Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults

Development of stress markers for the invader freshwater zebra mussel (Dreissena polymorpha) is of great interest for both conservation and biomonitoring purposes. Gene expression profiles of several putative or already established gene expression stress markers (Metallothionein, Superoxide dismutase, Catalase, Glutathione S transferase, Glutathione peroxidase, Cytochrome c oxidase, the multixenobiotic resistance P-gp1, and heat shock proteins HSP70 and HSP90) were analyzed by quantitative Real-Time PCR in adults and pediveliger larvae after exposure to metals (Hg, Cu, Cd). A defined pattern of coordinated responses to metal exposure and, presumably, to oxidative stress was observed in gills and digestive gland from adults. A similar, albeit partial response was observed in larvae, indicating an early development of stress-related gene responses in zebra mussel. The tools developed in this study may be useful both for future control strategies and for the use of zebra mussel as sentinel species in water courses with stable populations.     

DDT contamination in Lake Maggiore (N. Italy) and effects on zebra mussel spawning.

Zebra mussel (Dreissena polymorpha) is commonly used as contaminant bioindicator in Europe and North America. We used the zebra mussel to follow DDT pollution trends from 1996 to 1997 in Pallanza bay, Lake Maggiore, near the inlet of the River Toce, after a DDT-manufacturing plant discharging residues into a tributary of the River Toce had closed down. DDT contamination fell off sharply outside the bay, but tissue concentrations of the parent compound and residues remained high in molluscs sampled within the bay a year later. Molluscs collected in Pallanza bay in June 1997 released gametes earlier than those sampled at a nearby reference station. Histological studies showed that a significant percentage of these specimens showed marked oocyte degeneration suggesting that DDTs have endocrine-disrupting effects in this species.     

Use of zebra mussel (Dreissena polymorpha) to assess trace metal contamination in the largest Italian subalpine lakes.

Trace metal (Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn) contamination was evaluated in zebra mussels from the lakes Maggiore, Lugano, Como, Iseo and Garda, which are located in the most highly populated and industrialised area in Italy. Zebra mussels from Lake Maggiore contained the highest concentrations (3.44, 1.51, 4.97, 0.158, 5.87, 346 microg g(-1) for Cd, Co, Cr, Hg, Pb, Zn, respectively) of all metals analysed except Cu and Ni. The lowest levels of most metals were in animals from Garda and Lugano (0.78 and 0.60 microg g(-1) for Cd, 2.87 and 2.03 microg g(-1) for Cr, 0.065 and 0.049 microg g(-1) for Hg, 12.1 and 11.9 microg g(-1) for Ni, 1.96 and 2.46 microg g(-1) for Pb, 158 and 163 microg g(-1) for Zn). The most contaminated sites and possible local sources of metals were identified for each lake, and the lakes classified into quality classes concerning metal pollution.     

Effects of the pharmaceuticals gemfibrozil and diclofenac on biomarker expression in the zebra mussel (Dreissena polymorpha) and their comparison with standardised toxicity tests

Pharmaceuticals, including the lipid regulator gemfibrozil and the non-steroidal anti-inflammatory drug diclofenac have been identified in waste water treatment plant effluents and receiving waters throughout the western world. The acute and chronic toxicity of these compounds was assessed for three freshwater species (Daphnia magna,Pseudokirchneriella subcapitata, Lemna minor) using standardised toxicity tests with toxicity found in the non-environmentally relevant mid mg L⁻¹ concentration range. For the acute endpoints (IC₅₀ and EC₅₀) gemfibrozil showed higher toxicity ranging from 29 to 59 mg L⁻¹ (diclofenac 47-67 mg L⁻¹), while diclofenac was more toxic for the chronic D. magna 21 d endpoints ranging from 10 to 56 mg L⁻¹ (gemfibrozil 32-100 mg L⁻¹). These results were compared with the expression of several biomarkers in the zebra mussel (Dreissena polymorpha) 24 and 96 h after exposure by injection to concentrations of 21 and 21,000 μg L⁻¹ corresponding to nominal concentrations of 1 and 1000 μg L⁻¹. Exposure to gemfibrozil and diclofenac at both concentrations significantly increased the level of lipid peroxidation, a biomarker of damage. At the elevated nominal concentration of 1000 μg L⁻¹ the biomarkers of defence glutathione transferase and metallothionein were significantly elevated for gemfibrozil and diclofenac respectively, as was DNA damage after 96 h exposure to gemfibrozil. No evidence of endocrine disruption was observed using the alkali-labile phosphate technique. Results from this suite of biomarkers indicate these compounds can cause significant stress at environmentally relevant concentrations acting primarily through oxidation pathways with significant destabilization of the lysosomal membrane and that biomarker expression is a more sensitive endpoint than standardised toxicity tests.     

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

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

Tissue-specific heavy metal (Cd, Pb, Cu, Zn) deposition in a natural population of the zebra mussel Dreissena polymorpha Pallas.

The zebra mussel Dreissena polymorpha was tested as an indicator of heavy metal exposure in urban waters of Vienna, Austria. Mussels, two sediment fractions, suspended matter, and filtrate were collected over one annual cycle at five sampling stations. Dreissena was dissected into five body parts: viscera, gill, foot, byssus, and shell, to determine tissue-specific metal accumulation. Cadmium, lead, copper, and zinc were measured by AAS. There was no clear relationship between (relatively low) metal concentrations in ambient compartments and in zebra mussel bodies. Therefore, D. polymorpha must be regarded as a poor suitable indicator tool under near-background contamination situations. Tissue-specific metal accumulation showed that cadmium was mainly stored in soft body parts. Lead, copper, and zinc showed significantly highest concentrations in the byssal threads. Metal concentrations and distribution patterns within the mussel's body must be interpreted as a result of (unknown) internal metal treatment/regulation. Excretion of lead, copper, and zinc via the byssus complex probably is an effective strategy for preventing toxic injury in D. polymorpha.     

Evaluation of spatial distribution and accumulation of novel brominated flame retardants,HBCD and PBDEs in an Italian subalpine lake using zebra mussel (Dreissena polymorpha)

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.     

Influence of cadmium on antioxidant capacity and four microelement concentrations in tomato seedlings (Lycopersicon esculentum)

Tomato (Lycopersicon esculentum) seedlings were grown in four cadmium (Cd) levels of 0-10 microM in a hydroponic system to analyze the antioxidative enzymes, Cd concentration in the plants, and the interaction between Cd and four microelements. The results showed that there was a significant increase in malondialdehyde (MDA) concentration, and superoxide dismutase (SOD) and peroxidase (POD) activities in the plants subjected to 1-10 microM Cd. This indicates that Cd stress induces an oxidative stress response in tomato plants, characterized by an accumulation of MDA and increase in activities of SOD and POD. Root, stem and leaf Cd concentrations increased with its exposure Cd level, and the highest Cd concentration occurred in roots, followed by leaves and stems. A concentration- and tissue-dependent response was found in the four microelement concentrations to Cd stress in the tomato leaves, stems and roots. Regression analysis showed that there was a significantly negative correlation between Cd and Mn, implying the antagonistic effect of Cd on Mn absorption and translocation. The correlation between Cd and Zn, Cu and Fe were inconsistent among leaves, stems and roots.     

Effects of some environmental parameters on catalase activity measured in the mussel (Mytilus galloprovincialis) exposed to lindane

Mussels (Mytilus galloprovincialis), collected from the Bizerta lagoon, were acclimated for four days to various conditions of temperature, salinity, photoperiod and food supply and then exposed to lindane at a concentration of 40 microg l(-1). Catalase activity, which is a biomarker of exposure to an oxidative stress, was measured in the whole soft tissues of control and assay groups. In control mussels, high temperature, high salinity and light duration significantly increased catalase activity whereas this activity decreased when food, composed of freeze-dried, algae was available. When mussels were treated with lindane, catalase activities were higher than in controls. This increase was significant with temperature, salinity and light duration. The food supply did not change catalase activity, which was always higher compared to controls. Oxidative stress was shown in mussels exposed to lindane. The results highlight the need of considering abiotic parameters in biomonitoring studies, and especially when using catalase as a biomarker.     

Bioaccumulation and effects of perfluorinated compounds (PFCs) in zebra mussels (Dreissena polymorpha)

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

Use of sedimentary metals to predict metal concentrations in black mussel (Mytilus galloprovincialis) tissue and risk to human health (Sydney estuary, Australia)

Filter-feeding bivalves have been used extensively as an indicator of ecosystem condition and in management of estuarine environments. The current study aimed to determine whether sedimentary metals could predict metal concentrations in tissue of filter-feeding mussels (Mytilus galloprovincialis) and to identify areas of the estuary where mussel consumption posed a human health risk. Mussel tissue Cu and Zn concentrations (wet weight) were below guideline values for human consumption in all parts of the waterway, whereas Pb tissue concentrations exceed these guidelines (2.0 μg?g^?1 wet weight) in the upper reaches of some embayments of the estuary. Concentrations of Cu and Pb in the fine fraction (<62.5 μm) of bottom sediment reasonably predicted concentrations (dry weight) of these metals in mussel tissue (r ²?=?0.460 and p?=?0.001 and r ²?=?0.669 and p?<?0.0001, respectively) as these materials are resuspendable and available to filter-feeding estuarine animals, whereas total sediment and mussel tissue were poorly related. Lead concentrations (>350 μg?g^?1) in fine sediments indicated areas of this estuary where human health was at risk due to high tissue concentrations of this metal. These results give encouragement for the use of the metal concentration in fine sediments as an indicator of estuarine condition and risk to human health in this waterway. Mussels were distributed in all parts of the estuary, even in areas where metal concentrations exceeded sediment quality guidelines.     

Copper and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in the microalga Pavlova viridis (Prymnesiophyceae)

The metal-induced lipid peroxidation and response of antioxidative enzymes have been investigated in the marine microalga Pavlova viridis to understand the mechanisms of metal resistance in algal cells. We have analyzed superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) activities and glutathione (GSH) contents in microalgal cells grown at different concentrations of copper and zinc. In response to each metal, lipid peroxidation was enhanced with the increase of concentrations, as an indication of the oxidative damage caused by metal concentration assayed in the microalgae cells. Exposure of P. viridis to the two metals caused changes in enzyme activities in a different manner, depending on the metal assayed: after copper treatments, total SOD activity was enhanced, while it was reduced after zinc exposure. Copper and zinc stimulated the activities of CAT and GSH whereas GPX showed a remarkable increase in activity in response to copper treatments and decrease after zinc treatments. These results suggest that an activation of some antioxidant enzymes was enhanced to counteract the oxidative stress induced by the two metals.     

Effects of Cd and Zn waterborne exposure on the uptake and depuration of 57Co, 110mAg and 134Cs by the Asiatic clam (Corbicula fluminea) and the zebra mussel (Dreissena polymorpha)--whole organism study

Groups of zebra mussels (Dreissena polymorpha) and asiatic clams (Corbicula fluminea) were exposed to cadmium and zinc with the aim of studying the effect of these metals on the 57Co, 110Ag and 134Cs uptake and depuration by these freshwater bivalves. In the presence of zinc, the 57Co concentration factor for the whole organism of the two species was halved, notably because of a decrease of the uptake parameter. Conversely, Zinc and the Cd + Zn mixture increased the 110mAg uptake process by clams and mussels. The two metals also increased the depuration of this radionuclide in mussels, whereas this phenomenon was only observed in clams exposed to cadmium. In comparison with 57Co and 110mAg, the 134Cs bioconcentration was 5-10 times lower in D. polymorpha and not detected in C. fluminea. This weak contamination by this radionuclide resulted from a lower uptake and a higher depuration parameters.     

Lipid peroxidation and antioxidant enzyme activity in different organs of mice exposed to low level of mercury

The effects of mercuric chloride (Hg) on lipid peroxidation (LPO), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD) and glutathione (GSH) levels in different organs of mice (CD-1) were evaluated. Mice were exposed (2 days/week) to 0.0 (control), 0.8 (low) and 8.0 (mid) and 80.0 (high) gHg/kg/day for 2 weeks. The high dose group was excluded from the study due to high mortality. LPO levels in kidney, testis and epididymus at low and mid doses; GR and GPx levels in testis at mid dose; SOD levels in brain and testis at both doses, liver and epididymus at mid dose; GSH levels in testis at both doses were significantly increased compared to their controls. However, the GR levels in kidney at both doses and in epididymus at mid dose; GPx levels in kidney and epididymus and SOD levels in kidney at both the doses; GSH levels in epididymus at mid dose were significantly decreased compared to their control. Body weight gain and food efficiency were significantly reduced (p<0.05) in mid dose. These results indicated that Hg treatment enhanced LPO in all tissues, but showed significant enhancement only in kidney, testis and epididymus suggesting that these organs were more susceptible to Hg toxicity. The increase in antioxidant enzyme levels in testis could be a mechanism protecting the cells against reactive oxygen species.     

Elucidation of lead-induced oxidative stress in Talinum triangulare roots by analysis of antioxidant responses and DNA damage at cellular level

Hydroponic experiments were performed with Talinum triangulare (Jacq.) Willd. focusing the root cellular biochemistry with special emphasis on DNA damage, structural, and elemental analyses in Pb(NO₃)₂ exposed with 0, 0.25, 0.5, 0.75, 1.0, and 1.25 mM for 7 days. Lead (Pb) increased reactive oxygen species production, lipid peroxidation, protein oxidation, cell death, and DNA damage and decreased the protein content in a dose-dependent manner. Likewise, a dose-dependent induction of antioxidative enzymes superoxide dismutase and catalase by Pb was evident. Ascorbate peroxidase on the other hand responded biphasically to Pb treatments by showing induction at low (0.25 and 0.50) and repression at high (0.75–1.25 mM) concentrations. The estimation of proline content also indicated a similar biphasic trend. Scanning electron microscope and energy-dispersive X-ray spectroscopy analysis showed that 1.25 mM Pb treatment resulted in ultrastructural modifications in roots and stem tissue that was marked by the change in the elemental profile. The findings pointed to the role of oxidative stress in the underlying Pb phytotoxicity and genotoxicity in T. triangulare.     

Effects of long-term alachlor exposure on hepatic antioxidant defense and detoxifying enzyme activities in crucian carp (Carassius auratus)

Alachlor has been widely used in agriculture all over the world. It is suggested that it may be a carcinogen and also an environmental estrogen. In this paper, the physiological and biochemical perturbations of crucian carp (Carassius auratus) exposed to alachlor at different concentrations over 60 days were investigated. The gonadosomatic index (GSI) and hepatosomatic index (HSI) were measured. The activity of hepatic antioxidant defense and detoxifying enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) and the content of glutathione (GSH) were determined and compared with the control group. The result showed that GSI and HSI decreased significantly (P<0.05) in almost all treatments. The activities of SOD, CAT and GST were induced continuously (P<0.05), while the content of reduced glutathione (GSH) was inhibited on the whole. These changes reflect that the antioxidant systems of the tested fishes were affected. The possible defense mechanistic implications about the changes were thus discussed. Furthermore, hepatic SOD and GST were sensitive to alachlor at low concentration, indicating that they might be potential biomarkers in early detection of alachlor contamination in aquatic ecosystems.     

Influence of several fungicides on the antioxidant activity of red wines (var. Monastrell)

The antioxidant activity of wines obtained from grapes treated with six fungicides (famoxadone, fenhexamid, fluquinconazole, kresoxim-methyl, quinoxyfen and trifloxystrobin) was investigated. Two field trials in triplicate were carried out for each formulation of the fungicide at the recommended dose of the manufacturer. The first trial was carried out under good agricultural practices (GAP), following the recommended pre-harvest interval, and the second one under critical agricultural practices (CAP) that involves treating the same field just before the harvest. The residue levels were determined by gas and liquid chromatography coupled to mass detectors (GC-MS and LC-MS). The antioxidant activity was determined in the wines obtained from the thirteen trials including one control, six from treated grapes obeying the pre-harvest interval, and six from grapes treated at the day of harvest or at most unfavorable conditions. Elimination of 40–100% of the initial fungicide residues present in grapes was observed during the wine-making process. It can be inferred from the results that the use of these fungicides did not produce any decrease of the antioxidant activity in the wines (7.19 ± 0.22 mmol Trolox/L for the blank wine versus a range of 6.45 ± 0.82 mmol Trolox/L to 10.06 ± 0.59 mmol Trolox/L for the treated wines) at the pre-harvest interval and most unfavorable conditions. Nevertheless, the presence of famoxadone, kresoxim-methyl and quinoxyfen increased the antioxidant activity and this was directly related to their residue levels in the grapes. Also, the wine phenolic composition was altered in variable intensity by the presence of the fungicide residues.     

Short-time exposure to mono-n-butyl phthalate (MBP)-induced oxidative stress associated with DNA damage and the atrophy of the testis in pubertal rats

Phthalates are widely used as plasticizer in various consumer domestic products and are known to disturb the male reproductive function in rodents. This study investigated the involvement of oxidative stress and the atrophy of the testes in pubertal rats exposed to mono-n-butyl phthalate (MBP). Four-week-old pubertal male rats were separated into three groups. In group I, 21 rats were fed rat chow containing 2 % MBP for 3 days. In group II, 21 rats were fed rat chow containing 2 % MBP for 3 days and antioxidant vitamins C (250 mg/kg/day) and E (50 mg/kg/day) were injected daily. In group III, 21 rats were fed standard rat chow and used as controls. After 3 days, each testis was weighed and the germ cell development was evaluated using the Johnsen score. The urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels were measured as a biological marker of oxidative DNA damage. The mean testis weight was significantly lower for group I than groups II or III (p?<?0.05). The mean Johnsen score was significantly lower for group I than for groups II or III (p?<?0.05). Urinary 8-OHdG concentrations were higher in group I than in groups II or III. Short-time exposure to MBP may therefore induce oxidative DNA damage in rat testes, while antioxidant vitamins administered during exposure may protect against this stress.     

The detoxifying effect of Polygonum equisetiforme extracts against dichlorvos (DDVP)-induced oxidative stress and neurotoxicity in the commercial clam Ruditapes decussatus

Environmental Science and Pollution Research - Effects of Polygonum equisetiforme extracts against dichlorvos were investigated in the commercial clam Ruditapes decussatus. The toxicity of this...