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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.
▶ 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).
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.
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