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Polycyclic aromatic hydrocarbons (PAHs) are pervasive pollutants globally, and exposure has been linked to developmental toxicity in the early life stages of many species. Oil spill disasters such as Deepwater Horizon can result in a large and concentrated release of PAHs directly into aquatic ecosystems. Previous studies have demonstrated that exposure to crude oil or phenanthrene (a reference PAH found in oil) produces an array of gross morphological abnormalities in developing fish embryos, including edema and craniofacial defects. However, our understanding of the molecular mechanisms that lead to these phenotypes is still limited. Recently, studies utilizing transcriptomic analyses in several oil-exposed marine fish species including red drum (Sciaenops ocellatus) and mahi-mahi (Coryphaena hippurus) found significant changes in the abundance of transcripts involved in cholesterol biosynthesis. Given the vital role of cholesterol in embryonic development, we hypothesized that disruption of these metabolic pathways following exposure to crude oil or phenanthrene alone may contribute to abnormal development of fish embryos. Aim 1 is the first study in any fish species to investigate the impact of crude oil exposure on cholesterol concentrations in fish embryos. Here we found that exposure to the highest tested High Energy Water Accommodated Fraction (HEWAF) concentration ∑PAH 8.3 μg L-1 significantly reduced total cholesterol in mahi-mahi larval homogenates. Aim 2 built upon our initial findings and assessed the role of cholesterol on phenanthrene-induced cardiotoxicity in zebrafish (Danio rerio). While free cholesterol concentrations in the larval body were not affected in zebrafish exposed to phenanthrene, treatment with cholesterol prior to phenanthrene exposure significantly mitigated phenanthrene-induced bradycardia. This suggests that cholesterol may play a role in advancing PAH-driven cardiotoxicity. In aim 3, we investigated the effects of crude oil on embryos of the Gulf native species, red drum. Here we found that while expression of several key genes in the synthesis pathway were not affected, red drum larvae displayed significant increases in free cholesterol, detected by whole-mount staining (∑PAH 4.71-16.15 μg L-1). Overall, our findings demonstrate that cholesterol in developing fish embryos can be impacted by crude oil exposure. Future work should investigate the mechanisms underlying changes to cholesterol homeostasis.