Generational effects of priority emerging pollutants

Abstract

Aquatic ecosystems are continually facing pressures imposed by human activities, particularly those associated with chemical contamination. In recent years, the scientific community has become increasingly interested on the environmental impacts of pharmaceutically active compounds (PhACs) as contaminants of emerging concern. This interest resulted from the potent bioactivity of these compounds even at very low concentrations, as well as, the development of highly sensitive analytical techniques that have improved detection of PhACs in environmental samples worldwide. Therefore, although their low environmental concentrations were previously believed to pose little or no threat to non-target organisms, PhACs are now recognized for their long-term environmental concern. Despite the growing number of studies evaluating the short-term effects of PhACs, there is an urgent need to improve the methodologies commonly used. Short-term bioassays, mostly using high concentrations of PhACs, have limited environmental relevance. Consequently, current toxicity testing often underestimates the potential threats posed by PhACs to living organisms. Given that natural populations are continuously exposed to low concentrations of PhACs throughout their entire life-cycles, the use of long-term bioassays is crucial to achieve a more realistic evaluation of the associated risks. Unfortunately, this kind of data is frequently unavailable for PhACs. However, this type of test approach would allow to investigate a fundamental aspect of PhACs, as bioactive compounds, namely their mode of action (MoA) in non-target organisms. Furthermore, by using multi-parametric analyses that span multiple levels of biological organization, it is possible to assess the signaling pathways affected by long-tern exposures to PhACs and to establish links between their MoA with the effects observed at the apical level. To address this need, the current thesis focused on the effects of three widely-used PhACs commonly detected in surface waters: simvastatin (SIM), metformin (MET), and naproxen (NPX), using zebrafish as a model organism. A partial life-cycle exposure with SIM and generational exposures with NPX and MET (a full generational exposure and inter-generational exposure, respectively) were conducted using environmentally relevant concentrations of each PhAC. Different developmental phases of zebrafish were evaluated, using a multidisciplinary approach that enabled the assessment of several levels of biological organization. Gene expression (qRT-PCR and RNA-seq), biomarkers of lipid content (cholesterol and triglycerides), histopathology, survival, growth and reproductive output were assessed. Together, these analyses allowed for a better evaluation of the PhACs bioactive nature by linking key biological responses with their MoA. In this thesis, valuable data is provided to fill the existing gaps on the long-term effects of these PhACs. It was determined that, even at environmental concentrations, SIM, MET and NPX consistently induced important adverse effects on zebrafish (Danio rerio) across several biological functions, including lipid/carbohydrate homeostasis, brain and liver energy metabolism, growth and reproduction. Furthermore, the molecular analyses performed provided compelling evidence that MET and NPX may act as endocrine disrupting chemicals (EDCs) by interfering with steroid hormone biosynthesis at concentrations in the range of the ng/L. The RNA-seq analysis performed also indicated disruption of zebrafish epigenetic machinery, suggesting that exposure to these PhACs may have deep impacts on population structure across generations. Lastly, non-monotonic dose-response curves (NMDRC) were frequently observed in this research which challenge the conventional understanding of dose-response relationships. Therefore, it is crucial to conduct long-term bioassays with environmental concentrations of PhACs to determine safe levels of exposure. Notably, none of the studies presented in this thesis were able to determine a No-Observed Effect Concentration (NOEC) for the selected PhACs, as all of the lowest concentrations tested (8 ng/L for SIM, 390 ng/L for MET, and 100 ng/L for NPX) negatively affected zebrafish, even at apical level. In fact, the environmentally relevant concentrations of the PhACs here analyzed, which induced noteworthy adverse effects, are several orders of magnitude lower than the current predicted no-effect concentrations (PNEC) and environmental quality standards (EQS) used in environmental risk assessment for these PhACs. Thus, this thesis results show that the toxicological risks of the selected PhACs may have been underestimated, and suggest that their current PNECs/EQS should be revised. Together these findings highlight the need for long-term assessments, especially in the context of generational and/or multi/trans-generational effects, focusing on environmentally relevant concentrations of PhACs. This approach is crucial to accurately evaluate the effects on non-target organisms and the extent of toxicological risks to aquatic ecosystems. It is therefore proposed that regulatory agencies give priority to long-term toxicity assessment strategies by establishing appropriate test guidelines for PhACs, to ensure an accurate assessment of their impacts on ecosystems under more realistic scenarios.