Josephine A. Hagger

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

To answer the requirement of the European Commissions 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.

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.

Tributyltin induces cytogenetic damage in the early life stages of the marine mussel, Mytilus edulis

Using an integrated approach, the genotoxic potential of bis(tri‐n‐butyltin), an antifouling agent known to disrupt endocrine system in marine invertebrates, has been evaluated in the embryo‐larval stages of the edible mussel, Mytilus edulis. While evaluating the genotoxic potential, the toxicity of tributyltin was also taken into account by determining the maximum tolerated dose. The study suggested that tributyltin is capable of inducing cytogenetic damage (sister chromatid exchanges and chromosomal aberrations) in this target species. The study emphasises the need for further investigations of the potential genotoxic effects of this and other endocrine disrupters on ecologically relevant aquatic invertebrates, which contribute to the maintenance of ecosystems and that could potentially be harmful to human health via the food chain. Environ. Mol. Mutagen. 35:343–350, 2000

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.

Genotoxic, cytotoxic and developmental effects of tributyltin oxide (TBTO): an integrated approach to the evaluation of the relative sensitivities of two marine species

Adopting an integrated approach the potential genotoxic, cytotoxic and developmental effects of tributyltin oxide (TBTO), a known endocrine-disrupting agent for neogastropods, have been evaluated in two ecologically relevant invertebrates: Mytilus edulis (blue mussel) and Platynereis dumerilli (rag worm). Following determination of the maximum tolerated dose (MTD) in terms of developmental and survival effects, the embryo-larval stages of these organisms were exposed to a range of concentrations of TBTO, and analysed for cytotoxic (proliferation rate index) and genotoxic (sister chromatid exchanges and chromosomal aberrations) effects. The study suggested that: (1) TBTO is both toxic and genotoxic to embryo-larval stages of both species; (2) at comparable concentrations, for developmental and genotoxic effects, P. dumerilii (non-target species) is more sensitive compared to M. edulis (target species); and (3) genotoxic effects are more closely tied with the development and survival of the organisms. The study emphasises the need of the evaluation of genotoxic potential of other endocrine-disrupting agents in different taxonomic groups.

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.

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.