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.     

The use of biomarkers in environmental monitoring programmes

The monitoring of biological effects has recently become an integral component of environmental monitoring programmes as a supplement to the commonly used contaminant monitoring. Over the years, many biomarkers have been developed that are claimed to be efficient at providing an early warning of deleterious effects on biological systems and for estimating biological effects due to contaminants. Although biomarkers are potentially useful, they have a number of important limitations. In this paper, we examine some of the key assumptions behind the theory and practice of use of biomarkers, and propose a scheme, which may facilitate decisions by environmental managers as to how and when to use biomarkers in their monitoring programmes.     

A multibiomarker approach in barramundi (Lates calcarifer) to measure exposure to contaminants in estuaries of tropical North Queensland

A suite of biomarkers were measured in barramundi (Lates calcarifer) from five North Queensland estuaries along a perceived pollution gradient. The biomarkers selected were 7-ethoxyresorufin-O-deethylase (EROD), cytochrome P450, fluorescent aromatic compounds (FACs), DNA integrity, RNA:DNA ratio, cholinesterase activity (ChE), condition factor and hepatosomatic index. The resulting database was subjected to uni- and multi-variate analyses in order to assess the most suitable biomarkers to assess pollution in North Queensland estuaries and to classify the environmental quality of the sites. Principal components analysis (PCA) on the biochemical markers revealed that EROD, EROD/P450, DNA damage and to a lesser extent ChE and FACs were found to be responsive to contaminants in the environment while cytochrome P450, condition factor and the hepatosomatic index were found to be less responsive biomarkers. This study has demonstrated the utility of applying a multibiomarker approach in conjunction with traditional analysis of contaminants in providing valuable information in environmental risk assessment.     

Oxidative stress, genotoxicity and histopathology biomarker responses in mullet (Mugil cephalus) and sea bass (Dicentrarchus labrax) liver from Bizerte Lagoon (Tunisia)

The aim of the study was to evaluate the impact of environmental contaminants on oxidative stress, genotoxic and histopathologic biomarkers in liver of mullet (Mugil cephalus) and sea bass (Dicentrarchus labrax) collected from a polluted coastal lagoon (Bizerte Lagoon) in comparison to a reference site (the Mediterranean Sea). Antioxidant enzyme activities were lower in fish from the polluted site compared with fish from the reference site, suggesting deficiency of the antioxidant system to compensate for oxidative stress. DNA damage was higher in both fish species from the contaminated site indicating genotoxic effects. The liver histopathological analysis revealed alterations in fish from Bizerte Lagoon. Hepatocytes from both fish species featured extensive lipid-type vacuolation and membrane disruption. Results suggest that the selected biomarkers in both fish species are useful for the assessment of pollution impacts in coastal environments influenced by multiple pollution sources.     

Induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis

Biomarkers are generally applied to detect pollution in environmental monitoring. Such biological responses should accurately reflect the stress over time in a quantitative manner. As such, the initial and maximum responses induced by stress, as well as adaptation and recovery of these biomarkers, need to be fully understood or else erroneous false-negative or false-positive may be arrived. However, most of the biomarker studies only provided information on initially induced responses under different concentrations of toxicants, while biological adaptation and recovery were poorly known. In this study, the time required for induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis upon exposure to benzo[a]pyrene was investigated over a period of 62 days. Maximum induction occurred on day 6 when lysosomal integrity was significantly reduced by 51%, and no further change or adaptation was detected thereafter. When mussels were depurated in clean seawater after 18 days of exposure to benzo[a]pyrene, a gradual recovery was observed, with lysosomal integrity returning to its background level and showing a complete recovery after 20 days of depuration. Lysosomal integrity was significantly correlated with the body burden concentrations of benzo[a]pyrene and condition index of the mussels. The relatively fast induction (6 days) and recovery (20 days) without apparent adaptation suggested that lysosomal integrity in P. viridis can serve as a good biomarker in biomonitoring, as its response is not likely to generate both false-negative and false-positive results.     

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.     

Cytological and Physiological Biomarker Responses from Green Mussels, Perna viridis (L.) Transplanted to Contaminated Sites in Hong Kong Coastal Waters

Biological monitoring of marine contaminants often involves the analysis of metals accumulated in mussel tissues. Critiques of this type of monitoring suggest that although relatively good indications of ambient metal concentrations are determined, actual biological harm is not elucidated. Biomarkers, techniques originally developed in medicine, have recently been employed in an attempt to ascertain the health of marine mussels. Biomarkers have been used with good effect in temperate marine pollution monitoring programmes. The use of biomarkers in the toxicological evaluation of tropical and subtropical waters is, however, generally lacking, which is somewhat surprising, as considerable industrialisation and environmental degradation is taking place in the Pacific-rim countries of Southeast Asia. In the present study, a suite of cytological and physiological biomarkers have been measured from mussels (Perna viridis: Mytilidae) transplanted within the coastal waters of Hong Kong. Mussels from contaminated sites, had labile lysosomal membranes, whereas animals from a cleaner reference site had more stable membranes. These cytological responses have been related to metal-induced stress and are simple, cost-effective markers of pollution. Cardiac activity from mussels transplanted to polluted sites was generally similar to that of individuals from the reference site, although a mild tachycardia was associated with one site. The cause of the tachycardia and the use of heart rate in mussels as a biomarker is discussed. Integrated physiological responses to stress, condition indices, revealed mussels from a clean reference site had higher indices than animals from contaminated sites. The relative sensitivities of the cytological and physiological biomarkers deployed in this study and their application to pollution monitoring is discussed.     

Toxicological impact assessment of heavy metal contamination on macrobenthic communities in southern coastal sediments of Korea

In the heavily industrialized Masan Bay of southern coast, Korea, the potential harmful effects of heavy metals (Cd, Co, Cu, Ni, Pb, Sn, Zn, and Hg) were evaluated in terms of the pollution load index (PLI) and ecological risk assessment index (ERI) methods, and the results obtained were considered alongside the health of the macrobenthic fauna communities. The results revealed that the bay sediments, especially in the inner bay and the outfall area of a sewage treatment plant, are exposed to moderate to serious levels of metal pollution. Hg and Cd contributed the most to the potential toxicity response indices in sediments recently deposited in the bay. The potential ecological risk assessment of heavy metals in the bay was highlighted by the use of the benthic biological pollution index (BPI), suggesting that the ERI is a useful toxicity response index, which can quantify the overall ecological risk level to a target environment.     

Induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis

Biomarkers are generally applied to detect pollution in environmental monitoring. Such biological responses should accurately reflect the stress over time in a quantitative manner. As such, the initial and maximum responses induced by stress, as well as adaptation and recovery of these biomarkers, need to be fully understood or else erroneous false-negative or false-positive may be arrived. However, most of the biomarker studies only provided information on initially induced responses under different concentrations of toxicants, while biological adaptation and recovery were poorly known. In this study, the time required for induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis upon exposure to benzo[a]pyrene was investigated over a period of 62 days. Maximum induction occurred on day 6 when lysosomal integrity was significantly reduced by 51%, and no further change or adaptation was detected thereafter. When mussels were depurated in clean seawater after 18 days of exposure to benzo[a]pyrene, a gradual recovery was observed, with lysosomal integrity returning to its background level and showing a complete recovery after 20 days of depuration. Lysosomal integrity was significantly correlated with the body burden concentrations of benzo[a]pyrene and condition index of the mussels. The relatively fast induction (6 days) and recovery (20 days) without apparent adaptation suggested that lysosomal integrity in P. viridis can serve as a good biomarker in biomonitoring, as its response is not likely to generate both false-negative and false-positive results.     

Application of biomarkers for assessing the biological impact of dredged materials in the Mediterranean: the relationship between antioxidant responses and susceptibility to oxidative stress in the red mullet (Mullus barbatus)

In the period 1997-2000, approximately 1,800,000 m3 of material dredged from the Port of Leghorn was discharged into a sea dumping site located 14 miles from the coast. The red mullet (Mullus barbatus) was used as a bioindicator species for monitoring the biological impact of these discharges on a geographical and temporal scale. Organisms were sampled over three years (1998-2000) at different stations and several biomarkers, both of exposure and effect, were analyzed. Bioavailability of specific classes of pollutants was evaluated by analyzing levels of metallothioneins, the activity of cytochrome P450 1A (CYPIA) and of glutathione S-transferases. Among biomarkers of effect, special attention was paid to the balance between prooxidant challenge and antioxidant defenses, and to the appearance of damage caused by oxidative stress. The analyses of the main components of the antioxidant system included superoxide dismutase, catalase, glutathione peroxidases, glutathione reductase, glyoxalase I and II, and total glutathione. These data were integrated with the measurement of total oxyradical scavenging capacity (TOSC) as an indication of the overall biological resistance to toxicity of different forms of oxyradicals (peroxyl radicals, hydroxyl radicals and peroxynitrite). Results indicated a biological impact in organisms sampled near the disposal site; the impact was particularly evident during 1999 and mainly related to organic chemicals such as PAH. Exposure to these pollutants also caused variations in the levels and activity of several antioxidants. The analysis of TOSC, however, revealed that the overall capacity of specific tissues of organisms to absorb various oxidants was not seriously compromised when challenged with increased prooxidant pressures. Variations of single antioxidants were useful in revealing early warning "biological responses", while integration with TOSC analyses indicated if such changes also reflect a more integrated and functional "biological effect" with possible consequences at the organisms level. The red mullet appears to be a useful sentinel species for a biomarker approach to monitoring impact caused by dredged materials.     

Multivariate discriminant analysis distinguishes metal- from non metal-related biomarker responses in the clam Chamaelea gallina

Molecular biomarkers are among the most sensitive and earliest responses to pollutants. However, lack of detailed knowledge on variability of responses and their possible seasonal variation limit their use. In addition, the seasonality of biological processes modulates the response of organisms to pollutant stressors. Using multivariate statistics, we have studied the influence of environmental and biological factors on the response of a battery of molecular biomarkers in the clam Chamaelea gallina collected along the South-Spanish littoral. Multivariate discriminant analysis clearly distinguished biomarker response between clean and polluted areas, using heavy metals as indicator of pollution. Such differences disappeared when the dataset was normalised for metal content, thus indicating that pollution was the main significant cause of the changes observed between clean and polluted sites. In conclusion, this work shows that, when applying a complete biomarker panel, multivariate statistical tools can be used to discern pollutant- from non pollutant-related responses.     

Metal exposure and biological responses in resident and transplanted blue mussels (Mytilus edulis) from the Scheldt estuary

The Western Scheldt river and estuary is known to be highly polluted as it receives industrial, agricultural and domestic effluents from one of the most populated and industrialised zones in Europe. Aquatic organisms are exposed to pollutants, specifically metals that are present in different environmental phases, e.g. dissolved, suspended material or sediment phases. The objective of this study was to study the relationship that exists between environmental metal levels, the degree of metal uptake by aquatic organisms with the concomitant biological responses. For this purpose the bivalve mollusk, Mytilus edulis, was selected as bioaccumulation indicator organism. Environmental (water and sediment) and mussel samples were collected during the late winter (March 2000) from sampling sites in the Scheldt estuary. Sites were selected to represent a salinity gradient from the mouth of the estuary to the furthest distribution area of mussels upstream in the system. Together with standard water quality parameters (e.g. salinity, dissolved oxygen, dissolved organic carbon, etc.) concentrations of twelve metals were analysed in the water (dissolved and suspended matter) and sediments. Levels of these metals were also measured in the soft tissue of M. edulis, together with concomitant biomarker responses in resident mussel populations at each site. The biomarkers that were included in this study were condition index, scope for growth, survival in air, cell membrane stability, DNA damage, and metallothioneins. Data were subjected to multivariate statistical analysis. The physico-chemical parameters and metals in the environmental samples clustered the sites to reflect the distribution based on the salinity gradient. Bioaccumulation results revealed increased metal uptake along a pollution gradient with the highest metal bioaccumulation occurring at the upstream most sites and therefore closest to the in the industrial activities. However, the biomarker responses clustered the sites in a manner that reflected the influence of combination of internal exposure (bioaccumulation) and external exposure (physico-chemical conditions). These differences in biomarker responses clearly demonstrated were attributed to abiotic factors other than metal pollution alone e.g. localized short-term increases in increased suspended sediment concentrations and decreased dissolved oxygen concentrations.     

Metal exposure and biological responses in resident and transplanted blue mussels (Mytilus edulis) from the Scheldt estuary

The Western Scheldt river and estuary is known to be highly polluted as it receives industrial, agricultural and domestic effluents from one of the most populated and industrialised zones in Europe. Aquatic organisms are exposed to pollutants, specifically metals that are present in different environmental phases, e.g. dissolved, suspended material or sediment phases. The objective of this study was to study the relationship that exists between environmental metal levels, the degree of metal uptake by aquatic organisms with the concomitant biological responses. For this purpose the bivalve mollusk, Mytilus edulis, was selected as bioaccumulation indicator organism. Environmental (water and sediment) and mussel samples were collected during the late winter (March 2000) from sampling sites in the Scheldt estuary. Sites were selected to represent a salinity gradient from the mouth of the estuary to the furthest distribution area of mussels upstream in the system. Together with standard water quality parameters (e.g. salinity, dissolved oxygen, dissolved organic carbon, etc.) concentrations of twelve metals were analysed in the water (dissolved and suspended matter) and sediments. Levels of these metals were also measured in the soft tissue of M. edulis, together with concomitant biomarker responses in resident mussel populations at each site. The biomarkers that were included in this study were condition index, scope for growth, survival in air, cell membrane stability, DNA damage, and metallothioneins. Data were subjected to multivariate statistical analysis. The physico-chemical parameters and metals in the environmental samples clustered the sites to reflect the distribution based on the salinity gradient. Bioaccumulation results revealed increased metal uptake along a pollution gradient with the highest metal bioaccumulation occurring at the upstream most sites and therefore closest to the in the industrial activities. However, the biomarker responses clustered the sites in a manner that reflected the influence of combination of internal exposure (bioaccumulation) and external exposure (physico-chemical conditions). These differences in biomarker responses clearly demonstrated were attributed to abiotic factors other than metal pollution alone e.g. localized short-term increases in increased suspended sediment concentrations and decreased dissolved oxygen concentrations.     

Metal exposure and biological responses in resident and transplanted blue mussels (Mytilus edulis) from the Scheldt estuary

The Western Scheldt river and estuary is known to be highly polluted as it receives industrial, agricultural and domestic effluents from one of the most populated and industrialised zones in Europe. Aquatic organisms are exposed to pollutants, specifically metals that are present in different environmental phases, e.g. dissolved, suspended material or sediment phases. The objective of this study was to study the relationship that exists between environmental metal levels, the degree of metal uptake by aquatic organisms with the concomitant biological responses. For this purpose the bivalve mollusk, Mytilus edulis, was selected as bioaccumulation indicator organism. Environmental (water and sediment) and mussel samples were collected during the late winter (March 2000) from sampling sites in the Scheldt estuary. Sites were selected to represent a salinity gradient from the mouth of the estuary to the furthest distribution area of mussels upstream in the system. Together with standard water quality parameters (e.g. salinity, dissolved oxygen, dissolved organic carbon, etc.) concentrations of twelve metals were analysed in the water (dissolved and suspended matter) and sediments. Levels of these metals were also measured in the soft tissue of M. edulis, together with concomitant biomarker responses in resident mussel populations at each site. The biomarkers that were included in this study were condition index, scope for growth, survival in air, cell membrane stability, DNA damage, and metallothioneins. Data were subjected to multivariate statistical analysis. The physico-chemical parameters and metals in the environmental samples clustered the sites to reflect the distribution based on the salinity gradient. Bioaccumulation results revealed increased metal uptake along a pollution gradient with the highest metal bioaccumulation occurring at the upstream most sites and therefore closest to the in the industrial activities. However, the biomarker responses clustered the sites in a manner that reflected the influence of combination of internal exposure (bioaccumulation) and external exposure (physico-chemical conditions). These differences in biomarker responses clearly demonstrated were attributed to abiotic factors other than metal pollution alone e.g. localized short-term increases in increased suspended sediment concentrations and decreased dissolved oxygen concentrations.     

Monitoring biomarkers in fish (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf after the Prestige oil spill

Hepatic biomarker responses were measured in two demersal fish species (Lepidorhombus boscii and Callionymus lyra) from the northern Iberian shelf associated with the massive Prestige oil spill (POS), five months after the accident. The biomarkers selected were 7-ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT), and DNA integrity. Interspecies differences and spatial variations in biomarker responses were observed along the shelf. GST, GR and CAT activities were significantly elevated in L. boscii in the most oil impacted area (Finisterre) and positively correlated (p<0.05) with POS tar aggregate densities. The lack of previous data from the area together with the existence of chronic background pollution of the shelf implies that the observed biomarker responses cannot be solely attributed to the petroleum hydrocarbon components of the spilled oil. This first biological effect assessment showed that L. boscii is a potentially suitable target species to be used in future biomonitoring programmes along the northern Iberian shelf.     

Development of a fish-based index to assess the ecological quality of transitional waters: The case of French estuaries

The Water Framework Directive requires the assessment of the ecological status of transitional waters considering the fish component. An original methodology, based on a pressure-impact approach, was established to develop a multimetric fish-based index to characterize the ecological quality of French estuaries. An index of contamination, based on the chemical pollution affecting aquatic systems, was used as a proxy of anthropogenic pressure. The fish metric selection was based on their response to disturbances tested via statistical models (generalized linear models) taking into account sampling strategy and estuarine features. Four metrics, for which discriminating responses to level of pressure were demonstrated, were retained to constitute the estuarine multimetric fish index. This new tool appeared particularly relevant to detect the contamination effects on fish communities in estuaries. It could help managers to take decisions in order to maintain or reach the good status required by the Water Framework Directive for 2015.     

Assessing streams in Germany with benthic invertebrates: Development of a multimetric invertebrate based assessment system

The main aim of the study was to develop a multimetric assessment system for macroinvertebrate communities of small and mid-sized streams in Germany in accordance with the Water Framework Directive of the European Union. The system should be applicable by water authorities, capable of distinguishing between different impacts, and specifically adapted to all types of small and mid-sized rivers in Germany. An assessment system has been developed consisting of two modules based on the established indices for saprobic degradation and acidity, and a third module covering general degradation. For the third module a new multimetric index (MMI) was developed and tested using preclassifications for stream hydromorphological degradation, land use and general impairment as impact measures. More information on sampling method, season and the type of dominating human impairment allowed to further analyse the index' capabilities and sensitivity. It performed well in all seasons and detected all types of impairment except acidity. The usefulness of the impact measures used in this study is discussed with regard to the development of a multimetric index. The comparison of the new index with other indices showed that it is especially sensitive in detecting macroinvertebrate community responses to hydromorphological degradation and pollution such as the German AQEM index, while IBI 12 and Renkonen's similarity more reflect general community shifts caused by any type of disturbance. The new assessment system will be proposed for future use by state agencies for nationwide monitoring in the context of the Water Framework Directive implementation.     

Biochemical biomarkers in adult female perch (Perca fluviatilis) in a chronically polluted gradient in the Stockholm recipient (Sweden)

A battery of biochemical biomarkers and the SigmaPCB concentration in adult female perch (Perca fluviatilis) verified an aquatic pollution gradient with the city of Stockholm (Sweden) as a point source of anthropogenic substances. The investigation included both an upstream gradient, 46 km westwards through Lake M?laren, and a downstream gradient, 84 km eastwards through the Stockholm archipelago. Besides the main gradient from Stockholm, there were strong indications of pollution coming from the Baltic Sea. The results indicated a severe pollution situation in central Stockholm, with poor health status of the perch, characterised by increased specific EROD activity in the liver, increased liver EROD somatic index, decreased AChE activity in the muscle, increased amount of DNA adducts in the liver, and a high concentration of biliary 1-pyrenol. In addition, laboratory exposure to common EROD inducers elicited an abnormal response, suggestive of chronic intoxication.     

Cell and tissue biomarkers in mussel, and histopathology in hake and anchovy from Bay of Biscay after the Prestige oil spill (Monitoring Campaign 2003)

In order to assess the biological effects of the Prestige oil spill (POS), mussels (Mytilus galloprovincialis), European hake (Merluccius merluccius) and European anchovy (Engraulis encrasicolus) were sampled between April and September 2003 in various geographical areas of Bay of Biscay: Galicia, Central Cantabrian and East Cantabrian. In mussels, several cell and tissue biomarkers were measured: peroxisome proliferation as induction of acyl-CoA oxidase (AOX) activity, lysosomal responses as changes in the structure (lysosomal volume density, V(V(L)), surface-to-volume ratio, S/V(L), and numerical density, N(V(L))) and in membrane stability (labilization period, LP), cell type replacement as relative proportion of basophilic cells (volume density of basophilic cells, V(V(BAS))) in digestive gland epithelium, and changes in the morphology of digestive alveoli as mean luminal radius to mean epithelial thickness (MLR/MET). Additionally, flesh condition index (FCI) and gonad index (GI) were measured as supporting parameters. In hake and anchovy, liver histopathology was examined to determine the prevalence of parasites, melanomacrophage centers, non-specific lesions (inflammatory changes, atrophy, necrosis, apoptosis), early non-neoplastic toxicopathic lesions (i.e., hepatocellular nuclear polymorphism), foci of cellular alteration, benign and malignant neoplasms. In mussels, AOX induction was noticeable in April except in Galicia. LP values were low in all the geographical areas studied, indicating disturbed health, especially in Galicia. Alike, lysosomal enlargement was observed in most stations as shown by the extremely low S/V(L) values. V(V(BAS)) and MLR/MET values were markedly high. Overall, employed biomarkers detected exposure to toxic chemicals and disturbed health in mussels from Bay of Biscay, with impact decreasing from April to September. Although hepatocellular nuclear polymorphism and nematode parasitization in fish liver were remarkably prominent in some areas, they cannot be hitherto related to POS, since we lack historical data to determine whether the prevalences found were normal or significantly raised after POS.     

Bioassessment of water quality and defining of critical levels of multicontamination for aquatic life

The existing approaches to and methods for bioassessment of water quality are critically analyzed. The significance of the methodological approach under the concept of ecosystem “health” as the most integral numerical method for assessing pollution effects. Comparative analysis is given to the significance of criteria based on the state of individuals, populations, and communities. The results of practical testing of the methods developed for biological assessment of water quality for some water bodies are presented. Critical levels are established based on the construction of dose-effect dependences. The basic principles for limiting surface water pollution are formulated.