Field application of biochemical markers and a physiological index in the mussel,Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy)

A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter.     

Comparison of the antioxidant defense system in Crassostrea rhizophorae and Crassostrea gigas exposed to domestic sewage discharges

Oysters Crassostrea rhizophorae and Crassostrea gigas were kept for fourteen days at four sites in S?o José, SC, Brazil, chosen according to a sewage discharge contamination gradient. Enzymatic activities of CAT, GST, G6PDH and GR were evaluated in gills and digestive glands. Higher CAT activity was observed in tissues of C. rhizophorae in response to sewage contamination possibly indicating peroxisome proliferation induction. C. gigas showed elevated G6PDH activity in digestive gland, and GR in gills, after domestic sewage exposure. In conclusion, C. rhizophorae and C. gigas showed different biochemical responses after in situ exposure to domestic sewage. However, C. rhizophorae showed more significant changes in CAT suggesting that this organism could be a better monitor to this kind of effluent.     

Biochemical and histochemical responses to environmental contaminants in clam, Tapes philippinarum, transplanted to different polluted areas of Venice Lagoon, Italy.

In this study the clam, Tapes philippinarum, a filter-feeding bivalve living in soft bottoms, was used to investigate the water/sediment pollution in Venice Lagoon (Venice, Italy), a heavily urbanised and industrialised area. To this end, clams collected from indigenous populations in a clean farming area (Val Dogà) were transplanted into a relatively low contaminated site (Palude della Rosa) and in a heavily polluted area (Porto Marghera), for 5 weeks. A range of possible induced stress indices were measured, on the basis of potential toxicity mechanisms, including specific enzymatic activities [Bap hydroxylase and NADPH cytochrome c reductase, superoxide dismutase, catalase, glutathione peroxidase and propionaldehyde dehydrogenase (PDH)] and general cellular responses (lysosomal latency). Moreover, chemical analyses (polychlorinated biphenyls, polycyclic aromatic hydrocarbon, DDTs) were carried out on sediments and animal tissues at the beginning and end of the transplantation experiments. The chemical results show an active bio-accumulation activity from sediment in clams translocated to the most polluted area. Correspondingly, biological data indicate marked effects on PDH activity and latency in T. philippinarum from the same site. In contrast, no similar pattern has been observed in the other considered indices, possibly due to antagonistic effects of the complex contaminant mixture present in the environment.     

Biomarkers and trace metals in the digestive gland of indigenous and transplanted mussels, Mytilus galloprovincialis, in Venice Lagoon, Italy.

The aim of this study was to investigate the cellular and biochemical response of mussels, Mytilus galloprovincialis, transplanted from a relatively pristine site to a polluted one and vice versa in the Lagoon of Venice (northeast of Italy) and to apply auto-metallography, a rapid and sensitive histochemical technique, to determine the bio-available fraction of heavy metals accumulated in the body tissues of organisms. Animal digestive glands have been used for morphological analyses (lysosomal volume, surface and numerical density, mean epithelial thickness, mean diverticular and luminar radius), autometallographical black silver deposits quantification and biochemical assays (superoxide dismutase and catalase activity). Furthermore, heavy metal content was determined by atomic absorption spectrophotometry (AAS) using standard procedures. The overall results indicate a direct influence of the environment in the thinning of the digestive cells and in the increasing number of lysosomes in mussels from the more polluted site. These data are in agreement with the metal content in digestive cell lysosomes as determined by autometallography, whereas AAS measurements show less significant differences.     

Effects of acid mine drainage from an abandoned copper mine, Britannia Mines, Howe Sound, British Columbia, Canada, on transplanted blue mussels (Mytilus edulis)

Juvenile mussels (Mytilus edulis) were transplanted to Howe Sound, British Columbia, Canada, along an apparent pollution gradient of acid mine drainage (AMD) from an abandoned copper (Cu) mine. Cages containing 75 mussels each were placed at a total of 15 stations and were exposed to concentrations of dissolved Cu in surface waters ranging from 5 to 1009 micrograms/l for a period of 41 days. Mussels located at stations closer to the source of AMD at the mouth of Britannia Creek bioaccumulated higher concentrations of Cu and zinc (Zn) in their tissues. Mussel growth was adversely affected by Cu tissue concentrations above 20 micrograms/g dry wt., while declines in survival and condition index occurred in mussels that bioaccumulated greater than 40 micrograms/g dry wt. Cu. Tissue Zn concentrations (117-192 micrograms/g dry wt.) were likely not high enough to have a direct impact on mussel health. Reduced survival of transplanted mussels was supported by an absence of natural mussels in contaminated areas. Phytoplankton was also severely reduced in areas contaminated by mine waters. Based on the weight of evidence, AMD from the Britannia mine had a deleterious impact on mussel survival in a zone extending at least 2.1 km to the north and 1.7 km to the south of Britannia Creek on the east shore of Howe Sound.     

Oxidative stress responses in two populations of Laeonereis acuta (Polychaeta, Nereididae) after acute and chronic exposure to copper

Acute (4 days) and chronic (14 days) effects of copper were evaluated on the antioxidant defenses of Laeonereis acuta (Polychaeta) collected in unpolluted (UP) and polluted (P) sites. In the acute assay (125 and 250 micro g Cu/l) superoxide dismutase (SOD) and glutathione S-transferase (GST) activities did not change, whereas catalase (CAT) increased in worms from both the sites. Lipid peroxidation was higher in copper exposed worms from the P site. In the chronic assay (62.5 micro g Cu/l) polychaetes from the P site showed enhanced activities of SOD, GST and CAT and higher contents of metallothionein-like proteins and sulfhydrils compared to worms from UP. Differences in responses between polychaetes from UP and P sites suggest that organisms from the polluted site, P, are more susceptible to oxidative stress conditions.     

Effects of furadan in the brown mussel Perna perna and in the mangrove oyster Crassostrea rhizophorae

Furadan is a carbamate pesticide used widely to combat agricultural pests. However little information is available about the toxicity of furadan in aquatic macroinvertebrates. The in vivo effects of furadan were evaluated in mussels, Perna perna, and oysters, Crassostrea rhizophorae. Glutathione S-transferase (GST), catalase (CAT) and cholinesterase (ChE) activities were measured in the gills of both species exposed to furadan (100 microg/l) for 96 h. No changes were observed in GST activity in the exposed groups. CAT activity was higher (9%) in the oysters exposed to furadan. ChE activity was inhibited by 64 and 35%, respectively, in C. rhizophorae and P. perna exposed to furadan, suggesting that the former is more susceptible to the toxic effects of furadan.     

Phase I and II biotransformation enzymes and polycyclic aromatic hydrocarbons in the Mediterranean mussel (Mytilus galloprovincialis,Lamarck, 1819) collected in front of an oil refinery

The aim of the present study was to investigate the responses of phase I and II biotransformation enzymes and levels of PAHs in the Mediterranean mussel (Mytilus galloprovincialis, Lamarck, 1819) collected from three sites at different distance from an oil refinery. Phase I enzyme activities as NAD(P)H-cyt c red, NADH ferry red, B(a)PMO and phase II as UDPGT, GST were measured in digestive gland while 16 PAHs (US-EPA) in whole soft tissue. An added value to the data obtained in the present study rely on the RDA analysis which showed close correlations between PAHs levels and phase I enzyme activities in mussels collected in front of the refinery. And again a significant spatial correlation between B(a)P levels and NADPH-cyt c red activities was observed using linear models. No differences among sites for B(a)PMO and phase II GST activities were observed, while the application of UDPGT as biomarkers requires further investigation.     

Effects of linear alkylbenzene sulphonate (LAS) and cadmium in the digestive gland of mussel, Mytilus sp.

A laboratory study was carried out exposing mussels (Mytilus sp.) to linear alkylbenzene sulphonate (LAS) (6 mg litre⁻¹), Cd (0.05 mg litre⁻¹) and LAS plus Cd at the same concentrations. The aim was to assess the use of several histopathological and biochemical indices as potential biomarkers of the impact of these xenobiotics in the digestive gland of molluscs. Treated mussels actively accumulated Cd in the digestive gland compared with controls (p 0.01), the highest levels occurring after 30 days of exposure in the group treated with Cd plus LAS. Among several histological alterations screened in digestive gland tissues the thickness of digestive tubules in Cd treated animals decreased more markedly (p 0.01) than in LAS exposed mussels. As for biochemical parameters, the investigated antioxidant enzyme activities, superoxide dismutase (SOD), catalase, DT-diaphorase and glutathione peroxidase (GPX) did not show any significant induction due to these xenobiotics. However, a slight decrease of the antioxidant defences of the animals was detected after 30 days of exposure to contaminants.     

Biochemical biomarkers in Scinax fuscovarius tadpoles exposed to a commercial formulation of the pesticide fipronil

One of the main pesticides used in the cultivation of sugarcane in São Paulo State, Brazil, is Regent^®800WG, the main active compound of which is fipronil. Fipronil is a potent insecticide that eliminates pests, including insects resistant to pyrethroids, organophosphates (OP) and carbamates (CA). There is little known on the toxic effects of fipronil on non-target organisms, such as tadpoles of frogs. It is possible that this compound carries a high toxicity for these organisms, since the pesticide can be incorporated into aquatic environments during the rainy season, a time which coincides with the time of amphibian reproduction and the occurrence of tadpoles in the aquatic environment in this region. Thus, the pesticide could be contributing to the decline of amphibians in the northwest region of São Paulo state due to its wide use. This study aimed to test the influence of Regent^®800WG on some biochemical systems of tadpoles (such as antioxidant defense systems) at different stages of development. The results of analysis from in vivo exposures demonstrated that only a few parameters in the groups exposed to fipronil responded to exposure to Regent^®800WG, results which indicate that the pesticide instigates biochemical responses in tadpoles. Although catalase and glucose-6-phosphate dehydrogenase (G6PDH) were unchanged during the experiments, glutathione-S-transferase (GST) was inhibited in tadpoles, and the activity of glutathione reductase (GR) varied according to the exposure period and pesticide concentration. This data demonstrated the influence of the fipronil formulation on the metabolism of tadpoles, and showed that it can increase their susceptibility to environmental contaminants.     

DNA strand breakage in mussels (Mytilus edulis L.) deployed in intertidal and subtidal zone in Reykjavík harbour

DNA single-strand breaks were analysed in the blue mussel (Mytilus edulis L.) deployed in intertidal and subtidal zones in the PAH contaminated Reykjavík harbour and at a reference site, Hvalfj?rethur, Iceland. DNA strand breaks were analysed by Comet assay in isolated gill and haemocyte cells from six mussels from each site and depth. Increased DNA damage in both gill cells and haemocytes were observed in mussels deployed in Reykjavík harbour compared to the reference site. Intertidal mussels from Reykjavík harbour had higher DNA damage in haemocytes compared to subtidal mussels. The Comet assay seems to be useful for measuring genotoxic exposure in mussels from the field, and that DNA damage might be higher in the intertidal zone either due to higher exposure to contaminants or because of physiological and biochemical responses to variations in oxygen availability.     

Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels

An aluminium smelter on the west coast of Scotland discharges an aqueous effluent containing polycyclic aromatic hydrocarbons (PAHs) at the head of Loch Leven. The loch also supports two mussel (Mytilus edulis) farms. Data are presented on burdens of PAHs in the soft tissues of mussels and the effect of these contaminants on glutathione S-transferase (GST) activity in mussel hepatopancreas. GST activity is shown to be correlated with total PAH burden and also with the concentrations of certain individual PAHs. These field data show that high molecular weight PAHs are closely correlated to GST activity, whereas low molecular weight PAHs are not. This suggests that 5- and 6-ring PAHs have a more pronounced role than 2- to 4-ring compounds in inducing GST activity in mussels from Loch Leven. It is proposed that it may be more appropriate to link GST activity with 5- and 6-ring compounds only, rather than with the total PAH burden.     

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, for monitoring the impact of an offshore platform in the Adriatic Sea

An ecotoxicological protocol with caged mussels, Mytilus galloprovincialis, was developed to evaluate the potential impact of an offshore gas platform in the central Adriatic Sea. Reference organisms were collected on a seasonal basis from an unpolluted site and transplanted for four weeks in both the sampling area and to the investigated platform. Chemical analyses of trace metals in mussel tissues were integrated with a multi-biomarker approach for the early detection of biological responses at several cellular targets. Induction of metallothioneins, peroxisomal proliferation and activity of acetylcholinesterase were measured as markers for specific classes of chemicals. Special attention was given to oxyradical metabolism and appearance of oxidative-mediated toxicity to reveal a more general onset of cellular disturbance. In addition to individual antioxidants (superoxide dismutase, catalase, glutathione S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, and levels of total glutathione), the total oxyradical scavenging capacity (TOSC) allowed a quantification of the overall capability to neutralize specific forms of intracellular reactive oxygen species (ROS; i.e. peroxyl and hydroxyl radicals). Cellular damages were evaluated as lysosomal destabilization (membrane stability, accumulation of lipofuscin and neutral lipids), lipid peroxidation products (malondialdehyde) and DNA integrity (strand breaks and micronuclei); the air survival test was finally applied to evaluate the overall physiological condition of mussels. Concentration of trace metals (As, Ba, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Zn) revealed only limited variations in transplanted mussels during various experimental periods and such changes appeared partly related to natural fluctuations. Among biological responses, variations of antioxidants and lysosomal stability were confirmed as sensitive early warning signals for biological disturbance of both natural and anthropogenic origin. The presented protocol with caged mussels allowed marked biological effects caused by the investigated platform to be excluded, and represented a useful approach that is easy to extend for monitoring the impact of offshore activities in the Adriatic sea.     

Short-term variability of multiple biomarker response in fish from estuaries: Influence of environmental dynamics

Short-term variability in biomarker responses and the effects of temperature and salinity variation were explored in three fish species (Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps) occurring in the Tejo estuary. Short-term variability in biomarkers was observed in all species although no pattern was discerned over time (days to weeks). Antioxidant enzymes activity (superoxide dismutase (SOD) and catalase (CAT)) showed low temporal variability, indicating some constancy or baseline level in antioxidant responses. Only CAT activity in S.?senegalensis was correlated with temperature, suggesting that exposure to contaminants triggered antioxidant acclimation. Higher short-term variability was observed in xenobiotic biotransformation enzymes activity (phase I ethoxyresorufin O-deethylase (EROD) and phase II glutathione S-transferase (GST)). Yet a significant correlation between EROD and GST in D.?labrax and S.?senegalensis suggests a concomitant response to contaminants. Moreover the lack of correlation between xenobiotic biotransformation enzymes and abiotic variables on concordant time scales, suggest a high specificity of these biomarkers to chemical exposure, rather than high variability due to environmental dynamics.     

Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels

An aluminium smelter on the west coast of Scotland discharges an aqueous effluent containing polycyclic aromatic hydrocarbons (PAHs) at the head of Loch Leven. The loch also supports two mussel (Mytilus edulis) farms. Data are presented on burdens of PAHs in the soft tissues of mussels and the effect of these contaminants on glutathione S-transferase (GST) activity in mussel hepatopancreas. GST activity is shown to be correlated with total PAH burden and also with the concentrations of certain individual PAHs. These field data show that high molecular weight PAHs are closely correlated to GST activity, whereas low molecular weight PAHs are not. This suggests that 5- and 6-ring PAHs have a more pronounced role than 2- to 4-ring compounds in inducing GST activity in mussels from Loch Leven. It is proposed that it may be more appropriate to link GST activity with 5- and 6-ring compounds only, rather than with the total PAH burden.     

Seasonal variations of exposure biomarkers, oxidative stress responses and cell damage in the clams, Tapes philippinarum, and mussels, Mytilus galloprovincialis, from Adriatic sea

This work investigated the natural variability of several biomarkers in Tapes philippinarum and Mytilus galloprovincialis, sampled from Northern Adriatic where these organisms are important sentinel species for future environmental impact assessment. Levels of metallothioneins, peroxisomal enzymes and acetylcholinesterase, showed a significant seasonality and marked differences between clams and mussels. Among antioxidant enzymes, catalase and GST decreased during the warmer period, the latter enzyme activity resulting particularly high in clams. The total oxyradical scavenging capacity toward peroxyl radicals decreased in mussels from winter to summer, indicating a prooxidant challenge due to higher seawater temperature and intensity of light irradiance. Lysosomal membrane stability did not exhibit significant seasonal variations, while some variations were observed for DNA damages. Overall results indicated a significant influence of seasonal variability on several biomarkers and species-specific differences which should be considered to discriminate the appearance of anthropogenic disturbance.     

Bioaccumulation and depuration of Zn and Cd in mangrove oysters (Crassostrea rhizophorae, Guilding, 1828) transplanted to and from a contaminated tropical coastal lagoon

In order to study Zn and Cd accumulation and depuration, a set of oysters, Crassostrea rhizophorae, were transplanted to a metal contaminated coastal lagoon and another one was harvested there and transplanted to a non-polluted site. C. rhizophorae oysters and Perna perna mussels native from both sites were collected in order to monitor variability of metal concentration in resident populations. After three months exposure, oysters transplanted to the polluted site accumulated fourfold Zn (307-1319 microgg(-1)) without reaching the concentration level of resident oysters (9770 microgg(-1)). Cadmium concentrations had a slight but significant decrease during the same period (1.25-0.54 microgg(-1)). Oysters transplanted to the non-polluted site, showed threefold Zn depuration (6727-2404 microgg(-1)), while Cd had no significant variation (0.90-1.45 microgg(-1)). Results showed that transplanted oysters do not reach heavy metal concentrations in indigenous populations suggesting transplanted organisms would be better used to evaluate bioavailability instead of environmental concentrations.     

Seasonal variation of antioxidant and biotransformation enzymes in barnacle, Balanus balanoides, and their relation with polyaromatic hydrocarbons

Seasonal variations in the antioxidant enzymes (catalase, superoxide dismutase [SOD], NADH-DT diaphorase), biotransformation enzyme, glutathione-S-transferase (GST) and microsomal lipid peroxidation in digestive tissue of barnacle, Balanus balanoides, from polluted and non-polluted populations have been evaluated. Relationships with accumulated polyaromatic hydrocarbon (PAH) concentration in barnacle tissues and environmental parameters (water temperature, salinity, dissolved oxygen concentration, water pH) were determined. As a general trend, maximum antioxidant enzyme and GST activities were detected in the pre-monsoon period or summer (March-June) followed by a gradual decrease during the monsoon (July October) with a minimum in the post-monsoon period or winter (November February). This pattern was similar to tissue concentrations of PAHs, resulting in a significant positive correlation with antioxidant enzymes, mainly catalase and SOD. Microsomal lipid peroxidation exhibited an almost reverse trend of seasonal variation to that of antioxidant enzyme activities indicating an enhanced susceptibility of barnacle tissues to oxidative stress. Among the environmental parameters, only water temperature seemed to have a significant effect on observed variations of antioxidant enzymes and GST activities. The barnacles from polluted and non-polluted populations exhibited seasonal differences in the activities of all the enzymes studied, particularly catalase, SOD and GST, suggesting the possibility of some biochemical adaptation in organisms from a chronically polluted environment. The results indicated that antioxidant defense components, catalase and SOD, are sensitive parameters that could be useful biomarkers for the evaluation of contaminated aquatic ecosystems. The results also suggested the potentiality of barnacle, B. balanoides, as a bioindicator organism against organic pollution.     

Adaptation of the antioxidant defence system in hydrothermal-vent mussels (Bathymodiolus azoricus) transplanted between two Mid-Atlantic Ridge sites

The vent mussel Bathymodiolus azoricus is the dominant member of the Northern Mid‐Atlantic Ridge (MAR) hydrothermal megafauna, and lives in an environment characterized by temporal and spatial variations in the levels of heavy metals, methane and hydrogen sulphide, substances which are known to increase reactive oxygen species levels in the tissues of exposed organisms. To evaluate the effects of two contrasting hydrothermal environments on the antioxidant defence system of this vent mussel species, a 2‐week transplant experiment was carried out involving mussels collected from the relatively deep (2300 m), and chemical rich, Rainbow vent field. These were transplanted to the shallower (1700 m), and relatively less toxic, Lucky Strike vent field. To achieve this objective, levels of superoxide dismutase, catalase (CAT), total glutathione peroxidase (GPx), selenium‐dependent glutathione peroxidase and lipid peroxidation (LPO) were measured in the gills and mantle tissues of resident and transplant mussels before and after the transplant experiment. With the exception of CAT, the gills of the transplanted mussels had significantly higher antioxidant enzyme activity compared with the basal levels in the donor (Rainbow) and recipient (Lucky Strike) populations; whereas the antioxidant enzyme levels in the mantle tissues of the transplants reflected the baseline levels of activity in the native Lucky Strike mussels after 2 weeks. In contrast, LPO levels were significantly higher in both tissue types in the transplants than in either the source or the recipient populations, which suggested a response to hydrostatic pressure change (note, the transplant animals were brought to the surface for transportation between the two vent fields). The fact that the Rainbow mussels survived the transplant experience indicates that B. azoricus has a very robust constitution, which enables it to cope behaviourally, physiologically and genetically with the extreme conditions found in its naturally contaminated deep‐sea environment.     

Biomonitoring of trace metals using transplanted mussels, <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis>, in coastal areas around Ulsan and Onsan Bays,Korea

Mediterranean (blue) mussels (Mytilus galloprovincialis) collected from a reference site were transplanted to 15 stations in coastal areas around Ulsan and Onsan Bays, an extensively metal polluted area in Korean coastal waters, to assess metal contamination in the coastal oceans of Korea. During the biomonitoring periods (June 30 to July 20, 2003; 21 days), transplanted mussels, seawater, and particulate materials were collected for analysis of 15 metals (Ag, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sb, Se, Sn, and Zn). Transplanted mussels showed metal enrichment compared to initial concentrations and spatial gradients consistent with dissolved and/or particulate metal concentrations in seawaters. Based on Q mode factor analysis, stations were clustered into three groups. The first group, located on Onsan Bay, showed high Ag, Cd, Cu, Hg, Pb, Sb and Zn enrichment, presumably arising from non-ferrous metal refineries and chemical industries in this area. The second group was located near the mouth of the Oehwang River and was enriched in Co from petrochemical industries. The third group comprised a site intermediate between Group 1 and Group 2, an isolated station with independent metal sources located in Jangsaengpo harbor, where a number of ship repairing and building companies operate, and a less contaminated station near a small fishing village. Metal accumulation rates (%·day^-1) in mussels were estimated to be between 8% (Cr) and 281% (Pb), based on accumulated metal concentrations over 21 days. The active biomonitoring technique using M. galloprovincialis demonstrated here is a useful monitoring method because it reflects the present status of seawaters; furthermore, physiological factors can be standardized, and bioavailable and time-integrated metal concentrations can be obtained. Furthermore, this method can be applied even in coastal seawaters so heavily contaminated that living organisms would not normally survive.