P09-16 Neuroactive Behavioral Fingerprinting of Crude Oil-Derived Water Accommodated Fractions in Larval Zebrafish Using a New Approach Method

Petroleum spills introduce chemically complex mixtures into aquatic environments, posing neurodevelopmental risks to aquatic species. Water Accommodated Fractions (WAF), which contain bioavailable petroleum constituents, are of particular concern, yet the specific neuroactive properties of their chemical classes remain poorly understood. Here, we applied a behavior-based new approach method (NAM) to identify neuroactive effects of a crude oil-derived WAF and its fractions in larval zebrafish. Zebrafish larvae (5 days post fertilization) were exposed to WAF concentrations (9.8–100%, 1 g/L WAF loading) and evaluated using a 26-endpoint behavioral battery, the Visual and Acoustic Motor Response (VAMR) assay. WAF exposure elicited concentration-dependent effects where lower concentrations induced hypoactivity and reduced startle responses and higher concentrations (≥ 56.6%) triggered lightphase hyperactivity, seizure-like motor patterns, and impaired habituation learning. To identify chemical drivers, WAF was fractionated into five classes: PAHs, resins, naphthalenes, monoaromatics, and saturates. All fractions disrupted behavior, with PAH and resin fractions closely mimicking total WAF profiles. GCxGC-MS analysis identified phenanthrene as the dominant PAH (50%). The resin fraction was chemically diverse with no single compound exceeding 5% abundance. The three most abundant compounds from each fraction were tested individually. In the PAH group, phenanthrene reproduced habituation deficits, while 2-methylanthracene and ethyl 4-ethoxybenzoate contributed to late dark-phase hypoactivity. In contrast, resin compounds including 1H-phenalen-1-one, 7-methyl-1-indanone, and dihydrocarvone produced varied behavioral effects, suggesting that the resin fraction's behavioral profile likely emerges from the combined action of numerous low-abundance constituents. Behavioral profiles were mapped against a neuroactivity fingerprint library of 63 reference compounds. The resin fraction clustered near GABA A re-ceptor antagonists, indicating disrupted inhibitory signaling. High-concentration WAF clustered with known neurodevelopmental disruptors affecting mTOR, PPAR δ, RXR, and BMP pathways. These findings demonstrate that potential neurotoxicity effects derived from crude oil arise from chemically diverse mixtures targeting distinct molecular pathways. Importantly, this study highlights the power of neurobehavioral fingerprinting as a scalable, mechanistically-informative strategy for mixture toxicity analysis. By capturing fraction-specific neurobehavioral signatures and aligning them with known reference profiles, the VAMR fingerprinting system enables mode-of-action-based hypothesis testing for complex mixtures. This framework supports chemical hazard characterization, facilitates hypothesis generation, and has broad relevance for advancing new approach methodologies in environmental toxicology.