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.     

Biomarker responses as indication of contaminant effects in blue mussel (Mytilus edulis) and female eelpout (Zoarces viviparus) from the southwestern Baltic Sea

During a field study performed in spring and autumn 2001 and 2002, blue mussels (Mytilus edulis) and female eelpout (Zoarces viviparus) were collected at three locations in the Wismar Bay (Baltic Sea), and several biomarkers of contaminant effects were analysed. Besides seasonal and inter-annual variations, biomarker signals were most pronounced at the location closest to Wismar Harbour (Wendorf) in both species. Lysosomal membrane stability (LMS) was lowest and acetylcholinesterase activity (AChE) was significantly reduced. Frequency of micronuclei (MN) was significantly higher (in blue mussels), indicating mutagenic effects. In eelpout elevated levels of DNA adducts, EROD induction and PAH-metabolites were measured. Metallothionein (MT), biomarker for trace metal exposure, showed a gradient only in spring. Organochlorine contaminant analyses (PCBs, DDTs) corresponded to the observed biomarker levels. The results obtained clearly demonstrate pollution effects in the southwestern Baltic Sea. Moreover, they show that a multibiomarker approach is also applicable in a brackish water environment.     

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.     

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.     

Hydrocarbons in water,sediment and mussels from the southern Baltic Sea

Samples of water, sediment and mussels (Mytilus edulis) from the southern Baltic Sea have been analysed for total hydrocarbons by fluorescence spectroscopy and capillary gas chromatography. The sediment and mussel samples have also been analysed for specific aliphatic and aromatic hydrocarbons by computerized capillary gas chromatography-mass spectrometry. The highest hydrocarbon concentrations in all samples occurred either inshore (particularly in Gdansk Bay) or in deep offshore basins where fine sediment accumulates.     

Selection of bioindicators for monitoring marine environmental quality in the Bay of Fundy,Atlantic Canada

Distribution of metals, PAH's and PCB's in lobsters, mussels, and sediments were used to assess marine environmental quality of the Bay of Fundy. This study demonstrates that the lobster (Homarus americanus) is a better bioindicator for monitoring contaminants in the marine environment and has a greater capacity for the uptake and accumulation of contaminants than the mussel (Mytilus edulis) and sediments. A definite pattern in the spatial distribution of lobster Cu, Cd, and Ag was evident. The distribution of organic contaminants for both mussels and lobsters in the Bay of Fundy lacked a spatial trend, and organic contaminants were undetectable in sediments from all sites. The Gulf Watch Programme, which monitors chemicals in mussels in the Bay of Fundy, did not indicate a problem with high levels of Cu, Cd, and Zn in the ecosystem. Analytes below the detection limit, such as in mussels and sediments, increase the difficulties of chemical analysis and detection for environmental monitoring. Deficiencies of mussels in monitoring the Bay of Fundy were discussed.     

Concentrations of organotin compounds in blue mussels from the wharves of Tokyo Bay

Organotin compounds (tributyltin (TBT), dibutyltin (DBT), monobutyltin (MBT), triphenyltin (TPT), diphenyltin (DPT), and monophenyltin (MPT)) were quantified in blue mussels, Mytilus edulis, in 1989 at 26 wharves along Tokyo Bay. Organotin compounds, other than DPT, were detected in all mussel samples (in case of DPT: 23 of 26). The concentration of DBT was highest among butyltin compounds and that of TPT was highest among phenyltin compounds. The results showed that relatively high concentrations of organotin compounds persist in blue mussels of Tokyo Bay, although the use of the paints containing these compounds has been limited for several years by regulations.     

Bioassay‐directed fractionation for analyzing estrogens in surface waters of the German Baltic Sea

A reversed‐phase fractionation method with subsequent biological and chemical analysis has been developed to estimate the contributions of the most potent estrogens to observed estrogenic effect potentials. Surface water samples were taken in the German Baltic Sea (Inner Wismar Bay and Darss Peninsula, sampling campaign July 2003) and were separated into seven individual fractions. Three fractions showed significant estrogenic activities and clear dose‐dependant responses were obtained in the yeast estrogen screen (YES). In the 2nd fractions liquid chromatographic‐electrospray‐tandem mass spectrometric (LC‐ESI‐MS‐MS) analyses showed the presence of bisphenol A (Inner Wismar Bay: 4.8 ng L^–1 and 6 ng L^–1; Darss Peninsula: 0.91 ng L^–1 and 1.7 ng L^–1) and ethinylestradiol (Inner Wismar Bay: 2.0 ng L^–1 and 6.0 ng L^–1; Darss Peninsula: < MDL and 1.7 ng L^–1), whereas estrogenic activities in the YES were only around 10% of the positive control E2. Although not identified prior in the total extract the natural hormones estradiol (Inner Wismar Bay: 0.13 ng L^–1 and 0.19 ng L^–1; Darss Peninsula: 0.12 ng L^–1 and 0.16 ng L^–1) and estriol (Inner Wismar Bay: < MDL and 0.33 ng L^–1; Darss Peninsula: < MDL) could be detected in the 3rd fractions, where high estrogenic potentials could be observed. The 4th fractions showed high responses as well and estrone were herein quantified with concentrations of 0.16 ng L^–1 and 0.18 ng L^–1 (Darss Peninsula) up to 0.37 ng L^–1 (Inner Wismar Bay). Measured and calculated estradiol equivalents for individual fractions correlated very well (R² = 0.78), when disregarding results of the 2nd fraction, where high deviations occurred.     

PCB-containing paint and plaster caused extreme PCB-concentrations in biota from the Sørfjord (Western Norway)--a case study

The Sørfjord (Western Norway) has a long history of industry and pollution, and has been monitored for several decades. The environmental monitoring has comprised analyses of different contaminants in sea water, mussel, fish, seaweed and sediments. Measurements of polychlorinated biphenyls (PCBs) in blue mussels (Mytilus edulis) some 17 years ago indicated a local source. In 2001 severe concentrations were measured in blue mussels and further investigations disclosed the primary source of PCBs in the Sørfjord. In 2002, extreme PCB-concentrations were found in cod (Gadus morhua) from the same area. However, no induction of EROD activity was detected, indicating the limitations of this assay as marker for PCB contamination. A future scientific challenge will be to understand the specific mechanisms and effects of such PCB accumulation in fish.     

Alkylbenzenes in mussels from South and South East Asian coasts as a molecular tool to assess sewage impact

Alkylbenzenes, molecular markers of sewage, were measured in 34 green mussels collected from India, Indonesia, Malaysia, Thailand, Cambodia, Vietnam, and the Philippines together with blue mussels collected from Tokyo Bay, Japan. Linear alkylbenzene (LAB) concentrations in South and South East Asian countries ranged from 10 to 1640 ng-∑LAB/g-dry tissue. In some populous cities, LAB concentrations were similar or higher than those found in northern Tokyo Bay which is heavily impacted by sewage effluents. I/E ratios (a ratio of internal to external isomers of LABs) in the South and South East Asian countries (1–3) were much lower than those in Tokyo Bay (3–8), indicating sewage discharged in the coastal zone is poorly treated (e.g., raw sewage and/or primary effluents). Alkylbenzenes with branched alkyl chains, tetrapropylene-based alkylbenzenes, were also detected in mussels from Indonesia and Philippines. This “tell-tale” sign indicates that poorly degradable detergents are still in use in this area, although they have long been phased-out in many industrialized countries.     

Lysosomal membrane stability and metallothionein content in Mytilus galloprovincialis (L.), as biomarkers. Combination with trace metal concentrations

The simultaneous study of two biomarkers, the lysosomal membrane stability (LMS) of the digestive gland and the metallothionein (MT) content of the digestive gland, the gills and the mantle/gonad complex of the mussel Mytilus galloprovincialis was examined in an enclosed estuarine system in June and October 2001. Mussels were collected along the Gulf of Thermaikos (northern Greece) from stations displaying a pollution gradient, while Olympiada in the Gulf of Strymonikos was used as a reference station. In addition, the heavy metal (Cd, Pb, Cu and Zn) content, using atomic absorption spectrophotometry (AAS), were evaluated in the digestive gland, the gills and the mantle/gonad complex of mussels collected from the same sites and seasons. LMS values were significantly greater, and the MT content of the studied tissues were significantly less in mussels collected from the reference station compared to mussels from more polluted stations located in the Gulf of Thermaikos. Significant correlation was observed between the MT content of the gills and the mantle/gonad complex with the LMS values.     

Characterizing the effect of heavy metal contamination on marine mussels using metabolomics

Marine mussels (Mytilus) are widely used as bioindicators to measure pollution in marine environments. In this study, (1)H NMR spectroscopy and multivariate statistical analyses were used to differentiate mussel groups from a heavy metal-polluted area (Onsan Bay) and a clean area (Dokdo area). Principal component analysis and orthogonal projection to latent structure-discriminant analysis revealed significant separation between extracts of mussels from Onsan Bay and from the Dokdo area. Organic osmolytes (betaine and taurine) and free amino acids (alanine, arginine, glutamine, phenylalanine, and threonine) were more highly accumulated in Onsan Bay mussels compared with Dokdo mussels. These results demonstrate that NMR-based metabolomics can be used as an efficient method for characterizing heavy metal contamination derived from polluted area compared to clean area and to identify metabolites related to environments that are contaminated with heavy metals.     

Assessing health of the Bay of Fundy--concepts and framework

A discussion of health and ecosystem health (EH) concepts and a conceptual framework for assessing health of the Bay of Fundy are presented. The framework includes:Concepts--What is health? What is EH and marine ecosystem health (MEH)? How does EH relate to other closely related concepts and principles i.e. environmental quality (especially marine or MEQ), ecosystem integrity, and ecosystem sustainability?Importance--Why is EH important, and what are the linkages to people i.e. human health?Approaches and techniques--How do we monitor and measure EH, and in that context, ecosystem or ecological change? i.e. What are the monitoring approaches and tools? What is an appropriate set of EH indicators and indices for the Bay of Fundy and Gulf of Maine? At the present time, can we make unequivocal statements about the status and trends in EH measures of the Bay of Fundy? Do we have adequate guidelines, objectives and standards for assessing environmental quality and EH?Connecting with management needs--Do we have adequate mechanisms in place to address MEH, marine environmental quality (MEQ), and environmental sustainability in the Bay of Fundy? What is the role of periodic marine assessments (state of the marine environment reports) in this activity? What kinds of new directions and new ecosystem science should be given priority? What kinds of other new activities should be pursued?For the Bay of Fundy Coastal Forum at the recent 5th BOFEP Bay of Fundy Science Workshop (May 2002), five core questions flowing from the health framework served to initiate and focus the discussions:Current state--What is the present health or condition of the Bay of Fundy?Changes--Are conditions improving or deteriorating?Indicators--What kinds of indicators do we consider most useful in trying to answer these questions?Existing resources--Are there adequate resources (e.g. institutional, financial, scientific, regulatory) to protect or restore the health of the Bay?Needs--What kinds of new information and approaches do we need to protect the health of the Bay?The background information and ideas of this paper were intended to assist discussion at the Fundy workshop, and to help identify the next steps, both individual and collective, for assessing the health of the Bay of Fundy.     

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

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

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.     

Metal pollution in the Saronikos Gulf

Sediment samples from the Saronikos Gulf and Elefsis Bay were analysed for weak acid extractable Zn, Fe and Cu, as well as for organic carbon. The main sources of pollution (fertilizers, factory, sewage outfall, Piraeus Port and the heavily industrialized part of Elefsis Bay) showed levels that were higher than background by a ratio of 1000 for Cu, 3500 for Fe, 700 for Zn and 10 for organic carbon. The concentrations fell rapidly to background at a distance of 5 km and the affected area was estimated at 40 km².     

Integrated use of biomarkers (superoxide dismutase, catalase and lipid peroxidation) in mussels Mytilus galloprovincialis for assessing heavy metals' pollution in coastal areas from the Saronikos Gulf of Greece

Mussels are used as sentinel organisms and bioindicators to evaluate the toxic effects of chemical pollutants in marine organisms, especially heavy metals, representing an important tool for biomonitoring environmental pollution in coastal areas. Antioxidant defence enzymes play an important role in cellular antioxidant defence systems and protect from oxidative damage by reactive oxygen species (ROS). Indigenous mussels Mytilus galloprovincialis of the Saronikos Gulf of Greece were used for monitoring heavy metal pollution in three polluted sites in the area and in one unpolluted site. Seasonal variations of the activity of antioxidant defence enzymes, superoxide dismutase (SOD) and catalase (CAT), as well as lipid peroxidation (LP) were measured as biomarkers in a period of three years in relation to concentrations of trace metals in their gills and mantle and compared to mussels from an unpolluted sampling site. SOD activity increased at least 2 fold at the polluted sites when compared to the control site (the high activity was recorded in the spring time). CAT activity was increased 2-3 times at the polluted sites, with high activity in the winter and spring time, compared to the control site. LP concentration was twice higher at the polluted sites, following the same seasonal pattern. Trace metals contents in mussels collected at polluted sites were 3-4 fold higher compared to the control site and showed moderate variations along the months, with a winter maximum followed by a summer pre-spawning minimum matching the seasonal trends of temperature and salinity. Our results showed that metal pollution in the Elefsis Bay (the most polluted coastal area) causes relatively medium levels of oxidative stress in tissues of mussels due to cellular oxy-radical generation. This study, which is the first in the area, showed that seasonal variations of the activity of antioxidant defence enzymes and LP concentrations in mussels can be used as potential biomarkers of toxicity for long-term monitoring in marine coastal ecosystems.     

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.     

Stable carbon and nitrogen isotope signatures indicate recovery of marine biota from sewage pollution at Moa Point,New Zealand

Stable carbon and nitrogen isotopes have been used to assess sewage contamination of a sewage outfall, discharging milli-screened effluent into Moa Point Bay, New Zealand, and monitor the recovery of flora and fauna after the outfall's closure. An initial study characterising the extent of the discharge and the effects on seaweed (Ulva lactuca L.), blue mussels (Mytilus galloprovincialis) and limpets (Cellana denticulata) from the area, showed effects of the sewage discharge on flora and fauna were localised within in the bay. The immediate area surrounding the discharge area was found to contain limited biodiversity, with an abundance of Ulva lactuca, a bright green lettuce-like seaweed, typically found in areas with high nutrient input, limpets and small blue mussels. The nitrogen isotopic signature (delta15N) is shown to be a good tracer of sewage pollution in seaweed and associated grazers (i.e. limpets) as a result of the increased contribution of urea and ammonia to seawater nitrogen derived from the effluent. The carbon isotopic signature (delta13C) is suggested as a more appropriate sewage tracer for mussels, which filter feed the effluent's particulate organic matter from the water. Lower carbon:nitrogen ratios were found in Ulva lactuca sampled from around the outfall region compared to uncontaminated control sites. However carbon:nitrogen ratios do not vary significantly amongst shellfish species.After closure, monitoring continued for 9 months and showed that the carbon and nitrogen isotopic signatures of algae (Ulva lactuca L.) returned to similar control site levels within 3 months. Limpet and blue mussels (Cellana denticulata and Mytilus galloprovincialis) showed slower recovery times than the Ulva lactuca, with detectable levels of the sewage-derived carbon and nitrogen remaining in the animal's tissue for up to 9 months.     

Mercury content of mussels from the St. Lawrence Estuary and northwestern Gulf of St. Lawrence, Canada

A first survey of the mercury content of intertidal mussels in the St. Lawrence Estuary and part of the Gulf of St. Lawrence shows means values ranging from 0.160 to 0.629 ppm (dry weight). Highest values (grand mean 0.430 ppm) were found in the upper part of the lower estuary, lowest values (grand mean 0.172 ppm) on the north shore of the estuary and Gulf, and intermediate values along the Gaspé Peninsula. The results indicate a decreasing gradient in mercury levels from the mouth of the Saguenay Fjord seaward.