An enhanced, multiple lines of evidence approach was applied to assess potential toxicological effects associated with polluted sediments. Two in vitro bioassays (H4IIE-luc and Vibrio f ischeri) and three in vivo bioassays (microalgae: Isochrysis galbana and Phaeodactylum tricornutum; zebrafish embryo: Danio rerio) were applied. To identify causative chemicals in samples, targeted analyses (polycyclic aromatic hydrocarbons (PAHs), styrene oligomers (SOs), and alkylphenols) and nontargeted full-scan screening analyses (FSA; GC-and LC-QTOFMS) were performed. First, great AhR-mediated potencies were observed in midpolar and polar fractions of sediment extracts, but known and previously characterized AhR agonists, including PAHs and SOs could not fully explain the total potencies of samples. Enoxolone was identified as a novel AhR agonist in a highly potent sediment fraction by use of FSA. Enoxolone has a relative potency of 0.13 compared to benzo[a]pyrene (1.0) in the H4IIE-luc bioassay. Nonylphenols associated with membrane damage that influenced the viability of the microalgae were also observed. Finally, inhibitions of bioluminescence of V. f ischeri and lethality of D. rerio embryos were strongly related to nonpolar compounds. Overall, the present work addressed assay-and end point-specific variations and sensitivities for potential toxicities of mixture samples, warranting a significant utility of the "multiple lines of evidence" approach in ecological risk assessment.

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