Field application of biochemical markers and a physiological index in the mussel,Mytilus galloprovincialis: transplantation and biomonitoring studies in the lagoon of Venice (NE Italy)

A number of biochemical markers and a physiological index were measured in mussels, Mytilus galloprovincialis, transplanted or native to five different contaminated sites in the lagoon of Venice. Mussels from Pellestrina, a reference site in the adjacent Adriatic Sea, were transplanted for 6 weeks to areas of the lagoon where indigenous mussels were also collected. As biochemical indices, superoxide dismutase (SOD), catalase, aldehyde dehydrogenase (ADH) and NADPH cytochrome c reductase (NADPHcred) were measured in mussel digestive gland; survival in air as a physiological index was also determined. Biomarker responses varied among sites and between indigenous and transplanted animals. Significant induction of catalase and SOD was shown in animals transplanted to the urban sites of Salute and Chioggia, respectively. In indigenous mussels, induction of SOD and NADPHcred was seen in animals from the polluted site of Treporti and the heavily contaminated industrial area of Marghera. The overall biochemical data indicate significantly higher activity for ADH in transplanted animals in comparison with indigenous ones which, in contrast, present an increase in SOD. As regard survival in air, control mussels did not seem to be healthier in comparison either with transplanted or indigenous ones, suggesting that pollution has no effect on this parameter.     

Evaluation of general and specific stress indices in mussels collected from populations subjected to different levels of heavy metal pollution

It has been shown that the rate of protein synthesis is significantly decreased in various tissues of mussels gathered from a coastal area, characterised by a high level of pollution, when compared with animals collected in the reference unpolluted area. Such a decrease may represent a general stress index. Additional biological parameters, such as amino acid uptake and RNA synthesis, are similarly decreased in the mussels collected from the polluted area.Moreover, it has been demonstrated that in the digestive gland of the animals sampled from the heavy metal polluted area the level of low molecular weight thionein-like Cu-binding proteins is three times higher than in the digestive gland of the control mussels. The increase in the level of metallothionein-like proteins in the digestive gland of the mussels appears to represent a specific stress index directly related to the presence in the sea water of high concentrations of heavy metals.     

Seasonal variation of histopathological and histochemical markers of PAH exposure in blue mussel (Mytilus edulis L.)

The aim of this work was to study seasonal variation of histopathological and histochemical markers in blue mussels (Mytilus edulis L.) exposed to pyrogenic PAH contaminants. Mussels were collected in January, June, September and October from a sampling site in the vicinity of the discharge from an aluminium smelter and from a clean reference site. Histopathological analysis was carried out on the digestive gland (DG) and the gonads, lipofuscin and neutral lipids were analysed in the DG. Clear responses in lipofuscin and neutral lipids were detected in the DG of mussels collected from the polluted site at some sampling times. Moreover, these mussels presented atrophy in digestive tubules and haemocytic aggregates in the gonad and DG. However, in all parameters studied, the magnitude of the response showed clear seasonal variation.     

The role of oxyradicals in intracellular proteolysis and toxicity in mussels

Studies were performed on the common mussel, M. edulis L., to determine whether copper (Cu) exposure can affect the extent to which digestive cell proteins are oxidised and whether such oxidative damage is mediated by free radicals. Three age groups of mussels were exposed for 6 ^-days to environmentally realistic concentrations of Cu and then digestive gland homogenates were examined for evidence of protein carbonyl formation. Significant increases in carbonyls relative to untreated control mussels were seen for the youngest (2–4 year-old) and oldest (≥ 10 year-old) mussels only after exposure for 6 days, followed by recovery from exposure for a further 6 days. Untreated mussels also showed an age-related difference in protein oxidation, with a significantly lower concentration in the youngest animals (2–4 year olds). Copper did not affect the levels of modified tryptophan or tyrosine residues or the extent of total lipid peroxidation in digestive gland homogenate. Significant depletion of total vitamin E (a-tocopherol) was seen only in young and medium-aged mussels following exposure for 6 days. The levels of protein carbonyl groups were increased in digestive cell cytosol and lighter lysosomes but not in heavier lysosomes or digestive gland microsomes following 5 days exposure to Cu. Dihydrohodamine-123 was converted to fluorescent rhodamine-123 following sequestration into digestive cell lysosomes. The results suggest a link between the lysosomal sequestration of copper, a concomitant increase in the production of oxyradicals and the potential for intracellular oxidative damage, as well as an increased capacity for oxidative damage in older animals.     

Anthropogenic pollution stimulates oxidative stress in soft tissues of mussel <Emphasis Type="Italic">Crenomytilus grayanus</Emphasis> (Dunker1853)

The digestive gland and gills of the mussel Crenomytilus grayanus extracted from three locations — (i) sampled from a clean and (ii) polluted site and (iii) transplanted from the nonpolluted to polluted site - were analysed for antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase), total oxyradical scavenging capacity and levels of lipid peroxidation products (malondialdehyde, conjugated dienes and lipofuscin). Perturbation of redox status was found in both digestive gland and gill tissues of mussels living in the polluted area. As the activities of superoxide dismutase and catalase were 1.2–3 times higher, the total oxyradical scavenging capacity was lower by 20–35% and the levels of lipid peroxidation products were 2–7 times higher compared to mussels from the reference site. In transplanted mussels, the lipid peroxidation process in both tissues was significantly stimulated (the level of conjugated dienes was increased 1.7–2.5-fold; malondialdehyde and lipofuscin contents were increased 3.5–5-fold) and the total oxyradical scavenging capacity fell by 50–70%. In addition, the transplantation generally resulted in transient and variable responses of antioxidant enzymes for both tissues. Complex response-behaviour of the antioxidant enzymes strongly points to the necessity of employing a combined approach that takes into account activities of antioxidant enzymes and the total oxyradical scavenging capacity, as well as measurement of oxidative damage (e.g., lipid peroxidation) to evaluate the physiological health of molluscs.     

Elevation of cytochrome P450-immunopositive protein and DNA damage in mussels (Mytilus edulis) transplanted to a contaminated site

Mytilus edulis were collected from a reference site (Port Quin) and an urban/industrial contaminated site (New Brighton) in the UK during June 1999. Levels of PCBs (sigma7 congeners) and CB-138 were determined to be, respectively, 21 fold and 16 fold higher in the mussel digestive glands from New Brighton. Levels of CYPIA-immunopositive protein were 1.5 fold higher (P < 0.05) at the polluted site but the levels of DNA strand breaks were 1.3 fold higher (P<0.05) at the reference site. Mussels from Port Quin were placed in cages at both sites and both transplanted and indigenous populations sampled in September (13 weeks). Mussels transplanted from the reference site to the industrial site, reported elevated levels of CYP1A-immunopositive protein (1.4 fold; P < 0.05) and higher levels of DNA damage (1.2 fold; P < 0.05) compared to caged populations at the reference site and a PCB loading similar to the populations from the polluted site. Moreover, transplanted mussels had DNA damage 1.8 fold greater (P < 0.05) than indigenous mussels at the transplant site. These changes were small but significant when compared to the observed temporal changes in the indigenous populations.     

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.     

Bioaccumulation and depuration of Zn and Cd in mangrove oysters (Crassostrea rhizophorae, Guilding, 1828) transplanted to and from a contaminated tropical coastal lagoon

In order to study Zn and Cd accumulation and depuration, a set of oysters, Crassostrea rhizophorae, were transplanted to a metal contaminated coastal lagoon and another one was harvested there and transplanted to a non-polluted site. C. rhizophorae oysters and Perna perna mussels native from both sites were collected in order to monitor variability of metal concentration in resident populations. After three months exposure, oysters transplanted to the polluted site accumulated fourfold Zn (307-1319 microgg(-1)) without reaching the concentration level of resident oysters (9770 microgg(-1)). Cadmium concentrations had a slight but significant decrease during the same period (1.25-0.54 microgg(-1)). Oysters transplanted to the non-polluted site, showed threefold Zn depuration (6727-2404 microgg(-1)), while Cd had no significant variation (0.90-1.45 microgg(-1)). Results showed that transplanted oysters do not reach heavy metal concentrations in indigenous populations suggesting transplanted organisms would be better used to evaluate bioavailability instead of environmental concentrations.     

Some responses of transplanted Mytilus edulis to metal-enriched sediments and sewage sludge

Approximately 25 % of the sewage sludge produced in the United Kingdom (about 10 million wet tonnes anually) is disposed of to British coastal waters at 15 sites. A series of tests is being evaluated in order to determine the effects of this practice in relation to the effects due to other polluting inputs, both natural and man-made, to the marine environment. The results presented here are for one site on the south-west coast of Britain. They indicate that the increased metal content of the tissue and the presence of sulphydryl-rich metal-binding proteins in the lysosomes of digestive cells in transplanted mussels can be correlated with exposure at sites overlying metal-rich sediments and to a lesser extent an area used for the disposal of sewage sludge containing metallic contaminants.     

Biochemical responses in farmed mussel Perna perna transplanted to contaminated sites on Santa Catarina Island, SC, Brazil.

The effects of contaminants on the biochemical parameters of the intensively farmed mussel Perna perna, are unknown. The aim of this study was to compare biochemical responses in mussels held in clean and contaminated sites in Santa Catarina Island, Brazil. Mussels were transplanted from a farming area, Ratones Grande Island (RGI), to two contaminated sites, Itacorubi (ITAC) and Hercílio Luz Bridge (HLB). A reference group was kept at RGI. After 150 and 180 days of exposure, the digestive glands of the mussels were analyzed for catalase, glucose 6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST) activities. No changes were observed in the catalase activity, in both periods. Low G6PDH activity was observed in mussels transplanted for 150 days at the ITAC site. Increased GST activity was observed in mussels from ITAC and HLG sites after 180 days. These responses are probably related to the augmented discharges of domestic effluents associated with elevated rainfall index.     

Field application of lysosomal destabilisation indices in the mussel Mytilus galloprovincialis: biomonitoring and transplantation in the Lagoon of Venice (north-east Italy)

A field study was carried out in the Lagoon of Venice (north-east Italy) with the aim of evaluating the potential use of lysosomal destabilisation as a biomarker of anthropogenic stress in the autochthonous mussel Mytilus galloprovincialis. Two different approaches were adopted in biomonitoring six sites in the Lagoon, evaluating indigenous populations of mussels and organisms transplanted from a reference site and checked at several points in time. Lysosomal membrane stability was investigated by means of two tests: neutral red retention assay (NRRA) for evaluating haemocyte lysosomes and lysosomal latency test (LLT) for digestive cell lysosomes. Results indicate that the lysosomal response measured in haemocytes according to NRRA is a more valuable biomarker of anthropogenic stress in the framework both of passive and active biomonitoring in marine coastal environments.     

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.     

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.     

渤海湾表层沉积物重金属与污染评价

用原子吸收法测定了渤海湾表层沉积物中重金属的含量,同时用2种评价方法对该海域底质环境进行了评价。结果表明:沉积物中汞、锌含量平均值超过标准,其余项目未发现超标;其分布特征由于受沿岸不同污染源的污染影响,重金属污染物难以形成一定规律的分布状态。同时该海域底质环境质量总指数已达到污染程度和很强的生态危害,其中主要污染元素是Hg,已达重污染,并形成极强的生态危害。     

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

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

Immuno-localisations (GSSP) of subcellular accumulation sites of phenanthrene, aroclor 1254 and lead (Pb) in relation to cytopathologies in the gills and digestive gland of the mussel Mytilus edulis

Cell and tissue pathology of both, gill and digestive tissue, has been the subject of many studies for the elucidation of contaminant-induced biological effects. In the present study, cellular pathological alterations were linked to subcellular sites of chemical accumulation in gills and digestive gland tissues. For this purpose, mussels (Mytilus edulis) were exposed to the organic contaminants aroclor 1254 (PCB) (20 microg/L), phenanthrene (PAH) (150 microg/L) or the metal lead (Pb) (2.5mg/L). The localization of chemicals at the subcellular level was analysed by an antibody-based detection system (GSSP) by the use of commercially available antibodies specifically directed against the chemicals. Pathological changes were analysed in parallel in identical samples by transmission electron microscopy. After exposure to the different contaminants, cell organelles such as mitochondria, the endo-lysosomal system as well as endoplasmic reticulum showed clear evidence of chemically-induced alterations. Large numbers of crystalloid inclusions were found in mitochondria and in autophagic lysosomes as well as multi-lamellated whorls after PAH and aroclor exposure. Immunocytochemical detection of the chemicals showed their accumulation inside of various cell organelles such as lysosomes, mitochondria, and nuclei. Additionally, chemicals were localized in association to membranes, cilia and microvilli of gill and digestive gland cells. Furthermore, the chitinous rod and mucus secretions of gill epithelial cells were positively labelled for contaminants indicating their role in protection. Localization of contaminants by immuno-detection in combination with pathological diagnosis gives insights into the cellular targets of chemical attack.     

Effects of linear alkylbenzene sulphonate (LAS) and cadmium in the digestive gland of mussel, Mytilus sp.

A laboratory study was carried out exposing mussels (Mytilus sp.) to linear alkylbenzene sulphonate (LAS) (6 mg litre⁻¹), Cd (0.05 mg litre⁻¹) and LAS plus Cd at the same concentrations. The aim was to assess the use of several histopathological and biochemical indices as potential biomarkers of the impact of these xenobiotics in the digestive gland of molluscs. Treated mussels actively accumulated Cd in the digestive gland compared with controls (p 0.01), the highest levels occurring after 30 days of exposure in the group treated with Cd plus LAS. Among several histological alterations screened in digestive gland tissues the thickness of digestive tubules in Cd treated animals decreased more markedly (p 0.01) than in LAS exposed mussels. As for biochemical parameters, the investigated antioxidant enzyme activities, superoxide dismutase (SOD), catalase, DT-diaphorase and glutathione peroxidase (GPX) did not show any significant induction due to these xenobiotics. However, a slight decrease of the antioxidant defences of the animals was detected after 30 days of exposure to contaminants.     

Role of metallothioneins in Cu and Cd accumulation and elimination in the g gill and digestive gland cells of mytilus galloprovincialis lam.

The data presented in this paper demonstrate that exposure of mussels to Cu (40 μg/liter) or Cd (200 μg/liter) for a period of 2 days is sufficient to induce Cu or Cd thioneins in the gills of the metal-exposed animals. The concentrations of Cu and Cd thioneins increase during the period of metal exposure (3–4 weeks), confirming the fact that metallothioneins play a fundamental role in the accumulation of these metals in the tissues examined (gills and digestive gland). Moreover, it has been demonstrated that when metal-loaded mussels are returned to the field for the recovery period, Cu is rapidly eliminated from the gill and digestive gland cells, showing a biological half-life of 9–10 days, whereas Cd is released much more slowly from the tissues, only about 50% of the total metal being lost after 4 months of detoxification. Interestingly enough, during the detoxification period the concentration of the metal bound to thioneins also follows the same pattern as the total metal present in the tissues, in fact the concentration of Cu thioneins decreases near the control level in about 24 days but, in contrast, the Cd-thionein concentration in the cytisol of the gill and digestive gland cells remaains high, decreasing only by about 40% at the end of the 4 month recovery period.The data presented also demonstrate that when Cd-loaded, detoxified mussels are exposed to Cu (100 μg/liter) for 6 h, the metal taken up by the gill cells is able to displace the Zn which is always present in the Cd-thionein fraction, but it does not, however, displace the Cd present. In this case also, period (20 days), whereas the Cd-thionein content decreases slightly. Such data seem to indicate that the metabolic characteristics of thioneins are closely related to the metal bound to them, particularly where the elimination rate of the metals from the cells is concerned.     

渤海湾(天津段)柱样沉积物重金属污染研究

用原子吸收法测定了渤海湾柱样沉积物中重金属的含量,分析了各元素在各站位的分布特征,同时对各重金属元素和有机质做了相关分析并对该海域底质环境进行了评价,结果表明：各重金属含量随深度的分布特征由于受沿岸不同污染源的污染影响,重金属污染物难以形成一定规律的分布状态,总体上呈波折型分布;且该海域底质环境质量总指数已达很强的生态危害,其中主要污染元素是Hg,已达极强的生态危害。     

Molecular investigations into pollutant impact on roundnose grenadier (C. rupestris) and transplanted common mussel (M. edulis) in Skagerrak, the North Sea

The use of a suite of different biochemical markers in the deep sea fish Roundnose grenadier and caged common mussels, and analyses of contaminant levels in the mussels, indicate that Skagerrak are more polluted than waters around the Faroe Islands. In Roundnose, induction of ethoxyresorufin-O-deethylase (EROD) is consistent with increased benzo(a)pyrene hydroxylase (BPH) in mussels and elevated levels of polynuclear aromatic hydrocarbons (PAHs) in mussels and sediment from Skagerrak. This evidence of a large-scale environmental impact of pollutants should lead to further efforts to decrease transport into, and deposition of, waste compounds in the Skagerrak area.