Chemical structure drives developmental toxicity of alkyl-substituted naphthalenes in zebrafish

Naphthalene and its alkyl-substituted derivatives are among the most abundant polycyclic aromatic hydrocarbons (PAHs) in environmental and human exposure studies, yet their developmental toxicity and mode of action remain poorly understood due to challenges in testing semi-volatile compounds. This study developed a vial based, high throughput method to effectively assess the activity of naphthalenes and a set of 24 alkyl-substituted naphthalenes. Early life stage zebrafish were exposed to a concentration series of each chemical (0-50 µM) in rotating sealed glass vials to minimize volatilization. Benchmark concentration (BMC₅₀) values were calculated for morphological endpoints and lowest effect levels were determined for behavioral effects. The data were assessed for evidence of a narcotic mode of action using body burden measurements for select chemicals and logK_ow modeling. Targeted transcriptomics at a single concentration and timepoint as well as in silico molecular docking were conducted to generate mode of action hypotheses. The vial method enabled detection of highly variable developmental toxicity not previously observed using standard 96-well plate exposures. LogK_ow and body burden were poor predictors of toxicity, suggesting a non-narcotic mode of action. Transcriptomic analysis revealed evidence for the disruption of glucocorticoid signaling pathways. Molecular docking identified potential protein targets (e.g., CYP1A2, NT5E, FOLR1) that may mediate observed effects. This study demonstrates the importance of appropriate exposure methods for semi-volatile compounds, reveals structure-dependent toxicity among alkyl-substituted naphthalenes, and provides a foundation for further mechanistic studies and improved risk assessment of alkyl-substituted PAHs.