Technical guidance on biodegradation testing of difficult substances and mixtures in surface water.

The aim of this article is to address critical challenges in the OECD 309 "Aerobic mineralization in surface water - simulation biodegradation test" for volatile chemicals, highly hydrophobic chemicals, mixtures or UVCBs (unknown or variable composition, complex reaction products or biological materials). Several modifications are presented to address technical challenges (minimize and account for losses), make testing more environmentally relevant (lower concentrations) and generate data for multiple substances (more and better aligned data):•Minimizing and accounting for test substance losses: Aqueous solutions are handled using gas tight syringes, tests are conducted in gas tight vials, and automated analysis is performed directly on unopened test vials. Abiotic losses are accounted for via concentration ratios between test systems and abiotic controls that are incubated and measured in parallel.•Testing at low environmentally relevant concentrations: Substances are tested at low concentrations to avoid toxicity and solubility artefacts and analyzed using a sensitive analytical method. Substances are added without co-solvent (using passive dosing) or with a minimum of co-solvent (using microvolume spiking).•Testing of multiple chemicals in mixtures combined with constituent specific analysis: Primary biodegradation kinetics of chemicals are determined in tests of multi-constituent mixtures or UVCBs using constituent specific analysis.