New evidences in the complexity of contamination of the lagoon of Venice: polybrominated diphenyl ethers (PBDEs) pollution.

This study presents the first evaluation of the current pollution by polybrominated diphenyl ethers (PBDEs) of surface sediments from the Lagoon of Venice. We focused the research on tri-to hepta-BDEs, the main components of penta- and octa-mixtures, which are considered to be the most toxic for the biocoenosis. The results pointed out a quite homogeneous contamination of this keystone European transitional environment, with ∑ (13)PBDEs values ranging from 0.39 to 6.78 ng/g dry weight; these values reflect low to moderate pollution levels, which is in conformity to other coastal European ecosystems. The average PBDE profile of the lagoon sediments follows this decreasing trend of congeners: BDE-47>BDE-99> >BDE-190>BDE-28>BDE-153>BDE-154>BDE-138, BDE-183, and BDE-17, which is similar to the worldwide distribution pattern. BDE-47 and BDE-99 revealed a recent use of a penta-BDE mixture, while the presence of hepta-BDEs (BDE-183 and BDE-190) in all of the sites can indicate the actual use of a deca-BDE formulation, because these congeners are considered to be debrominated byproducts of BDE-209 degradation.

Chronic effects induced by ibuprofen on the freshwater bivalve Dreissena polymorpha.

The sub-lethal effects induced by the non-steroidal anti-inflammatory drug ((NSAID) ibuprofen (IBU; ((±)-2-(p-isobutylphenyl) propionic acid))) were investigated using a battery of biomarkers on the freshwater bivalve Dreissena polymorpha. According to the results from a semi-static in vivo approach, mussels were exposed for 96 h to increasing levels of environmentally relevant IBU concentrations (0.2, 2 and 8 µg/l, corresponding to 1, 9 and 35 nM, respectively). Cyto-genotoxicity was evaluated via the single cell gel electrophoresis (SCGE) assay, the DNA diffusion assay, the micronucleus test (MN test) and lysosome membrane stability (Neutral Red Retention Assay) in mussel hemocytes. In addition, the activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and the phase II detoxifying enzyme glutathione S-transferase (GST) were measured in the cytosolic fraction that was extracted from a pool of entire bivalves to determine whether the oxidative status was imbalanced. The biomarker battery pointed out a slight cyto-genotoxicity on zebra mussel hemocytes at the IBU concentration of 0.2 µg/l, with higher IBU concentrations able to significantly increase both genetic and cellular damage. In addition, IBU seems to have a considerable effect on the activities of antioxidant and detoxifying enzymes as shown in the exposed specimens' notable oxidative status imbalances.

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.

Cytotoxicity assessment of four pharmaceutical compounds on the zebra mussel (Dreissena polymorpha) haemocytes, gill and digestive gland primary cell cultures.

Pharmaceutical compounds are considered the new environmental pollutants but at present few studies have evaluated their ecotoxicity on aquatic invertebrates. This study was aimed to investigate the in vitro cytotoxicity of four common drugs, namely atenolol (ATL), carbamazepine (CBZ), diclofenac (DCF) and gemfibrozil (GEM), on three different cell typologies from the zebra mussel (Dreissena polymorpha): haemocytes, gill and digestive gland cells. Results obtained by the Trypan blue exclusion test revealed that exposure to increasing concentrations (0.001; 0.01; 0.1; 1 and 10 mg L(-1)) of CBZ, DCF and GEM were able to significantly decrease the viability of each cell type, while the MTT (3(4,5-dimethyl-2thiazholyl)-2,5-diphenyl-2H-tetrazolium bromide) reduction assay highlighted only a slight reduction of mitochondrial activity of gill and digestive gland cells. Overall, DCF was the most cytotoxic drug for zebra mussel cells, followed by GEM, CBZ, while ATL has not a noteworthy toxic potential. Our preliminary results lay the groundwork for further in vitro evaluations, which will allow a better definition of the potential toxicity of these drugs.

Evaluation of 4-nonylphenol in vivo exposure in Dreissena polymorpha: Bioaccumulation, steroid levels and oxidative stress.

Nonylphenol (NP) represents the most critical metabolite of alkylphenols (APs) and alkylphenol ethoxylates (APEs), non-ionic surfactants widely used in the formulation of domestic and industrial products. On the basis of in vitro and in vivo animal studies 4-nonylphenol (4-NP) is considered an endocrine disrupting chemical (EDC). The evidence to date indicates that mollusks are able to synthesize sex steroids from the precursor cholesterol and their endocrine pathways are theoretically susceptible to disruption. The aim of this study was to investigate the endocrine modulating potency of 4-NP in the freshwater mussel Dreissena polymorpha by looking at endogenous steroid levels in control and exposed individuals. 4-NP bioaccumulation in mussels tissues and alterations in the activity of enzymes related both to oxidative stress (catalase - CAT- and glutathione peroxidase - GPX-) and phase II metabolism (glutathione-S-transferase - GST-) were also assessed. The results highlighted a build-up of 4-NP in exposed mussels and an overall decrease of 17-beta-estradiol and testosterone levels. On the other hand this chemical at the tested concentrations does not interfere with the antioxidant defense mechanisms in D. polymorpha. The mechanisms by which 4-NP alter steroids levels are unknown and require more in-depth investigations.

Multi-biomarker approach for the evaluation of the cyto-genotoxicity of paracetamol on the zebra mussel (Dreissena polymorpha).

Paracetamol (PCM; N-(4-hydroxyphenyl)acetamide) is a widely used analgesic and antipyretic agent that is utilized in human medicine. Its use is so widespread that it is constantly being introduced into global water bodies where it reaches concentrations up to several microgL(-1). A battery of eight biomarkers was applied in the freshwater bivalve Dreissena polymorpha in order to evaluate its potential sub-lethal effect. Mussels were exposed for 96h to increasing environmental concentrations (1, 5, 10nM) of PCM. Cyto-genotoxicity was determined in mussel hemocytes by the lysosomal membrane stability (Neutral Red Retention Assay), the single cell gel electrophoresis (SCGE) assay, the micronucleus test (MN test) and assessments of the apoptotic frequency (DNA diffusion assay). Moreover, in order to evaluate the probable alterations to the mussels' oxidative status, measurements of the activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and the detoxifying enzyme glutathione S-transferase (GST) were performed using the cytosolic fraction extracted from a pool of entire mussels. The biomarker battery demonstrated moderate cyto-genotoxicity in zebra mussel hemocytes since no primary DNA fragmentation was measured by the SCGE assay and only a slight increase in fixed DNA damage was registered by apoptotic and MN frequencies. Significant destabilization of the lysosomal membrane from baseline levels was evident at 5 and 10nM at the end of the exposures, as was a high induction capacity of the activities of CAT and GST.

Preferential retention of POPs on the northern aspect of mountains.

Soils are the main reservoirs of POPs in mountain ecosystems, but the great variability of the concentrations, also at small scale, leaves some uncertainties in the evaluation of environmental burdens and exposure. The role of the aspect of the mountain side and the seasonal variation in the contamination levels was analysed by means of several soil samples taken from central Italian Alps. A greater contamination content was present in northern soils with a mean ratio between the north vs. south normalised concentration of around a factor of 2 (North-South Enrichment Factor). Experimental factors agreed with theoretical calculations based on temperature-specific calculated K(sa) values. From May to November consistent differences in normalised concentrations up to 5-fold were observed. A dynamic picture of the POP contamination in high altitudinal soils is derived from the data in this work, with spring-summer half-lives between 60 and 120 days for most of the compounds.

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

Seasonal changes and temperature-dependent accumulation of polycyclic aromatic hydrocarbons in high-altitude soils.

Polycyclic Aromatic Hydrocarbons (PAHs) are a major group of pollutants whose occurrence in the environment is mainly of anthropogenic origin. In this paper, we examine the effect of topographical slope exposure on PAH contamination and seasonal change in PAH concentrations in soils. We collected soil samples on three dates in 2007 (early May, end of July and beginning of November) from south- and north-facing aspects at 1900 m a.s.l. in the central Italian Alps. We found greater PAH contamination in soils from a north-facing slope than in those from a south-facing slope at all seasons. We calculated North-South Enrichment Factors as the ratio between the concentrations measured in soils from northern and southern aspects. These ratios ranged from 1.4 to 1.9 for lighter PAHs (from 2 to 4 rings). These values are consistent with theoretical calculations based on temperature-specific octanol-air partition coefficients (predicted North-South Enrichment Factors range from 1.6 to 2.0). For heavier PAHs (from 5 to 6 rings), smaller differences were observed between soils from northern and southern aspects, due to the gas/particle distribution of these compounds. We also found consistent differences in normalised PAH concentrations across the three sampling periods. The majority of compounds showed a significant decreasing trend from the beginning of May to the end of July, due to the annual cycles of physical processes (deposition vs. volatilisation) and biological processes (uptake and/or biotransformation). Only a few compounds showed different trends, presumably due to season-specific local emission sources.

Genotoxic effects of p,p'-DDT (1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane) and its metabolites in Zebra mussel (D. polymorpha) by SCGE assay and micronucleus test.

This is the first study to evaluate the potential genotoxicity of p,p'-DDT (1,1,1-trichloro-2,2-bis-(chlorophenyl)ethane) and its metabolites (p,p'-DDD and p,p'-DDE) in the sentinel mollusc Zebra mussel (Dreissena polymorpha). DNA damage was measured using the single cell gel electrophoresis (SCGE) assay and the micronucleus test (MN test), which represent two of the more sensitive biomarkers for genotoxicity evaluation. Three different concentrations (0.1, 2, and 10 mug/L) of each compound were administered in water for 168 hr, maintaining mussels at constant laboratory conditions and collecting several specimens every 48 hr for biochemical analyses. At the same time, the bioaccumulation process and the concentration/effect relationship were checked by GC-MS/MS analyses of mussel soft tissues. The SCGE assay results showed a clear and significant (P < 0.05) relationship between DNA injuries and tested doses for all the homologues throughout the 7-day exposure period. The final DNA damage due to p,p'-DDE was almost double that of the other two homologues that showed the same toxicity pattern. The micronucleus frequency analysis confirmed the genotoxicity potential of the three homologues and p,p'-DDE showed the highest irreversible DNA damage. The capability of Zebra mussels to biotransform the administered compound in the other homologues was demonstrated by multiple regression analyses carried out between the MN frequencies and the concentrations of the different homologues in the mussel soft tissues. A greater genotoxic potential of the p,p'-DDE with respect to the other two chemicals was revealed.

Evaluation of several priority pollutants in zebra mussels (Dreissena polymorpha) in the largest Italian subalpine lakes.

Zebra mussel (Dreissena polymorpha) has been used for the biomonitoring of several POPs (PCBs, DDTs, HCB and HCHs) in the largest Italian subalpine great lakes (Lake Maggiore, Garda, Como, Iseo and Lugano). Samplings were carried out in April 2003 at 15 locations selected according to industrial and anthropic levels of lakes. Results have pointed out high DDT levels in D. polymorpha specimens from Lake Maggiore (700-1400 ng/g lipids, 5-9 times higher than those measured in mussels of other Italian lakes), due to a contamination from a chemical plant located on one of the main lake inlet that occurred in 1996. On the contrary, PCB levels (400-2509 ng/g lipids) highlighted an overall pollution, with some sporadic peaks of contamination. Data showed a moderate increase trend compared to those found in a previous monitoring campaign carried out in 1996. Future monitoring is needed in order to confirm this tendency.

Assessment of the genotoxic potential of benzo(a)pyrene and pp'-dichlorodiphenyldichloroethylene in Zebra mussel (Dreissena polymorpha).

The single-cell gel electrophoresis (SCGE) assay and the micronucleus (MN) test were carried out with haemocytes of Zebra mussel (Dreissena polymorpha) specimens to evaluate the potential genotoxicity of benzo(a)pyrene (BaP) and pp'-dichlorodiphenyldichloroethylene (pp'-DDE, a metabolite of pp'-DDT). Mussels were exposed to three different concentrations (0.1 microg/L, 2 microg/L, 10 microg/L) of each chemical in water during 168 h (SCGE assay) and 96 h (MN test) of exposure under laboratory conditions. These levels correspond to nominal molar concentrations of 0.4 nM, 7.9 nM and 40 nM for BaP and 0.3 nM, 6.2 nM and 31 nM for pp'-DDE, respectively. Concurrently, the levels of toxicants were measured in soft tissues of the mussels by gas-chromatographic analyses, to evaluate their temporal trends and the dose/response relationships. Significant increases of the ratio between the comet length and the diameter of the comet head (LDR) and of micronucleus frequencies in comparison with baseline levels were observed not only for all concentrations of BaP, but also for pp'-DDE (except 0.3 nM). The concentration above which DNA damage starts to be significantly increased was 0.8 nmol/g lipids for BaP and 1.6 nmol/g lipids for pp'-DDE, respectively. The results of these experiments show a clear genotoxic effect on this non-target organism not only for the well-known genotoxicant BaP, but also for the final metabolite of pp'-DDT at soft-tissue concentrations that have been found in several aquatic ecosystems worldwide.

Evaluation of DNA damage induced by decabromodiphenyl ether (BDE-209) in hemocytes of Dreissena polymorpha using the comet and micronucleus assays.

The recent widespread production and use of flame retardants, polybrominated diphenyl ethers (PBDEs), is one of the reason of the increasing contamination observed worldwide. At the present, deca-BDE mixture, in which the decabromodiphenyl ether (BDE-209) is the major congener (98%), dominates the EU market. The potential genotoxicity of BDE-209 was examined in the freshwater bivalve zebra mussel (Dreissena polymorpha) by means of Comet assay and micronucleus assay (MN assay). Mussels were exposed in vivo to BDE-209 at nominal concentration of 0.1, 2, and 10 mug/l under laboratory conditions. The assays were performed on the bivalve hemocytes monitoring the levels of DNA strand breaks and the percentage of micronuclei until 168 and 96 hr of exposure, respectively. At the same time, BDE-209 levels were measured daily in mussel soft tissues to evaluate the bioaccumulation. Results of the Comet assay showed a significant increase of DNA damages compared to controls, but a lack of dose/effect relationship probably due to the formation of less-brominated congeners. By contrast, no significant changes in MN frequency from baseline levels were observed. These preliminary results about the potential genotoxicity of this compound in invertebrates indicated a clear BDE-209 capability to induce DNA damage, but no irreversible effects on DNA hemocytes. Furthermore, bioaccumulation of this high-molecular-weight substance and its uptake mechanism in zebra mussel are also discussed.

Use of two biomarkers (CYP450 and acetylcholinesterase) in zebra mussel for the biomonitoring of Lake Maggiore (northern Italy).

The use of zebra mussel Dreissena polymorpha as a bioaccumulator for lipophilic compounds is nowadays standardized, but its employment in early warning systems by the biomarker approach is much less frequent. One of the main problems with the biomarker approach is due to natural variation of abiotic factors such as temperature that influence the activity of several enzymes. In this study, we investigated the influence of this environmental parameter on the activities of two different biomarkers: acetylcholinesterase (AChE) (inhibited by organophosphorus compounds) and CYP450 (inversely influenced by planar compounds and heavy metals). We used these two biomarkers to evaluate the environmental pollution of Lake Maggiore (northern Italy). Results showed a strong AChE inhibition in mussel specimens collected in some sampling sites of the lake, indicating heavy pollution by neurotoxic compounds. We also found a twofold effect on CYP450 activity, probably due to the activating effect of planar compounds and the inhibiting effect of trace metals.

Screening of POP pollution by AChE and EROD activities in Zebra mussels from the Italian Great Lakes.

The increase of ethoxyresorufin dealkylation (EROD) and the inhibition of acetylcholinesterase (AChE) as biomarkers have been commonly used in vertebrates for the persistent organic pollutants (POPs) biomonitoring of aquatic environments, but very few studies have been performed for invertebrates. Previous researches demonstrated the interference due to some chemicals on EROD and AChE activities of the freshwater bivalve Zebra mussel (Dreissena polymorpha) in laboratory and field studies, showing its possible use for the screening of POP effects. We investigated the contamination of the Italian sub-alpine great lakes (Maggiore, Lugano, Como, Iseo, Garda) by the biomarker approach on Zebra mussel specimens collected at 17 sampling sites with different morphometric characteristics and anthropization levels. Results showed a homogeneous contamination of AChE inhibitors in Lake Garda, Maggiore, Como and Iseo with values ranging from 0.5 to 3 nmol/min/mg proteins and with an average inhibition of about 66% to controls. The planar compounds pollution, able to activate the EROD activity, seems higher in some sampling stations of Lake Garda, Como and Iseo (2-4 pmol/min/mg proteins) than that measured in Lake Lugano (1.5-3 pmol/min/mg proteins). On the contrary, the enzyme activity in Lake Maggiore showed an interesting opposite effect of AhR-binding compounds and trace metals. Finally, the possible use of Zebra mussel specimens maintained at laboratory conditions as controls against the selection of the less polluted sampling site is discussed.

DDT in zebra mussels from Lake Maggiore (N. Italy): level of contamination and endocrine disruptions.

The DDT contamination of Lake Maggiore (Northern Italy) has been monitored since a serious pollution event occurred in 1996. To assess the environmental risk associated with this contamination, bioaccumulation data coupled with histopathological markers were evaluated on zebra mussel populations from two different contaminated sites from April 2001 to April 2002. Biomonitoring results showed high DDT pollution in 2001 because of a flood which transported DDTs still contained in the sediments of a polluted river to the lake. DDT concentrations reached values of 4-5 microg/g lipids, higher than those recorded in other industrialized countries but comparable to levels measured in developing ones. In the ovaries of the most highly polluted mussels, histological analyses showed a delay in oocyte maturation and a high incidence of pathological pictures mainly referable to oocyte degeneration and haemocytic infiltration. Moreover, despite the presence of mature sperms, in 2001 first male gamete release occurred about 2 months later than in females. These results indicated a neuroendocrine interference of DDT on Dreissena polymorpha reproduction and also showed that these invertebrates can be successfully used to evaluate ecological implications due to exposure to endocrine disruptors in freshwater environments.