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 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 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.     

Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers

Over the past decade, molecular, biochemical and cellular markers have been extensively used in pollution monitoring of aquatic environments. Biochemical markers have been selected among early molecular events occurring in the toxicological mechanisms of main contaminants. This paper assesses the marine environment quality along the Tunisian coasts using a statistical approach. Clams (Ruditapes decussatus) were collected during the four seasons of 2003 on seven different sites from the Tunisian coasts. Oxidative stress was evaluated in gills using catalase activity (Cat), neutral lipids and malonedialdehyde accumulation. Glutathione S-transferase activity is related to the conjugation of organic compounds and was evaluated in both, gills and digestive glands. Acetylcholinesterase activity was evaluated as the biomarker of exposure to organophosphorous, carbamate pesticides and heavy metals. For each biomarker, a discriminatory factor was calculated and a response index allocated. For each site, a global response index was calculated as the sum of the response index of each biomarker. Discriminant analysis shows significant differences between sites and seasons compared with control sample. Faroua (site 1) and Menzel Jemile (site 2) seem to be the less polluted with respect to the other sites for all seasons. Gargour (site 6) shows the highest Multimarker Pollution Index during the four seasons, indicating higher contamination level.     

Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers.

Over the past decade, molecular, biochemical and cellular markers have been extensively used in pollution monitoring of aquatic environments. Biochemical markers have been selected among early molecular events occurring in the toxicological mechanisms of main contaminants. This paper assesses the marine environment quality along the Tunisian coasts using a statistical approach. Clams (Ruditapes decussatus) were collected during the four seasons of 2003 on seven different sites from the Tunisian coasts. Oxidative stress was evaluated in gills using catalase activity (Cat), neutral lipids and malonedialdehyde accumulation. Glutathione S-transferase activity is related to the conjugation of organic compounds and was evaluated in both, gills and digestive glands. Acetylcholinesterase activity was evaluated as the biomarker of exposure to organophosphorous, carbamate pesticides and heavy metals. For each biomarker, a discriminatory factor was calculated and a response index allocated. For each site, a global response index was calculated as the sum of the response index of each biomarker. Discriminant analysis shows significant differences between sites and seasons compared with control sample. Faroua (site 1) and Menzel Jemile (site 2) seem to be the less polluted with respect to the other sites for all seasons. Gargour (site 6) shows the highest Multimarker Pollution Index during the four seasons, indicating higher contamination level.     

Monitoring pollution in Tunisian coasts: application of a classification scale based on biochemical markers

Over the past decade, molecular, biochemical and cellular markers have been extensively used in pollution monitoring of aquatic environments. Biochemical markers have been selected among early molecular events occurring in the toxicological mechanisms of main contaminants. This paper assesses the marine environment quality along the Tunisian coasts using a statistical approach. Clams (Ruditapes decussatus) were collected during the four seasons of 2003 on seven different sites from the Tunisian coasts. Oxidative stress was evaluated in gills using catalase activity (Cat), neutral lipids and malonedialdehyde accumulation. Glutathione S-transferase activity is related to the conjugation of organic compounds and was evaluated in both, gills and digestive glands. Acetylcholinesterase activity was evaluated as the biomarker of exposure to organophosphorous, carbamate pesticides and heavy metals. For each biomarker, a discriminatory factor was calculated and a response index allocated. For each site, a global response index was calculated as the sum of the response index of each biomarker. Discriminant analysis shows significant differences between sites and seasons compared with control sample. Faroua (site 1) and Menzel Jemile (site 2) seem to be the less polluted with respect to the other sites for all seasons. Gargour (site 6) shows the highest Multimarker Pollution Index during the four seasons, indicating higher contamination level.     

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.     

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.     

The role of abiotic factors and pesticide levels on enzymatic activity in the freshwater mussel Anodonta cygnea at three different exposure sites

Natural variation in abiotic factors, such as temperature and pH, probably influence the activity of enzymes used as potential biomarkers in bivalve mollusks to assess environmental contamination in the field. Changes in levels of an enzymatic biomarker may thus merely reflect natural variation in the annual physiological cycle of a species rather than exposure to contaminants. To investigate this issue, we documented the relationship between pesticide levels in water and three different enzymatic biomarkers over 1 year in enclosed populations of the freshwater unionid mussel Anodonta cygnea at three different sites of exposure. We considered the natural variation in temperature, pH and dissolved oxygen over the year and across the different sites as a potential correlate of enzymatic activity to disentangle the relative contribution of abiotic factors and pesticide levels. Pesticide levels varied among the three sites and over the course of the year. Catalase (CAT) and acetylcholinesterase activity (AChE) varied as a function of abiotic factors but showed no relation to pesticide levels. Glutathione S-transferase (GST) activity was also related to abiotic factors but also decreased with increases in total pesticide levels. The lack of activity induction or inhibition by pesticides and the natural variation in abiotic factors among sites and across time limits the use of CAT and AChE to assess environmental contamination in this species.     

Evaluation of biomarkers in oyster larvae in natural and polluted conditions

Crassostrea gigas D-shaped larvae were subjected to different conditions of temperature and salinity for 24 h and four biomarkers (acetylcholinesterase (AChE) activity, thiobarbituric acid reactive substances (TBARS) levels, glutathione S-transferase (GST) and catalase (CAT) activities) were measured. AChE activity decreased when salinity increased from 25 to 30 and 35 psu at 20 and 25 degrees C. Temperature did not seem to have an influence on AChE activity. TBARS levels increased as a function of salinity when the temperature was maintained at 20 degrees C, whereas at 25 degrees C no effect of salinity could be observed. Variations in GST and CAT activities were not significant with salinity and temperature except that catalase activity was higher at 25 degrees C than at 20 degrees C. Exposure experiments were conducted at 23 degrees C and 30 psu with carbofuran (100 and 1000 microg/l) and malathion (100 and 300 microg/l). There was an inhibition of AChE activity with carbofuran, and a toxic effect shown by an increase in TBARS levels counteracted by increases in GST and CAT activities which protected the larvae. When two pairs of adults producing larvae were taken into consideration, significant differences in biomarker levels were noted between the larval offspring of each pair. Malathion induced a decrease in AChE activity and an increase in CAT activity.     

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.     

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.     

Effects of dibutyl phthalate and di‐ethylhexyl phthalate on acetylcholinesterase activity in bagrid catfish,Pseudobagrus fulvidraco (Richardson)

Acetylcholinesterase (AChE) activity is a well‐known biomarker for exposure to organophosphate or carbamate compounds in aquatic organisms. However, the effect of dibutyl phthalate (DBP) and di‐ethylhexyl phthalate (DEHP), widely used as a plasticizer, on the change of AChE activity is not yet known. Bagrid catfish Pseudobagrus fulvidraco were administrated with 100, 500 and 1000 mg kg^?1 diet of DBP or DEHP and the effects on AChE activity were assessed in the liver, gill, kidney, heart, brain, muscle and eye of the exposed fish. All tissues contained different background AChE activity in non‐treated bagrid catfish: the highest was observed in the brain, followed by muscle, heart, and kidney. The enzyme activities in various tissues were significantly inhibited after exposure to DBP or DEHP in a concentration‐dependent manner, especially in brain and muscle. A similar, but less pronounced, inhibition was also observed in liver and kidney when exposed to DBP and DEHP. Although AChE activity in gill and heart was also affected by DBP and DEHP, the decrease in these organs was least marked in these organs. Exposure to 1000 mg kg^?1 led to mortalities of 8.0% with DBH and 14% with DEHP; both seemed to be ascribable to phthalate toxicity. This study is the first report that the measurement of AChE activity in bagrid catfish is a valuable biomarker of DBP and DEHP exposure. This biomarker could be incorporated into a battery of biomarkers to strengthen the confidence with which ecotoxicologists can assess the impact of phthalate ester pollution in the aquatic environment.     

基于生物标志物指数法的海洋环境评价方法综述

生物标志物对化学污染物具有“早期预警”功能,在海洋环境评价中应用广泛.以生物标志物为基础的综合指数能够整合多种标志物对环境状况的响应,因而成为评价环境质量的有用工具.这些综合指数方法包括多生物标志物污染指数(MPI)、综合生物标志物响应指数(IBR)、生物效应评价指数(BAI)、生物标志物响应指数(BRI)、健康状态指数(HIS)等.本文从生物标志物指标体系确定、综合指数计算方法、污染程度分级、应用效果等方面对这些评价方法进行综述,并对基于生物标志物指数的海洋环境评价方法存在的问题和改进建议进行了探讨.     

Biochemical biomarkers in gills of mangrove oyster Crassostrea rhizophorae from three Brazilian estuaries

Responses of biochemical biomarkers were evaluated in gills of immature adult mangrove oyster Crassostrea rhizophorae collected in three estuarine regions along the Brazilian coast. In each region, ten oysters were collected in one reference site (R) located far from pollution sources, and in two polluted sites (P-I and P-II sites) located in another water body with similar characteristics. P-I site is located close to recognized pollution sources while P-II site is in the same water body, but far from pollution sources. At the Paranaguá Bay (Southern Brazil), polluted sites receive domestic, harbor and phosphate fertilizer plant discharges. High lipid peroxides (LPO) content was observed in winter oysters from the P-I site. In summer, higher catalase activity was observed in these oysters. In the Piraquê region (Southeastern Brazil), polluted sites receive domestic and agricultural effluents. Lower total oxyradical scavenging capacity (TOSC) towards peroxyradicals was observed in summer oysters from both P-I and P-II sites. In the Itamaracá region (Northeastern Brazil), polluted sites receive paper mill and caustic soda and chlorine factories effluents. Increased glutathione S-transferase (GST) activity was observed in oysters from the P-I site in both summer and winter. At Paranaguá Bay (higher latitude), no seasonal differences were observed in oysters from the R site, suggesting that temperature was not an important factor influencing biomarkers levels. Lower GST activity was observed in oysters from the R site of the Itamaracá Bay (lower latitude) in winter and summer. Taken together, data obtained point to responses of biomarkers in oysters from polluted sites of the three estuarine regions analyzed, indicating the need for future monitoring of the biological effects of contaminants in these environments. They also point to the relevance to consider both season and latitude as factors influencing biomarker responses in environmental contamination monitoring programs.     

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.     

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.     

Biomarker responses of the estuarine brown shrimp Crangon crangon L. to non-toxic stressors: Temperature, salinity and handling stress effects

Biochemical biomarkers in common estuarine species, such as the brown shrimp Crangon crangon, have the potential to provide early warning of contaminant exposure from field collected samples and through the development of in situ tests. The biomarkers acetylcholinesterase (AChE), lactate dehydrogenase (LDH) and glutathione S-transferase (GST) have been shown to provide evidence of exposure to contaminants in a number of species and field situations. As they may naturally respond to the marked physicochemical changes found in estuaries (thus confounding contaminant-induced effects), this work aims to determine the effects of salinity, temperature and handling stressors on these biomarkers in C. crangon.AChE recovery in field-collected shrimp transplanted to clean laboratory conditions suggests the presence of inhibiting factors at the sampling site (River Minho estuary). Maintenance time in stock tanks had effects that led to the choice of a minimum 15-day maintenance period of C. crangon in the laboratory before subsequent use of the enzymes as effect criterions in toxicity assays. Field levels of biomarker activity were unaffected following field-laboratory transportation of C. crangon, making this factor unlikely to jeopardize detection of contaminant associated effects. LDH levels were significantly lower under conditions that mimic a diurnal salinity fluctuation, increasing under low salinity conditions; this potentially indicates increased energy costs associated with raised osmoregulatory demands. It is recommended that a lower limit to field exposure of in situ tests should be based on salinity. Higher temperatures led to higher AChE activities and this is in agreement with the existing evidence of increases of endogenous AChE levels as a function of temperature (within a certain range). To avoid misinterpretation of biomarker responses, studies such as this are an important contribution to the establishment of reference activity levels against which biomarker changes can be estimated and are therefore essential preliminary steps in the development of in situ bioassays using biomarkers.     

Assessment of a glutathione S-transferase and related proteins in the gill and digestive gland of Mytilus edulis (L.), as potential organic pollution biomarkers

The response of the glutathione S-transferase (GST, EC 2.5.1.18) and related proteins of Mytilus edulis to environmental pollution load was assessed. Mussels were reciprocally transplanted between an industrial estuary (Douglas), a rural estuary (Youghal) and a m arine site (Bantry). In addition, m ussels were sam pled along a pollution gradient in an estuary receiving leather tannery effluent (Colligan). These latter m ussels were previously shown to be subject to oxidative stress resulting from the discharges. GST specific activity of cytosolic extracts from the gill and digestive gland tissues was determ ined for all anim als. Specific activity was shown to vary significantly in anim als from different sites, with highest specific activity always observed in sam ples (local and transplanted) taken from the industrial site. By com parison, the m ussels exposed to tannery discharges displayed no significant alteration in GST specific activity. Total intracellular glutathione (GSH) was also determ ined for sam ples taken from the Douglas and Youghal estuaries but no correlation with pollution load was observed. Using FPLC analysis, we observed no specific effect on the relative levels of the GST and the individual GST related proteins in gill or digestive gland sam ples from local or reciprocally transplanted anim als from Douglas or Youghal. The increase in GST specific activity observed in samples from the industrial estuary are indicative of a possible, specific inductive agent at this site. The results from the tannery site, by com parison, indicate that general oxidative stress does not result in elevated G ST specific activity in M. edulis.