Andrea Binelli

In vivo experiments for the evaluation of genotoxic and cytotoxic effects of Triclosan in Zebra mussel hemocytes.

In this work, we investigated the possible genotoxic and cytotoxic effects of the antibacterial agent Triclosan in hemocytes of the freshwater bivalve Zebra mussel (Dreissena polymorpha). For this study, we used several biomarkers for in vivo experiments (96h of exposure) carried out at three possible environmental Triclosan concentrations (1, 2, 3nM). We used the single cell gel electrophoresis (SCGE) assay, the micronucleus test (MN test) and the measure of the apoptotic frequency (Halo assay) to measure the genotoxic potential of Triclosan, and the neutral red retention assay (NRRA) as a measure of lysosomal membrane stability to identify general cellular stress. We observed significant increases in all of the genotoxic biomarkers examined as early as 24h after initial exposure, as well as a clear destabilization of lysosomal membranes (after 48h), indicating that this chemical is potentially dangerous for the entire aquatic biocoenosis. A comparison of these in vivo data with existing data from in vitro experiments allowed us to suggest possible mechanisms of action for Triclosan in this bivalve. Although further studies are needed to confirm the possible modes of action, our study is the first to report on the effects of this widespread antibiotic on freshwater invertebrates.

POPs in edible clams from different Italian and European markets and possible human health risk

Human intake of persistent organic pollutants (POPs) occurs primarily through diet and fish and other seafood represent the principal means of contamination. Despite this, few legal limits have been established to protect human health from the various chemicals that are alleged to be carcinogenic or endocrine disruptor compounds. Several pools of edible clams (Tapes philippinarum and Venus gallina) bought in different Italian and European markets were examined in order to perform a risk assessment of some of these contaminants and also to identify possible health issues. POP concentrations in the soft tissues of mollusks varied considerably among source farming sites, particularly in the Venice Lagoon. Some POPs (DDTs, HCB and HCHs) showed very low concentrations and no risk for human health, in line with the limits established by European law. In contrast, PAHs could represent a potential hazard for human health and PCB levels in the most contaminated sites reached or even exceed the Minimal Risk Level as set by the ATSDR for human consumption, even if all the samples did not exceed the PCB limits in edible seafood as set by the FDA.

Cytotoxic and genotoxic effects of in vitro exposure to Triclosan and Trimethoprim on zebra mussel (Dreissena polymorpha) hemocytes

Pharmaceuticals and personal care products (PPCPs) have been detected in several aquatic ecosystems for a number of years, but the potential for biological effects in exposed non-target organisms is only now being reported. In this study the potential cellular damage due to two of the main PPCPs found in aquatic environments was investigated by in vitro exposures. Hemolymph samples of the freshwater bivalve Dreissena polymorpha were collected and treated with increasing concentrations of the antibacterial agent Triclosan (TCS) and the antibiotic Trimethoprim (TMP). Doses selected for TCS were 0.1, 0.15, 0.2, and 0.3 microM, while 0.2, 1, and 5 microM for TMP exposures, respectively. We evaluated the potential genotoxicity on hemocytes by the SCGE (single cell gel electrophoresis) assay and apoptosis frequency evaluation, while the cytotoxicity was measured by the lysosomal membranes stability test (NRRA, neutral red retention assay). TCS genotoxicity increased in a dose-dependent manner and this pharmaceutical significantly affects hemocyte functionality due to severe DNA injuries at very low doses. In contrast, TMP seems to be less dangerous than TCS for D. polymorpha because the cytotoxic and the moderate genotoxic effects noticed were obtained only at very high concentration levels.

Use of zebra mussel (Dreissena polymorpha) to assess trace metal contamination in the largest Italian subalpine lakes.

Trace metal (Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn) contamination was evaluated in zebra mussels from the lakes Maggiore, Lugano, Como, Iseo and Garda, which are located in the most highly populated and industrialised area in Italy. Zebra mussels from Lake Maggiore contained the highest concentrations (3.44, 1.51, 4.97, 0.158, 5.87, 346 microg g(-1) for Cd, Co, Cr, Hg, Pb, Zn, respectively) of all metals analysed except Cu and Ni. The lowest levels of most metals were in animals from Garda and Lugano (0.78 and 0.60 microg g(-1) for Cd, 2.87 and 2.03 microg g(-1) for Cr, 0.065 and 0.049 microg g(-1) for Hg, 12.1 and 11.9 microg g(-1) for Ni, 1.96 and 2.46 microg g(-1) for Pb, 158 and 163 microg g(-1) for Zn). The most contaminated sites and possible local sources of metals were identified for each lake, and the lakes classified into quality classes concerning metal pollution.

DDT contamination in Lake Maggiore (N. Italy) and effects on zebra mussel spawning.

Zebra mussel (Dreissena polymorpha) is commonly used as contaminant bioindicator in Europe and North America. We used the zebra mussel to follow DDT pollution trends from 1996 to 1997 in Pallanza bay, Lake Maggiore, near the inlet of the River Toce, after a DDT-manufacturing plant discharging residues into a tributary of the River Toce had closed down. DDT contamination fell off sharply outside the bay, but tissue concentrations of the parent compound and residues remained high in molluscs sampled within the bay a year later. Molluscs collected in Pallanza bay in June 1997 released gametes earlier than those sampled at a nearby reference station. Histological studies showed that a significant percentage of these specimens showed marked oocyte degeneration suggesting that DDTs have endocrine-disrupting effects in this species.

A comparison of sediment quality guidelines for toxicity assessment in the Sunderban wetlands (Bay of Bengal, India).

The aim of this paper was to obtain the first screening ecotoxicological risk evaluation in the Sunderban wetlands, the largest prograding delta in the estuarine phase of the River Ganges. The characterization of exposure was conducted by means of an extensive survey of several persistent organic pollutants (PAHs, PCBs, DDTs, PBDEs, HCHs, HCB) measured in seven core sediments from the Sunderban wetlands, obtaining a dataset with more than 2200 analyses. The pollutant effects were assessed by the use of three different sediment quality guidelines (SQGs) previously developed in the literature to evaluate toxicity induced in sediment-dwelling organisms. The three different approaches chosen for risk assessment of the Sunderban were the consensus SQGs obtained by TEC (threshold effect concentration), PEC (probable effect concentration) and EEC (extreme effect concentration), the threshold/probable effect level (TEL/PEL) approach and, finally, the ERL-ERM guidelines, including the m-ERM-Q (mean ERM quotient). The evaluation of the toxicity induced by a mixture of the target pollutants indicated the importance of gamma-HCH contamination in the Sunderban sediments despite the very low concentrations measured in core sediments. A different sensitivity for toxicity assessment due to quality guidelines was obtained, as the consensus SQGs based on TEC were less conservative and protective than the TEL and ERL approaches, while the use of m-ERM-Q seems to be the most powerful tool to predict the toxicity related to a contaminant mixture.

The PCB pollution of Lake Iseo (N. Italy) and the role of biomagnification in the pelagic food web.

Several models of varying complexity have been used to predict pollutant concentrations in the higher levels of the food web from those in lower levels, but the role of the biomagnification process in aquatic food chains is still controversial. We used the fugacity-based approach to verify the transfer of PCBs through the pelagic food chain of Lake Iseo (N. Italy), sampling several zebra mussel specimens and some fish belonging of different trophic levels. The zebra mussel seems to be a suitable starting species for modelling the bioaccumulation process through the trophic web, not only because its physiological characteristics and population size do not change much with time (as do algae and zooplankton) but also because it takes up toxicants exclusively from the water, as shown by the application of two predictive trophic models commonly used. The data provided by one of those models were in good agreement with our experimental data on fish in Lake Iseo, that show a not negligible uptake from food for the top predator species (pike and perch) with an increase of about three times in comparison with the PCB levels measured in the zebra mussel specimens.

An in vitro biomarker approach for the evaluation of the ecotoxicity of non-steroidal anti-inflammatory drugs (NSAIDs).

Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most frequently detected pharmaceuticals in aquatic environments. They are the sixth most sold drugs worldwide and are usually found in significant quantities in municipal effluents. The aim of this study was to assess a first screening evaluation of the cytogenotoxicity of three common NSAIDs (diclofenac, ibuprofen and paracetamol) using an in vitro biomarker approach on the haemocytes of the freshwater bivalve zebra mussel (Dreissena polymorpha). Genotoxicity was evaluated by SCGE (single cell gel electrophoresis) and DNA diffusion assay while cytotoxicity was evaluated by neutral red retention assay (NRRA). The exposure of the haemocytes to increasing concentrations of the three drugs, chosen based on the results of a viability test, revealed high cytogenotoxic potential and allowed the creation of the first toxicity scale for zebra mussel haemocytes (paracetamol<diclofenac<ibuprofen). The present results lay the groundwork for in vivo exposures, which will allow for a better definition of the observed cytogenotoxicity of these molecules in a setting miming real environmental exposure.

A multi-biomarker assessment of the impact of the antibacterial trimethoprim on the non-target organism Zebra mussel (Dreissena polymorpha)

A battery of eight biomarkers was applied in the freshwater mussel Dreissena polymorpha to evaluate potential sub-lethal effects of the antimicrobial trimethoprim (TMP, 5-[3,4,5-trimethoxybenzyl]pyrimidine-2,4-diamine). Mussels were exposed for 96 h to increasing concentrations (1, 3, 10 nM) of TMP in in vivo experiments. We determined the single cell gel electrophoresis (SCGE) assay, the micronucleus test (MN test), the apoptotic frequency (Halo assay) and the lysosomal membrane stability (Neutral Red Retention Assay) in mussel hemocytes. Moreover, to reveal whether the oxidative status was altered, measurements of the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and the phase II detoxifying enzyme glutathione S-transferase (GST) were performed using the cytosolic fraction extracted from a pool of entire mussels. The biomarker battery pointed out only a moderate cyto- and genotoxicity on Zebra mussel hemocytes since only a slight increase in DNA damage was registered by apoptosis induction and MN frequency, while significant differences of lysosomal membrane stability from baseline levels were measured at 3 and 10 nM at the end of exposures only. Finally, TMP seems to have a very low induction capability or even an inhibitory effect on the activities of antioxidant enzymes, but a clear significant induction on GST.

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy)

Abstract Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.