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.

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.

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.

Is there a causal association between genotoxicity and the imposex effect?

There is a growing body of evidence that indicates common environmental pollutants are capable of disrupting reproductive and developmental processes by interfering with the actions of endogenous hormones. Many reports of endocrine disruption describe changes in the normal development of organs and tissues that are consistent with genetic damage, and recent studies confirm that many chemicals classified to have hormone-modulating effects also possess carcinogenic and mutagenic potential. To date, however, there have been no conclusive examples linking genetic damage with perturbation of endocrine function and adverse effects in vivo. Here, we provide the first evidence of DNA damage associated with the development of imposex (the masculinization of female gastropods considered to be the result of alterations to endocrine-mediated pathways) in the dog-whelk Nucella lapillus. Animals (n = 257) that displayed various stages of tributyltin (TBT) -induced imposex were collected from sites in southwest England, and their imposex status was determined by physical examination. Linear regression analysis revealed a very strong relationship (correlation coefficient of 0.935, p < 0.0001) between the degree of imposex and the extent of DNA damage (micronucleus formation) in hemocytes. Moreover, histological examination of a larger number of dog-whelks collected from sites throughout Europe confirmed the presence of hyperplastic growths, primarily on the vas deferens and penis in both TBT-exposed male snails and in females that exhibited imposex. A strong association was found between TBT body burden and the prevalence of abnormal growths, thereby providing compelling evidence to support the hypothesis that environmental chemicals that affect reproductive processes do so partly through DNA damage pathways.

Health effects in fish of long-term exposure to effluents from wastewater treatment works.

Concern has been raised in recent years that exposure to wastewater treatment effluents containing estrogenic chemicals can disrupt the endocrine functioning of riverine fish and cause permanent alterations in the structure and function of the reproductive system. Reproductive disorders may not necessarily arise as a result of estrogenic effects alone, and there is a need for a better understanding of the relative importance of endocrine disruption in relation to other forms of toxicity. Here, the integrated health effects of long-term effluent exposure are reported (reproductive, endocrine, immune, genotoxic, nephrotoxic) . Early life-stage roach, Rutilus rutilus, were exposed for 300 days to treated wastewater effluent at concentrations of 0, 15.2, 34.8, and 78.7% (with dechlorinated tap water as diluent). Concentrations of treated effluents that induced feminization of male roach, measured as vitellogenin induction and histological alteration to gonads, also caused statistically significant alterations in kidney development (tubule diameter), modulated immune function (differential cell count, total number of thrombocytes), and caused genotoxic damage (micronucleus induction and single-strand breaks in gill and blood cells). Genotoxic and immunotoxic effects occurred at concentrations of wastewater effluent lower than those required to induce recognizable changes in the structure and function of the reproductive endocrine system. These findings emphasize the need for multiple biological end points in tests that assess the potential health effects of wastewater effluents. They also suggest that for some effluents, genotoxic and immune end points may be more sensitive than estrogenic (endocrine-mediated) end points as indicators of exposure in fish.

Toxicity of tributyltin in the marine mollusc Mytilus edulis.

Our previous studies have demonstrated that tributyltin (TBT) is genotoxic to the early life stages of marine mussels and worms. Here, the toxicity of TBT to adult organisms was determined using a suite of biomarkers designed to detect cytotoxic, immunotoxic and genotoxic effects. Exposure of adult mussels, Mytilus edulis, to environmentally realistic concentrations of TBTO for 7 days resulted in a statistically significant decrease in cell viability at concentrations of 0.5 microg/l and above. TBT had no effect on phagocytic activity or antioxidant capacity (FRAP assay). There was a statistically significant increase in DNA damage detected using the comet and micronucleus assays between the controls and 0.5, 1 and 5 microg/l of TBTO (P > 0.0005). Furthermore there was a strong correlation between DNA strand breaks (comet assay) and formation of micronuclei (P = 0.0005; R2 = 61.5%). Possible mechanisms by which TBT could damage DNA either directly or indirectly are discussed including the possibility that TBT is genotoxic due to its ability to disrupt calcium homeostasis.

Genotoxic, cytotoxic, developmental and survival effects of tritiated water in the early life stages of the marine mollusc, Mytilus edulis.

Using an integrated approach linking different levels of biological organisation, the genotoxic, cytotoxic, developmental and survival impact of tritiated water (HTO) were investigated in the embryo-larvae of marine mollusc Mytilus edulis. One-hour-old embryos were exposed to a range of concentrations (0.37-370 kBq ml(-1)) of HTO, which delivered a dose between 0.02 and 21.41 mGy over the exposure period for different end points. Detrimental effects, if any, were monitored at different levels of biological organisation (i.e. DNA, chromosomal, cellular and individual). Genotoxic effects were assessed using molecular and cytogenetic approaches which included analysis of random amplified polymorphic DNA (RAPD), induction of sister chromatid exchanges (SCEs) and chromosomal aberrations (Cabs). Cytotoxic effects were evaluated by determining the proliferative rate index (PRI) of the embryo-larval cells. Developmental and survival effects were also monitored every 24 h up to 72 h. Results in general indicated that HTO significantly increased cytogenetic damage, cytotoxicity, developmental abnormalities and mortality of the embryo-larvae as a function of concentration or radiation dose. The analysis of RAPD profiles also revealed qualitative effects in the HTO exposed population compared to controls. However, while the embryo-larvae showed dose or concentration dependent effects for mortality, developmental abnormalities and induction of SCEs, the dose-dependent effects were not apparent for Cabs and PRI at higher doses. The study contributes to our limited understanding of the impact of environmentally relevant radionuclides on non-human biota and emphasises the need for further investigations to elucidate potentially long term damage induced by persistent, low levels of other radionuclides on commercially and ecologically important species, in order to protect human and ecosystem health.

Esterase activities in the bivalve mollusc Adamussium colbecki as a biomarker for pollution monitoring in the Antarctic marine environment.

Marine environments are continuously being threatened by a large number of xenobiotics from anthropogenic sources. Even in sparsely populated and relatively pristine areas, such as Antarctica, hazardous chemicals can pose a serious environmental problem. The main aims of the present study were to (1) validate and optimize an analytical technique utilizing a microtitre-plate photometer to ascertain background levels of esterase activities in the Antarctic bivalve Adamussium colbecki, (2) carry out in situ monitoring of esterase activities to assess any potential environmental impacts of the Italian Scientific Antarctic Base "Terra Nova Bay" on the surrounding marine area. Results showed the presence of organophosphorous-sensitive cholinesterase (ChE) and carboxylesterase (CbE) activities in the gills of A. colbecki and optimal assay conditions were comparable with those found for bivalve species from temperate areas. A higher sensitivity of ChE versus acetylthiocholine activity in A. colbecki to chlorpyrifos compared to species from temperate areas may also be inferred. The in situ study indicated no differences in the environmental quality of the three study sites located around the Italian Base.

Genotoxic, cytotoxic and ontogenetic effects of tri-n-butyltin on the marine worm, Platynereis dumerilii (Polychaeta: Nereidae).

The genotoxic, cytotoxic and ontogenetic (embryo-larval) or developmental effects of tri-n-butyltin (TBT), were investigated in Platynereis dumerilii. Following the determination of maximum tolerated dose with regard to ontogenetic effects and mortality, early life stages of P. dumerilii were exposed to a range of TBT concentrations. Subsequently, the embryo-larvae were analysed for evidence of genotoxicity and cytotoxicity. Genotoxicity was assessed using cytogenetic endpoints that included the frequency of sister chromatid exchanges and chromosomal aberrations from metaphase spreads. Cytotoxicity was evaluated by determining the proliferative rate index of the growing embryo-larval cells using 5-bromodeoxyuridine labelling of the chromosomes or fluorescence plus Giemsa staining technique. TBT-exposed embryo-larvae of P. dumerilii exhibited sensitivity similar to that of other invertebrates, indicating that P. dumerilii is a suitable ecotoxicity test species. The results also suggested dose-dependent effects for genotoxic and cytotoxic end points in relation to TBT exposure. The present study highlights the need to elucidate the relative importance of direct genotoxic and indirect effects through production of genotoxic hormonal derivatives.