Genotoxicity in two bivalve species from a coastal lagoon in the south of Portugal

DNA damage was evaluated by comet assay in the haemolymph of two bivalve species Ruditapes decussatus and Mytilus galloprovincialis from the Ria Formosa lagoon (south Coast of Portugal). Clams and mussels were sampled from sites close to each other to determine interspecific responses to similar environmental conditions, considering also seasonal and gender differences. Coupled with genotoxic effect, another damage biomarker (lipid peroxidation) was analysed to verify if the conditions that instigate DNA damage can be related with injury to cell membranes. For both species, DNA damage was low, reflecting the low levels of genotoxic contaminants in the lagoon, and no interspecific differences were found. However, seasonal differences were established for both bivalve species, reflecting higher environmental stress in summer. Regarding gender susceptibility, only clams showed differences in percentage of Tail DNA, with females more sensitive to DNA damage than males. Additionally, results for clams point out that factors responsible for LPO may not be the same as those causing genotoxicity. This study demonstrated that DNA damage is a sensitive biomarker to discriminate spatial, temporal and gender differences, being an appropriate biomarker for genotoxicity evaluation even in places of low contamination, such as the Ria Formosa lagoon.     

Spatial and temporal variation of biomarkers in mussels (Mytilus galloprovincialis) from the Lagoon of Venice, Italy

A multiple biomarker approach was adopted in a seasonal study carried out in the Lagoon of Venice, with the double aim of evaluating the natural and anthropic stresses influencing the biological responses of mussel (Mytilus galloprovincialis) and of assessing the effects due to spatial rather than temporal variations.Biochemical (aldehyde dehydrogenase and catalase activities), cellular (neutral red retention time) and physiological (survival in air and condition index) biomarkers were determined in mussels collected in four differently impacted lagoon areas.Multivariate analysis showed that samples were distributed mainly according to temperature and four seasonal groups were identified. The combination of spatial and temporal information enabled us to distinguish physiological variations due to natural causes from those due to anthropic stress, and to identify the sampling period when several biomarkers are less influenced by both natural and endogenous factors.     

Dominance of blue mussels versus consumer-mediated enhancement of benthic diversity

In the shallow subtidal of Kiel Fjord (western Baltic Sea), the blue mussel Mytilus edulis is the dominant competitor for space. Vertically suspended settlement substrata in the upper 6 m of the water column almost invariably become dominated by M. edulis within a few summer months. However, not all naturally available hard substrata bear mussel monocultures.In three in situ experiments we investigated the dominance of mussels and the influence of local consumers on establishment and dynamics of a benthic community: (a) the natural course of succession in the absence of benthic consumers was followed on vertically suspended settlement substrata, (b) settling plates were exposed to natural recruitment being either accessible or inaccessible to all benthic consumers, and (c) the three major local consumer species, viz. the shore crab Carcinus maenas, the starfish Asterias rubens and the periwinkle Littorina littorea, were enclosed separately in cages containing a settling panel to assess species-specific consumer effects on recruitment.The results illustrate that in this region of the Baltic Sea mussels do have the potential to dominate ungrazed substrata within a few weeks and that top-down effects (predation and grazing) may control community structure. While some species - mussels in particular - were suppressed by consumption, others seemed to benefit from the presence of consumers. Thus, barnacles and algae thrived when consumers were present. Blue mussels being the locally dominant competitor, the beneficial effects of consumers on barnacles and algae were presumably indirect ones through consumer-caused release from asymmetrical competition.The isolated effects of C. maenas, A. rubens and L. littorea, on recruitment differed in quality (positive vs. negative), quantity (strength of effect) and specificity. Barnacle recruitment was significantly reduced in the presence of both starfish and shore crabs. Diatom recruitment was significantly reduced by snails and shore crabs. Scyphozoan recruitment was significantly reduced by crabs only, but was significantly increased by snails. The influence of the shore crabs was most remarkable: recruitment by any potential coloniser species was impeded to a point where in the presence of single individuals of C. maenas the plates stayed perfectly clean macroscopically.     

Preliminary evaluation of the toxic effects of the antifouling biocide Sea-Nine 211™ in the soft coral Sarcophyton cf. glaucum (Octocorallia,Alcyonacea) based on PAM fluorometry and biomarkers

Sea-Nine 211™ is a new biocide specifically formulated for antifouling paints and being considered to have a low environmental impact. Even with a short environmental half-life, this compound can cause toxic effects on marine organisms. This study used PAM fluorometry and biomarkers of oxidative stress (GST, CAT and LPO) to monitor potential toxic effects of Sea-Nine 211™ on fragments of the soft coral Sarcophyton cf. glaucum. After exposure to concentrations of 1–100 μg l⁻¹ for 72 h, CAT activity was inhibited under the two highest concentrations, being in accordance with the activity of GST. LPO activity (as TBARS) and photosynthetic efficiency of endosymbiotic zooxanthellae were not significantly affected. These results show that PAM fluorometry alone cannot detect the full effects of Sea-Nine 211™ on Sarcophyton cf. glaucum and should be used together with other biomarkers. This holobiont driven approach to evaluate chemical toxicity in photosynthetic corals is therefore recommended for biocides which are not photosystem II inhibitors.     

Temporal variation in the antioxidant defence system and lipid peroxidation in the gills and mantle of hydrothermal vent mussel Bathymodiolus azoricus

Hydrothermal vent mussels are exposed continually to toxic compounds, including high metal concentrations and other substances like dissolved sulphide, methane and natural radioactivity. Fluctuations in these parameters appear to be common because of the characteristic instability of the hydrothermal environment. Temporal variation in the antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), total glutathione peroxidases (Total GPx), selenium dependent glutathione peroxidases (Se-GPx)), metallothioneins and lipid peroxidation (LPO) in the gills and mantle of the mussel Bathymodiolus azoricus from Menez–Gwen hydrothermal vent site was evaluated and related to the accumulated metal concentrations (Ag, Cu, Cd, Fe, Mn and Zn) in the tissues. Maximum antioxidant enzyme activities in the gills were detected in the beginning of summer, followed by a gradual decrease throughout the following months. One year after, the levels of antioxidant enzyme activities were similar to those reported one year before. LPO in this tissue exhibited a similar temporal variation trend. A different pattern of temporal variation in antioxidant enzyme activities was observed in the mantle, with a gradual increase from summer to the end of autumn (November). LPO in the mantle exhibited an almost reverse trend of temporal variation to that of antioxidant enzyme activities in this tissue. Antioxidant defences in the gills of B. azoricus were significantly enhanced with increasing concentrations of Ag, Cu and Mn, while negative relationships between antioxidant enzymes and Cd, Cu, Mn and Zn concentrations in the mantle were observed, suggesting different pathways of reactive oxygen species (ROS) production and that these tissues responded differently to the metal accumulation. However, temporal variation in biomarkers of defence and damage were in general similar to coastal bivalve species and can be associated with temporal variations of the physiological status due to reproduction. These variations might also be linked to the highly unstable nature of the hydrothermal environment.     

Oxygen radical formation in anoxic transgression and anoxia-reoxygenation: Foe or phantom? Experiments with a hypoxia tolerant bivalve

Intertidal blue mussels, Mytilus edulis, experience hypoxia reoxygenation during tidal emersion and resubmersion cycles, and this is often suggested to represent a major stress for the animals, especially for their respiratory tissues, the gills. We exposed mussels to experimental short and prolonged anoxia and subsequent reoxygenation and analyzed the respiratory response in excised gill tissue and the effects of treatment on reactive oxygen species (mainly ROS: superoxide anion, O₂^·− and hydrogen peroxide, H₂O₂), formation using live imaging techniques and confocal microscopy. Our aim was to understand if this “natural stress” would indeed produce oxidative damage and whether antioxidant defenses are induced under anoxia, to prevent oxidative damage during reoxygenation. Exposure to declining pO₂ in the respiration chamber caused an increase of gill metabolic rate between 21 and 10 kPa, a pO₂ range in which whole animal respiration is reported to be oxyregulating. Exposure of the animals to severe anoxia caused an onset of anaerobiosis (succinate accumulation) and shifted high and low critical p_c values (p_c1: onset of oxyregulation in gills, p_c2: switch from oxyregulation to oxyconformity) to higher pO₂. Concentrations of both ROS decreased strongly during anoxic exposure of the mussels and increased upon reoxygenation. This ROS burst induced lipid peroxidation in the mantle, but neither were protein carbonyl levels increased (oxidative damage in the protein fraction), nor did the tissue glutathione concentration change in the gills. Further, analysis of apoptosis markers indicated no induction of cell death in the gills. To our knowledge, this is the first paper that directly measures ROS formation during anoxia reoxygenation in mussels. We conclude that hypoxia tolerant intertidal mussels do not suffer major oxidative stress in gill and mantle tissues under these experimental conditions.     

Genotoxicity of copper oxide and silver nanoparticles in the mussel Mytilus galloprovincialis

Though there is some information on cytotoxicity of copper nanoparticles and silver nanoparticles on human cell lines, there is no information on their genotoxic and cytotoxic behaviour in bivalve molluscs. The aim of this study was to investigate the genotoxic impact of copper oxide and silver nanoparticles using mussels Mytilus galloprovincialis. Mussels were exposed to 10 μg L⁻¹ of CuO nanoparticles and Cu²⁺ and Ag nanoparticles and Ag⁺ for 15 days to assess genotoxic effects in hemocytes using the comet assay. The results obtained indicated that copper and silver forms (nanoparticles and ionic) induced DNA damage in hemolymph cells and a time-response effect was evident when compared to unexposed mussels. Ionic forms presented higher genotoxicity than nanoparticles, suggesting different mechanisms of action that may be mediated through oxidative stress. DNA strand breaks proved to be a useful biomarker of exposure to genotoxic effects of CuO and Ag nanoparticles in marine molluscs.     

Effects of environmental pollution in caged mussels (Mytilus galloprovincialis)

Biological effects of environmental pollution, mainly related to presence of PAHs, were assessed in mussels Mytilus galloprovincialis caged in Priolo, an anthropogenically-impacted area, and Vendicari, a reference site, both located along the eastern coastline of Sicily (Italy). PAHs concentration and histopathological changes were measured in digestive gland tissues. Expression of cytochrome P4504Y1 (CYP4Y1) and glutathione S-transferase (GST), indicative of xenobiotic detoxification, and activity of catalase (CAT) as oxidative stress index, were evaluated.The results show a direct correlation between the high concentrations of PAHs in digestive glands of mussels from Priolo and the significantly altered activity of phase I (P < 0.001) and phase II (P < 0.0001) biotransformation enzymes, along with increased levels of CAT activity (P < 0.05). These findings show the enhancement of the detoxification and antioxidant defense systems. The mussel caging approach and selected biomarkers demonstrated to be reliable for the assessment of environmental pollution effects on aquatic organisms.     

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.     

Effects of the MV Rena oil spill on intertidal rocky reefs in the Bay of Plenty,New Zealand

In the weeks following the 2011 Rena oil spill, a series of surveys was initiated on eight rocky intertidal reefs to describe the distribution of oil and to assess the impacts of oil on ecological communities. Consistent but relatively low cover of oil occurred at two sites (Mt Maunganui and Moturiki). The area covered by oil had decreased by c. 90% after 5 months due to natural weathering processes. There were immediate effects of oil fouling on the mussel Limnoperna pulex and its associated fauna, with reductions in the number of mussels and infaunal taxonomic richness. However, no ecological effects on any of the communities were detectable after 1 month. Overall, the ecological effects of the Rena oil spill on rocky shore intertidal communities were small and not long-lasting, but we stress that this does not consider potential sublethal effects and their consequences on organisms.     

Enhancement of polycyclic aromatic hydrocarbons in estuarine invertebrates by surface runoff at a decommissioned military fuel depot

Accumulation of polycyclic aromatic hydrocarbons (PAHs) was determined in blue mussels (Mytilus spp.) and shore crabs (Hemigrapsus sp.) at a recently closed military fuel depot in central San Francisco Bay, California. In April 1996, during a period of above average precipitation, specimens were collected at the depot, near the depot, and at sites 10 and 20 km south of the depot. Four weeks after the rains ended, blue mussels were again collected at the depot, and at two additional sites in the central Bay region. In April, total PAHs in mussels from the depot were significantly higher only than that in mussels collected 20 km from the depot; however, seven specific, substituted PAHs were higher at the depot than at all other sites. In June, only two of the 38 PAHs common in mussels in April were detected at the depot; these concentrations were comparable to ambient concentrations in mussels at the Bay. It seemed that bioavailability of PAHs at the depot was enhanced by rainfall, probably due to the mobilization of PAHs via groundwater into the Bay. Concentrations in mussels from chronically contaminated sites were about five times higher than mussels collected from the depot. Low PAH concentrations were detected in shore crabs near the depot, and the highest levels were not associated with the depot. Observed PAH concentrations are discussed in relation to upper trophic organisms.     

Integration of biochemical, histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary, U.K

The aim of this study was to examine whether a combination of biochemical, histopathological and toxicogenomic data could be used as a valuable tool for the assessment of biological risk associated with pollutants within the Tamar River and Estuary, S.W. England, U.K. Accordingly, biochemical and histopathological biomarkers (protein carbonyls, lipofuscin, neutral lipids, lysosomal stability [N-acetyl-β-hexosaminidase and neutral red], lysosomal volume, ferric reducing antioxidant power [FRAP] and malonaldehyde [MDA]) and gene expression profiles were assessed in 5 sites from the Tamar River and Estuary (Neal Point, Town Quay, Wilcove, Cremyll Ferry and Whitsand; and a reference site, Trebarwith Strand, N. Cornwall). PAHs were measured in mussel tissue and sediment and metals were measured in mussel tissue only. Data from the biomarkers was integrated into a Mussel Expert System (MES) model to produce a simple assessment of mussel stress. Clear gradients of mussel toxicity were identified by the biomarkers (with the exception of neutral lipids) with the highest impacted animals found furthest up the Tamar, whilst the MES was unable to identify a gradient of effect. Gene expression profiles also indicated a gradient of stress with the greatest number of significantly up- or down- regulated genes found at the uppermost 2 sites. The MES did, however, determine that mussels from all sites, except the reference site, were highly stressed; a conclusion that could not be inferred from the biomarker data alone. It is concluded that the MES is a valuable tool that permits integration and interpretation of complex sets of biomarker data by identifying the biological meaning of biomarker changes.     

Effect of seasonality on oxidative stress responses and metal accumulation in soft tissues of Aulacomya atra,a mussel from the South Atlantic Patagonian coast

This study investigated the effects of pollution and its interaction with temperature on the oxidative status of the ribbed mussel Aulacomya atra in the southern Atlantic Patagonian coast. Animals were collected from four sites with different degree and type of human activity impact, during the summer and winter of 2011. Seawater chromium, copper, manganese, nickel and zinc concentrations were measured, as well as metal accumulation, lipid peroxidation, protein oxidation, reduced glutathione levels, and enzymatic activities of superoxide dismutase and glutathione-S-transferase in gills and digestive glands.Metal bioaccumulation and oxidative stress responses in both tissues were generally higher in mussels from harbor areas. Water temperature had a remarkable effect on gill SOD activity and protein oxidation during winter in mussels from all locations.Methodologically, we conclude that measuring both metal bioaccumulation and oxidative stress responses allowed for a more accurate assessment of the biological effects of metal present in seawater.     

Shore exposure affects mussel population structure and mediates the effect of epibiotic algae on mussel survival in SW Ireland

On rocky shores, the relative importance of abiotic and biotic processes that regulate community structure are thought to vary with levels of shore exposure. This can lead to characteristic features found on sheltered and exposed shores. This study identified differences in the population structure of mussels on exposed and sheltered rocky shores on Atlantic coasts of south-west Ireland. Direct interactions between epibiotic algae and their host mussels were also examined to test if potential effects varied with shore exposure. Mussel beds on sheltered shores were less dense and comprised larger mussels with greater rates of individual survival and growth than those on exposed shores. The results of a field experiment showed that algal epibionts had a negative effect on mussel survival on sheltered shores but not on exposed shores. Surprisingly, the presence of algal epibionts had no effect on mussel growth on either shore type. These findings contrast with those of previous studies. The effects of shore exposure and algal epibionts on mussels may be species-specific and may interact with other factors across different regions. This study shows that predictions of effects of exposure on mussel populations and their epibionts should only be based on specific experimental evidence and cannot be generalised across regions.     

Biomarkers in mangrove root crab Goniopsis cruentata for evaluating quality of tropical estuaries

The present study reports the use of biomarkers analyzes in mangrove root crab Goniopsis cruentata tissues to assess the environmental quality of two tropical estuarine areas. Animals from Ceará River estuary presented inhibition of ChE and GST enzymatic activities and higher rates of DNA damage with respect to those sampled in a pristine environment. G. cruentata appears to represent a proper species to monitor the quality of tropical estuaries. Since Ceará River is a legally protected area, this survey highlight the needs to implement actions to control pollution loads and improve the protection of natural ecosystems and resources.     

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.     

An example of drastic natural changes in the intertidal biota and the implications for monitoring programmes

During a period of about two years at Parkmore Point, Co. Kerry, Ireland, fucoids drastically decreased in the mid and upper shore, meanwhile barnacle abundance increased on the upper shore and mussels increased on the lower shore. Causes could not be determined but human interference was excluded due to remoteness, and changes were so localized as to exclude climatic factors. These drastic natural changes emphasize the need for guarded interpretation of monitoring data from areas suspected of pollution.     

Application of the biomarker responses in scallop (Chlamys farreri) to assess metals and PAHs pollution in Jiaozhou Bay,China

Scallops (Chlamys farreri) were collected at three sites in the Jiaozhou Bay, China in April and July 2007. The responses of biomarkers were evaluated for assessment of physiological status of scallops and pollution in the areas. Compared with site S1, the activity of aryl hydrocarbon hydroxylase was induced notably (P < 0.05) and glutathione S-transferase activity was inhibited significantly (P < 0.05) in S2 and S3. The levels of DNA alkaline unwinding, DNA protein crosslinks, protein carbonyl and lipid peroxidation were tested and indicated the oxidative stress situation of scallops. There was significant difference of biomarker levels between sampling seasons (P < 0.05), and between digestive gland and gill (P < 0.05). The results provided the reference data for multiple-pollution assessment in the marine environment and indicated that tissue type and seasons affect biomarkers, therefore these factors should be taken into consideration when biomarkers are used for environmental assessment purposes.     

Sewage-derived bacteria monitored in a marine water column by means of captive mussels

Batches of mussels, Mytilus edulis, obtained from a sewage-free shore were introduced into the water column at a sewage polluted site and the changed content (Escherichia coli, faecal streptococci) was assessed after immersion periods of 2 to 40 h. Rapid bacterial uptake was demonstrated; changes were analysed with respect to immersion period, revealing that 2h was satisfactory. The numbers of bacteria varied with height in the water column; mussels from the sea bed contained higher numbers of E. coli than mussels immersed just below the surface except for mussels withdrawn at, and for about 3h after, low water (LW). These observations were shown to be related to the characteristics of the e effluent and tidal systems operative at the site.     

Comparing quality of estuarine and nearshore intertidal habitats for Carcinus maenas

Estuarine and nearshore marine areas are vital habitats for several fish and benthic invertebrates. The shore crab Carcinus maenas (Crustacea: Brachyura: Portunidae) inhabits a variety of coastal, estuarine and lagoon habitats. At low tide, habitat structural complexity may be most important for crabs in the intertidal, providing refuge from predation and desiccation. The quality of different vegetated and nonvegetated estuarine and rocky shore habitats in SW Portugal and SW England was evaluated for intertidal C. maenas populations. We estimated population density, size–structure, and potential growth (RNA/DNA ratios) to investigate habitat quality. Vegetated estuarine habitats supported higher crab densities, than nonvegetated estuarine and rocky shore habitats. Investigation of population size–structure revealed that all habitats seem important recruitment and nursery areas although estuarine habitats in SW Portugal appeared to support higher densities of new recruits than equivalent habitats in SW England. Significant variation was found in RNA/DNA ratios among habitats. Ratios were highest in the rocky shore suggesting a high quality habitat where growth potential is high. We speculate that competition from other top-predators (Pachygrapsus spp.) rather than low habitat quality may limit the occurrence of C. maenas in intertidal rocky shore habitats in SW Portugal. In estuarine environments RNA/DNA ratios were significantly higher in the vegetated than in the nonvegetated estuarine habitats in SW Portugal but not in SW England, suggesting geographic differences in the extent to which highly structure habitats represent high quality. Our results challenge the current paradigm that structured habitats are necessarily those of higher quality for C. maenas.