Chemicals’ half-lives derived from biotransformation simulation studies are central metrics for persistence assessment in international regulatory frameworks. To determine the persistence of chemicals released to the aquatic environment, paradigm shifts in recent and ongoing revisions of chemical legislation assign increasing importance to OECD 309 simulation studies. OECD 309 studies were designed to target biotransformation in natural water (pelagic test) or in water amended with sediment (suspension test). Suspension tests bear several advantages over the pelagic test, most importantly, employing higher bacterial cell densities, which promote biotransformation of various chemicals at observable rates. However, experience with suspension tests is limited. In this study, we followed the fate of 43 pharmaceuticals, pesticides, and industrial chemicals in various suspension test setups and elucidated parameters influencing biotransformation kinetics and half-lives derived thereof. Besides striking intrastudy variability between replicates, we found that differences in sediment origin and bacterial cell density resulted in chemical half-lives that were different by up to 2 orders of magnitude, making persistence classification rather uncertain. However, data suggested that test systems employing bacterial cell densities close to the upper limit of what is commonly observed in natural surface waters (i.e., 107 cells mL−1) yielded increased and more uniform biotransformation of chemicals.