CYTO-GENOTOXIC EFFECTS OF SOME PHARMACEUTICAL AND PERSONAL CARE PRODUCTS (PPCPS) ON THE FRESHWATER BIVALVE ZEBRA MUSSEL (DREISSENA POLYMORPHA)

Pharmaceutical and personal care products (PPCPs) are an emerging class of environmental pollutants that are extensively and increasingly being used in human and veterinary medicine. Due to their continuous production, consumption and often abuse, many studies have shown worldwide measurable concentrations of about 100 of these drugs in the aquatic environment in the high ng L-1 to low µg L-1 range. Among these, antimicrobial, antibiotics and non-steroidal anti-inflammatory drugs (NSAIDs) are three of the most used and frequently detected PPCP classes in aquatic environments. Since these chemicals have physico-chemical characteristics similar to those of harmful xenobiotics, they could be potentially dangerous to aquatic non-target organisms, as they are exposed to contaminants over their whole life span. At present, few studies were carried out in order to evaluate the PPCPs potential toxicity on aquatic organisms. The major part of these researches was aimed to acute toxicity evaluation of few drugs on organisms belonging to different trophic levels. Nonetheless, considering the current low environmental levels, these data are not suitable for an accurate risk assessment since chronic effects are much more probable. In order to enlarge this topic, we decided to evaluate the potential cyto-genotoxicity of a common antimicrobial agent (triclosan; TCS), a widespread antibiotic (trimethoprim; TMP) and three largely used NSAIDs (paracetamol; PCM – diclofenac; DCF – ibuprofen; IBU) on the freshwater bivalve zebra mussel (Dreissena polymorpha). This mussel was chosen as biological model because the invertebrates constitute more the 90% of living species, they play an important role in freshwater ecosystems and are particularly susceptible to environmental stressors. Additionally, previous studies have revealed that D. polymorpha is useful and sensible organism capable of highlighting sub-lethal effects when exposed to synthetic chemicals. In detail, a stepwise in vitro-in vivo multi-biomarker approach was planned to study the potential environmental risk due to these PPCPs. The first screening evaluation was carried out by using an in vitro approach on the mussel hemocytes. Genotoxicity was evaluated by SCGE (single cell gel electrophoresis) and DNA diffusion assay, while cytotoxicity was checked by Neutral Red Retention Assay (NRRA). By analyzing the obtained results we drawn the first toxicity scale for zebra mussel hemocytes (TCS<PCM< DCF<IBU<TMP). These data lay the groundwork for in vivo exposures, which will allow for a better definition of the observed cyto-genotoxicity of these molecules in a setting miming real environmental exposure. Starting from the most toxic PPCP, an in vivo multi-biomarker battery was applied on D. polymorpha to evaluate their real potential sub-lethal effect. In order to give a marked ecological relevance to our research, mussels were exposed for 96 h to increasing environmentally relevant drug concentrations. Cyto-genotoxicity was determined in mussel hemocytes by the lysosome membrane stability (NRRA), the single cell gel electrophoresis (SCGE) assay, the micronucleus test (MN test) and the assessment of the apoptotic frequency (DNA diffusion assay). Moreover, the probable unbalance of mussels’ oxidative status was evaluated by analyzing the activity of three antioxidant phase I enzymes, namely superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), as well as the detoxifying phase II enzyme glutathione S-transferase (GST) in the cytosolic fraction extracted from a pool of entire mussels. By analyzing the final biomarker data, the PPCP in vitro toxicity scale was partially confirmed. In fact, according to in vitro screening results, TCS was the most toxic drug, followed by PCM, IBU and TMP. On opposite, nonetheless its remarkable in vitro cyto-genotoxicity, in vivo results noticed that current DCF levels do not induce significant adverse effects on D. polymorpha. Our data point out that the use of a tied in vitro/in vivo approach is a useful method to study in depth the toxicity of new environmental pollutants. In vitro studies, thanks to their simplicity, reliability and speed, provide basic information on the toxicity of xenobiotics and often assist in hypothesizing or testing the probable mechanism of action of pollutants. In addition, they can act as a guide to direct additional research and could be used in first environmental risk assessment (ERA) evaluations. On this basis, in vivo approach, miming the real environmental conditions of exposure (concentrations, uptake pathways, defense mechanisms, metabolism) allow us to evaluate the effective environmental hazard of single PPCPs and/or their mixtures and it furnish more precise and in-depth information of their toxicity on aquatic species for further ERA operations. Considering the relevance of in vitro tests in ecotoxicological preliminary analyses, in vitro cultures of zebra mussel hemocytes, gill and digestive gland cells were developed in collaboration with the Irish Centre for Environmental Toxicology (ICET - Galway, Ireland). These innovatory techniques were applied to investigate the potential in vitro cytotoxicity of atenolol (ATL), carbamazepine (CBZ), diclofenac (DCF) and gemfibrozil (GEM) and to discriminate the most sensitive cell type for further applications. On overall, DCF was the more cytotoxic compound for zebra mussel cells, followed by GEM, CBZ and ATL, while gill cells and hemocytes seemed to be the most sensitive targets for tested PPCPs.