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.     

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.     

Power analysis for biomarkers in mussels for use in coastal pollution monitoring

Data from literature on neutral red retention time (NRRT) in lysosomes, micronucleus (MN) frequency and condition index (CI) in mussel Mytilus, especially Mytilus edulis and Mytilus galloprovincialis, were re-analyzed to ascertain their statistical power in detecting a minimum 20% spatial/temporal change in field studies. Results showed that CI largely displayed higher statistical power (>90%) than lysosomal NRRT and MN frequency (<50%), suggesting that data from the latter two biomarkers may lead to erroneous conclusions if sample size is inadequate. Samples of green-lipped mussel Perna viridis were also analyzed in Hong Kong. To achieve statistically valid power, the optimal sample sizes for monitoring lysosomal NRRT, MN frequency, CI and gonosomatic index (GSI) were determined as ?34, ?90, ?16 and ?29, respectively. Natural variability of lysosomal NRRT and MN frequency was significantly greater than CI and/or GSI in mussels, rejecting the general belief in the greater variability of higher-tiered hierarchical biomarkers.     

Field observations on correlation of fatty acid profiles between suspended particulate matter and green-lipped mussels in subtropical waters of Hong Kong

By analysis of the fatty acid profiles in mussel tissues and suspended particulate matter (SPM) in water, the present study showed a significant relationship of the trophic linkage between mussels and the SPM. At seven locations from inner to outer areas along the eutrophic Tolo Harbour and Tolo Channel, Hong Kong, the composition (as percentage of total fatty acids) of both monoenoic and polyunsaturated fatty acids in the tissues of green-lipped mussels Perna viridis and SPM in water had significant correlation (p < 0.01). In particular, the composition of docosahexaenoic acid (DHA, 22:6n3) (as percentage of total fatty acids) in mussel tissues was statistically correlated with that in SPM (p < 0.01), implying that mussels incorporate DHA, the biomarker of dinoflagellates. Principal component analysis further demonstrated that the fatty acid profiles of SPM were different among locations in the harbour, mid and outer channel of the study area, so were the mussel tissue fatty acid profiles. Cluster analysis of phytoplankton data also revealed the dominance of diatoms in the inner harbour and channel areas, whereas dinoflagellates were abundant in the outer channel waters. The possible implications of using benthic suspension feeders such as green-lipped mussels P. viridis for controlling phytoplankton abundance in coastal waters are discussed.     

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.     

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.     

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.     

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.     

Experimental studies on the accumulation of copper and zinc in the green mussel

Toxicity of copper and zinc to the green mussel, Mytilus viridis was evaluated by short-term bioassays. Probit analysis was used for calculating 48 h median tolerance limits, 95% fiducial limits and slope functions (S). The rates of copper and zinc accumulation were determined by analysing the metal content in the soft parts of the mussels at fixed intervals of time by atomic absorption spectrometry. Bioaccumulation was determined as a function of metal concentration in the experimental medium. The correlations for the regression lines (P < 0.1%) indicate that the uptake of metal is linear at least for the first five weeks. During a more extended experimental period, adjustment to a steady state probably occurs.     

Quality assessment of the blue mussel (Mytilus edulis): Comparison between commercial and wild types

This study compared species identity, microplastics, chemical and microbial contamination between consumption mussels and wild type mussels, collected at Belgian department stores and Belgian groynes and quaysides, respectively. Species identification based on genetic analysis showed a high number of Mytilus (M.) edulis compared to M. galloprovincialis and M. edulis/galloprovincialis hybrid mussels. The number of total microplastics varied from 2.6 to 5.1 fibres/10 g of mussel. A higher prevalence of orange fibres at quaysides is related to fisheries activities. Chemical contamination of polycyclic aromatic hydrocarbons and polychlorobiphenyls could be related to industrial activities and water turbidity, with maximum concentrations at the quayside of port Zeebrugge. The inverse was noted for Escherichia coli contamination, which was relatively low at Zeebrugge quayside with a total count of 3.9 × 10² CFU/100 g tissue, due to limited agricultural effluents. Results of this complementary analysis stress the importance of integrated monitoring and quality assessment.     

Field validation,in Scotland and Iceland,of the artificial mussel for monitoring trace metals in temperate seas

The artificial mussel (AM), a novel chemical sampling device, has been developed for monitoring dissolved trace metals in marine environments. The AM consists of Chelex-100 suspended in artificial seawater within Perspex tubing and enclosed with semi-permeable polyacrylamide gel at both ends. To validate the field performance of the AM in temperate waters, we deployed AMs alongside transplanted blue mussels Mytilus edulis in coastal environments in Scotland (Holy Loch, Loch Fyne, Loch Striven and Millport) and Iceland (Reykjavikurhöfn, Gufunes, South of þerney, Hofsvik, Hvalfjörður and Sandgerði) for monitoring trace metals. While uptake patterns of Cd between the AM and M. edulis were highly comparable, discrepancies were found in the accumulation profiles of the other metals (Cu, Cr, Pb and Zn), in particular Zn. Nonetheless, the AMs gave a better resolution to accurately reveal the spatial difference in dissolved metal contamination when compared with M. edulis. AMs complement the use of mussels since AMs indicate dissolved metals in seawater, whereas uptake by mussels indicates a mixture of dissolved and particulate metals. Our results also indicated that historical metal exposure of the transplanted M. edulis could significantly confound their metal concentrations especially when the deployment period was short (i.e. <34 d). This study suggested that the AM can overcome problems associated with variable biological attributes and pre-exposure history in the mussel, and provides a standardized and representative time-integrated estimate of dissolved metal concentrations in different marine environments.     

Comparative petroleum hydrocarbons levels and biochemical responses in mussels from hydrothermal vents (Bathymodiolus azoricus) and coastal environments (Mytilus galloprovincialis)

Aliphatic hydrocarbons and PAHs in the whole soft tissues of Bathymodiolus azoricus from three Mid-Atlantic Ridge hydrothermal vents (Menez-Gwen, Lucky Strike and Rainbow), and Mytilus galloprovincialis from three contaminated coastal sites in South Portugal were analysed, and its effects on the digestive gland microsomes mixed-function oxygenase system (MFO) were assessed.Aliphatic hydrocarbons levels were present in the same magnitude in both coastal and hydrothermal environments, while the UCM (unresolved complex mixture) for coastal mussels were higher than in vent mussels. In general, significantly higher PAHs concentrations were found in coastal mussels, compared to B. azoricus where low molecular weight PAHs (2–3 rings) represented the majority of PAHs contrarily to what was observed in M. galloprovincialis. The MFO components were present in both mussel species, and were detected in vent mussels for the first time. However this system seems to have different roles in species from these contrasting environments. In coastal mussels MFO responded to hydrocarbon contamination while response in hydrothermal organisms appeared to be related mainly to endogenous factors.     

Allometric relationships of Po and Pb in mussels and their application to environmental monitoring

Mussels from the Portuguese coast collected during several seasons of the year have shown ²¹⁰Po and ²¹⁰Pb body burdens (Bq mussel⁻¹) that increased with mussels’ body size but displayed significant decrease in radionuclide concentrations (Bq kg⁻¹). For example, the increase of mussel size from 2.5 cm to 5.0 cm maximum shell length corresponded in average to a 50% decrease of ²¹⁰Po activity concentration in soft tissues from 1065 Bq kg⁻¹ (dw) to 540 Bq kg⁻¹ (dw). A similar reduction in concentration was observed for ²¹⁰Pb. The physiological condition of mussels, relating to fat and glycogen storage, had an effect on radionuclide concentrations, although the total body burden of radionuclide in mussels remained nearly constant throughout the year. These factors may play an important role in data interpretation for environmental monitoring programmes. Besides the mussel size and condition index, due to the inter-individual variation even inside narrow mussel size classes, the sample size, i.e., the number of specimens in one mussel sample is another key factor to be considered when obtaining environmentally representative radionuclide concentrations.     

Okadaic acid, a causative toxin of diarrhetic shellfish poisoning, in green-lipped mussels Perna viridis from Hong Kong fish culture zones: Method development and monitoring

Green-lipped mussels (Perna viridis) were collected from seven fish culture zones (FCZs) in Hong Kong and analyzed for okadaic acid (OA). A conventional HPLC method was modified by incorporating a proteinase K digestion step. Results suggest that a higher recovery (2.5 times higher) of OA was obtained from spiked samples after the addition of 1.08 mg proteinase K in comparison with samples incubated without the proteolytic enzyme. For the hepatopancreas (HP) of individual field-collected mussels, the additional digestion step can enhance OA extraction by 3.1 times. Spatial and temporal variations in OA concentrations in the mussels from various FCZs were investigated. The highest concentration of OA in mussel HP samples was 1164.9 ng/g HP wet wt. With respect to OA concentrations in whole mussel tissues from seven sites and four seasonal samplings, the concentrations were between 70.0 and 131.0 ng/g wet wt., which did not exceed the generally recognized international regulatory criteria (>200 ng/g) for OA.     

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.     

Seasonal and pollution-induced variations in biomarkers of transplanted mussels within the Beagle Channel

The occurrence of biomarker variations linked to environmental factors makes it difficult to distinguish the effect of pollution. In an attempt to evaluate spatial and seasonal effects of environmental parameters on biomarker responses, mussels Mytilus edulis chilensis coming from an aquaculture farm were transplanted to several points within Ushuaia Bay (Beagle Channel) for 6 weeks in summer and winter. Activities of superoxide dismutase, catalase, glutathione-S-transferase and levels of lipid peroxidation were measured in gills and digestive gland. Cu, Zn, Fe, Cd and Pb concentrations were also assessed. Results indicated a significant effect of seasons on biological responses as well as in metal bioaccumulation showing the influence of natural factors such as dissolved oxygen, temperature and food availability. The interdependence of those environmental factors is important for the homeostasis of thermoconformers, especially regarding their oxidative metabolism and should also be taken into consideration to distinguish natural from pollution-induced variations.     

Polycyclic musks in green-lipped mussels (Perna viridis) from Hong Kong

Six polycyclic musk compounds [Cashmeran (DPMI), Celestolide (ADBI), Phantolide (AHMI), Traseolide (ATII), Tonalide (AHTN), and Galaxolide (HHCB)] were analysed in marine green-lipped mussels (Perna viridis) from Hong Kong. ADBI, HHCB and AHTN were detected in almost all samples, while AHMI, ATII and DPMI were not detected. Concentrations of ADBI, HHCB and AHTN in mussels ranged from below detection limit–0.0743 (mean: 0.0246), 0.247–6.08 (mean: 1.15) and 0.0591–0.738 (mean: 0.190) mg/kg lipid weight, respectively. Mussels from two sampling sites in central Victoria Harbour contained the highest total polycyclic musk levels, suggesting that these waters are heavily influenced by domestic sewage. Concentrations of HHCB and AHTN detected in the mussel samples were the second highest and the highest levels, respectively, compared to global concentrations. A preliminary risk assessment indicated that HHCB and AHTN in mussels pose little or no threat to the health of shellfish consumers. Nevertheless, more comprehensive studies are required to further assess the ecological and human health risks associated with polycyclic musks.     

Adaptation strategy of bivalve Modiolus modiolus from upwelling regions of the Kuril Islands shelf (Sea of Okhotsk) to heavy metal effects

Cd, Cu, Fe, Mn, Ni, Pb, Zn concentration in the organs (muscle, gonads, mantle, gills, digestive gland, kidney) of mussel Modiolus modiolus was studied. Mussels were collected from shelf upwelling sites of Kuril Is. Sea of Okhotsk (104 and 85 m) and, for comparison, coastal high contaminated and clean sites (Peter the Great Bay, Sea of Japan). It was found that mussels from shelf upwelling regions accumulated unusually high Cd concentration in studied organs, excepting digestive gland. Shelf mussels have evolved passive adaptation to elevated level of ambient heavy metals (distribution of heavy metal load between all organs, especially, muscle). In contrast, mussels from high contaminated sites have evolved active adaptation (strong regulation of metal concentration in the organs at the maximal permissible concentration due to increased kidney function). We supported that under natural condition, passive adaptation of aquatic organisms to increased ambient metal level have been evolved only under constant low-water temperature.     

Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): Do mussels “integrate” biomarker responses?

Polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCs) are generally present in the marine environment in complex mixtures. The ecotoxicological nature of contaminant interactions, however, is poorly understood, with most scientific observations derived from single contaminant exposure experiments. The objective of this experiment was to examine dose-response relationships between antioxidant parameters and body contaminant levels in mussels exposed to different exposure regimes under laboratory conditions. Accordingly, the green-lipped mussel, Perna viridis, was challenged with a mixture of PAHs (anthracene, fluoranthene, pyrene, benzo[a]pyrene) and OC pesticides (α-HCH, aldrin, dieldrin, p,p′-DDT) over a 4 week period. Contaminants were delivered under four different dosing regimes, with all treatments receiving the same total contaminant load by the end of the exposure period. Antioxidant biomarkers were measured after 1, 2, 3 and 4 weeks, including glutathione (GSH), gluathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and lipid peroxidase (LPO). GST and CAT were induced in hepatic tissues in most of the exposure regimes, with the majority of significant induction occurring in a constant exposure regime and a two-step alternate exposure regime. Significant differences among exposure regimes were detected in the body burden of contaminants after 28 days. Hepatic CAT and GSH are proposed as potentially useful biomarkers as they showed good correlation with target contaminants and were not readily affected by different dosing patterns.     

A River Water Quality Model for Chlorophyll and Dissolved Oxygen that Accommodates Zebra Mussel Metabolism

The development, testing and application of a dynamic two-dimensional (longitudinal-vertical) mass balance model for dissolved oxygen (DO) and chlorophyll (Chl) for rivers is documented that for the first time accommodates both the oxygen demand and filtering effects of zebra mussels. The test system is a phytoplankton-rich section ( 15 km long) of the Seneca River, NY, that is believed to represent an upper bound of the impact of this exotic invader. Changes in common measures of water quality of the river brought about by the zebra mussel invasion are reviewed and related longitudinal patterns in DO, Chl, and Secchi disc transparency are documented. Model testing is supported by comprehensive measurements of DO, Chl, and various forcing conditions over a three-month period, and independent determinations of several model coefficients. Wide variations in the areal consumption rate of DO (ZOD; g·m^–2·d^–1) and filtering rate (m³·m^–2·d^–1) of zebra mussels, as determined through model calibration, occurred over the study period. Values of ZOD in areas with dense zebra mussel populations at times (e.g., > 50 g·m^–2·d^–1) were an order of magnitude greater than the sediment oxygen demand associated with organically enriched deposits. The value of determinations of these fluxes from model calibration procedures is evaluated within the context of the limitations of protocols presently available to support independent specification of these rates. Model analyses are conducted to evaluate the relative magnitude of source and sink processes for DO and Chl, the potential operation and implications of feedback from low DO levels on oxygen consumption by zebra mussels, and the sensitivity of model simulations to selected sources of uncertainty and variability. Model projections of oxygen resources of the river are presented in a probabilistic format in evaluating reductions in zebra mussel biomass that would be necessary to eliminate violations of standards and regain assimilative capacity.