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.

Behavioural Responses of Unio tumidus Freshwater Mussels to Pesticide Contamination.

A pesticide is a chemical substance used for the disposal of pests, such as insects, weeds, invertebrates, or rodents. Pesticides interfere with the normal metabolism of the target species; however, some of them may inadvertently affect organisms other than those targeted. Increased quantities of pesticides in water disturb various ecological processes and may increase the mortality rate of various native species of flora and fauna. One of the groups of organisms that are at the greatest risk from the adverse effects of pesticides is the bivalves. This study was designed to assess the behavioural reaction of bivalves to widespread pesticides. As a representative example, the Polish native Unio tumidus (Philipsson 1788) was used. The study investigated different groups of toxic pesticides, such as herbicides (lenacil), insecticides (thiacloprid, DDT and dichlorvos), and fungicides (tebuconazole), in concentrations of 10 mg L-1. The results showed various behavioural reactions of bivalves to the pesticides. The most evident were activity time and shell opening rate. Moreover, as a result of DDVP contamination, effects were recorded in terms of shell opening level as well as rapid onset of death. Among the five analysed plant protection products, the most toxic was DDVP. Its presence caused adductor muscle paralysis in all analysed individuals. The least toxic pesticides were DDT and thiacloprid. A strong reaction to lenacil was observed especially in the shell opening rate. Tebuconazole caused significant reductions in activity. Despite the fact that the impact of pesticides on ecosystems is under regular observation, with the use of a wide range of scientific techniques, the use of bivalves was shown to have considerable potential for water quality monitoring.

Biochemical and molecular alterations in freshwater mollusks as biomarkers for petroleum product, domestic heating oil.

To investigate the effect one of the oil products, domestic heating oil (DHO), on freshwater mollusks, Unio tigridis and Viviparous bengalensis were exposed to three DHO concentrations for each species (5.8, 8.7, and 17.4 ml L-1 for mussels; 6.5, 9.7, and 19.5 mlL-1 for snails, respectively). Antioxidant enzymes (superoxide dismutase, catalase), malondialdehyde, acetylcholinesterase and DNA damage in both species tissues were monitored over 21 days. The results showed that both antioxidant enzymes concentration (SOD and CAT) increased in the lowest DHO concentrations (5.8, and 8.7 ml L-1), and then decreased in the highest concentration (17.4 ml L-1) as the same pattern for Unio tigridis, but this not occurred for Viviparous bengalensis. MDA values recorded significantly increased compared to control. No reduction was observed in AChE concentrations in soft tissues of both mollusks may due to that DHO was a non-neurotoxicant to Unio tigridis and Viviparous bengalensis. The results of DNA damage parameters were showed significant differences (p≤ 0.05) between control and DHO concentrations except lowest concentration for each parameter measured in digestive gland of Unio tigridis. As well as, these significant differences were recorded between control and three concentrations of DHO exposure for comet length, and tail length parameters, and between control and highest oil concentration for tail moment in Viviparous bengalensis. DHO has the ability to prevent the reproduction of Viviparous bengalensis snail relation to control, that is what we considered strong evidence of the toxicity properties of DHO on the reproductive status of this species of snails. SOD, CAT, and MDA were useful biomarkers for evaluating the toxicity of DHO in mussel and snails, and comet assay was a good tool to assess the potential genotoxicity of DHO.

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.

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.

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.

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.

The impact of in vivo and in vitro exposure to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater mussels Unio pictorum and Unio tumidus.

The impact of in vivo and in vitro exposure to anticancer drug 5-Fluorouracil (5-FU) on the level of DNA damage was evaluated using comet assay on haemocytes of freshwater mussels Unio pictorum and Unio tumidus. For the in vivo experiment, the groups of 5 mussels per concentration were exposed for 72 h to 5-FU (0.04, 0.4, 4, 40 and 100 µM) with 0.4 µM being the lowest concentration to induce significant DNA damage. For the in vitro experiment, the primary cultures of haemocytes were treated with 0.04, 0.4, 4 and 40 µM 5-FU for 22 h and the treatment with CdCl2 was used as a positive control. In contrast to in vivo exposure, 5-FU did not induce significant increase of DNA damage in vitro, possibly because of the absence of haemocytes proliferation in primary cultures.

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.

Aquatic predicted no-effect-concentration derivation for perfluorooctane sulfonic acid.

Perfluorooctane sulfonic acid (PFOS), a representative perfluorinated surfactant, is an anthropogenic pollutant detected in various environmental and biological matrices. Some laboratory and field work has been conducted to assess the aquatic toxicity of PFOS, but little is known regarding its toxicity threshold to the aquatic ecosystem. In the present study, predicted no-effect concentrations (PNECs) were derived by four different approaches. The interspecies correlation estimation (ICE) program and final acute-to-chronic ratio (FACR) were applied to the development of PNEC based on the toxic mode of action (MOA) of PFOS. By comparison of the different PNECs, the recommended aquatic toxicity thresholds for PFOS are in the range of 0.61 to 6.66 µg/L. Based on comparison of PNEC values, microcosm results, and reported environmental concentrations, PFOS appears not to pose a serious threat to aquatic organisms. The present results demonstrate that MOA is an important consideration for the derivation of reliable PNECs; moreover, the ICE-based species sensitivity distribution (SSD) method can be used to derive PNECs when toxicological data are limited. The application of MOA and ICE for deriving PNEC values in the present study may facilitate studies on using a combination of quantitative structure-activity relationship (QSAR) models and ICE to estimate PNECs.

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.

Comparative toxicity of single and combined mixtures of selected pollutants among larval stages of the native freshwater mussels (Unio elongatulus) and the invasive zebra mussel (Dreissena polymorpha).

This study evaluated the impact of biocides (tributyltin, chlorthalonil and Irgarol 1051) and of pollutants (copper, inorganic and methyl mercury and 4-nonylphenol) occurring in Ebro River (NE Spain) on early developmental stages of native Spanish freshwater and invasive zebra mussels. Toxicity tests were conducted with embryos and glochidia of zebra mussel (Dreissena polymorpha) and the naiad species Unio elongatulus, respectively. Toxicity was quantified in terms of median effective concentration (EC50) impairing embryogenesis and glochidia viability in single and combined mixture exposures. Irgarol 1051 was not toxic at concentrations below 40x10(3)nM. Zebra mussel embryos were on average 50 fold more sensitive to the studied pollutants than glochidia. Tributyltin was the most toxic compound with EC50s for zebra mussel embryos and glochidia, respectively, of 1.24 and 47.93 nM, followed by chlorothalonil (3.65, 176.58 nM), methyl mercury (7.06, 156.4 nM), inorganic mercury (3.64, 518.28 nM), copper (19.73, 1358.55 nM) and 4-nonylphenol (33.99, 1221.48 nM). Combined toxicity of Ebro River pollutants (copper, inorganic and methyl mercury and 4-nonylphenol) was greater than additive in zebra mussel embryos and additive in glochidia. These results indicated that contaminant levels that affect zebra mussel embryos are not toxic to early life stages of the naiad mussel species U. elongatulus.

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.