Ecocytological and toxicological responses to copper in Perna viridis (L.) (Bivalvia: Mytilidae) haemocyte lysosomal membranes.

Bivalve lysosomes are sites of intense intracellular digestion. Lysosomes accumulate many pollutants to high concentrations resulting in membrane destabilisation. Consequently, the elucidation of lysosomal membrane integrity utilising the neutral red assay has been used to good effect in pollution monitoring. Naturally occurring environmental stressors also have the potential to destabilise the membrane. Exposure to elevated copper concentrations and extremes of temperature, salinity, hypoxia, emersion and inadequate ration were investigated in haemocyte lysosomes from the tropical bivalve, Perna viridis. Elevated copper concentrations destabilised the membrane although responses were not entirely related to the exposure-concentration. Environmental stressors induced through higher thermal regimes (29 degrees C and 35 degrees C), hyposalinity (10-25/1000) and prolonged emersion elicited significant lysosomal membrane destabilisation. Hypoxia and inadequate ration did not significantly effect membrane stability. The haemocyte lysosomal membranes were generally resistant to exogenous alterations within normal ranges and only showed significant labilisation at environmental extremes. P. viridis haemocyte lysosomal membrane biomarkers should, therefore, prove robust to natural stressors when deployed in marine monitoring programmes and thus prove a valuable, rapid, cost-effective cytological marker of pollution.     

Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index

Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density - Vv - and labilisation period -LP) and combined (lysosomal response index - LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference = 0; critical threshold = 1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists.     

Biochemical and lysosomal biomarkers in the mussel <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> from the Mar Piccolo of Taranto (Ionian Sea,Southern Italy)

Biomarkers are internationally recognized as useful tools in marine coastal biomonitoring, in particular, as early-warning signals at the level of individual organisms to assess biological effects of pollutants and other stressors. In the present study, Mytilus galloprovincialis has been employed as a sentinel organism to assess biological pollution effects in the Mar Piccolo of Taranto (Southern Italy), a coastal lagoon divided into two small inlets, connected to the open sea through one natural and one artificial narrow openings. Mussels were collected in June 2013 at three sites located within each of the two inlets of the Mar Piccolo. Biological effects were investigated through a suite of biomarkers suitable to reflect effects and/or exposure to contaminants at biochemical and cellular levels. Biochemical biomarkers included glutathione-S-transferase (GST) and acetylcholinesterase (AChE) enzyme activities; as histochemical biomarkers, lysosomal membrane stability, lipofuscin and neutral lipid accumulation, and lysosomal structural changes were considered. As a whole, results highlighted differences among the three study sites, particularly for GST, AChE, and lipofuscins, which are consistent with the variations of the chemical pollutants in sediments. The applied biomarkers showed that a stress syndrome likely to be ascribed to environmental pollutants is occurring in mussels living in the Mar Piccolo of Taranto, in particular, the ones inhabiting the first inlet.     

Biological effects of palytoxin-like compounds from Ostreopsis cf. ovata: A multibiomarkers approach with mussels Mytilus galloprovincialis

Massive blooms of the harmful benthic dinoflagellate Ostreopsis cf. ovata are of growing environmental concern in the Mediterranean, having recently caused adverse effects on benthic invertebrates and also some intoxication episodes to humans.The toxicological potential of produced palytoxin-like compounds was investigated in the present study on a typical marine sentinel species, the mussel Mytilus galloprovincialis. Organisms were sampled during various phases of a O. cf. ovata bloom, in two differently impacted sites. The presence of the algal toxins was indirectly assessed in mussels tissues (mouse test and hemolysis neutralization assay), while biological and toxicological effects were evaluated through the measurement of osmoregulatory and neurotoxic alterations (Na⁺/K⁺-ATPase and acetylcholinesterase activities), oxidative stress responses (antioxidant defences and total oxyradical scavenging capacity), lipid peroxidation processes (level of malondialdehyde), peroxisomal proliferation, organelle dysfunctions (lysosomal membrane stability, accumulation of lipofuscin and neutral lipids), immunological impairment (granulocytes percentage).Obtained results demonstrated a significant accumulation of algal toxins in mussels exposed to O. cf. ovata. These organisms exhibited a marked inhibition of the Na⁺/K⁺-ATPase activity and alterations of immunological, lysosomal and neurotoxic responses. Markers of oxidative stress showed more limited variations suggesting that toxicity of the O. cf. ovata toxins is not primarily mediated by an over production of reactive oxygen species. This study provided preliminary results on the usefulness of a multi-biomarker approach to assess biological alterations and toxicological events associated to blooms of O. cf. ovata in marine organisms.     

Effects of dioxin exposure in Eisenia andrei: integration of biomarker data by an Expert System to rank the development of pollutant-induced stress syndrome in earthworms

A battery of biomarkers has recently been developed in the earthworm Eisenia andrei. In this study, different biomarkers (i.e. Ca²⁺-ATPase activity, lysosomal membrane stability-LMS, lysosomal lipofuscin and neutral lipid content) were utilized to evaluate the alterations in the physiological status of animals, induced by exposure for 3 d to different sublethal concentrations of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) (1.5 × 10⁻³, 1.5 × 10⁻², 1.5 × 10⁻¹ ng mL⁻¹) utilizing the paper contact toxicity test. Lysosome/cytoplasm volume ratio and DNA damage were also evaluated as a biomarker at the tissue level and as a biomarker of genotoxicity, respectively. Moreover, the NR retention time assay conditions were optimized for the determination of in vivo LMS in earthworm coelomocytes. The results demonstrate that LMS and Ca²⁺-ATPase activity were early warning biomarkers able to detect the effects of minimal amounts of TCDD and that biomarkers evaluated at the tissue level are important for following the evolution of the stress syndrome in earthworms. To evaluate the health status of the animals, an Earthworm Expert System (EES) for biomarker data integration and interpretation was developed. The EES proved to be a suitable tool able to rank, objectively, the different levels of the stress syndrome in E. andrei induced by the different concentrations of TCDD.     

Effects of blood lipid lowering pharmaceuticals (bezafibrate and gemfibrozil) on immune and digestive gland functions of the bivalve mollusc, Mytilus galloprovincialis

Fibrates are hypolipidemic pharmaceuticals that have been detected as contaminants in wastewaters and surface waters. In this work, the possible effects of two fibrates, Bezafibrate (BEZA) and Gemfibrozil (GEM) in the bivalve mollusc Mytilus spp were investigated. In the immune cells, the hemocytes, addition of both compounds in vitro induced rapid lysosomal membrane destabilization, extracellular lysozyme release, NO production and decreased phagocytic activity. The effect of fibrates were partly mediated by activation of ERK and p38 MAPKs (Mitogen Activated Protein Kinases), as demonstrated by the use of specific inhibitors of different kinases. The effects of fibrates on hemocyte function were confirmed in vivo, in the hemocytes of mussels injected with 0.01, 0.1 and 1 nmol/animal (corresponding to nominal concentrations of 3.61, 36.18 and 361.8ng/g dry weight for BEZA and of 2.50, 25.03 and 250.35 ng/g dry weight for GEM, respectively) and sampled at 24h post-injection. Both compounds induced a concentration-dependent lysosomal destabilization and extracellular lysozyme release; an increase in phagocytosis was observed at the highest concentration. In vivo exposure to fibrates also induced significant effects on mussel digestive gland, the key metabolic organ in bivalves. Both BEZA and GEM increased the activity of the glycolytic enzymes phosphofructokinase (PFK) and pyruvate kinase (PK), and of Glutathione transferase (GST) glutathione reductase (GSR), and total glutathione content. A significant increase in the peroxisomal enzyme catalase was observed; however, BEZA exposure decreased Palmytoyl CoA oxidase activity, whereas GEM was ineffective. The results indicate that in mussels environmental concentrations of hypolipidemic drugs can affect the immune function, as well as glycolysis, redox balance and peroxisomal function.     

Lysosomal and lipid alterations in the digestive gland of mussels, Mytilus galloprovincialis (L.) as biomarkers of environmental stress

Lysosomal and lipid alterations in the digestive gland cells of the common mussel Mytilus galloprovincialis collected from six stations along Thermaikos Gulf (Northern Greece) were monitored through a 1-year period. The ultrastructural observations of the digestive cells of sampled mussels indicated a higher incidence of "granular" or "dark" residual bodies in the relatively more polluted stations compared to less polluted ones. Alterations in the lysosomal system of digestive cells were assessed: (1) using the lysosomal membrane stability test (2) quantifying lysosomal structural alterations of the residual bodies and the heterolysosomes of the digestive cells at the ultrastructural level, and (3) determining the elemental composition of lysosomes by means of X-ray microanalysis on epoxy resin sections. Evaluation of neutral lipids was realized using image analysis on histochemical resin sections. Mussels from the most heavily polluted stations exhibited reduced lysosomal membrane stability, higher volume density and diameter of heterolysosomes and residual bodies, and, in some cases, relatively higher lysosomal heavy metal content compared to mussels from less polluted stations. The results showed that the lysosomal membrane stability and the volume density of the residual bodies are more sensitive to general stress compared to the other biomarkers used, and could be used successfully as early warning tools in biomonitoring programs.     

Aporrectodea caliginosa, a suitable earthworm species for field based genotoxicity assessment?

There is a growing interest for the application of biomakers to field-collected earthworms. Therefore we have evaluated the usability of native populations of endogeic, widely distributed earthworm Aporrectodea caliginosa in the assessment of soil genotoxicity using the Comet assay. Validation of the Comet assay on earthworm coelomocytes has been established using commercially available Eisenia fetida exposed to copper, cadmium, and pentachlorophenol, along with A. caliginosa exposed to copper in a filter paper contact test. Neutral red retention time (NRRT) assay was conducted on copper exposed and field-collected earthworms. Significant DNA and lysosomal damage was measured using Comet and NRRT assays in native populations of A. caliginosa sampled from the polluted soils in the urban area in comparison to the earthworms from the reference site. The results of this study confirm the employment of A. caliginosa as a suitable species for the in situ soil toxicity and genotoxicity field surveys.     

Seasonal variability of oxidative biomarkers, lysosomal parameters, metallothioneins and peroxisomal enzymes in the Mediterranean mussel Mytilus galloprovincialis from Adriatic Sea

The Mediterranean mussel, Mytilus galloprovincialis, is a classical sentinel organism for monitoring the biological effects of contaminants through the use of molecular and cellular biomarkers. These biological responses can be modulated also by seasonal changes of both environmental and biological factors, potentially influencing responsiveness and sensitivity to pollutants. The aim of this study was to characterize in a reference mussel population from the Adriatic Sea, the natural fluctuations of several oxidative stress biomarkers widely used in ecotoxicological applications. Analyses of individual antioxidant defenses (catalase, glutathione S-transferases, glutathione reductase, glutathione peroxidases, levels of glutathione) were integrated with those of the total oxyradical scavenging capacity (TOSC-assay), which quantify the overall capability of a tissue to neutralize different forms of oxyradicals. Due to the close relationship between antioxidant efficiency and onset of various cellular alteration, the seasonal characterization was carried out also on lysosomal membrane stability, accumulation of malondialdehyde, neutral lipids and lipofuscin, levels of metallothioneins and activity of peroxisomal enzymes (acyl-CoA oxidase and d-aminoacid oxidase). Results indicated a significant seasonality for several biological responses; major variations occurred especially in spring and summer months suggesting the influence of natural factors, such as temperature, reproductive cycle and food availability. The observed seasonal oscillations revealed both similarities and differences with those reported for other Mediterranean mussel populations suggesting that opposite trends can occur when the same environmental prooxidant factors have a different regional influence.     

Illicit drugs as new environmental pollutants: cyto-genotoxic effects of cocaine on the biological model Dreissena polymorpha

The increase in global consumption of illicit drugs has produced not only social and medical problems but also a potential new environmental danger. Indeed, it has been established that drugs consumed by humans end up in surface waters, after being carried through the sewage system. Although many studies to measure concentrations of several drugs of abuse in freshwater worldwide have been conducted, no data have been available to evaluate their potentially harmful effects on non-target organisms until now. The present study represents the first attempt to investigate the cyto-genotoxic effects of cocaine, one of the primary drugs consumed in Western Countries, in the biological model Dreissena polymorpha by the use of a biomarker battery. We performed the following tests on Zebra mussel hemocytes: the single cell gel electrophoresis (SCGE) assay, the apoptosis frequency evaluation and the micronucleus assay (MN test) for the evaluation of genotoxicity and the lysosomal membranes stability test (neutral red retention assay; NRRA) to identify the cocaine cytotoxicity. We exposed the molluscs for 96 h to three different nominal concentrations in water (40 ng L⁻¹; 220 ng L⁻¹; and 10 μg L⁻¹).Cocaine caused significant (p < 0.05) primary DNA damage in this short-term experiment, but it also caused a clear increase in micronucleated cells and a marked rise in apoptosis, which was evident in samples from even the lowest environmental cocaine concentration. Because cocaine decreased the stability of lysosomal membranes, we also highlighted its cytotoxicity and the possible implications of oxidative stress for the observed genotoxic effects.     

Molecular probes for the detection and identification of ichthyotoxic marine microalgae of the genus Pseudochattonella (Dictyochophyceae, Ochrophyta)

Phytoflagellates of the genus Pseudochattonella (Dictyochophyceae, Ochrophyta) form blooms in marine coastal waters in northern Europe, Japan, and New Zealand that at times cause fish kills with severe losses for the aquaculture industry. The aim of this study was to develop molecular probes for the detection and identification of Pseudochattonella at the genus and species level. A variety of probes were developed and applied to either dot blot hybridization, (q)PCR, or microarray format. In the dot blot hybridization assay, five different oligonucleotide probes targeting the small subunit (SSU) rDNA were tested against DNA from 18 microalgal strains and shown to be specific to the genus Pseudochattonella. A genus-specific PCR assay was developed by identifying an appropriate primer pair in the SSU—internal transcribed spacer 1 (ITS1) rDNA region. Its specificity was tested by screening against both target and non-target strains, and the assay was used to confirm the presence or absence of Pseudochattonella species in environmental samples. In order to distinguish between the two species of the genus, two PCR primer pairs each biased towards one of the species were designed in the large subunit (LSU) rDNA D1 domain and used for quantitative real-time PCR. Five selected probes (three SSU and two LSU rDNA) were adapted for the use on microarrays and included on a prototype multi-species microarray for the detection of harmful algae (http://www.midtal.com). Finally, microarrays and qPCR were used for the monthly monitoring of a sampling site in outer Oslofjorden during a 1-year period. Members of Pseudochattonella are difficult to identify by light microscopy in Lugol’s preserved samples, and the two species Pseudochattonella verruculosa and Pseudochattonella farcimen can be morphologically distinguished only by transmission electron microscopy. The molecular probes designed in this study will be a valuable asset to microscopical detection methods in the monitoring of harmful algae and for biogeographical and ecological studies of this genus.     

Evaluation of biomarkers in caged fishes and mussels to assess the quality of waters in a bay of the NW Mediterranean Sea

Specimens of sea bass (Dicentrarchus labrax) were placed in cages for 1 month in spring and autumn at different locations in the Bay of Cannes (NW Mediterranean). Biochemical markers evaluated were: ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities in fish livers and acetylcholinesterase (AChE) activity in fish muscle. EROD and GST activities were higher in front of the outlet for the wastewater plant of Cannes and in the harbour than outside the marina. High EROD and GST activities may be induced by petrol hydrocarbons and/or polychlorinated biphenyls (PCBs). AChE was low in the muscles of the fish caged in the harbour compared with samples from the other cages. Low AChE activity could suggest the presence of organophosphorus and carbamate compounds in seawater from the harbour. Mussels (Mytilus galloprovincialis) were caged off Cannes for the same periods as the fishes. Heavy metal, metallothionein (MT) concentrations and lysosomal membrane stability were evaluated in the digestive gland of the mussels. Results show low heavy metal and MT concentrations, implying low metal concentrations in the surrounding waters. High lysosomal membrane stability revealed a good physiological status of these animals after caging. The whole set of data indicates that seawater in the Bay of Cannes appeared to be unpolluted as regards pollutants which may induce the measured biomarkers, except in restricted areas.     

Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using mussels as sentinel organisms

With the aim of assessing the biological effects of pollution along three gradients of pollution in the NW Mediterranean Sea, a biomonitoring survey was implemented using a battery of biomarkers (lysosomal membrane stability, lysosomal structural changes, metallothionein (MT) induction and peroxisome proliferation) in mussels over a period of two years as part of the EU-funded BEEP project. Mussels from the most impacted zones (Fos, Genova and Barcelona harbours) showed enlarged lysosomes accompanied by reduced labilisation period of lysosomal membranes, indicating disturbed health. MT levels did not reveal significant differences between stations and were significantly correlated with gonad index, suggesting that they were influenced by gamete development. Peroxisomal acyl-CoA oxidase (AOX) activity was significantly inhibited in polluted stations possibly due to interactions among mixtures of pollutants. In conclusion, the application of a battery of effect and exposure biomarkers provided relevant data for the assessment of biological effects of environmental pollution along the NW Mediterranean Sea.     

Differential expression profile of membrane proteins in Aplysia pleural–pedal ganglia under the stress of methyl parathion

This study was aimed to analyze the alteration of membrane protein profiles in Aplysia juliana Quoy & Gaimard (A. juliana) pleural–pedal ganglia under MP exposure. Both the results of GC–MS analysis and the activity assay of acetylcholinesterase (AChE), superoxide dismutase (SOD), catalase (CAT) reveal that MP toxicological effects on Aplysia left and right pleural–pedal ganglia are different under 7 and 14 days of exposure. Therefore, Aplysia were subjected for exposure at two concentrations (1 and 2 mg/l) of MP for 7 and 14 days for membrane proteomic study. As a result, 19 and 14 protein spots were differentially expressed in A. juliana left pleural–pedal ganglia under 7 and 14 days treatment, and 20 and 14 protein spots found with differential expressions in their right ganglia under the same treatment, respectively. Several proteins with expression variations were detected from both the left and right pleural–pedal ganglia; however, most proteins have distinctive expressions, indicating different mechanisms might be involved in initiating MP toxicology in left and right ganglia. Among the total differential protein spots obtained, 29 proteins were classed as membrane proteins. These proteins are mainly involved in the metabolism process, cell redox homeostasis, signal transduction, immunology, intracellular transport and catalysis, indicating MP toxicity in mollusks seems to be complex and diverse. Some differentially expressed proteins were further confirmed by Western blotting and quantitative real-time PCR. These results might provide renovated insights to reveal the mechanism of MP-induced neurotoxicity, and the novel candidate biomarkers might have potential application for environmental evaluation of MP pollution level.     

Mutagenicity of particulate matter fractions in areas under the impact of urban and industrial activities

Organisms in the environment are exposed to a mixture of pollutants. Therefore the purpose of this study was to analyze the mutagenicity of organic and inorganic responses in two fractions of particulates (TSP and PM2.5) and extracts (organic and aqueous). The mutagenicity of organic and aqueous particulate matter extracts from urban-industrial and urban-residential areas was evaluated by Salmonella/microsome assay, through the microsuspension method, using strain TA98 with and without liver metabolization. Additionally, strains YG1021 and YG1024 (nitro-sensitive) were used for organic extracts. Aqueous extracts presented negative responses for mutagenesis and cytotoxicity was detected in 50% of the samples. In these extracts the presence of potential bioavailable metals was identified. All organic extracts presented mutagens with a higher potential associated with PM2.5. This study presents a first characterization of PM2.5 in Brazil, through the Salmonella/microsome assay. The evaluation strategy detected the anthropic influence of groups of compounds characteristically found in urban and industrial areas, even in samples with PM values in accordance with quality standards. Thus, the use of a genotoxic approach in areas under different anthropic influences will favor the adoption of preventive measures in the health/environment relation.     

The role of algae (Isochrysis galbana) enrichment on the bioaccumulation of benzo[a]pyrene and its effects on the blue mussel Mytilus edulis

The role of algal concentration in the transfer of organic contaminants in a food chain has been studied using the ubiquitous model polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) as the contaminant, Isochrysis galbana as the phytoplankton food source, and the common mussel (Mytilus edulis) as the primary consumer. The effect of algal concentration on BaP uptake by M. edulis was determined by feeding M. edulis daily with I. galbana which had previously been kept in the presence of BaP for 24 h. Four combinations of concentrations of algae and BaP were used to give final exposure concentrations of 30,000 or 150,000 algal cells ml(-1) in combination with either 2 or 50 microg BaP l(-1). BaP concentrations were determined fluorometrically in rest tissues (excluding digestive glands) and digestive gland microsomal fractions of M. edulis after 1, 7 and 15 days exposure, and also in isolated algae. Potentially toxic effects of BaP on M. edulis were examined in terms of blood cell lysosomal membrane damage (neutral red dye retention assay) and induction of digestive gland microsomal mixed-function oxygenase (MFO) parameters [BaP hydroxylase (BPH) and NADPH-cytochrome c (P450) reductase activities]. BaP bioaccumulation in rest tissues (and to a lesser extent in digestive gland microsomes) of M. edulis increased with both increasing BaP and algal exposure concentrations, and over time, producing maximal bioconcentration factors in rest tissues after 15 days exposure to 150,000 algal cells ml(-1) and 50 microg BaP l(-1) of 250,000. The five-fold higher concentration of algae increased BaP bioaccumulation by a factor of approximately 2 for 50 microg BaP l(-1) at day 15. Blood cell neutral red dye retention time decreased linearly with increasing log(10) tissue BaP body burden, indicating an increased biological impact on M. edulis with increasing BaP exposure possibly due to a direct effect of BaP on blood cell lysosomal membrane integrity. An increase was seen in NADPH-cytochrome c reductase activity, and indicated in BPH activity, with 1 but not 7 or 15 days exposure to BaP, indicating a transient response of the digestive gland microsomal MFO system to BaP exposure.     

A Cr(VI)-reducing Microbacterium sp. strain SUCR140 enhances growth and yield of Zea mays in Cr(VI) amended soil through reduced chromium toxicity and improves colonization of arbuscular mycorrhizal fungi

Pot culture experiments were conducted in a glasshouse to evaluate the effects of four efficient Cr(VI)-reducing bacterial strains (SUCR44, SUCR140, SUCR186, and SUCR188) isolated from rhizospheric soil, and four arbuscular mycorrhizal fungi (AMF—Glomus mosseae, G. aggregatum, G. fasciculatum, and G. intraradices) alone or in combination, on Zea mays in artificially Cr(VI)-amended soil. Presence of a strain of Microbacterium sp. SUCR140 reduced the chromate toxicity resulting in improved growth and yields of plants compared to control. The bioavailability of Cr(VI) in soil and its uptake by the plant reduced significantly in SUCR140-treated plants; the effects of AMF, however, either alone or in presence of SUCR140 were not significant. On the other hand, presence of AMF significantly restricted the transport of chromium from root to the aerial parts of plants. The populations of AMF chlamydospores in soil and its root colonization improved in presence of SUCR140. This study demonstrates the usefulness of an efficient Cr(VI)-reducing bacterial strain SUCR140 in improving yields probably through reducing toxicity to plants by lowering bioavailability and uptake of Cr(VI) and improving nutrient availability through increased mycorrhizal colonization which also restricted the transport of chromium to the aerial parts.     

Monitoring of DNA damage in haemocytes of freshwater mussel Sinanodonta woodiana sampled from the Velika Morava River in Serbia with the comet assay

This study was undertaken to investigate the potential of the freshwater mussel Sinanodonta woodiana for detection of genotoxic pollution of the environment. Study was performed at two sites in the Velika Morava River, from May 2010 to February 2011. The alkaline comet assay on haemocytes was used, and the olive tail moment (OTM) was chosen as a measure of DNA damage. The specimens held on acclimation under controlled laboratory conditions for 10 d were used as a control. Chemical analysis revealed the presence of phosphates and increased concentrations of zinc, copper and nickel at both sites during the entire sampling period. The values of OTM in mussels collected from the environment, significantly correlated with the concentration of zinc (r = 0.6248), temperature (r = 0.7006) and dissolved oxygen (r = 0.7738). Seasonal variations in genotoxic response were observed, with the highest OTM values obtained during summer months. Preliminary results of the in vitro study indicated the effect of water temperature on genotoxic response to zinc and cadmium in S. woodiana suggesting that the presence of genotoxic pollutants during months with lower temperature could be under-estimated. Obtained results indicate that S. woodiana could be a valuable tool for active biomonitoring of aquatic environments and emphasizes the importance of seasonal genotoxic monitoring with this organism.