Enantioselective toxicokinetics drive the differential toxicity of chiral 6PPD-quinone in rainbow trout.

The tire-derived contaminant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6PPD-Q) is widespread in aquatic ecosystems and poses significant ecological risk. As a chiral compound, its enantiomers may exhibit differential toxicity, yet the underlying mechanisms remain limited. Here, we found that (+)-6PPD-Q was 2.4-fold more toxic to rainbow trout than (-)-6PPD-Q (p < 0.05). In addition, in vitro metabolism assays using liver S9 fraction showed that (+)-6PPD-Q was metabolized 2.5-fold faster than (-)-6PPD-Q. Semi-quantitative nontarget analysis revealed that hydroxylated metabolites dominated the phase I metabolism. We identified metabolites with 3.5- and 1.5-fold lower toxicity than (+)-6PPD-Q and (-)-6PPD-Q, respectively, indicating that metabolism primarily serves as a detoxification pathway and does not explain enantioselective toxicity in vivo. To resolve this discrepancy, we systemically characterized the in vivo enantioselective toxicokinetics of 6PPD-Q enantiomers for the first time. (+)-6PPD-Q exhibited a significantly higher uptake rate and slower elimination rate, leading to 4-9-fold greater bioaccumulation. This predominant accumulation outweighed faster metabolism than (-)-6PPD-Q, resulting in substantially higher internal concentrations and ultimately greater toxicity of (+)-6PPD-Q. Neglecting enantiomer-specific bioaccumulation and elimination could substantially underestimate risks of chiral pollutants such as 6PPD-Q in aquatic systems. Our findings demonstrate that enantioselective toxicity of 6PPD-Q is governed by whole toxicokinetics rather than metabolism alone, highlighting the necessity of incorporating enantioselective toxicokinetic analysis into ecological risk assessment of chiral contaminants.