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Metformin (MET), an anti-diabetic pharmaceutical of large-scale consumption, is increasingly detected in surface waters. However, current knowledge on the long-term effects of MET on non-target organisms is limited. The present study aimed to investigate the effects of MET in the model freshwater teleost Danio rerio, following a full life-cycle exposure to environmentally relevant concentrations (390 to 14 423 ng/L). Considering that the mode of action (MoA) of MET on non-target organisms remains underexplored and that MET may act through similar human pathways, i.e., lipid and energy metabolisms, biochemical markers were used to determine cholesterol and triglycerides levels, as well as mitochondrial complex I activity in zebrafish liver. Also, the hepatosomatic index as an indication of metabolic disruption, and the expression levels of genes involved in MET's putative MoA, i.e. acaca, acadm, cox5aa, idh3a, hmgcra, prkaa1, were determined, the last by qRT-PCR. A screening of mRNA transcripts, associated with lipid and energy metabolisms, and other signaling pathways potentially involved in MET-induced toxicity were also assessed using an exploratory RNA-seq analysis. The findings here reported indicate that MET significantly disrupted critical biochemical and molecular processes involved in zebrafish metabolism, such as cholesterol and fatty acid biosynthesis, mitochondrial electron transport chain and tricarboxylic acid cycle, concomitantly to changes on the hepatosomatic index. Likewise, MET impacted other relevant pathways mainly associated with cell cycle, DNA repair and steroid hormone biosynthesis, here reported for the first time in a non-target aquatic organism. Non-monotonic dose response curves were frequently detected in biochemical and qRT-PCR data, with higher effects observed at 390 and 2 929 ng/L MET treatments. Collectively, the results suggest that environmentally relevant concentrations of MET severely disrupt D. rerio metabolism and other import

The primary focus of environmental toxicological studies is to address the direct effects of chemicals on exposed organisms (parental generation – F0), mostly overlooking effects on subsequent non-exposed generations (F1 and F2 – intergenerational and F3 transgenerational, respectively). Here, we addressed the effects of simvastatin (SIM), one of the most widely prescribed human pharmaceuticals for the primary treatment of hypercholesterolemia, using the keystone crustacean Gammarus locusta. We demonstrate that SIM, at environmentally relevant concentrations, has significant inter and transgenerational (F1 and F3) effects in key signaling pathways involved in crustaceans’ neuroendocrine regulation (Ecdysteroids, Catecholamines, NO/cGMP/PKG, GABAergic and Cholinergic signaling pathways), concomitantly with changes in apical endpoints, such as depressed reproduction and growth. These findings are an essential step to improve hazard and risk assessment of biological active compounds, such as SIM, and highlight the importance of studying the transgenerational effects of environmental chemicals in animals’ neuroendocrine regulation

The hypothesis that exposure to certain environmental chemicals during early life stages may disrupt reproduction across multiple non-exposed generations has significant implications for understanding disease etiology and adverse outcomes. We demonstrate here reproductive multi and transgenerational effects, at environmentally relevant levels, of one of the most prescribed human pharmaceuticals, simvastatin, in a keystone species, the amphipod Gammarus locusta. The transgenerational findings has major implications for hazard and risk assessment of pharmaceuticals and other contaminants of emerging concern given that transgenerational effects of environmental chemicals are not addressed in current hazard and risk assessment schemes. Considering that the mevalonate synthesis, one of the key metabolic pathways targeted by simvastatin, is highly conserved among metazoans, these results may also shed light on the potential transgenerational effects of simvastatin on other animals, including humans.

The use of transcriptomics data brings new insights and works as a powerful tool to explore the molecular mode of action (MoA) of transgenerational inheritance effects of contaminants of emerging concern. Therefore, in this dataset, we present the transcriptomic data of the transgenerational effects of environmentally relevant simvastatin levels, one of the most prescribed human pharmaceuticals, in the keystone amphipod species Gammarus locusta. In summary, G. locusta juveniles were maintained under simvastatin exposure up to adulthood (exposed group - F0E) and the offspring of F0E were transferred to control water for the three subsequent generations (transgenerational group - F1T, F2T and F3T). To gain insights into the biological functions and canonical pathways transgenerationally disrupted by simvastatin, a G. locusta de novo transcriptome assembly was produced and the transcriptomic profiles of three individual G. locusta females, per group, over the four generations (F0 to F3) - solvent control groups (F0.C, F1.C, F2.C and F3.C), F0 320 ng/L simvastatin exposed group (F0.320E) and F1 to F3 320 transgenerational group (F1.320T; F2.320T and F3.320T) - were analyzed. Briefly, Illumina HiSeq™ 2500 platform was used to perform RNA sequencing, and due to the unavailability of G. locusta genome, the RNA-seq datasets were assembled de novo using Trinity and annotated with Trinotate software. After assembly and post-processing steps, 106093 transcripts with N50 of 2371 bp and mean sequence length of 1343.98 bp was produced. BUSCO analyses showed a transcriptome with gene completeness of 97.5 % Arthropoda library profile. The Bowtie2, RSEM and edgeR tools were used for the differential gene expression (DEGs) analyses that allowed the identification of a high quantity of genes differentially expressed in all generations. Finally, to identify the main metabolic pathways affected by the transgenerational effects of SIM across all generations, the DGEs genes were blasted o

Simvastatin (SIM), a hypocholesterolaemic compound, is among the most prescribed pharmaceuticals for cardiovascular disease prevention worldwide. Several studies have shown that acute exposure to SIM causes multiple adverse effects in aquatic organisms. However, uncertainties still remain regarding the chronic effects of SIM in aquatic ecosystems. Therefore, the present study aimed to investigate the effects of SIM in the model freshwater teleost zebrafish (Danio rerio) following a chronic exposure (90 days) to environmentally relevant concentrations ranging from 8 ng/L to 1000 ng/L. This study used a multi-parameter approach integrating distinct ecologically-relevant endpoints, i.e. survival, growth, reproduction and embryonic development, with biochemical markers (cholesterol and triglycerides). Real Time PCR was used to analyse the transcription levels of key genes involved in the mevalonate pathway (hmgcra, cyp51, and dhcr7). Globally, SIM induced several effects that did not follow a dose-response relationship; embryonic development, biochemical and molecular markers, were significantly impacted in the lower concentrations, 8 ng/L, 40 ng/L and/or 200 ng/L, whereas no effects were recorded for the highest tested SIM levels (1000 ng/L). Taken together, these findings expand our understanding of statin effects in teleosts, demonstrating significant impacts at environmentally relevant concentrations and highlight the importance of addressing the effects of chemicals under chronic low-level concentrations

Oil spills are an importance source of polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. Intertidal communities are particularly sensitive since most organisms from these ecosystems are sessile or present reduced mobility. Hence, it is important to validate the use of resident species as sentinels to characterize the impact of oil spills on the rocky shores and the improvement during the restoration process. Recently the advantages of using the shanny Lipophrys pholis in pollution monitoring within the northwestern Atlantic coast has been pointed out. Therefore, with the aim of further validating the use of L. pholis in pollution monitoring associated with petrogenic hydrocarbon contamination, a multi-biomarker approach study was carried out 1 week after a moderate oil spill from the waste treatment plant (WTP) of the major Portuguese refinery in the north of Portugal (Petrogal). Fish collected at 2km from the accident displayed a significant induction of ethoxyresorufin-O-deethylase activity (EROD) and fluorescent aromatic compounds (FACs) in bile (up to a 5-fold induction) in comparison with the pre-spill scenario, and a 15% induction in erythrocytic nuclear abnormalities (ENA), a biomarker of genotoxicity. In contrast, no significant differences were recorded in the reference site. In order to better characterize the time-course accumulation of FACs in bile after a PAH insult, laboratory exposure of L. pholis to benzo[a]pyrene (B[a]P) was performed. A clear dose-response accumulation of B[a]P metabolites was observed that closely reflected nominal exposure concentrations already after 3. d. Overall, the findings of the present study highlight the potential of L. pholis in pollution monitoring dealing not only with chronic contamination, but also with oil spill accidents of a moderate scale. Taking into consideration that EROD and FACs determinations in L. pholis are cost effective, rapid and easy to use, they offer a great potential to be incorp

The "Prestige" oil spill occurred in November 2002 off the Galician Coast (NW Spain). Soon after the black tide episode, that caused an ecological disaster, several monitoring programs and projects evaluating the acute and longterm toxicity of the crude oil were initiated (see monograph Marine Pollution Bulletin 2006; vol. 53). Characteristics of the "Prestige" oil are described in Albaigés and Bayona (2003). Briefly, it consists of 50% polycyclic aromatic hydrocarbons (PAHs), mostly dominated by 2–3 ringed compounds which, contrarily in contrast to the higher molecular weight PAHs, are not reported as very toxic to fish (Seruto et al. 2005). Also, due to the heavy nature of this oil and its low solubility in water, the dispersion in sea water was very low and it mostly remained in situ forming oil patches adhered to rocks and sediment., Fish, as with all aerobic aquatic organisms, face natural stress conditions when oxygen is not fully reduced, and intermediate reactive oxygen species (ROS) are formed. Enhanced ROS formation can also take place under exposure to some toxicants such as crude oil (Livingstone 2001). In particular, the water-accommodated fraction (WAF) of some crude oils has been responsible for oxidative stress situations in aquatic organisms (Cajaraville et al. 1992). Hence, in order to evaluate if exposure to the WAF of the "Prestige" crude would interfere with the antioxidant defense system of juvenile sole, Solea senegalensis, the activities of indicative enzymes were analyzed. These included: catalase (CAT), total glutathione peroxidase (t-GPX; sum of Selenium dependent and independent forms), glutathione reductase (GR) and DT-diaphorase (DTD), as part of the antioxidant defense system. However, when this natural protective system is saturated, damage to key molecules such as DNA, proteins or lipids can occur. The latter, termed lipid peroxidation (LP), was included in the present study as a biomarker of effect, indicating damage caused by ROS. The enzyme acetylcholinesterase (AChE) regulates nerve impulse transmissions and is used as a marker of exposure to neurotoxic substances (Galloway et al. 2006). Carboxylesterases (CbE) constitute an heterogeneous group of isozymes, dominant in the liver, that play a role in the metabolism and subsequent detoxification of many xenobiotics. In marine fish AChE and CbE are biomarkers used as indicators of neurotoxicity (Lionetto et al. 2004) and enhanced hepatic metabolism (Wheelock et al. 2005). The phase II conjugating enzyme glutathione S-transferase (GST) has also been included in the present work as a measure of the detoxification metabolism., This study is part of a project aiming to evaluate the toxicity of the water-accommodated fraction (WAF) of the "Prestige" oil in juvenile Solea senegalensis after short-term (24, 48 and 72 h) exposure to environmentally realistic PAHs levels. The choice of this species was based on (1) its high economic interest in the affected region, (2) its benthic nature and, therefore, its adequacy for pollution monitoring programs and (3) its ease to rear under laboratory conditions.