Beatriz Diniz Lopes¹, Antônia Duciene Feitosa Lima¹, Flávia Yoshie Yamamoto¹, Mario Machado Martins², Arlene Bispo dos Santos Nossol², Hebreia Oliveira Almeida Souza², Luciana Machado Bastos², Rivelino Martins Cavalcante¹

Metabolomics applied to ecotoxicology has emerged as a promising tool for understanding how stressors affect the metabolic profile of organisms. Fish are effective bioindicators, and bile is a strategic matrix because it allows the detection of metabolites derived from contaminants and provides information on hepatic biotransformation processes and chronic exposure. Based on this, this research sought to identify metabolites associated with exposure to multiple pollutants and analyze possible spatio-temporal variations in coastal pollution patterns. To this end, the metabolic profiles of bile from fish of the species Stegastes fuscus, collected at three locations on the northern coast of Alagoas (Japaratinga, Maragogi, and Paripueira) during 2020 and 2021, were characterized. The organisms were caught by local fishermen and euthanized by immersion in a 0.1% benzocaine solution. The bile was extracted from the gallbladder, stored at -80°C, and subsequently analyzed by HPLC-MS/MS (QTOF). The data were processed with MS-DIAL software, resulting in the detection of 25,828 features. Among these, 107 metabolites were annotated with greater confidence, and another 6,694 were suggested based on spectral similarity. Statistical analysis by ANOVA revealed 24 differentially regulated annotated metabolites between the groups, a number that rose to 1,575 when metabolites with suggested identification were also considered. The results showed several compounds associated with pollution, mainly from plastics, solvents, and derivatives, such as dibutylphthalate, phthalic anhydride, erucamide, and 2-hydroxybenzonitrile. Endogenous metabolites were also identified, such as bile acids and hexadecanedioic acid, the latter being associated with oxidative stress and changes in lipid metabolism, as well as bioactive compounds such as loliolide and a highly acetylated flavonoid, whose presence may be related to the ingestion of plant matter or eutrophication. Comparing the two sampling periods, the 2021 samples showed greater diversity and concentration of metabolites related to pollution, possibly due to increased tourist activity and anthropic pressure on the sites during this period. Among the locations evaluated, Maragogi, especially in 2021, had the highest levels of metabolites associated with urban contaminants and liver changes. Low levels of bile acids, such as cholic acid, may indicate liver impairment and dysfunction in biotransformation pathways. In Paripueira, in both 2020 and 2021, high concentrations of metabolites such as dibutylphthalate, terbuthylazine-TP, which is a metabolite of a triazine herbicide, and phenanthrene carboxylic acid, associated with HPAs and complex phenols, which suggest the burning of fossil fuels and domestic sewage. Japaratinga showed lower levels of metabolites associated with pollution, but a significant presence of erucamide was observed in both years, indicating recurrent exposure to industrial plastic additives. Thus, it can be concluded that environmental metabolomics demonstrates great relevance and applicability for assessing sublethal effects and early signs of contamination, contributing to the conservation of coastal ecosystems and the health of aquatic organisms.

Agradecimentos: Avaliação do derramamento de óleo na costa semiárida do Brasil (Processo CNPq 440880/2020-3).