A partitioning-based experimental platform was developed and applied to determine primary biodegradation kinetics of 53 hydrocarbons at ng/L to μg/L concentrations covering C8-C20, 11 structural classes, and several orders of magnitude in hydrophobicity and volatility: (1) Passive dosing from a loaded silicone donor was used to set the concentration of each hydrocarbon in mixture stock solutions; (2) these solutions were combined with environmental water samples in gastight auto sampler vials for 1-100 days incubation, and (3) automated solid phase microextraction (SPME) coupled to GC-MS was applied directly on these test systems for measuring primary biodegradation relative to abiotic controls. First order biodegradation kinetics were obtained for 40 hydrocarbons in activated sludge filtrate, 18 in seawater, and 21 in lake water. Water phase half-lives in seawater and lake water were poorly related to hydrophobicity and volatility but were, with a few exceptions, within a factor of 10 or shorter than BioHCwin predictions. The most persistent hydrocarbons, 1,1,4,4,6-pentamethyldecalin, perhydropyrene, 1,2,3,6,7,8-hexahydropyrene, and 2,2,4,4,6,8,8-heptamethylnonane, showed limited or inconsistent degradation in all three environmental media. This biodegradation approach can cover a large chemical space at low substrate concentrations, which makes it highly suited for optimizing predictive models for environmental biodegradation.