Metabolomics to Study the Sublethal Effects of Diazepam and Irbesartan on Glass Eels (Anguilla Anguilla)

Abstract

Since glass eels are continuously exposed to contamination throughout their migratory journey in estuaries, to a certain extent the fall in the population of this endangered species can be attributed to this exposure, which is especially acute in estuaries under high urban pressure. In this work, metabolomics was used to assess the effects that two pharmaceuticals (diazepam and irbesartan) have on glass eels. An exposure experiment to diazepam, irbesartan and their mixture was carried out over 7 days followed by 7 days of depuration phase. After exposure, glass eels were individually sacrificed using a lethal bath of anaesthesia, and then an unbiased sample extraction method was used to extract separately the polar metabolome and the lipidome. The polar metabolome was submitted to targeted and non-targeted analysis, whereas for the lipidome only the non-targeted analysis was carried out. A combined strategy using partial least squares discriminant analysis and univariate and multivariate statistical analysis (ANOVA, ASCA, t-test, and fold-change analysis) was used to identify the metabolites altered in the exposed groups with respect to the control group. The results of the polar metabolome analysis revealed that glass eels exposed to the diazepam-irbesartan mixture were the most impacted ones, with altered levels for 11 metabolites, some of them belonging to the energetic metabolism, which was confirmed to be sensitive to these contaminants. Additionally, the dysregulation of the levels of twelve lipids, most of them with energetic and structural functions, was also found after exposure to the mixture, which might be related to oxidative stress, inflammation, or alteration of the energetic metabolism.

Keywords: Glass eels, Metabolomics, Mass Spectrometry, non-target analysis, Exposomics, Lipidomics