Search

Illicit drugs represent not only a great social problem but are also considered an environmental problem because their use and, often, abuse release large amounts of parent compounds, and especially their metabolites, into freshwaters. One of the most commonly used drugs is cocaine, which is the second most prevalent drug in Europe (accounting for almost 30% of all cocaine users worldwide). Cocaine is rapidly metabolised in humans to benzoylecgonine (35-54%), ecgonine methyl ester (32-49%) and norcocaine (5%), which are eliminated in the urine and are only partially removed by wastewater treatment plants (WWTPs). Because no studies have previously been carried out to evaluate the possible risks due to cocaine and its metabolites in non-target organisms, we applied a multi-disciplinary approach to investigate the possible environmental risk related to benzoylecgonine (BE), the main metabolite of cocaine. Previous studies carried out by means of a biomarker suite and the redox-proteomic approach showed an imbalance of anti-oxidant enzyme activities and several genotoxic effects to be caused by environmental BE concentrations in the freshwater bivalve Zebra mussel (Dreissena polymorpha). This report presents the results obtained in the last step of this study, based on a proteomics analysis. We analysed the protein expression profile in the gills of Zebra mussels exposed to two different concentrations (0.5 and 1 μg/L) of BE for 14 days through 2-DE and mass spectrometry analysis (RP-UPLC ESI-LTQ-Orbitrap). Our results highlight significant changes in some proteins in gill cells whose functions are crucial for overall metabolism. In particular, we detected a probable effect of BE on calcium homeostasis and a consequent imbalance of oxidative stress, as verified for vertebrates., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

The question of whether cell death by apoptosis plays a biological function during infection is key to understanding host-parasite interactions. We investigated the involvement of apoptosis in several host-parasite systems, using zebra mussels Dreissena polymorpha as test organisms and their micro- and macroparasites. As a stress response associated with parasitism, heat shock proteins (Hsp) can be induced. In this protein family, Hsp70 are known to be apoptosis inhibitors. Mussels were diagnosed for their respective infections by standard histological methods; apoptosis was detected using the TUNEL methods on paraffin sections and Hsp70 by immunohistochemistry on cryosections. Circulating hemocytes were the main cells observed in apoptosis whereas infected tissues displayed no or few apoptotic cells. Parasitism by intracellular bacteria Rickettsiales-like and the trematode Bucephalus polymorphus were associated with the inhibition of apoptosis whereas ciliates Ophryoglena spp. or the trematode Phyllodistomum folium did not involve significant differences in apoptosis. Even if some parasites were able to modulate apoptosis in zebra mussels, we did not see evidence of any involvement of Hsp70 on this mechanism.

The zebra mussel Dreissena polymorpha is a Caspian Sea bivalve that colonized freshwater bodies worldwide during the XX century. To analyze the impact of seasonal and environmental variations on the physiology and metabolism of this invasive species, we developed a custom microarray using 4057 publicly available DNA sequences from Dreissena and other related genera. Transcriptome profiles were analyzed using half-body samples from a relatively clean site (Riba-Roja, low Ebro River, N.E. Spain), at three different stages of the annual cycle: Pre-spawning (February), spawning (June), and gonad resorption (September). Transcripts from a total of 745 unique sequences showed significant changes among these three groups of samples. Functional characterization of these transcripts based on their closest known homologues showed that genes involved in stress defense (oxidative and infection) were overrepresented in September, whereas genes related to reproductive functions were overrepresented in the spawning and pre-spawning periods. This transcriptomic information can help to identify developmental stages at which the organism is more vulnerable for future control strategies. These data will also contribute to the implementation of gene expression-based assays for pollution monitoring in water bodies harboring stable zebra mussel populations., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Pseudomonas fluorescens strain CL0145A was discovered at the New York State Museum Field Research Laboratory as an effective agent against the environmentally destructive zebra mussel, which has contaminated US waters. Dried cells of the microbe are being commercialized as an environmentally friendly solution to the problem. We found that antibiotic activity against the Gram-positive bacterium Bacillus subtilis is produced and excreted by this strain. We have carried out studies to optimize production of the antibiotic. Studies were begun in a complex corn meal medium. Activity was found in both cells and culture supernates and was maximal after one day of fermentation. Static fermentation conditions were found to be superior to shaken culture. Production of extracellular antibiotic in complex medium was found to be dependent on the content of sucrose and enzymehydrolyzed casein. Indeed, production was greater in sucrose plus enzyme-hydrolyzed casein than in the complex medium. Of a large number of carbon sources studied as improvements over sucrose, the best was glycerol. An examination of nitrogen sources showed that production was improved by replacement of enzymehydrolyzed casein with soy hydrolysates. Production in the simple glycerol-Hy-Soy medium was not improved by addition of an inorganic salt mixture or by complex nitrogen sources, with the exception of malt extract. In an attempt to keep the medium more defined, we studied the effect of amino acids and vitamins as replacements for malt extract. Of 21 amino acids and 7 vitamins, we found tryptophan, glutamine, biotin, and riboflavin to be stimulatory. The final medium contained glycerol, Hy- Soy, tryptophan, glutamine, biotin, and riboflavin.

Zebra mussels (Dreissena polymorpha) are filter feeders located near the base of the foodweb and these animals are able to utilize a variety of carbon sources that may also vary seasonally. We conducted both a spatial and a temporal study in order to test the hypotheses: (1) dissolved organic carbon (DOC) concentrations influence Hg accumulation in zebra mussels sampled from a series of lakes and (2) seasonal variations in diet influence Hg accumulation. In the spatial study, we found a significant negative relationship between Hg concentrations and DOC concentrations, suggesting an influence of DOC on Hg bioaccumulation. In the temporal study, we used stable isotope ratios of nitrogen (δ(15)N) and carbon (δ(13)C) as ecological tools to provide a temporally integrated description of the feeding ecology of zebra mussels. Both δ(15)N and δ(13)C varied seasonally in a similar manner: more depleted values occurred in the summer and more enriched values occurred in the fall. Mercury concentrations also varied significantly over the year, with highest concentrations occurring in the summer, followed by a progressive decrease in concentrations into the fall. The C/N ratio of zebra mussels also varied significantly over the year with the lowest values occurring mid-summer and then values increased in the fall and winter, suggesting that there was significant variation in lipid stores. These results indicate that in addition to any effect of seasonal dietary changes, seasonal variation in energy stores also appeared to be related to Hg levels in the zebra mussels. Collectively results from this study suggest that DOC concentrations, seasonal variation in diet and seasonal depletion of energy stores are all important variables to consider when understanding Hg accumulation in zebra mussels., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Perfluorinated chemicals (PFCs) have been used for many years in numerous industrial products and are known to accumulate in organisms. A recent survey showed that tissue levels of PFCs in aquatic organisms varied among compounds and species being undetected in freshwater zebra mussels Dreissena polymorpha. Here we studied the bioaccumulation kinetics and effects of two major PFCs, perfluorooctane sulfonic acid compound (PFOS) and perfluorooctanoic acid (PFOA), in multixenobiotic transporter activity (MXR) and filtration and oxygen consumption rates in zebra mussel exposed to a range of concentrations of a PCF mixture (1-1,000 μg/L) during 10 days. Results indicate a low potential of the studied PFCs to bioaccumulate in zebra mussel tissues. PFCs altered mussel MXR transporter activity being inhibited at day 1 but not at day 10. Bioaccumulation kinetics of PFCs were inversely related with MXR transporter activity above 9 ng/g wet weight and unrelated at tissue concentration lower than 2 ng/g wet weight suggesting that at high tissue concentrations, these type of compounds may be effluxed out by MXR transporters and as a result have a low potential to be bioaccumulated in zebra mussels. Oxygen consumption rates but not filtering rates were increased in all exposure levels and periods indicating that at environmental relevant concentrations of 1 μg/L, the studied PFCs enhanced oxidative metabolism of mussels. Overall, the results obtained in this study confirm previous findings in the field indicating that an important fraction of PFC accumulated in mussel tissues is eliminated actively by MXR transporters or other processes that are metabolically costly.

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.

Zebra mussels have invaded many lakes in the United States and could be a useful tool for monitoring responses of aquatic biota to changes in mercury loading. The goal of the present study was to evaluate zebra mussels for use as a biomonitor of mercury contamination by comparing zebra mussel mercury concentrations between a lake with only indirect atmospheric mercury contamination (Otisco Lake, NY, USA) and a lake that was directly contaminated by mercury discharges (Onondaga Lake, NY, USA). Zebra mussels were sampled in both the spring and fall of 2004 and 2005. Total mercury (THg) concentrations in zebra mussels were approximately seven times greater in Onondaga Lake than in Otisco Lake, and water column mercury concentrations differed by an order of magnitude between the two lakes. Seasonal differences resulted in significantly higher zebra mussel THg concentrations during the fall for both lakes. There was also significant variation among different sampling sites in Onondaga Lake. Mussel methylmercury concentrations averaged 53% of THg concentrations but were highly variable. Strong relationships between water column THg and zebra mussel THg suggest that zebra mussels are a good indicator of aquatic mercury concentrations and could be used as an effective biomonitor of mercury contamination in aquatic ecosystems., (Copyright © 2013 SETAC.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The freshwater zebra mussel (Dreissena polymorpha) is a notorious biofouling organism. It adheres to a variety of substrata underwater by means of a proteinaceous structure called the byssus, which consists of a number of threads with adhesive plaques at the tips. The byssal proteins are difficult to characterize due to extensive cross-linking of 3,4-dihydroxyphenylalanine (DOPA), which renders the mature structure largely resistant to protein extraction and immunolocalization. By inducing secretion of fresh threads and plaques in which cross-linking is minimized, three novel zebra mussel byssal proteins were identified following extraction and separation by gel electrophoresis. Peptide fragment fingerprinting was used to match tryptic digests of several gel bands against a cDNA library of genes expressed uniquely in the mussel foot, the organ which secretes the byssus. This allowed identification of a more complete sequence of Dpfp2 (D. polymorpha foot protein 2), a known DOPA-containing byssal protein, and a partial sequence of Dpfp5, a novel protein with several typical characteristics of mussel adhesive proteins.

The present study investigated to what extent accumulated metal levels in aquatic invertebrates can reflect environmental contamination and how these tissue levels can be related to alterations in macroinvertebrate communities in the dredged River Dommel. Metal accumulation was measured in translocated zebra mussels (Dreissena polymorpha) and resident Chironomidae. Furthermore, macroinvertebrate community composition was assessed. Our results indicated that trends of total metal concentrations in surface water of the Dommel in time are reflected well by metal levels in tissue of D. polymorpha. In contrast, sediment-bound metals were the most dominant exposure route for Chironomidae. Alterations in macroinvertebrate community composition were observed during dredging and significant relations between metal levels in invertebrate tissues and ecological responses were found. Our results demonstrated that metal accumulation in both zebra mussels and Chironomidae can be used as an integrated measure of metal bioavailability and to predict ecological effects of metal toxicity on macroinvertebrate communities., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The use of DNA adduct analysis has previously focused on the use of marine organisms for biomonitoring, whereas similar investigations in freshwater organisms are sparse. In that context, we have investigated the relevance of DNA adducts as biomarkers of genotoxicity in the freshwater mussels Dreissena polymorpha. The objective of the present study is to determine the stability of DNA adducts induced by benzo[a]pyrene (B[a]P) in zebra mussels. Mussels were exposed to dissolved B[a]P (10-100 µg/l) for 4 days. Afterwards, mussels were kept in clean water for 28 days and DNA adduct levels were subsequently measured in two different organs, the digestive glands and the gills, using the (32)P-postlabelling technique. In parallel, the expression of genes involved in the detoxification system was assessed by qPCR (catalase, superoxide dismutase, glutathione S transferase, HSP70, aryl hydrocarbon receptor, P glycoprotein). We observed a higher level of DNA adducts in the digestive glands compared to the gills. Moreover, in gills, the level of DNA adduct was dependent on the B[a]P concentration. The levels of adducts tended to decrease in both organs after 28 days in clean water. In addition, an early induction of HSP70, PgP, AHR and SOD mRNA levels was noticed in the gills compared to the digestive glands. CAT and GST gene expression increased from 12h exposure in both organs. A higher gene expression level of those genes was observed in the gills, except for AHR and CAT genes. Data converge towards the fact that DNA adducts hence represent a very promising biomarker of B[a]P contamination and potentially of exposure to polycyclic aromatic hydrocarbons. In addition, for the first time in this study, B[a]P detoxification system was characterised in D. polymorpha.

Fluoride toxicity and bioaccumulation tests (short- and long-term) were performed on the nonindigenous freshwater mollusk Dreissena polymorpha at two different temperatures: 17 ± 0.5°C and 22 ± 0.5°C. Concentrations that did not result in toxicity in short-term experiments (96 h) induced effects over a longer period (17 weeks), especially at the warmest temperature, highlighting the role of this parameter. Fluoride bioaccumulation increased linearly with increasing concentration and temperature, reaching 4,202 µg F(-)/g dry weight in soft tissues only after 48 h of exposure at 22°C at a concentration of 640 mg F(-)/L. Comparing tolerance to fluoride and bioaccumulation values of this species with those of other freshwater invertebrates, D. polymorpha was much more resistant and revealed its capacity to accumulate a great quantity of this xenobiotic substance. The results of the present study demonstrated that fluoride accumulation in the soft tissue of this animal was much higher (up to 1,409.6 µg F(-)/g dry wt) than that in its shell (up to 706.4 µg F(-)/g dry wt). If we consider this datum and the fact that D. polymorpha is widespread in many aquatic ecosystems around the world, representing a food source for many birds and other vertebrates, we must acknowledge the possibility that it can represent a serious danger in view of fluoride biomagnification in the aquatic environment., (Copyright © 2012 SETAC.)

This study examines spatial and temporal variability of bioaccumulation of cadmium, copper, and zinc in tissues of zebra mussels in the upper reach of the St. Lawrence River which originates at the outflow of Lake Ontario. It was hypothesized that concentrations of these metals in mussel tissues would decline over time and decrease with increasing distance from the outlet of Lake Ontario as a result of on-going efforts to reduce contaminant discharges into the lake. Size of mussels was also evaluated as a factor influencing bioaccumulation. Mussels were collected annually in October from six sites from 1994 to 2005, including one site near a local industry. Individuals were grouped into five or more size classes per site in each year. Soft tissues were analyzed for total cadmium, copper and zinc. Concentrations of cadmium and copper in tissues varied significantly both spatially and temporally. Cadmium concentrations were elevated at most sites; copper concentrations were moderately elevated compared with other studies in the Lake Ontario basin and St. Lawrence River. Zinc showed the most uniformity in mussels possibly due to internal regulation and to low levels of environmental exposure. Animal size correlated with copper concentrations of tissues in approximately 30% of samples but infrequently for cadmium and zinc. Cadmium and copper levels were found to decline downstream over time. Inter-annual variability of metal concentrations in mussel tissues suggests utilization in long-term monitoring programs to discern significant trends.

Triclosan (TCS, 5-chloro-2-(2,4-dichlorophenoxy)phenol) is commonly used in several personal care products, textiles, and children's toys. Because the removal of TCS by wastewater treatment plants is incomplete, its environmental fate is to be discharged into freshwater ecosystems, where its ecotoxicological impact is still largely unexplored. Previously, we began a structured multi-tiered approach in order to evaluate TCS toxicity in the freshwater mussel Dreissena polymorpha. The results of our previous studies, based on in vitro and in vivo experiments, highlighted a pronounced cytogenotoxic effect exerted by TCS, and showed that an increase in oxidative stress was likely to be one of its main toxic mechanisms. In this work, in order to investigate TCS toxicity mechanisms in aquatic non-target species in greater depth, we decided to use a proteomic approach, analysing changes in protein expression profiles in gills of D. polymorpha exposed for seven days to TCS. Moreover, thiobarbituric acid reactive substances (TBARS) were measured to investigate further the role played by TCS in inducing oxidative stress. Finally, TCS bioaccumulation in mussel tissues was also assessed, to ensure an effective accumulation of the toxicant. Our results not only confirmed the role played by TCS in inducing oxidative stress, but furthered knowledge about the mechanism exerted by TCS in inducing toxicity in an aquatic non-target organisms. TCS induced significant alterations in protein expression profiles in gills of D. polymorpha. The wide range of proteins affected suggested that this chemical has marked effects on various biological processes, especially those involved in calcium binding or stress response. We also confirmed that the proteomic analysis, using 2-DE and de novo sequencing, is a reliable and powerful approach to investigate cellular responses to pollutants in a non-model organism with few genomic sequences available in databases., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Nitrification in shell biofilms and denitrification in the gut of the animal accounted for N(2)O emission by Dreissena polymorpha (Bivalvia), as shown by gas chromatography and gene expression analysis. The mussel's ammonium excretion was sufficient to sustain N(2)O production and thus potentially uncouples invertebrate N(2)O production from environmental N concentrations.

Polybrominated diphenyl ethers (PBDEs) are a group of highly hydrophobic and persistent chemicals that has been used as flame retardants in several industrial applications. They have been detected in various environmental matrices worldwide and an increasing number of studies have recently been carried out to investigate their potential toxicity on ecosystem communities. Although a variety of biological damage has been documented in vertebrates, the effects on invertebrates are largely unknown. The objective of the present study was to determine the cyto-genotoxic effects induced by single exposure to three concentrations of 2,4,2',4'-tetra BDE (BDE 47), 2,2',4,4',6-penta BDE (BDE-100) and 2,2',4,4',5,6'-hexa BDE (BDE-154) on the freshwater mussel Dreissena polymorpha by a multi-biomarker approach. We performed on bivalve hemocytes the Single Cell Gel Electrophoresis (SCGE) assay, the DNA Diffusion assay and the Micronucleus test (MN test) to assess genotoxicity, while the Neutral Red Retention Assay (NRRA) was used to evaluate cytotoxic effects. Results showed that BDE-47 did not produce any genetic damage at the tested concentrations (0.1 μg/L, 0.5 μg/L and 1 μg/L), while BDE-100 and BDE-154 can be considered moderately genotoxic, since both primary and fixed DNA injuries were induced. The NRRA indicated a moderate increase in cellular stress in BDEs-treated bivalves. Thus, our data seems to suggest that investigated BDEs may pose a low risk to freshwater mussels at environmental concentrations., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The zebra mussel Dreissena polymorpha is widely used as sentinel organism for the assessment of environmental contamination in freshwater environments. However, in the River Rhine (Germany), the D. polymorpha population is declining, whereas the closely related quagga mussel D. bugensis is found in high numbers at some sites. In the present laboratory study, D. polymorpha and D. bugensis were exposed to resuspended native sediments for ≤2 weeks. Wet sediments (<63 μm, 100 mg l(-1) dry weight) were used as surrogate suspended particulate matter to mimic one of the mussels' main uptake route for chemicals. The sediments were sampled in (1) the River Elbe in Dessau, a site known to be highly polluted with, e.g., organochlorine (OC) pesticides and (2) at a relatively unpolluted site in Havelberg in the River Havel, one of the Elbe's tributaries. Chemical analysis of persistent OC compounds (seven polychlorinated biphenyls [PCBs], DDT and its metabolites (DDX), hexachlorocylohexanes [HCHs], and hexachlorobenzene [HCB]) in soft tissue of mussels showed significantly greater values of PCBs 101, 118, 153, 138, 180, the sum of seven PCBs, and p,p'-DDD in D. bugensis compared with D. polymorpha. Fourteen days of exposure to Dessau sediment increased the concentration of p,p'-DDE and p,p'-DDD, as well as the sum of DDX, in both species compared with Havelberg sediment. Interspecific differences were less pronounced when regarding chemical concentrations with lipid content instead of dry-weight of tissue because D. bugensis had greater levels of total lipid than D. polymorpha. DNA damage in gills, as measured with the comet assay, was greater in D. bugensis compared with D. polymorpha. Simultaneously, the content of heat-shock protein (hsp70) in gills was greater in D. polymorpha than in D. bugensis. DNA damage and hsp70 were not induced by exposure time or sediment type. This study shows that D. bugensis and D. polymorpha may differ in their bioaccumulation potential of OC pesticides as well as their levels of DNA damage and hsp70. Therefore, more investigations are needed before quagga mussel can be used as alternative test organism for the zebra mussel.

The effect of Ca, Zn, and dissolved natural organic matter (NOM) on waterborne Ni accumulation was investigated in a freshwater mussel. An enriched stable metal isotope tracer was required to measure the Ni uptake rate accurately. Zebra mussels were exposed to environmentally relevant concentrations of (62) Ni (from 0.5 to 8 µg/L) for 48 h in media spiked with Ca, Zn, or dissolved NOM. The (62)Ni uptake was inhibited by Ca (from 0.138 ± 0.021 to 0.061 ± 0.010 L/g/d for Ca concentrations ranging from 43 to 133 mg/L) and enhanced by Zn (from 0.051 ± 0.006 to 0.109 ± 0.007 L/g/d for Zn concentrations ranging from 6.6 to 38.3 µg/L). The mechanisms behind the synergistic effect of Zn remain unclear, yet it can be hypothesized that Ni uptake is facilitated by Zn-dependent transport sites. To formalize the effects of Ca and Zn, a model was proposed to express the Ni uptake rate as a function of the mussels' filtration rate and of Ca and Zn concentrations. The (62)Ni uptake increased at low NOM concentrations and decreased at higher concentrations. This could be explained by the influence of NOM on both the speciation of Ni and the filtration activity of mussels. At high NOM concentrations, a modification of the membrane's permeability might also have favored Ni uptake, although this was not clearly established in this study. Therefore, the effect of water composition on Ni bioavailability to zebra mussels cannot be predicted by competition and complexation models alone, because it also influences the animal's physiology., (Copyright © 2012 SETAC.)

Aquatic organisms are exposed to pollution which may make them more susceptible to infections and diseases. The present investigation evaluated effects of nickel contamination and parasitism (ciliates Ophryoglena spp. and intracellular bacteria Rickettsiales-like organisms), alone and in combination, on biological responses of the zebra mussel Dreissena polymorpha, and also the infestation abilities of parasites, under laboratory controlled conditions. Results showed that after 48 h, more organisms were infected in nickel-exposed groups, which could be related to weakening of their immune system. Acting separately, nickel contamination and infections were already stressful conditions; however, their combined action caused stronger biological responses in zebra mussels. Our data, therefore, confirm that the parasitism in D. polymorpha represents a potential confounding factor in ecotoxicological studies that involve this bivalve.

The freshwater zebra mussel (Dreissena polymorpha) owes a large part of its success as an invasive species to its ability to attach to a wide variety of substrates. As in marine mussels, this attachment is achieved by a proteinaceous byssus, a series of threads joined at a stem that connect the mussel to adhesive plaques secreted onto the substrate. Although the zebra mussel byssus is superficially similar to marine mussels, significant structural and compositional differences suggest that further investigation of the adhesion mechanisms in this freshwater species is warranted. Here we present an ultrastructural examination of the zebra mussel byssus, with emphasis on interfaces that are critical to its adhesive function. By examining the attached plaques, we show that adhesion is mediated by a uniform electron dense layer on the underside of the plaque. This layer is only 10-20 nm thick and makes direct and continuous contact with the substrate. The plaque itself is fibrous, and curiously can exhibit either a dense or porous morphology. In zebra mussels, a graded interface between the animal and the substrate mussels is achieved by interdigitation of uniform threads with the stem, in contrast to marine mussels, where the threads themselves are non-uniform. Our observations of several novel aspects of zebra mussel byssal ultrastructure may have important implications not only for preventing biofouling by the zebra mussel, but for the development of new bioadhesives as well., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The pharmaceutical fluoxetine, a selective serotonin reuptake inhibitor (SSRI), is often detected in municipal wastewater treatment plant effluents and surface waters within the ng/l range. There is, however, insufficient research evaluating potential hazards of fluoxetine in aquatic organisms at environmentally relevant concentrations. Taking into account that several SSRIs (fluoxetine, fluvoxamine) act as spawning inducers in bivalves, this study aimed at investigating the effects of fluoxetine exposure in the zebra mussel (Dreissena polymorpha) by assessing its potential to induce spawning at environmentally relevant concentrations (20 and 200 ng/l), as well as alterations of endogenous levels of testosterone and estradiol. Histological analyses of female and male gonads showed a concentration dependent decrease of oocyte and spermatozoan density, with a reduction in the number of oocytes per follicle of 40-70%, and spermatozoan density of 21-25%, relative to controls, following exposure to 20 and 200 ng/l of fluoxetine for 6 days, respectively. There was also a significant increase (1.5-fold) in the endogenous level of esterified estradiol in organisms exposed to 200 ng/l fluoxetine. Overall, the study shows that exposure to low levels of fluoxetine may effectively induce gamete liberation in the zebra mussel as well as alter endogenous levels of estradiol, and evidences the need of further investigating the potential of fluoxetine to alter the endocrine system of molluscs at environmentally relevant concentrations., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The quagga mussel (Dreissena rostriformis bugensis) and zebra mussel (Dreissena polymorpha) are invasive freshwater bivalves in Europe and North America. The distribution range of both Dreissena species is still expanding and both species cause major biofouling and ecological effects, in particular when they invade new areas. In order to assess the effect of temperature, salinity and light on the initial byssogenesis of both species, 24 h re-attachment experiments in standing water were conducted. At a water temperature of 25°C and a salinity of 0.2 psu, the rate of byssogenesis of D. polymorpha was significantly higher than that of D. rostriformis bugensis. In addition, byssal thread production by the latter levelled out between 15°C and 25°C. The rate of byssogenesis at temperatures<25°C was similar for both species. Neither species produced any byssal threads at salinities of 4 psu or higher. At a salinity of 1 psu and a water temperature of 15°C, D. polymorpha produced significantly more byssal threads than D. rostriformis bugensis. There was no significant effect of the length of illumination on the byssogenesis of either species. Overall, D. polymorpha produced slightly more byssal threads than D. rostriformis bugensis at almost all experimental conditions in 24 h re-attachment experiments, but both species had essentially similar initial re-attachment abilities. The data imply that D. rostriformis bugensis causes biofouling problems identical to those of D. polymorpha.

This study investigates the ability of the biodynamic model to predict the trophic bioaccumulation of cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni) and zinc (Zn) in a freshwater bivalve. Zebra mussels were transplanted to three sites along the Seine River (France) and collected monthly for 11 months. Measurements of the metal body burdens in mussels were compared with the predictions from the biodynamic model. The exchangeable fraction of metal particles did not account for the bioavailability of particulate metals, since it did not capture the differences between sites. The assimilation efficiency (AE) parameter is necessary to take into account biotic factors influencing particulate metal bioavailability. The biodynamic model, applied with AEs from the literature, overestimated the measured concentrations in zebra mussels, the extent of overestimation being site-specific. Therefore, an original methodology was proposed for in situ AE measurements for each site and metal., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The metal-specific covalent index and the species-specific size-based filtration rate were integrated into a biokinetic model estimating metal bioaccumulation in mussels from the dissolved phase and phytoplankton. The model was validated for zebra (Dreissena polymorpha) and quagga (Dreissena rostriformis bugensis) mussels in the rivers Rhine and Meuse, the Netherlands. The model performed well in predicting tissue concentrations in different-sized zebra mussels from various sampling sites for (55) Mn, (56) Fe, (59) Co, (60) Ni, (82) Se, (111) Cd, (118) Sn, and (208) Pb (r(2) =0.71-0.99). Performance for (52) Cr, (63) Cu, (66) Zn, (68) Zn, and (112) Cd was moderate (r(2) <0.20). In quagga mussels, approximately 73 to 94% of the variability in concentrations of (82) Se, (111) Cd, (112) Cd, and (208) Pb was explained by the model (r(2) =0.73-0.94), followed by (52) Cr, (55) Mn, (56) Fe, (60) Ni, and (63) Cu (r(2) =0.48-0.61). Additionally, in both zebra and quagga mussels, average modeled concentrations were within approximately one order of magnitude of the measured values. In particular, in zebra mussels, estimations of (60) Ni and (82) Se concentrations were equal to 51 and 76% of the measurements, respectively. Higher deviations were observed for (52) Cr, (59) Co, (55) Mn, (56) Fe, (111) Cd, (63) Cu, and (112) Cd (underestimation), and (66) Zn, (68) Zn, (208) Pb, and (118) Sn (overestimation). For quagga mussels, modeled concentrations of (66) Zn and (68) Zn differed approximately 14% from the measured levels. Differences between predictions and measurements were higher for other metals., (Copyright © 2011 SETAC.)

Carbamazepine (CBZ), Ibuprofen (IBU) and Bezafibrate (BEZ) were tested for their potential to bioaccumulate and provoke molecular changes in the non-target organism Dreissena polymorpha. mRNA changes of enzymes and other proteins involved in the prevention from protein damage (heat shock protein 70, hsp70) and oxidative stress (superoxide dismutase, SOD; catalase, CAT; metallothionein, MT), biotransformation (pi-class glutathione S-transferase, piGST; aryl hydrocarbon receptor, AH-R), elimination (P-glycoprotein, P-gp) and reversible protein posttranslational modification (protein phosphatase 2A, PP2A) served as molecular biomarkers. Mussels were exposed in a flow-through system to increasing concentrations of the three substances (1, 10, 100 and 1000 nM). The two lower concentrations correspond to environmentally relevant concentrations detected in surface and effluent waters, respectively. Measuring tissue concentration after one, four and seven days the uptake of CBZ and IBU by the mussels could be evidenced, whereas no accumulation data could be achieved for BEZ. The bioconcentration factor was highest for mussels exposed to the lowest CBZ and IBU concentrations, with 90 and 460-fold higher tissue concentration, respectively, after seven days. CBZ was the only substance tested which caused a significant increase in gill mRNA level of hsp70 after only one day exposure, evidencing the potential of CBZ to immediately provoke a stress condition and assumingly protein damage in gills. After longer exposure, mussels displayed down-regulated mRNA levels of hsp70 and SOD in gills, as well as of MT and P-gp in the digestive gland, hinting on an inhibitory character of CBZ. In IBU exposed mussels increased oxidant stress conditions were evidenced by induced mRNA levels in the digestive gland of CAT and MT, as well as SOD after one and four days, respectively. A concentration as found at sewage treatment plant effluents provoked an increase in transcript levels of piGST, suggesting enhanced need for biotransformation of IBU or by-products derived from oxidative stress. Also exposure to an environmentally relevant BEZ concentration provoked an immediate increase in piGST transcript level in the digestive gland followed by up-regulated hsp70 after four and seven days evidencing a chronic stress condition for the mussels., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Pharmaceuticals, including the lipid regulator gemfibrozil and the non-steroidal anti-inflammatory drug diclofenac have been identified in waste water treatment plant effluents and receiving waters throughout the western world. The acute and chronic toxicity of these compounds was assessed for three freshwater species (Daphnia magna, Pseudokirchneriella subcapitata, Lemna minor) using standardised toxicity tests with toxicity found in the non-environmentally relevant mid mg L(-1) concentration range. For the acute endpoints (IC(50) and EC(50)) gemfibrozil showed higher toxicity ranging from 29 to 59 mg L(-1) (diclofenac 47-67 mg L(-1)), while diclofenac was more toxic for the chronic D. magna 21 d endpoints ranging from 10 to 56 mg L(-1) (gemfibrozil 32-100 mg L(-1)). These results were compared with the expression of several biomarkers in the zebra mussel (Dreissena polymorpha) 24 and 96 h after exposure by injection to concentrations of 21 and 21,000 μg L(-1) corresponding to nominal concentrations of 1 and 1000 μg L(-1). Exposure to gemfibrozil and diclofenac at both concentrations significantly increased the level of lipid peroxidation, a biomarker of damage. At the elevated nominal concentration of 1000 μg L(-1) the biomarkers of defence glutathione transferase and metallothionein were significantly elevated for gemfibrozil and diclofenac respectively, as was DNA damage after 96 h exposure to gemfibrozil. No evidence of endocrine disruption was observed using the alkali-labile phosphate technique. Results from this suite of biomarkers indicate these compounds can cause significant stress at environmentally relevant concentrations acting primarily through oxidation pathways with significant destabilization of the lysosomal membrane and that biomarker expression is a more sensitive endpoint than standardised toxicity tests., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

A 12-month active biomonitoring study was performed in 2008-2009 on a northern French river system using the freshwater mussel Dreissena polymorpha as a sentinel species. Allochtonous mussels originating from a reference site (Commercy) were caged at four sites (Bouy, Sept-Saulx, Fismes, Ardre) within the Vesle River basin. The main objective of the study was to characterize the influence of biotic (sex, food availability) and abiotic (temperature, chemicals) factors on the reproductive and energy reserve (glycogen, lipids) cycles of exposed mussels. Both cycles were markedly disturbed at the Bouy and Sept-Saulx sites where the lowest chlorophyll a levels were recorded during the study. At these sites, mussels obviously faced a negative energy balance, as confirmed by the impairment of their physiological state and byssal attachment. At other exposure sites, reproductive and energy reserves cycles were less impacted but were still dependent on the nutritional state of mussels. The latter appeared as a significant natural confounding factor in ecotoxicological survey performed in low polluted areas., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In the Flix Reservoir (Ebro River, Spain), ca. 300,000tons of industrial waste were dumped because of the activity of a factory plant in Flix. Within the recovery program implemented, this exceptional situation provides a unique opportunity to test the value of zebra mussel as sentinel organism. Ten metal concentrations were measured in mussels from different sites to assess spatial redistribution of metals and bioavailability to the food web. Our results showed an important metal uptake by mussels; metal concentrations (except As) measured in impacted sites were up to 10 times higher than in control sites, and Mn and Hg exceeded several times the levels previously reported for polluted waters. Concentrations increased downstream showing the metal mobilization from polluted sediments in Flix Reservoir. The higher metal concentrations measured in zebra mussel individuals clearly indicated their bioavailability to the food web, allowing the toxics transfer to predators and occasionally to humans. Thus, zebra mussel is a valuable sentinel organism to identify highly polluted waters, transport routes and trophic transfer., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Bioaccumulation and effects of the contraceptive hormone levonorgestrel were examined in the non-target organism Dreissena polymorpha. Molecular biomarkers of biotransformation, elimination, antioxidant defence and protein damage were analyzed after exposure to increasing concentrations of levonorgestrel in a flow-through system. The lowest concentration (0.312 μg L(-1)) was 100-fold bioconcentrated within four days. A decrease of the bioconcentration factor was observed within one week for the highest test concentrations (3.12 and 6.24 μg L(-1)) suggesting enhanced excretory processes. The immediate mRNA up-regulation of pi class glutathione S-transferase proved that phase II biotransformation processes were induced. Disturbance of fundamental cell functions was assumed since the aryl hydrocarbon receptor has been permanently down-regulated. mRNA up-regulation of P-glycoprotein, superoxide dismutase and metallothioneine suggested enhanced elimination processes and ongoing oxidative stress. mRNA up-regulation of heat shock protein 70 in mussels exposed to the two highest concentrations clearly indicated impacts on protein damage., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

It is often difficult to evaluate the level of contamination in small urban rivers because pollution is mainly diffuse, with low levels of numerous substances. The use of a coupled approach using both chemical and biological measurements may provide an integrated evaluation of the impact of micro-pollution on the river. Zebra mussels were transplanted along a metal and organic pollution gradient in spring 2008. For two months, mussels and water samples were collected from two sites every two weeks and analyzed for metal and PAH content as well as water physicochemical parameters. Diffusive gradients in thin film (DGT) were also used to assess levels of labile metals. Exposure of mussels to contaminants and potential impact were evaluated using physiological indices and various biomarkers including condition index (CI), defense mechanisms (glutathione-S-transferase: GST), digestive enzymes (amylase and cellulase) and genotoxicity (micronucleus test: MN and comet assay: CA). For most contaminants, the water contamination was significantly higher downstream. Bioaccumulation in zebra mussels was related to water contamination in the framework of the biodynamic model, which allowed us to take into account the biological dilution that was caused by the growth of soft tissue downstream. Thus, metal influxes were on average two times higher downstream than upstream in particular for Zn, Cr, Cu and Cd. Significant differences in condition index were observed (final CI was 0.42 ± 0.03 downstream and 0.31 ± 0.03 upstream) reflecting a better food availability downstream. Moreover a significant decrease of GST activity and digestive enzymes activity in the cristalline style was observed downstream. Interpreting this decrease requires considering not only micro-pollution but also the trophic status related to the water's physicochemistry. The MN test and the CA on gill cells highlighted genotoxicity in mussels transplanted downstream compared to upstream., (© 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.)

The present study aims at investigating the effects of Zn, Ca, and dissolved organic carbon (DOC) on the waterborne Cd bioaccumulation of a freshwater bivalve (Dreissena polymorpha). Mussels were exposed for 48 h at 3 µg/L of Cd in different media. Their physiological activities were assessed by separately measuring the filtration rate in the same exposure water. Increased Zn (from 3 to 89 µg/L) and Ca (from 37 to 131 mg/L) concentrations in water led to a threefold and sevenfold reduction of Cd bioaccumulation, whereas the effect of DOC varied greatly depending on its concentration. At low DOC concentrations (from 0.2 to 1.1 mg/L), the uptake of Cd increased, whereas at higher concentrations (from 1.1 to 17.1 mg/L), the uptake decreased. The filtration activity was not strongly influenced by either Zn or Ca concentration, whereas it was modified in enriched DOC media in the same manner as Cd uptake. A competitive model was built to predict the waterborne uptake rate constant of Cd (k (u)) as a function of Zn and Ca concentrations in the water. Over the range of DOC concentrations we tested, organic matter was shown to influence Cd bioaccumulation in two ways: by modifying Cd speciation and thus its bioavailability and its interaction with the biological membrane, and by affecting the mussel's physiology and therefore its sensitivity to metal. The present study provides a useful means of adjusting the toxicokinetic constant to the water's physicochemical characteristics and proposes a unifying model that takes into account the different geochemical and biological influences on bioaccumulation., (Environ. Toxicol. Chem. 2010;29:2182-2189. © 2010 SETAC.)

The validation of a suite of cellular biomarkers for biomonitoring studies necessitates a good knowledge of the meaning of these early responses to environmental stress in terms of individual health. This requires confirmation (i) of linkages between the cellular and higher levels of the biological organisation, (ii) of temporal persistence of the stress symptoms and (iii) of their reversibility after a return to more favourable conditions. Besides, (iv) the sensitivity of the biomarker suite towards subtle variations of environmental contamination has to be assessed. With this aim, field experiments were performed on deployed freshwater zebra mussels (Dreissena polymorpha) in the vicinity of the confluence of a small heavily anthropized stream with a larger river. We examined the persistence of the responses over a 90-day period and their reversibility after a depuration-transplantation. A second experiment was conducted later by adding a study site at an increased distance from the confluence. Decreased digestive lysosomal volume and neutral lipid contents, and lipofuscin accumulation preceded effects on the mussels' condition. The following experiment confirmed that the cellular biomarkers were more sensitive than both individual endpoints to reflect the effects of subtler variations of environmental contamination. Integration of the results with multivariate analysis and the Integrated Biomarker Response tended to confirm the relevance of the biomarker suite., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

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., (Copyright 2010 Elsevier Inc. All rights reserved.)

Due to increasing amounts of pharmaceutically active compounds (PhACs) in the aquatic environment, their largely unknown effects to non-target organisms need to be assessed. This study examined physiological changes in the freshwater mussel Dreissena polymorpha exposed to increasing concentrations (0.534, 5.34, 53.4 and 534 microg L(-1)) of the beta-blocker metoprolol in a flow-through system for seven days. The two lower concentrations represent the environmentally relevant range. Surprisingly, metallothionein mRNA was immediately up-regulated in all treatments. For the two higher concentrations mRNA up-regulation in gills was found for P-glycoprotein after one day, and after four days for pi class glutathione S-transferase, demonstrating elimination and biotransformation processes, respectively. Additionally, catalase and superoxide dismutase were up-regulated in the digestive gland indicating oxidative stress. In all treated mussels a significant up-regulation of heat shock protein mRNA was observed in gills after four days, which suggests protein damage and the requirement for repair processes. Metoprolol was 20-fold bioaccumulated for environmentally relevant concentrations., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: The zebra mussel (Dreissena polymorpha) has been well known for its expertise in attaching to substances under the water. Studies in past decades on this underwater adhesion focused on the adhesive protein isolated from the byssogenesis apparatus of the zebra mussel. However, the mechanism of the initiation, maintenance, and determination of the attachment process remains largely unknown., Results: In this study, we used a zebra mussel cDNA microarray previously developed in our lab and a factorial analysis to identify the genes that were involved in response to the changes of four factors: temperature (Factor A), current velocity (Factor B), dissolved oxygen (Factor C), and byssogenesis status (Factor D). Twenty probes in the microarray were found to be modified by one of the factors. The transcription products of four selected genes, DPFP-BG20&#95;A01, EGP-BG97/192&#95;B06, EGP-BG13&#95;G05, and NH-BG17&#95;C09 were unique to the zebra mussel foot based on the results of quantitative reverse transcription PCR (qRT-PCR). The expression profiles of these four genes under the attachment and non-attachment were also confirmed by qRT-PCR and the result is accordant to that from microarray assay. The in situ hybridization with the RNA probes of two identified genes DPFP-BG20&#95;A01 and EGP-BG97/192&#95;B06 indicated that both of them were expressed by a type of exocrine gland cell located in the middle part of the zebra mussel foot., Conclusions: The results of this study suggested that the changes of D. polymorpha byssogenesis status and the environmental factors can dramatically affect the expression profiles of the genes unique to the foot. It turns out that the factorial design and analysis of the microarray experiment is a reliable method to identify the influence of multiple factors on the expression profiles of the probesets in the microarray; therein it provides a powerful tool to reveal the mechanism of zebra mussel underwater attachment.

This study assesses the response of the zebra mussel (Dreissena polymorpha) to chemical pollution derived from the R. Lambro/R. Po confluence, which is one of the most polluted aquatic environments in Europe. The mussels were tested under laboratory conditions to water sampled in the spring and fall at three sites located upstream or downstream of the confluence or directly in R. Lambro alone. We performed on mussel specimens a biomarker battery composed by eight different assays: single cell gel electrophoresis, apoptosis determination, the micronucleus test and Neutral Red retention, as well as catalase, superoxide dismutase, glutathione peroxidase and glutathione transferase activities. We also evaluated the bioaccumulation of several organic pollutants (PAHs, PCBs, DDTs, HCHs and HCB) to characterize the sampling sites. Significant increases in DNA strand breaks, apoptosis and micronuclei were observed, with no significant seasonal differences. We observed a clear induction of the enzyme activities measured in the spring, but the enzymatic activity trend in the fall was very complex, with several enzymes returning to baseline levels of activity, suggesting a possible seasonal change in chemical mixture characteristics.

Freshwater mussels Dreissena polymorpha (Pallas, 1771) were exposed to the elevated concentrations of Cd (10, 50, 100, and 500 microg/L), Cu (10, 30, 50, and 80 microg/L), and an organochlorinated pesticide, pentachlorophenol (PCP) (1, 10, and 100 microg/L). Induced synthesis of biomarker metallothionein (MT) and changes in concentrations of cytosolic Cd, Cu, and Zn in the whole soft tissue of mussels were monitored after a 7-day laboratory exposure to the contaminants. A clear dose-dependent elevation in the MT concentration was observed after exposure to Cd at doses of 10-100 microg/L, and this increase of MT content was accompanied with a linear increase of cytosolic Cd. Cd concentration of 500 microg/L caused no additional increase of MT and Cd in mussel cytosol, suggesting possible toxic effects due to exceeding cellular inducible/defense capacity. Cu exposure resulted with variable changes in MT concentrations, with no clear linear relationship between MT and Cu concentrations in water, although a progressive dose-dependent accumulation of Cu in the soluble fraction of mussel tissues was recorded. A decrease of cytosolic Zn was evident at higher exposure concentrations of both metals used. PCP in concentrations applied was unable to induce MT synthesis, but the higher concentrations of PCP influenced the cytosolic metal concentrations. In conclusion, the results obtained confirm the specificity of MT induction in D. polymorpha as an biological response on metal stimulation, especially by cadmium, being more closely correlated to MT than copper within the ecologically relevant concentration range. The strong induction potential of cadmium as well as an absence of MT induction following exposure to PCP as an organic chemical contaminant are supporting evidences for usage of zebra mussel MT as a specific biomarker of Cd exposure in biomonitoring programs.

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.

Large amounts of industrial waste containing high concentrations of mercury, cadmium and organochlorine residues were dumped in a reservoir adjacent to a chlorine-alkali plant in the village of Flix(Catalonia, Spain), situated at the shore of the lower Ebro river. Effects of these contaminants to aquatic river invertebrates were assessed by integrating analyses of metals and organochlorine residues in field collected zebra mussels and crayfish with a wide range of biomarkers. Biological responses included levels of metallothioneins, activities of ethoxyresorufin-O-deethylase, oxidative stress biomarkers (glutathione content, enzymatic activities of superoxide dismutase, catalase, glutathione s-transferase, glutathione peroxidise and glutathione reductase), levels of lipid peroxidation and of DNA strand breaks. The results obtained evidenced similar response patterns in mussels and crayfish with increasing toxic stress levels from upper parts of the river towards the meander located immediately downstream from the most polluted site, close to the waste dumps. The aforementioned stress levels could be related with concentrations of mercury, cadmium, hexachlorobenzene, polychlorobiphenyls and dichlorodiphenyltrichloroethanes from 4- to 195-fold greater than local background levels. The response of biomarkers to these pollutant concentrations differences was reflected in high activities and levels of antioxidant enzymes, metallothioneins, lipid peroxidation and DNA strand breaks and decreased levels of glutathione., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Given that virtually all organisms are hosts for parasites, the investigation of the combined effects of contamination and parasitism is important in the framework of aquatic bioindication procedures. To assess the impact of such multistresses at the host cellular level, we sampled parasitized zebra mussel (Dreissena polymorpha) populations from two sites in northeast France that presented different levels of contamination. Experimental groups were formed based on parasite species and host gender and tested by histochemistry and automated image analysis for biological responses, such as structural changes of the lysosomal system and neutral lipid accumulation. Infected organisms displayed smaller and more numerous lysosomes compared with uninfected congeners, and infection further elevated the effect of the chemical contamination on this biomarker. In contrast, co-infection of females with selected parasites did produce inverse results, i.e. a more developed lysosomal system and neutral lipid depletion. Our data, therefore, suggest that parasitism in zebra mussels represents a potential confounding factor in ecotoxicological studies and must be taken into account in environmental risk assessment studies.

Organisms are able to detoxify accumulated metals by, e.g. binding them to metallothionein (MT) and/or sequestering them in metal-rich granules (MRG). The different factors involved in determining the capacity or efficiency with which metals are detoxified are not yet known. In this work we studied how the sub-cellular distribution pattern of cadmium, copper and zinc in whole tissue of zebra mussels from clean and polluted surface waters is influenced by the total accumulated metal concentration and by its physiological condition. Additionally we measured the metallothionein concentration in the mussel tissue. Metal concentration increased gradually in the metal-sensitive and detoxified sub-cellular fractions with increasing whole tissue concentrations. However, metal concentrations in the sensitive fractions did not increase to the same extent as metal concentrations in whole tissues. In more polluted mussels the contribution of MRG and MT became more important. Nevertheless, metal detoxification was not sufficient to prevent metal binding to heat-sensitive low molecular weight proteins (HDP fraction). Finally we found an indication that metal detoxification was influenced by the condition of the zebra mussels. MT content could be explained for up to 83% by variations in Zn concentration and physiological condition of the mussels.

This study investigated whether biotransformation and antioxidant enzymes of an organism tolerant to pollution such as the zebra mussel Dreissena polymorpha can be employed to evaluate the extent of urban water pollution. Activity changes of soluble and membrane bound glutathione S-transferase (s- and mGST), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) to environmental stress were explored in precultured mussels exposed for one day to one week at selected sites with reasonable anthropogenic impact. The enzymatic response of whole mussel tissue was compared to gill tissue. Changes in enzyme activity were accordant with the different pollution scenarios, as charges with polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethane (DDT) and other pesticides, as well as contamination with metals. Whereas in whole mussel tissue all analyzed enzymes responded with elevation, in gills inhibition took place with the exception of mGST. The results confirm the high sensitivity of gills, nevertheless enzymatic changes measured in whole mussel tissue provided the clearest results. Significant changes in GST, CAT and GPx enzyme activity were only observed at water temperatures above 20 degrees C. SOD activity was not restricted by temperatures and serves for this reason as a biomarker for oxidative stress at lower water temperatures. Discrepancies between biological and chemical evaluation of the sampling sites are presented and biomarker responses appraised.

Contaminant related changes in behavioral, phase I and II metabolizing enzymes and pro-oxidant/antioxidant processes in the freshwater mussels Dreissena polymorpha exposed to metals and PCBs were assessed. Behavioral and biochemical responses including filtering rates, key phase I, II and antioxidant enzymes and levels of metallothioneins, glutathione, lipid peroxidation and DNA strand breaks were determined in digestive glands of mussels after being exposed to sublethal levels of mercury chloride, methyl mercury, cadmium and Aroclor 1260 during 5 days. In 7 out of 12 responses analyzed, mussels showed significant differences across treatments. Unusual properties of measured ethoxyresorufin-O-deethylase (EROD) activities indicated that mussels lack an inducible CYP1A enzymatic activity. Despite of using similar exposure levels, inorganic and organic mercury showed different biomarker patterns of response with methyl mercury being more bio-available and unable to induce metallothionein proteins. Mussels exposed to Cd presented higher levels of metallothioneins and an enhanced metabolism of glutathione, whereas those exposed to Aroclor showed their antioxidant glutathione peroxidase related enzyme activities inhibited. Although there was evidence for increased lipid peroxidation under exposure to inorganic and organic mercury, only mussels exposed to Aroclor had significant greater levels than those in controls.

Background: Elemental imbalances of carbon (C): nitrogen (N): phosphorus (P) ratios in food resources can constrain the growth of grazers owning to tight coupling between growth rate, RNA allocation and biomass P content in animals. Testing for stoichiometric constraints among invasive species is a novel challenge in invasion ecology to unravel how a successful invader tackles ecological barriers in novel ecosystems., Methodology/principal Findings: We examined the C:P and N:P ratios and the condition factor of a successful invader in lakes, the zebra mussel (Dreissena polymorpha), collected from two Swedish lakes. Concurrently, we analyzed the elemental composition of the food (seston) and tissue of the mussels in which nutrient composition of food and mussels varied over time. Zebra mussel condition factor was weakly related to the their own tissue N:P and C:P ratios, although the relation with the later ratio was not significant. Smaller mussels had relatively lower tissue N:P ratio and higher condition factor. There was no difference in C:P and N:P ratios between seston and mussels' tissues. Our results indicated that the variation in nutrient stoichiometry of zebra mussels can be explained by food quality and quantity., Conclusions/significance: Our study suggests that fitness of invasive zebra mussels is not constrained by nutrient stoichiometry which is likely to be important for their proliferation in novel ecosystems. The lack of imbalance in C:P and N:P ratios between seston and mussels along with high tissue C:P ratio of the mussel allow them to tolerate potential P limitation and maintain high growth rate. Moreover, zebra mussels are able to change their tissue C:P and N:P ratios in response to the variation in elemental composition of their food. This can also help them to bypass potential nutrient stoichiometric constraints. Our finding is an important step towards understanding the mechanisms contributing to the success of exotic species from stoichiometric principles.