Marine water quality assessment using transplanted oyster larvae.

Active bio-monitoring in terms of biomarkers was attempted using Crassostrea gigas larvae produced in the laboratory and transplanted using special containers to two sites at the entrance (A) and inner part (P) of the harbour of Arcachon (French Atlantic Coast). The larvae were kept in the medium for 48 h. Their physiological status and their biomarker levels : acetylcholinesterase AChE, catalase CAT and glutathione S-transferase GST activities were determined together with metallothionein MT and Thiobarbituric Acid Reactive Substances TBARS concentrations. Copper and PAH (polycyclic aromatic hydrocarbon) concentrations were determined in the exposed larvae and in the sediments collected under the containers. Cadmium, lead and zinc could be also analyzed in the sediments. Toxicity tests demonstrate that the larvae are in better physiological conditions in A compared to P. Larvae transplanted in the inner harbour (P) present relatively high GST activity (869.1+/-39.3 nmol min(-1)mg protein(-1)), TBARS (2.74+/-0.19 nmol mg protein(-1)), compared to those exposed at the harbour entrance (A). Copper measured in the sediments (65+/-1 mg kg(-1) d.w.) collected under the cages at P is higher than at A. Larvae placed in A present higher total PAH concentrations compared to the inner part. The data tend to reveal a lower copper and higher PAH contamination in A than in P. Therefore larvae, developing in the natural medium, show different responses according to their immersion sites. These responses, obtained within 48 h, may be related to the chemical contamination of the environment and may be used for seawater quality assessment in future studies.

Integrated biomarker response index as a useful tool for environmental assessment evaluated using transplanted mussels.

Mytilus galloprovincialis mussels from a clean area were transplanted from 2003 to 2005 to several stations in the Bay of Cannes (North-Western Mediterranean Sea) including a site considered as reference, for 1 month at the end of spring (May). Several biomarkers (AChE, GST and CAT activities, TBARS and MT concentrations) were measured in the transplanted organisms. The concentrations of metals (Cd, Cu and Zn) were determined in the transplanted mussels, PAH and PCB analyses were performed in the mussels caged in 2004. The integrated biomarker response (IBR) was calculated; pollutant concentrations in mussels were displayed as star plots and compared to IBR star plots. Visualization was thus possible between sites for comparison with exposure conditions. Results demonstrated that the mussels from the Old harbour site (VP) are characterized by elevated copper and PCB concentrations, those from Canto harbour (PC) presented high PCB contents and those from the mouth of the Siagne River (ES) high PAH concentrations compared to the animals transplanted in the reference site (IL). In 2003, there was a visual correlation between the copper gradient measured in the transplanted mussels and the IBR variation. In 2004, the agreement between the copper gradient and the PCB gradient measured in the caged mussels and the IBR variation was good whereas the PAH gradient did not seem to contribute to the IBR demonstrating that the chosen biomarkers did not respond to PAHs. In 2005, IBR showed that other contaminants, not measured might be present in VP, PC and ES compared to the reference station (IL).

Responses of Hexaplex (Murex) trunculus to selected pollutants.

Cadmium, copper and zinc concentrations (in whole soft body and in tissues) were measured in Hexaplex trunculus collected from the Bizerta lagoon in Tunisia. An evaluation of the biological effects of the most toxic metals (cadmium and copper) and of two organics (carbofuran and lindane), present in the sediments of the Bizerta lagoon, was attempted by measuring biomarkers (acetylcholinesterase: AChE, catalase: CAT and glutathione S-transferase: GST activities) in animals experimentally exposed for 48 or 72 h. The concentration ranges as follows: Zn>Cu>Cd. Copper concentrations are highly variable (8.0 to 235 microg g(-1) d.w.) whereas cadmium (range 1.35-4.86 microg g(-1)) and zinc (range 360-1320 microg g(-1)) concentrations are less variable. The digestive gland and the gill take up more metal than the muscle. AChE activity in H. trunculus is decreased by exposure to carbofuran or the mixture carbofuran and cadmium, in the digestive gland and muscle and by copper and by lindane in the digestive gland. AChE is generally inhibited by carbamates but some other compounds may also decrease this activity as observed in this paper. An increase in CAT activity associated with a decrease in GST activity is noted in the muscle of H. trunculus exposed to cadmium, to carbofuran and to the mixture of cadmium and carbofuran, and in the digestive gland of animals exposed to lindane. These pollutants may act upon glutathione and decrease the GST activity that cannot detoxify them and CAT activity has a protective effect. On the contrary, copper increases CAT and GST activities in the digestive gland of exposed gastropods; these enzymes seem to cooperate and play together their rôle of anti-oxidant enzymes. If H. trunculus is not a bioindicator species for metal concentrations, due to a high variability in metal concentrations, nevertheless the biochemical responses to pollutants (cadmium, copper, carbofuran and lindane) represented by AChE, CAT and GST activities may act as biomarkers of exposure in this species.

Heavy metal concentrations in molluscs from the Senegal coast.

Bivalve molluscs from the Western coast of Senegal were sampled and measured for their cadmium, copper and zinc concentrations. This part of Africa has not been studied as regards heavy metal concentrations in the molluscs. The collected species are two small African bivalve molluscs, living in the sand: Cardita ajar and Dosinia isocardia; the African mussel Perna perna which is the only mussel of this genus in the Western coast of Africa and the oyster Crassostrea gasar, which lives in mangroves attached to the rhizophores, in the intertidal zone. C. gasar and C. ajar present higher cadmium concentrations, respectively 6.82+/-0.54 and 13.77+/-0.80 microg Cd/g (d.w.) than the two other species (D. isocardia: 3.88+/-0.31 microg/g and P. perna 2.37+/-0.22 microg/g. Copper and zinc concentrations are in the range of the published values for C. gasar and P. perna collected elsewhere in Africa. The results suggest that cadmium may be present in high concentrations in Senegalese waters where upwellings occur.

Biomonitoring of trace metals in the Black Sea (Romania) using mussels Mytilus galloprovincialis.

In a preliminary biomonitoring study, accumulated trace metal concentrations (Cd, Cu, Zn, Hg, Fe, Mn) have been measured in the mussel Mytilus galloprovincialis collected in the Black Sea, at 100 km from the Danube Delta in February 2001 and February 2002. Mussels were collected from four sites. In 2001 metal concentrations were determined in the whole soft body whereas, in 2002, the distribution of metals was evaluated in three different organs (gills, visceral mass and remaining tissues). The results obtained in 2002 confirmed those of 2001. For a given site, concentrations were always higher in the gills and visceral mass of mussels than in the remaining tissues. Principal component analysis allowed separating stations as a function of the metal concentrations in the organs. It is concluded that the mussels M. galloprovincialis are suitable biomonitors to assess changes in metal pollution in this coastal area of the Black Sea.

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.

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.

Multimarker approach in transplanted mussels for evaluating water quality in Charentes, France, coast areas exposed to different anthropogenic conditions.

An active biomonitoring experiment was performed using mussels collected at a clean site, Fier d'Ars, and transplanted to two locations, outside the harbor of La Rochelle and in the Baie de L'Aiguillon along the coast of Charentes (French Atlantic coast) beginning in April for several months. Mussels were collected in June and October. The cadmium, copper, and zinc concentrations of all resident and transplanted mussel samples and the polycyclic aromatic hydrocarbon and polychlorinated biphenyl concentrations in some mussel samples and in the sediment samples were determined. Mussel response was evaluated for several biochemical biomarkers: concentrations of metallothionein, activities of glutathione S-transferase and acetylcholinesterase (AChE) and levels of thiobarbituric reactive substance (TBARS). The physiological status of the animals was assessed using the condition index. A principal component analysis performed with the chemical and biochemical results of the evaluations of the resident and transplanted mussels collected in June allowed them to be separated into three groups: resident mussels from la Rochelle with high metal and TBARS levels, resident mussels from Baie de L'Aiguillon with a very high condition index, and resident mussels from Fier d'Ars and transplanted mussels at La Rochelle and Baie de L'Aiguillon with low TBARS and AChE activities. Strong seasonal variation from June to October of all parameters was noted. Mussels transplanted to La Rochelle appeared to be the most "polluted" in their pollutant concentrations and biochemical responses; moreover, the La Rochelle site had the highest concentration of organics in sediments of all the sites. The choice of Fier d'Ars as a reference site may be questionable because some of the biomarker responses of the mussels were higher than expected there, although these pollutants in mussels and sediment were present at the lowest concentrations measured.

Effects of mercury on antioxidant mechanisms in the marine phanerogam Posidonia oceanica.

Biochemical markers of oxidative stress such as catalase activity, glutathione S-transferase (GST) activity and levels of lipid peroxidation evaluated in terms of thiobarbituric acid reactive substances (TBARS) were measured in the sheaths of the marine phanerogam Posidonia oceanica (L.) Delile experimentally exposed to 0.01, 0.1 and 1 microgHg l(-1) for 48 h. Up to a threshold concentration of 0.1 microg Hg l(-1), an increase in catalase and GST activities and TBARS levels was observed, indicating that the antioxidant mechanisms were overtaxed and could not prevent membrane lipid peroxidation. Paradoxically, at 1 microg Hg l(-1), the damage seemed to decrease, as the lipid peroxidation levels of exposed sheaths were lower than those of controls and as catalase and GST activities were not different from those of controls. A possible rapid induction of phytochelatins detoxifying mercury could occur at this high level of mercury.

The use of acetylcholinesterase activity in Ruditapes decussatus and Mytilus galloprovincialis in the biomonitoring of Bizerta lagoon.

The variations of acetylcholinesterase activity, considered to be a biochemical biomarker of organophosphorus and carbamate compounds, was followed in Mediterranean clams (Ruditapes decussatus) and mussels (Mytilus galloprovincialis) as a function of season and sampling sites in the lagoon of Bizerta (Tunisia). Results show that acetylcholinesterase activity in clams collected from station A was the lowest and was so throughout the year. This low activity may be due to the input of non-treated waste waters in the lagoon. Heavy metal contamination has been reported in the sediments of this area, suggesting that acetylcholinesterase activity in clams may be inhibited by this type of pollution. Clams harvested from station F, a site adjacent to an agricultural area, were characterized by a decrease in acetylcholinesterase activity, especially after rain periods. Clams from station J presented the highest activity throughout the greater part of the year. This phenomenon may imply that this location is less affected by pollutants inhibiting AChE activity than the others. In the mussels from station C, located in the channel from the lagoon to the Mediterranean and submitted to pollutants (urban wastes from Bizerta and hydrocarbons from the maritime traffic), acetylcholinesterase activities were lower than in those from station J. In conclusion, the variations in acetylcholinesterase activity observed between stations in both species may be the result of pollution and of the environmental conditions.

Differential induction of glutathione S-transferases in the clam Ruditapes decussatus exposed to organic compounds.

Studies of glutathione S-transferase (GST) induction were performed in the Mediterranean clam Ruditapes decussatus after controlled exposure to organics in holding tanks. Clams were treated with phenobarbital (PB), benzo[a]pyrene (BaP), and 2,2-bis-(p-chlorophenyl)-1,1-dichlorethylene (p,p'-DDE). Three different substrates, i.e., 1-chloro-2,4-dinitrobenzene (CDNB), ethacrynic acid (ETHA), and paranitrobenzene chloride (PNBC), were used to determine GST activities in order to distinguish the isoenzymes induced by contamination. The isoforms conjugating ETHA were significantly induced by treatment with PB and BaP, whereas exposure to p,p'-DDE induced isoforms conjugating CDNB and ETHA. An antibody against affinity-purified GSTs from R. decussatus was prepared by injection into rabbit. The serum containing the antibody gave a positive reaction with both the purified GSTs from R. decussatus and the low molecular weight GSTs from rat. Subcellular fractions from both control and treated animals were analyzed by Western blot. Cytosolic extracts from clams contaminated with PB and p,p'-DDE showed a 24-kDa band in addition to the 26-kDa band recognized by the antibody. Results of these studies suggest that, in R. decussatus, organics may induce GSTs belonging to the pi class.

Biochemical response of the mussel Mytilus galloprovincialis from Bizerta (Tunisia) to chemical pollutant exposure.

Three biomarkers (glutathione S-transferase [GST] activity, catalase [CAT] activity, and malonedialdehyde [MDA] levels) were measured in specimens of the mussel Mytilus galloprovincialis collected from two different stations (BC and MJ) in the lagoon of Bizerta (Tunisia). Animals were allowed to acclimate in the laboratory for some days. They were then exposed for 48 h to two concentrations of pp'DDE and two doses of mercury chloride. The acclimation period increased CAT activities and MDA levels in control mussels from both sites. GST activities were not modified during the acclimation period, whereas the sampling site of mussels appeared to exert a significant influence (higher values in control mussels from MJ than in those from BC). The treatment with both contaminants also increased GST activities of mussels from BC and not from MJ. It is hypothesized that animals from this last location are more exposed to the urban waste waster disposal, their biochemical response (GST activity) to pollutant exposure will be less marked. The treatment with pp'DDE or mercury did not show significant trend in CAT activities or MDA levels due to the variation of controls, and comparison of sites for mussels exposed to either pollutant therefore seems difficult. The acclimation period in the laboratory and the origin of mussels must be taken into consideration when studying the biochemical responses of mussels experimentally exposed to chemical pollutants.

Cadmium and copper display different responses towards oxidative stress in the kidney of the sea bass Dicentrarchus labrax.

Copper and cadmium were i.p. injected into the fish Dicentrarchus labrax. Cu as Cd-treated fish showed an enlargement of the lysosomal membrane of the kidney (pronephros), Cu being more toxic than Cd. Following injection, metal uptake, measured in muscle, liver and kidney, was much higher with cadmium treatment than with copper, the kidney being the main accumulating organ of cadmium in which metal accumulation is correlated with increased zinc level, suggesting metallothionein induction. In vivo exposure to metal decreases the lysosomal membrane stability of pronephros with a half dose of 127 ng g(-1) Cu and 735 ng g(-1) Cd. Lipid peroxidation, expressed as malondialdehyde equivalents (MDA), and catalase activity were measured in kidney subcellular fractions. When added in vitro, Cu significantly raises the MDA level (365% at 200 µM), Cd having a lower effect (20% at 500 µM). Catalase activity is significantly reduced by Cd whereas Cu does not produce any significant effect at the tested concentrations. Results suggest that although both metals cause in vivo damage to pronephros lysosomal membrane, Cu activates the redox process generating oxyradicals but does not affect in vitro the protective catalase activity unlike Cd which appears to weakly participate in oxyradical generation but alters in vitro protective catalase activity.

Heavy metal distribution in different fish species from the Mauritania coast.

Cadmium, copper, zinc and mercury concentrations were determined in pelagic and benthic fishes from the Mauritania coast. The pelagic fishes consisted of the round sardinella Sardinella aurita (Clupeidae), the chub mackerel Scomber japonicus (Scombridae) and the Atlantic horse mackerel Trachurus trachurus (Carangidae). Four benthic species were considered, three Serranidae: the painted comber Serranus scriba, the golden grouper Epinephelus costae, and the Niger hind Cephalopholis nigri and one Mullidae: the West African goatfish Pseudupeneus prayensis. Cadmium, copper, zinc and mercury concentrations are low in the edible muscles of the pelagic species. In the benthic fishes, metals could be determined also in the gills and liver. Cadmium and copper were present in relatively low levels in the muscle (< or = 0.06 microgram Cd g-1 dry wt., < or = 1.6 micrograms Cu g-1 dry wt.) and gills (< or = 0.23 microgram Cd g-1, < or = 3.1 micrograms Cu g-1) and higher levels are encountered in the livers (< or = 51 micrograms Cd g-1, < or = 49.1 micrograms Cu g-1). Zinc concentrations in the muscle are low (< or = 20 micrograms Zn g-1 dry wt.), the concentrations in the gills (< or = 120 micrograms Zn g-1) approach those of the livers for C. nigri and P. prayensis or are higher in the case of S. scriba and E. costae. Mercury concentrations in the gills and muscle of the pelagic species are very low, higher quantities are found in the livers of the benthic species. The metal concentrations in all the fishes analysed, which are restricted to approximately 40 specimens, are low except those found for cadmium in the livers of the benthic fishes.

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.