OECD 310, Regulation (EC) No 440/2008, C.29, and DIN EN ISO 14593
The CO2 headspace test describes a procedure to determine the biodegradability of test items by measuring the CO2-evolution in sealed serum flasks with a headspace volume of about a third of the total flask volume. The test item, as sole source of carbon corresponding to 2 - 40 mg C/l, is added to a mineral salts medium, and is inoculated with activated sludge (4 mg d.s. /l). The oxygen needed for biodegradation is supplied from the headspace-volume. The test flasks are sealed and incubated at 20 - 25 °C in diffuse light for 28 days while being shaken. Degradation is followed by determining the carbon dioxide produced via TIC-measurement (TIC= total inorganic carbon) after absorption to sodium hydroxide.
For doing this, at least weekly about three flasks each from the test flasks, blanks and reference flasks were taken for TIC-measurements. As a control of the procedure the biodegradation of the reference items potassium phthalate or sodium benzoate is determined in parallel flasks.
The amount of carbon dioxide produced from the test item less the amount derived from the blank inoculum is expressed as a percentage of ThIC (theoretical amount of inorganic carbon), which corresponds to the TOC (total organic carbon). Additionally the DOC-elimination can optionally be determined for water soluble test items. According to ISO 14593 the mean value of biodegradation reached in the plateau phase or the highest level of biodegradation (if the curve decreases in the plateau phase) indicates the "degree of biodegradation of the test compound". Additionally the criterion for the ready biodegradability of OECD 310 is considered. Here the pass level for ready biodegradability is 60% of ThIC and this must be reached within a 10-days window (10 days after the degree of biodegradation has reached 10%). The test method is suitable for water soluble and non-soluble as well as foaming and volatile test items which are not toxic under the test conditions. The method has been chosen as a reference method for biodegradability testing in the (EC) Regulation 648/2004 on detergents.