Chemical and biomarker responses for site-specific quality assessment of the Lake Maggiore (Northern Italy).

Since the 1990s, the Lake Maggiore (Northern Italy) has been recognized as an aquatic environment contaminated by DDTs and other persistent organic pollutants, but to date just few studies were carried out to investigate the effects of pollution to aquatic organisms. The aim of this study was the application of a stepwise approach based on chemical data, a suite of biomarkers and the integration of their responses into a biomarker response index (BRI) to evaluate the site-specific quality assessment in different sampling stations of Lake Maggiore, one of the largest European lakes. We used as biological model the freshwater bivalve Zebra mussel (Dreissena polymorpha). Several hundred bivalve specimens were sampled on May 2011 from eight sampling sites located along the lake shoreline. We measured levels of DDTs, PCBs, HCHs, HCB, and PAHs accumulated in D. polymorpha soft tissues by GC/MSn, while the activities of catalase, superoxide dismutase, glutathione peroxidase, and glutathione S-transferase, as well as the lipid peroxidation and protein carbonyl content were evaluated in homogenates from native bivalves as oxidative stress indices. Moreover, DNA damage was investigated by the alkaline precipitation assay. Significant imbalances of enzymatic activity were found in mussels from most of the sampling sites, as well as notable increases of damage to macromolecules. Health status of mussels from Baveno was greatly affected by lake pollution, probably due to high levels of DDTs measured in this site, while a wide variability in biomarker responses was found in all the other stations. The application of a BRI allowed distinguishing impacts of pollution to bivalves, confirming mussels from Baveno as the most threatened and revealing that also the health status of bivalves from Suna, Brissago, Pallanza, and Laveno is affected. These evidences suggest the usefulness of a specific index to integrate all the biomarker endpoints in order to provide a correct environmental risk assessment.

A redox proteomic investigation of oxidative stress caused by benzoylecgonine in the freshwater bivalve Dreissena polymorpha.

Drugs of abuse and their human metabolites have been recently recognized as emerging environmental contaminants. Notwithstanding the fact that these kinds of compounds share some features with pharmaceuticals, their ecotoxicology has not yet been extensively investigated, although some of their characteristics may potentially threaten aquatic ecosystems. One of the most abundant drugs found in rivers and wastewaters is benzoylecgonine (BE), the main metabolite of cocaine. We applied a redox proteomics approach to evaluate changes in the proteome of Dreissena polymorpha exposed to two different concentrations of BE (0.5 and 1 µg/l). Exposures were performed in vivo for a period of 14 days and the effect of oxidative stress on protein thiol and carbonyl groups in mussel gills were evaluated. One-dimensional electrophoresis did not reveal a reduction in protein thiol content but showed a significant increase of protein carbonylation at both doses tested. Then, protein profiling using two-dimensional gel electrophoresis was performed with subsequent matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) and TOF/TOF with LIFT technique and linear ion trap combined with orbitrap mass spectrometer (LTQ-Orbitrap). This yielded de novo protein sequences suitable for database searching. These preliminary results and protein identifications obtained suggest that BE causes oxidative stress. Oxidative modifications were detected in differing classes of proteins such as those of the cytoskeleton, energetic metabolism and stress response.

Application of a biomarker response index for ranking the toxicity of five pharmaceutical and personal care products (PPCPs) to the bivalve Dreissena polymorpha.

Pharmaceuticals and personal care products (PPCPs) have been detected in several aquatic ecosystems during the last two decades, but their potential for biological effects to nontarget organisms is only now being studied. The aim of this study was to compare and rank the cyto-genetic effects induced by 96-hour exposure to an environmental concentration (1 nM) of triclosan (TCS), trimethoprim (TMP), diclofenac (DCF), ibuprofen (IBU), and paracetamol (PCM) on the freshwater bivalve Dreissena polymorpha by integrating biological responses of eight biomarker into a simple biomarker response index (BRI). The application of the BRI decreased the wide biomarker variability and enabled toxicity ranking of the tested PPCPs as follow: TCS > TMP > IBU > DCF = PCM. This approach allowed us to draw an accurate PPCP scale of toxicity of the most dangerous drug and to address further in-depth investigations.

Sub-lethal effects caused by the cocaine metabolite benzoylecgonine to the freshwater mussel Dreissena polymorpha.

Illicit drugs have been recognized as emerging environmental pollutants that could represent a potential risk for aquatic communities. Even if many studies have shown the occurrence of several drugs of abuse and their metabolites in freshwaters in the High ng/L to Low µg/L range worldwide, no information on their potentially harmful effects on non-target organisms is available. The aim of this study was to investigate sub-lethal effects induced by the main metabolite of cocaine, the benzoylecgonine (BE), on the freshwater bivalve Dreissena polymorpha. Mussels were exposed under semi-static conditions for 14 days to two environmentally relevant BE concentrations (0.5 µg/L and 1 µg/L) and induced adverse effects were evaluated through the application of a suite of ten different biomarkers. We applied on bivalve hemocytes the single cell gel electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test) to investigate DNA injuries, while the neutral red retention assay (NRRA) was used to assess BE cytotoxicity. Catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione S-transferase (GST) activities, as well as the lipid peroxidation (LPO) and protein carbonyl content (PCC), were measured as oxidative stress indices in zebra mussel homogenates. Significant decrease in lysosomal membrane stability and imbalances of defense enzyme activities were found at both exposure concentrations, suggesting the involvement of oxidative stress in BE toxicity. Significant increases in LPO and PCC, as well as in primary (DNA strand breaks) and fixed DNA damage (apoptotic and micronucleated cell frequency), were found at the highest BE treatment, confirming that adverse effects to macromolecules were due to the increase of BE-induced oxidative stress.

A proteomic study using zebra mussels (D. polymorpha) exposed to benzo(α)pyrene: the role of gender and exposure concentrations.

It has recently been established that the use of proteomics can be a useful tool in the field of ecotoxicology. Despite the fact that the mussel Dreissena polymorpha is a valuable bioindicator for freshwater ecosystems, the application of a proteomic approach with this organism has not been deeply investigated. To this end, several zebra mussel specimens were subjected to a 7-day exposure of two different concentrations (0.1 and 2 µg L⁻¹) of the model pollutant benzo[α]pyrene (B[α]P). Changes in protein expression profiles were investigated in gill cytosolic fractions from control/exposed male and female mussels using 2-DE electrophoresis. B[α]P bioaccumulation in mussel soft tissue was also assessed to validate exposure to the selected chemical. We evaluated overall changes in expression profiles for 28 proteins in exposed mussels, 16 and 12 of which were, respectively, over- and under-expressed. Surprisingly, the comparative analysis of protein data sets showed no proteins that varied commonly between the two different B[α]P concentrations. Spots of interest were manually excised and analysed by MALDI-TOF/TOF mass spectrometry. The most significant proteins that were identified as altered were related to oxidative stress, signal transduction, cellular structure and metabolism. This preliminary study indicates the feasibility of a proteomic approach with the freshwater mussel D. polymorpha and provides a starting point for similar investigations. Our results confirm the need to increase the number of invertebrate proteomic studies in order to increase the following: their representation in databases and the successful identification of their most relevant proteins. Finally, additional studies investigating the role of gender and protein modulation are warranted.