Time-course evaluation of ROS-mediated toxicity in mussels, Mytilus galloprovincialis, during a field translocation experiment

Harbours can be considered as model environments for developing and validating field monitoring procedures and to investigate mechanistic relationships between different biological responses. In this study several biomarkers were investigated in marine mussels caged for 4 weeks (June–July 2001) into an industrialized harbour of NW Italy. Organisms were collected at different time-intervals to better characterize the sensitivity, temporal variations and interactions of analysed responses. Besides single antioxidants the total oxyradical scavenging capacity (TOSC) assay was used to analyse the capability of the whole antioxidant system to neutralize specific forms of radicals: these data were further integrated by measurement of DNA integrity, oxidized bases and the impairment of lysosomal membrane stability in haemocytes. Results showed a biphasic trend for single antioxidants and TOSC, with an increase during the first 2 weeks of exposure to the polluted site followed by a progressive decrease up to a severe depletion in the final part of the experiment.     

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.     

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.     

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

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

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

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

Heavy metals in mussels (Mytilus galloprovincialis) from the gulf of la spezia and from the promontory of Portofino,Italy

In different samples of Mytilus galloprovincialis Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb and Zn were determined by AAS. The mussels were taken from 11 sites in the Port of La Spezia. Typical concentration ranges (μ/g dry weight) were as follows: Cd, 2.0–6.8; Co, 0.8–3.2; Cr, 5.8–19.5; Cu, 6.9–33.7; Hg, 0.15–0.38; Mn, 11.8–37.8; Ni, 1.3–10.9; Pb, 13.9–44.6 and Zn, 203–379.Other samples were collected at 4 sites on the Promontory of Portofino and the following values were obtained: Cd, 0.8–1.1; Co, 2.6–2.9; Cu, 3.3–4.2; Hg, 0.09–0.15; Mn, 6.3–9.1; Ni, 1.2–2.4; Zn, 120–140.     

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 17beta-estradiol exposure in the mussel Mytilus galloprovincialis

Mussels Mytilus galloprovincialis were exposed to different concentrations of estradiol (20, 200, and 2000 ng/l) in a semi-static regime (1-day dosing intervals) for up to 7 days in an attempt to see how mussels dealt with exogenous estrogenic compounds. Sex hormone levels were determined in whole tissue. Free-estradiol was only significantly elevated at the highest exposure dose (up to 10-fold). Most of the estradiol was in the tissues as fatty acid esters (> 78%), which sharply increased in a dose-dependent manner (from 4 ng/g in controls to 258 ng/g at the high exposure group). In contrast, neither free nor esterified testosterone levels showed significant differences between control and exposure groups. The results suggest the existence of mechanisms that allow mussels to maintain their hormonal status, and the important role that fatty acid esterification may play within those mechanisms. Synthesis and conjugation rates of estradiol were further investigated by measuring the activity of P450 aromatase, and palmitoyl-CoA:estradiol acyltransferase, in digestive gland microsomal fractions. Overall, the study contributes to the better knowledge of molluscan endocrinology, and defines new mechanisms of regulation of free steroid-levels in mussels.     

Tissue sampling from live blue mussels,Mytilus edulis. A field study from the Swedish west coast

Histological techniques are often used to study environmental effects on mussels, but since these techniques include killing of the individuals, rare or endangered populations cannot be studied using conventional tissue sampling. This study is an attempt to find a method that can be used repeatedly with the same mussel individual and which does not affect growth and survival. From 200 mussels, Mytilus edulis, tissue was sampled in different ways, such as drilling a hole in the shell or prising apart the shell valves. Two kind of instruments were used, an injection needle and surgery forceps. Some of the drilled mussels had their holes sealed again with cement.Drilling a hole in the shell, removing tissue sample with surgical forceps and then leaving the holes open did not seriously harm the mussels during the two months the experiment lasted. But if the holes were sealed with cement, both length and weight growth were negatively affected (35% lower length growth and 36% lower weight growth compared to the control mussels). Mortality was highest among the drilled and sealed mussels (80% higher than among the other treatments). The vulnerability of the population, the aim of the study and the duration of the experiment should decide what method to use for tissue sampling. For long-term experiments and repeated sampling, opening the mussels by prizing apart the valves is a better alternative than drilling holes in the shells, but depending on the morphology of the species it could be difficult to sample the anterior part of the mussel body. For a short experiment and to sample anterior parts, drilling the shells, leaving the holes open and using surgical forceps, seems to be an acceptable compromise between the different treatments used.     

Effects of okadaic acid on haemocytes from Mytilus galloprovincialis: A comparison between field and laboratory studies

Individuals of Mytilus galloprovincialis, contaminated with Diarrheic Shellfish Poisoning (DSP) toxins, were studied with the aim to correlate the okadaic acid (OA) body burden and the percentage of damaged haemocytes by quantifying annexin V positive cells by flow cytometry. Results showed less percentage of damaged haemocytes in high OA contaminated samples. These data were compared with results of in vitro assays of mussel haemocytes exposed to increased concentrations of OA. Similarly, haemocytes exposed to the most concentrated OA solution were less damaged.     

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.     

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.     

Monitoring a Marine Coastal Area: Use of Mytilus galloprovincialis and Mullus barbatus as Bioindicators

Abstract. Samples of Mytilus galloprovincialis and Mullus barbatus were collected in eight coastal sites along the South Adriatic and Ionic coasts of Italy in spring 2000 for a survey of coastal pollution in the Mediterranean basin. Specimens were analysed using an integrated approach based on residue analysis of common aquatic pollutants like organochlorines such as hexachlorobenzene (HCB), DDTs and polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), heavy metals and nonylphenols (NPnE) and biomarker responses such as acetylcholinesterase (AChE) activity, and the two specific P450 activities benzo(a)pyrene monooxygenase (BPMO) and 7-ethoxyre-sorufin-(9-deethylase (EROD). Biological and morphological parameters like somatic liver index (SLI), gonadosomatic index (GSI) and gonadal and gamete histology (eggs and sperms) were also evaluated in red mullet samples. A contamination gradient in which several hot spots occur were revealed in this study. The hot spots account for high levels of organochlorines in both species near incinerators and of PAHs in harbour areas. Levels of both NPnE and AChE activity were highest in two protected marine areas and were within detectable limits in others. This finding was confirmed by P450 activities, in which maximum levels were detected in harbours and protected marine areas. No morphological alterations of male and female gonads were observed on the histological level.     

Distribution of PACs in surficial sediments and bioavailability to mussels, Mytilus edulis of Halifax Harbour

Surficial sediments and blue mussels were collected around the perimeter of Halifax Harbour. Samples were analysed for the levels and fingerprint of polycyclic aromatic compounds (PACs). The objective was to explore the relationship between contaminant profiles in sediments and in mussels and to explore the possibility of deducing their sources, pattern of deposition and circulation in the harbour. Combustion derived compounds predominated in both sets of samples, with more variability observed in the level of petroleum derived hydrocarbons in mussel samples. Biota-sediment accumulation factors (BSAF, dry/dry wt.) varied throughout the harbour, ranging from means of 0.006 to 1.26, where generally fluoranthene displayed the highest BSAF. Sediments and mussels give complementary views of the geographical distribution of contaminants, they reflect the state of benthic and pelagic habitats. Results observed for Halifax Harbour are discussed from the perspective of published data.     

Aggregational behavior of the blue mussels Mytilus edulis and Mytilus trossulus: a potential pre‐zygotic reproductive isolation mechanism

The blue mussels Mytilus edulis and Mytilus trossulus occur sympatrically and are able to hybridize in populations on the eastern coast of Newfoundland, Canada, presenting an opportunity to study their aggregational behavior. Aggregation behavior may therefore provide insight into post‐settlement interactions and pre‐zygotic reproductive isolation between the species. Three treatments were designed using M. edulis and M. trossulus to investigate their intraspecific and interspecific spatial distribution patterns. With Ripley’s K‐function and Monte Carlo simulation analysis, we found that in the single‐species treatment, M. edulis aggregated significantly but not M. trossulus. Based on results of two‐way ANOVAs, both the number of aggregations and the moving distance were significantly affected by the treatments (single‐species or mixed‐species treatment) and times (24, 48, 72 and 96 h). In further pairwise comparisons using Tukey’s test, M. edulis aggregated differently with or without M. trossulus occupying the same tank, suggesting that the aggregational behavior of M. edulis could be driven by species‐specific chemical cues. The result that M. edulis aggregates intraspecifically may increase the probability of intraspecific fertilization of the spawned gametes and thus function as a pre‐zygotic reproductive isolation mechanism maintaining the blue mussel hybrid zone.     

Assessment of coastal marine pollution in Galicia (NW Iberian Peninsula); metal concentrations in seawater,sediments and mussels (Mytilus galloprovincialis) versus embryo-larval bioassays using Paracentrotus lividus and Ciona intestinalis

Sediments from three Galician Rias were tested for toxicity using sea-urchin and ascidian sediment elutriate embryo-larval bioassays. Trace metal contents in seawater, sediments and mussels were also determined and subjected to multidimensional scaling methods which grouped stations according to chemical contamination. High metal contents were found in seawater, sediments and mussels from the Ria of Pontevedra, and moderate levels were detected in the Ria of Vigo and Ria of Arousa. The results revealed that samples assessed as toxic, according to the sea-urchin and ascidian embryo-larval bioassays, were among the most polluted by trace metals. A good agreement was reported between ordination plots resulting from applying multidimensional scaling to the chemical data, and the results of the biological endpoints tested.     

舟山海域外来物种地中海贻贝的自然分布现状及生态影响

以筛选的2个可鉴别的地中海贻贝（Mytilus galloprovincialis）、厚壳贻贝（M.coruscus）及其杂交后代的PCR标记（扩增核基因Glu-5’特异片段PCR标记Me14/Me17和延伸因子1α第一个内含子区段PCR标记引物EFbisF/EFbisR）,对舟山海域3个贻贝主产区（马鞍列岛海域、浪岗山列岛海域、中街山列岛海域）采集的贻贝样本的种类进行鉴别.研究结果显示：100个马鞍列岛海域贻贝个体和30个中街山列岛海域贻贝个体均为厚壳贻贝;而54个浪岗山列岛海域贻贝样本中,16个个体（29.6%）为厚壳贻贝,38个个体（70.4%）为地中海贻贝;3个群体中均未检测到2种贻贝的杂交个体.因此,推测舟山海域地中海贻贝自然分布仅局限于局部海区,但在适宜的海区地中海贻贝可取代厚壳贻贝成为贻贝床的优势物种,舟山海域贻贝床的动态变化值得进一步关注;贻贝样本中没有检测到地中海贻贝与厚壳贻贝的杂交个体,表明调查海域可能没有杂交贻贝分布或数量极少,地中海贻贝通过杂交、基因渐渗污染厚壳贻贝基因组的风险较小.