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.     

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.     

Measurement of DNA single-strand breaks in gill and hemolymph cells of mussels, Mytilus sp., collected on the French Atlantic Coast

DNA single-strand breaks were measured by the comet assay in both gill and hemolymph cells of mussels collected in 3 sampling areas of the French coast (Pointe du Castelli, Pen Bron and Saint-Nazaire Harbour). Whole mussel tissue samples were also collected for the chemical determination of PAH, PCB and heavy metal concentrations. In mussel, a higher level of DNA strand breaks was measured in gill than in hemolymph cells (p < 0.01). Despite a factor of contamination from 2 to 3 between sites, no difference in the extent of mussel DNA strand breaks was shown between sampling locations (p > 0.05), questioning the sensitivity of the assays used in biomonitoring studies.     

Long-term environmental exposure to metals (Cu,Cd, Pb,Zn) activates the immune cell stress response in the common European sea star (Asterias rubens)

The common sea star Asterias rubens represents a key-species of the North-Eastern Atlantic macro benthic community. The cells of their immune system, known as coelomocytes, are the first line of defence against environmental hazards. Here, we report the results of investigations on the immune cells response of sea stars exposed to marine environmental pollution for long periods. We show that levels of the heat shock cognate protein 70 (HSC70) in coelomocytes from A. rubens, which were collected during a field study in the Sǿrfjord (North Sea, SW coast of Norway) along a contamination gradient, are directly associated with the long-term accumulation of Cd, Cu heavy metals exclusively in the tegument. Conversely, Pb and Zn accumulation in the tegument did not relate to HSC70 levels and none of the metals were found accumulated in the pyloric coeca. In addition the coelomocytes from A. rubens, collected in high and low metal impacted stations were examined by a proteomic approach using two-dimensional electrophoresis (2DE). By comparison of the proteomic maps, we observed that 31 protein spots differed in their relative abundance, indicating a gene expression response to the metal mixture exposure. All together, our results confirm that the echinoderm immune cells are a suitable model for the assessment of long-term exposure to environmental pollution, moreover that the increased level of HSC70 can be considered a signal of an acquired tolerance within a large spectrum of protein profile changes occurring in response to metal contamination.     

Evaluation of neutral red retention assay,micronucleus test,acetylcholinesterase activity and a signal transduction molecule (cAMP) in tissues of Mytilus galloprovincialis (L.), in pollution monitoring

The neutral red lysosomal retention assay (NRR) of the haemocytes, and the acetylcholinesterase activity (AChE) in the haemolymph, the digestive gland, the gills and the mantle/gonad complex have been evaluated on mussels Mytilus galloprovincialis collected from Thermaikos and Strymonikos gulfs (northern Greece) in June and October 2001. The validity of performing the above core biomarkers is supported, firstly by their ability to respond to different pollution levels and, secondly, by the significant linear correlation among them. The evaluation of the micronuclei frequency (MN) has been performed in gill tissue and haemocytes of the same mussels and, according to the results, it needs more research in order its use as stress indices to be validated. In addition, the first results on cAMP levels in the gills, the mantle/gonad complex and the digestive gland, whose concentrations correlated to both, NRR and AChE introduce this signal transduction molecule as a new, promising biomarker.     

Biomarker responses in Macoma nasuta (Bivalvia) exposed to sediments from northern San Francisco Bay

Our study investigates biomarker responses and survival of Macoma nasuta exposed to sediments collected from six locations in northern San Francisco Bay. Biomarkers analyzed were stress proteins (hsp70) in gill, mantle and digestive gland, lysosomal membrane damage and histopathologic lesions. Sediments and clam tissues were analyzed for a comprehensive suite of heavy metals and trace organic pollutants. Sediment grain size and organic carbon content were determined. Clams accumulated metals, polyaromatic hydrocarbons (PAHs) and organochlorine pesticides (aldrin and p,p^′-DDT and its metabolites p,p^′-DDD and p,p^′-DDE). Pearson and Spearman correlation analysis revealed that mortality, hsp70 in gill and histopathologic lesion scores in gonads, and lysosomal membrane damage were significantly correlated with tissue concentrations of DDT and/or its metabolites. Tissue concentrations of metals, in particular nickel, chromium, and copper, were associated with macrophage aggregates in digestive gland and germ cell necrosis. Cadmium was linked to mortality and lysosomal membrane damage.     

Histopathology of mussels (Mytilus sp.) from the Tamar estuary, UK

This study assessed numerous histological parameters as markers of health status in mussels (Mytilus spp.) collected from several locations along a contamination gradient on the River Tamar and two coastal sites on the north and south coast of Cornwall, UK. Twenty-seven health parameters were assessed including the presence of pathogens, inflammatory lesions, non-specific pathologies and reproductive condition. Logistic regression showed that reproductive condition and inflammatory lesions showed changes indicative of a contamination gradient response. Mussel populations further upstream within the Tamar were relatively less developed compared to mussels situated downstream in the estuary. Compared to coastal locations, mussels sampled within the Tamar also exhibited a higher prevalence of inflammatory lesions that increased at locations situated furthest upstream. Similarly, levels of lipofuscin within the kidney epithelium were highest in mussels sampled within the estuary and Whitsand Bay compared to Trebarwith Strand. The most prevalent pathogens observed during this survey included Rickettsia/Chlamydia-like organisms (R/CLO), gill ciliates including Ancistrum mytili, the copepod Mytilicola intestinalis, Ciliophora-like organisms and Marteilia sp. Marteilia sp. was observed in 31% of the mussels sampled from the Cremyll Ferry site. Generally, the range and prevalence of pathogens was highest from the two estuarine locations at Cremyll Ferry and Wilcove. No significant differences were observed between estuarine sampling sites and Whitsand Bay with respect to median epithelial cell height of digestive tubules, however mussels from Trebarwith Strand did exhibit a reduced median epithelial cell height compared to mussels sampled from other sampling sites. Species genotyping was also undertaken during this study in order to consider any histological differences in relation to species. Results are discussed in relation to histopathology health parameters and biological effects monitoring.     

CDOM and its contribution to the underwater light climate of a shallow, microtidal estuary in south-western Australia

Light attenuation (K_d) of photosynthetically active radiation (PAR) by chromophoric dissolved organic matter (CDOM), total suspended solids (TSS) and chlorophyll a (Chl a) were measured at nine stations along an estuarine gradient in the Swan River, Western Australia, over 15 months. There were strong spatial gradients associated with the marine-freshwater transition along the 32 km of estuary sampled, as well as seasonal gradients mainly associated with rainfall, 80% of which occurs between May and September. CDOM absorbances at 440 nm reached a maximum of 10.9 m⁻¹ with the freshwater inflow but concentrations of suspended matter remained low throughout the sampling period (1.0–21.0 mg l⁻¹) under the diurnal tides of the estuary. CDOM was the dominant constituent of K_d and a stepwise multiple regression showed that 66% (p < 0.0001) of the variation in K_d can be explained by CDOM and an additional 8% (p < 0.0001) by TSS. As a consequence of this result, analysis into the influence of river discharge rates on CDOM absorbance levels was examined for 2002 using data collected during this study, and for 2000 and 2001 using historical dissolved organic carbon (DOC) and river discharge data. The outcome of this analysis infers that greater river discharge rates result in increased CDOM absorbances in the Swan River estuary.     

Metallothionein and stress protein levels in shore crabs (Carcinus maenas) along a trace metal gradient in the Fal estuary (UK)

A field study was conducted to assess the potential use of metallothionein (MT) and stress proteins as biomarkers of trace metal exposure. Shore crabs (Carcinus maenas) were collected from five sites: three located in the metal-contaminated Fal estuary (Southwest England) and two control sites located on the south coast of England. Two methods for determining MT levels in crab midgut gland were compared. A direct method in which MT was quantified by spectrophotometric measurement of SH content, and an indirect method in which the metal content (Cu and Zn) of partially purified MT was measured. Stress protein levels (stress-70) were quantified in crab gills using an improved western blotting technique. Elevated Cu and Cd concentrations compared to values in control crabs were found in the midgut glands of crabs from the Fal estuary. No significant variation was seen in Zn concentrations among the five sites. The two techniques used for MT analysis yielded similar results. Metallothionein concentrations were slightly higher in crabs from Restronguet Creek (the most metal-contaminated site) than in crabs from the other four sites, however this difference was not significant (p > 0.05). Stress protein levels did not reflect the metal gradient.     

Monitoring trace metals in seawater using a diffusive gradient in thin film probe in Ulsan Bay,East Sea,Korea: Comparison with transplanted mussels

To evaluate the applicability of a diffusive gradient in thin film (DGT) probe for monitoring dissolved metals in coastal seawater, DGT-labile metal concentrations were compared with total dissolved metal concentrations using spiked and natural seawater samples in the laboratory and transplanted mussels (Mytilus galloprovincialis). This was achieved through the simultaneous deployment of DGT probes and transplanted mussels in Ulsan Bay during winter and summer. DGT-labile metal concentrations were 45% (Cu) ~ 90% (Zn) of total dissolved concentrations, and the order of non-labile concentrations was Cu > Pb > Co ~ Ni > Cd ~ Zn in both metal-contaminated and non-contaminated seawater samples, which was similar to the order of stability of metal complexes in the Irving–Williams series. The overall variability of the DGT probe results within and between tanks was less than 10% (relative standard deviation: RSD) for all the metals tested during a 48-h deployment. The accumulation of metals, as determined by DGT probes, represented the spatial gradients better than the transplanted mussels did for all of the metals tested, and the extent of metal accumulation in mussels differed depending on the metal. The comparison of results for the DGT probe and the transplanted mussels in two seasons (winter and summer) suggested that metal accumulation in mussels was controlled by the physiological factors of mussels and partly by their diet (particulate metal loadings). The DGT probe could be used as a monitoring tool for dissolved metals in coastal seawater because its results explained only labile species. When using the DGT probe, slightly more than half of the total dissolved concentration in seawater samples for all the metals investigated displayed timeintegrated properties and distinct spatial gradients from pristine to metal-contaminated seawater.     

Quantitative determination of lysozyme in the hemolymph of Mytilus edulis by the enzyme-linked immunosorbent assay (ELISA)

The defensive responses of the bay mussel, Mytilus edulis, to invading organisms and toxicants is primarily cellular. Hemolymph cells as well as the other organs and tissues of the mussel through their normal functioning and interactions with toxic substances influence the chemical composition of the animal's hemolymph. It has been found that the levels of specific molecules in the hemolymph fluctuate as a consequence of trace metal insult. It was proposed that one way in which trace metal toxicity may be expressed is through an influx of free lysosomal hydrolases in the hemolymph.Reported here is the development of an enzyme-linked immunosorbent assay (ELISA) with which micro-samples of mussel hemolymph can be analyzed quantitatively for the lysosomal hydrolytic enzyme, lysozyme. During development comparisons were made of the results acquired by spectrophotometric determinations of lysozyme activity and ELISA-determined lysozyme concentrations in the hemolymph of mussels. The hemolymph from unstressed and copper-stressed mussels was examined. It was found that the overall trends in lysozyme fluctuations were indicated by both techniques. However, on occasion in copper-stressed populations a divergence between the two sets of data was observed. This suggested an effect of copper on lysozyme molecular configuration.     

Pattern and annual rates of Scrobicularia plana mercury bioaccumulation in a human induced mercury gradient (Ria de Aveiro, Portugal)

Due to the lack of knowledge regarding annual bioaccumulation rates in estuarine and marine fauna, the main aim of this work was to study the annual mercury bioaccumulation in the well-documented bivalve species Scrobicularia plana along a human induced mercury gradient in the Ria de Aveiro coastal lagoon (Portugal) and in a nearby, non-polluted system (Mondego estuary), parallel to the risks associated with its consumption by humans.Minimum total mercury concentration was as low as 0.019 mg kg⁻¹ (wwt) in 4+ year old organisms in the reference site, where a significant negative correlation (p < 0.05) was found between total mercury concentrations and size, resulting in negative bioaccumulation rates (detoxification). On the other hand, values reached 1.8 mg kg⁻¹ (wwt) in 3+ year old bivalves from the most contaminated area, where a strong positive correlation with size was found (p < 0.01) and annual bioaccumulation rates were as high as 0.25 mg kg⁻¹ yr⁻¹. Annual bioaccumulation rates were highly correlated with suspended particulate matter mercury concentrations. Even though the levels of organic mercury contents increased parallel to the contamination gradient, at each sampling station, no increment was found with age, which corresponded to a decrease in organic mercury percentage with age.In terms of ecological management and public health, the ratio of 0.01 consistently found between Scrobicularia plana annual mercury accumulation rates and SPM mercury levels for most sites may permit to roughly estimate S. plana contamination of commercial sized individuals (>2.5 cm) and, if verified and confirmed in other systems, be used as a simple management tool.     

Biomarkers of biochemical and cellular stress in Carcinus maenas: an in situ field study

Biomarkers are useful diagnostic tools for identifying exposure to physical, chemical, and environmental stresses. In this study a suite of techniques—measurements of metallothionein, lysosomal integrity, and osmoregulatory ability—have been used in an attempt to provide an in situ assessment of environmental quality. Five estuarine sites were studied, two clean sites, two urban sites, and one intermediate. Shore crabs, Carcinus maenas, were collected and haemolymph taken for the lysosomal and osmoregulatory assays. Animals were killed and tissue preserved for metallothionein analysis. Lysosomal integrity was significantly different in crabs from clean sites and the urban sites (n = 40, p < 0.05). The potential value of the biomarker approach is that it could provide data for impact assessment and is applicable in the field.     

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.     

Protein expression from zooplankton communities in a metal contaminated NW mediterranean coastal ecosystem

Bidimensional and monodimensional polyacrylamide gel electrophoresis were used to study protein expression from zooplankton collected in thirteen stations of Toulon Bay (NW Mediterranean). In this ecosystem, Little Bay showed higher trace metal concentrations (13.5–23.8 nM for Cu, 0.73–1.24 nM for Pb, 27.8–58.7 nM for Zn) than Large Bay (Cu 2.2–15.6 nM; Pb 0.19–0.78 nM; Zn 9.0–38.8 nM). Trace metals positively correlated (p < 0.05) with expression of four zooplankton proteins (MW in kDa/pI: 25.0/5.6; 48.8/4.1; 38.2/4.4; 38.3/5.8) and with biomass of Oithona nana, predominant copepod in Little Bay. Sequencing by LC–MS/MS putatively provided zooplankton identity of these proteins: they were cytoskeleton actin, except one protein that was the chaperone calreticulin. We suggest that actin and calreticulin could be regarded as zooplankton markers of metal stress and be involved in a possible tolerance of O. nana to contamination, contributing to its development in a marine perturbed ecosystem.     

Lysosomal reaction to xenobiotics in mussel hemocytes using BODIPY-FL-verapamil

Many cellular and sub-cellular biomarkers associated with mussel (Mytilus edulis) digestive gland and kidney have been characterised. The lysosomal compartment of these tissues have been recognised as being particularly sensitive, exhibiting pollutant induced responses which could be potentially used as a ‘biomarker’. However, relatively few studies have investigated the lysosomal response within molluscan hemocytes. This study was conducted to test whether lysosomal reactions, in live hemocytes isolated from mussels, can be used as a biomarker of pollutant exposure and deleterious effect. Lysosomal responses to a number of hydrocarbons, including anthracene and phenanthrene, and to the amphiphilic heterocylic chemical, chlorpromazine, were examined. The supravital dye neutral red (NR) was used to examine lysosomal membrane fragility, following xenobiotic exposure. NR was also used to verify the lysosomal compartment as the reported accumulation site of a new molecular probe, BODIPY-FL-verapamil (BFLV). The use of BFLV, with confocal laser microscopy and image analysis enabled visualisation and quantification of lysosomal distribution and perturbation. BFLV showed that exposure of molluscan hemocytes to xenobiotics (20 ppb–10 ppm) induced the formation of pathologically enlarged lysosomes. The internal trafficking of lysosomes was shown to be severely compromised after exposure to chlorpromazine. Exposed molluscan hemocytes exhibited significantly reduced lysosomal retention times, for neutral red. Preliminary data is presented demonstrating the opportunity for these non-destructive biomarker techniques to detect pollution gradients in situ.     

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.     

Teneurs en benzo 3,4 pyrène chez Mytilus edulis L. de l'estuaire et du golfe du Saint-Laurent

Blue mussels (Mytilus edulis L.) taken from 27 stations along the estuary and gulf of St. Lawrence were analyzed for their content in 3,4-benzopyrene. Most of the concentrations are below the detection limit for the analytical method, i.e. 0·15 μg kg^?1 (dry weight) and probably reflect the natural baseline level. However, mussels from two stations located at the mouth of Saguenay Fjord exhibit very high contents: 24 and 28·5μg kg^?1 (dry weight). A mussel transplantation experiment has shown that the entire Fjord is contaminated by anthropogenic 3,4-benzopyrene.     

Assessment of lysosomal membrane stability and peroxisome proliferation in the head kidney of Atlantic cod (Gadus morhua) following long-term exposure to produced water components

There is a need for sensitive biological effect methods by which to detect impacts of chronic exposure to low concentrations of contaminants. Two methods shown to be potentially useful for monitoring purposes in fish include lysosomal membrane stability and peroxisome proliferation. These biological endpoints were assessed in Atlantic cod (Gadus morhua) head kidney following exposure to a mixture of produced water components including polycyclic aromatic hydrocarbons, phenol, and alkylphenols. Lysosomal damage of head kidney cells occurred within the first two weeks and did not recover during the entire exposure period (32 weeks). Lysosomal membrane stability was not affected by gender and was responsive at low concentrations of contamination, indicating that lysosomal membrane stability measured in the head kidney could be a useful biomarker for effects of offshore pollution. Peroxisome proliferation, measured as acyl-CoA oxidase activity in the head kidney, appeared to be a potential biomarker in male cod exposed less than 16 weeks.     

Surveys of mussels (Mollusca: Lamellibranchia) in the firth of Thames, 1961–67

Surveys are described which were made to determine distribution and relative density of stocks of mussels, Perna canaliculus Gmelin (Lamellibranchia: Anisomyaria; Mytilidae), in the Firth of Thames, North Island, New Zealand, during the late autumn‐early winter of each year 1961–63 and 1965–67.

Two dredges available for sampling were tested against one another over similar ground. Both gave comparable results, but although the one chosen was never actually calibrated, it gave consistent results which allowed relative changes of abundance to be studied.

In 1961, 206 stations were sampled on a grid of 1 square mile which covered most of the Firth. Early results showed that the densest beds were along the western shore line; there were only small patches of mussels off Coromandel and along the eastern shore. A well‐stocked and readily identifiable area of 2 square miles off Matingarahi Point was selected for more intensive sampling on a 1/16 square‐mile grid. Samples collected there annually showed a rapid decline of the population after commercial dredging in 1961–62; there was no substantial regeneration on the bottom after this, though satisfactory settlement occurred above the bottom on experimental moorings. Declining yields are shown for other areas (Ponui Island and New Brighton) which were also sampled on a 1/16 square‐mile grid.

Commercial landings of mussels decreased rapidly after 1961, and have not recovered. There have been no regular landings since 1966 but a promising private experiment in small‐scale cultivation is described.