Sub-lethal effects induced by a mixture of three non-steroidal anti-inflammatory drugs (NSAIDs) on the freshwater bivalve Dreissena polymorpha

Abstract

Non-steroidal anti-inflammatory drugs (NSAIDs) are the sixth top-selling drugs worldwide and are commonly found in freshwater ecosystems in the high ng/l to low μg/l range. Recent studies have investigated both the acute and the chronic toxicity of single NSAIDs on different biological models, but these studies have completely neglected the fact that, in the environment, non-target organisms are exposed to mixtures of drugs that have unforeseeable toxicological behavior. This work investigated the sub-lethal effects induced by a mixture of three common NSAIDs, namely, diclofenac, ibuprofen and paracetamol, on the freshwater bivalve, the zebra mussel (Dreissena polymorpha). The mussels were exposed to three different environmental concentrations of the mixture (Low, Mid and High). A multi-biomarker approach was used to highlight cyto-genotoxic effects and the imbalance of the oxidative status of the treated specimens. The Neutral Red Retention Assay (NRRA) was used as a biomarker of cytotoxicity, whereas the activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferase were measured to assess the role played by the oxidative stress enzymes. In addition, the single cell gel electrophoresis assay, the DNA Diffusion assay and the micronucleus test were used to investigate possible genotoxic effects. According to our NRRA results, each treatment was able to induce a significant cellular stress in bivalves, probably due to the raise of oxidative stress, as indicated by the alteration of enzyme activities measured in treated specimens. Moreover, the mixture induced significant enhancements of DNA fragmentation, which preluded fixed genetic damage, as highlighted by the increase of both apoptotic and micronucleated cells.

Evaluation of ecotoxicological effects of drugs on Daphnia magna using different enzymatic biomarkers

“…Similarly, increases in GPx activity have been described by Parolini et al (2010) after acutely exposing zebra mussel (Dreissena polymorpha) to acetaminophen, but also in other antioxidant enzymes (catalase, superoxide dismutase, and GSTs). The work described by Parolini and Binelli (2012) showed that GPx activity could also be enhanced after paracetamol exposure in the freshwater bivalve D. polymorpha. According to Aouacheri et al (2009), GPx enhancement caused by paracetamol is not restricted to aquatic biota; blood GPx activity of mammals was also responsive to acetaminophen oral administration.…”

“…The pathway of conjugation of paracetamol with glutathione vis GSTs activity was observed in mammals, where high drug levels are oxidized by enzymes of the CYP 450 mixed-function oxidase (MFO) system, forming NAPQI (Bowles et al, 2005), which promptly reacts with glutathione (Huber et al, 2009). In addition, Parolini and Binelli (2012) described the combined effects of paracetamol in a mixture with other non-steroidal anti-inflammatory drugs (such as diclofenac and ibuprofen) on the freshwater bivalve D. polymorpha; the obtained results evidenced a significant increase in GSTs activities after acute exposure to the mixture. The involvement of GSTs, in our case, did not seem to occur, reinforcing the lack of a strong oxidative-based response.…”

“…Glutathione peroxidase (GPx) plays an important protective role by acting as a scavenger for high levels of hydrogen peroxide; during this process, glutathione is oxidized and loses its protective antioxidant potential (Nunes et al, 2006). Some pharmaceutical compounds may stimulate the production of ROS, eliciting oxidative damage to aquatic organisms including fish and invertebrates as verified in studies with acetaminophen (Solé et al, 2010;Gómez-Oliván et al, 2012;Antunes et al, 2013;Sung et al, 2014), chlorpromazine (Li et al, 2008), sodium diclofenac (Oviedo-Gómez et al, 2010;Parolini and Binelli, 2012;Parolini et al, 2013;Nava-Álvarez et al, 2014) and propranolol (Solé et al, 2010;Franzellitti et al, 2011).…”

Chronic toxicity of the antiepileptic carbamazepine on the clam Ruditapes philippinarum

“…However few studies addressed the effects of those contaminants on these organisms (e.g. Gagné et al, 2006a;Canesi et al, 2007;Parolini and Binelli, 2012), being necessary additional information to better understand the toxicity that drugs may pose to this group of marine invertebrates.…”

Preliminary evaluation of Diopatra neapolitana regenerative capacity as a biomarker for paracetamol exposure

“…Given the large number of studies describing similar outcomes in many distinct organisms (please see references above), it is believed that this mechanism of paracetamol toxicity can be widely conserved and is likely to also occur in aquatic organisms. In fact, previous studies aiming to assess the effects of paracetamol in aquatic invertebrate species revealed the effects of this drug to bivalves (Corbicula fluminea, Brandão et al 2011;Ruditapes decussatus and Ruditapes philippinarum, Antunes et al 2013;Dreissena polymorpha, Parolini et al 2010;Parolini and Binelli 2012;Parolini et al 2013) and to crustaceans (Daphnia magna, Nunes et al 2014a). Additionally, similar effects were also reported for vertebrates, such as fish (Oncorhynchus mykiss, Ramos et al 2014; Anguilla anguilla, Nunes et al 2015), and for plants (Lemna gibba and Lemna minor, Nunes et al 2014b).…”