Integrated biomarker assessment of the effects exerted by treated produced water from an onshore natural gas processing plant in the North Sea on the mussel Mytilus edulis

The biological impact of a treated produced water (PW) was investigated under controlled laboratory conditions in the blue mussel, Mytilus edulis. Mussel health status was assessed using an integrated biomarker approach in combination with chemical analysis of both water (with SPMDs), and mussel tissues. Acyl-CoA oxidase activity, neutral lipid accumulation, catalase activity, micronuclei formation, lysosomal membrane stability in digestive cells and haemocytes, cell-type composition in digestive gland epithelium, and the integrity of the digestive gland tissue were measured after 5 week exposure to 0%, 0.01%, 0.1%, 0.5% and 1% PW. The suite of biomarkers employed were sensitive to treated PW exposure with significant sublethal responses found at 0.01-0.5% PW, even though individual chemical compounds of PW were at extremely low concentrations in both water and mussel tissues. The study highlights the benefits of an integrated biomarker approach for determining the potential effects of exposure to complex mixtures at low concentrations. Biomarkers were integrated in the Integrative Biological Response (IBR/n) index.     

Bioaccumulation depuration kinetics and effects of benzo(a)anthracene on Mytilus galloprovincialis

Polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants due to their persistence and bioaccumulation potential both in organisms and in sediments. In this study, bioaccumulation and depuration experiments were performed employing local Mediterranean mussel species, Mytilus galloprovincialis, with two biomarkers: filtration rate and lysosomal stability (neutral red retention) assay. Benzo(a)anthracene (BaA) was chosen as the model PAH compound due to its common presence in several matrices in the marine environment. Bioconcentration Factors (BCFs) for the mussels exposed to different BaA concentrations were calculated from both kinetic rate constants and from the experimental data and found between 27-3184 and 16-2745, respectively. Experimental QSAR values were found comparable to the previous QSAR models created for BaA. The effect of BaA exposure on the mussels showed a more clear dose–response relationship according to the results of lysosomal stability compared to the filtration rate.     

Cytogenetic and cytotoxic effects in gill cells of the blue mussel Mytilus spp. from different zones of the Baltic Sea

Frequency of micronuclei (MN) and other nuclear abnormalities (nuclear buds, bi-nucleated and fragmented-apoptotic cells) was analysed in gill cells of the blue mussel (Mytilus spp.) from selected coastal sites in the Baltic Sea--Kv?d?fj?rden (Sweden), Klaipeda-Būtinge zone (Lithuania), Gulf of Gdansk (Poland) and Wismar Bay (Germany). Samples were collected from 650 specimens during bi-annual sampling campaigns in 2001 and 2002. The lowest frequency of MN (0.37 MN/1000 cells) was found in blue mussels from the reference site (Kv?d?fj?rden). The highest MN values (up to 6.7 MN/1000 cells) were registered in blue mussels from the Gulf of Gdansk in autumn 2001 and 2002, and at Wismar Bay in spring 2001 (up to 5.06 MN/1000 cells). Gradients of MN incidences were observed when comparing the three studied locations in Wismar Bay, and at the Lithuanian coast before the crude oil spill in the Būtinge oil terminal. Moreover, significant seasonal and inter-location differences in the responses were documented (P<0.0001). Nuclear abnormalities were observed most frequently in blue mussels from the Gulf of Gdansk.     

PAH body burden and biomarker responses in mussels (Mytilus edulis) exposed to produced water from a North Sea oil field: laboratory and field assessments

In order to study the impact of produced water (PW) from a North Sea oil field on blue mussels (Mytilus edulis), chemical and biological markers were selected. A laboratory exposure (0.125%, 0.25% and 0.5% of PW) and a field study (6 stations 0.2-2 km from a PW discharge point) were conducted. In the laboratory study, PAH bioaccumulation increased in mussel soft tissue even at the lowest exposure dose. Micronuclei frequency demonstrated a dose-response pattern, whereas lysosomal membrane stability showed tendency towards a dose-response pattern. The same markers were assessed in the field study, biomarker analyses were consistent with the contamination level, as evaluated by mussel polycyclic aromatic hydrocarbons body burden. Overall, obtained results confirmed the value of an ecotoxicological approach for a scientifically sound characterisation of biological effects induced by offshore oilfield operational discharges.     

Influences of ammonia-nitrogen and dissolved oxygen on lysosomal integrity in green-lipped mussel Perna viridis: laboratory evaluation and field validation in Victoria Harbour, Hong Kong

Lysosomal integrity in mussels has been applied as a biomarker to detect the pollution of trace organics and metals in the natural environments. However, few studies have examined the effects of water quality on the response of lysosomal integrity, in particular total ammonia-nitrogen (TAN) and dissolved oxygen (DO). This study demonstrated that high level of TAN (2.0 mg/l) and low DO (2.5 mg O₂/l) could significantly reduce the lysosomal integrity in green-lipped mussel Perna viridis, respectively by 33% and 38%, whereas the mussel lysosomal integrity decreased by 70% in the combined treatment of TAN and low DO under laboratory conditions after one week. The mussel lysosomal integrity of all treatment groups could return to the control level after a three week recovery period. In the field validation in Victoria Harbour, Hong Kong during an one-year study period, lysosomal integrity in P. viridis identified the cleanest site east to the harbour, where the lowest TAN and highest DO concentrations were found. While lysosomal integrity in mussels seemed not affected by seasonal changes, approximately 40% of the variation of this biomarker could be attributable to the changes in TAN and DO in seawater. In conclusion, the response of the mussel lysosomal integrity can be confounded by both TAN and DO prevailing in the natural environments and thus caution must be exercised in relating the observed changes in lysosomal integrity to any specific pollutant in coastal water quality monitoring studies.     

An ecotoxicological approach for the Boka Kotorska Bay (south-eastern Adriatic Sea): First evaluation of lysosomal responses and metallothionein induction in mussels

Mytilus galloprovincialis was used as a biomonitoring organism in Boka Kotorska Bay, a coastal transitional ecosystem in south-western Montenegro. Native mussels were collected in June 2008 at four sites thought to be differently impacted. Biological effects were investigated analysing both generic and specific biomarkers at cellular level (metallothionein content, lysosomal membrane stability, lipofuscin and neutral lipid accumulation, lysosomal structural changes). Trace element levels in mussels were quite low, only Cu and Zn exhibiting slight increases in the two sampling sites of Tivat Bay, the first one in front of a small working dockyard and the other in the water area of a former Naval dockyard. Mussels collected from these sites exhibited the highest values in neutral lipid and lysosomal volume density and the lowest neutral red retention times. Metallothionein content was always low, reflecting the minor body burden of the trace elements and suggesting a non-relevant environmental induction.     

Assessment of the effects of a marine urban outfall discharge on caged mussels using chemical and biomarker analysis

To assess the presence of endocrine disruptors in treated marine outfall discharges and their possible effects, mussels (Mytilus galloprovincialis) were caged in the environmental mixing zone of the outfall of the Santander sanitation system and in one control area. After 30, 60 and 90 days, samples were collected to perform chemical analyses (metals, anionic surfactants, alkylphenols, bisphenol A, phthalates and estrogenic hormones), biomarkers of general stress (lysosomal membrane stability-LMS, histopathology) and biomarkers of endocrine disruption (vitellogenin-like proteins and gonad index). There were no significant differences between outfall and control sites on contaminant levels, except for 4-tert-octylphenol which was higher in the outfall site. Bacteriological counts were higher in the outfall area. No relevant differences in biomarkers were detected between treated and control mussels. A significant reduction in LMS occurred in both groups after 90 days caging, indicating a stress situation possibly related to caging or to post-spawning reproductive state.     

Metal exposure and biological responses in resident and transplanted blue mussels (Mytilus edulis) from the Scheldt estuary

The Western Scheldt river and estuary is known to be highly polluted as it receives industrial, agricultural and domestic effluents from one of the most populated and industrialised zones in Europe. Aquatic organisms are exposed to pollutants, specifically metals that are present in different environmental phases, e.g. dissolved, suspended material or sediment phases. The objective of this study was to study the relationship that exists between environmental metal levels, the degree of metal uptake by aquatic organisms with the concomitant biological responses. For this purpose the bivalve mollusk, Mytilus edulis, was selected as bioaccumulation indicator organism. Environmental (water and sediment) and mussel samples were collected during the late winter (March 2000) from sampling sites in the Scheldt estuary. Sites were selected to represent a salinity gradient from the mouth of the estuary to the furthest distribution area of mussels upstream in the system. Together with standard water quality parameters (e.g. salinity, dissolved oxygen, dissolved organic carbon, etc.) concentrations of twelve metals were analysed in the water (dissolved and suspended matter) and sediments. Levels of these metals were also measured in the soft tissue of M. edulis, together with concomitant biomarker responses in resident mussel populations at each site. The biomarkers that were included in this study were condition index, scope for growth, survival in air, cell membrane stability, DNA damage, and metallothioneins. Data were subjected to multivariate statistical analysis. The physico-chemical parameters and metals in the environmental samples clustered the sites to reflect the distribution based on the salinity gradient. Bioaccumulation results revealed increased metal uptake along a pollution gradient with the highest metal bioaccumulation occurring at the upstream most sites and therefore closest to the in the industrial activities. However, the biomarker responses clustered the sites in a manner that reflected the influence of combination of internal exposure (bioaccumulation) and external exposure (physico-chemical conditions). These differences in biomarker responses clearly demonstrated were attributed to abiotic factors other than metal pollution alone e.g. localized short-term increases in increased suspended sediment concentrations and decreased dissolved oxygen concentrations.     

Evaluation of the Mytilus galloprovincialis Lam. digestive gland metallothionein as a biomarker in a long-term field study: seasonal and spatial variability

A study was made of seasonal and spatial variability of metallothionein (MT) concentrations, determined spectrophotometrically in the soluble fraction of the digestive gland of mussels Mytilus galloprovincialis, collected between 1999 and 2001 from several coastal and estuarine locations along the central Eastern Adriatic coast (Croatia). The seasonal influence on the MT and metal concentrations (higher values in winter-spring season, than in summer-autumn season) is more pronounced than the local site-specific influence. Furthermore, within each season a significant site-specific dependence on the MT and trace metal variations can be detected. An inverse relationship of mussel condition index (CI) and temperature with MT and trace metals levels indicates the influence of food abundance and mussel annual reproductive cycle. Substantially higher concentrations of both MT and Cd were recorded in mussels inhabiting estuarine locations, possibly indicating a markedly higher Cd bioavailability at these locations. The positive correlations obtained between MT and Cd in all seasons except autumn support an argument for application of digestive gland MT as a biomarker of Cd exposure, providing evidence for assessing the most appropriate season for mussel sampling.     

Lysosomal Membrane Stability and Metallothioneins in Digestive Gland of Mussels (Mytilus galloprovincialis Lam.) as Biomarkers in a Field Study

The lysosomal membrane destabilization and the metallothionein content in the digestive gland cells of mussels (Mytilus galloprovincialis Lam.), collected along the east coast of the North Adriatic (Istrian and Kvarnerine coast, Croatia), were examined over a period of four years (1996–1999). The lysosomal membrane stability, as a biomarker of general stress, showed that the membrane labilization period in mussels from polluted, urban- and industrial-related areas was significantly decreased (p<0.05) when compared to mussels from control, clean sea water sites. In the harbour of Rijeka, the most contaminated site, the lysosomal membrane stability was reduced by more than 70% compared to the control. This method also proved to be a useful biomarker for detection of additional stress caused by short-term hypoxia that occurred once during this study inside the polluted and periodically quite eutrophic Pula Harbour. The concentration of metallothioneins in the mussel digestive gland, as a specific biomarker of exposure to heavy metals, did not reveal significant differences (p<0.05) between sites covered by this study.     

Bioaccumulation depuration kinetics and effects of benzo(a)anthracene on Mytilus galloprovincialis

Polycyclic aromatic hydrocarbons (PAHs) are important environmental pollutants due to their persistence and bioaccumulation potential both in organisms and in sediments. In this study, bioaccumulation and depuration experiments were performed employing local Mediterranean mussel species, Mytilus galloprovincialis, with two biomarkers: filtration rate and lysosomal stability (neutral red retention) assay. Benzo(a)anthracene (BaA) was chosen as the model PAH compound due to its common presence in several matrices in the marine environment. Bioconcentration Factors (BCFs) for the mussels exposed to different BaA concentrations were calculated from both kinetic rate constants and from the experimental data and found between 27-3184 and 16-2745, respectively. Experimental QSAR values were found comparable to the previous QSAR models created for BaA. The effect of BaA exposure on the mussels showed a more clear dose-response relationship according to the results of lysosomal stability compared to the filtration rate.     

Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae

Pacific oyster (Crassostrea gigas Thunberg) embryos, bay mussel (Mytilus edulis Linnaeus) embryos, and Dungeness crab (Cancer magister Dana) zoea I stage larvae were exposed to arsenic, cadmium, chromium, copper, lead, mercury, nickel, selenium, silver, and zinc salts. The two bivalve species were exposed through the completion of embryogenesis (48 h), and the effects of the metals upon abnormal development were monitored. The crab larval stages were exposed to the metal solutions for 96 h. Concentrations (μg l_.^?1 as metal) which caused 50% abnormal development in the two species of bivalve larvae and which were fatal to 50% of the crab larvae ranged from 5.3 (oyster) to 49 (crab) for copper; 5.8 (mussel) to 8.2 (crab) for mercury; 14 (mussel) to 55 (crab) for silver; 119 (oyster) to 456 (crab) for zinc; 232 (crab) to 3000 (mussel) for arsenic; 247 (crab) to 1200 (mussel) for cadmium; 349 (oyster) to 4360 (crab) for nickel; 476 (mussel) to 758 (oyster) for lead; 3440 (crab) to 4538 (oyster) for hexavalent chromium; and selenium greater than 10 000 for the three species. The acute toxicity testing of C. gigas embryo together with two California native species confirmed its usefulness for predicting toxicity to the latter species.     

Hydrocarbons in water,sediment and mussels from the southern Baltic Sea

Samples of water, sediment and mussels (Mytilus edulis) from the southern Baltic Sea have been analysed for total hydrocarbons by fluorescence spectroscopy and capillary gas chromatography. The sediment and mussel samples have also been analysed for specific aliphatic and aromatic hydrocarbons by computerized capillary gas chromatography-mass spectrometry. The highest hydrocarbon concentrations in all samples occurred either inshore (particularly in Gdansk Bay) or in deep offshore basins where fine sediment accumulates.     

Evaluation of the mussel Perna perna as a biomonitor of polycyclic aromatic hydrocarbon (PAH) exposure and effects

The mussel Perna perna was used to monitor and assess areas suspected of oil contamination by transplanting animals from unimpacted to impacted sites and vice versa. The deployment lasted for three months during which total PAH were measured in seawater samples collected every 15 days. PAH concentrations were determined in mussel tissue monthly. The lysosomal neutral red assay and the condition index were used as biomarkers of the adverse biological effects. The PAH concentration in mussels at unimpacted sites, prior to deployment was ca. 100 microg kg(-1) but increased to 300 microg kg(-1) after three months at the contaminated site. Mussels transplanted to the uncontaminated site showed a decrease from 380 to 80 microg kg(-1). Significant changes in lysosomal stability occurred within one month of the beginning of the transposition; in contrast condition indexes were not correlated with the observed changes in PAH concentration.     

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The stability of the lysosomes and the latency of lysosomal enzymes of Mytilus are responsive to temperature and nutritive stressors. An empirical biochemical index of the molar concentration ratio of taurine: glycine in the mussel's tissue is also sensitive to stress. Changes in both of these indices correlate with the amount of energy available to the mussel for growth and are useful indicators of the degree of stress experienced by Mytilus in the natural environment.     

Monitoring trace elements in the mussel,Mytilus edulis using X-ray energy spectroscopy

X-ray energy spectroscopy has been successfully utilized to provide rapid, multi-elemental, quantitative analyses of the intertidal mussel, Mytilus edulis, found in southern coastal waters of British Columbia (Canada). Since no a priori decision needed to be made on which elements required monitoring, concentrations of the seven dominant elements (Mn, Fe, Cu, Zn, As, Se and Pb) were determined in the gills and viscera of these organisms. The potential usefulness of XES for providing relevant data on aquatic bioaccumulators for environmental monitoring studies is discussed.     

Stable isotopes and metal contamination in caged marine mussel Mytilus galloprovincialis

Metal concentrations and isotopic composition were measured in different tissues of the mussel Mytilus galloprovincialis in waters of the Balearic Islands (Western Mediterranean) in order to assess pollution levels. The isotopic composition was correlated with lead, cadmium, selenium and nickel obtained from the digestive gland and foot of the mussels. Significant negative correlations were found between cadmium, selenium and zinc and the mussel foot, mainly for ¹³C. Significant correlations were also found between lead and cadmium and the digestive gland. Pearson correlations indicated that the ¹³C isotopic signal in foot is a good proxy for the concentration of metals such as lead, cadmium, selenium and zinc. Similarly, ¹⁵N isotopic signatures in the digestive gland reflected the lead and cadmium concentration.     

Assessment of genotoxic and humoral immune system alterations in silica exposed workers from pottery industries in South India

Chemicals, especially silica, have been suspected to cause genetic alterations in pottery industry workers. The present study aims to analyze the frequency of chromosomal aberrations (CA), micronucleus (MN) and DNA damage (comet assay) in the peripheral blood lymphocytes and the immunological alterations workers chronically exposed to silica and in control subjects. In our study, 50 silica exposed workers and 35 control subjects were recruited and silica level was measured by respirable dust and respirable quartz concentrations of badge dosimeter. The serum immunoglobulins (IgM, IgG, and IgA) of all the subjects were measured by using ELISA method. The individuals exposed to silica have a significant increase in the frequency of CA, MN and the total DNA damage (p < 0.05). Immunoglobulin elevation on silica exposed workers was statistically significant (p < 0.05) on comparison with their respective controls. Investigation of the smoking and alcohol habitats coupled with silica exposure in exposed and control subjects represents alcohol consumption and smoking as additional risk factors and must be avoided. Multiple linear regression analysis obtained for CA, MN and comet assay confirm these tests as biomarkers for silica exposed pottery workers. Some confounding factors also showed significant influence on exposed subjects. These results indicate the mutagenic risk in the working environment has a high probability of association with the silica dust exposure in pottery industries. Nevertheless, the present study will create awareness and public concern not only among the silica exposed workers but also to the welfare of their progeny.     

Mortality,growth and fecundity of transplanted mussel and barnacle populations near a pulp mill outfall

The mortality, growth and fecundity of transplanted adult mussel (Mytilus edulis) and barnacle (Balanus glandula) populations near a kraft pulp mill outfall at Port Mellon, British Columbia, were determined. The results indicated that adults of both species were tolerant of bleached kraft pulp mill effluent and survived near the pulp mill outfall. However, the growth rates of both species were retarded when compared with those at the control station. The reproductive activities of the transplanted barnacles were also impaired.     

Induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis

Biomarkers are generally applied to detect pollution in environmental monitoring. Such biological responses should accurately reflect the stress over time in a quantitative manner. As such, the initial and maximum responses induced by stress, as well as adaptation and recovery of these biomarkers, need to be fully understood or else erroneous false-negative or false-positive may be arrived. However, most of the biomarker studies only provided information on initially induced responses under different concentrations of toxicants, while biological adaptation and recovery were poorly known. In this study, the time required for induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis upon exposure to benzo[a]pyrene was investigated over a period of 62 days. Maximum induction occurred on day 6 when lysosomal integrity was significantly reduced by 51%, and no further change or adaptation was detected thereafter. When mussels were depurated in clean seawater after 18 days of exposure to benzo[a]pyrene, a gradual recovery was observed, with lysosomal integrity returning to its background level and showing a complete recovery after 20 days of depuration. Lysosomal integrity was significantly correlated with the body burden concentrations of benzo[a]pyrene and condition index of the mussels. The relatively fast induction (6 days) and recovery (20 days) without apparent adaptation suggested that lysosomal integrity in P. viridis can serve as a good biomarker in biomonitoring, as its response is not likely to generate both false-negative and false-positive results.