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.     

Monitoring biological effects of pollution in the Baltic Sea: neglected--but still wanted?

In the Baltic Sea, studies regarding biological effects of contaminants are relatively few, partly due to political and economical reasons, specific hydrographic characteristics, and a strong eutrophication-targeted research focus during the past three decades. The development of a monitoring strategy concerning biological effects and its implementation into environmental monitoring programmes in the Baltic Sea is lagging behind the progress currently taking place in most of western and southern Europe. The pan-European project BEEP (Biological Effects of Environmental Pollution on Marine Coastal Ecosystems, 2001-2004) included the Baltic Sea as one of the target areas for the evaluation of a suite of biological effects indicators in European coastal waters. The main aims of the BEEP project are described and how the expected outcome for the Baltic Sea could provide the needed "baseline" information and expertise for a biological effect monitoring and contribute to harmonise environmental monitoring programmes within the EU.     

Accidental spills at sea - Risk, impact, mitigation and the need for co-ordinated post-incident monitoring

A fully integrated and effective response to an oil or chemical spill at sea must include a well planned and executed post-incident assessment of environmental contamination and damage. While salvage, rescue and clean-up operations are generally well considered, including reviews and exercises, the expertise, resources, networks and logistical planning required to achieve prompt and effective post-spill impact assessment and monitoring are not generally well established.The arrangement and co-ordination of post-incident monitoring and impact assessment need to consider sampling design, biological effects, chemical analysis and collection/interpretation of expert local knowledge. This paper discusses the risks, impacts and mitigation options associated with accidental spills and considers the importance of pre-considered impact assessment and monitoring programmes in the wider response cycle. The PREMIAM (Pollution Response in Emergencies: Marine Impact Assessment and Monitoring; www.premiam.org) project is considered as an example of an improved approach to the planning, co-ordination and conduct of post-incident monitoring.     

Evaluating legacy contaminants and emerging chemicals in marine environments using adverse outcome pathways and biological effects-directed analysis

Natural and synthetic chemicals are essential to our daily lives, food supplies, health care, industries and safe sanitation. At the same time protecting marine ecosystems and seafood resources from the adverse effects of chemical contaminants remains an important issue. Since the 1970s, monitoring of persistent, bioaccumulative and toxic (PBT) chemicals using analytical chemistry has provided important spatial and temporal trend data in three important contexts; relating to human health protection from seafood contamination, addressing threats to marine top predators and finally providing essential evidence to better protect the biodiversity of commercial and non-commercial marine species. A number of regional conventions have led to controls on certain PBT chemicals over several years (termed ‘legacy contaminants’; e.g. cadmium, lindane, polycyclic aromatic hydrocarbons [PAHs] and polychlorinated biphenyls [PCBs]). Analytical chemistry plays a key role in evaluating to what extent such regulatory steps have been effective in leading to reduced emissions of these legacy contaminants into marine environments. In parallel, the application of biomarkers (e.g. DNA adducts, CYP1A-EROD, vitellogenin) and bioassays integrated with analytical chemistry has strengthened the evidence base to support an ecosystem approach to manage marine pollution problems. In recent years, however, the increased sensitivity of analytical chemistry, toxicity alerts and wider environmental awareness has led to a focus on emerging chemical contaminants (defined as chemicals that have been detected in the environment, but which are currently not included in regulatory monitoring programmes and whose fate and biological impacts are poorly understood). It is also known that natural chemicals (e.g. algal biotoxins) may also pose a threat to marine species and seafood quality. Hence complex mixtures of legacy contaminants, emerging chemicals and natural biotoxins in marine ecosystems represent important scientific, economic and health challenges. In order to meet these challenges and pursue cost-effective scientific approaches that can provide evidence necessary to support policy needs (e.g. the European Marine Strategy Framework Directive), it is widely recognised that there is a need to (i) provide marine exposure assessments for priority contaminants using a range of validated models, passive samplers and biomarkers; (ii) integrate chemical monitoring data with biological effects data across spatial and temporal scales (including quality controls); and (iii) strengthen the evidence base to understand the relationship between exposure to complex chemical mixtures, biological and ecological impacts through integrated approaches and molecular data (e.g. genomics, proteomics and metabolomics). Additionally, we support the widely held view that (iv) that rather than increasing the analytical chemistry monitoring of large number of emerging contaminants, it will be important to target analytical chemistry towards key groups of chemicals of concern using effects-directed analysis. It is also important to evaluate to what extent existing biomarkers and bioassays can address various classes of emerging chemicals using the adverse outcome pathway (AOP) approach now being developed by the Organization for Economic Cooperation and Development (OECD) with respect to human toxicology and ecotoxicology.     

The application of histo-cytopathological biomarkers in marine pollution monitoring: a review

During the past two decades, a variety of histopathological alterations in fish and bivalves have been developed and used as biomarkers in pollution monitoring. Some of these have been successfully adopted in major national monitoring programmes, while others, although show promise, are still in the experimental stage. This paper critically reviews the scientific basis, cause and effect relationship, reliability, advantages and limitations of 14 histo-cytopathological biomarkers. The usefulness and practical application of each biomarker have been evaluated against a number of objective criteria including: ecological relevance, sensitivity, specificity, dose-response relationship, confounding factors, technical difficulties and cost-effectiveness.     

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.     

Biomarkers and environmental risk assessment: guiding principles from the human health field

Although the potential use of biomarkers within environmental risk assessment (ERA) has long been recognised their routine use is less advanced compared with clinical human health risk assessment, where a number of familiar biomarkers (such as blood pressure and serum cholesterol) are in common usage. We have examined how biomarkers are incorporated into human health risk assessment and have identified several 'required elements'. These include identification of the (clinical) assessment endpoint at the outset, rational selection of the biomarker(s) (the measurement endpoint), biomarker 'validation' (e.g. QA/QC) and biomarker 'qualification' (evidence linking the measurement and assessment endpoints). We discuss these elements in detail and propose that their adoption will facilitate the routine use of biomarkers in environmental risk assessment. Furthermore, our analysis highlights the need for cooperation between those working with biomarkers within human and environmental risk assessment to exchange best practice between common disciplines for mutual advantage.     

Molecular and biochemical biomarkers responses in the mussel Mytilus edulis collected from Southern Brazil coast

Marine ecosystems are typically subjected to a variety of stressors containing complex xenobiotics mixtures. This study aims to evaluate the responses of molecular and biochemical biomarkers in the mussel Mytilus edulis providing data for environmental monitoring programs development in Southern Brazil. Mussels were collected at a polluted site, near Patos Lagoon outfall, and at a control site. Gills, muscle and mantle samples were used for biomarker determinations. Mussels collected at the polluted site significantly increased superoxide dismutase (SOD) and glutathione S-transferase (GST) activities and decreased catalase (CAT) activity. Moreover, an increase in sod1, gstπ and hsp70 mRNA expression was observed. Overall, biochemical and molecular biomarkers responses were observed, but these responses varied depending on the analyzed tissue. These results indicate possible contaminants effects on organisms and the need for effective environmental monitoring programs in this ecosystem.     

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.     

Review of oil and HNS accidental spills in Europe: identifying major environmental monitoring gaps and drawing priorities

The European Atlantic area has been the scene of a number of extensive shipping incidents with immediate and potential long-term impacts to marine ecosystems. The occurrence of accidental spills at sea requires an effective response that must include a well executed monitoring programme to assess the environmental contamination and damage of the affected marine habitats. Despite a number of conventions and protocols developed by international and national authorities that focused on the preparedness and response to oil and HNS spills, much remains to be done, particularly in relation to the effectiveness of the environmental monitoring programmes implemented after oil and HNS spills. Hence, the present study reviews the status of the environmental monitoring programmes established following the major spill incidents over the last years in European waters, aiming at identifying the key monitoring gaps and drawing priorities for an effective environmental monitoring of accidental spills.     

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.     

Environmental effects of the use of oil-based drilling muds in the North Sea

The environmental effects of oil-based drilling mud cuttings have been evaluated using all the data available from monitoring around North Sea platforms. Beneath the platforms making extensive use of oil-based drilling muds the natural sediment is buried by cuttings and the hydrocarbon concentration 250 m from the platforms may be 1000 times background, but the concentration gradient is very steep and background levels are usually reached 2000–3000 m from the platform.The extent of the biological effects appear to be greater from the use of oil-based muds than from water-based muds. Beyond the area of physical smothering the effects of oil-based mud cuttings may be due to organic enrichment or to the toxicity of the aromatic fraction of the oil. Despite the large scale of inputs, in all the fields studied the major deleterious biological effects occurred within 500 m of the platform. Surrounding the area of major impact was a transition zone in which subtle biological effects could be detected as community parameters returned to normal, generally within 400–1000 m.The shape and extent of this zone varied and was largely determined by the current regime and scale of the drilling operation. Elevated hydrocarbon concentrations were detected beyond the areas of biological effects.     

Induction, adaptation and recovery of biological responses: implications for environmental monitoring

A wide range of biological responses have been used to identify exposure to contaminants, monitor spatial and temporal changes in contamination levels, provide early warning of environmental deterioration and indicate occurrences of adverse ecological consequences. To be useful in environmental monitoring, a biological response must reflect the environmental stress over time in a quantitative way. We here argue that the time required for initial induction, maximum induction, adaptation and recovery of these stress responses must first be fully understood and considered before they can be used in environmental monitoring, or else erroneous conclusions (both false-negative and false-positive) may be drawn when interpreting results. In this study, data on initial induction, maximum induction, adaptation and recovery of stress responses at various biological hierarchies (i.e., molecular, biochemical, physiological, behavioral, cytological, population and community responses) upon exposure to environmentally relevant levels of contaminants (i.e., metals, oil, polycyclic aromatic hydrocarbons (PAHs), organochlorines, organophosphates, endocrine disruptors) were extracted from 922 papers in the biomarker literature and analyzed. Statistical analyses showed that: (a) many stress responses may decline with time after induction (i.e., adaptation), even if the level of stress remains constant; (b) times for maximum induction and recovery of biochemical responses are positively related; (c) there is no evidence to support the general belief that time for induction of responses at a lower biological hierarchy (i.e., molecular responses and biochemical responses) is shorter than that at higher hierarchy (i.e., physiological, cytological and behavioral responses), although longer recovery time is found for population and community responses; (d) there are significant differences in times required for induction and adaptation of biological responses caused by different types of contaminants; (e) times required for initial and maximum induction of physiological responses in fish are significantly longer than those in crustaceans; and (f) there is a paucity of data on adaptation and recovery of responses, especially those at population and community levels. The above analyses highlight: (1) the limitations and possible erroneous conclusions in the present use of biomarkers in biomonitoring programs, (2) the importance of understanding the details of temporal changes of biological responses before employing them in environmental management, and (3) the suitability of using specific animal groups as bioindicator species.     

The "bioeffect assessment index" (BAI). A concept for the quantification of effects of marine pollution by an integrated biomarker approach

The "bioeffect assessment index" (BAI) is based on the integration of several pathological endpoints measured in the liver of European flounder (Platichthys flesus (L.)) during a long term study of biological effects of pollution in the German Bight. The BAI represents a modification of the "health assessment index" since it includes solely validated biomarkers reflecting toxically induced alterations at different levels of biological organisation in order to quantify the effects of environmental pollution. The concept of the BAI is based on the observation of progressive deleterious effects from early responses to late effects. Specific "key events" were detected, representing progressive stages of functional deterioration. The biomarkers selected from a whole battery of cellular markers for the BAI calculation reflect deleterious effects of various classes of contaminants such as heavy metals, organochlorines, pesticides, PAHs, and therefore reflect general toxicity in an integrative manner. Selected biomarkers were: lysosomal perturbations (reduced membrane stability), storage disorders (lipid accumulation) as early markers for toxic effects of liver cells, and the size of macrophage aggregates and their acid phosphatase activity. The latter two markers are indicative for the modulation of non-specific immune response which represents longer time scale responses after chronic exposure.     

Cardiac and Lysosomal Responses to Periodic Copper in the Mussel Perna viridis (Bivalvia: Mytilidae)

Biological monitoring of metals in marine waters often employs tissue residue analyses from bivalves. Chemical residues in tissues do not elucidate biological injury, however, whereas biomarkers, do afford sensitive stress indices. Physiological and cytological responses were evaluated in laboratory experiments from the mussel Perna viridis in order to assess the species’ suitability for pollution monitoring in the tropics. Periodic copper exposure increased tissue residues but failed to perturb cardiac activity. Copper destabilized haemocyte lysosomal membranes in a exposure-dependent relationship showing a sensitive, rapid, indication of stress. Cytological biomarkers from P. viridis have potential as indices of pollutant exposure and could facilitate effective biomonitoring of water quality throughout South-east Asia.     

Seasonal baseline levels of physiological and biochemical parameters in polar cod (Boreogadus saida): Implications for environmental monitoring

Seasonality of biomarker baseline levels were studied in polar cod (Boreogadus saida), caught in Kongsfjorden, Svalbard, in April, July, September and December, 2006-2007. Physiological parameters (condition factor, gonado- and hepato-somatic indexes, energy reserves, potential metabolic activity and antifreeze activity) in polar cod were used to interpret the seasonality of potential biomarkers. The highest levels of ethoxyresorufin-O-deethylase (EROD) activity occurred concomitantly with the highest potential metabolic activity in July due to e.g. intense feeding. During pre-spawning, EROD showed significant inhibition and gender differences. Hence, its potential use in environmental monitoring should imply gender differentiation at least during this period. Glutathione S-transferase and catalase activities were stable from April to September, but changed in December suggesting a link to low biological activity. Knowledge of the biomarker baseline levels and their seasonal trends in polar cod is essential for a trustworthy interpretation of forthcoming toxicity data and environmental monitoring in the Arctic.     

Measuring and monitoring persistent organic pollutants in the context of risk assessment

Due to growing concerns regarding persistent organic pollutants (POPs) in the environment, extensive studies and monitoring programs have been carried out in the last two decades to determine their concentrations in water, sediment, and more recently, in biota. An extensive review and analysis of the existing literature shows that whilst the vast majority of these efforts either attempt to compare (a) spatial changes (to identify “hot spots”), or (b) temporal changes to detect deterioration/improvement occurring in the environment, most studies could not provide sufficient statistical power to estimate concentrations of POPs in the environment and detect spatial and temporal changes. Despite various national POPs standards having been established, there has been a surprising paucity of emphasis in establishing accurate threshold concentrations that indicate potential significant threats to ecosystems and public health. Although most monitoring programs attempt to check compliance through reference to certain “environmental quality objectives”, it should be pointed out that many of these established standards are typically associated with a large degree of uncertainty and rely on a large number of assumptions, some of which may be arbitrary. Non-compliance should trigger concern, so that the problem can be tracked down and rectified, but non-compliance must not be interpreted in a simplistic and mechanical way. Contaminants occurring in the physical environment may not necessarily be biologically available, and even when they are bioavailable, they may not necessarily elicit adverse biological effects at the individual or population levels. As such, we here argue that routine monitoring and reporting of abiotic and biotic POPs concentrations could be of limited use, unless such data can be related directly to the assessment of public health and ecological risks. Risk can be inferred from the ratio of predicted environmental concentration (PEC) and the predicted no effect concentration (PNEC). Currently, the paucity of data does not allow accurate estimation of PNEC, and future endeavors should therefore, be devoted to determine the threshold concentrations of POPs that can cause undesirable biological effects on sensitive receivers and important biological components in the receiving environment (e.g. keystone species, populations with high energy flow values, etc.), to enable derivation of PNECs based on solid scientific evidence and reduce uncertainty. Using the threshold body burden of POPs required to elicit damages of lysosomal integrity in the green mussel (Perna virvidis) as an example, we illustrate how measurement of POPs in body tissue could be used in predicting environmental risk in a meaningful way.     

Measuring and monitoring persistent organic pollutants in the context of risk assessment

Due to growing concerns regarding persistent organic pollutants (POPs) in the environment, extensive studies and monitoring programs have been carried out in the last two decades to determine their concentrations in water, sediment, and more recently, in biota. An extensive review and analysis of the existing literature shows that whilst the vast majority of these efforts either attempt to compare (a) spatial changes (to identify "hot spots"), or (b) temporal changes to detect deterioration/improvement occurring in the environment, most studies could not provide sufficient statistical power to estimate concentrations of POPs in the environment and detect spatial and temporal changes. Despite various national POPs standards having been established, there has been a surprising paucity of emphasis in establishing accurate threshold concentrations that indicate potential significant threats to ecosystems and public health. Although most monitoring programs attempt to check compliance through reference to certain "environmental quality objectives", it should be pointed out that many of these established standards are typically associated with a large degree of uncertainty and rely on a large number of assumptions, some of which may be arbitrary. Non-compliance should trigger concern, so that the problem can be tracked down and rectified, but non-compliance must not be interpreted in a simplistic and mechanical way. Contaminants occurring in the physical environment may not necessarily be biologically available, and even when they are bioavailable, they may not necessarily elicit adverse biological effects at the individual or population levels. As such, we here argue that routine monitoring and reporting of abiotic and biotic POPs concentrations could be of limited use, unless such data can be related directly to the assessment of public health and ecological risks. Risk can be inferred from the ratio of predicted environmental concentration (PEC) and the predicted no effect concentration (PNEC). Currently, the paucity of data does not allow accurate estimation of PNEC, and future endeavors should therefore, be devoted to determine the threshold concentrations of POPs that can cause undesirable biological effects on sensitive receivers and important biological components in the receiving environment (e.g. keystone species, populations with high energy flow values, etc.), to enable derivation of PNECs based on solid scientific evidence and reduce uncertainty. Using the threshold body burden of POPs required to elicit damages of lysosomal integrity in the green mussel (Perna virvidis) as an example, we illustrate how measurement of POPs in body tissue could be used in predicting environmental risk in a meaningful way.     

The detection and recognition of underground nuclear explosions

This paper reports on a joint meeting of the Royal Astronomical Society's Joint Association for Geophysics and VERTIC (the Verification Technology Information Centre) held in London in 1992. The topics presented focused on the detection and recognition of underground nuclear explosions. The objective of the meeting was to emphasize the multi-methodological approach that is important in verifying compliance with test-ban treaties. An overview of seismological monitoring was followed by a discussion of the technical and scientific aspects of a global seismic monitoring network, and in particular of the 1991 experiment to test the large-scale international exchange of seismic data between recording stations and data centres world-wide. The current capabilities of satellite remote-sensing were presented, and their use explained in terms of both the provision of information for monitoring the development of foreign nuclear testing programmes and also for providing sufficient information for the evaluation of treaty compliance. A review of radio-isotope sampling showed how the isotopic signature of both air and ground based sampling programmes can be diagnostic of the nuclear source. Finally, previously classified research on the ionospheric effects of underground nuclear explosions was presented, the generated acoustic waves disturbing the ionosphere and producing detectable changes in the reflection of radio and radar signals which have potential as a monitoring technique.