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OECD 302: Inherent Biodegradability Tests
Biodegradation is one of the most important factors in assessing the environmental fate of chemicals. Substances coming into the aquatic environment via waste water get in contact with the sewage treatment microorganisms, which is therefore the basis for the assessment of aquatic biodegradation. Standard OECD 302 tests performed at ibacon are the 302B: Zahn-Wellens/EMPA-Test and the 302C: Modified MITI Test (II).
Study Design
Selection of adequate test
Depending on the test item specific properties such as water solubility, different tests are applicable:
| Guideline | Test | Water solubility / volatility | Measured Parameter |
|---|---|---|---|
| OECD 302B | Zahn-Wellens-Test |
Water Soluble Non-Volatile, non-foaming |
DOC (Dissolved Organic Carbon) |
| OECD 302C | Modified MITI Test (II) |
Not Water Soluble / Soluble Non-Volatile / Volatile |
CO2 pressure decrease (BSB-Sensomat flasks) and DOC (Dissolved Organic Carbon) or additional analytical method |
Course of the test
Activated sludge is taken from the aeration tank of a domestic waste water treatment plant. After a conditioning procedure defined suspensions of the activated sludge microorganisms are introduced in the test vessels. Beside the test item treatments a series of controls can be performed: control, reference item and toxicity control. The control contains only activated sludge. The reference item is used to check the activity of the sludge microorganisms. The toxicity control contains the test item and the reference item to detect inhibitory effects of the test item. Both tests are carried out in controlled environment rooms with constant temperature in the dark with a standard test duration of 28 days.
OECD 302B: Zahn-Wellens/EMPA-Test
The purpose of this study is to follow the degree of bio-elimination of the a test item in an inherent biodegradation test. The test is ususally performed only after failure to pass a test for ready biodegradability. The test item is exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for normally 28 days. The biodegradation is determined by following the DOC (Dissolved Organic Carbon) of the test item in the incubation flasks during exposure. The percentage of degradation at the sampling time is determined by correction for the blank after each time interval and under consideration of the measured DOC of the test item at test start (after 3 hours).
OECD 302C: Modified MITI Test (II)
The purpose of this study is to determine the inherent biodegradability of a test item which has been found to indicate low degradability. The test item is exposed to activated sludge from the aeration tank of a domestic waste water treatment plant for normally 28 days. The biodegradation is followed by the oxygen uptake of the microorganisms during exposure. The percentage of degradation is determined by correction for the blank and under consideration of the theoretical oxygen demand of the test item. For the assessment of primary degradation, the test item concentration is determined in aqueous phase as well as the adsorption on the sludge. The grade of primary degradation is assessed by measurement of the test item concentration in the test vessels by a substance-specific analysis.
Endpoints
Test items that reach 70 % biodegradation based on DOC-measurement can be clasified as inherently biodegradable. With the Modiefied MITI Test (II) it can be additionally determined if the test substance will be transformed in an aqueous system to hydrolysis products or not and not used in bacterial metabolismt with the grade of ultimate biodegradation.
Guidelines and literature
- OECD Guideline for Testing of Chemicals No. 302 B: "Inherent Biodegradability: Zahn-Wellens/EMPA Test", adopted July 17, 1992.
- Commission Regulation 440/2008/EC, Method C.9 of May 30, 2008: Zahn-Wellens Test (EEC Publication No. L 142/538, May 2008)
- OECD Guideline for Testing of Chemicals No. 302 C: "Inher-ent Biodegradability: Modified MITI Test (II)", adopted May 12, 1981.