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OECD 303A: Simulated Biodegradation Test - Activated Sludge Units

OECD 303A is an aqueous aerobic biodegradation test that determines the biodegradability of a material by measuring dissolved organic carbon or chemical oxygen demand under environmentally realistic conditions (municipal wastewater treatment plant with an activated sludge unit). As one of the third-tier tests among OECD 301-303 standard methods, it is typically used for materials that failed one or both of the first two tiers of tests (301 and 302). The test normally lasts for 9-12 weeks.


Less commonly used than OECD 301 and 302, OECD 303 is designed to evaluate the primary and/or ultimate biodegradation of water soluble organic materials in a continuously operated activated sludge system simulating the realistic wastewater treatment process. The dosage of the test compound is generally between 10 and 20 mg DOC/L, and the biodegradation percentage is calculated based on the DOC/COD removal.

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OECD 303A Key Summary

The OECD 303A Activated Sludge Unit measures the DOC/COD removal during biodegradation. Two parallel units are generally used, one containing organic medium only as a blank control, while the other containing both organic medium and the test compound. The difference of the DOC removal between the two units is believed to be the contribution of the test compound. This method cannot be used for testing insoluble or volatile materials. 

▶ Measurement: DOC (or COD) and/or substance itself;
▶ Carbon source: easily biodegradable organic medium (synthetic sewage) and test compound;
▶ Duration: normally 9 weeks, no more than 12 weeks;
▶ Two systems: one test, one blank control (to determine the degradation of organic medium);
▶ Blank control is not required if the substance itself can be measured;
▶ Reference control is not required but the reference (e.g., adipic acid, 2-phenyl phenol, 1-naphthol, diphenic acid, and 1-naphthoic acid) may be added occasionally together with the test substance;
▶ Mean hydraulic retention time: 6 h;
▶ Mean sludge age (retention time): 6-10 day;
▶ Test substance dosage: 10-20 mg/L of DOC;
▶ Initial activated sludge dosage: 2.5 g/L suspended solids;
▶ Distinguish from bioadsorption: by examining the elimination-time curve, and confirming with ready biodegradation test using an acclimatized inoculum from the unit;
▶ Sampling: filtration with membrane filters (0.45 μm) or centrifugation (40,000 m/s2);
▶ Test substance requirement: normally involatile and soluble, able to calculate DOC or COD;
▶ Reproducibility: errors usually within 10-15% for substances degraded more than 80%. Lower degradation, larger error;
▶ Little difference for the two apparatus (Husmann unit vs porous pot), but the real sewage water may be better than the synthetic one.

OECD 303A Requirement and Applicability

Different testing methods are applicable for materials with different properties. Below is a summary of the applicability of OECD 303A. Find the applicability for all other methods on Aropha Resource Center

TestAnalytical methodSample info required*Poorly solubleVolatileAdsorbing
OECD 303A (Activated sludge unit)
DOC and/or COD
Organic carbon content* and/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).

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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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