Evaluation of transcriptional biomarkers using a high-resolution regression approach: Concentration-dependence of selected transcripts in copper-exposed freshwater mussels (Anodonta anatina).

We tested concentration-dependence of selected gene transcripts (cat, gst, hsp70, hsp90, mt and sod) for evaluation as biomarkers of chemical stress. Contrary to the common approach of factorial designs and few exposure concentrations, we used regression across a high-resolution concentration series. Specifically, freshwater mussels (Anodonta anatina) were acutely (96 h) exposed to Cu (13 nominal concentrations, measuring 0.13-1 600 µg/L), and transcripts were measured by RT-qPCR. In digestive glands, cat, hsp90 and mt decreased with water Cu (p < 0.05), but response magnitudes saturated at < 2-fold decreases. In gills, gst, hsp70, hsp90 and mt increased with water Cu (p < 0.05). While hsp70, hsp90 and mt exceeded 2-fold increases within the exposure range, high Cu concentrations were required (38-160 µg/L). Although gill responses were generally more robust compared to digestive glands, overall small response magnitudes and moderate sensitivity may set limit for potential application as general biomarkers of chemical stress.

Host-parasite interaction as a toxicity test endpoint using asymmetrical exposures.

Interspecific relationships frequently determine the effect a pollutant can have on an organism, and this is especially true in closely interacting species such as hosts and parasites. The high spatial and temporal variability of contaminant concentrations combined with the movement of aquatic biota can further influence the consequences that are associated with contamination. We used a full factorial design for the exposed and unexposed partners of the relationship between the parasitic larvae (glochidia) of the European freshwater mussel (Anodonta anatina) and its host fish (Squalius cephalus) to identify the sources of variation in the sublethal endpoints of species interaction (the intensity of parasite attachment, the spatial position of glochidia on the host body, and encapsulation success). We used the water-borne human pharmaceutical compounds methamphetamine (a central nervous system stimulant) and tramadol (an opioid) at environmentally relevant concentrations (˜ 6.7 and 3.8 nmol L-1 of methamphetamine and tramadol, respectively) as a proxy for contaminant exposure because these compounds are emerging aquatic stressors that are known for high spatial and temporal variability in their detected concentration levels. The relationship between the bivalve and the fish species was influenced by the preceding contact with both methamphetamine and tramadol, but this effect was highly asymmetric. Our experimental design enabled us to identify the specific changes in the relationship outcome that are elicited by the exposure of individual partners, such as the significant increase in glochidia infection success rate from 59.6 ± 3.9% to 78.7 ± 2.8% (means ± s.e.) that was associated with host exposure to methamphetamine. Additionally, the significant interaction effect of the exposure was demonstrated by the lowered proportion of glochidia attached to gills after the coexposure of both partners to tramadol. The impact of pharmaceuticals on wild aquatic host-parasite relationships provides an example of the risks that are associated with the unintentional discharge of biologically active compounds into freshwater habitats. Given the increasing evidence showing the ecological impact of waste pharmaceuticals, the use of multitrophic interaction endpoints after joint and unilateral exposures provides an important step towards the realistic risk assessment of these compounds.

Copper Bioaccumulation Kinetics in Swan Mussel, Anodonta cygnea (Linnaeus, 1758) During Waterborne Exposure to CuO Nanoparticles.

This study was conducted to investigate bioaccumulation of copper in two internal organs (mantle and foot) of swan mussel, Anodonta cygnea (Linnaeus, 1758) in exposure to copper oxide nanoparticles (CuO NPs). Basal concentration of Cu in the mantle (3.15 ± 1.09 µg g-1 DW) was significantly (p < 0.05) lower than the foot (5.43 ± 1.54 µg g-1 DW). At the end of the exposure period, the highest concentration of Cu in both organs belonged to the highest exposure concentration. Calculated bioconcentration factor (BCF) values showed significant (p < 0.05) higher values for the mantle in each day and each exposure concentration (except the lowest exposure concentration) than the foot. For both organs, the highest and lowest BCFs occurred at the lowest and highest exposure concentrations, respectively. Cu concentration in both organs was significantly (p < 0.05) decreased after day 4. Based on the results, it was obvious that exposure to sub-lethal concentrations of CuO NPs would lead to the significant accumulation of copper in mantle and foot that may have adverse effects on this organism.

Isolation and characterization of two glutathione S-transferases from freshwater bivalve Anodonta woodiana: Chronic effects of pentachlorophenol on gene expression profiles.

Glutathione S-transferases (GST) play a prominent role in protecting cells against oxidative stress. Our previous study showed that the reactive oxygen species (ROS) generated from pentachlorophenol (PCP) could cause an acute impact on freshwater bivalve Anodonta Woodiana, but its chronic toxicity remain unclear. In order to investigate the chronic effect of PCP, clams A. Woodiana were randomly grouped into PCP treated group in which animals were administrated with 13.9 µg/L concentrations of PCP, and control group those with similar volume dimethyl sulfoxide. In addition, two complete GST sequences were isolated from A. Woodianaa and respectively named AwGST1 and AwGST2. The full-length cDNA of AwGST1 was consisted of a 5' untranslated region (UTR) of 132 bp, a 3' UTR of 80 bp and an open reading frame (ORF) of 609 bp encoding a polypeptide of 203 amino acids. The full-length cDNA of AwGST2 contained a 5' UTR of 57 bp, a 3' UTR of 291 bp and an ORF of 678 bp encoding a polypeptide of 226 amino acids. The constitutive expression levels of AwGST1 and AwGST2 were examined in different tissues including foot, mantle, adductor muscle, heart, hepatopancreas, hemocytes and gill. Administration of PCP could result in a significant increase of AwGST1 and AwGST2 expression in the hepatopancreas, gill and hemocytes. In the hepatopancreas, AwGST1 mRNA levels of PCP treated group increased more than 28.73% at day 1, then 70.37% (P < 0.05) at day 3, reach to 6.64 times (P < 0.01) at day 15 in contrasted with that of control group. AwGST2 increased more 18.18%, 82.88% (P < 0.05) and 2.43 times (P < 0.01) at day 1, 3 and 15, respectively. In the gill, AwGST1 expression showed a significant up-regulation in the PCP treated group during experiment observed compared with that of control group, mRNA level of AwGST2 increased more than 1.44 times (P < 0.05). In addition, expressions of AwGST1 and AwGST2 were significantly induced after PCP treatment in the hemocytes. These results indicated that up-regulations of AwGST1 and AwGST2 expression in bivalve A. woodiana are contribute to against oxidative stress derived from PCP treatment during experiment observed.

Effects of Calcium and Magnesium Ions on Acute Copper Toxicity to Glochidia and Early Juveniles of the Chinese Pond Mussel Anodonta woodiana.

We evaluated the effects of calcium (Ca(2+)) and magnesium (Mg(2+)) ions on copper (Cu) toxicity to glochidia and newly-transformed juvenile mussels (age 1-2 days) of the Chinese pond mussel (Anodonta woodiana). Acute Cu toxicity tests were performed with glochidia for 24 h and juveniles for 96 h with measured Ca(2+) concentrations of 1.1, 14, 26, 51, and 99 mg L(-1), or measured Mg(2+) concentrations of 2.6, 11, 21, and 39 mg L(-1). The Ca(2+) and Mg(2+) cations provided no statistically significant protection against Cu toxicity to glochidia or juveniles. The 24-h EC50 value for glochidia was 82 µg L(-1) Cu, and contrastly, 96-h EC50 value for newly-transformed juvenile mussels was as low as 12 µg L(-1) Cu, implying that the juveniles of A. woodiana are more vulnerable to Cu contamination at concentrations close to currently-accepted levels.

Response of selenium-dependent glutathione peroxidase in the freshwater bivalve Anodonta woodiana exposed to 2,4-dichlorophenol,2,4,6-trichlorophenol and pentachlorophenol.

2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP) pose a health risk to aquatic organism and humans, and are recognized as persistent priority pollutants. Selenium dependent glutathione peroxidase (Se-GPx) belongs to the family of selenoprotein, which acts mainly as an antioxidant role in the cellular defense system. In the current study, a Se-GPx full length cDNA was cloned from Anodonta woodiana and named as AwSeGPx. It had a characteristic codon at 165TGA167 that corresponds to selenocysteine(Sec) amino acid as U44. The full length cDNA consists of 870 bp, an open reading frame (ORF) of 585 bp encoded a polypeptide of 195 amino in which conserved domain (68LGFPCNQF75) and a glutathione peroxide-1 GPx active site (32GKVILVENVASLUGTT47) were observed. Additionally, the eukaryotic selenocysteine insertion sequence (SECIS) was conserved in the 3'UTR. The AwSeGPx amino acid sequence exhibited a high similarity with that of other Se-GPx. Real-time PCR analysis revealed that AwSeGPx mRNA had a widely distribution, but the highest level was observed in hepatopancreas. AwSeGPx mRNA expression was significantly up-regulated in hepatopancreas, gill and hemocytes after 2,4-DCP, 2,4,6-TCP and PCP exposure. Under similar environment, clams A. woodiana showed a more sensitive to PCP than that of 2,4-DCP and 2,4,6-TCP. These results indicate that AwSeGPx plays a protective role in eliminating oxidative stress derived from 2,4-DCP, 2,4,6-TCP and PCP treatment.

Molecular cloning, characterization, and the response of Cu/Zn superoxide dismutase and catalase to PBDE-47 and -209 from the freshwater bivalve Anodonta woodiana.

Polybrominated diphenyl ethers-47 (PBDE-47) and -209 are significant components of total PBDEs in water and can catalyze the production of reactive oxygen species (ROS) in the organisms. Anti-oxidant enzymes play an important role in scavenging the high level of ROS. In the current study, two full-length cDNAs of Cu/Zn superoxide dismutase (CuZnSODs) and catalase (CAT) were isolated from freshwater bivalve Anodonta woodiana by rapid amplification of cDNA ends approach and respectively named as AwSOD and AwCAT. The nucleotide sequence of AwSOD cDNA had an open reading frame (ORF) of 465 bp encoding a polypeptide of 155 amino acids in which signature 1 GKHGFHVHEFGDNT and signature 2 GNAGARSACGVI of SODs were observed. Deduced amino acid sequence of AwSOD showed a significant similarity with that of CuZnSODs. AwCAT had an ORF 1536 bp encoding a polypeptide of 512 amino acids which contains a conserved catalytic site motif, and a proximal heme-ligand signature motif of CATs. The time-course expressions of AwSOD and AwCAT in hepatopancreas were measured by quantitative real-time PCR. Expressions of AwSOD and AwCAT showed a significant up-regulation in groups at a low concentration treatment of PBDE-47, a biphasic pattern in groups with a high concentration treatment. Administration of PBDE-209 could result in an up-regulation of AwSOD and AwCAT expressions with time- and dose-dependent matter. These results indicate that up-regulations of AwSOD and AwCAT expression of hepatopancreas of freshwater bivalve A. woodiana contribute to eliminate oxidative stress derived from PBDE-47 and -209 treated.

2,4-Dichlorophenoxyacetic acid alters intracellular pH and ion transport in the outer mantle epithelium of the bivalve Anodonta cygnea.

Bivalve molluscs, due to their sedentary mode of life and filter-feeding behavior, are very susceptible to pollutant bioaccumulation and used as sentinel organisms in the assessment of environment pollution. Herein we aimed to determine the in vivo, ex vivo and in vitro effects of 2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, in Anodonta cygnea shell growth mechanisms. For that, we evaluated the effect of 2,4-D (100 µM) exposure on the transepithelial short-circuit current (Isc), potential (Vt) and conductance (Gt), as well as on OME ion transport systems and intracellular pH (pHi). In vivo exposure to 2,4-D caused an increase of 50% on the Isc generated by OME and ex vivo addition of that compound to the apical side of OME also induced an Isc increase. Furthermore, 2,4-D was able to cause a pHi increase in isolated cells of OME. Noteworthy, when 2,4-D was added following the exposure to specific inhibitors of several membrane transporters identified as responsible for pHi maintenance in these cells, no significant effect was observed on pHi except when the V-type ATPase inhibitor was used, indicating an overlap with the effect of 2,4-D. Thus, we concluded that 2,4-D is able of enhancing the activity of the V-ATPases present on the OME of A. cygnea and that this effect seems to be due to a direct stimulation of those H(+) transporters present on the apical portion of the membrane of OME cells, which are vital for shell maintenance and growth. This study allows us to better understand the molecular mechanisms behind 2,4-D toxicity and its deleterious effect in aquatic ecosystems, with particular emphasis on those involved in shell formation of bivalves.

Effects of non-steroidal estrogen diethylstilbestrol on pH and ion transport in the mantle epithelium of a bivalve Anodonta cygnea.

Freshwater bivalves are used as sentinel organisms to detect pollutants effects in the aquatic environment due to their sedentary nature, filter-feeding behaviour. We aimed to determine the in vivo, ex vivo and in vitro influence of Diethylstilbestrol (DES), a widely used synthetic non-steroidal estrogen and endocrine disruptor, in Anodonta cygnea shell growth mechanisms. For that, in vivo exposure to DES (0.75µM) during 15 days, in vitro and ex vivo exposure of outer mantle epithelium (OME) cells to DES (0.75µM), were performed followed by study of short-circuit current (Isc), transepithelial potential (Vt) and transepithelial conductance (Gt) as well as identification of membrane transport systems and intracellular pH (pHi). Our results show that in vivo exposure to DES decreases in 30% the OME Isc and ex vivo addition of DES to the basolateral side of OME also induced Isc decrease. Several membrane transporters such as V-type ATPases, Na(+)/H(+) exchangers, Na(+)-K(+) pump, Na(+)-driven and Na(+)-independent HCO3(-)/Cl(-) transporters and Na(+)/HCO3(-) co-transporter were identified as responsible for pHi maintenance in OME and noteworthy, DES caused a pHi decrease in OME cells similar to the effect observed when OME cells were exposed to 4,4'-diisothiocyanostilbene disulfonic acid (DIDS), an inhibitor of several bicarbonate membrane transporters. The addition of DIDS after OME cells exposure to DES did not cause any alteration. We concluded that DES is able to modulate membrane ion transport and pHi in the OME of A. cygnea and that this effect seems to be due to inhibition of HCO3(-)/Cl(-) co-transporters present on the basolateral membrane.

[Regulation of adenylyl cyclase signaling system by insulin, biogenic amines, and glucagon at their separate and combined action in the muscle membranes of the mollusc Anodonta cygnea].

In smooth muscles of mollusc Anodonta cygnea, hormones produce regulatory effects on the adenylyl cyclase (AC) signaling system via receptors of the serpentine (biogenic amine, isoproterenol, glucagon) and of tyrosine kinase (insulin) types. Intracellular mechanisms of their action are interconnected. Use of hormones, their antagonists, and pertussis toxin at the combined action of insulin and biogenic amines or of glucagon on the AC activity allows revealing possible intersection points in mechanisms of their action. The combined effect of insulin and serotonin or of glucagon leads to a decrease of stimulation of AC by these hormones, whereas at action of insulin and isoproterenol the AC-stimulatory effect of insulin is blocked, while the AC-inhibitory effect of isoproterenol is preserved both in the presence and in the absence of the non-hydrolyzed GTP analog - guanylylimidodiphosphate (GppNHp). Specific blocking of the AC-stimulatory serotonin effect by cyproheptadine - an antagonist of serotonin receptors - did not affect stimulation of AC by insulin. Beta-adrenoblockers (propranolol and alprenolol) interfered with inhibition of the AC activity by isoproterenol, but did not change the AC stimulation by insulin. Pertussis toxin blocked the AC-inhibitory effect of isoproterenol and attenuated the AC-stimulatory effect of insulin. Thus, in muscles of the mollusc Anodonta cygnea there have been revealed negative interrelations between the AC system, which are realized at the combined effect of insulin and serotonin or of glucagon, probably at the level of receptor of the serpentine type (serotonin, glucagon), while at action of insulin and isoproterenol - at the level of interaction of G1 protein and AC.

In situ exposure history modulates the molecular responses to carbamate fungicide Tattoo in bivalve mollusk.

The aim of the present study was the investigation of the effect of in situ exposure history on the responses of freshwater mussels to thiocarbamate fungicide. Male bivalve mollusks Anodonta anatina (Unionidae) from polluted (A) and unpolluted (F) sites were subjected to 14 days of exposure to fungicide Tattoo (mixture of propamocarb and mancozeb, 91 µg L(-1)). When unexposed mussels were compared, chronic effect of toxic environment in site A was confirmed by oxidative stress indices (high levels of superoxide dismutase and catalase activities, lipid peroxidation, protein carbonyls and oxyradical production, low level of total glutathione (GSH)), genotoxicity (high levels of DNA-strand breaks and caspase-3 activity in digestive gland), and cytotoxicity (low lysosomal membrane stability in hemocytes), elevated vitellogenin-like proteins (Vtg-LP) concentration in gonads, high levels of Cu, Zn, Cd, metallothionein (MT)-bound metals (MT-Me) and MT-related thiol (MT-SH), and low ethoxyresorufin-O-deethylase (EROD) activity in digestive gland. The major differences in the responses of the two exposed groups were related to antioxidant defense and MT: in the group A, prominent oxidative stress response with the participation of MT-SH and GSH in the gills, EROD activation, but decrease of MT-Me level was shown, whereas in group F exposure provoked the elevation of MT-Me, caspase-3 and Vtg-LP values. Carbamate did not cause cholinesterase depletion and cytotoxicity. However, genotoxic and pro-oxidant effects (increased levels of hemocytes with micronuclei and nuclear abnormalities, DNA-strand breaks and oxyradical in digestive gland), were common responses for both the exposed groups.

Effect of in situ exposure history on the molecular responses of freshwater bivalve Anodonta anatina (Unionidae) to trace metals.

The goal of the study was to assess the adequacy of molecular responses in mollusks in relation to their in situ exposure history. Freshwater male bivalve mollusks Anadonta anatina (Unionidae) from polluted (A) and unpolluted (F) sites were subjected to 14 days of exposure to copper (Cu(2+), 10 µg L(-1)), zinc (Zn(2+), 130 µg L(-1)) or cadmium (Cd(2+), 15 µg L(-1)). The comparison of two control groups showed that the specimens from site A had higher levels of Cu, Zn and Cd and metallothionein (measured both through metal (MT-Me), and protein (MT-SH) levels) in the tissues. Cytotoxicity (low lysosomal membrane stability), low glutathione level, high antioxidant and apoptotic enzymes activities, lipid and protein oxidative injury, depletion of ethoxyresorufin-O-deethylase (EROD) in digestive gland, high vitellogenin-like protein (Vtg-LP) concentration in gonads confirmed the effect of toxic environment on this group. Exposures provoked increased number of hemocytes with micronuclei (by 100-500%) and nuclear abnormalities (by 50-400%) (genotoxicity), elevation of caspase-3 (in 1.5-10 times) and/or Vtg-LP (by 70-310%) levels in all groups. However, the responses were strongly dependent on the origin of mussels. Exposed mussels from site F demonstrated typical for the effect of toxic metals elevation of MT-SH (by 100-380%) and MT-Me (up to seven times) levels and accumulation of metals (with a few exceptions) in the tissues. Conversely, in the mussels inhabiting site A, exposures caused the decrease of metal (by 37% for Cu, by 62% for Zn, by 50% for Cd), MT-SH (by 68% in ZnA group) and MT-Me (by 50-68%) levels. That was accompanied with increase of cytotoxicity and EROD activity (by 144-240%). High level of protein carbonyls was the distinguished feature of all groups from site A. Hence, despite high efficiency of metal detoxification and oxidative stress responses in the mussels, in the specimens from spontaneously polluted site they were impaired.

Main partitioning criteria for the characterization of the health status in the freshwater mussel Anodonta cygnea from spontaneously polluted area in Western Ukraine.

The aim of this study was to appreciate the consequences of spontaneous human activity for freshwater mollusks in the generally ecologically sustainable area in Western Ukraine. For this, bivalve mollusk, Anodonta cygnea, at three sites, with mixed agricultural and municipal activities (A), close to a municipal water inlet (F) and the cooling pond of a nuclear power plant (N), were studied in spring, summer, and autumn. The set of parameters included the characteristics of oxidative stress (activity of catalase (CAT), levels of protein carbonyls (PC)), levels of reduced and oxidized glutathione (GSH, GSSG, respectively), activities of lactate dehydrogenase (LD), cholinesterase (ChE), ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST) in the digestive gland, and concentrations of vitellogenin-like proteins (Vtg-LP) in gonads and also morphological indices. Although the discriminant functional analysis confirmed the general seasonal regularities for studied groups, it allowed to discriminate between sites (P < 0.05). At site A, oxidative stress; high levels of LD, EROD, and GST; and low levels of ChE and condition factor were reflected. This demonstrated the sensitivity of mussels to constant effect of mixed pollution. At site N, oxidative injury was shown that might be explained by the constantly high temperature. At site F, abrupt elevations of Vtg-LP and EROD levels in autumn were probably related to an emergency situation on the nearby dump. So, both chronic and temporal environmental effects were reflected by a set of markers in mollusk. The classification and regression tree (CART) algorithm selected GSH and PC in the digestive gland and Vtg-LP as partitioning criteria for the characterization of mussel health status. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

[Evaluation of biological effects of cobalt-nanocomposites with the use of biochemical markers of bivalve mollusk Anodonta cygnea].

Intensive implementation of nanomaterials requires development of novel methods for evaluation of their potential ecotoxicity. The aim of our study was to identify specific characteristics of the effect of cobalt-nanocomposite (Co-NC) on the molecular stress-responsive system in the digestive gland of bivalve mollusk Anodonta cygnea. Nanocomposite was synthesized by mixing alcohol solution of copolymer N-vinylpirrolidone, 5-(tret-butylperoxy)-5-methyl-1-hexene-3-yne and dimethylaminoethylmetacrylate and cobalt (II) chloride. After 14 days of the mollusk exposure in the presence of Co-NC, CoCl, or corresponding polymer substance it was shown that the Co-NC, in contrast to other agents, does not cause an oxidative stress due to the superoxide dismutase activity, metallotioneins (MTs) level, glutathione redox index and oxyradical production. Multivariate analysis confirmed specific features of the Co-NC's effect related to an enhanced expression of MTs, while CoCl2 activated lactate dehydrogenate and oxyradical production, and polymer substance enhanced glutathione transferase activity.

Vulnerability of biomarkers in the indigenous mollusk Anodonta cygnea to spontaneous pollution in a transition country.

The aim of this study was to estimate the sensitivity of biomarkers of stress and exposure in the bivalve mollusk Anodonta cygnea to spontaneous anthropogenic activities typical for the Western Ukraine. Three sites were examined during spring, summer and autumn: an agricultural site (A); the cooling pond of nuclear power plant (N) and a forestry close to the municipal water inlet (F). Common temporal changes of a battery of biochemical markers in the gills and hemolymph and morphological characteristics were shown by discriminant functional analysis. Classification trees built on the basis of the screened biomarkers demonstrated persistent peculiarities at each site: genotoxicity (nuclear abnormalities) at site A and endocrine disruption (high levels of vitellogenin-like proteins (Vtg-LP) in hemolymph) at site F. Interim local effects were best characterized by metallothionein (MT) concentrations, lipid peroxidation (LPO), activities of glutathione S-transferase and lactate dehydrogenase, and the conditional index of the gills. In autumn, the mollusks from the three sites revealed the highest differences in pollution status: the activation of antioxidant defense and cholinesterase were typical for site A, highest levels of MT related to high levels of Cu and Cd in the water at site B, and a steep increase in the level of Vtg-LP and the decrease of lysosomal membrane stability were recorded at the site selected as reference (F). The biomarker alterations recorded at site F were later related to an emergency event at the municipal dump located nearby. Thus, our case study demonstrated the reliability of using biomarkers of exposure to assess both long-term and accidental environmental pollution loads.

A comparative study on the uptake of polycyclic aromatic hydrocarbons by Anodonta californiensis.

Uptake of polycyclic aromatic hydrocarbons (PAHs) by the freshwater bivalve mollusc Anodonta californiensis was examined in the presence and absence of surfactant in order to gain further insight into mixture toxicity and to predict whether certain mixtures have negative and/or positive effects on aquatic organisms. In the presence of surfactant, the uptake of anthracene or chrysene was higher than that of naphthalene, given the same concentration in the solution. In the absence of surfactant, the trend was similar, but the uptakes were increased by approximately 100% compared to those in the presence of surfactant. On the uptake of naphthalene, the presence of anthracene showed only minor influence. The uptake of anthracene was affected by both naphthalene and chrysene. The uptake of chrysene was influenced by neither naphthalene nor anthracene. There was no observable displacement of divalent cations from the surface of the gill membrane by any of the PAHs studied.

Multi-biomarkers approach in different organs of Anodonta cygnea from the Dnister Basin (Ukraine).

The aim of this study was to compare environmental quality in two sites in western Ukraine-rural (R) and urbanized (U)-with the usage of the resident bivalve mollusk Anodonta cygnea. The study was realized during three seasons. The metal uptake and a set of biochemical markers were determined. For each season, Cd and metallothioneins (MTs) contents in the digestive gland and gills of the mollusc were higher at the U site, reflecting its chronic pollution. The oxidative stress in the mollusk was observed at the U site during spring and at the R site during summer and autumn according to the differences in Mn-superoxide dismutase and catalase activities, O (2) (*-) production, lipid peroxidation, and glutathione levels. The elevated vitellogenin-like protein levels in the hemolymph and the ethoxyresorufin-O-deethylase activity in the digestive gland in summer-autumn suggested pollutions by organic substances at the R site. The acetylcholinesterase activity was similar in both groups. The centroid grouping analysis of biomarkers and morphological and water indexes demonstrated the clear differentiation of general response in each group in spring and, at the R site, in summer and autumn but its similarity at the U site in summer and autumn.

[The effect of pesticide acetamiprid on biochemical markers in tissues of fresh water bivalve mussels Anodonta cygnea L. (Unionidae)].

The effects of ecological real concentration of pesticide "Mospilan" (active substance - acetamiprid) on the status of oxidative stress biomarkers, neurotoxicity and metallothioneins' (MTs) characteristics of freshwater bivalve mussels Anodonta cygnea were investigated. The prooxidative changes (decrease of superoxide dismutase activity, reduced glutathione content, increase of content of lipid and proteins peroxidation products) and decrease of the MTs content in gills and especially in digestive gland in the experimental group were reflected, whereas acetylcholinesterase activity was not changed. The effect of acetamiprid induces the appearance of additional chromatographic MTs form.

Effects of aqueous aluminium on four fish ectoparasites.

Effects of aqueous aluminium on four species of fish ectoparasites were studied. Fish infected by Gyrodactylus derjavini, G. macronychus and Anodonta anatina glochidia, and free living Argulus foliaceus were exposed to acidic Al-rich water (pH 5.8), acidic Al-poor water (pH 5.8) and control water (pH 6.3). The results showed that aqueous aluminium had a negative effect on the ectoparasites tested, without any apparent negative effect on the fish hosts. The effect of aluminium was dependent on the kind of organism tested. The gyrodactylids were eliminated, while the duck mussel glochidia showed a decrease in abundance when exposed to 200-260 mug Al/l at pH 5.8. The little fish louse showed increased mortality rate when exposed to aluminium. The effects of aluminium on the parasites in the present study confirm the importance of water quality in general, and acidification in particular, for the distribution and population dynamics of ectoparasites in freshwater environments.

The presence of microcystins and other cyanobacterial bioactive peptides in aquatic fauna collected from Greek freshwaters.

Toxic bloom-forming cyanobacteria can cause animal death and adversely affect human health. Blooms may contain microcystins (MCs), cyanobacterial heptapeptide hepatotoxins and other peptides such as anabaenopeptins and anabaenopeptilides. MCs have been shown to occur in various aquatic organisms including mussels, water snails, crustaceans and fish. Muscle and viscera samples from eight species of fish (Acipenser gueldenstaedtii, Carassius auratus, Carassius gibelio, Cyprinus carpio, Perca fluviatilis, Rutilus rubilio, Silurus aristotelis and Silurus glanis), a frog (Rana eperotica), a mussel (Anodonta sp.) and a water snail (Viviparus contectus) were analyzed by high-performance liquid chromatography (HPLC), protein phosphatase 1 (PP1) inhibition assay (PP1IA) and ELISA. MC(s) was detected in all fish, frog, mussel and water snail samples tested by PP1IA and ELISA, including the frog R. eperotica and the freshwater snail V. contectus, in which the occurrence of MCs was not previously known. MC concentration ranged from 20 to 1500 ng g(-1)dw and from 25 to 5400 ng g(-1)dw in muscle and visceral tissue of fishes and frogs, respectively. In mussel and water snail tissue MC concentration ranged from 1650 to 3495 ng g(-1)dw. HPLC analysis revealed peaks having the same UV spectrum as anabaenopeptin- or anabaenopeptilide-like compounds, not previously known to occur in aquatic fauna tissue. The concentrations of the compounds detected ranged from 1.5 to 230 microg g(-1)dw. Comparison of the PP1IA and ELISA showed that values obtained with PP1IA where higher than those obtained with ELISA. Anabaenopeptins and/or anabaenopeptilides occurring in faunal tissue may account for the higher PP1IA values as we found that PP1 activity was inhibited by the purified anabaenopeptins A (45-60% inhibition) and B (5-75% inhibition). Purified anabaenopeptilides 90A and 90B exhibited weaker PP1 inhibition activity (5-35 and 5-23% inhibition, respectively). This is the first report of MC occurrence in aquatic animals collected from freshwaters of southern Europe.