Modified MITI (II) test

OECD 302 C



The inherent biodegradability is measured by the consumption of oxygen. It is the only OECD standardised test, which measures biodegradation by mineralization and allows the testing of insoluble chemicals. The Zahn-Wellens Combination test could be an alternative, by additionally measuring carbon dioxide and using it as the endpoint of mineralization.


The test item (30 mg/L) is placed in a closed vessel as the only carbon source along with mineral medium and activated sludge (100 mg/L dry solids) from a municipal sewage treatment plant and incubated for a period of 14 till 28 days. The evolved carbon dioxide is absorbed into sodium hydroxide. The changed pressure in the vessel indicates the oxygen consumption. The biodegradation is the result of the ratio of oxygen uptake by test item (corrected with blank value of the inoculum) and the theoretical oxygen demand (ThOD). Diethylene glycol, an inherent biodegradable substance, is dosed to parallel reference vessels. At the end of the test the rest of dissolved organic carbon (DOC) is measured in the bottles. Optionally, substance specific analyses of the test item or its transformation products could be performed.


Contrary to the OECD 302 C Guideline, activated sludge from a municipal wastewater treatment plant is used as inoculum and the mineral medium according to OECD 302 B is used.

The original OECD 302 C guideline describes a mixed inoculum derived from at least 10 different sources that is further grown in the laboratory with a synthetic sewage water for one to five months until an artificial activated sludge has developed. Several publications indicated that the resulting inoculum is highly artificial and has low microbial potency.

While for the MITI (I) test there is a counterpart under Reach via Regulation (EC) No. 440/2008, C.4-F for testing ready biodegradability, the inherent MITI(II) test has not yet been integrated into the REACH Test Methods Regulation.


A degradation level of > 70% of TOD within 28 days is considered as evidence of ultimate inherent biodegradability.

Substance characteristics

The MITI (II) test is suitable for evaluating the inherent degradability of water-soluble, semi-volatile and sparingly soluble substances.