Combination test

OECD 302 B, Regulation (EC) No. 440/2008, C.9, DIN EN ISO 9888 with additional CO2-quantification


The Zahn-Wellens test is currently the most important test for assessing inherent biodegradability. However, it is not suitable for substances with low water solubility or substances that strongly adsorb to the activated sludge, since it only records the DOC elimination and does not differentiate between biological degradation and abiotic elimination. In the combination test, in addition to DOC elimination, the CO2 development is also determined by continuous aeration of the reactors with CO2-free air, analogous to the CO2 evolution test (OECD 301 B). The CO2 released during biodegradation is then absorbed in sodium hydroxide and subsequently quantified using IC (inorganic carbon) measurements. In this way, in addition to the DOC elimination, the mineralization (ultimate degradability) of the substance is also determined.


The test item as the sole carbon source is incubated in a mineral medium together with activated sludge from a municipal sewage treatment plant for up to 28 days. In order to limit the CO2 release of the control vessels (blank values), the lower activated sludge concentration according to OECD 302 B is preferred (200 mg/L TS). The biological degradation is monitored by determining the DOC in the reactors and the IC in the absorbers. According to OECD 302 B, the first sampling takes place 3 hours after the start of the test and then once or twice a week. The total carbon introduced at the start of the test (ThCO2) at the beginning of the test serves as a reference point (and not the three-hour value as with the OECD 302 B).


A biodegradation degree of > 70% of ThCO2 within 28 days is taken as evidence of ultimate inherent biodegradability. The DOC elimination only serves as additional information.


Although the CO2/DOC-combination test is widely accepted and is also carried out under GLP, a corresponding extension of the OECD 302 B guideline has not yet agreed on, so that acceptance by the regulatory authorities should be clarified before it is performed.

Substance characteristics

The test is suitable for water-soluble or emulsifiable, non-volatile, non-foaming and non-toxic organic substances that are only adsorbed to a limited extent in the test system. In the case of poorly water-soluble substances, it may be possible to waive the DOC determination. On the other hand, the increase or decrease in the DOC indicates changes of the or the water solubility due to other effects such as hydrolysis and provides additional information on the course of the test.

Further information

Gartiser, S., Urich, E., Alexy, R., Kümmerer, K. (2007): Ultimate biodegradation and elimination of antibiotics in inherent tests. Chemosphere 67, S. 604-613

Gartiser, S. (2009): Bestimmung der Mineralisierung und DOC-Elimination von Abwasserproben im Zahn-Wellens-Test. gwf-Wasser-Abwasser 150 (9), S. 700-710.