Caffeine (CAF) exposures have been shown to cause several pharmacological and biological effects in target and non-target organisms. Although there are already several ecotoxicological studies with CAF in non-target organisms, they are focused on marine organisms, with relevant concentrations in these ecosystems, therefore, less ecologically relevant to freshwater ecosystems (the main ecoreceptor of this type of anthropogenic contaminant). The present study aimed to assess the chronic effects (28 days) of sub-lethal and environmentally relevant concentrations of CAF (0.16, 0.42, 1.09, 2.84, 7.40, 19.23, and 50 μg/L) in Danio rerio. Biochemical endpoints as biomarkers of antioxidant defense, biotransformation, lipid peroxidation, energy sources, and neurotransmission were assessed. CAF exposure induced alterations in antioxidant defenses (superoxide dismutase and glutathione reductase activities, and glutathione content) preventing lipid peroxidation. Lactate dehydrogenase activity decreased in all the concentrations tested, while acetylcholinesterase activity was only affected by the highest concentrations tested (19.23 and 50 μg/L). We also utilized a multi-biomarker approach (Integrated Biomarker Response version 2, IBRv2) to investigate the effects of CAF in the dispersion scope of individual biochemical responses of D. rerio. IBRv2 showed that the concentration of 50 μg/L promotes the highest stress. However, the results showed that CAF induced disturbances in the metabolic pathways studied in D. rerio. These results demonstrated the toxic effects of CAF on freshwater fish, compromising their physiological functions and evidencing the need for monitoring the residues of CAF released into the inland aquatic environments. Furthermore, this research evidence that phylogenetically and physiologically different species may present different biological responses with concern for ecologically relevant environmental conditions. In this sense, the present study generated ecotoxicologically relevant data, that can be considered by environment regulators, since the here-endpoints evaluated showed sensitivity and consistency in the evaluation of caffeine risks in freshwater environments.

CIMAR CIIMAR Centro Interdisciplinar de Investigação Marinha e Ambiental Universidade do Porto Terminal de Cruzeiros do Porto de Leixões Avenida General Norton de Matos S N 4450 208 Matosinhos Portugal

Department of Aquatic Sciences and Assessment Swedish University of Agricultural Sciences SE 75007 Uppsala Sweden

FCUP Departamento de Biologia Faculdade de Ciências Universidade do Porto Rua do Campo Alegre S N 4169 007 Porto Portugal

ICBAS Instituto de Ciências Biomédicas de Abel Salazar Universidade do Porto Rua de Jorge Viterbo Ferreira 228 4050 313 Porto Portugal

UMIB ICBAS Unidade Multidisciplinar de Investigação Biomédica Instituto Ciências Abel Salazar da Universidade do Porto Rua de Jorge Viterbo Ferreira n ° 228 4050 313 Porto Portugal

University of South Bohemia in České Budějovice Faculty of Fisheries and Protection of Waters South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Zátiší 728 2 CZ 389 25 Vodňany Czech Republic

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