Immunotoxicity and oxidative stress in the Arctic scallop Chlamys islandica: effects of acute oil exposure.

With increasing oil exploration in Arctic regions, the risk of an accidental oil spill into the environment is inevitably elevated. As a result, concerns have been raised over the potential impact of oil exposure on Arctic organisms. This study assessed the effects of an acute oil exposure (mimicking an accidental spill) on the immune function and oxidative stress status of the Arctic scallop Chlamys islandica. Scallops were exposed to the water accommodated fraction of crude oil over 21 d (maximum SigmaPAH 163 microg l(-1)) and immune endpoints and oxidative stress parameters were measured. Mortalities were recorded during the exposure and reductions in immunocompetence were observed, with significant impairment of phagocytosis and cell membrane stability. Scallops were also subjected to oxidative stress, with a significant reduction in glutathione levels and induction of lipid peroxidation. After the acute oil exposure had subsided, no recovery of immune function was observed indicating potential for prolonged sublethal effects.

The influence of seasonality on biomarker responses in Mytilus edulis.

The utility of some biomarkers in environmental monitoring may be limited due to the lack of knowledge that exists on how they respond to extrinsic abiotic and intrinsic biotic factors. During the present study we investigated the seasonal responses of three biomarkers, Neutral Red Retention, clearance/filtration rate and heart rate in the common blue mussel Mytilus edulis located in the Exe Estuary, UK during September 2006-September 2007. During the current study, a significant decrease in feeding rate was observed in mussels during June, July and August 2007, coinciding with the period following spawning when the mussels lay down nutrient reserves. Heart rate also increased between April and September 2007 and corresponded with times when mussels were spawning and laying down nutrient reserves. By integrating the individual biomarker responses into a Biomarker Response Index (BRI) we were able to identify times of the year when environmental impact was highest and hence when the timing of monitoring programmes using biomarkers should be carried out. For many years the lack of knowledge of normal physiological ranges of biomarkers has impeded their applied use, however by integrating biomarker responses into the BRI and creating an index of health, we have shown that we can limit the natural variability of individual responses; and thus we are better able to make informed judgements on the overall health status of these populations of mussels.

Effects of the model PAH phenanthrene on immune function and oxidative stress in the haemolymph of the temperate scallop Pecten maximus.

Phenanthrene, a major component of crude oil, is one of the most abundant PAHs in aquatic ecosystems, and is readily bioavailable and toxic to a range of marine invertebrates. Within bivalves, the haemolymph acts as a transfer medium for these pollutants and their metabolic products, leaving haemocytes susceptible to deleterious effects. Using a suite of biological endpoints, this study determined the sublethal (7-d exposure to 50, 100 and 200microgL(-1)) effects of phenanthrene on several oxidative stress and immunological parameters in the haemolymph of the commercially-important scallop Pecten maximus. Phenanthrene exposure (200microgL(-1)) resulted in immune modulation with significant reductions in cell membrane stability (P<0.05) and phagocytosis (P<0.05), and a significant increase in the number of total haemocytes (P<0.05). Oxidative stress was also observed with a significant decrease in total glutathione (P<0.05) and significantly increased levels of lipid peroxidation in the haemolymph (P<0.05). Changes in the cellular and biochemical endpoints observed in this study illustrate their potential use in assessing the subtle effects of contaminant exposure. Whilst previous reports have suggested a link between free radical generation and immune suppression in vertebrates, this is the first instance where oxidative stress and immune function have been measured together in the haemolymph of a bivalve mollusc, demonstrating a possible link between PAH-induced oxidative stress and the subsequent inhibition in haemocyte immune function.

Functional immune response in Pecten maximus: combined effects of a pathogen-associated molecular pattern and PAH exposure.

Pathogen-associated molecular patterns (PAMPs) enable recognition of structures present in microorganisms such as lipopolysaccharides (LPS). LPS are an essential constituent of the outer membrane of Gram-negative bacteria, stimulating the innate immune system of invertebrates. Here, LPS from Escherichia coli (055:B5) were used to investigate the functional immune response of Pecten maximus after stimulation with a PAMP and to determine the combined effect of a phenanthrene exposure and LPS challenge. Organisms were exposed to 200 mug l(-1) phenanthrene and after 7 d were injected with either physiological saline (injection controls) or LPS solution, and returned to their respective exposure tanks. Haemolymph was sampled from the scallops 48 h post-injection and immune function was assessed using a combination of cellular biological responses. The LPS challenge significantly altered the immune response in P. maximus with increased cell counts and phagocytic activity. An immunosuppressive effect of phenanthrene was also observed in this study; however, exposure to phenanthrene did not significantly impair the organism's ability to respond to a PAMP challenge. The overall level of phagocytosis and cytotoxic capability following the LPS challenge was lower in phenanthrene exposed scallops and may have consequences for disease resistance in this commercially-exploited species.

Linking genotoxic responses with cytotoxic and behavioural or physiological consequences: differential sensitivity of echinoderms (Asterias rubens) and marine molluscs (Mytilus edulis).

Integrated laboratory studies addressed multiple biomarker responses in the sea star (Asterias rubens) and the blue mussel (Mytilus edulis) exposed to a range of concentrations of direct and indirect acting genotoxins: methyl methane sulfonate (MMS) and cyclophosphamide (CP; an environmentally relevant anti-cancer pharmaceutical), respectively, in order to determine if the expressed genotoxicity has knock-on effects at the higher levels of biological organisation. The experimental design aimed to concurrently evaluate biomarkers of behavioural and physiological conditions (i.e. 'righting time' and 'clearance rate' for sea stars and mussels, respectively) in addition to cytotoxicity (neutral red retention assay), induction of micronuclei (Mn) and DNA strand breaks (as determined by the Comet assay). The protocol also included the determination of the maximum tolerated concentration (MTC), prior to genotoxic evaluation. The 3d MTC, as determined by the survival of the organisms, showed sea stars to be more sensitive than mussels to MMS (18 and 32 mg L(-1), respectively) and CP (56 and 180 mg L(-1), respectively). For both species and chemicals, cytotoxicity was not found to be significantly different compared to controls. Apart from the MMS exposure to sea stars (which showed 100% mortality at higher concentrations after 5d exposure), clear dose-response relationships were observed for both genotoxicity endpoints in each species. Following exposure to CP, good correlations were also found between the behavioural and physiological responses and genetic damage in each species (sea stars-MN vs. RT: R=0.73; Comet vs. RT: R=0.91; mussels-MN vs. CR: R=0.69; Comet vs. CR: R=0.72). This integrated approach, applying non-invasive assays to simultaneously determine the responses at different levels of biological organisation, indicates the potential value of behavioural and physiological measures in determining the toxicity of chemicals to marine organisms and highlights also the relevance of including adult echinoderms in environmental studies.

Application of biomarkers to assess the condition of European Marine Sites.

A series of European Marine Sites has been designated as Special Areas of Conservation (SAC) in England. The aim of this study was to develop a practical methodology to assess the condition of SACs by applying a suite of biomarkers. Biomarkers were applied to the blue mussel Mytilus edulis and the shore crab Carcinus maenas from the Fal and Helford SAC (Cornwall). Individual biomarkers provided useful diagnostic information on the activity of certain classes of contaminants and an integrated Biomarker Response Index (BRI) was used to achieve a more holistic understanding of the condition of the SAC. The BRI indicated that the general health of both organisms was impacted in the upper part of the SAC (Fal Estuary) which correlated well with known chemical hotspots and sources of contamination. The BRI allows a pragmatic way to prioritise SAC sites that may require further investigative studies.

Immune function in the Arctic Scallop, Chlamys islandica, following dispersed oil exposure.

With the current expansion of offshore oil activities in Arctic regions, there is an urgent need to establish the potential effects of oil-related compounds on Arctic organisms. As susceptibility to growth, disease and survival is determined partly by the condition of an organism's immune system, measurement of endpoints linked to the latter system provide important early warning signals of the sub-lethal effects of exposure to contaminants. This study assessed the impact of dispersed oil exposure on immune endpoints in the Arctic Scallop Chlamys islandica, using a combination of cellular and humoral biological responses. Laboratory exposures of C. islandica to sub-lethal dispersed oil concentrations (0.06 and 0.25 mg l(-1)) were conducted over 15 days, followed by a 7-day recovery period in clean, filtered seawater. Cellular endpoints were significantly altered following dispersed oil exposure: haemocyte counts (P<0.01) and protein levels (P<0.01) were significantly elevated, whilst cell membrane stability (P<0.001) and phagocytosis (P<0.01) demonstrated a significant reduction. Whilst these results indicate alteration in the immune endpoints measured, this appears to be reversible upon removal of the contaminant stress. However, the impact of long-term continuous exposure and high-level acute exposure to oil is still unknown, and may have consequences for disease resistance and hence survival.

Physiological responses of juvenile and adult shore crabs Carcinus maenas (Crustacea: Decapoda) to pyrene exposure.

This study measured aspects of the physiology of juvenile [(<35mm carapace width (CW)] and adult (>60mm CW) Carcinus maenas to test the hypothesis that these different life-history stages exhibit different sensitivities to environmental contamination. Newly-collected juveniles had significantly (P<0.05) lower immunocompetence (phagocytosis and cellular integrity), lower metabolic energy (haemolymph glucose) and increased scope for growth compared with adults. Seven day exposure to a sub-lethal concentration (200microgL(-1)) of pyrene significantly (P<0.01) reduced immunocompetence, elevated basal heart rate and decreased respiration (at rest) for juveniles but had no overall impact on adult crab physiology. Results confirm that physiological differences exist between juvenile and adult shore crabs, and cause juveniles to be more susceptible to the effects of pyrene exposure than adults. Such differences in sensitivity to contamination between life-cycle stages of the same species have to be taken into account during the risk assessment process.

Nutritional status of Carcinus maenas (Crustacea: Decapoda) influences susceptibility to contaminant exposure.

Using the shore crab Carcinus maenas as a model, this study tested the hypothesis that nutritional status influences susceptibility of adult crabs (>60mm carapace width (CW)) to environmental contamination. In the laboratory, crabs were either starved, given a restricted diet (fed on alternate days) or fully fed (fed each day). In addition, crabs under each feeding regime were exposed to a sublethal concentration (200microgl(-1)) of pyrene (PYR) as a model organic (PAH (polyaromatic hydrocarbon)) contaminant. Various physiological end points were measured after 7 and 14 days. Results indicated that adult shore crab physiology was relatively robust to short-term (7 days) nutritional changes as multivariate analysis (ANOSIM) showed no significant difference in shore crab physiological condition between control and pyrene-exposed crabs, irrespective of dietary feeding regime [Global R=0.018, P (%)=19.2]. After 14 days, however, starved crabs showed significant impacts to physiological condition (as revealed by multivariate analysis) [Global R=0.134, P (%)=0.1], [R=0.209, P (%)=0.1]; starved individuals had significantly lower antioxidant status (F(2,48)=5.35, P<0.01) compared to crabs under both types of feeding regime. Exposure to pyrene resulted in significantly elevated pyrene metabolite concentrations in the urine at 7 and 14 days compared with control individuals (P<0.001), validating contaminant bioavailability, and this was found for all dietary treatments. Also, exposed crabs had significantly increased protein levels (proteinuria) than controls (P<0.001) in their urine after 7 and 14 days, irrespective of dietary regime. After 7 days, pyrene-exposed crabs showed significantly increased antioxidant status (P<0.001) and cellular functioning (increased cellular viability and decreased phagocytosis) (P<0.001) compared to control crabs; however, after 14 days, antioxidant status (P<0.01) and cellular viability (P<0.001) were significantly decreased in pyrene-exposed compared to unexposed crabs. Results indicate that differences in nutritional status of adult crabs result in shore crabs being robust to short-term sublethal (7 days) pyrene exposure. Susceptibility to contaminant exposure, however, was measured after prolonged exposure (14 days) as indicated by reduced ability to combat oxidative stress. These results indicate that ecotoxicological studies need to take into account the nutritional state of the test organism to achieve the full assessment of contaminant impact. In addition, the results highlight that subtle seasonal biotic features of an organism can influence biomarker responses, and these need to be considered when interpreting field data and during the routine application of biological-effects tools in environmental monitoring.

Application of biomarkers for improving risk assessments of chemicals under the Water Framework Directive: a case study.

To answer the requirement of the European Commission's Water Framework Directive (WFD) for biological-effects endpoints to classify the ecological health of aquatic ecosystems, we propose the biomarker response index (BRI). The BRI, based on a suite of biomarkers at different levels of biological response at the individual level, provides an integrated relative measure of the general health status of coastal invertebrates. Using the BRI, the health of mussels (Mytilus edulis) from 10 estuaries classified by the Environment Agency of England and Wales under the WFD was compared. Eight sites were healthier than predicted and two showed a similar health status to that of the predicted point-source pollution risk classification. Results indicate that the BRI offers a potential measure of organism health that can be used in monitoring under the WFD as an additional aid to reduce uncertainty in defining risk classification and to provide better evidence of existing impact.

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.

Characterisation of esterases as potential biomarkers of pesticide exposure in the lugworm Arenicola marina (Annelida: Polychaeta).

Here, we identify and characterise cholinesterase (ChE) and carboxylesterase (CbE) activities in the body tissues of the sediment dwelling worm Arenicola marina. Exposure to the organophosphorus pesticide azamethiphos yielded an in vitro IC50 of 5 microg l(-1) for propionylcholinesterase (PChE). PChE was significantly inhibited in vivo after a 10 day exposure to 100 microg l(-1) azamethiphos, equivalent to the recommended aquatic application rate (ANOVA; F=2.75, P=0.033). To determine sensitivity to environmental conditions, A. marina were exposed for 10 days to field collected sediments. PChE activity was significantly lower in worms exposed to sediments from an estuary classified to be at high risk from point source pollution by the UK Environment Agency (ANOVA; F=15.33, P<0.001). Whilst causality cannot be directly attributed from these latter exposures, they provide an important illustration of the potential utility of esterase activity as a biomarker of environmental quality in this ecologically relevant sentinel species.

Biomarkers and integrated environmental risk assessment: are there more questions than answers?

The introduction of the European Commission's Water Framework Directive (WFD; 2000/60/EC) established a new era in environmental risk assessment. In addition to incorporating the compliance of chemical quality standards, the key objective of the WFD is the general protection of the aquatic environment in its entirety. This new approach emphasizes the need for an integrated environmental risk assessment and offers the potential for the incorporation of biological effects measures, including the use of biomarkers in this process. Biomarkers have been suggested as practical tools for environmental management for a number of decades, but their inclusion has not been universally accepted because of a number of unanswered questions regarding sensitivity, practicality, and reproducibility. With this in mind, this paper addresses these potential questions and shows how, by taking a weight-of-evidence approach, biomarkers may be successfully incorporated within environmental risk assessment frameworks such as the WFD.

The ECOMAN project: A novel approach to defining sustainable ecosystem function.

The ECOMAN was initiated in 2001 by the University of Plymouth, UK, Plymouth Marine Laboratory and the Department of the Environment, Fisheries and Rural Affairs (DEFRA) to address the need for more pragmatic assessment techniques linking environmental degradation with its causes. The primary aim of the project was to develop an evidence-based approach in which suites of easy-to-use, cost-effective and environmentally valid biological responses (biomarkers) could be used together to assess the health of coastal systems through the general condition of individuals. A range of sub-lethal endpoints, chosen to reflect successive levels of biological organisation (molecular, cellular, physiological), was evaluated in common coastal organisms showing different feeding types (filter feeding, grazing, predation) and habitat requirements (estuary, rocky shore). Initially, the suite of biomarkers was used in laboratory studies to determine the relative sensitivities of key species within different functional groups to common contaminants. These results were then validated in field studies performed in a range of ecosystems exhibiting different degrees/signatures of contamination. Here, an example is provided of a field study in the Humber Estuary, UK, which illustrates how multivariate statistical analysis can be used to identify patterns of response to discriminate between contaminated and clean sites. The use of a holistic, integrated approach of this kind is advocated as a practical means of assessing the impact of chemical contamination on organismal health and of ranking the status of marine ecosystems.

A microplate freshwater copepod bioassay for evaluating acute and chronic effects of chemicals.

Toxicity test protocols for establishing the effect of zinc on the freshwater copepod Bryocamptus zschokkei are reported. In the absence of food, larval life-history stages were more sensitive than adult females to acute zinc exposure with a 96-h median lethal concentration of 0.62 mg Zn/L (0.52-0.73 mg Zn/L, 95% confidence intervals) for copepodids. The acute toxicity of zinc to adult females was also significantly reduced in the presence of food (a leaf disc). The main toxic effect of zinc in a life-cycle test was a reduction in the number of offspring per brood at 0.48 mg Zn/L. As this endpoint corresponded with zinc concentrations causing reduced juvenile survival, prolonged embryonic development times, and mortality during hatching, it appears that zinc had a direct toxic effect on the current brood rather than an indirect effect on egg production via maternal reallocation of resources. The lowest-observed-effect concentration for reproduction of measured zinc concentrations of 0.48 mg Zn/L corresponded with environmental concentrations of zinc causing reduced population densities of this copepod in the field. It is proposed that B. zschokkei is an ecologically relevant test species for evaluating the potential effects of contaminants on freshwater invertebrates and for giving insight into the mode of action of a test chemical.

Mysid crustaceans as potential test organisms for the evaluation of environmental endocrine disruption: a review.

Anthropogenic chemicals that disrupt the hormonal systems (endocrine disruptors) of wildlife species recently have become a widely investigated and politically charged issue. Invertebrates account for roughly 95% of all animals, yet surprisingly little effort has been made to understand their value in signaling potential environmental endocrine disruption. This omission largely can be attributed to the high diversity of invertebrates and the shortage of fundamental knowledge of their endocrine systems. Insects and crustaceans are exceptions and, as such, appear to be excellent candidates for evaluating the environmental consequences of chemically induced endocrine disruption. Mysid shrimp (Crustacea: Mysidacea) may serve as a viable surrogate for many crustaceans and have been put forward as suitable test organisms for the evaluation of endocrine disruption by several researchers and regulatory bodies (e.g., the U.S. Environmental Protection Agency). Despite the long-standing use of mysids in toxicity testing, little information exists on their endocrinology, and few studies have focused on the potential of these animals for evaluating the effects of hormone-disrupting compounds. Therefore, the question remains as to whether the current standardized mysid endpoints can be used or adapted to detect endocrine disruption, or if new procedures must be developed, specifically directed at evaluating hormone-regulated endpoints in these animals. This review summarizes the ecological importance of mysids in estuarine and marine ecosystems, their use in toxicity testing and environmental monitoring, and their endocrinology and important hormone-regulated processes to highlight their potential use in assessing environmental endocrine disruption.

Ecosystem management bioindicators: the ECOMAN project--a multi-biomarker approach to ecosystem management.

The ECOMAN project was initiated from an awareness of the complexity of the functioning of coastal marine systems and the clear need for more pragmatic environmental assessment techniques linking environmental degradation with its causes. The aim of the project is to develop a suite of easy to use, cost effective and environmentally valid biological responses (biomarkers) to assess the general health of coastal systems, including estuaries. To achieve this aim, various sublethal endpoints are being measured and evaluated from a range of common coastal organisms showing different feeding types (filter feeding, grazing and predation) and habitat requirements (estuary and rocky shore) and at different levels of biological response (cellular, physiological and behavioural). This holistic integrated approach is essential to identify the full impact of chemical contamination on organisms, and enables the sensitivity of organisms to be ranked and key sentinel species for specific habitats to be identified.

A multibiomarker approach to environmental assessment.

Incorporation of ecologically relevant biomarkers into routine environmental management programs has been advocated as a pragmatic means of linking environmental degradation with its causes. Here, suites of biomarkers, devised to measure molecular damage, developmental abnormality and physiological impairment, were combined with chemical analysis to determine exposure to and the effects of pollution at sites within Southampton Water (UK). Test species included a filter feeder, a grazer, and an omnivore to determine the sensitivity of organisms occupying different trophic levels. Linear regression confirmed a significant association between incidence of intersex in Littorina littorea and tributyltin (TBT) concentrations (R2 = 0.954) and between PAH metabolites in Carcinus maenas urine and PAHs in sediments (R2 = 0.754). Principal component analysis revealed a gradient of detrimental impact to biota from the head to the mouth of the estuary, coincident with high sediment concentrations of heavy metals, PAHs, and biocides. Multidimensional scaling identified C. maenas as the organism most sensitive to contamination. Carboxylesterase activity, metallothionein and total haemolymph protein were the most discriminating biomarkers among sites. This holistic approach to environmental assessment is encouraged as it helps to identify the integrated impact of chemical contamination on organisms and to provide a realistic measure of environmental quality.

Behavioural responses of estuarine mysids to hypoxia and disruption by cadmium.

Neomysis integer (Peracarida: Mysidacea) occupies the upper, low-salinity regions of estuaries in Europe, where it can experience periods of reduced oxygen concentration associated with the maximum turbidity zone. The present study reports the distribution of N. integer in response to gradients of dissolved oxygen in combination with environmentally realistic cadmium concentrations (0.1, 0.5 and 1.0 microg Cd2+ l(-1). Control and 0.1 microg Cd2+ l(-1) mysids were distributed evenly along a flume containing an oxygen gradient, and demonstrated no avoidance to hypoxia (down to 30% air saturation). In contrast, mysids exposed to 1.0 microg Cd2+ l(-1) migrated away from areas of low dissolved oxygen and, after 90 min, no mysids were found in regions of <75% air saturation. These results indicate that cadmium poses an increased risk to estuarine organisms inhabiting upper estuarine areas subjected to reduced dissolved oxygen concentrations.