percentages. This must be available for a selected method.
*"Organic carbon content" is the ratio of the organic carbon weight to the weight of the sample. It can be calculated by the sample formula (e.g., acetic acid C2H4O2, carbon content is 12*2/(12*2+1*4+16*2)=40%). Try our Online C% Calculator. If the formula is unknown, we can send the sample out to a third party lab for you for analysis (normally $110 with a 10-day turnaround time).
*"COD": we provide COD analysis at Aropha.

Aropha Resource Center

To learn more about different types of biodegradation tests, their applicability, biodegradation mechanisms, and many other information such as case studies, publications, and blogs, please check our Aropha Resource Center
.

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