The cerium oxide nanoparticles toxicity induced physiological, histological and biochemical alterations in freshwater mussels, Unio crassus.

INTRODUCTION: Releasing of cerium oxide nanoparticles (nano-CeO2) to the nature has increased due to the widespread use in many fields ranging from cosmetics to the food industry. Therefore, nano-CeO2 has been included in the Organization for Economic Co-operation and Development's (OECD) priority list for engineering nanomaterials. In this study, the effects of nano-CeO2 on the freshwater mussels were investigated to reveal the impact on the freshwater systems on model organism. METHODS: First, the chemical and structural properties of nano-CeO2 were characterized in details. Second, the freshwater mussels were exposed to environmentally relevant concentrations of nano-CeO2 as 10 mg, 25 mg and 50 mg/L during 48-h and 7-d. Third, after the exposure periods, hemolymph and tissue samples were taken to analyse the Total Hemocyte Counts (THCs) histology and oxidative stress parameters (total antioxidant status, glutathione, glutathione-S-transferase, and advanced oxidative protein products). RESULTS: Significant decrease of the THCs was observed in the nano-CeO2 exposed mussels compared to the control group (P < 0.05). The histological results showed a positive association between nano-CeO2 exposure concentration in the water and level of tissue damage and histopathological alterations were detected in the gill and the digestive gland tissues. Oxidative stress parameters were slightly affected after exposure to nano-CeO2 (P > 0.05). In conclusion, this study showed that acute exposure of freshwater mussels to nano-CeO2 did not pose significant biological risk. However, it has been proven that mussels are able to accumulate nano-CeO2 significantly in their bodies. CONCLUSION: This suggests that nano-CeO2 may be a potential risk to other organisms in the ecosystem through trophic transfer in the food-web based on their habitat and niche in the ecosystem.

Combined effect of microplastic, salinomycin and heating on Unio tumidus.

Microplastic (MP) and heating (T) suspected to modulate biological effects of aquatic contaminants. Salinomycin (Sal) is veterinary antibiotic and anticancer agent. The goal of this study was to examine the multistress effect of MP, Sal and T on the bioindicator bivalve mollusc. The Unio tumidus were treated with MP (1 mg L-1), Sal (0.6 µg L-1), their combination under 18° C (Mix) and 25° C (MixT) for 14 days. The digestive glands were analyzed. MP and Sal did not cause changes of Mn- and Cu,Zn-SOD, lipid peroxidation and Cyp-450-depended EROD levels, whereas catalase, GST and protein carbonyls (Sal-group) increased compared to control. In the Mix-group, enzymes, particularly EROD and GST (by 34% and 115% respectively) were up-regulated. However, in the MixT-group, they were corresponding to control or lesser (EROD, catalase). Our findings emphasize the need to take into account multistress interactions in the MP environmental risk assessment.

Stress responses of bivalve mollusc Unio tumidus from two areas to ibuprofen, microplastic and their mixture.

Even though bivalve molluscs are recognized as bioindicators of freshwater quality, their responses to multiple stressors are unpredictable. This study aims to elucidate the inter-population peculiarities of the effect in the sub-chronic environmentally relevant exposure to novel contaminants. The specimens of Unio tumidus from reference (Pr) and contaminated (Ct) areas were treated with ibuprofen (IBU, 0.8 µg L-1), microplastic (MP, 1.0 mg L-1, size 0.1-0.5 mm), or their combination (Mix) for 14 days. Untreated mussels (PrC- and CtC-groups) served as controls. The PrC-group had higher levels of antioxidants Mn-SOD, Cu,Zn-SOD, catalase, and cholinesterase (AChE) as well as lesser levels of oxidative lesions (TBARS and protein carbonyls) in digestive glands, indicating lower environmental impact than in the CtC-group. However, lysosomal stability was similar in both control groups. Among antioxidants, Mn-SOD activity was affected most prominently, increasing in all exposed Ct-groups. TBARS level was increased only in PrMP-group compared to responsive control. IBU and Mix enhanced protein carbonyl concentration in the Pr-groups, and decreased it in the Ct-groups. AChE was induced in the CtIBU- and PrMix-groups, and lysosomal integrity increased in the CtIBU and CtMix-groups. Discriminant analyses indicated lesser differences between Pr-groups, demonstrating lower cumulative stress compared to Ct-groups. Generally, the most remarkable response was revealed in the CtIBU-group, and distortion of individual effects was established in combined exposures. The qualification of stress-neutral and stress-positive populations was proposed for Pr- and Ct-populations correspondingly. Inter-site peculiarities must be taken into consideration when the environmental impact of MP and pharmaceuticals is evaluated.

Indication of the impact of environmental stress on the responses of the bivalve mollusk Unio tumidus to ibuprofen and microplastics based on biomarkers of reductive stress and apoptosis.

The vulnerability of bivalve mollusks to micropollutants is estimated mainly in single model exposures. However, chronic environmental stress and complex exposures can modulate their responses. To evaluate the impact of population-dependent adaptations on the ability to react to common micropollutants, we compared freshwater bivalves Unio tumidus from two distinct populations, pure (Pr) and contaminated (Ct), in their exposures to microplastics (MP, 1 mg L-1, size 0.1-0.5 mm), pharmaceutical ibuprofen (IBU, 0.8 µg L-1), or their combination (Mix) for 14 days. Control groups from both sites showed remarkable differences, with lower levels of total antioxidant capacity (TAC), metallothionein protein (MTSH), NADH and NAD+, cytochrome P450-related EROD, glutathione-S transferase (GST), and citrate synthase (CS) but higher levels of GSH, GSSG, caspase-3 and cathepsin D (CTD) in the Ct-control group. These data indicate a chronic stress impact in the Ct population. Under exposures, we found an almost common strategy in both populations for NAD+/NADH and MTSH suppression and CTD induction. Additionally, Mix exposure caused an increase in CS, and IBU did not change GSH in both populations. However, the expected response to IBU - the suppression of caspase-3 - was indicated only in PrIBU- and PrMix-mollusks. CTD efflux increased dramatically only in PrMP- and PrMix- groups, and suppression of EROD and GST was detected in the PrMix-group. According to discriminant analysis, exposed Pr-groups were highly differentiated from control, whereas Ct-control and exposed groups had common localization demonstrating high resistance to environmental stress. Thus, the same exposures resulted in different adverse outcome pathways depending on the population.

Characterization of ATPases in the gill of freshwater mussel (Unio tigridis) and effects of ionic and nanoparticle forms of aluminium and copper.

Mussels are often used to monitor the aquatic systems for different ecological aims, as they are one of the best bioindicator animals. However, the characterization of biomarkers should be known before using them in environmental monitoring and toxicology studies. There is no study to our knowledge on the characterization of Na-ATPase and Ca-ATPase in tissues of freshwater mussel (Unio tigridis). Thus, this study was undertaken to characterize the optimum working conditions of Na-ATPase and Ca-ATPase in the gill of mussels, determining the highest levels of parameters (Na+, Mg2+, Ca2+, ATP, pH, temperature, enzyme amount, incubation time) to obtain maximum activity. The present study also aimed to investigate in vitro effects of ionic and nanoparticle (Al2O3, CuO) forms of aluminium and copper (0, 30, 90, 270 µg/L) on the activities of Na-ATPase and Ca-ATPase. Data showed that there was no ouabain-sensitive ATPase activity in the gill up to 10 mM ouabain concentrations. Na-ATPase and Ca-ATPase activities in the gill of control mussels were 5.124 ± 0.373 and 3.750 ± 0.211 µmol Pi/mg pro./h, respectively. Exposure to different concentrations of nanoparticles did not alter significantly (P > 0.05) the activities of Na-ATPase and Ca-ATPase in vitro, whereas the same concentrations of ionic aluminium and copper significantly decreased (P < 0.05) the enzyme activities. Data emphasized that there were different modes of action between ionic and nanoparticle forms of aluminium and copper. Data also suggested that in vivo studies should also be carried out to estimate better the effects of nanoparticle and ionic forms of metals on ATPases of U. tigridis.

Antioxidant system biomarkers of freshwater mussel (Unio tigridis) respond to nanoparticle (Al2O3, CuO, TiO2) exposures.

PURPOSE: Mussels are known as the natural filters of the aquatic systems and are accepted as one of the best bioindicator organism. There is no data on the response of Unio tigridis to metal-oxide nanoparticle (NP) exposures. This study aimed to investigate the response of the antioxidant enzymes of U. tigridis following exposure to NPs. MATERIALS AND METHODS: The mussels were exposed to different concentrations (0, 1, 3, 9 mg NP/L) of Al2O3, CuO and TiO2 NPs for 14 days and subsequently, the activities of CAT (catalase), SOD (superoxide dismutase), GPx (glutathione peroxidase), GST (glutathione S-transferase) and GR (glutathione reductase) were measured in the gill and digestive gland. Mussels were fed with cultured unicellular algae (Chlorella vulgaris) during experiments. RESULTS: Data showed that algae consumptions of mussels were not significantly (p>0.05) altered by NPs. However, all enzyme activities in the digestive gland and gill altered significantly (p<0.05) after NP exposures. The activities of CAT and SOD decreased, while the activities of enzymes belonging to glutathione metabolism (GPx and GST) increased in both tissues. CONCLUSION: This study representing the first record on the antioxidant system response of U. tigridis toward NP exposures suggests that NP toxicity should be investigated thoroughly in organisms and some regulations must be done on their usages.

Common and particular biochemical responses of Unio tumidus to herbicide, pharmaceuticals and their combined exposure with heating.

The priority list of freshwater pollutants is increasingly amended by pharmaceuticals. Their impact on the aquatic biota can be modulated by the presence of typical pollutants, like pesticides, and/or abnormal heating. The aim of this study was to elucidate potentially hazardous impact of combined environmental factors on the freshwater mussels by analyzing various sets of biochemical markers. We treated the bivalve molluscs of Unio tumidus with non-steroidal anti-inflammatory drug diclofenac (Dc, 2 nM), calcium antagonist and antihypertensive drug nifedipine (Nf, 2 nM) or organophosphonate glyphosate-based herbicide Roundup MAX (Rn, 79 nM of glyphosate) at 18 °C as well as with the mixture of these substances at 18 °C (Mix) or 25 °C (MixT) during 14 days. The concentrations used were correspondent to the environmentally relevant levels. The biomarkers of stress and toxicity were evaluated in digestive gland, except the lysosomal membrane stability measured in hemocytes. Exposures caused an oxidative stress due to the decreased SOD and GST activities and GSH/GSSG ratio, increased levels of thiobarbituric acid-reactive substances and protein carbonyls (with some exceptions). Dc increased cathepsin D activity in lysosomes. Nf increased lysosomal membrane stability and caspase-3 activity. Rn caused a dramatic distortion of metallo-thiolome due to increased levels of GSH and metallothionein-related thiols (MTSH) as well as depletion of Zn, Cu and Cd in the composition of metallothioneins, and decreased Zn/Cu molar ratio in the tissue. The particular toxicity of Rn was also attested by decreased lysosomal membrane stability and cholinesterase activity. Canonical discriminant analysis separated Rn-, Mix- and MixT-groups from the joint set of C-, Dc- and Nf-groups. Generally, compound-specific effects were expressed in U. tumidus responses to the mixtures, but in MixT-group some effects were particular or extremely strong. Multi-marker approach and integrative analysis proved to be a useful tool for understanding possible future risks to freshwater mussels under a combination of xenobiotics and warming climate.

Biochemical Responses of the Bivalve Mollusk Unio tumidus Inhabiting a Small Power Plant Reservoir on the Dniester River Basin, Ukraine.

Hydropower plants (HPPs) can affect the hydrological regime. However, biochemical responses of aquatic animals for the evaluation of this disturbing are not applied yet. The specimens of Unio tumidus were sampled in a reservoir (R) of a small HPP as well as downstream from the dam (DS). Biochemical indexes in the digestive gland and alkali labile phosphates (ALP) in the gonads were examined. The R-mollusks showed low cholinesterase, catalase and caspase-3 activities, and metallothionein concentration, but elevated levels of zinc and copper, oxidized glutathione and protein carbonyls. Concentrations of lactate, pyruvate and ALP, activity of superoxide dismutase and glutathione S-transferase, and lipid peroxidation level were similar in both groups. Integrated biomarker response (IBR/n) index (n = 13) was 2.17 and 0.29 in the R- and DS-groups correspondingly. We suggest that using integrative biological response based on the biochemical markers of bivalve mollusks can be a valid early warning step in assessing 'environmental flow' impact.

Biomonitoring of Trace Metals in the Keban Dam Reservoir (Turkey) Using Mussels (Unio elongatulus eucirrus) and Crayfish (Astacus leptodactylus).

Freshwater mussels and crayfish are commonly used as biomonitors of trace metals. In the present study, the concentrations of ten metals were determined in mussels (Unio elongatulus eucirrus) and crayfish (Astacus leptodactylus) collected from the Keban Dam Reservoir in Turkey. The significant spatial differences in concentrations of studied metals except As in mussels were not found. However, Co, Cr, Cu, and Zn concentrations in mussels and As, Co, Cu, Fe, Pb, and Zn concentrations in crayfish showed significant seasonal differences. As, Cd, and Mn levels in mussels were about nine times higher than those in crayfish. The concentrations of Cd, Cr, Cu, Pb, Zn, and inorganic As in crayfish and mussels were lower than maximum permissible levels. When compared with other biomonitoring studies using mussels and crayfish, high concentrations of As, Cd, Co, Cr, and Ni in mussels and Cr and Ni in crayfish were observed due to lithogenic sources and anthropogenic activities in the basin. Bioconcentration factor values of Fe, Mn, Cd, and Zn in mussels and Zn, Cu, Fe, and Co in crayfish were > 1000, which indicates that both U. e. eucirrus and A. leptodactylus have potential to bioaccumulate these metals. Therefore, attention should be paid to mussels and crayfish from ecological and human health perspective, because they are potential vectors of metals to higher trophic levels.

A field study of hemolymph yolk protein levels in a bivalve (Unio tumidus) and future considerations for bivalve yolk protein as endocrine biomarker.

Induction of yolk protein in male fish is a recognized biomarker for estrogenic exposure because the estrogen-dependent induction mechanism is well investigated and there is a clear difference in yolk protein levels of unexposed males and females. Attempts have been made to use induction of bivalve yolk protein as biomarker for estrogenic exposure. However, several biomarker validation criteria have not yet been investigated e.g. an in-depth understanding of the induction mechanism and background variability is needed and reliable detection assays are yet to be developed. To obtain background knowledge about yolk protein levels freshwater bivalves (Unio tumidus) were collected in an uncontaminated Danish lake over the course of a year (33 collection dates). The hemolymph yolk protein concentration of 569 individuals was determined by a species specific enzyme-linked immunosorbent assay (ELISA) and male and female gonadal development cycles were established. The average yolk protein levels of males and females collected at each sampling date overlapped in some periods; the male and female range was 66,946 - 169,692 ng/mL and 88,731 - 681,667 ng/mL, respectively. Because male and female hemolymph yolk protein levels overlap, great care should be taken if yolk protein induction in bivalve hemolymph is considered as endocrine biomarker.

The effects of zinc nanooxide on cellular stress responses of the freshwater mussels Unio tumidus are modulated by elevated temperature and organic pollutants.

Nanoparticle toxicity is a growing concern in freshwater habitats. However, understanding of the nanoparticle effects on aquatic organisms is impeded by the lack of the studies of the nanoparticles effects in the environmentally relevant context of multiple stress exposures. Zinc oxide nanoparticles (n-ZnO) are widely used metal-based nanoparticles in electronics and personal care products that accumulate in aquatic environments from multiple non-point sources. In this study, we evaluated the effects of n-ZnO in a model organism, a mussel Unio tumidus, and the potential modulation of these effects by common co-occurring environmental stressors. Male U. tumidus were exposed for 14 days to n-ZnO (3.1 µM), Zn(2+) (3.1 µM), Ca-channel blocker nifedipine (Nfd 10 µM), combinations of n-ZnO and Nfd or n-ZnO and thiocarbamate fungicide Tattoo (Ta, 91 µg L(-1)) at 18 °C, and n-ZnO at 25 °C (n-ZnO+t°). Total and metallothionein-bound Zn levels as well as levels of metallothioneins (MT), cellular stress responses and cytotoxicity biomarkers were assessed in the mussels. The key biomarkers that showed differential responses to different single and combined stressors in this study were activities of caspase-3 and lysosomal cathepsin D, as well as protein carbonyl content. At 18 °C, exposures to n-ZnO, organic pollutants and their combinations led to a prominent up-regulation of MT levels (by ∼30%) and oxidative stress response including up-regulation of superoxide dismutase activity, an increase in oxyradical production, and a 2-3-fold decrease in the levels of protein carbonyls in all exposures except nZnO+Ta. Expos ure to n-ZnO in the absence of other stressors also led to a strong (∼7-fold) elevation of cathepsin D activity. Cellular responses to Zn(2+) and n-ZnO were different indicating that n-ZnO was not due exclusively to Zn release. Ca-channel blocker Nfd affected intracellular Zn distribution (reflected in the prominent elevation of Zn-MT levels) and caused reductive stress indicated by elevated levels of reduced glutathione levels and an increase in lactate/pyruvate ratio (reflecting higher NADH/NAD ratio). Elevated temperature (25 °C) abolished most of the typical responses to n-ZnO and induced oxidative injury, DNA fragmentation and caspase-3 mediated apoptosis in n-ZnO-exposed mussels. DNA fragmentation was also induced by exposure to organic toxins (alone and in combination with n-ZnO) but not by n-ZnO alone. These data indicate that n-ZnO toxicity to freshwater organisms is modulated by organic pollutants and enhanced by elevated temperatures.

Acute toxicity of cypermethrin on the freshwater mussel Unio gibbus.

Cypermethrin is a synthetic pyrethroid insecticide used worldwide in agriculture, home pest control, food stuff protection and disease vector control. We investigate the potential of cypermethrin to induce oxidative stress and enzyme activities within the gills of freshwater mussel Unio gibbus. This study was carried out under laboratory conditions using two nominal cypermethrin concentrations C1 (100µg/L) and C2 (150µg/L) during 96h. The measured concentrations of cypermethrin using GC-MS-MS in the treatment aquariums were respectively 59.7 µg/L and 97.5µg/L. Antioxidant enzyme activities (superoxide dismutase (SOD) and catalase (CAT)) as well as H2O2, malondialdehyde (MDA) and protein carbonyl (PCO) levels were assessed. An exposure during 96h induced the SOD activity at the highest concentration. The CAT activity and H2O2 level were increased significantly (P<0.05) in gills following a dose-dependent profile. Cypermethrin also generated an increase in malondialdehyde (MDA) levels reaching the highest value at the high concentration. The considered parameters can be used as biomarkers of exposure to cypermethrin. Freshwater mussel U. gibbus can be potentially employed in biomonitoring surveys of such threatened ecosystems.

Uptake from water, internal distribution and bioaccumulation of selenium in Scenedesmus obliquus, Unio mancus and Rattus norvegicus: part A.

The (75)Se internal bioavailability was investigated in microalgae, mussels and rats as biological experimental models. The (75)Se accumulation from freshwater to microalgae [Scenedesmus obliquus (Turpin) Kützing], from freshwater to mussels (Unio mancus Lamark) and, finally, per os to rats (Rattus norvegicus Berkenhout) was followed using (75)Se-labelled selenite looking at (75)Se uptake, retention, intracellular distribution and binding with cellular biocomplexes. After exposure to 10, 50 and 500 µg Se L(-1), the microalgae showed an inhibitory effect on population growth only at the highest concentration. Mussels exposed to 105 µg Se L(-1) showed an accumulation of the element with time in all tissues. Intracellularly, Se was present in all subcellular fractions, especially in the cytosol. Rats were treated via oral administration with 5 µg Se rat(-1). After 24 h, liver and kidney showed the highest Se concentration.

Uptake from water, internal distribution and bioaccumulation of selenium in Scenedesmus obliquus, Unio mancus and Rattus norvegicus: part B.

(75)Se-selenite transfer was investigated in a phytoplankton-mussel-rat food chain model consisting of Scenedesmus obliquus (Turpin) Kützing, Unio mancus Lamark and Rattus norvegicus Berkenhout. (75)Se-metabolized forms were investigated in order to identify potential critical steps in the food chain, as well as its relative bioavailability looking also at intracellular, cellular and organ partitioning. Tissue and intracellular distribution of (75)Se in mussels fed with (75)Se-S. obliquus was different compared to those exposed only to inorganic (75)Se-selenite. The intracellular distribution of (75)Se in the hepatopancreas and mantle of mussels fed (75)Se-microalgae was similar to hepatic and renal distributions in rats, suggesting that their stomach dissociated larger (75)Se-containing molecules. The (75)Se partitioned from water (culture medium) to microalgae showing a bioconcentration factor of 435. The bottleneck in the trophic transfer of (75)Se occurred between S. obliquus-U. mancus. From microalgae to mussels and subsequently to rats no bioaccumulation was verified.

Evaluation of yolk protein levels as estrogenic biomarker in bivalves; comparison of the alkali-labile phosphate method (ALP) and a species-specific immunoassay (ELISA).

Altered concentration of the vertebrate yolk protein precursor vitellogenin is a recognized biomarker for endocrine disruption in fish, and within recent years yolk protein alteration has also been associated with endocrine disruption in bivalves. Species-specific, direct and sensitive methods for quantification of vitellogenin in fish have been available for years whereas bivalve yolk protein levels have been estimated indirectly by alkali-labile phosphate (ALP) liberated from high molecular weight proteins because the sequence and biochemical structure of most bivalve yolk proteins are unknown. By applying a species-specific enzyme-linked immunosorbent assay (ELISA) for accurate determination of yolk protein level the impact of 17ß-estradiol (57, 164 and 512 ng/L) on the freshwater bivalve Unio tumidus was investigated and compared with ALP estimations. Seven weeks of exposure during the pre-spawning and spawning period had no consistent effect on yolk protein concentration in hemolymph, and ALP levels in hemolymph also remained unchanged in both males and females. Further, basal male and female ALP levels were indistinguishable whereas the ELISA demonstrated that yolk protein levels of females exceeded male levels at the time of sampling, although male basal levels were high compared to fish. Altogether it is shown that individual ALP levels do not reflect yolk protein levels and hence hemolymph ALP levels cannot serve as biomarker for estrogenic exposure during the pre-spawning and spawning period in U. tumidus. The necessity of sensitive and validated biomarkers for reliable interpretation of data and the utility of ALP and yolk protein levels as biomarkers in bivalves are discussed.

Specific proteomic response of Unio pictorum mussel to a mixture of glyphosate and microcystin-LR.

Cyanobacterial toxins and pesticides regularly impact freshwaters. Microcystin-LR is one of the most toxic and common cyanobacterial toxins whereas glyphosate is the active ingredient of a widely use herbicide. As filter feeders, freshwater mussels are particularly exposed. Like many native bivalve species, Unio pictorum suffers from a continuous decline in Europe. In order to get a deeper insight of its response to contaminants, U. pictorum was exposed to either 10 µg L(-1) of microcystin-LR or 10 µg L(-1) of glyphosate or a mixture of both. Proteins of the digestive glands were extracted and analyzed by DIGE. Gel analysis revealed 103 spots with statistical variations, and the response seems to be less toward glyphosate than to microcystin-LR. Specific spots have variations only when exposed to the mixture, showing that there is an interaction of both contaminants in the responses triggered. The proteins of 30 spots have been identified. They belong mostly to the cytoskeleton family, but proteins of the oxidative pathway, detoxification, and energetic metabolism were affected either by glyphosate or microcystin-LR or by the mixture. These results demonstrate the importance to study contaminants at low concentrations representative of those found in the field and that multicontaminations can lead to different response pathways.

Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin.

The freshwater painter's mussel (Unio pictorum) was used as sentinel species to assess the chemical disturbance in an Italian river (the river Cecina) characterized by elevated levels of trace metals of both natural and anthropogenic origin. Organisms were transplanted for 4 weeks in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses. Such parameters included the levels of individual antioxidants (catalase, glutathione-S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, total glutathione), the total oxyradical scavenging capacity (TOSC), metallothionein-like proteins, the assessment of DNA integrity, chromosomal damages and lysosomal membrane stability. Elevated levels of several metals were measured in sediments, but the relatively low tissue concentrations suggested a moderate bioaccumulation, possibly due to a high excretion efficiency, of U. pictorum and/or to a limited bioavailability of these elements, partly deriving from erosion of bedrocks. Among antioxidant responses, those based on glutathione metabolism and the activity of catalase were mostly affected in bivalves showing a significant accumulation of arsenic, mercury and/or nickel. In these specimens, the content of glutathione and the activities of glutathione reductase and glutathione peroxidases (H2O2) were respectively 9-, 6- and 4-fold lower than in controls, while a 3-fold increase was observed for catalase. Despite some differences in the response of individual antioxidants, a significant reduction of the capability to neutralize peroxyl radicals was observed in bivalves caged in all the impacted sites of the river basin; these organisms also exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. Considering the mild contamination gradient in the investigated area, the overall results suggested that some oxidative biomarkers, as well as those evaluating chromosomal and cell damages, are highly sensitive and could be profitably applied to caged painter's mussels for environmental quality assessment in freshwater.

SOD and CAT cDNA cloning, and expression pattern of detoxification genes in the freshwater bivalve Unio tumidus transplanted into the Moselle river.

The cDNA sequences encoding manganese superoxide dismutase (Mn-SOD) and catalase (CAT) were isolated in the freshwater bivalve Unio tumidus by reverse-transcription polymerase chain reaction (RT-PCR) using degenerate primers. Quantitative real-time PCR approach was used to evaluate the mRNA expression patterns of SOD, CAT, selenium-dependent glutathione peroxidase (Se-GPx), pi class glutathione S-transferase (pi-GST) and metallothionein (MT), in the digestive gland of Unio tumidus transplanted from a control site to four stations in the Moselle River (M1-M4), for periods of 8 and 21 days. These sites were chosen upstream and downstream of populated areas. Chemical analysis performed on sediments from the Moselle river sites did not show high levels of pollutants. Decrease of SOD, CAT, Se-GPx and MT mRNA levels were observed at M3 site after a 21-day exposure compared to control site. These results suggest inefficiency of antioxidant systems affected by cytotoxic mechanisms and confirm an environmental perturbation. Organisms transplanted at M4 site showed a strong increase of biomarkers transcription levels after 21 days of exposure. These inductions could correspond to an adaptive response to an altered environment. Our results showed that biological approaches using multibiomarkers appear as essential tools complementary to measurement of contaminants, to detect environmental degradations.

Cadmium availability to freshwater mussel (Unio tumidus) in the presence of organic matter and UV radiation.

The influence of ultraviolet radiation (UV) on cadmium availability to freshwater mussel (Unio tumidus) in the presence of organic matter was investigated. Water solutions containing 1000, 500, and 250 microg x L(-1) of cadmium and 28.8 mg x L(-1) of total organic carbon (TOC) were exposed to ecologically relevant biologically effective UV-A and UV-B irradiances of 1.73 x 10(-6) W x cm(-2) and 1.50 x 10(-5) W x cm(-2) respectively for 12 h. The availability of cadmium was measured directly as Cd concentration in gills, mantle, digestive gland and foot, and indirectly as its impact on the content of essential cations. The concentration of cadmium ions, soluble forms of cadmium, pH and conductivity in UV irradiated solutions were also measured. UV exposures resulted in a decrease of the TOC to 25.9 mg x L(-1), followed by a decrease in the total concentration of Cd soluble forms, increase in the concentration of cadmium bound to particulate matter, and increase in the concentration of free cadmium ions in comparison to non-irradiated mixtures. Mussels from mixtures exposed to UV accumulated more cadmium than specimens from non-irradiated mixtures. This was confirmed by the fluctuations of essential cations, i.e. decreases in the contents of Zn, Ca, Fe and increase of the Cu content. Na/K molar ratios in gills were negatively correlated with cadmium content, while a positive trend occurred in other organs. We suggest that in UV-exposed water more ionic forms of cadmium occur. On the other hand more cadmium is also bound to particulate matter. Our research indicate that the increase of cadmium accumulation in mussels grown in humic acid solutions exposed to UV were most probably related to the increase in the concentration of cadmium bound to particulate matter.

Effects of deltamethrin on antioxidant status and oxidative stress biomarkers in freshwater mussel, Unio elongatulus eucirrus.

In this study, the effects of deltamethrin on antioxidant status and oxidative stress biomarkers in digestive gland and gill of freshwater mussel, Unio elongatulus eucirrus, was examined. Deltamethrin was applied at concentrations of 25, 50, 100, 200, 400, 800 and 1,600 microg L(-1), for 1, 24, 48, 72 and 96 h. With increasing deltamethrin concentrations, the mussels exposed duration 1-96 h significantly increased lipid peroxidation, which might be associated to decreased levels of reduced glutathione and catalase activity in digestive gland and gill of freshwater mussel (p<0.05 for each cases). Negative correlations were observed between the lipid peroxidation and glutathione or catalase levels after deltamethrin exposure, indicating a protective role of glutathione and catalase against lipid peroxidation, suggesting the use of these antioxidants as a potential biomarker of toxicity associated with contaminant exposure in freshwater mussels.