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

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

Biomarker responses in flounder (Platichthys flesus) and mussel (Mytilus edulis) in the Klaipeda-Būtinge area (Baltic Sea)

During the EU project BEEP a battery of biomarkers was applied in flounder (Platichthys flesus) and the blue mussel (Mytilus edulis) collected at three locations off the Lithuanian coast (Baltic Sea) in June and September 2001 and 2002. The elevated biomarker responses in specimens sampled in September 2001 were apparently related to the extensive dredging activities in the Klaipeda port area and subsequent dumping of contaminated sediments. High concentrations of organic pollutants (organochlorines and PBDEs) were also measured in the tissues of both indicator species. In addition, response levels of genotoxicity, cytotoxicity, immunotoxicity as well as concentrations of PAH metabolites in the bile of flounder showed elevations in 2002 after an oil spill in the Būtinge oil terminal in November 2001. In flounder, biomarker measurements 10 months after the spill indicated recovery processes but in mussels a high level of genotoxicity could still be observed 22 months later. The present study illustrates the usefulness of the multi-biomarker approach in the detection of biological effects of pollution in this region of the Baltic Sea.     

Liver histopathology in Baltic flounder (Platichthys flesus) as indicator of biological effects of contaminants

Results are presented of a study on liver histopathology in Baltic flounder (Platichthys flesus) carried out in 2001 and 2002 in four coastal sampling areas of the Baltic Sea: Kv?d?fj?rden (Swedish east coast, reference area), Klaipeda-Butinge (Lithuanian coast), Gulf of Gdansk (Polish coast), and Wismar Bay (German coast) within the framework of the EU-funded BEEP project. Liver lesions were diagnosed and categorised using standardised methodologies and, for a spatial and temporal assessment of the prevalence and types of lesions detected, a scoring system was applied, involving the calculation of mean histopathology lesion scores. 83.0% of the 436 female flounder examined (size range: 20-43 cm total length, age range: 2-8 years) were affected by liver lesions, out of which 74.3% were assigned to the category of non-specific, 3.4% to the category of early toxicopathic non-neoplastic, 4.6% to the category of pre-neoplastic and 0.7% to the category of neoplastic lesions. Mean lesions scores were highest in the areas at the Lithuanian and Swedish coast and there is indication of an impact of the age structure of the flounder populations studied, the sampling season as well as of contaminant effects.     

Use of biliary PAH metabolites as a biomarker of pollution in fish from the Baltic Sea

During field campaigns of the BEEP project (Biological Effects of Environmental Pollution in Marine Coastal Ecosystems) in 2001-2002, metabolites of polycyclic aromatic hydrocarbons (PAHs) were determined in bile samples from three fish species, flounder (Platichthys flesus), perch (Perca fluviatilis) and eelpout (Zoarces viviparus), from four separate areas in the Baltic Sea. Two determination methods were applied: fixed wavelength fluorescence (FF) for pyrene-type metabolites and high-performance liquid chromatography with fluorescence detection (HPLC). There was a good correlation between the FF method and 1-OH pyrene determined by HPLC. Normalisation of the FF data for absorbance at 380 nm or bile protein concentrations greatly increased variance in one third and decreased it in two thirds of the cases and resulted in a loss of significant differences (protein normalisation) between the sampling stations, but normalisation of the HPLC data had little effect on the results. The biliary PAH metabolite content was usually higher in males than in females. In perch and eelpout the biliary PAH contents were at similar levels, whereas in flounder the levels were lower. The sampling areas arranged in decreasing order of biliary PAH contents were: Wismar Bay > Gulf of Gdansk > Lithuanian coast > Kvadofj?rden (reference area). It is concluded that FF with un-normalised data is a reliable and simple method for monitoring purposes and only one sex of a selected species should be used.     

Indices for the assessment of environmental pollution of the Baltic Sea coasts: integrated assessment of a multi-biomarker approach

Two mathematical methods to assess the "health status" of flounder (Platichthys flesus), eelpout (Zoarces viviparus) and blue mussel (Mytilus spp.) populations of the Baltic Sea were applied on selected biomarker data collected during the EU project "BEEP" (Biological Effects of Environmental Pollution on Marine Coastal Ecosystems). The Bioeffect Assessment Index (BAI) and the Integrated Biomarker Index (IBR) combine different biomarkers to single values, which can be used to describe the toxically-induced stress level of populations in different areas. Both indices determined here produced essentially similar results, which in most cases agreed with the known contamination levels in the different study areas. Advantages and limitations of index applications and interpretations are critically discussed. The use of indices provides comprehensive information about biological effects of pollution in marine organisms and may therefore serve as a useful tool for environmental management by ranking the pollution status of marine coastal areas.     

Evidence of chromosomal damage in common eiders (Somateria mollissima) from the Baltic Sea

Common eiders nesting in the Baltic Sea are exposed to generally high levels of contaminants including potentially genotoxic polycyclic aromatic hydrocarbons and organochlorines. Blood samples were collected from eiders at eight sites in the Baltic Sea and two sites in the Beaufort Sea. DNA content variation was estimated using the flow cytometric method, and subsequently utilized as a biomarker of genetic damage. We observed no significant differences in genetic damage among populations within either the Baltic or Beaufort Seas. However, eider populations from the Baltic Sea had significantly elevated estimates of genetic damage compared to populations from the Beaufort Sea.     

The BEEP project in the Baltic Sea: overview of results and outline for a regional biological effects monitoring strategy

Field studies in the framework of the EU funded BEEP project (Biological Effects of Environmental Pollution in Marine Coastal Ecosystems, 2001-2004) aimed at validating and intercalibrating a battery of biomarkers of contaminant exposure and effects in selected indicator species in the Mediterranean, the North Atlantic and the Baltic Seas. Major strategic goals of the BEEP project were the development of a sensitive and cost-efficient biological effects monitoring approach, delivery of information and advice to end-user groups, and the implementation of a network of biomarker researchers around Europe. Based on the main results obtained in the Baltic Sea component of the BEEP the present paper summarises and assesses the applicability of biomarkers for different regions and species in this sea area. Moreover, a general strategy and some practical considerations for the monitoring of biological effects in the Baltic Sea are outlined.     

Eelpout (Zoarces viviparus) in marine environmental monitoring

The implementation of the EU Marine Strategy Framework Directive necessitates the development of common criteria and methodological standards for marine environmental monitoring and assessment across Europe. Eelpout (Zoarces viviparus) is proposed as a key indicator organism in the Baltic and North Sea regions. This benthic fish species is widely used in ecotoxicological studies and as a bioindicator of local pollution due to its stationary behavior. Eelpout is included in the environmental monitoring program of several Baltic States, covering both chemical and biological effects measurements, and samples have been archived in environmental specimen banks for >15 years. A method for evaluating the frequency of larval aberrations has been suggested as a standardized assessment tool. The large scientific knowledge-base and considerable experience of long-term chemical and biological effects monitoring and specimen banking, make eelpout a suitable species for the assessment of Good Environmental Status in the Baltic and North Seas.     

Patterns and Levels of Organochlorines (DDTs, PCBs, non-ortho PCBs and PCDD/Fs) in Male Harbour Porpoises (Phocoena phocoena) from the Baltic Sea, the Kattegat-Skagerrak Seas and the West Coast of Norway

Patterns and levels of chlorinated aromatic contaminants (DDTs, PCBs, non-ortho PCBs and PCDD/Fs) in blubber tissue were compared among six sample groups of male harbour porpoises (Phocoena phocoena) from the Baltic Sea, the Kattegat-Skagerrak Seas and the west coast of Norway. A principal component and classification analysis showed that mature harbour porpoises from the Baltic had significantly different contaminant patterns than animals from the Kattegat-Skagerrak and Norway. ANOVAs showed that mature porpoises from the Baltic had higher levels of ΣPCB and several individual PCBs and PCDD/Fs than the Kattegat-Skagerrak and the Norwegian samples and higher ΣDDT than the Norwegian. A comparison between immature porpoises showed that Baltic animals had higher levels of ΣPCDD/F than the corresponding sample from the Kattegat-Skagerrak. The levels of ΣDDT, ΣPCB and Σnon-ortho PCB were significantly higher in animals collected during 1978–81 compared to animals collected in 1988–90 indicating a temporal decline of these organochlorines in the Kattegat-Skagerrak Seas. The contaminant levels recorded in the Baltic Sea are a serious cause for concern and could have management implications for the already threatened harbour porpoises in this area.     

The Elbe flood 2002--toxic effects of transported contaminants in flatfish and mussels of the Wadden Sea

Cellular changes in livers from flounder (Platichthys flesus L.) and digestive glands of blue mussels (Mytilus edulis) were studied to analyze whether the Elbe flood catastrophe in August 2002 had any deleterious effects on animal health. Flatfish and mussels were sampled at different locations in the Elbe estuary and the Wadden Sea after the flood wave had reached the German Bight. Internationally used core biomarkers (lysosomal membrane stability, biotransformation enzymes) were applied to assess the toxic effects of putatively increased pollution levels. In comparison to earlier data from long-term studies at the same stations, we found a significant impairment in the function of cell organelles (lysosomes) involved in the detoxification and elimination of pollutants in fish liver. Concentrations of relevant contaminants (organochlorines, polychlorinated biphenyls) were analyzed in parallel with cellular biomarkers, and conspicuously raised concentrations of insecticides metabolites were detected. Cell recovery and a clear reduction in contaminant concentrations were observed in fish livers five months after the flood at all sampling sites except the Helgolander Tiefe Rinne.     

Water column monitoring near oil installations in the North Sea 2001-2004

Fisheries have been vital to coastal communities around the North Sea for centuries, but this semi-enclosed sea also receives large amounts of waste. It is therefore important to monitor and control inputs of contaminants into the North Sea. Inputs of effluents from offshore oil and gas production platforms (produced water) in the Norwegian sector have been monitored through an integrated chemical and biological effects programme since 2001. The programme has used caged Atlantic cod and blue mussels. PAH tissue residues in blue mussels and PAH bile metabolites in cod have confirmed exposure to effluents, but there was variation between years. Results for a range of biological effects methods reflected exposure gradients and indicated that exposure levels were low and caused minor environmental impact at the deployment locations. There is a need to develop methods that are sufficiently sensitive to components in produced water at levels found in marine ecosystems.     

Protective effect of phospholipid hydroperoxide glutathione peroxidase (PHGPx) against lipid peroxidation in mussels Perna perna exposed to different metals

Levels of antioxidant defenses and lipid peroxidation were evaluated in mussels exposed to lead (200 mg/l), iron (500 microg/l), cadmium (200 microg/l) and copper (40 microg/l), for 12, 24, 72 and 120 h. Glutathione S-transferase (GST) activity was unchanged with all treatments. Catalase (CAT) increased after 120 h of exposure to all metals. Mussels exposed to Cd for 12 h, and to Cu and Fe for 120 h had increased lipid peroxidation, which might be associated to decreased levels of reduced glutathione (GSH) and glutathione peroxidase (GPx) activity. Pb exposure caused GSH depletion after 12 h and increased GPx activity after 120 h. Negative correlations were observed between the enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) and malonaldehyde (MDA) levels after Fe and Cu exposure, indicating a protective role of PHGPx against lipid peroxidation, and suggesting the use of this enzyme as a new potential biomarker of toxicity associated with contaminant exposure in mussels.     

Chlorinated compounds and polybrominated diphenyl ethers (PBDEs) in mussels (Mytilus galloprovincialis) collected from Apulia Region coasts

This project was carried out to assess the levels and spatial distribution of organochlorine compounds in the coastal marine environment, using mussels as bioindicators to evaluate the coastal water quality. Levels of polychlorobiphenils (PCB), chlorinated pesticides (DDT isomers, HCH isomers, Aldrin, Dieldrin, alfa-Endosulfan, Hexachlorobenzene, Pentachlorobenzene) and polybrominated diphenyl ethers (PBDEs) were determined in tissues from mussels (Mytilus galloprovincialis) collected along the Apulia Region coasts (Mediterranean Sea). Results indicate that contamination by organochlorine compounds is higher in mussels sampled in the Ionian Sea than in those from the Adriatic Sea, with PCB levels up to seven times higher in mussels from Ionian than from the Adriatic Sea. Although PCB levels were above the maximum values indicated by both European Community (EC) and National regulation in several sample sites, the PCB concentrations were particularly high in some stations, suggesting that these locations require a much specific attention. Conversely, results on the mussel contamination by PBDEs highlight their ubiquitous environmental distribution, and underline the need to establish the maximum level for these compounds in foodstuff, according to European Regulations.     

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.     

Izmit Bay (Turkey) ecosystem after Marmara earthquake and subsequent refinery fire: the long-term data.

As a part of Marmara Sea, Izmit Bay (Turkey) has been one of the most polluted sites in the region for the last 25 years. On 17 August 1999, a powerful earthquake along the North Anatolian Fault struck the eastern part of the Marmara region including Izmit Bay. The earthquake destroyed many coastal cities. The Bay was also affected by the quake and subsequent fire in the refinery situated on the north-eastern coast of the Bay. Oceanographic characteristics and polycyclic aromatic hydrocarbon (PAH) levels of Izmit Bay (Marmara Sea) have been investigated to find out the degree of contamination. Seawater samples were collected at nine stations of the Bay in April and September 1999 and the results were compared with those obtained in the previous years (1984 and 1994). Monitoring data are presented for plant nutrients (nitrate + nitrate, ortho-phosphate and silicate), dissolved oxygen and chlorophyll a. Surface sediments and mussels, Mytilus galloprovincialis, have been analysed for total PAH (T-PAH) contents in April and September 1999 (before and after the Marmara Earthquake) for the samples collected from eight coastal stations of the Bay. Biomarker (Lysosomal stability and feeding rate) studies at three different sites of the Bay have also been performed to investigate the effect of pollution on mussels. Nitrate + nitrite levels in the upper layer of the eastern part of the Bay increased significantly compared to those measured before the earthquake. Of the samples analysed, the highest o-phosphate concentrations were found in September 1999 in the bottom waters of the Bay. The concentration of chlorophyll a reached its minimum value of the last 15 years. Dissolved oxygen decreased dramatically from 1984 to 1999. Total PAH concentrations measured in April 1999 at both offshore and coastal sites of the Bay were more or less the same (2 micrograms l-1). The subsequent fire after the earthquake caused an increase in the total PAH levels in water column, in sediment and in mussels. Seawater total PAH concentrations ranged between 3.5 and 11 micrograms l-1 at open coast stations and 5-17.5 micrograms l-1 at coastal stations in September 1999. A 2- to 3-fold increase in sediment PAH concentrations (200-5220 mg kg-1 dry weight) was detected after the earthquake. This increase was much more significant in the sediments located around the refinery. More contaminated mussels were detected around the refinery area (110-170 mg kg-1 dry weight). Overall, sediment and mussel PAH concentrations in Izmit Bay are much higher than those found in the other marine systems. In general, the feeding rate and the neutral red retention times of the mussels decreased in some sites of the Bay after the earthquake, but no direct correlation could be detected between the body burden of mussels and biomarkers or between the two biomarker techniques.     

Mutagenicity of Baltic seawater and the relation to certain chemical and microbiological parameters.

The presented work investigates the mutagenicity of seawater with regard to its distribution along the German Baltic Sea coastline. Further on in this paper the relationship between mutagenic activity and certain chemical, microbiological and physical parameters is analysed. Water samples were drawn from eight places between Eckernf?rde and Ribnitz-Damgarten. Seawater was concentrated using XAD-2/7 resin. Extracts of up to 3-l per plate were tested with the Salmonella mutagenesis assay employing the strains TA 98 and TA 100 with and without addition of S9-mixture for metabolic activation. Samples from Weissenhaus, Travemünde and Wismar demonstrated a mutagenic activity with the strain TA 98. Tested filter sediment was not mutagenic. The sum of six polycyclic aromatic hydrocarbons (PAH) concentrated by XAD resin and obtained by high performance liquid chromatography (HPLC) were between 0.03 and 34.11 ng/l seawater. No correlation of measured mutagenicity and chemical or physical parameters could be established. The sampling place Wismar with one mutagenic sample had the highest number of colony forming units of Escherichia coli.     

Developmental disorders in larvae of eelpout (Zoarces viviparus) from German and Swedish Baltic coastal waters

Pregnant eelpout were collected for several years at German and Swedish Baltic coastal stations with different impact of pollutants and the prevalence of fry abnormalities compared between sites. Mortality of apparently normal developed larvae was observed with high variability in both German and Swedish sites. Malformation of larvae was a prominent disorder at all German stations with a prevalence ranging from about 50% up to a maximum of 90% in almost all analysed samples. At the Swedish stations malformation rates were significantly lower (range: 0-6%). Growth retardation of fry was clearly more prominent in eelpout from German coastal waters as well. Taken together the field data suggests that impaired larval development was associated with the state of environmental pollution at the sampling locations.     

The use of selected genotoxicity assays in green-lipped mussels (Perna viridis): a validation study in Hong Kong coastal waters

In Hong Kong, discharges of domestic and industrial wastewaters and disposal of contaminated muds into coastal waters have resulted in high levels of persistent organic contaminants in the water column, biota and bottom sediments. Although the acute toxicity of effluents has been tested by government regulators using mortality-based bioassays, the sublethal effects of many of the pollutants, including environmental carcinogens, have not been well studied. The objective of the present work was to examine the efficiency and efficacy of using: (1) DNA strand breaks (comet assay and alkaline agarose gel electrophoresis assay); (2) oxidative DNA damage (levels of 8-hydroxy-2-deoxyguanosine in cellular DNA) and (3) micronuclei (MN) formation as early warning toxicological indicators of potential environmental carcinogen exposure. Green-lipped mussels (Perna viridis) were transplanted to study sites and retrieved after 4, 8, 12, 16 and 30 days. The bioaccumulation of organic pollutants, including the polycyclic aromatic hydrocarbons (PAHs), organochlorine pesticides (OC pesticides) and polychlorinated biphenyls (PCBs) was observed, and compared with biomarker assays. Some significant dose- and time-dependent inductions of the genotoxic biomarkers, including 8-OHdG, DNA strand breaks and micronuclei were observed. In particular, several very strong (r>0.9) and significantly positive relationships were found at all sites during days 0-12. In addition, significant strong relationships between certain organic contaminants (carcinogenic PAHs and 4 common OC pesticides) and micronuclei were detected at the polluted sites, Tsim Sha Tsui and Sai Wan Ho. The results suggest that the use of micronuclei as a biomarker is potentially effective, especially at the more polluted sites. Nonetheless, further research is required to validate their effectiveness under varying field conditions, especially to elucidate responses under lower contaminant loads, interactions between the environmental and physiological factors affecting organisms in differing coastal environments, and changes in biotransformation and DNA repair enzymes in test organisms.     

(137)Cs baseline levels in the Mediterranean and Black Sea: a cross-basin survey of the CIESM Mediterranean Mussel Watch programme

The common mussel Mytilus galloprovincialis was selected as unique biomonitor species to implement a regional monitoring programme, the CIESM Mediterranean Mussel Watch (MMW), in the Mediterranean and Black Seas. As of today, and upon standardization of the methodological approach, the MMW Network has been able to quantify (137)Cs levels in mussels from 60 coastal stations and to produce the first distribution map of this artificial radionuclide at the scale of the entire Mediterranean and Black Seas. While measured (137)Cs levels were found to be very low (usually < 1 Bq kg(-1) wet wt) (137)Cs activity concentrations in the Black Sea and North Aegean Sea were up to two orders of magnitude higher than those in the western Mediterranean Basin. Such effects, far from representing a threat to human populations or the environment, reflect a persistent signature of the Chernobyl fallout in this area.