Scale of classification based on biochemical markers in mussels: application to pollution monitoring in European coasts

A battery of biochemical parameters is used to evaluate the response of mussels to a contaminated coastal environment. In the European BIOMAR programme, a multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites. AChE activity is highly sensitive to organophosphorus and carbamate insecticides but also to heavy metals. Catalase activity and lipid oxidation (evaluated as MDA) are markers of oxidative stress, GST activity is related to the conjugation of organic compounds and BPH activity is a marker of planar compounds (e.g. PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). Contamination levels were estimated by measurement of PAHs and heavy metals in animals. For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a global response index was calculated as the sum of the response index of each of the five biomarkers. As a result of our calculation method, the quality of the coastal environment at each site can be classified with a five level scale. Mussels were collected during five cruises in 1995-1996 on the Baltic and Mediterranean coasts. The results show that water quality ranged from class 1 (clean areas in some sites of the French Riviera, Spanish Costa Brava and the Baltic coast) to class 5 (high pollution in main harbours, e.g. Kiel and Toulon). Some areas fall into class 4, e.g. Carteau, Cortiou, Barcelona, Warnemunde, Swinemunde, Ebro delta. The global Biomarker Index was positively correlated with the level of PAHs in mussels in Baltic transects. A number of other contaminants or stressors may be present in the marine environment and the Biomarker Index appeared to be relevant to classify coastal environmental pollution.     

Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends

A battery of biochemical parameters was used to evaluate the response of mussels to a contaminated coastal environment. A multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites (BIOMAR European programme). This study allows the evaluation of the temporal trends of this scale when applied to selected sites of European Mediterranean coast (BEEP Biological Effects of Environmental Pollution in Marine Coastal Ecosystems: European programme). Acetylcholinesterase activity (AChE) is highly sensitive to organophosphorus and carbamate insecticides and, to some extent, also to heavy metals. Catalase activity (CAT) and lipid oxidation (evaluated as malonedialdehyde) are markers of oxidative stress, glutathione S-transferase (GST) activity is related to conjugation of organic compounds and benzo(a)pyrene hydroxylase activity (BPH) is a marker of effect of certain planar organic compounds (e.g. polycyclic aromatic hydrocarbons, PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a Multimarker Pollution Index (MPI) was calculated as the sum of the response index of each of the five more discriminating biomarkers. As the result of our calculation method, the quality of the coastal environment at each site can be classified according to a five levels scale. Samples collected for five cruises in May 2001, 2002, 2003, and September 2001 and 2002 showed MPI evolutions. The results show that water quality can be classified from class 1 (clean areas in some sites of France, Italy and Spain) to class 4 (high pollution in main harbours). Results of the use of the biomarker scale in WP3 (Work Package Concernant Biomonitoring Programmes in Mediterranean Sea) during the BEEP programme make a strong contribution to the establishment of standardized strategies and methods for internationally agreed protocols for biomarker-based monitoring programmes. In comparison with scale pollution methodology used in the BIOMAR programme, the main contribution of BEEP was (1) to select from discriminatory analysis the biomarkers to be included in calculation of scale pollution; (2) to improve the use of the biomarker index in order to identify the main contaminants by analysis of individual contributions to the MPI; and (3) to apply methodology for temporal trends at sampled sites.     

Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends.

A battery of biochemical parameters was used to evaluate the response of mussels to a contaminated coastal environment. A multimarker approach was developed, establishing a scale for the classification of the water quality in European coastal sites (BIOMAR European programme). This study allows the evaluation of the temporal trends of this scale when applied to selected sites of European Mediterranean coast (BEEP Biological Effects of Environmental Pollution in Marine Coastal Ecosystems: European programme). Acetylcholinesterase activity (AChE) is highly sensitive to organophosphorus and carbamate insecticides and, to some extent, also to heavy metals. Catalase activity (CAT) and lipid oxidation (evaluated as malonedialdehyde) are markers of oxidative stress, glutathione S-transferase (GST) activity is related to conjugation of organic compounds and benzo(a)pyrene hydroxylase activity (BPH) is a marker of effect of certain planar organic compounds (e.g. polycyclic aromatic hydrocarbons, PAHs). These parameters were measured either in gills (AChE, GST) or digestive gland (BPH, GST, CAT, MDA). For each biomarker, a discriminatory factor was calculated (maximum variation range/confidence interval) and a response index was allocated. For each site, a Multimarker Pollution Index (MPI) was calculated as the sum of the response index of each of the five more discriminating biomarkers. As the result of our calculation method, the quality of the coastal environment at each site can be classified according to a five levels scale. Samples collected for five cruises in May 2001, 2002, 2003, and September 2001 and 2002 showed MPI evolutions. The results show that water quality can be classified from class 1 (clean areas in some sites of France, Italy and Spain) to class 4 (high pollution in main harbours). Results of the use of the biomarker scale in WP3 (Work Package Concernant Biomonitoring Programmes in Mediterranean Sea) during the BEEP programme make a strong contribution to the establishment of standardized strategies and methods for internationally agreed protocols for biomarker-based monitoring programmes. In comparison with scale pollution methodology used in the BIOMAR programme, the main contribution of BEEP was (1) to select from discriminatory analysis the biomarkers to be included in calculation of scale pollution; (2) to improve the use of the biomarker index in order to identify the main contaminants by analysis of individual contributions to the MPI; and (3) to apply methodology for temporal trends at sampled sites.     

Biomarker responses and accumulation of hazardous substances in mussels (Mytilus trossulus) transplanted along a pollution gradient close to an oil terminal in the Gulf of Finland (Baltic Sea)

Baltic Sea blue mussels (Mytilus trossulus) were used as sentinel organisms to detect the biological effects of chemical contamination in the low salinity environment. Mussels naturally adapted to a salinity of ca. 6.0 PSU were caged for 30 days at four sites along an assumed pollution gradient (salinity ca. 4.5 PSU) in the vicinity of Finland's largest oil refinery and harbor Kilpilahti in the Gulf of Finland. Tissue concentrations and accumulation rates of especially organic contaminants (PAHs, PCBs and organotins) were clearly elevated at the innermost coastal stations near the harbor area. Biological effects of contaminant exposure on caged mussels were evaluated by measuring a suite of biomarkers including catalase, glutathione S-transferase, superoxide dismutase, glutathione reductase, lipid peroxidation, acetylcholinesterase activity and lysosomal membrane stability. Mussels transplanted near the harbor area were able to elevate their antioxidant defense in response to environmental contamination. Reduced morphometric condition index and soft tissue growth rate together with increased lipid peroxidation and low lysosomal membrane stability were also observed at the most contaminated site. The results suggest that caging of M. trossulus for four weeks at lower salinity is a feasible method for the detection of environmental pollution also in low salinity areas of the Baltic Sea.     

Biomarker-enhanced assessment of reproductive disorders in Monoporeia affinis exposed to contaminated sediment in the Baltic Sea

Introducing biomarkers into monitoring programs requires understanding of their responses in relation to higher-level biological effects as well as modulating effects of confounding environmental factors. We evaluated relationships between the general toxicity biomarkers (acetylcholinesterase [AChE], lysosomal membrane stability [LMS], oxygen radical absorbance capacity [ORAC]) and reproductive performance (fecundity and embryo aberrations) in the amphipod Monoporeia affinis in the Baltic Sea. To further link biomarker response to contaminant (PCBs, PAHs and metals) levels in the surrounding sediments as well as environmental factors (salinity, bottom depth and total organic carbon in sediments [TOC]), correlation and partial least square regression (PLSR) analyses were applied. The observed contaminants levels were frequently elevated for heavy metals and PAHs, but not PCBs. In the amphipod populations, female ORAC values were positively related to the occurrence of females carrying malformed or membrane-damaged embryos and to the percentage of such embryos in their broods, but also to the fecundity. Female AChE activity was negatively related to the frequency of the membrane-damaged embryos, and positively to the frequency of embryos with arrested development in the broods. Moreover, higher AChE activity and ORAC values in the females occurred at elevated concentrations of metals and PAHs, while there was a negative correlation between embryo ORAC and some PCB congeners. The PLSR models explained over 80% of the variation in the female ORAC and AChE values by variation in contaminant concentrations in combination with environmental variables. Specifically, CB180 and PAM4,9 were identified as negative predictors for ORAC, whereas many PAHs and some metals were positive predictors. The AChE activity was positively related to some metals and negatively to PCBs. In the PLSR models, environmental factors had significant modulating effects, with positive effect of salinity on female ORAC and AChE, and negative effect of TOC on the AChE. The LMS data were less informative, with no apparent relation to any of the contaminants. Linking subcellular responses to the reproduction effects facilitates environmental stress assessment and understanding of the response mechanisms, but also calls for more experimental and field data providing a mechanistic understanding to these linkages.     

Biomarkers in Ruditapes decussatus: a potential bioindicator species.

The clam Ruditapes decussatus is distributed worldwide and due to its ecological and economical interest has been proposed as a bioindicator in areas where mussels are not available. The accumulation of several anthropogenic compounds in their tissues suggests that they possess mechanisms that allow them to cope with the toxic effects of these contaminants. Besides pollutant uptake, the use of biomarkers is pointed out in this paper since it is a promising approach to monitor the effect of these contaminants in the marine environment. Biomarkers complement the information of the direct chemical characterization of different types of contaminants. Therefore, the aim of this paper is to review the role of several biomarkers: (metallothioneins (MT), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and selenium-dependent), lipid peroxidation (measured as MDA, one of the final products of lipid peroxidation), glutathione S-transferase (GST) and acetylcholinesterase (AChE), measured in different tissues of the clam R. decussatus, in laboratory conditions and under various environmental stresses, in two ecosystems (Ria Formosa lagoon- Portugal) and Bizerta lagoon (Tunisia) in a perspective of a multibiomarker approach to assess environmental changes. Experiment and field studies are in good agreement since MT levels, especially in the gills, the first target tissue of these contaminants, can be used as biomarker of exposure to Cd. GPx and MDA may also be determined in this respect. AChE activity is inhibited by pesticide and, to a less extent, by metal exposure in the gills and whole soft body of clams. However, the induction of GST isoforms experimentally demonstrated is not observed in the field because only global GST activity was determined. The whole set of results opens new research perspectives for the use of this species to assess the effect of mixtures of pollutants in the aquatic environment.     

Multiple biomarkers of pollution effects in caged mussels on the Greek coastline

A suite of biomarkers was measured in caged mussels at areas impacted by different anthropogenic activities along the Greek coastline to assess biological effects of environmental pollution. Mussels were caged at coastal sites in the vicinity of major cities, in areas influenced by major industries, agricultural practices and in islands away from known sources of pollution. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AchE), oxidative stress (catalase, CAT), phase II biotransformation of xenobiotics (glutathione S-transferase, GST), metal exposure (metallothioneins, MTs) and protein synthesis (RNA:DNA ratio) were measured to assess effects of various types of pollutants. AchE activity proved to be the most responsive biomarker with decreased values at sites influenced by agricultural, urban and industrial activities. Decreased CAT and GST activities and increased MTs levels were recorded at a number of anthropogenic-impacted sites. RNA:DNA ratio showed a biphasic response as both high and low values were found at impacted sites. Principal component analysis clearly distinguished sites receiving pollution inputs from non-polluted sites. The combination of the selected biomarkers used in caged mussels resulted useful in the assessment of the effects of environmental pollution.     

Biomarkers in Ruditapes decussatus: a potential bioindicator species

The clam Ruditapes decussatus is distributed worldwide and due to its ecological and economical interest has been proposed as a bioindicator in areas where mussels are not available. The accumulation of several anthropogenic compounds in their tissues suggests that they possess mechanisms that allow them to cope with the toxic effects of these contaminants. Besides pollutant uptake, the use of biomarkers is pointed out in this paper since it is a promising approach to monitor the effect of these contaminants in the marine environment. Biomarkers complement the information of the direct chemical characterization of different types of contaminants. Therefore, the aim of this paper is to review the role of several biomarkers: (metallothioneins (MT), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and selenium-dependent), lipid peroxidation (measured as MDA, one of the final products of lipid peroxidation), glutathione S-transferase (GST) and acetylcholinesterase (AChE), measured in different tissues of the clam R. decussatus, in laboratory conditions and under various environmental stresses, in two ecosystems (Ria Formosa lagoon- Portugal) and Bizerta lagoon (Tunisia) in a perspective of a multibiomarker approach to assess environmental changes. Experiment and field studies are in good agreement since MT levels, especially in the gills, the first target tissue of these contaminants, can be used as biomarker of exposure to Cd. GPx and MDA may also be determined in this respect. AChE activity is inhibited by pesticide and, to a less extent, by metal exposure in the gills and whole soft body of clams. However, the induction of GST isoforms experimentally demonstrated is not observed in the field because only global GST activity was determined. The whole set of results opens new research perspectives for the use of this species to assess the effect of mixtures of pollutants in the aquatic environment.     

Biomarkers in Ruditapes decussatus: a potential bioindicator species

The clam Ruditapes decussatus is distributed worldwide and due to its ecological and economical interest has been proposed as a bioindicator in areas where mussels are not available. The accumulation of several anthropogenic compounds in their tissues suggests that they possess mechanisms that allow them to cope with the toxic effects of these contaminants. Besides pollutant uptake, the use of biomarkers is pointed out in this paper since it is a promising approach to monitor the effect of these contaminants in the marine environment. Biomarkers complement the information of the direct chemical characterization of different types of contaminants. Therefore, the aim of this paper is to review the role of several biomarkers: (metallothioneins (MT), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx) (total and selenium-dependent), lipid peroxidation (measured as MDA, one of the final products of lipid peroxidation), glutathione S-transferase (GST) and acetylcholinesterase (AChE), measured in different tissues of the clam R. decussatus, in laboratory conditions and under various environmental stresses, in two ecosystems (Ria Formosa lagoon- Portugal) and Bizerta lagoon (Tunisia) in a perspective of a multibiomarker approach to assess environmental changes. Experiment and field studies are in good agreement since MT levels, especially in the gills, the first target tissue of these contaminants, can be used as biomarker of exposure to Cd. GPx and MDA may also be determined in this respect. AChE activity is inhibited by pesticide and, to a less extent, by metal exposure in the gills and whole soft body of clams. However, the induction of GST isoforms experimentally demonstrated is not observed in the field because only global GST activity was determined. The whole set of results opens new research perspectives for the use of this species to assess the effect of mixtures of pollutants in the aquatic environment.     

Seasonal variability in biomarkers in the bivalves Mytilus edulis and Macoma balthica from the northern Baltic Sea

Metallothionein level (MT), and acetylcholinesterase (AChE), catalase (CAT) and glutathione-S-transferase (GST) enzyme activities in the bivalves Mytilus edulis and Macoma balthica were investigated for seasonal variations from an inshore and an offshore site in the northern Baltic Sea. All the biomarkers showed variability, following mostly a similar pattern at both sites. Relationships between biomarkers and environmental factors and protein concentration and weight of target tissues were examined. In M. edulis, GST activity was related to Secchi depth, while in M. balthica a correlation with near-bottom oxygen saturation was observed. AChE activity correlated with the weight of the foot tissue of M. balthica. In both species, an integrated biomarker index indicated a stressed condition during the spring/early summer period. Strong seasonal variability in temperature and a concentrated period of food availability in spring-both governing the reproductive cycle of the bivalves-probably explains most of the observed natural variability in biomarkers in this sea area.     

Effect of temperature and salinity on acetylcholinesterase activity, a common pollution biomarker, in Mytilus sp. from the south-western Baltic Sea

Acetylcholinesterase (AChE) activity is a well established biomarker to monitor environmental pollution caused by neurotoxic compounds, such as organophosphorus and carbamate pesticides. The presence of these compounds results in a measurable inhibition of the enzyme. This has been shown for different marine species including blue mussels. Besides pollution, environmental variables may also have a direct or indirect effect on AChE activity, particularly in estuarine and brackish water environments. To assess the impact of abiotic factors on the AChE activity the seasonal course of gill AChE activity was followed in relation to temperature and salinity in Mytilus sp. collected from the south-western Baltic Sea. In addition, the effect of salinity on AChE activity was investigated in an experimental study. The AChE activity showed significant seasonal differences with maximum activities during the summer period and minimum activities in winter. These changes correlate significantly (p<0.001) and positively with water temperature. The experimental exposure of Mytilus sp. to different salinities (5, 7, 16 or 20 psu) resulted in changes in the gill AChE activity. Empirical orthogonal function (EOF) analysis revealed that AChE activity was significantly and negatively correlated with salinity (p<0.01). These results clearly demonstrate the need to consider the potential influence of temperature and salinity on AChE activity when applying this biomarker to monitor exposure to and effect of neurotoxic substances in estuarine and brackish water blue mussels.     

Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplanted into the northwest Mediterranean Sea

Seasonal variations of six mussel (Mytilus galloprovincialis) biomarkers at two sites in the Mediterranean Sea were compared with physiological indices (condition, growth and gonad maturation), environmental parameters (temperature, salinity and turbidity), and chemical contamination levels. The basal levels of acetylcholinesterase (AChE), DNA adducts, benzo[a]pyrene hydroxylase (BPH), heat-shock proteins (HSP70), metallothioneins (MT) and P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR) were estimated as early warning signals in caged mussels sampled at Carteau (native site) and La Fourcade (transplantation site) over a 2-year period. The Carteau and La Fourcade mussels have specific chemical contamination profiles but a similar range of values. For example, both are highly contaminated by heavy metals (201 and 258.4 mg kg(-1) dw, respectively) and considered as moderately impacted for polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). However, contamination levels at Carteau are twice as high for PAHs (101.5 mg g(-1) dw) and PCBs (90.2 mg g(-1) dw) than La Fourcade. The seasonal contamination trend at Carteau showed six-fold higher levels of pyrolytic pollutants in winter. Although few tissue lesions were detected in individuals studied at either site, greater parasitic infestation was observed at Carteau. The results of findings from the two Mediterranean pilot studies support the adaptability of transplanted mussels to be used as biomarkers and to establish physiological endpoints for chemical contaminant exposure.     

Contamination of a dredged material disposal site La Rochelle Bay,France .The use of the acetylcholinesterase activity of Mytilus edulis L. as a biomarker of pesticides: the need for a critical approach

A single aspect of the toxic impact of a dredged material disposal site located near a mussel-farming zone was followed for eight months. Acetylcholinesterase activity (AChE) of Mytilus edulis was investigated as a biomarker for possible contamination by neurotoxic compounds (carbamates and/or organophosphorous pesticides). Our observations showed that the enzymatic activities (including AChE) of these harbour mussels were decreased in sites directly and indirectly influenced (according to hydrodynamic conditions) by the dumping of dredged sediments, suggesting possible contamination by pesticides. The strong correlations observed between AChE activity and growth parameters (length and weight) seems to show, however, that the enzyme activity is also indirectly controlled through growth restriction, which may imply limitation of the development of the nervous system in juveniles. The concentration of total proteins, as well as the spawning process also seem to disturb the assessment of AChE activity. These field observations clearly indicate that the use of this enzyme activity as a biomarker should proceed with caution. For example, the seasonal variability of such activity should be taken into account in a biomonitoring programme.     

Spatial and seasonal biomarker responses in the clam Ruditapes decussatus

The clam Ruditapes decussatus is an important resource to preserve in coastal lagoon systems around the Mediterranean including the South Portugal. To assess spatial and temporal biomarker responses to contamination in the species, a multibiomarker approach was conducted using antioxidant enzymes, MFO system phase I and II; acetylcholinesterase, metallothionein (MT), δ-aminolevulinic acid dehydratase and lipid peroxidation (LPO). The condition index (CI), metals and polycyclic aromatic hydrocarbons (PAHs) were also determined. The levels of contaminants were not particularly high and the antioxidant enzymes, acetylcholinesterase (AChE), MT in the digestive gland, and δ-aminolevulinic acid dehydratase (ALAD) do not provide a suitable seasonal and spatial discrimination reversely to that regarding CYP450, glutathione-S-transferase (GST), MT in the gills, and LPO in both tissues. However, even those could vary with natural variables that may act as confounding factors. Thus, seasonal variability and natural range of biomarker responses must be carefully and accurately taken into account in ecotoxicological approaches of environmental quality assessment programmes.     

First record of the occurrence of "gill disease" in Mytilus edulis trossulus from the Gulf of Gdańsk (Baltic Sea, Poland)

We are presenting the first report of the occurrence of "gill disease" in Mytilus edulis trossulus from the southern Baltic Sea, Gulf of Gdańsk (Poland). The disease preliminary diagnosis was based on the presence of white and yellow "spots" causing deep indentations in the gills with degeneration, destruction and necrosis of gill filaments. Average prevalence of gill erosion in the blue mussels population was 15.8%. According to other authors, gill erosion may affect over 80% of the bivalve population resulting in mortality rates of up to 40%. The origin of the gill erosion remains unknown, but viruses are most likely involved in the etiology of these pathological conditions. The disease as such may indicate a decrease in the immunological resistance of organisms to infections and inflammations directly or indirectly caused by harmful factors in the ambient environment. Furthermore, the occurrence of numerous pathologies in bivalves is a particular problem in the Gulf of Gdańsk being a low biodiversity ecosystem. Pathologies reduce bilvalves' reproduction ability, worsen their physiological condition and increase their mortality rate. Those factors may also pose a significant ecological danger and lead to negative alterations of the ecosystem.     

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy)

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.     

The use of Fulvia fragilis (Mollusca: Cardiidae) in the biomonitoring of Bizerta lagoon: A mutimarkers approach

Five biomarkers, catalase (CAT) activity, glutathione-S-transferase (GST) activity, the neural transmitter enzyme acetylcholinesterase (AChE), reduced glutathione (GSH) and malondialdehyde (MDA) levels were measured in specimens of Fulvia fragilis collected from Bizerta lagoon (Tunisia). Results demonstrated that F. fragilis showed differential biomarker response according to the importance of the anthropogenic pressure and the nature of pollutants that affect the lagoon. A clear organotropism was also observed with a higher biomarker response in digestive gland than in gills of this bivalve. These results indicate that F. fragilis constitutes a useful tool as sentinel organism for biomonitoring of aquatic pollution.     

Biomarkers in Zebra mussel for monitoring and quality assessment of Lake Maggiore (Italy)

Three different biomarkers (acetylcholinesterase (AChE), ethoxy resorufin-O-deethylase (EROD) and DNA strand breaks) were measured in Zebra mussel (Dreissena polymorpha) specimens collected in April 2005 at six different sampling sites on Lake Maggiore, the second largest Italian lake in terms of depth and volume, in order to assess the spatial variation of exposure to man-made contaminants. Mussels maintained at fixed laboratory conditions were used as controls to eliminate potential interference due to environmental factors. Biomarker data were also supported by the analysis of several chemicals (six dichlorodiphenyltrichloroethane (DDTs), 23 polychlorinated biphenyls (PCBs), 14 polybrominated diphenyl ethers (PBDEs), 11 polycyclic aromatic hydrocarbons (PAHs) and hexachlorobenzene (HCB)) measured in the mussel soft tissues by gas chromatographic analyses. We found a negative correlation between temperature and AChE activity, while any measured environmental or physiological factor seemed to influence EROD activity and DNA strand breaks. A positive relationship was found between EROD activity and all of the measured chemicals, except for PAHs, which correlated with the amount of DNA strand breaks. Significant differences were noted for all biomarkers, both among sampling stations and between control and experimental data, even if the general level of variability was low. The biomarkers showed a distinct pattern of spatial variation, but the evaluation of DNA strand breaks was the strongest discriminating power between sites. In addition, the comparison between AChE and EROD activity measured in 2005 was compared with results obtained in a previous study carried out over the same sampling period in 2003. Results indicated a strong influence of temperature on AChE activity and probable interference of substrate inhibition of EROD activity, pointing out the need to take care in the interpretation of data comparisons. The results obtained with two different metrics used for the measure of DNA strand breaks is also discussed, as well as the relationship between EROD activity data and potential genotoxicity.     

Genetic divergence and a hybrid zone between Baltic and North Sea Mytilus populations (Mytilidae: Mollusca)

Populations of the common mussel ( Mytilus edulis ) from the North Sea area (Skakerrak-Kattegat) and those from the Baltic Sea are almost diagnostically differentiated at five out of 22 studied allozyme loci; at a further seven loci, alleles predominant or common in one area are nearly absent in the other. Genetic distance was estimated at 0.28; this is similar to the distances of these populations to the Mediterranean mussel M. galloprovincialis. The three mussel types obviously represent equal evolutionary divergence from one another, and should also be taxonomically equally separated; a semispecies rank within a more comprehensive M. edulis complex or superspecies is suggested. The age of the Baltic mussel type ( 'M. trossulus' ), as an independent evolutionary lineage, is probably far greater than that of the post-glacial Baltic Sea.
Allele frequencies change gradually and in parallel when entering from the Kattegat through the Sound into the Baltic. Only a slight Wahlund effect at the strongly diverged Gpi and Pgm loci was found in intermediate populations, indicating that extensive hybridization of the two taxa takes place in the area. However, strong interlocus genotypic associations suggest that selection against hybrids is intense in later generations; the c. 100 km wide hybrid zone is narrow relative to the dispersal distance. The genotypic structure of the Lap locus does not conform with those of the other loci studied in the hybrid zone; it cannot be viewed merely as a neutral marker of the process of hybridization.