Characterization of the Immunotoxic Effects and Underlying Mechanism of Butylated Hydroxytoluene and Its Metabolites to Early Life Stage Fish

Butylated hydroxytoluene (BHT), a widely used synthetic phenolic antioxidant, undergoes metabolic transformation in aquatic environments and organisms. However, the immunotoxic potential of its metabolites remains largely unexplored. Here, we combined density functional theory calculations, chemical analyses, and zebrafish bioassays to investigate the metabolism and immunotoxicity of BHT and its representative metabolites. Gas chromatography–mass spectrometry confirmed the presence of 3,5-di tert -butyl-4-hydroxybenzoic acid (BHT-COOH) and 2,6-di tert -butyl-p-benzoquinone (BHT-Q) in exposure water and larvae, whereas 2,6-di tert -butyl-4-hydroxymethylphenol (BHT-OH) was primarily detected in larvae. Exposure to environmentally relevant concentrations of BHT, BHT-COOH, and BHT-Q significantly increased macrophage and neutrophil numbers, weakened antibacterial resistance against Vibrio parahemolyticus, and induced oxidative stress with dysregulated immune markers and elevated tumor necrosis factor-α (TNF-α) levels. Comparative analyses showed that BHT-COOH and BHT-Q induced stronger immunotoxic responses than the parent compound, BHT. Additionally, high-throughput screening of the immune-related receptors using molecular docking identified the Interleukin-1β (IL-1β) signaling pathway as a critical mediator of these effects, as coexposure with an IL-1β inhibitor markedly attenuated immune disruption. Collectively, these findings demonstrate that BHT metabolites, especially BHT-COOH and BHT-Q, exhibit stronger immunotoxic responses than the parent compound, highlighting the importance of incorporating metabolite pathways into ecological risk assessments of synthetic phenolic antioxidants.