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One of the top ecological priorities is to find sensitive indicators for pollution monitoring. This study focuses on the bioconcentration and responses (condition index, survival, oxygen consumption, heart rates, and oxidative stress and neurotoxic effect biomarkers) of mussels from the Volga River basin, Dreissena polymorpha and Dreissena bugensis, to long-term exposure to toxic chemicals such as tributyltin (TBT, 25 and 100 ng/L) and copper (Cu, 100 and 1000 μg/L). We found that TBT was present in the tissues of zebra and quagga mussels in comparable amounts, whereas the bioconcentration factor of Cu varied depending on its concentration in water. Differences in responses between the two species were revealed. When exposed to high Cu concentrations or a Cu-TBT mixture, quagga mussels had a lower survival rate and a longer heart rate recovery time than zebra mussels. TBT treatment caused neurotoxicity (decreased acetylcholinesterase activity) and oxidative stress (increased levels of thiobarbituric acid reactive substances) in both species. TBT and Cu levels in mussel tissues correlated positively with the condition index, but correlated with the level of acetylcholinesterase in the mussel gills. The principal component analysis revealed three main components: the first consists of linear combinations of 14 variables reflecting TBT water pollution, TBT and Cu levels in mussel tissues, and biochemical indicators; the second includes Cu water concentration, cardiac tolerance, and mussel size; and the third combines weight, metabolic rate, and heart rates. Quagga mussels are less tolerable to contaminants than zebra mussels, so they may be used as a sensitive indicator., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dreissena polymorpha is a sentinel freshwater mussel providing key functional ecosystemic services like nutrient recycling and suspended matter filtration. Global warming and especially extreme events imply rapid fluctuations of environmental parameters that sessile organisms could not escape. The increase occurrence of heat waves and the subsequent expansion of hypoxic areas could challenge the survival of mussels. This study provided a deeper knowledge of energy management and cellular function during thermal (+15 °C) or hypoxic (30% of dissolved oxygen saturation) stress for 7 days. A potential metabolic rate depression was highlighted in D. polymorpha under hypoxia through a decline in the mitochondrial activity and a constant AMP content over time. A contrasted pattern of response was observed in thermal-stressed mussels between 24 h and 7 days of exposure. A global increase of metabolic activity was noticed in mussels after 24 h while a return to control level was noticed at the end of the experiment. Although D. polymorpha is considered as a temperature tolerant species, a significant increase of ADP:ATP ratio, related to a decrease of mitochondrial activity and density, suggested an overwhelming of organisms. This study pointed to the importance of considering time of exposure to natural factor variations in tolerance window of organisms in a long-term changing environment. The apparent short-term tolerance of D. polymorpha could hide much more deleterious consequences, i.e. mortality, if abiotic stresses persist, as suggested by climate change models., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Carbamazepine (CBZ) and Hg are widespread and persistent micropollutants in aquatic environments. Both pollutants are known to trigger similar toxicity mechanisms, e.g. reactive oxygen species (ROS) production. Here, their effects were assessed in the zebra mussel Dreissena polymorpha, frequently used as a freshwater model in ecotoxicology and biomonitoring. Single and co-exposures to CBZ (3.9 μg L -1 ) and MeHg (280 ng L -1 ) were performed for 1 and 7 days. Metabolomics analyses evidenced that the co-exposure was the most disturbing after 7 days, reducing the amount of 25 metabolites involved in protein synthesis, energy metabolism, antioxidant response and osmoregulation, and significantly altering cells and organelles' structure supporting a reduction of functions of gills and digestive glands. CBZ alone after 7 days decreased the amount of α-aminobutyric acid and had a moderate effect on the structure of mitochondria in digestive glands. MeHg alone had no effect on mussels' metabolome, but caused a significant alteration of cells and organelles' structure in gills and digestive glands. Single exposures and the co-exposure increased antioxidant responses vs control in gills and digestive glands, without resulting in lipid peroxidation, suggesting an increased ROS production caused by both pollutants. Data globally supported that a higher number of hyperactive cells compensated cellular alterations in the digestive gland of mussels exposed to CBZ or MeHg alone, while CBZ + MeHg co-exposure overwhelmed this compensation after 7 days. Those effects were unpredictable based on cellular responses to CBZ and MeHg alone, highlighting the need to consider molecular toxicity pathways for a better anticipation of effects of pollutants in biota in complex environmental conditions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Like marine mussels, freshwater zebra and quagga mussels adhere via the byssus, a proteinaceous attachment apparatus. Attachment to various surfaces allows these invasive mussels to rapidly spread, however the adhesion mechanism is not fully understood. While marine mussel adhesion mechanics has been studied at the individual byssal-strand level, freshwater mussel adhesion has only been characterized through whole-mussel detachment, without direct interspecies comparisons on different substrates. Here, adhesive strength of individual quagga and zebra mussel byssal plaques were measured on smooth substrates with varying hydrophobicity-glass, PVC, and PDMS. With increased hydrophobicity of substrates, adhesive failures occurred more frequently, and mussel adhesion strength decreased. A new failure mode termed 'footprint failure' was identified, where failure appeared to be adhesive macroscopically, but a microscopic residue remained on the surface. Zebra mussels adhered stronger and more frequently on PDMS than quagga mussels. While their adhesion strengths were similar on PVC, there were differences in the failure mode and the plaque-substrate interface ultrastructure. Comparisons with previous marine mussel studies demonstrated that freshwater mussels adhere with comparable strength despite known differences in protein composition. An improved understanding of freshwater mussel adhesion mechanics may help explain spreading dynamics and will be important in developing effective antifouling surfaces., (© 2021. The Author(s).)

Metal trace elements such as cadmium (Cd) are commonly present in ecosystems and could lead to impairment of mitochondrial functions and energy imbalance in aquatic organisms including molluscs. Combined exposure to increasing temperatures and Cd could enhance such an impact on animals. Seasonal fluctuations, such as temperature, and the corresponding reproduction cycle can affect biomarker responses. However, the reproduction cycle stage is rarely taken into account in ecotoxicological studies. Thus, this work aimed at understanding energy metabolism responses in a sentinel species, Dreissena polymorpha. Mussels were collected during the rest and the reproduction periods and were exposed to 10 μg.L -1 of cadmium (Cd) at two temperatures (in situ temperature and in situ temperature + 5°C) during 7 days. Energy metabolism was monitored by measuring reserves and energy nucleotides charge and by assessing aerobic and anaerobic metabolism markers, and upstream regulation pathways. Markers related to OXPHOS activity revealed seasonal variations under laboratory conditions. Conversely, adenylate nucleotides, glycogen, lipid and transcript levels of AMP-activated protein kinase, citrate synthase, ATP synthase and cytochrome b encoding genes remained steady after the acclimation period. No evident effect of Cd on energy metabolism markers was noticed for both exposures although the transcript level of succinate dehydrogenase and citrate synthase encoding genes decreased with Cd during the rest period. Cellular stress, revealed by lipid peroxidation and catalase mRNA levels, only occurred in Cd and warming co-exposed mussels during the reproduction period. These results suggest that contaminant impact might differ according to the reproduction cycle stage. The effect of confounding factors on biomarker variations should be further investigated to have a deeper knowledge of metabolism responses under laboratory conditions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A highly multiplexed liquid chromatography mass spectrometry-multiple reaction monitoring (MRM)-based assay has been developed for evaluating 107 candidate immune biomarkers in both hemocytes and plasma of the zebra mussel Dreissena polymorpha. The Scout-MRM strategy was employed for the first time, shortening the implementation of a targeted MRM bottom-up proteomics assay using selected immune protein-related peptides identified by shotgun discovery proteogenomics. This strategy relies on spiking scout peptides during the discovery phase and using them to build and deploy the MRM targeted proteomics method. It proved to be highly relevant, since about 90% of the targeted peptides and proteins were monitored and rapidly measured in both hemocyte and plasma samples. The sample preparation protocol was optimized by evaluating the digestion efficiency of tryptic peptides over time. The accuracy and precision of 50 stable isotope-labeled peptides were evaluated for use as internal standards. Finally, the specificity of the transitions was thoroughly assessed to ensure the reliable measurement of protein biomarkers. Several analytical and biological validation criteria were evaluated across hemocytes and plasma samples exposed ex vivo to biological contaminants, resulting in the validation of two Scout-MRM assays for the relative quantitation of 85 and 89 proteins in hemocytes and plasma, respectively. Graphical abstract.

Mechanistic models based on chemical properties of metals and body size have received substantial attention for their potential application to various metals and to different conditions without required calibration. This advantage has been demonstrated for a number of metals, such as Cd and Ag. However, the capacity of metal-specific chemical properties to explain variations in the accumulation for platinum-group elements (PGEs) has not been investigated yet, although emission of these metals is of increasing concern. Once being released, PGEs exist in the environment in mixtures with other metals. The present study attempted to model the accumulation of Pd and Pt in mixtures with Ag and Cd in the zebra mussel (Dreissena polymorpha) from the aqueous phase; and to investigate the potential application of mechanistic models to Pd and Pt. The present study showed statistically insignificant differences in metal accumulation among size groups in a narrow range of shell length (16-22 mm). Kinetic models could simulate well the accumulation of Cd, Ag, and Pt when metal-specific responses of zebra mussels are taken into consideration. These responses include enhanced immobilisation as a detoxifying mechanism and exchange between soft tissues and shells via the extrapallial fluid. Environmental conditions, e.g. the presence of abiotic ligands such as chloride, might also play an important role in metal accumulation. Significant relationships between the absorption efficiency and the covalent index indicate the potential application of mechanistic models based on this chemical property to Pt., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Quagga mussels (Dreissena rostriformis burgensis) are a highly invasive aquatic species to North America, capable of filtering large volumes of water and causing severe ecological and economic impacts. Their range has been expanding since they first invaded the Great Lakes in the 1980s. To predict their spread, it is crucial to understand environmental parameters, which facilitate their range expansion. Two factors likely to influence their distribution include calcium and temperature, because the former is vital for shell development and the latter for metabolic activity. When these factors are optimal for mussels' fitness, the filtration rate has the potential to be maximized if other environmental conditions are also favorable, thus enabling mussels to exploit their growth potential. Deviations from optimal conditions likely result in filtration-rate decline. We identify calcium concentrations and temperatures that maximize the mussel filtration rate for 2 phytoplankton species: Ankistrodesmus facaltus, a common food source for quagga mussels, and a less palatable Microcystis icthyoblabe. In laboratory experiments, filtration rates were measured through cell counts after 24 h of filtration when exposed to a range of temperatures between 2 and 30 °C, and calcium concentrations between 0 and 180 mg/L. Response surface methodology was used to identify a maximum filtration rate, which occurred at 22 mL/mg/h at 137 mg/L of calcium carbonate and 26 °C when fed Ankistrodesmus. To establish a quagga mussel population in a new water source, optimum conditions are required; thus, this information can be used to rank the relative susceptibility of water bodies to invasion by quagga mussels. Environ Toxicol Chem 2020;39:410-418. © 2019 SETAC., (© 2019 SETAC.)

The environmental neurotoxin β-methylamino-l-alanine (BMAA) may represent a risk for human health. BMAA accumulates in freshwater and marine organisms consumed by humans. However, few data are available about the kinetics of BMAA accumulation and detoxification in exposed organisms, as well as the organ distribution and the fractions in which BMAA is present in tissues (free, soluble bound or precipitated bound cellular fractions). Here, we exposed the bivalve mussel Dreissena polymorpha to 7.5 µg of dissolved BMAA/mussel/3 days for 21 days, followed by 21 days of depuration in clear water. At 1, 3, 8, 14 and 21 days of exposure and depuration, the hemolymph and organs (digestive gland, the gills, the mantle, the gonad and muscles/foot) were sampled. Total BMAA as well as free BMAA, soluble bound and precipitated bound BMAA were quantified by tandem mass spectrometry. Free and soluble bound BMAA spread throughout all tissues from the first day of exposure to the last day of depuration, without a specific target organ. However, precipitated bound BMAA was detected only in muscles and foot from the last day of exposure to day 8 of depuration, at a lower concentration compared to free and soluble bound BMAA. In soft tissues (digestive gland, gonad, gills, mantle and muscles/foot), BMAA mostly accumulated as a free molecule and in the soluble bound fraction, with variations occurring between the two fractions among tissues and over time. The results suggest that the assessment of bivalve contamination by BMAA may require the quantification of total BMAA in whole individuals when possible.

This study examined to what degree bioaccumulated pesticides in transplanted zebra mussels can give an insight to pesticide bioavailability in the environment. In addition, it was investigated if pesticide body residues could be related to ecological responses (changes in macroinvertebrate community composition). For this at 17 locations, 14 pesticide concentrations and nine dissolved metals were measured in translocated zebra mussels and the results were related to the structure of the macroinvertebrate community. Critical body burdens in zebra mussel, above which the ecological status was always low, could be estimated for chlorpyrifos, terbuthylazine and dimethoate being respectively 8.0, 2.08 and 2.0 ng/g dry weight. With multivariate analysis, changes in the community structure of the macroinvertebrates were related to accumulated pesticides and dissolved metals. From this analysis, it was clear that the composition of the macroinvertebrate communities was not only affected by pesticides but also by metal pollution. Two different regions could be clearly separated, one dominated by metal pollution, and one where pesticide pollution was more important. The results of this study demonstrated that zebra mussel body burdens can be used to measure pesticide bioavailability and that pesticide body burdens might give insight in the ecological impacts of pesticide contamination. Given the interrelated impacts of pesticides and heavy metals, it is important to further validate all threshold values before they can be used by regulators., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Microplastics (MPs) are now one of the major environmental problems due to the large amount released in aquatic and terrestrial ecosystems, as well as their diffuse sources and potential impacts on organisms and human health. Still the molecular and cellular targets of microplastics' toxicity have not yet been identified and their mechanism of actions in aquatic organisms are largely unknown. In order to partially fill this gap, we used a mass spectrometry based functional proteomics to evaluate the modulation of protein profiling in zebra mussel (Dreissena polymorpha), one of the most useful freshwater biological model. Mussels were exposed for 6 days in static conditions to two different microplastic mixtures, composed by two types of virgin polystyrene microbeads (size = 1 and 10 μm) each one. The mixture at the lowest concentration contained 5 × 105 MP/L of 1 μm and 5 × 105 MP/L of 10 μm, while the higher one was arranged with 2 × 106 MP/L of 1 μm and 2 × 106 MP/L of 10 μm. Proteomics' analyses of gills showed the complete lack of proteins' modulation after the exposure to the low-concentrated mixture, while even 78 proteins were differentially modulated after the exposure to the high-concentrated one, suggesting the presence of an effect-threshold. The modulated proteins belong to 5 different classes mainly involved in the structure and function of ribosomes, energy metabolism, cellular trafficking, RNA-binding and cytoskeleton, all related to the response against the oxidative stress., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Due to the increasing presence of platinum (Pt) in the environment, the caveat arises to identify its toxic potential in species at risk of being exposed - especially those found in aquatic environments where pollutants tend to accumulate. Comprehensive characterisation of possible adverse effects following exposure of aquatic organisms to Pt remains elusive. To address this, Zebra mussels (Dreissena polymorpha) were exposed to a range of Pt(IV) concentrations (0.1, 1, 10, 100 and 1000 μg/L) for one and four days, respectively, after which bioaccumulation was quantified and compared to alterations in biomarker profiles relevant to metal toxicity i.e. glutathione-S-transferase (GST) and catalase (CAT) activity, lipid peroxidation and metallothionein (MT) induction. Despite pre-conditioning of the tanks, Pt recovery in the exposure media was found to be 36% (0.1 μg/L), 42% (1 μg/L), 47% (10 μg/L), 68% (100 μg/L) and 111% (1000 μg/L) due to biological and non-biological processes. Pt concentrations in dried mussel soft tissue increased with exposure concentrations and were 20-153 times higher compared to quantified Pt concentrations in the exposure media. CAT activity was significantly increased in the tissue of mussels exposed to 0.1-100 μg/L Pt after Day 1 while the lowest effect concentration (LOC) for this response on both Day 1 and Day 4 was 0.1 μg/L. The effect on the GST activity was less pronounced but demonstrated a similar trend. However, enhanced lipid peroxidation was measured in the tissue of mussels exposed to ≥0.1 μg/L on Day 4. Bioaccumulation of Pt was also associated with a concentration-dependent increase in Pt-MT. Although these effects occurred at Pt levels higher than those present in the environment, it indicates that Pt has the ability to cause aberrancies in metal-associated biomarker profiles., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biological organisms evolved to take advantage of recurring environmental factors which enabled them to assimilate and process metabolic energy for survival. Mitochondria display non-linear oscillations in NADH levels (i.e. wave behavior) that result from the balance between NADH production (aerobic glycolysis) and oxidation for ATP synthesis. The purpose of this study was to examine the effects of cadmium (Cd) on mitochondrial NADH oscillations in quagga mussels Dreissena bugensis exposed to 50 and 100 μg/L CdCl2 for 7 days at 15 °C. Metallothionein (MT) levels, thioredoxin reductase (TrxR) activity and NADH oxidation rate were also determined, as were oscillations in NADH and the formation of dissipative structures (turbidity), in isolated mitochondria suspensions. The results show that exposure to Cd readily induced MT levels at both concentrations tested and that TrxR and NADH oxidase activity was induced at 100 μg/L Cd only. In control mussels, NADH levels oscillated in mitochondria suspensions with a natural period of 2 to 2.5 min for up to 40 min. Exposure to Cd increased the complexity of the frequency profile of NADH oscillations and reduced the amplitudes of the natural signal with a period of 2 to 2.5 min. The formation of dissipative structures decreased in response to a Cd concentration of 100 μg/L but increased at a level of 50 μg/L. The amplitudes at the natural frequency were significantly correlated with NADH oxidase activity (r = -0.91) and with the formation of dissipative structures (r = -0.59). We conclude that Cd could alter the natural frequency in oscillations of NADH in mitochondria, thereby contributing to an increase in NADH oxidation rate and disruption of the spatial organization of mitochondria in suspension. In conclusion, changes in the wave behavior of NADH in mitochondria are proposed as a novel biomarker of toxicity in aquatic organisms., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

Untreated organic contaminants in municipal wastewater, such as endocrine-disrupting chemicals (EDCs), have become a significant issue in aquatic ecosystems, particularly in freshwater bodies that receive wastewater discharge. This has raised concerns about the accumulation of EDCs in aquatic species via continuous exposure. This study evaluated the uptake of EDCs by quagga mussels (Dreissena bugensis), an invasive species in a water supply reservoir. The field sampling results showed that steroid hormones were not detected in the water samples, and only pharmaceuticals and personal care products were present (0.49 to 36 ng/L). Additionally, testosterone was the most abundant steroid in the mussel tissue (6.3 to 20 ng/g dry weight), and other synthetic chemicals (i.e., bisphenol A, triclosan, and salicylic acid) were also detected in the mussel tissue (24 to 47 ng/g dry weight). After being exposed to exogenous EDCs for 7, 21, and 42 days under controlled laboratory conditions, testosterone was not detected in the mussel anymore, but bisphenol A, triclosan, and salicylic acid were found at relatively high levels in the mussel tissue, although the concentrations did not increase over time. Overall, the study demonstrated the uptake of EDCs in quagga mussels, which suggests that this species can be used to reflect water quality deterioration in aquatic ecosystems.

Samarium (Sm) and yttrium (Y) are commonly used rare earth elements (REEs) but there is a scarcity of information concerning their biological effects in non-target aquatic organisms. The purpose of this study was to determine the bioavailability of those REEs and their toxicity on Dreissena polymorpha after exposure to increasing concentration of Sm and Y for 28 days at 15 °C. At the end of the exposure period, the gene expression of superoxide dismutase (SOD), catalase (CAT), metallothionein (MT), glutathione-S-transferase (GST), cytochrome c oxidase 1 (CO1) and cyclin D (Cyc D) were analysed. In addition, we examined lipid peroxidation (LPO), DNA strand breaks (DSB), GST and prostaglandin cyclooxygenase (COX) activities. Results showed a concentration dependent increase in the level of the REEs accumulated in the soft tissue of mussels. Both REEs decreased CAT but did not significantly modulated SOD and MT expressions. Furthermore, Sm 3+ up-regulated GST, CO1 and Cyc D, while Y 3+ increased and decreased GST and CO1 transcripts levels, respectively. Biomarker activities showed no oxidative damage as evidenced by LPO, while COX activity was decreased and DNA strand breaks levels were changed suggesting that Sm and Y exhibit anti-inflammatory and genotoxic effects. Factorial analysis revealed that the major impacted biomarkers by Sm were LPO, CAT, CO1 and COX, while GST gene expression, COX, Cyc D and CAT as the major biomarkers affected by Y. We conclude that these REEs display different mode of action but further investigations are required in order to define the exact mechanism involved in their toxicity., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

Mussels are among the most frequently used invertebrate animals in aquatic toxicology to detect toxic exposure in the environment. The presence and activity of a cellular defence system, the multixenobiotic resistance (MXR) mechanism, was also established in these organisms. In isolated gill tissues of dreissenid mussels (D. bugensis) the MXR activity was assayed after treatment by commercially available insecticides (formulated products) which contain neonicotinoids as their active ingredients: Actara (thiamethoxam), Apacs (clothianidin), Calypso (thiacloprid) and Kohinor (imidacloprid), respectively. While applying the accumulation assay method, 0.5 μM rhodamine B was used as model substrate and 20 μM verapamil as model inhibitor of the MXR mechanism. In acute (in vitro) experiments when isolated gills were co-incubated in graded concentrations of insecticides and rhodamine B simultaneously, Calypso and Kohinor treatment resulted increasing rhodamine accumulation. Chemical analysis of gills in vitro incubated in insecticides demonstrated higher tissue concentrations of thiamethoxam, clothianidin and thiacloprid in the presence of verapamil suggesting that the active ingredients of Actara, Apacs and Calypso are potential substrates of the MXR mediated cellular efflux. In contrast, verapamil did significantly alter the accumulated imidacloprid concentrations in gills, suggesting that the active component of Kohinor is not transported by the MXR mechanism. Chronic (in vivo) exposures of the intact animals in lower, 1, 10 mg/L concentration of neonicotinoid products, resulted in a decreased level of both rhodamine accumulation and verapamil inhibition by the 12th-14th days of treatment. These results suggest an enhancement of MXR activity (chemostimulation), building up gradually in the animals exposed to Actara, Apacs and Kohinor, respectively. Neonicotinoid-type insecticides are generally considered as selective neurotoxins for insects, targeting the nicotinic type acetylcholine receptors (nAChRs) in their central nervous system. Our present results provide the first evidences that neonicotinoid insecticides are also able to alter the transmembrane transport mechanisms related to the MXR system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Dreissenid mussels Dreissena bugensis (quagga mussel) and Dreissena polymorpha (zebra mussel) are prolific invasive species to the freshwaters of the United States and Western Europe. In the Great Lakes, D. polymorpha has initially dominated the system since its invasion in the mid-1980s; however, recently D. bugensis has displaced D. polymorpha as the dominant species. Dreissena bugensis has several competitive advantages over D. polymorpha, including greater tolerances to deeper and colder waters and lower respiration rates. Nevertheless, physiological differences between the species remain largely unknown. The oxidative stress response is a mechanism used by all organisms to mitigate environmental stress by reducing oxygen radicals in the body, and comparing this mechanism between similar species can be useful for understanding how different species compete in aquatic environments. We compared oxidative stress biomarkers (lipid peroxidation [LPO] and catalase [CAT] activity) in mussels after exposure to 4 stressors (i.e., high densities, temperature, hypoxia, and polychlorinated biphenyls [PCBs]) independently and in combinations of 2 stressors. Overall, D. bugensis had lower LPO and CAT activity than D. polymorpha when exposed to single stressors; however, in multiple stressor treatments D. bugensis had increased LPO, especially with high temperatures and PCBs. The lower lipid damage in D. bugensis compared with D. polymorpha under single stressor conditions may come at the cost of the ability to respond to multiple stressors. Environ Toxicol Chem 2018;37:1643-1654. © 2018 SETAC., (© 2018 SETAC.)

The global dynamic spread of chemical contamination through the aquatic environment calls for the development of biomarkers of interest. Reproduction is a key element to be considered because it is related to the sustainability of species. Spermatogenesis is a complex process that leads to the formation of mature germ cells, whose steps and impairments need to be finely described in ecotoxicological analyses. The physiological process has been commonly described by histological analyses of gonads in different taxa. In the present paper, we describe the development of a novel technique to characterize spermatogenesis based on the analysis of the DNA content of germ cells by flow cytometry, using a DNA-intercalating agent. This new biomarker, referred to as an index of sexual maturity, proved relevant to describe the seasonal reproductive cycle of the zebra mussel, Dreissena polymorpha (Pallas, 1771), used as a sentinel species in the biomonitoring of continental waters and sensitive to highlight the reprotoxicity of carbamazepine (an anti-epileptic pharmaceutical) tested under ecosystemic conditions (mesocosms).

Municipal effluents are major source of pharmaceutical products in the environment. The purpose of this study was to examine the toxicity of a largely used drug, ibuprofen (Ibu), in Dresseina polymorpha mussels and its impact on air survival time. The mussels were exposed to increasing concentration of Ibu (0, 1, 10 and 100μg/L) for 96 at 15°C and a sub-group of mussels was maintain in air for another 96h. Post-exposure mussels (Ibu and Ibu+Air) were analyzed for weight loss, total triglycerides, neutral lipids, lipid peroxidation (LPO), arachidonate-dependent cyclooxygenase (COX) and glutathione S-transferase activity. Lipid extracts of mussel tissues were also analyzed by 1 H-nuclear resonance spectroscopy. The data revealed that mussels exposed to Ibu had increased signs of lipid oxidation, neutral lipids and decreased triglycerides, LPO and GST activity. COX activity was significantly reduced by Ibu in keeping with mode of action of the drug. Following exposure to air, increased weight loss, neutral lipids (lipid degradation), were observed in mussels exposed to Ibu but no changes in COX activity were observed. Air stress limited the decrease in triglycerides and the increase in GST in mussels exposed to 100μg/L Ibu indicating decreased anti-oxidant response/phase II biotransformation and limited lipid metabolism. In conclusion, exposure to Ibu has some anti-inflammatory effects to mussels based on COX activity but resulted in increased oxidative damage and lipid catabolism. Exposure to air stress could enhance some of these responses and contribute to decreased resistance to air exposures., (Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.)

Organic sediments in aquatic ecosystems are well known sinks for nutrients, silicon, and metal(loid)s. Organic matter-consuming organisms like invertebrate shredders, grazers, and bioturbators significantly affect element fixation or remobilization by changing redox conditions or binding properties of organic sediments. Little is known about the effect of filter feeders, like the zebra mussel Dreissena polymorpha, an invasive organism in North American and European freshwater ecosystems. A laboratory batch experiment exposing D. polymorpha (∼1200 organisms per m 2 ) to organic sediment from a site contaminated with arsenic, copper, lead, and uranium revealed a significant uptake and accumulation of arsenic, copper, iron, and especially uranium both into the soft body tissues and the seashell. This is in line with previous observations of metal(loid) accumulation from biomonitoring studies. Regarding its environmental impact, D. polymorpha significantly contributed to mobilization of silicon, iron, phosphorus, arsenic, and copper and to immobilization of uranium (p < 0.001), probably driven by redox conditions, microbial activity within the gut system, or active control of element homeostasis. No net mobilization or immobilization was observed for zinc and lead, because of their low mobility at the prevailing pH of 7.5-8.5. The present results suggest that D. polymorpha can both ameliorate (nutrient mobilization, immobilization of toxicants mobile under oxic conditions) or aggravate negative effects (mobilization of toxicants mobile under reducing conditions) in ecosystems. Relating the results of the present study to observed population densities in natural freshwater ecosystems suggests a significant influence of D. polymorpha on element cycling and needs to be considered in future studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Zebra mussels (Dreissena polymorpha) were exposed to polyvinylpyrrolidone (PVP)-coated silver nanoparticles (AgNP; hydrodynamic diameter 80 nm; solid diameter 50 nm) to investigate the behavior of Ag in the tank water with respect to its uptake, bioaccumulation, elimination and subcellular distribution in the mussel soft tissue. Parallel experiments were performed with ionic Ag (AgNO 3 ) to unravel possible differences between the metal forms. The recovery of the applied Ag concentration (500 μg/L) in the tank water was clearly affected by the metal source (AgNP < AgNO 3 ) and water type (reconstituted water < tap water). Filtration (<0.45 μm) of water samples showed different effects on the quantified metal concentration depending on the water type and Ag form. Ag accumulation in the mussel soft tissue was neither influenced by the metal source nor by the water type. Ag concentrations in the mussel soft tissue did not decrease during 14 days of depuration. For both metal forms the Ag distribution within different subcellular fractions, i.e. metal-rich granules (MRG), cellular debris, organelles, heat-sensitive proteins (HSP) and metallothionein-like proteins (MTLP), revealed time-dependent changes which can be referred to intracellular Ag translocation processes. The results provide clear evidence for the uptake of Ag by the mussel soft tissue in nanoparticulate as well as in ionic form. Thus, zebra mussels could be used as effective accumulation indicators for environmental monitoring of both Ag forms., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Increasing discharge of industrial wastes into the environment results in pollution transfer towards hydrosystems. These activities release heavy metals such as cadmium, known as persistent pollutant that is accumulated by molluscs and exercise immunotoxicological effects. Among molluscs, the zebra mussel, Dreissena polymorpha constitutes a suitable support for freshwater ecotoxicological studies. In molluscs, homeostasis maintain is ensured in part by hemocytes that are composed of several cell populations involved in multiple physiological processes such as cell-mediated immune response or metal metabolism. Thus, hemocytes constitute a target of concern to study adverse effects of heavy metals. The objectives of this work were to determine whether immune-related endpoints assessed were of different sensitivity to cadmium and whether hemocyte functionalities were differentially affected depending on hemocyte subpopulation considered. Hemocytes were exposed ex vivo to concentrations of cadmium ranging from 10 -6 M to 10 -3 M for 21h prior flow cytometric analysis of cellular markers. Measured parameters (viability, phagocytosis, oxidative activity, lysosomal content) decreased in a dose-dependent manner with sensitivity differences depending on endpoint and cell type considered. Our results indicated that phagocytosis related endpoints were the most sensitive studied mechanisms to cadmium compared to other markers with EC 50 of 3.71±0.53×10 -4 M for phagocytic activity and 2.79±0.19×10 -4 M considering mean number of beads per phagocytic cell. Lysosomal content of granulocytes was less affected compared to other cell types, indicating lower sensitivity to cadmium. This suggests that granulocyte population is greatly involved in metal metabolism. Mitochondrial activity was reduced only in blast-like hemocytes that are considered to be cell precursors. Impairment of these cell functionalities may potentially compromise functions ensured by differentiated cells. We concluded that analysis of hemocyte activities should be performed at sub-population scale for more accurate results in ecotoxicological studies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cannabis is the most used illicit drug worldwide and in some countries a new regulatory policy makes it legal under some restrictions. This situation could lead to a substantial increase in environmental levels of the cannabis active principle (Δ-9-tetrahydrocannabinol [Δ-9-THC]) and its main metabolite, 11-nor-9-carboxy-Δ 9 -tetrahydrocannabinol (THC-COOH). Although previous studies have highlighted the toxicity of Δ-9-THC, the adverse effects of THC-COOH on aquatic organisms is completely unknown, even though such effects could be more significant because the environmental concentrations of THC-COOH are higher than those of the parent compound. The present study aimed to assess oxidative and genetic damage to the zebra mussel (Dreissena polymorpha) because of 14-d exposures to 3 THC-COOH concentrations, mimicking a current environmental situation (100 ng/L), as well as exposure to 2 possible worst-case scenarios (500 ng/L and 1000 ng/L), because of the potential increase in THC-COOH in surface waters. Variations in the activity of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione S-transferase (GST) were measured, as well as levels of lipid peroxidation and protein carbonyl content. Genetic injuries were investigated by single-cell gel electrophoresis assay, DNA diffusion assay, and the micronucleus test. A significant imbalance in antioxidant defense enzymes was noted in response to the 3 tested concentrations, whereas oxidative damage was noted only at the higher one. Moreover, an increase in DNA fragmentation in zebra mussel hemocytes, but no fixed genetic damage, was found. Although the results showed that THC-COOH toxicity was lower than that of Δ-9-THC, the increase in cannabis use might increase its levels in freshwaters, enhancing its hazard to bivalves and likely to the whole aquatic community. Environ Toxicol Chem 2017;36:472-479. © 2016 SETAC., (© 2016 SETAC.)

Understanding body size-dependent metal accumulation in aquatic organisms (i.e., metal allometry) is critical in interpreting biomonitoring data. While growth has received the most attention, little is known about controls of metal exposure routes on metal allometry. Here, size-dependent Cd accumulation in zebra mussels (Dreissena polymorpha) from different routes were investigated by exposing mussels to A.( 111 Cd spiked algae+ 113 Cd spiked river water) or B.( 111 Cd spiked sediments+ 113 Cd spiked river water). After exposure, 111 Cd or 113 Cd levels in mussel tissue were found to be negatively correlated with tissue weight, while Cd allometry coefficients (b values) were dependent on Cd exposure routes: -0.664 for algae, -0.241 for sediments and -0.379 for river water, compared to -0.582 in un-exposed mussels. By comparing different Cd exposure routes, we found that size-dependent Cd bioaccumulation from algae or river water could be more responsible for the overall size-dependent Cd accumulation in mussels, and the relative importance of the two sources was dependent on mussel size ranges: Cadmium obtained from algae (algae-Cd) was more important in size-dependent Cd accumulation in smaller mussels (tissue dry weight < 5 mg), while river water-Cd became more important in larger individuals (tissue dry weight > 5 mg). In contrast, sediment-Cd contributed only a small amount to Cd accumulation in zebra mussels and may have little effect on size-dependent Cd bioaccumulation. Our results suggest that size-dependent Cd accumulation in mussels could be largely affected by exposure routes, which should be considered when trying to interpret Cd biomonitoring data of zebra mussels., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

With current global changes, the combination of several stressors such as temperature and contaminants may impact species distribution and ecosystem functioning. In this study, we evaluated the combined impact of two metals (Ni and Cr) with a thermal stress (from 12 to 17 °C) on biomarker responses in two bivalves, Dreissena rostriformis bugensis and Dreissena polymorpha. Biomarkers are informative tools to evaluate exposure and effects of stressors on organisms. The set of 14 biomarkers measured here was representative of both physiologic (filtration activity) and cellular antioxidant and detoxification mechanisms. Our aim was to study the response pattern of both species, and its meaning in terms of invasive potential. The implications for the use of these mussels in environmental monitoring are also discussed. Results evidenced that the two species do not respond to multiple stressors in the same way. Indeed, the effects of contamination on biomarker responses were more marked for D. polymorpha, especially under nickel exposure. While we cannot conclude as to the effect of temperature, invasiveness could be influenced by species sensitivity to contaminants. The physiological and cellular differences between D. polymorpha and D. r. bugensis might also be of concern for environmental risk assessment. The two species present differential bioaccumulation patterns, filtration activity and cellular biomarker responses. If D. polymorpha populations decline, their substitution by D. r. bugensis for biomonitoring or laboratory studies will not be possible without a deeper understanding of biomarker responses of the new invasive., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Exposures in realistic environmental conditions are essential to properly assess the effects of emerging pollutants on ecosystems. While ceria nanoparticles (nCeO2) production and use are expanding quickly, ecotoxicity studies remain very scarce. In this study, we set up experimental systems reproducing a simplified ecosystem to assess the effects of a chronic exposure to citrate-coated nCeO2 (ci-CeO2) and bare nCeO2 (ba-CeO2) on the freshwater mussel Dreissena polymorpha using an integrated multibiomarker approach. The fate of nanoparticles was tightly monitored to properly characterize the exposure. Organisms were exposed for 3 weeks and sampled weekly for biomarker analysis. Mussel filter-feeding activity resulted in significant removal of nCeO2 from the water column. At the same time, bioaccumulation was low, reaching its maximum in the first week. Mussels bioaccumulated ci-CeO2 three times more than ba-CeO2, probably due to coating-related differences in their behavior in the water column and in organisms. Meanwhile, biomarker results were integrated and synthesized using linear discriminant analysis, highlighting that pi-glutathione-S-transferase (piGST) mRNA, catalase (CAT) activity and lysosomal system were the most impacted of the seven biomarkers singled out by the discriminant analysis. These biomarker responses indicated that mussels exposed to both forms of nCeO2 were stressed and differentiate from the controls. Moreover, they responded differently to ba-CeO2 and ci-CeO2 exposure. However, biomarkers used in the experimental conditions of this study did not indicate severe nCeO2 toxicity on mussels, as cellular damage biomarkers and mussel filtering activity were left unimpaired. However, further studies are needed to investigate if the slight perturbations observed could lead to populational impacts in the long term.

The size-related differences in subcellular biomarker responses were assessed in Dreissena bugensis mussels inhabiting harbours moderately affected by pollution with complex mixtures of heavy metals and polycyclic aromatic hydrocarbons (PAHs). Adult D. bugensis samples were collected from three harbours of Lake Balaton (Hungary) characterized by moderate shipping activity, and as reference site, from a highly protected remote area of the lake. Biomarkers of exposure (metallothioneins (MTs), ethoxyresorufin-o-deethylase (EROD)), oxidative stress (lipid peroxidation (LPO), DNA strand breaks (DNAsb)) and possible endocrine disruption (vitellogenin-like proteins (VTG)) were analysed in whole-tissue homogenates of differently sized groups of mussels in relation to environmental parameters and priority pollutants (heavy metals and polycyclic aromatic hydrocarbons). Integrated biomarker response (IBR) indices were calculated for biomarker responses gained through in situ measurements to signalize critical sites and to better distinguish natural tendencies from biological effects of contaminants. Biomarker responses showed close positive correlation in case of MT, EROD, LPO, and DNAsb and negative correlation with VTG levels with mussel shell length in autumn, when higher levels of biomarkers appeared, possibly due to natural lifecycle changes of animals.

Aquatic organisms, such as bivalves, employ ATP binding cassette (ABC) transporters for efflux of potentially toxic chemicals. Anthropogenic water contaminants can, as chemosensitizers, disrupt efflux transporter function enabling other, putatively toxic compounds to enter the organism. Applying rapid amplification of cDNA ends (RACE) PCR we identified complete cDNAs encoding ABCB1- and ABCC1-type transporter homologs from zebra mussel providing the molecular basis for expression of both transporter types in zebra mussel gills. Further, efflux activities of both transporter types in gills were indicated with dye accumulation assays where efflux of the dye calcein-am was sensitive to both ABCB1- (reversin 205, verapamil) and ABCC1- (MK571) type specific inhibitors. The assumption that different inhibitors targeted different efflux pump types was confirmed when comparing measured effects of binary inhibitor compound mixtures in dye accumulation assays with predictions from mixture effect models. Effects by the MK571/reversin 205 mixture corresponded better with independent action, whereas reversin 205/verapamil joint effects were better predicted by the concentration addition model indicating different and equal targets, respectively. The binary mixture approach was further applied to identify the efflux pump type targeted by environmentally relevant chemosensitizing compounds. Pentachlorophenol and musk ketone, which were selected after a pre-screen of twelve compounds that previously had been identified as chemosensitizers, showed mixture effects that corresponded better with concentration addition when combined with reversine 205 but with independent action predictions when combined with MK571 indicating targeting of an ABCB1-type efflux pump by these compounds., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Sanitation is of crucial importance for the microbiological safety of drinking water. However, chlorination of water rich in organic material produces disinfection by-products (DBPs), many of which have been reported to be mutagenic and/or carcinogenic compounds such as haloacetic acids and trihalomethanes. Epidemiological studies have suggested a link between drinking water consumption and cancer. We previously observed that Cyprinus carpio fish exposed to DBPs, may be subject to epigenetic effects such as those referable to the up-regulation of cytochrome P450 (CYP) superfamily (ex. co-mutagenesis/co-carcinogenesis and oxidative stress) that has been associated to non-genotoxic carcinogenesis. Our goal was to study the xenobiotic metabolism in mollusks exposed in situ to surface water of Lake Trasimene (Central Italy) treated with several disinfectants such as the traditional chlorine dioxide (ClO2), sodium hypochlorite (NaClO) or the relatively new one peracetic acid (PAA). The freshwater bivalves (Dreissena polymorpha) being selected as biomarker, have the unique ability to accumulate pollutants. Freshwater bivalves were maintained in surface water containing each disinfectant individually (1-2 mg/L). Following an exposure period up to 20 days during the fall period, microsomes were collected from the mussels, then tested for various monooxygenases. Strong CYP inductions were observed. These data indicate that drinking water disinfection generates harmful DBP mixtures capable of determining a marked perturbation of CYP-supported reactions. This phenomenon, being associated to an increased pro-carcinogen bioactivation and persistent oxidative stress, could provide an explanation for the observational studies connecting the regular consumption of drinking water to increased risk of various cancers in humans., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Illicit drugs have been recognized as emerging aquatic pollutants due to their presence in aquatic ecosystems up to µg/L level. Among these, the synthetic psycho-stimulant drug amphetamine (AMPH) is commonly found in both surface and wastewaters worldwide. Even though the environmental occurrence of AMPH is well-known, the information on its toxicity towards non-target freshwater organisms is completely lacking. This study investigated the imbalance of the oxidative status and both oxidative and genetic damage induced by a 14-day exposure to two concentrations (500 ng/L and 5000 ng/L) of AMPH on the freshwater bivalve Dreissena polymorpha by the application of a biomarker suite. We investigated the activity of antioxidant enzymes (SOD, CAT and GPx), the phase II detoxifying enzyme GST, the lipid peroxidation level (LPO) and protein carbonyl content (PCC), as well as primary (Single Cell Gel Electrophoresis assay) and fixed (DNA diffusion assay and Micronucleus test) genetic damage. Our results showed that a current realistic AMPH concentration (500 ng/L) did neither cause notable imbalances in enzymatic activities, nor oxidative and genetic damage to cellular macromolecules. In contrast, the bell-shaped trend of antioxidants showed at the highest tested concentration (5000 ng/L) suggested an overproduction of reactive oxygen species, leading to oxidative damage, as confirmed by the significant increase of protein carbonylation and DNA fragmentation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The invasive freshwater mollusc Dreissena bugensis (quagga mussel) sticks to underwater surfaces via a proteinacious 'anchor' (byssus), consisting of a series of threads linked to adhesive plaques. This adhesion results in the biofouling of crucial underwater industry infrastructure, yet little is known about the proteins responsible for the adhesion. Here the identification of byssal proteins extracted from freshly secreted byssal material is described. Several new byssal proteins were observed by gel electrophoresis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to characterize proteins in different regions of the byssus, particularly those localized to the adhesive interface. Byssal plaques and threads contain in common a range of low molecular weight proteins, while several proteins with higher mass were observed only in the plaque. At the adhesive interface, a plaque-specific ~8.1 kDa protein had a relative increase in signal intensity compared to the bulk of the plaque, suggesting it may play a direct role in adhesion.

Opioids are considered as emerging contaminants in aquatic ecosystems, mainly due to their large illicit consume worldwide. Morphine (MOR) is the main opiate and it was commonly found at measurable concentrations in freshwaters. Even though its occurrence is well documented, just limited information is available regarding its hazard to nontarget organisms. The aim of this study was of the evaluation of sublethal effects induced by MOR to the freshwater bivalve Dreissena polymorpha. We exposed mussels to two MOR concentrations (0.05 µg/L and 0.5 µg/L) for 14 days and we investigated the sublethal effects by a suite of biomarkers. The Neutral Red Retention Assay (NRRA) was used as a test of cytotoxicity, while the oxidative stress was evaluated by the activity of antioxidant and detoxifying enzymes, namely catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and by measuring the levels of lipid peroxidation (LPO) and protein carbonylation (PCC). The genetic damage was assessed by the Single Cell Gel Electrophoresis (SCGE) assay, the DNA diffusion assay and the micronucleus test (MN test). Finally, the filtration rate of D. polymorpha was evaluated in order to investigate possible physiological effects. Both tested concentrations reduced the lysosome membrane stability of bivalves, but only the highest MOR concentration induced significant changes in the activity of antioxidant enzymes (SOD, CAT, and GPx) and increase in lipid peroxidation levels. Slight increase in primary DNA fragmentation was noticed, while no fixed genetic damage and alterations of the filtering rate were found., (© 2014 Wiley Periodicals, Inc.)

We carried out a project aimed to evaluate the possible role played by the freshwater zebra mussel (Dreissena polymorpha) in the possible decrease of some environmental pollutants recalcitrant to tradition wastewater treatments. By the help of a pilot-plant built in the largest wastewater treatment plant of Milan (Italy), we tested several waste mixtures in order to measure the chemicals' abatement made by mussels' biofiltration. This study represents the last step of the wider project and it aimed to evaluate if the decrease in the concentration of some urban pollutants measured in wastewaters was followed by a corresponding toxicity reduction. Thus, we performed 7-day exposures under laboratory conditions to test the toxicity of the raw wastewaters and those preliminary filtered by zebra mussels, through the measurement of different end-points of acute and chronic toxicity. Results showed a clear positive effect of mussels' biofiltration mainly to decrease the acute toxicity made by the two tested wastewater mixtures, while the biomarkers' suite used to evaluate the chronic toxicity showed contradictory results., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Freshwater bivalve molluscs are considered as effective indicators of environmental pollution. The comet assay allows the detection of DNA damage such as DNA strand breaks and alkali-labile sites. The main oxidative lesion, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), which is a pre-mutagenic lesion, can be detected by the comet assay coupled with the hOGG1 DNA repair enzyme. With this modified assay we recently observed that BaP induced 8-oxodG lesions and with the modified comet-Fpg assay we observed that Cd induced oxidative DNA damage. The aim of this study was to determine the stability of DNA lesions in Cd and BaP exposed zebra mussels using the comet-hOGG1 assay. Mussels were exposed for 24 h to these two chemicals and then placed in clean water for 6 days. We observed that BaP (7, 12 and 18 µg/L) induced an increase of DNA strand break levels as soon as 6 h of exposure and that the two highest concentrations of BaP induced a low level of hOGG1-sensitive sites. After 2 days of depuration, BaP induced DNA lesions returned to the basal level, indicating an effective DNA repair. Cd (3, 32 and 81 µg/L) induced an increase of the DNA strand break levels and a low level of hOGG1-sensitive sites. This study revealed that BaP-induced DNA lesions are repaired more efficiently than Cd-induced DNA lesions. As the level of hOGG1 sensitive sites was increased in Cd and BaP exposed mussels, it seems that these chemicals induce 8-oxo-dG.

Unprecedented food chain links between benthic and pelagic organisms are often thought to disrupt traditional contaminant transport and uptake due to changes in predation and mobilization of otherwise sequestered pollutants. A bioaccumulation model for polybrominated diphenyl ethers (PBDEs) is developed to simulate increases in biotic congener loads based upon trophic transfer through diet and gill uptake for a Lake Erie food chain including two invasive species as a benthic-pelagic link. The model utilizes species-specific bioenergetic parameters in a four-level food chain including the green alga Scenedesmus quadricauda, zebra mussels (Dreissena polymorpha), round goby (Appollonia melanostoma), and the smallmouth bass (Micropterus dolomiu). The model was calibrated to current biotic concentrations and predicts an increase in contaminant load by almost 48% in the upper trophic level in two years. Validation to archival data resulted in <2% error from reported values following a two-year simulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The transcriptional response of adult zebra mussels (Dreissena polymorpha) to heavy metals (mercury, copper, and cadmium) was analyzed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) to study the coordinated regulation of different metal-, oxidative stress- and xenobiotic defence-related genes in gills and digestive gland. Regulatory network analyses allowed the comparison of this response between different species and taxa., Results: Chemometric analyses allowed identifying the effects of these metals clearly separating control and treated samples of both tissues. Interactions between the different genes, either in the same or between both tissues, were analysed to identify correlations and to propose stress-related genes' regulatory networks. These networks were finally compared with existing data from human, mouse, zebrafish, Drosophila and the roundworm to evaluate their mechanistically-known response to metals (and to stressors in general) with the correlations observed in the still poorly-known, invasive zebra mussel., Conclusions: Our analyses found a general conservation of regulation genes and of their interactions among the different considered species, and may serve as a guide to extrapolate regulatory data from model species to lesser-known environmentally (or medically) relevant species.

The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Synthetic musk compounds (SMCs) are extensively used as fragrances in several personal care products and have been recognized as emerging aquatic pollutants. Among SMCs, galaxolide (HHCB) and tonalide (AHTN) are extensively used and have been measured in aquatic ecosystems worldwide. However, their potential risk to organisms remains largely unknown. The aim of this study was to investigate whether 21-day exposures to HHCB and AHTN concentrations frequently measured in aquatic ecosystems can induce oxidative and genetic damage in Dreissena polymorpha. The lipid peroxidation (LPO) and protein carbonyl content (PCC) were measured as oxidative stress indexes, while the DNA precipitation assay and the micronucleus test (MN test) were applied to investigate genetic injuries. HHCB induced significant increases in LPO and PCC levels, while AHTN enhanced only protein carbonylation. Moreover, significant increases in DNA strand breaks were caused by exposure to the highest concentrations of HHCB and AHTN tested in the present study, but no fixed genetic damage was observed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The ecotoxicity of nanoparticles (NPs) is a growing area of research with many challenges ahead. To be relevant, laboratory experiments must be performed with well-controlled and environmentally realistic (i.e., low) exposure doses. Moreover, when focusing on the intensively manufactured titanium dioxide (TiO2) NPs, sample preparations and chemical analysis are critical steps to meaningfully assay NP's bioaccumulation. To deal with these imperatives, we synthesized for the first time TiO2 NPs labeled with the stable isotope (47)Ti. Thanks to the (47)Ti labeling, we could detect the bioaccumulation of NPs in zebra mussels (Dreissena polymorpha) exposed for 1 h at environmental concentrations via water (7-120 μg/L of (47)TiO2 NPs) and via their food (4-830 μg/L of (47)TiO2 NPs mixed with 1 × 10(6) cells/mL of cyanobacteria) despite the high natural Ti background, which varied in individual mussels. The assimilation efficiency (AE) of TiO2 NPs by mussels from their diet was very low (AE = 3.0 ± 2.7%) suggesting that NPs are mainly captured in mussel gut, with little penetration in their internal organs. Thus, our methodology is particularly relevant in predicting NP's bioaccumulation and investigating the factors influencing their toxicokinetics in conditions mimicking real environments.

The invasive zebra mussel (Dreissena polymorpha) has become an accepted biomonitor organism for trace elements, but it has yet to be studied along the Lake Michigan shoreline. Likewise, the relationships between tissue concentrations of elements, organism size, and sediment concentrations of elements have not been fully explained. The present study found that a variety of allometric variables such as length, dry tissue mass, shell mass, organism condition indices, and shell thickness index were useful in explaining intrasite variability in elemental concentrations. The flesh condition index (grams of tissue dry mass per gram of shell mass) explained variability at the most sites for most elements. Once allometric intrasite variability was taken into account, additional significant differences were found between sites, although the net effect was small. Significant positive relationships between sediment and tissue concentrations were found for Pb and Zn, with a significant negative relationship for Cd. It was also found that Cu and Zn concentrations in tissues increased significantly along the shoreline in the southeasterly direction, whereas Hg increased in a northwesterly direction. Opportunistic sampling found that zebra mussels accumulate significantly higher concentrations of nearly all elements analyzed compared to Asian clams (Corbicula fluminea) at the same site. The present study demonstrates the need to fully explain natural sources of variability before using biomonitors to explain spatial distributions of trace elements., (© 2014 SETAC.)

Cerium nanoparticles (nCeO2) are widely used in everyday products, as fuel and paint additives. Meanwhile, very few studies on nCeO2 sublethal effects on aquatic organisms are available. We tried to fill this knowledge gap by investigating short-term effects of nCeO2 at environmentally realistic concentrations on two freshwater invertebrates; the amphipod Gammarus roeseli and the bivalve Dreissena polymorpha, using an integrated multibiomarker approach to detect early adverse effects of nCeO2 on organism biology. Differences in the behaviour of the organisms and of nanoparticles in the water column led to differential nCeO2 bioaccumulations, G. roeseli accumulating more cerium than D. polymorpha. Exposure to nCeO2 led to decreases in the size of the lysosomal system, catalase activity and lipoperoxidation in mussel digestive glands that could result from nCeO2 antioxidant properties, but also negatively impacted haemolymph ion concentrations. At the same time, no strong adverse effects of nCeO2 could be observed on G. roeseli. Further experiments will be necessary to confirm the absence of severe nCeO2 adverse effects in long-term environmentally realistic conditions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 μM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 μM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 μM. Surprisingly, at the higher concentration (500 μM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 μM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha., (© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In order to provide reliable information about responsiveness of biomarkers during environmental monitoring, there is a need to improve the understanding of inter-population differences. The present study focused on eight populations of zebra mussels and aimed to describe how variable are biomarkers in different sampling locations. Biomarkers were investigated and summarised through the Integrated Biomarker Response (IBR index). Inter-site differences in IBR index were analysed through comparisons with morphological data, proteomic profiles and genetic background of the studied populations. We found that the IBR index was a good tool to inform about the status of sites. It revealed higher stress in more polluted sites than in cleaner ones. It was neither correlated to proteomic profiles nor to genetic background, suggesting a stronger influence of environment than genes. Meanwhile, morphological traits were related to both environment and genetic background influence. Together these results attest the benefit of using biological tools to better illustrate the status of a population and highlight the need of consider inter-population difference in their baselines., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The relationship between the reproductive stage, the total lipid content and eight broadly used biochemical stress responses were used to assess seasonal and pollutant effects across eleven different zebra mussel (Dreissena polymorpha) populations from the Ebro and Mijares river basin, Spain. Biochemical markers included superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione S transferase (GST), multixenobiotic transporter activity (MXR), lactate dehydrogenase (LDH), lipid peroxidation (LPO) and single strand DNA breaks. Principal component analyses of zebra mussel responses across an annual cycle, showed a marked gonad stage component in total lipid content and biochemical responses. The same response pattern was observed across the populations sampled along a broad geographical and pollution gradient. Population differences on the gonad developmental stage were highly correlated with most of the measured responses and unrelated with the pollution gradient. Conversely, bioaccumulation of organic and inorganic contaminant residues was more related to pollution sources than with the reproductive cycle. These results indicate that the reproductive cycle is the major factor affecting the temporal and spatial variation of the studied markers in D. polymorpha., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The freshwater zebra mussel, Dreissena polymorpha, is an invasive, biofouling species that adheres to a variety of substrates underwater, using a proteinaceous anchor called the byssus. The byssus consists of a number of threads with adhesive plaques at the tips. It contains the unusual amino acid 3, 4-dihydroxyphenylalanine (DOPA), which is believed to play an important role in adhesion, in addition to providing structural integrity to the byssus through cross-linking. Extensive DOPA cross-linking, however, renders the zebra mussel byssus highly resistant to protein extraction, and therefore limits byssal protein identification. We report here on the identification of seven novel byssal proteins in the insoluble byssal matrix following protein extraction from induced, freshly secreted byssal threads with minimal cross-linking. These proteins were identified by LC-MS/MS analysis of tryptic digests of the matrix proteins by spectrum matching against a zebra mussel cDNA library of genes unique to the mussel foot, the organ that secretes the byssus. All seven proteins were present in both the plaque and thread. Comparisons of the protein sequences revealed common features of zebra mussel byssal proteins, and several recurring sequence motifs. Although their sequences are unique, many of the proteins display similarities to marine mussel byssal proteins, as well as to adhesive and structural proteins from other species. The large expansion of the byssal proteome reported here represents an important step towards understanding zebra mussel adhesion.

PCBs are a persistent environmental problem due to their high stability and lipophilicity. The non-ortho- and the mono-ortho-substituted PCBs (dioxin-like-PCBs) share a common and well-described toxicity mechanism in vertebrates, initially involving binding to cytosolic AhRs. Invertebrate AhRs, however, show a lack of dioxin binding, and little information is available regarding the mechanism of toxicity of dl-PCBs in invertebrates. In this study, a proteomic approach was applied to analyse the variations in the pattern of the gill proteome of the freshwater mussel Dreissena polymorpha. Mussels were exposed to a mixture of dl-PCBs, and to perform a more in-depth evaluation, we chose to investigate the role of gender in the proteome response by analysing male and female mussels separately. The results revealed significant modulation of the gill tissue proteome: glycolysis and Ca(2+) homeostasis appear to be the main pathways targeted by dl-PCBs. In light of the differences between the male and female gill proteome profiles following exposure to dl-PCBs, further in-depth investigations of the role of gender in the protein expression profiles of a selected biological model are required., (© 2013.)

The growing need to evaluate the quality of aquatic ecosystems led to the development of numerous monitoring tools. Among them, the development of biomarker-based procedures, that combine precocity and relevance, is recommended. However, multi-biomarker approaches are often hard to interpret, and produce results that are not easy to integrate in the environmental policies framework. Integrative index have been developed, and one of the most used is the integrated biomarker response (IBR). However, an analysis of available literature demonstrated that the IBR suffers from a frequent misuse and a bias in its calculation. Then, we propose here a new calculation method based on both a more simple formula and a permutation procedure. Together, these improvements should rightly avoid the misuse and bias that were recorded. Additionally, a case study illustrates how the new procedure enabled to perform a reliable classification of site along a pollution gradient based on biomarker responses used in the IBR calculations.

Because of the reduction in the use of polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD), novel brominated flame retardants (NBFRs), including 1,2-bis(2,4,6-tribromophenoxy) ethane (BTBPE), decabromodiphenyl ethane (DBDPE), hexabromobenzene (HBB), and pentabromoethylbenzene (PBEB), started to be marketed as alternatives to the banned formulations. In this study, the spatial distribution and accumulation of NBFRs, PBDEs, and HBCD in the biota have been investigated in the littoral compartment of a large and deep subalpine lake (Lake Maggiore, Northern Italy), using zebra mussel Dreissena polymorpha and roach (Rutilus rutilus) as bioindicators. To our knowledge, this is the first study reporting the contamination of NBFRs in the freshwater invertebrate D. polymorpha. Contamination of zebra mussel due to PBEB, HBB, and BTBPE was low, ranging from 0.9 to 2.9 ng/g lipid weight, from 1.1 to 2.9 ng/g l.w., and from 3.5 to 9.5 ng/g l.w., respectively. PBEB and BTBPE in roach were always below the detection limit, while the contamination of HBB ranged from < limits of detection (LOD) to 1.74 ng/g l.w., indicating a weak contamination. DBDPE was < LOD in all the considered biological samples. Finally, HBCD was detected in all organic tissues with mean concentrations up to 74.4 ng/g l.w. PBDE results, supported by principal component analysis elaboration, suggested a possible contamination due to the congeners composing the penta- and deca-BDE technical formulations, which are present in the Lake Maggiore basin. The biomagnification factor values showed that tetra- and penta-BDE biomagnified, while octa-, nona-, and deca-BDE were still bioavailable and detectable in the fish muscles, but they do not biomagnified. Considering the other BFRs, only HBCD showed a moderate biomagnification potential.

Size of organisms is critical in controlling metal bioavailability and bioaccumulation, while mechanisms of size-related metal bioaccumulation are not fully understood. To investigate the influences of different sources of particle-associated Cu on body size-related Cu bioavailability and bioaccumulation, zebra mussels (Dreissena polymorpha) of different sizes were exposed to stable Cu isotope ((65)Cu) spiked algae (Chlorella vulgaris) or sediments in the laboratory and the Cu tissue concentration-size relationships were compared with that in unexposed mussels. Copper tissue concentrations decreased with mussel size (tissue or shell dry weight) in both unexposed and algal-exposed mussels with similar decreasing patterns, but were independent of size in sediment-exposed mussels. Furthermore, the relative contribution of Cu uptake from algae (65-91%) to Cu bioaccumulation is always higher than that from sediments (9-35%), possibly due to the higher bioavailability of algal-Cu. Therefore, the size-related ingestion of algae could be more important in influencing the size-related variations in Cu bioaccumulation. However, the relative contribution of sediment-Cu to Cu bioaccumulation increased with body size and thus sediment ingestion may also affect the size-related Cu variations in larger mussels (tissue weight >7.5mg). This study highlights the importance of considering exposure pathways in normalization of metal concentration variation when using bivalves as biomonitors., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.)