Stress-70 proteins in marine mussel Mytilus galloprovincialis as biomarkers of environmental pollution: a field study

In the present work we have investigated levels of stress-70 proteins in the gills of mussel Mytilus galloprovincialis collected seasonally from subtidal rocky shores at 6 different sites of the Rovinj coastal area (Northern Adriatic, Croatia). 1-D analysis (SDS-PAGE) using monoclonal mouse antibodies anti-HSP70 detected two bands of stress-70 proteins, 70 and 72 kDa constitutively present during the year. 2-D analysis (IEF+SDS-PAGE) proved that the antibodies used detected HSP70 (pI 5.7-5.9) and HSP72 (pI 5.5-5.6). The quantification of stress-70 proteins was possible using 200 ng of external HSP70 protein standard included on every blot. Maximal levels of HSP72 and HSP70 were observed in mussels in summer (September), and minimal levels in winter (December), and only HSP70 showed significant correlation with the sea temperature (r=+0.822, p<0.05). Acclimatization of mussels to a different lower salinity under experimental conditions proved that small changes in sea salinity (Delta=2 psu) could not cause significant stress-70 proteins induction. Results indicated that there are significant differences in HSP70 and HSP72 content in mussels from the control site (S-1) and mussels from other sampling sites with urban and industrial pollution. The usefulness of stress-70 proteins as biomarkers of environmental pollution is discussed.     

Biologically available trace metals in Mytilus edulis from the coast of Northeast England

The concentrations of Fe, Mn, Zn, Cr, Cu, Pb, Ni, Cd, and Ag were determined in soft tissue and shell material of mussel Mytilus edulis collected in the winter season from five coastal sites in the northeast of England. The trace metal levels observed were significantly high, particularly at locations affected by past coal mining activities (Blackhall) and present industrial sources (Middleton). The mussels from the chosen control site (Holy Island), a shellfish-designated area, were particularly enriched in Cu and Ag. The metal pollution index for the Bran Sands site, a heavily industrialised area on the Tees Estuary, was relatively low. At four sites out of five, the levels of Fe and Cu were negatively correlated with mussel size. No significant correlation with size was found for the other metals. With few exceptions, the elements Fe, Zn, and Mn accumulated preferentially in the soft tissue, whereas Pb, Cd, Cr, Ag, and Ni were more abundant in the hard part of the mussels. The Ag values in shell material were remarkably similar (about 6 mg kg⁻¹ – 7 mg kg⁻¹, dry wt) at all sites studied. In general, the data show that the mussels from the coastline studied accumulate metal concentrations comparable to or higher than the most contaminated sites reported in the literature.     

Molecular biomarkers of energy metabolism in mussels under anthropogenic pollution of Peter the Great Bay, the Sea of Japan

The functional state of the indicator species, the Gray mussel Crenomytilus grayanus (Bivalvia), has been analyzed in five areas of Peter the Great Bay exposed to anthropogenic pollution. The following indices of the state of mussels have been used: molecular biomarkers of energy metabolism—Na⁺,K⁺-ATPase, Mg²⁺-ATPase, and total ATPase activity—as well as the level of lipid peroxidation (LPO) and glutathione concentration in the hepatopancreas, gills, and gonads of mussels. The activity of ATPases, LPO level, and glutathione concentration significantly change in mussels from polluted areas relative to those in mussels from a conventionally unpolluted area (a bay in the Far Eastern State Marine Reserve). The molecular biomarkers used in the study provide reliable information on animal metabolism in impact areas. With consideration of the data obtained, it is concluded that the state of mussels in polluted areas is impaired.     

Factors affecting 210Po and 210Pb activity concentrations in mussels and implications for environmental bio-monitoring programmes

The activity of ²¹⁰Po and ²¹⁰Pb was determined in mussels of the same size (3.5-4.0 cm shell length) sampled monthly over a 17-month period at the Atlantic coast of Portugal. Average radionuclide concentration values in mussels were 759 ± 277 Bq kg⁻¹ for ²¹⁰Po (range 460-1470 Bq kg⁻¹ dry weight), and 45 ± 19 Bq kg⁻¹ for ²¹⁰Pb (range 23-96 Bq kg⁻¹ dry weight). Environmental parameters and mussel biometric parameters were monitored during the same period. Although there was no seasonal variation of radionuclide concentrations in sea water during the study period, the concentration of radionuclide activity in mussels varied seasonally displaying peaks of high concentrations in winter and low concentrations in summer. Analysis of radionuclide data in relation to the physiological Condition Index of mussels revealed that ²¹⁰Po and ²¹⁰Pb activities in the mussel (average activity per individual) remained nearly constant during the investigation period, while mussel body weight fluctuated due to fat storage/expenditure in the soft tissues. Similar variation of radionuclide concentrations was observed in mussels transplanted from the sea coast into the Tejo Estuary. However, under estuarine environmental conditions and with higher food availability throughout the year, transplanted mussel Condition Index was higher than in coastal mussels and average radionuclide concentrations were 210 ± 75 Bq kg⁻¹ (dry weight) for ²¹⁰Po and 10 ± 4 Bq kg⁻¹ (dry weight) for ²¹⁰Pb, therefore lower than in coastal mussels with similar shell length. It is concluded that the apparent seasonal fluctuation and inter-site difference of radionuclide concentrations were mostly caused by mussel body weight fluctuation and not by radionuclide body burden fluctuation. This interpretation can be extended to the apparent seasonal fluctuation in concentrations of lipophilic and lipophobic contaminants in mussels, and provides an explanation for occasional high concentrations of ²¹⁰Po and man-made contaminants measured in mussels far from pollution sources.     

Evidence of genetic damage in grass gobies and mussels from the Venice lagoon

The presence of genetic damage has been investigated in two native species of the Venice lagoon: the common mussel Mytilus galloprovincialis and the grass goby Zosterisessor ophiocephalus. Two sampling campaigns were performed in summer 1998 and 1999. Aromatic-like DNA adducts were analysed in selected tissues of gobies and mussels by using the 32P-postlabelling assay. In 1999, micronuclei and other nuclear abnormalities were additionally scored on gill cells and haemocytes of individual mussels whereas inorganic (As, Cd, Cr, Hg, Ni, Pb, Sn) as well as organic contaminants (polycyclic aromatic hydrocarbons, polychlorinated biphenyls and other chlorinated compounds) were measured in the total mussel pulp. Compared to the lagoon inlet area, gobies and mussels from the industrial district (Marghera) showed significant DNA adduct levels and increased frequencies of cytogenetic alterations (evidence of genetic damage was absent or inconsistent in other sites). The substantial levels of aromatic and chlorinated contaminants detected in mussels from Marghera also support the exposure of native organisms to genotoxic agents.     

Immediate biomarker responses to benzo[a]pyrene in polluted and unpolluted populations of the blue mussel (Mytilus edulis L.) at high-latitudes

Immediate biomarker responses of two high-latitude populations of the blue mussel Mytilus edulis to benzo[a]pyrene (B[a]P) were evaluated. Mussels collected from a clean and a polluted site in southwest Iceland were exposed to the nominal dose of 100 microg B[a]P L(-l) for 3 h, after 4 days of acclimatization in clean seawater. To test the sensitivity to the toxicant and immediate biological responses, the following biomarkers were used: DNA single strand breaks, heart rate and feeding rate. All the biomarkers revealed differences between the study sites. Irrespective of the origin of the organisms, the short time exposure to the high B[a]P concentration did not induce DNA single strand breaks or significantly affect the feeding rate. However, the heart rate results showed significantly different responses. The mussels from the polluted site (Reykjavík harbour) increased their heart rate when exposed to B[a]P, while no difference was observed between the heart rate values of the individuals from the clean site (Hvassahraun). The mussels seem to sense the pollutant they have been previously exposed to, and their acute response indicates physiological adaptation to the polluted environment. The results indicate limited sensitivity and temporal predictivity, i.e. transient measurable changes of these biomarkers, as well as showing that the background of the organisms should be considered when evaluating short-term biomarker responses to contaminants.     

Allozyme polymorphisms and heavy metal levels in the green-lipped mussel Perna viridis (Linnaeus) collected from contaminated and uncontaminated sites in Malaysia

It has been widely reported that heavy metal contamination in coastal waters can modify the allozyme profiles of marine organisms. Previous studies have recorded elevated metal concentrations in sediments and mussel tissues off Peninsular Malaysia. In the present study, horizontal starch gel electrophoresis was carried out to estimate the levels of allelic variation of the green-lipped mussel, Perna viridis, collected from one contaminated and three relatively uncontaminated sites off Peninsular Malaysia. Fourteen polymorphic loci were observed. In addition, the concentrations of cadmium, copper, lead, mercury and zinc were determined in the sediments and in the soft tissues of the mussels. Mussels from contaminated site, evidenced by high metal pollution indices (MPI) of the sediment and the mussel tissues, showed the highest percentage of polymorphic loci (78.6%), while those collected from the uncontaminated sites had lower MPI of the sediment and mussel tissue, and exhibited lower percentages of polymorphic loci (35.7-57.1%). The population from the contaminated site showed the highest excess of heterozygosity (0.289) when compared to that of the populations from the three uncontaminated sites (0.108-0.149). Allozyme frequencies at the phosphoglucomutase (PGM; E.C. 2.7.5.1) locus also differed between the contaminated and uncontaminated populations. Previous studies have shown that exposure to heavy metals can select or counter-select for particular alleles at this locus. The present results suggest that allozyme polymorphism in P. viridis is a potential biomonitoring tool for heavy metal contamination but further validation is required.     

Radionuclides and metals in freshwater mussels of the upper South Alligator River, Australia

During an inspection of the old Rockhole Mine area in Kakadu National Park in 1999, it was found that a small amount of tailings from the former South Alligator uranium (U) mill had been uncovered by wet season rain and road works. Samples of sediment, water and freshwater mussels, Velesunio angasi, were collected from the South Alligator River, near and at the confluence of Rockhole Mine Creek, and adjacent to the exposed tailings. The 228Ra/226Ra activity ratios in sediments and mussel tissue indicate a small influence from the tailings and from Rockhole Mine adit water on 226Ra concentrations. The uptake of 226Ra in mussels does not correlate with other alkaline-earth metals. Mussel U concentrations are higher immediately downstream of Rockhole Mine Creek, but there is no noticeable increase in the immediate vicinity of the tailings area. A hypothetical ingestion of 2 kg of mussels from the sites was used to estimate the committed effective dose for a 10-year-old child resulting in a figure of 0.23 mSv per annum, of this total dose, 69% is attributed to 210Po. Only 0.03 mSv per annum can be directly linked to impacts of the tailings.     

A study of radium bioaccumulation in freshwater mussels, Velesunio angasi, in the Magela Creek catchment, Northern Territory, Australia

Freshwater mussels, Velesunio angasi, along Magela Creek in Australia’s Northern Territory were examined to study radionuclide activities in mussel flesh and to investigate whether the Ranger Uranium mine is contributing to the radium loads in mussels downstream of the mine. Radium loads in mussels of the same age were highest in Bowerbird Billabong, located 20 km upstream of the mine site. Variations in the ratio of [Ra]:[Ca] in filtered water at the sampling sites accounted for the variations found in mussel radium loads with natural increases in calcium (Ca) in surface waters in a downstream gradient along the Magela Creek catchment gradually reducing radium uptake in mussels. At Mudginberri Billabong, 12 km downstream of the mine, concentration factors for radium have not significantly changed over the past 25 years since the mine commenced operations and this, coupled with a gradual decrease of the ²²⁸Ra/²²⁶Ra activity ratios observed along the catchment, indicates that the ²²⁶Ra accumulated in mussels is of natural rather than mine origin. The ²²⁸Th/²²⁸Ra ratio has been used to model radium uptake and a radium biological half-life in mussels of approximately 13 years has been determined. The long biological half-life and the low Ca concentrations in the water account for the high radium concentration factor of 30,000-60,000 measured in mussels from the Magela Creek catchment.     

Monitoring DNA damage in <Emphasis Type="Italic">Mytilus galloprovincialis</Emphasis> from the Kaštela Bay in Croatia

The Kaštela Bay (Croatia) is known, by the use of Fast micromethod, as an area with the highest level of genotoxic agents along the Adriatic Sea. The genotoxic impacts in Kastela Bay and the neighbouring Trogir Bay using micronucleus test and Comet assay with mussel (Mytilus galloprovincialis) haemocytes were assesed during 2003 and 2004. This location received effluents from various industries (brewery, cement plant, etc.), ports (oil and general port), the Split shipyard, Vranjic shipworks along with domestic sewage and agricultural discharge that enters the bay without any treatment. In the meantime, a lot of industries were closed and the input of pollutants were discontinued. The tested mussels from the 2003–2004 confirmed that mussels from Kaštela Bay were affected by genotoxic contaminants. The aim of our study was to assess genotoxic impacts on Mytilus galloprovincialis in the same area after six years using Comet Assay test. Our survey showed decreses of genotoxic damage on mussel haemocytes.     

Histomorphological structure of the liver in roach (Rutilus rutilus) and pike (Esox lucius) from lakes with different levels of anthropogenic impact

Using histological techniques, changes in histomorphological characteristics of the liver were revealed in two fish species from lakes with different levels of anthropogenic impact. The most significant changes in the liver of roach from Kostomukshskoe Lake (used as a tailing dump of the Kostomuksha Mining and Ore-processing Plant) included initial stages of parenchymal protein dystrophy and, to a lesser extent, fatty dystrophy; in pike, fatty liver dystrophy prevailed. Compared to roach, pike proved to be more sensitive to ecological conditions in Kostomukshskoe lake, including increased water mineralization. In both fish species from Koivas Lake (20 km from the pollution source), only the initial stages of local dystrophic changes could be detected. The fish from clean Kamennoe Lake (located in a protected area) had no pathological changes in the liver.     

Distribution of organotin compounds in the bivalves of the Aegean Sea, Greece

Five bivalve species--Mytilus galloprovinciallis (Mediterranean mussels), Venus gallina (stripped venus), Modiola barbatus L. (bearded horse mussels), Pecten jacobeus (scallops) and Callista chione (hard clams)--were collected from seven areas in Aegean Sea, Greece, between August 2001 and January 2003 and analyzed for organotins (OTs). The concentrations (as geometric means) found were 17.1 ng g-1 for tributyltin (TBT), 18.8 ng g-1 for dibutytltin (DBT), 7.8 ng g-1 for monobutyltin (MBT) and 13.0 ng g-1 for triphenyltin (TPhT) (wet weight), which are at similar or lower levels than those reported worldwide. Studying OTs distribution between different bivalve species, lower concentrations were observed in mediterranean mussels, possibly due to their growth in water column (grown on sea net pens in mussel farms), in contrast to the free-ranging species, collected from fishing grounds. Concentrations of the OTs in the examined bivalves varied seasonally.     

Experimental studies on the biokinetics of 134Cs and 24Am in mussels (Mytilus galloprovincialis).

The biokinetics of 134Cs and 241Am in mussel species contaminated through water pathway has been studied under laboratory conditions. At equilibrium, the concentration factors for 134Cs and 241Am in small and large mussels were 2.80 and 2.57 and 200 and 150, respectively. The concentration factor of 134Cs in soft parts of the mussels was significantly high than whole body and shell tissue. However, the concentration factors of 241Am in soft parts and shell tissue samples were found to have similar rates. The depuration kinetics of the radionuclides were described by two-component exponential models. The biological half-lives at slow components between small and large mussels did not change significant, and were found to be 46.8-46.5 and 72.2-75.3 days for 134Cs and 241Am. The depuration kinetics of 134Cs and 241Am in soft parts described a single-component exponential model and the biological half-lives were found to be 29.4 and 41.1 days, respectively.     

Monitoring of the impact of Prestige oil spill on Mytilus galloprovincialis from Galician coast

In November 2002, the Prestige oil tanker was wrecked in front of Galician coast (NW of Spain), spilling near 63,000 tons of heavy oil until February 2003. Contamination produced was very extensive (70% of Galician beaches were reached by the oil) but heterogeneous, alternating intensely affected zones with neighbour locations where the repercussion was minimal. The objective of this study was to monitor sea environment contamination caused by Prestige oil spill during an 11-month period (August 2003-June 2004, nine samplings) in two locations of Galician coast with different geographical properties (Lira and Ancoradoiro beaches), by means of chemical determination of total polycyclic aromatic hydrocarbons (TPAH) in seawater, and using as exposure biomarker TPAH content in mussel (Mytilus galloprovincialis) tissues, and as effect biomarker DNA damage in mussel gills, evaluated by the comet assay. In addition, recovery ability of the mussels was determined after a 7-day stay in the laboratory. TPAH contents in seawater were very high in the earliest samplings, but then they maintained below 200 ng L(-)(1), similar to reference seawater. However, TPAH levels in mussel tissues were more variable: they increased again from January 2004, probably due to the adverse meteorological conditions that turned over the sea bottom and dispersed the oil accumulated in sediments. In most samplings, these levels decreased during the recovery stage. DNA damage in oil-exposed mussels was significantly higher than in reference mussels, both before and after the recovery phase, but they did not differ to one another. Comet tail length was slightly reduced during the recovery stage, indicative of a certain DNA repair in exposed mussels. This study showed up the importance of monitoring sea contamination events during an extended time, not only in evaluating the presence of the contaminants in the environment but also in determining their bioaccumulation and their effect on the exposed organisms.     

Metal and metallothionein level in the heat-treated cytosol of gills of transplanted mussels Mytilus galloprovincialis Lmk

A stock of mussels Mytilus galloprovincialis was transplanted over 1 year to four sites in a semi-enclosed bay in Croatia which is under the influence of various sources of pollution. The positive correlation of metal (Cd, Zn, Cu, Mn, Fe) and metallothionein (MT) tissue contents based on the analysis of heat-treated cytosol of gills with shell mass, as an indicator of mussel age, indicated to accumulation of metals, as well as the increase of MTs with mussel age. The principal component analysis (PCA) revealed that 74% of total variance of obtained results could be explained through two principal components. The first principal component was highly correlated with MT, Cd and Zn indices (metal or MT content/shell mass), and the second one with Cu, Mn, and Fe indices, as well as the gill index. High correlation of MTs with Zn and Cd is consistent with their affinity for binding to MTs.     

背角无齿蚌(Anodonta woodiana)在五里湖中的重金属富集

通过对2003年2～3月采自五里湖水域代表样点的背角无齿蚌(Anodonta woodiana) 及用于对照的水样、底泥中重金属的测试,初步得出了Zn、Cu、Pb、As和Cd在这3类样本中的浓度范围。虽然水样中各重金属均未超过国家渔业水质标准（GB11607 89）,但底泥和背角无齿蚌的结果显示出Pb,特别是Zn、As的污染比较明显。背角无齿蚌作为生物指示物观察和评价该水域Zn、Cu、As、Pb、Cd的污染状况是可行的。结果反映该水域已不适用于渔业生产。作为食用贝类,其Pb、Cd,特别是Zn、As的残留有可能带来毒性影响。     

Lysosomal responses and metallothionein induction in the blue mussel Mytilus edulis from the south-west coast of Iceland

It has recently been emphasized that high levels of inorganic and organic micropollutants (particularly organometals, POPs and PAHs) may be present in coastal waters at high latitudes, stressing the need to evaluate the effects of contaminants on marine organisms from sub-arctic zones. With this aim, specimens of the blue mussel Mytilus edulis were sampled in polluted and reference areas along the south-west coast of Iceland in July 2004. Samples were collected from the intertidal zone at three sites in Reykjavik harbour which are differently exposed to contaminants, and at three reference coastal sites, two located along the Reykjanes Peninsula and the third one on the northern part of Hvalfjordur fiord. Lipofuscin content, neutral lipid accumulation and lysosomal enlargement were evaluated in digestive cells from cryostat sections of the mussel hepatopancreas, and quantified by automated image analysis. Metallothionein induction was also determined in the same tissue. Results indicate that mussels from the inner part of Reykjavik harbour, which is the most sheltered and most influenced by extensive shipping traffic, were the worst affected, with the highest values in neutral lipids, lipofuscin and lysosomal swelling. At the other two harbour sites, mussels exhibited lower values, similar to those observed in organisms collected in Hvalfjordur fiord and in bay of Osar. Mussels from Kuagerdi had the lowest values.     

The Amoco Cadiz oil spill. Distribution and evolution of hydrocarbon concentrations in seawater and marine sediments

The shipwreck of the Amoco Cadiz supertanker on the rocks of the Brittany Coast of France (March, 1978) was one of the most disastrous oil spills ever recorded. The crude oils discharged in the marine environment were light petroleums. The distribution and the evolution of the oil pollution in the marine environment was examined. Chemical studies were limited to an overall estimate of the oil content to assess the importance and the extent of the pollution at the surface, in the water column, and in the sediment. UV fluorescence spectroscopy was used to detect oil in the seawater samples and IR spectrophotometry was used to analyze the sediment samples. Hydrocarbon concentrations in seawater ranged from 0.5 μ/1 to more than 100 μg/1. The oil spill has affected a very large section of the western English Channel. The diffusion of the hydrocarbons into the water column was observed. The evolution of the seawater pollution was followed, and the half-life of hydrocarbons in subsurface water was found to be between 11 and 28 days in different areas. One month after the disaster, the marine sediments were contaminated in the areas reached by the drifting slicks. The highest accumulation of petroleum in the sediments was located in the sheltered coastal environments. The natural decontamination process was found to be related to the nature of the sediment and the energy level of the geographic zone.     

Assessment of arsenic and heavy metal concentrations in water and sediments of the Rio Grande at El Paso-Juarez metroplex region

The Rio Grande located along the US-Mexico border is affected by anthropogenic activities along its geographical course. Runoff and wind deposition of smelting residues may contribute to the pollution of the Rio Grande in the El Paso-Ciudad Juarez area. Few studies have addressed the presence or impacts of heavy metals or arsenic in this ecosystem. This study reports a survey of heavy metals (Cr, Cu, Cd, Ni, Pb, and Zn) and arsenic (As) in water and sediments of the Rio Grande collected from seven sites in the El Paso-Juarez region. Since water quality influences metal content in water, physical (temperature, flow and conductivity), and chemical (pH, dissolved oxygen, nitrates, alkalinity, and water hardness) parameters were measured at each site. Arsenic and heavy metal levels were determined using Inductively Couple Plasma (ICP) emission spectroscopy following EPA procedures. Zinc and lead were found as both total and dissolved metals in most of the samples, with concentrations of total recoverable metals reaching up to 105 and 70 microg/l, respectively. Most metals were found in sediment samples collected from four of seven sites. The highest Cu concentration (35 mg/l) was found at the American Dam site. Concentrations of metals found through this survey will be used as a reference for future studies in monitoring arsenic, heavy metals, and their impacts in the Rio Grande.     

Toxicities and tolerances of Cd, Cu, Pb and Zn in a primary producer (Isochrysis galbana) and in a primary consumer (Perna viridis)

Studies on toxicities and tolerances of cadmium (Cd), copper (Cu), lead (Pb) and zinc (Zn) in the brown alga Isochrysis galbana and in the green-lipped mussel Perna viridis were conducted by short-term bioassays using endpoints growth production and mortality, respectively. The 5-day EC(50) and 24-h LC(50) of these heavy metals were determined in the brown alga and mussel, respectively. The EC(50) values calculated for the alga were 0.74 mg/l for Cd, 0.91 mg/l for Cu, 1.40 mg/l for Pb and 0.60 mg/l for Zn. The LC(50) values for the mussels were 1.53 mg/l for Cd, 0.25 mg/l for Cu, 4.12 mg/l for Pb and 3.20 mg/l for Zn. These LC(50) values were within the concentration ranges as reported by other authors who used P. viridis as the test organism. Based on these EC(50) and LC(50) values, the alga was most sensitive to Zn, followed by Cd, Cu and Pb while the mussel was most sensitive to Cu, followed by Cd, Zn and Pb. Differences in the trophic levels, metal handling strategies, biology and ecology of the primary producer (brown alga) and the primary consumer (mussel) are believed to be the plausible causes for the different toxicities and tolerances of the metals studied.