Multi-biomarker analysis of sub-chronic PAH mixture effects in fish at environmentally relevant levels

Polycyclic Aromatic Hydrocarbons (PAHs) are persistent pollutants in aquatic ecosystems, occurring as complex mixtures with unpredictable toxicity. Although PAHs are procarcinogenic, their harmful effects require metabolic activation, leading to reactive metabolites and reactive oxygen species (ROS) that can cause DNA damage. This study assessed the toxic effects of individual PAHs (Phenanthrene and Benzo[a]pyrene) and their mixtures (1:2 and 2:1 ratios) on juvenile seabream (Sparus aurata) after 42 days of exposure at 0.2nmol.L⁻¹. Biomarkers related to oxidative stress, detoxification, and lipid peroxidation were analysed in the liver and gills (e.g., glutathione (GSH), glutathione peroxidase (GPx), glutathione-S-transferase (GST), catalase (CAT), lipoperoxidation (LPO). Liver gene expression (Cytochromes P450 (CYP1A), GST3, tumour protein p53 (TP53) and blood cell DNA damage were also studied. Correlation analyses and Non-Metric Multidimensional Scaling (NMDS) were used to relate treatments and biomarkers. Results suggested differences in organ responses, with the gills generally showing the most significant changes in GSH levels, GST activity, and LPO compared to the control group. DNA repair mechanisms appeared to prevent significant genotoxicity as assessed by the comet assay. However, erythrocytic nuclear anomalies (ENAs) were notably higher in fish exposed to Phe, B[a]P, and the 2:1 B[a]P:Phe mixture compared to the control group. Interestingly, the 2:1 Phe:B[a]P mixture appeared to have an enhanced effect, showing a marked upregulation of GST3 mRNA (up to 7-fold), possibly influenced by the higher proportion of Phe. MDS analysis proved to be a valuable tool in identifying patterns among biological responses, offering insight into how fish cope with PAH exposure and helping to uncover the unpredictable effects of chemical mixtures. This study highlights the need for further research into the interactions of PAH mixtures, employing multi-analysis approach and underscores the importance of revising environmental guidelines to account for their effects.