Identification of Neuroactive Chemicals in Crude Oil-Derived Water-Accommodated Fractions

Crude oil-derived water-accommodated fractions (WAFs) are complex mixtures containing bioavailable constituents. We applied an automated zebrafish behavior-based assay to assess potential neuroactivity following exposure to WAF and chemical fractions including polycyclic aromatic hydrocarbons (PAHs), resin, naphthalene (NAP), monoaromatic hydrocarbon (MAH), and saturate fractions. Zebrafish larvae were exposed to WAF concentrations (9.8-100%, 1 g/L loading) and assessed using the 26-end point visual and acoustic motor response assay at 5 day postfertilization. WAF exposure elicited concentration-dependent behavioral effects, including reduced dark-period activity, impaired acoustic startle responses, and inappropriate activity during light and typically quiescent interstimulus periods. All WAF fractions were neuroactive, eliciting dark-phase hypoactivity and fraction-specific hyperactivity patterns. Exposure to PAH, MAH, or NAP fractions also impaired habituation learning. Comprehensive two-dimensional gas chromatography-mass spectrometry identified phenanthrene, 2-methylanthracene, and ethyl 4-ethoxybenzoate as the dominant PAHs. In contrast, the resin fraction was chemically diverse. Behavioral profiles of zebrafish exposed to PAH fraction constituents or an artificial mixture recapitulated WAF-like effects. Neurobehavioral fingerprinting predicted that WAF neuroactivity may arise from structurally diverse chemical classes disrupting multiple molecular targets, including disruption of peroxisome proliferator-activated receptor delta- or gamma-aminobutyric acid type A receptor-dependent signaling. Taken together, integration of chemical fractionation, high-resolution behavior phenotyping, and mode-of-action fingerprinting supports mechanistic dissection of environmentally relevant mixtures.