This Test Guideline is a screening method for the evaluation of ready biodegradability of chemicals.
The test substance, normally at 20 mg C/L, as the sole source of carbon and energy, is incubated (during 28 days normally) in sealed bottles with aerobic condition containing a buffer-mineral salts medium, which has been inoculated with a mixed population of micro-organisms. In order to check the test procedure, a reference substance (aniline, sodium benzoate or ethylene glycol and 1-octanol) of known biodegradability should be tested in parallel. It is recommended that triplicate bottles be analysed after a sufficient number of time intervals. Also at least five test bottles (from test vessels, blank controls, and vessels with the reference substance) are analysed at the end of the test, to enable 95% confidence intervals to be calculated for the mean percentage biodegradation value. The CO2 evolution resulting from the ultimate aerobic biodegradation of the test substance is determined by measuring the Inorganic Carbon (IC) produced in the test bottles in excess of that produced in blank vessels containing inoculated medium only. The extent of biodegradation is expressed as a percentage of the theoretical maximum IC production (ThIC), based on the quantity of test substance added initially. Biodegradation >60% ThIC within the 10-d window in this test demonstrates that the test substance is readily biodegradable under aerobic conditions.