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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

This form is in Word format and the boxes may be expanded or reduced, as appropriate.

ACCESS TO INFORMATIONThe information collected on this form will be stored electronically and may be sent to any part of Defra, or to individual researchers or organisations outside Defra for the purposes of reviewing the project. Defra may also disclose the information to any outside organisation acting as an agent authorised by Defra to process final research reports on its behalf. Defra intends to publish this form on its website, unless there are strong reasons not to, which fully comply with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.Defra may be required to release information, including personal data and commercial information, on request under the Environmental Information Regulations or the Freedom of Information Act 2000. However, Defra will not permit any unwarranted breach of confidentiality or act in contravention of its obligations under the Data Protection Act 1998. Defra or its appointed agents may use the name, address or other details on your form to contact you in connection with occasional customer research aimed at improving the processes through which Defra works with its contractors.

Project identification

1. Defra Project code AE1035

2. Project title

Development and application of biological effects techniques to support marine monitoring (DABSMAR)

3. Contractororganisation(s)

CefasBurnham LaboratoryRemembrance AvenueBurnham-on-CrouchEssexCM0 8HA

54. Total Defra project costs £ 847,601(agreed fixed price)

5. Project: start date................ 01 April 2002

end date................. 31 March 2007

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.Executive SummaryResearch and development from this project underpins the entire UK monitoring effort and provides vital knowledge and insight for developing a strategy for biological effects monitoring for the CSEMP (formerly the UK NMMP). The project also provides development and intercalibration of techniques to fulfil our international commitments within the OSPAR JAMP, and the CEMP. The main objectives were divided into three subsections, these include:

1) Developing the capability and competence in carrying out biological effects techniques.2) Development and application of biological effects techniques that can be applied to UK marine

monitoring programs. 3) Contributing to the development and application of biological effects techniques nationally and

internationally.

1) Developing capability and competenceOver the duration of the project, Cefas have initiated and taken part in various intercalibration exercises in order to demonstrate capability and competence in a range of biological effects techniques. These include:a) BEQUALM, which provides the AQC programme required for reporting of data to ICES and OSPAR and for the subsequent use of the data for assessments across the OSPAR area. Cefas administers and co-ordinates the self-funding scheme, which was set-up in 2004, and currently leads on the ‘whole organism’ (bioassay and fish disease) component. Cefas staff provide advice and consultation to the programme and project office. This programme is significant to OSPAR since it will now be able to raise many of the biological effects techniques to category 1 within the OSPAR CEMP (i.e. they will be raised to mandatory status). The biological effects techniques within the BEQUALM programme include: Ring-tests with the sediment bioassays: Corophium and Arenicola, and the water bioassays: Tisbe, Skeletonma, Crassostrea and Daphnia and biomarker intercalibration exercises with EROD, protein and VTG. In addition, the Microtox assay was successfully ring-tested within the BEQUALM programme during 2006. Overall Cefas performed well in the laboratory intercalibrations and were within the required tolerance limits for all tests.b) QUASIMEME: Cefas has successfully participated in two rounds of imposex ring tests using the Dogwhelk, Nucella lapillus and the common winkle, Littorina littorea. Cefas compared well with the other laboratories and were within the guideline values. Competency in these techniques has led to the successful delivery of a further Defra programme (e.g. TBT survey ME2105).c) EU intercalibration: Cefas has participated in three EU intercalibration exercises with DR-CALUX. The first intercalibration was inconclusive due to the inexperience of other laboratories in conducting the technique. However, the two later intercalibration exercises were successful and Cefas compared well with other laboratories and achieved a high quality output. In 2006, Cefas participated in an intercalibration

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with the YES assay under the EU swift programme. Seven laboratories took part and Cefas achieved a high quality output being one of the only laboratories to detect estrogenic activity in very mild estrogenic samples.In summary:AQC is a fundamental requirement for all biological effects methods that are used for monitoring and assessment purposes in a national and international context. AQC underpins the reliability and quality of the data and is a requirement for biological effects data submitted to the ICES database for OSPAR purposes and for UK national assessments under the CSEMP. This project has ensured that appropriate AQC has been developed and adhered to for all biological effects measurements conducted for UK monitoring purposes. The DR-CALUX and YES assays are used widely as screening tools for the testing of chemicals and in a monitoring context. The variability shown by some laboratories in the intercalibration exercises highlights the importance of developing consistent methodology and AQC procedures. Cefas have performed well in these exercises and has been able to influence the development of appropriate AQC procedures for these methods.

2) Development and application of biological effects techniques.The drivers behind the development of biological effects techniques for monitoring purposes comes from the UK CSEMP, OSPAR and in the future the WFD. Scientific advice on biological effects techniques comes through OSPAR and ICES and experience and expertise obtained through this programme enables Cefas to have a significant influence on the direction of biological effects techniques nationally and internationally. The biological effects techniques developed and validated under this programme include: a) DR-CALUX including development of extraction and clean up methods from biological samples; b) ER-CALUX; c) Echinoderm bioassay; d) MXR assay in mussels; e) COMET assay; f) Macroalgae reproduction and germling growth test; g) Genomics in flat fish; and h) Neutral Red Retention assay.a) DR CALUX: is one of a suite of in vitro assays that Cefas use as a generic screen for dioxin and dioxin like compounds. Cefas participated and scored well in the BICS 2005 intercalibration exercise, which assessed the assay as well as the extraction procedures using standard and spiked samples. The high level of competency in this technique has led to its use in other Cefas contracts such as ME1401 and ME2103, which also match funds the EU project MODELKEY. In addition, the compartmentalisation of contaminants in animal tissues has created difficulties for analytical techniques. In conjunction with ME2103, a new technique for the extraction and clean up of biota samples for the DR-CALUX assay has been developed. The technique has focussed on whole organism homogenates of mussels to complement the development of biological effects techniques in this species.b) ER CALUX cells, sourced from the USA, are designed to determine the estrogenic activity of environmental samples. Comparison between ER-CALUX and YES, which is routinely run in the laboratory, was carried out in conjunction with the EU MODELKEY programme for sensitivity to estrogenic samples. The limits of detection for YES were found to be more variable but slightly lower than the ER-CALUX.c) Echinoderm bioassay: The bioassay is a 48 h test that assesses the development of the sea urchin (Psammechinus miliaris) embryos into pluteus larvae. Development and validation of the echinoderm bioassay was carried out and the technique successfully applied to the current suite of bioassays used to measure water quality of concentrated offshore samples during the 2006 UK CSEMP research cruise. The sensitivity of the echinoderm bioassay was found to be comparable to the routinely used oyster embryo larvae developmental test.d) MXR assay: Cefas have developed the MXR assay using the gill tissue of mussels and will make a significant addition to the biological effects techniques in this species. The assay measures a cellular mechanism that is inducible by a wide range of environmental contaminants from metals to organics and is currently recommended for development by ICES. A Cefas protocol has been developed and the technique refined. Initial validation using a known MXR stimulator were encouraging, and the MXR assay has potential in assessing contaminant exposure in mussel populations. This assay was also successfully applied to an integrated monitoring programme using mussels as biological indicator species.e) The COMET assay is a sensitive test, which has great potential in estimating DNA damage in fish. Technical advantages of the COMET assay over traditional methods such as CA and (SCE) has increased its use in studies of pollution-induced DNA damage in both marine and freshwater fish. A series of preliminary studies on dab (Limanda limanda) and flounder (Platichthys flesus), have demonstrated that the comet assay can be readily applied to our current suite of coastal and offshore monitoring techniques. In addition, the COMET assay using haemocytes of the mussel has been successfully applied under this project to two UK monitoring studies.f) Macroalgae: A macroalgae reproduction and growth test has been developed with seaweeds (Fucus vesiculosus and Ulva intestinalis). The bioassays were combined with image analysis techniques to provide higher throughput and reproducible and statistically robust data. The development of the bioassay was combined with a Masters Thesis in collaboration with Essex University. This work was successful and was able to measure differences in germling growth between exposure to high and medium impacted areas. The successful development of this bioassay has led to its use in additional work such as an EU

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project investigating the effects of UV radiation and a study looking at the effects of copper toxicity in relation to the environmental consequences of using copper as a biocide in antifouling paint. g) Fish Genomics: Microarrays enable the expression profiles of thousands of genes to be fully characterised and allows for a comprehensive view of how an organism is responding to its environment at a given time point. As part of an integrated work program the flounder microarray was evaluated in experiments that investigated the effects of acute in vivo exposure to cadmium. Cadmium exposure resulted in the up and down regulation of 43 genes. This work successfully demonstrated that the flounder microarray could be used to identify characteristic patterns of differential gene expression due to cadmium exposure. This work has important implications for the development of techniques with the UK CSEMP and provides a potential molecular based approach for monitoring a broad spectrum of contaminants. The ICES WGBEC has recently promoted the development and use of genomic platforms alongside traditional biomonitoring assays.h) Neutral red retention: The NNR assay is used as a generic screen for the assessment of health status in mussel populations and is recommended by ICES as a useful monitoring technique for inclusion in the OSPAR JAMP. Under this project, training was provided for two Cefas scientific staff at PML.Application of these and other relevant biological effects techniques have been carried out as part of small and large scale monitoring programs under this project, these include i) determination of background vitellogenin concentrations in male fish species; j) WFD pilot program on the Ribble estuary; k) integrated mussel biomarker suite program; and l) application of bioassays to offshore extracted water samples.j) WFD Ribble estuary study: At Defra’s request, Cefas participated in the WFD pilot program and contributed at project meetings and in the design and implementation of the sampling programme. An integrated monitoring strategy using biological effects techniques developed under this project, were deployed using the flounder and mussel alongside histopathology and chemical analysis. The results of this study are reported. The overall environmental status of the Ribble estuary based on contaminant load histopathology and fish/mussel biomarkers was good. This study was successful in applying an integrated mussel and flounder biological and chemical sampling program to assess the environmental quality of the Ribble estuary.k) An integrated mussel biomarker suite program was carried out in order to apply methods developed under this project to environmental monitoring scenarios. Mussel populations from five locations around the UK were sampled for a selection of biomarkers, histopathology and tissue chemistry. The work was successful in providing an integrated sampling program, which was able to differentiate between relatively clean and contaminated sites based on mussel health status.l) Application of bioassays to offshore samples: The biological assessment of water quality in coastal and offshore water samples was achieved by measuring the toxicity of concentrated large volume seawater samples (obtained by extraction) to oyster embryo larvae, Skeletonema and Tisbe toxicity tests. This work program was successful in determining the toxicity of offshore waters and was able to differentiate between the more toxic inshore waters from the generally less toxic offshore samples.

In summary:The development of these techniques ensures that Cefas (Defra) can fulfil its obligations to the OSPAR JAMP CEMP programmes and to the UK CSEMP. Results can be generated with appropriate AQC that are fit for purpose and as such can be used for national and international assessment of data. This work also keeps the UK at the forefront of methodology for biological effects techniques and enables the UK to influence and advise in an authoritative manner on the uptake of new techniques. Conversely, we are also able to advise if recommended techniques are not fit for purpose and should be removed from monitoring programmes such as the JAMP, thus ensuring that resources are not wasted on ineffective techniques. The current demand for integrated monitoring programmes and integrated assessment tools such as those being developed by OPSAR WKIMON, and recognised as being essential for the QSR 2010, and needed for the UK CSEMP has been initiated in this work programme. The work using batteries of techniques on fish and mussels has addressed the practicalities of using multiple deployments of techniques at the same time on the same species. More research on this is needed to determine the base set of techniques that should be used and also to apply the integrated “health” assessment tools currently under development through OSPAR WKIMON. The integrated monitoring data generated to date in this programme will be fed into the OSPAR WKIMON initiatives to assist the process of defining and developing integrated assessment tools.

Cefas is an Executive Agency of Defra and can be called upon to respond to marine environmental emergencies such as the Sea Empress and more recently the Napoli grounding. The biological effects tools developed in this project and those already established in-house will allow Defra through Cefas to provide a rapid response to such scenarios. This will encompass a survey or monitoring strategy that is cost effective, uses tools that are fit for purpose and can be applied in a competent manner and generate data of the required standard.

3) Contributing to biological effects techniques nationally and internationally.Cefas have contributed to a whole host of international fora, such as biological effects workshops,

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MCERTS steering groups, ICES working groups and OSPAR workshops such as WKIMON and CSEMP AQC related activities. Cefas currently chairs many of these groups and have a strong influence in biological effects work at an international level. In addition, Cefas (Defra) reputation and profile has been enhanced by contributions to international conferences such as SETAC, SEB and Rsc.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

GlossaryADG Adipogranular tissue NaCl Sodium ChlorideANOVA Analysis of variance NADPH Nicotinamide Adenine Dinucleotide PhosphateAPEs Alkylphenols NERC National Environment Research Council AQC Analytical Quality Control NMEAQC National Marine Ecotoxicological Analytical Quality

Control GroupNMMP National Marine Monitoring Pprogram

AWI Alfred Wegner Institute NOECNP

No Observable Effect ConcentrationNonylphenol

BBSRC Biotechnology and Biological Sciences Research Council

NRR Neutral Red Retention

BDEs Brominated Diphenyl Ethers OCP Organochlorine PesticidesBDS BioDetection Systems OEB Oyster embryo bioassay BECPELAG Biological Effects of Contaminants in Pelagic

EcosystemsOSPAR Oslo and Paris

BEQUALM Biological Effects Quality Assurance in Marine Monitoring Programme

OSPAR JAMP

Oslo-Paris Joint Assessment and Monitoring Programme

CA Chromosomal aberration PAHs Polycyclic aromatic hydrocarbonsCB Chlorinated Biphenyls PBDEs Polybrominated Diphenyl EthersCSSEMP Clean and Safe Sea Environmental Monitoring

Program.cDNA Chromosomal DNA PCBs, Polychlorinated BiphenylsCEMP Co-ordinated Environmental Monitoring

ProgrammePCR Polymerase Chain Reaction

CYP1A Cytochrome P450 1A PGP Post Genomic and ProteomicDbA Dibenzylidene Acetone PML Plymouth Marine LaboratoryDMSO Dimethylsulphoxide QA Quality AssuranceDMSO Dimethyl sulfoxide QC Quality ControlDNA Deoxyribose nucleic acid QMS Quality Management SystemDR-CALUX Dioxin Response Chemical Activated Luciferase

gene expressionQSR Quality Status Report

DTA Direct Toxicity Assessment QUASIMEME

Quality Assurance of Information for Marine Environmental Monitoring in Europe

DVB Divinylbenzene RNase/DNase

Ribonuclease/ Deoxyribonuclease

EA Environment Agency ROS Reactive oxygen species EALs Environmental Assessment Levels Rsc

RVRoyal Society of ChemistryResearch Vessel

EC50 Effect concentration resulting in 50% of population

RYA Recombinant yeast assay

ELISA Enzyme-Linked ImmunoSorbent Assay SCE Sister chromatid exchangeEQSs Environmental Quality Standards SEB Society of Experimental BiologyER-CALUX Estrogen Response Chemical Activated

Luciferase gene expressionSETAC Society for Environmental Toxicology and Chemistry

EROD Ethoxyresorufin-O-deethylase SFG Scope for growthEU European Union SIME Substances in the Marine Environment GroupFRS Fisheries Research Service SOD Superoxide dismutaseFSW Filtered Seawater SOPs Standard Operating ProceduresGPC Gel permeation chromatography TBT Tri-butyl TinGST Glutathione-S-transferase TCDD 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

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HBCD Hexabromocyclododecane TIE Toxicity Identification and Evaluation ICES International Council for the Exploration of the

SeaUK CSEMP United Kingdon Clean and Safe Seas Environmental

Monitoring ProgramICES REGNS Regional Ecosystem Study Group for the North

SeaUK NMMP United Kingdon National Marine Monitoring Program

IP Intraperitoneal UV Ultra-violetISTA International Symposium on Toxicity

AssessmentVTG Vitellogenin

JAMP Joint Assessment and Monitoring Programme WFD Water Framework DirectiveLOECMCERTS

Lowest Observable Effect ConcentrationMonitoring Certification Scheme

WGBEC Working group on the biological effects of contaminants

mRNA Messenger Ribosomal Nucleic Acid WKIMON Workshop on Integrated Monitoring of Contaminants and their Effects in Coastal and Open-Sea Areas

MXR Multi-Xenobiotic resistance YES Yeast oestrogen Screen

IntroductionThis work programme supports the biological effects component of the UK marine monitoring programme undertaken by Defra. The UK has an obligation under OSPAR to undertake this monitoring in coastal and offshore waters. The monitoring is carried out through the OSPAR JAMP and of particular importance is that part of the JAMP known as the CEMP. Under the JAMP CEMP the biological effects of contaminants are monitored using techniques as prescribed in the guidelines for the specific JAMP issues. In order to be able to fulfil this commitment to OSPAR these techniques have to be developed and applied to ensure that the UK can undertake the required monitoring in an appropriate manner that is fit for purpose and design and also that the data is compliant with AQC requirement as prescribed by OSPAR. The programme outlined here aims to fulfil this requirement and in addition will help to influence and guide the uptake of biological effects methodology internationally via OSPAR and other fora. Currently, Cefas is the contracting monitoring authority undertaking this OSPAR work for Defra through the CSSEMP - formerly NMMP.

Main Objectives1. To develop the capability and competency in carrying out biological effects techniques within the OSPAR

JAMP and to fulfil UK commitments to the OSPAR CEMP and other obligations that will arise in the future e.g. WFD. This includes work on developing AQC of biological effects techniques, contributing to the BEQUALM in respect of new techniques within the OSPAR JAMP.

2. Develop and apply biological effects techniques that are of use or have potential use to the UK National Monitoring Programme (CSEMP, formerly the NMMP). This includes work on developing and validating promising ICES / OSPAR biological effects techniques and the use of these techniques in an integrated approach to marine monitoring.

3. Contribution to the development and application of biological effects techniques nationally and internationally: Contribution to ICES WGBEC annual meeting; Workshops e.g. BECPELAG, EA, etc.; Presentation of work at conferences e.g. SETAC; Development of biological indicators for UK CSEMP and European EA.

1. Developing the capability and competence in carrying out biological effects techniques within the OSPAR JAMP to fulfil our commitments to the OSPAR CEMP.

Biological effects techniques are only as good as their associated AQC. It is therefore essential that this is developed and reviewed, for techniques used in the OSPAR CEMP and the UK CSEMP. Throughout the course of the contract Cefas have initiated and taken part in a range of AQC programmes; in-house, within BEQUALM, QUASIMEME and EU inter-calibrations. Furthermore, Cefas has significantly influenced the direction of AQC schemes to bring them in line with meeting key objectives under UK statutory obligations.

a) BEQUALMIn order to ensure direct comparison of biological effects data across contracting parties, a quality assurance scheme BEQUALM was established. BEQUALM provides the AQC programme required for reporting of data to ICES and OSPAR and for the subsequent use of the data for assessments across the OSPAR area. In 2003/4 the BEQUALM Project Office was set up at the Cefas Burnham Laboratory. Uptake at other laboratories was poor and as a consequence there were funding difficulties. Time and resources were spent under this project to set up the scheme and liaise with ICES, as well as reporting to OSPAR SIME. The scheme started in its self-funding mode of operation in 2004 to 2005. Cefas currently administers and co-ordinates this scheme and leads on the ‘whole organism’ (bioassay and fish disease) component.

Under this project, Cefas staff have provided advice and consultation to the BEQUALM programme and Project Office and have assisted in the preparation of a website (www.bequalm.org). The programme has been significant to OSPAR since it has the ability to raise many of the biological effects techniques to category 1 within the OSPAR CEMP (i.e. they will be raised to mandatory status).

Cefas participated in the BEQUALM programme to demonstrate competency in conducting the biological effects techniques and also to underpin the reliability of the monitoring and R & D data produced. These biological effects techniques included;

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Ring-tests with the sediment bioassays, Corophium and Arenicola, and the water bioassays with Tisbe, Skeletonma, Crassostrea and Daphnia. The developed suite of techniques was presented as part of the BEQUALM programme at the ISTA conference 2005 in Greece. Overall, Cefas performed well in the laboratory intercalibrations and were within the required tolerance limits for all tests. However, this was not true for all participating laboratories and has highlighted a need for further intercalibrations in order to improve the quality of data produced by testing laboratories. In addition, Cefas has participated in intercalibrations with the biomarkers EROD, protein and VTG.

Intercalibration of Microtox assay The Microtox assay is a generic screen that is used widely in Europe for water quality monitoring. The assay measures inhibition of luminescence in the bacteria Vibrio scheri in response to contaminant exposure. Cefas took part in the intercalibration within the BEQUALM programme during 2006 [REF 1]. Cefas have also signed up to the 15th inter-laboratory comparison exercise in 2007. The Microtox assay provides Cefas with a useful addition to the current suite of in vitro assays and in particular those involving water/ contaminant extraction procedures and TIE.

b) QUASIMEMEOver the last few years Cefas have successfully participated in two rounds of QUASIMEME imposex ring tests. The two species that Cefas analysed were the Dogwhelk (Nucella lapillus) for imposex and the Common winkle (Littorina littorea) for intersex. Participation in the ring tests was important to demonstrate competence and maintain internal expertise. Cefas have always produced high quality data within these intercalibrations. The results of these ring tests showed that Cefas were within guideline values and compared well with other participating laboratories [REF 2]. Cefas expertise in imposex/ intersex has lead to the successful delivery of the Defra contract on the baseline survey of effects and concentrations of TBT in UK waters (Defra contract; ME2105). Recent funding has also been achieved to carry out a further baseline survey of TBT exposure in UK waters (Defra contract E2106).

c) EU IntercalibrationIn 2002/3 Cefas took part in a EU intercalibration exercise with the DR CALUX assay on fish oil. The DR-CALUX assay is an in vitro assay used as a generic screen for dioxins and dioxin–like compounds. Twenty European laboratories participated. The results of this ring study were inconclusive mainly because most of the laboratories were inexperienced in conducting the technique. Cefas have also been involved in two further international intercalibration studies for the DR-CALUX assay. In both these intercalibration studies Cefas data compared well with other laboratories and achieved a high quality output. The results of these studies have been made into reports [REF 3 &4].

In 2006, Cefas were invited to take part in an intercalibration exercise with the YES assay in water samples in conjunction with the EU SWIFT programme. The YES assay is an in vitro assay using genetically modified yeast cells to determine estrogenic activity. Seven laboratories took part in the exercise and Cefas scored very well in terms of intra and inter lab variation, being one of the only laboratories to detect activity in very mildly estrogenic samples. The work has been written into a report for the EU SWIFT programme and also into a peer reviewed paper [REF 5]. The expected versus measured estradiol equivalent values had a very good correlation and near to 1:1 relationship (Figure 1).

In summary:AQC is a fundamental requirement for all biological effects methods that are used for monitoring and assessment purposes in a national and international context. AQC underpins the reliability and quality of the data and is a requirement for biological effects data submitted to the ICES database for OSPAR purposes and for UK national assessments under the CSEMP. This project has ensured that appropriate AQC has been developed and adhered to for all biological effects measurements conducted for UK monitoring purposes. The DR Calux and YES assays are used widely as screening tools for the testing of chemicals and in a monitoring context. The variability shown by some laboratories in the intercalibration exercises highlights the importance of developing consistent methodology and AQC procedures. Cefas have performed well in these exercises and have been able to influence the development of appropriate AQC procedures for these methods.

2. Development of biological effects techniques that are used within the UK CSEMP.

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Laboratory 6y = 0.8617xR2 = 0.9402

0

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6

8

10

12

14

16

18

20

0 5 10 15 20 25

Expected values

Mea

sure

d va

lues

Figure 1: Correlation between expected and measured values for laboratory 6 (Cefas) for YES intercalibration. Figure used with permission from Brix et al., 2007.

The purpose of this part of the project was to develop and apply biological effects techniques that are required to fulfil the UKs’ obligation under OSPAR JAMP CEMP (and CSEMP) and to develop techniques that have projected value for monitoring purposes as recommended by ICES or for use within CSEMP. In addition, this project has also followed the development and requirements by OPSAR and elsewhere for biological effect – chemical integrated monitoring, e.g. ICES/OSPAR WKIMON. As a consequence, the project has applied the JAMP suite/ ICES recommended techniques in an integrated programme and also collaborated in a similar study with Plymouth University and the EA.

a) Development of DR-CaluxThe DR-CALUX assay is one of a suite of in vitro assays that Cefas use as a generic screen for dioxins and dioxin-like compounds. The DR-CALUX assay uses a cell-line (H4IIE rat hepatoma cells), which contains the luciferase reporter gene under the control of a dioxin responsive element (DRE). Consequently, when exposed to dioxins and dioxin-like chemicals, DR-CALUX cells synthesise luciferase in a dose-dependent manner. The increased luciferase expression can than be quantified in an enzymatic light producing reaction. There are a number of benefits in adopting the DR-CALUX assay as a screen for dioxin and dioxin-like compounds. It is significantly cheaper than conventional chemical analytical methods, sensitive to dioxin compounds with a high throughput of samples. The DR-CALUX assay is an existing technique that Cefas has gained competence through formal training of Cefas staff in the assay by staff at the BDS Laboratories in the Netherlands. DR-CALUX cells have been obtained under licence from BDS, who run regular intercalibration exercises with all of their licensees. Cefas successfully took part in the BICS 2005 intercalibration exercise. The exercise assessed the inter-lab comparability of the assay and the extraction methods used within each laboratory using standards and spiked samples [REF 6]. Cefas scored well for each of the individual samples tested and were within the required AQC criteria between laboratories. In terms of uptake and use of the assay, the DR-CALUX methodology has been successfully used in two Defra funded projects; The Coastal Development Thematic Programme (ME1401), where it is being used to assess dredge material alongside chemical methods, and the Hazardous Substances Thematic Programme (ME2103), which also match funds the EU project MODELKEY.

In addition to the existing DR CALUX cells used, we have obtained new cells from contacts within the USA. These include 2 types of DR-CALUX cell; the rat hepatoma cell line similar to the one already held, and a new mouse cell line. The new mouse cells will be cultured for intra-laboratory validation against our existing cell lines over the coming months. The new cells will allow us to explore different methods of determining dioxin-like activity, and assess whether one cell line is more responsive or reliable than the other. Early results showed that the two rat cell lines (from the Netherlands and USA) gave similar results using TCDD standards. However, over time the USA cells lost some of their activity, which has made the results very difficult to interpret. This is currently being investigated and updated protocols have been requested from the USA to ensure that the culturing conditions are the same as those required for the BDS cells. This has led to a temporary loss of capability in this area, and assessment of the five mussel tissue extracts (see next sub-section) taken as part of the mussel biomarker suite program (section 3l) are still pending.

Extraction of biota samples for the DR-CALUX assayThe compartmentalisation of contaminants such as dioxins in a variety of organism tissues and at different concentrations poses difficulties for current analytical techniques. In conjunction with project ME2103 (‘Hazardous substances thematic programme’), a new technique for the extraction and clean up of biota samples for the DR-CALUX assay has been developed by Cefas. The development has focussed on whole organism homogenates of mussels to complement the development of biological effects techniques in this species.

Mussels collected as part of the mussel biomarker suite program (see section 3l) were used for extraction and clean up in preparation for the DR–CALUX assay. In brief, extraction was performed with DCM/acetone 50/50 (v/v). The solvent extract was reduced in volume using evaporation and reconstituted in DCM prior to gel permeation chromatography (GPC). GPC clean-up was performed on a Agilent Series 1100 HPLC system. The GPC columns used contained a styrene/DVB polymeric material (10 µm particle diameter). The mobile phase was composed of dichloromethane heated to 25 °C before being run through the columns at a flow rate of 5 ml/min for 70 min. GPC clean-up was performed on exactly 1000 µl of sample extract, which was injected onto the calibrated GPC system. The collected fractions were concentrated to 1 ml using a TurboVap (Zymark, USA) at 30°C in preparation for the removal of the residual lipids. Following final clean-up, samples were solvent exchanged into 500 µl of DMSO prior to DR-CALUX analysis.

b) Development of ER-CaluxER-CALUX assay uses ovarian carcinoma BG1 cells to measure the estrogenic activity of environmental samples. The ER-CALUX cells contain the luciferase reporter gene under the control of an oestrogen responsive element (ERE). Consequently, when exposed to estrogens and oestrogen-like chemicals, ER-CALUX cells synthesise luciferase in a dose-dependent manner. The increased luciferase expression can than be quantified in an enzymatic light producing reaction. The main benefits of the ER-CALUX assay are its increased sensitivity to natural and synthetic estrogenic chemicals than the more established YES. Other benefits include, low costs compared to chemical analysis and high sample throughput. The ER CALUX cells have been sourced from the

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USA. This project provided the time and resources to develop the in-house capability of culturing the cell line and using protocols for conducting the ER-CALUX assay.

The ER CALUX and the YES, which is routinely run in the laboratory, where compared for sensitivity, cost and repeatability. This was done in a European intercalibration undertaken under the EU MODELKEY programme. A comparison was made between YES, ER-CALUX (using both the breast and ovarian carcinoma cells T47D and BG1) and RYA (recombinant yeast assay) [REF 7]. The Limits of detection for the YES and ER-CALUX assays averaged over the intercalibration period were found to be 66 and 133 ng/l respectively, although the limits of detection for YES were much more variable. Therefore, this suggests that the ER-CALUX assay was more sensitive and less variable to environmental estrogenic exposure than the YES, and shows potential as an oestrogen sensitive biological effect tool for monitoring purposes. However, it should be noted that the ER-CALUX assay is still in its infancy and further validation of the technique is required before it can be applied for regulatory monitoring purposes. Once established in the laboratory, the ER-CALUX will be validated for potential future use in the UK CSEMP.

c) Development of Echinoderm bioassayThe echinoderm bioassay is a generic screen that assesses the effects of waterborne contaminant exposure on the development of the embryo-larval of the sea urchin, Psammechinus miliaris. Eggs and sperm are collected from adult sea urchins and fertilised to produce developing embryos. The development of the embryos into pluteus larvae (Figure 2) over a 48 h period is then assessed in relation to exposure to a test concentration series. The use of the early life stages of the sea urchin provides the test with a high level of sensitivity. Other benefits of the test include:

1. It fills a current gap in the phyla of organisms currently used in marine water quality assessment;2. It has a definite endpoint that is assessed microscopically but has the potential to be determined by

current, in-house, image analysis techniques;3. It is a readily available species on the UK intertidal coastline that has the potential for year-round brood

stock culture;4. It is sensitive to a range of compounds including metals and PAHs (excluding pesticides);5. It is simple and inexpensive to carry out;6. It is an assay that is used and recognised internationally as being extremely sensitive and relevant.

Date EC50 95% confidence limits01/08/05 0.10 0.08 0.1409/08/05 0.12 0.09 0.1716/08/05 0.25 0.21 0.2823/08/05 0.20 0.16 0.23

Figure 2. Normal Pluteus larvae of the Sea urchin, P. miliaris.

Table 1. AQC data showing the toxicity of zinc to the developing embryos of the Sea urchin, P. miliaris (mg/L).

Table 2. Ecotoxicity data for concentrated offshore water samples to the developing embryos of the Sea urchin, P. miliaris. Statistical analysis carried out by ToxCalc scientific software.

Cefas have adopted this pre-existing bioassay and developed the in-house capability in order to carry out testing. Standard Operating Procedures (SOPs) and AQC have been developed and validated in-house. Following trials of the method four validation tests were conducted using zinc as a reference compound. The results show that the sensitivity of the assay, in our experience, is comparable to that of the oyster embryo larval development test (Table 1). The echinoderm bioassay has been added to the current suite of water quality tests and was applied to

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  EC50 95% confidence limits Statistical methodAmble 56.57 - - Graphical

Flamborough 55.05 52.08 58.2 Trimmed Spearman-KarberLyme Bay 73.8 57.85 94.13 Trimmed Spearman-KarberNE Dogger 17.9 - - Graphical

SE Eddystone 81.59 59.25 112.36 Trimmed Spearman-KarberSt Bees 56.57 - - Graphical

W Dogger 56.57 - - GraphicalBurbo Bight 51.18 44.57 58.77 Trimmed Spearman-Karber

assay concentrated offshore seawater samples collected during the 2006 UK CSEMP research cruise. The ecotoxicity data from nine selected offshore samples taken during the 2005 cruise programme are shown in Table 2. The bioassay has potential use as a sensitive biological effects tool in future marine monitoring programs and will represent an important marine phyla previously overlooked.

d) Development of MXR assay Multi-xenobiotic resistance (MXR) refers to the ability of cells to actively lower the intracellular concentration of many different structurally unrelated toxic compounds below their toxic level. The MXR assay is a generic screen, which measures a cellular mechanism that is inducible by a number of anthropogenic contaminants from metals (Cu and Pb) to organics (PAHs and Bisphenol A). The technique makes use of both fluorescence microscopy and digital image analysis techniques to determine the level of intracellular fluorescence by quantitative means (Figure 3). The level of fluorescence provides a measure of the environmental stress imposed on the mussel through contaminant exposure.

A Cefas protocol has been developed and the laboratory technique has been refined. In brief, gill segments of the blue mussel, Mytilus edulis are exposed to fluorescent calcein, which is then taken up inside the gill cells. After incubation of the gill cells the level of intracellular fluorescence is measured using image analysis and fluorescence microscopy. A low level of fluorescence signifies high MXR protein activity as a result of mussel exposure to chemical contaminants. Inversely, a relatively high level of fluorescence denotes low MXR activity from uncontaminated mussels.

The technique is currently on the ICES recommended list of techniques for monitoring and will be an important addition to the range of biological effect techniques in mussels as part of the ‘mussel tool box’ approach.

A literature review on the method and application of the MXR assay was firstly carried out by Cefas to provide adequate background knowledge for development of the assay [REF 8]. In addition, training of two Cefas staff members in the MXR assay has been carried out at the Alfred Wegner Institute (AWI) in Bremerhaven, Germany (specialists in this assay).

Initial laboratory validation studies have been conducted using a known MXR stimulator (lead), results are shown in Figure 4. The graph shows that the dose group (10mg/L lead and calcein (non-compound cleaved by cellular esterases to fluoresce)) had a significantly lower fluorescence than the control group (control + calcein) as a result of increased MXR protein expression. Verapamil (MXR inhibitor) was added as an inhibitory positive control. Overall, these data showed the potential of the MXR assay in assessing contaminant exposure. Mussels exposed to elevated contaminant levels were likely to exhibit increased MXR protein expression leading to a reduction in the intracellular concentration of calcein and reduced fluorescence, which could be measured. However, the method is still in its initial developmental stages and further laboratory validation will be required in order to ensure QA and QC of this important in vitro technique.

The MXR assay was successfully applied to assess the condition of native mussels in waters around the UK under the biomarker suite program (section 2l).

e) Development of COMET assay.The single cell gel electrophoresis (comet) assay is a sensitive test for measuring genotoxicity, investigating DNA repair, or monitoring populations for exposure against environmental mutagens. The technique requires individual

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Figure 3. Example of a fluorescing gill segment from a mussel using fluorescence microscopy and digital image analysis.

Figure 4. Normalised fluorescence of Mytilus edulis gills to 24 h exposure to lead at 10 mg/L (dose) and control treatments. The significant reduction in fluorescence for treatments exposed to 10 mg/L lead and calcein compared to the control calcein suggests an increased expression of MXR proteins as a result of lead exposure.

cells from the target tissues (i.e. erythrocytes-blood cells) to be isolated and embedded into agarose gel. These embedded cells are then lysed and exposed to high pH (alkaline unwinding) in order to relax the nuclear material. The cells are then micro-electrophoresed and stained with a florescent stain to reveal any broken fragments of the DNA, which have migrated towards the anode. The comet like appearance of these cells gives rise to the assays name and the size and staining intensity of the “comet tail” provides a quantitative measure of DNA damage (Figure 5).

Due to its high sensitivity to genotoxic contaminants the comet assay has been widely used in genotoxicity testing in vitro and in vivo, human biomonitoring and ecotoxicology research. In the aquatic environment the comet assay has great potential to estimate DNA damage in fish as the traditional cytological techniques used to assay DNA damage, such as chromosomal aberration (CA) and sister chromatid exchange (SCE) are hindered by the characteristically small size and high copy number of fish chromosomes. Due to these technical advantages, the comet test is increasingly being used in studies of pollution-induced DNA damage in both marine and freshwater fish. As such, the comet assay is on the ICES recommended list of techniques for monitoring purposes. Cefas has carried out a series of preliminary studies to assess the assay’s suitability when used in conjunction with dab (Limanda limanda) and flounder (Platichthys flesus), two species used routinely under the UK CSEMP. The results of the COMET assay for dab are shown in figures 6 & 7.

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Figure 6. Preliminary data obtained from erythrocytes of laboratory exposed flounder (control; 40 mg/L DbA; 40 mg/L DbA + 400 ng NP) examined for the induction of DNA strand breaks using the alkaline version of the comet assay. DNA damage was assessed using Euclid Comet Analysis software, (Euclid Analysis, USA).

Figure 7. Preliminary data obtained during the R.V. Endeavour 2004 Biological Effects CSEMP cruise. Erythrocytes from dab examined for the induction of DNA strand breaks using the alkaline version of the comet assay.

These preliminary studies have demonstrated that the comet assay can be readily applied to our current estuarine and offshore monitoring species, including dab and flounder. An additional advantage is that it is readily (and cheaply) adaptable for use with some of our current bioassay based monitoring tools. For example, the comet assay could be readily applied to bioassay species used in current sediment toxicity tests (e.g. Arenicola) thereby adding an additional sub-cellular biomarker to the existing suite of endpoints. Further successful application of the COMET assay using mussel haemocytes was made to two monitoring programs; 1) the Ribble estuary WFD pilot program, and 2) the integrated mussel biomarker suite program, both of which are discussed later in this report.

f) Development of macroalgae reproduction and germling growth test. The macroalgal growth and reproduction test has been developed by Cefas with the brown seaweed Fucus vesiculosus and the green seaweed Ulva intestinalis for use as a water quality bioassay. The test is a generic screen for the assessment of water quality criteria. The bioassays involve spawning the algae and then subjecting the resulting embryos or gametes (in the case of F. vesiculosus) and spores (in the case of U. intestinalis) to a standard dilution series. The endpoints are either germling growth or germination success. In the case of germling growth, germlings are attached to microscope slides, which can then be placed in the test media. Measurements of germling growth are then recorded using a microscope and image analysis tools at time intervals during a 21-day exposure.

Benefits of the assay are that it fills a gap in the phyla of organisms currently used in marine water quality assessment; it is sensitive to a wide range of contaminants at environmentally realistic concentrations, can be deployed in the field or controlled laboratory conditions and it uses image analysis techniques enabling high throughput, reproducibility and statistical robustness.

SID 5 (Rev. 3/06) Page 12 of 30

A BFigure 5. (A) Undamaged cells analysed by the comet assay with minimal migration of DNA into the comet tail. (B) Damaged cells with extensive DNA damage migrating into the tail region as a result of strand breakage.

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The development of the macroalgal bioassay was combined with a Masters Thesis in collaboration with the University of Essex [REF 9]. This work looked at the potential use of Fucus spiralis and Ulva intestinalis germlings in field deployments, and to assess the sensitivity of the germlings in measuring differences in environmental biocide concentrations from a relatively high (enclosed marina) and medium (outer estuary) impacted areas. The work was successful and was capable of measuring differences in growth between germlings exposed to high and medium impacted areas. The research also conducted laboratory tests with known antifouling contaminants TBT, Irgarol 1051 and diuron. An example of the results from these experiments is shown in table 3.

Gamma radiation effectsFollowing development of the bioassay under this project, the developed bioassays were adapted for use in experiments exposing macroalgal embryos and spores to gamma radiation (under EA project: C2154). In this study the mature adults were exposed to gamma radiation for 1 week prior to spawning and bioassay. The effects of the radiation exposure on germination success and germling growth were then studied. The main findings of these tests were that initial exposure of the adults to radiation gave further reduced levels of survival success of the progeny compared to radiation exposure of the progeny alone. The results also showed that both F. vesiculosus and U. intestinalis responded similar in magnitude. The results demonstrated that reproductive endpoints of marine macroalgae provided a good measure of radiation exposure. This further development of the reproductive stages has been included in the EA programme as representative of the phyla in a suite of bioassays for use in monitoring environmental radioactive contamination. This work was presented at the SETAC 2005 conference in Lille, France [REF 10].

Application of the techniques for measuring the effects of the biocide copperThe developed macroalgal bioassay was also applied to a work programme funded by the European Union Antifouling Copper Task Force. This involved an assessment of waterborne copper speciation and toxicity to germling growth. This study found F. vesiculosus germlings to be sensitive indicators of copper exposure. This work has been accepted for publication in Ecotoxicology and Environmental Safety [REF 11] and has environmental implications for the use of copper as a biocide in antifouling paints.

g) Development and use of genomics in biological effects monitoring: preliminary validation of the European Flounder (Platichthys flesus) microarray.With the advent of microarrays the expression profiles of thousands of genes can now be fully characterised. These experiments allow a comprehensive view of how an organism is responding to its environment by measuring the expression of multiple genes at a given time point. The technique offers an alternative approach and potential solutions to the limitations of the multi-biomarker approach. Furthermore, the development and use of genomic platforms, alongside traditional biomonitoring assays, has recently been recognised and recommended by ICES as an important tool for future monitoring purposes.

A multi-gene microarray has already been developed under a NERC post genomics thematic funded project1&2 Cefas is part of this consortia, which also includes FRS and the Universities of Birmingham, Exeter, Stirling and Glasgow. Work was conducted in parallel with a Cefas seedcorn funded project DP1592, which supported the development of the European Flounder microarray. As part of this integrated work programme, financially supported by this project, the flounder microarray platform was evaluated in an experiment that investigated the effect of acute exposure in vivo to the heavy metal cadmium. In addition, a suite of real time PCR markers for a number of contaminant related biomarker genes were employed in parallel.

Sexually immature P. flesus were obtained 8 months post-hatch and exposed to a single intraperitoneal injection of cadmium chloride up to 2 mg/kg body weight. The response of sexually immature P. flesus to a single intraperitoneal injection of cadmium chloride (up to 2 mg/kg body weight) was assessed by microarray analysis. A 500-clone microarray was used to measure gene expression (see Fig 8). In addition, 14 clones from the 1&2 Development of a flounder microarray was undertaken in collaboration with Cefas funded seedcorn project DP159: The development of a multi-gene array for detecting differential gene expression in aquatic species. Development of the flounder European microarray is now continuing under a UK Natural Environmental Research Council (NERC) Post Genomic and Proteomic (PGP) Directed programme under grant no. NE/C507661/1. Functional genomics facilities utilised at the University of Birmingham were funded by BBSRC grant 6/JIF 13209, bioinformatics by MRC grant G-4500017.

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Table 3. Growth inhibition of macroalgal germlings by exposure to antifouling compounds (mean ± SD).[Contamina

nt] U. intestinalis growth rate (mm/ day)(µg/ L) Irgarol Diuron TBT

0 26.1 ± 2.0 24.8 ± 2.5 26.4 ± 1.10.0033     22.1 ± 1.2 0.01 15.0 ± 2.3 15.2 ± 1.6 15.7 ± 1.0

0.056     No germination0.1 9.8 ± 0.6 10.8 ± 1.0  1 5.9 ± 1.01 6.1 ± 0.9  

3.3 No germination 5.9 ± 0.9  10   2.2 ± 0.7  

Figure 8. Custom 500-clone P. flesus microarray

plaice (Pleuronectes platessa) and 17 clones from the Winter flounder (Pseudopleuronectes americanus) were obtained. Microarrays were printed according to protocol (Sheader et al., 2006).

Exposure to cadmium resulted in a total of 43 genes, significantly, differentially expressed with 27 genes up-regulated and 14 genes down regulated by the exposure. An overview of the findings have been recently published (Sheader et al., 2006). Briefly, while it was not possible to explain all differentially expressed genes identified by the custom microarray employed, many gene expression changes could be rationalised against the known mechanisms of toxicity resulting from a particular chemical exposure. P. flesus showed induction of markers of oxidative stress as a characteristic response to cadmium 3 days post exposure. Markers of oxidative stress that were up-regulated included; ROS and Cu/Zn SOD (anti-oxidant enzyme). Other up-regulated genes following cadmium exposure included metallothionein and GSH, protein responsible for metal protection. One of the main down-regulators following cadmium exposure was CYP1A. This has important implications for the interpretation of CYP1A biomarker data from environmentally exposed animals that may be simultaneously exposed to CYP1A inducers and cadmium or other heavy metals.

This work has important implications for the development of techniques within the UK CSEMP and provides a potential molecular based approach for monitoring a broad spectrum of contaminants. The work demonstrates the ability to produce small-scale, ‘open’ microarray platforms for genomics analysis in non-model organisms, such as the European flounder. Significantly, this can be achieved within a relatively short time frame and at a relatively low cost. In addition, work is continuing to identify a number of genes worthy of further study as potential biomarkers. Overall, this work has contributed to three peer-reviewed papers [REF 12,13,14].

h) Development of the Neutral Red Retention Assay The NRR assay is used as a generic screen for the assessment of health status in mussel populations. The assay measures the ability of cellular lysosomes of mussel haemocytes to retain the neutral red dye. Mussels exposed to higher contaminant concentrations are more likely to have damaged membranes resulting in the inability to retain the neutral red dye. Longer retention times being an indication of increased health status.

Under this project training for two Cefas scientific staff was provided by PML in the NRR assay. This has provided Cefas with sufficient expertise in successfully carrying-out testing. The NRR is a quick and inexpensive technique that enables scientists to obtain an overall assessment of mussel health status, which can be used as an effective indicator of environmental condition. The main difficulty of the assay was in the assessment of lysosomal leakage of the neutral red dye into the cytoplasm. This at present has been carried out by eye using a microscope and requires a high degree of subjectivity. Methods of increasing the objectivity of the test should be sort, which would greatly improve the reliability and validity of the tests for monitoring purposes. This technique is currently on the ICES recommended list of techniques for biological monitoring and improvements of the technique, through increased objectivity, have been recommended for development by OSPAR WGBEC.

The NRR assay was successfully applied to assess the condition of native mussels in waters around the UK under the biomarker suite program (section 2l).

i) Application of VTG assay in cod and DR CALUX assay for offshore endocrine monitoring In January 2003 it was agreed with the Defra project officer that time and resource from this project could be allocated to complete additional work required under a previous Defra contract (Offshore Endocrine Programme). The reason for this reallocation was that additional work needed to be conducted to confirm and validate important findings resulting from that programme. This additional work involved the measurement of VTG in cod samples from Norwegian waters, the measurement of dioxin-like activity, using the DR CALUX assay, in sediments collected from the East Shetland Basin and the development of analytical methodologies for the analysis of alkyl phenols in produced water and environmental samples. The additional work was deemed necessary because of the scientific and political interest in the findings.

The analysis of 90 sediments from the East Shetland Basin was completed using the DR CALUX assay and showed levels of dioxin-like activity, or aryl hydrocarbon receptor-based activity to be at potentially harmful levels in some areas. In association with this work a method for the analysis of alkylphenols was developed and used to determine alkylphenol concentrations in produced waters and sediments. No alkylphenols were found to be present in the sediments analysed. Bioassay-directed fractionation of produced waters identified a range of alkyl-substituted phenols (C1 to C5 and C9) as the principal ER agonists present in the produced water. Details of this work have been published [REF 15,16,17].

j) Application of VTG assay to ascertain background levels in male cod and flounderVTG in male cod Vitellogenin (VTG) is an egg yolk precursor protein expressed in female fish. Although dormant in male fish, exposure to environmental estrogens or estrogen-mimicking chemicals has been found to increase the expression of VTG in a dose dependent manner. Despite the plethora of research on the expression of VTG in male fish, background concentrations are somewhat unknown. This work program was designed to determine the

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concentrations of VTG in male cod from Icelandic waters. These waters were considered to be a ‘clean’ site and therefore, provide data on “background” concentrations in cod plasma.

Cod were sampled 30 miles off Reykjavik, Iceland during the spring of 2004. Fifty male cod greater than 4.5 Kg in weight were caught for blood plasma VTG analysis. The results of this field study revealed Icelandic male cod plasma VTG concentrations to range between approximately 0.5 and 90 µg/ ml VTG per body weight. These concentrations were marginally higher than those found in male cod collected from the Irish Sea (Scott et al., 2006). It was unclear why male cod from a “clean” location should have such elevated concentrations but it may be related to spawning or diet. Further investigations into background VTG concentrations are needed before an accurate interpretation of fish VTG concentrations in sampling programs can be fully interpreted.

VTG in male flounderBackground VTG concentrations were determined in haemolymph plasma samples of male flounder collected during the 2006 CSSEMP research cruise (Figure 9). Lower concentrations of plasma VTG were found in male flounder collected from the Alde (0.015 µg/ ml/ body weight) compared to Rye and Off Boulogne populations (92.87 and 109.42 µg/ ml/ body weight respectively). These were comparable to the VTG concentrations found in Icelandic male cod populations. It suggests, from these two studies, that the Alde flounder were collected from a particularly clean site with respect to estrogenic exposure; with VTG concentrations significantly lower than those from all other flounder populations measured, including those from Icelandic waters. This highlights the potential use of the Alde flounder as a reference population in future monitoring programs and the difficulty in obtaining background VTG concentrations for comparative studies.

k) Application of biological effects techniques for the Ribble estuary WFD Pilot program.IntroductionThe Ribble estuary WFD demonstration program was initiated by Defra and the EA in November 2004. The purpose of the program was to determine whether surveyed estuaries have achieved good ecological status under a set of reference criteria. The principal driver of the program was the EU WFD. Defra requested that Cefas participate in the programme and agreed to support Cefas’ involvement under this project. Cefas have contributed at project meetings and in designing and implementing the sampling programme with the other participating and contracting organisations.

The Ribble estuary is located in the North West of England and discharges into the Irish Sea. The Ribble basin (with a catchment of 2128 km2) includes 5 main rivers (Ribble, Hodder, Calder, Darwen and Douglas). As part of the WFD there is a requirement that good ecological and chemical status will be reached by 2015. The main objectives of the monitoring program were to collaborate with the contracted organisations on the EU WFD pilot programme on the Ribble estuary and to deploy techniques within the JAMP suite of biological effects techniques and those developed under this project.Materials and MethodsThe flounder (Platicthys flesus) was identified as a suitable fish species for vertebrate biomarkers. Flounder are the recommended species in the UK CSSEMP for sampling in estuarine/transitional waters and have a range of validated techniques under the OSPAR JAMP that are suitable for environmental monitoring. Although immature flounder were present throughout the length of the tidal reaches of the Ribble estuary, only mature individuals were present in any number within the estuary mouth. In this study Flounder were caught at the mouth of the estuary (CSSEMP station 766) by modified otter trawl from the EA research vessel on the 19/10/05. Flounder were maintained in flowing seawater tanks and sampled within 3 hours of capture. The Alde estuary was

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Figure 9. Plasma VTG in male flounder collected from two locations during the CSSEMP 2006 cruise. (logarithmic scale). Alde used as reference site (mean ± SD).

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identified as a suitable reference for the Ribble. A range of biomarker techniques were used on both fish populations (see Table 4).

Table 4. Techniques deployed in Flounder and Mussels collected from the Ribble estuary.Technique Flounder Tissue Mussel Tissue Target Compounds/ effectsAnalytical Chemistry

Liver and muscle Whole body homogenates

PAHs, Organohalogens, Metals, HBCD

Histopathology Liver, Kidney, Gonad, Spleen

Whole section Whole organism/ tissue effects -

EROD Liver - PAHs, planar aromatic compounds

Bile metabolites Bile - PAHsVitellogenin Blood plasma - estrogenic compoundsCOMET assay - Haemocytes PAHs

Scope for Growth - Whole animals Whole organism effectsSpeciation - Gill -

Mussels (Mytilus edulis) were identified as a suitable invertebrate species for assessment of biomarker responses. This was mainly due to their widespread distribution on UK coastlines and estuaries, there ability to bioaccumulate a wide range of compounds as well as being a sentinel species with several biological effects techniques suitable for environmental monitoring. Mussels were identified as being available at one site within the estuary (Church scar) and at a site outside of the estuary (Blackpool Pier).

EROD activity was determined in liver samples of the flounder using a modification of the method described by Stagg et al. (1995). The reaction mixture consisted of 1.96 ml assay buffer (100 mM pH 7.5 TRIS, 100 mM NaCl), 20 μl liver homogenate supernatant, and 10 μl ethoxyresorufin substrate (0.4 μM in dimethylsulphoxide, DMSO). The assay was calibrated by the addition of 10 μl of resorufin standard (25 μM in DMSO). A spectrophotometer set at wavelengths of 580 nm emission was used to measure EROD activity. The reaction was initiated by the addition of 10 μl NADPH (0.25 mM) and fluorescence emission was recorded at 0 and 60 s post addition. EROD activity was normalised to protein content and expressed as pmol/ min/ mg protein.

Bile metabolites were measured in flounder in accordance with Cefas SOP 1310. A 20 l bile sample was diluted in 10ml of 50% ethanol. Detection of the PAH metabolite 1-hydroxy-pyrene glucuronide in all bile samples and standards was measured at wavelengths 344 and 350 on a Perkin-Elmer LS50 Fluorescence Spectrometer. Results were expressed as µg/L 1- hydroxy pyrene. Hydroxy pyrene (Sigma) was used as the standard.

Vitellogenin was determined in blood plasma of flounder using an established VTG specific ELISA technique previously described by Kirby et al (2004).

Comet: DNA damage was assessed using the single cell gel electrophoresis (comet) assay previously described.

Scope for Growth measurements were carried out in accordance with the manual ‘Practical Procedures for the measurement of scope for growth’ for marine mussels (Widdows and Staff, 1997). Clearance rates, respiration rates and overall scope for growth measurements were determined.

Speciation: DNA was extracted from all mussels following sterile maceration and vortexing with 1ml of tissue culture maintenance media (Sigma). One hundred µl of tissue supernatant from each vial was added to 1ml DNAzol. Total DNA was extracted using the standard DNAzol extraction protocol (Invitrogen) with nucleic acid being re-suspended in 100 µl of Rnase/Dnase free water and subsequently stored at –20oC pre PCR. For PCR, a standard 35 round PCR cycle was used to generate products from the 5’ end of the Glu gene employing the ME15 and ME16 primer. The Glu-5’ gene distinguishes alleles specific to M. edulis (180 base pair (bp)) and M. galloprovincialis (126 bp) by a 54 bp insertion/deletion polymorphism. Each assay contained negative controls and a 100bp DNA ladder as a marker. Post PCR, products were separated on a 4% agarose gel by electrophoresis and the images recorded using a Bio Rad Gel Doc 2000 imagine analysis system.

Results & DiscussionFish BiomarkersThe EROD response of male flounder from the Ribble estuary was found to be significantly lower than that from the Alde reference estuary (p<0.05, Figure 10). In contrast, bile metabolites, as measured by 1-OH pyrene equivalents, were not significantly different between the two estuaries (p>0.05, Figure 11).

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Figure 10. EROD response in Flounder collected from the Alde and Ribble estuaries. (mean SD, Alde n=20, Ribble n=10). * significant difference between means, p<0.05.

Figure 11. Bile metabolites measured by 1-OH pyrene equivalents in male Flounder collected from the Alde and Ribble estuaries (mean SD, n=20).

As expected, VTG concentrations were significantly higher in female flounder compared to the males of the same species (Figure 12). Low concentrations of VTG induction were found in male flounder collected from the Ribble estuary.

Liver ChemistryConcentrations of CBs in the liver of the flounder were relatively low compared to those previously recorded in the liver samples collected from the Rivers Dee and Mersey (Kleinkauf, et al. 2004). The highest CB congeners in the liver samples were congeners 138 and 153 (39 and 55 µg/kg ww); this is also in agreement with previous studies on liver contaminant burdens in flounder (Kleinkauf, et al. 2004). Low or undetected concentrations of the other organic contaminants were reported. Of the metal contaminants Fe, Zn and Cu were by the most abundant (298, 48 and 28.25 mg/kg respectively).

Flounder HistopathologyA total of 16 health index parameters were investigated in flounder collected from the Ribble estuary. Of these 16 parameters only 9 were detected in the fish sampled. With the exception of Lepeophtheirus pectoralis parasitic infection of the skin and fins in 36% of fish, the prevalence of the other health index parameters was low with most occurring in less than 5% of fish sampled. Overall, based on the health parameters measured, the flounder population sampled was unlikely to have been exposed to high contaminant concentrations.

Mussel BiomarkersAll mussels collected from the Ribble that were analysed for speciation were found to be of 100% Mytilus edulis. No hybrids or M. galloprovincialis species were found. Examples of the data are shown in Figure 13.

CometThe mean tail moment used as an indication of DNA damage shows clear differences between the mussel populations (Figure 14). As expected lowest tail moment was found in the reference mussels from Brancaster. Highest tail moment was found in mussels collected from the Church Scar within the Ribble estuary, which suggests that these mussels are in a worse condition and potentially exposed to high contaminant concentrations than the other two populations measured.

Scope for growth (SFG) measurements from two mussel populations inside and outside the Ribble estuary are shown in Table 5. Significant differences in clearance rates and SFG were found between the two mussel populations (p<0.05). Higher clearance rates and SFG values exhibited by the mussels within the Ribble suggest the better overall condition of this population over mussels from Blackpool Pier.

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Figure 12. Vitellogenin induction analysed in plasma of male and female Flounder collected from the Ribble estuary (mean SD, n=19♂, 31♀).

Figure 13. Mussel speciation: Ethidium bromide stained agarose gel. Samples 1-25 showing M. edulis positive staining. The +ve control contains both M.edulis (180bp) and M. galloprovincialis (126 bp).

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Figure 14. DNA damage determined by COMET assay in mussels collected from sites in and outside the Ribble estuary. Comparison with a ‘clean’ reference site -Brancaster. (mean SE).

Table 5. Comparison of Scope for growth measurements taken from mussels collected within (Lytham St Annes) and outside (Blackpool Pier) the Ribble estuary. * Significant difference between means p<0.05.

Analytical ChemistryWhole mussels collected from a site within the Ribble (Church Scar), outside the Ribble (Blackpool pier) and a ‘clean’ reference site (Brancaster) were pooled and analysed for the complete range of contaminant concentrations. The organic contaminants PCBs, PBDEs and organochlorine pesticides were either extremely low or undetected in the mussel tissue samples from all three sites. PAH concentrations and metals were found in low concentrations in all three-mussel populations. Overall, metal and PAH contaminant concentrations were marginally lower in the Brancaster reference mussels compared to the other two sites. Fe and Zn contributed greatest to the total metal concentration in all three populations.

Histopathology of musselsThirty-three health index parameters were recorded from fifty mussels collected from two sites inside and outside the Ribble estuary. The parameter groups included: reproductive markers, non-specific inflammatory lesions, infectious diseases and general pathology.

Reproductive Markers: Physiological state in terms of reproductive markers was assessed with respect to adipogranular (ADG) tissue and reproductive status. ADG cells fuel gametogenesis and therefore these measures are inversely correlated. Disruption to the annual reproductive cycle dynamic may be experienced in mussels that reside in impacted environments.

There was no significant difference between ADG rate and reproductive status for Church Scar or Blackpool Pier mussels (Figure 15). This would be expected in healthy mussel populations.

Figure 15. Reproductive markers Figure 16. Non-specific inflammatory lesions

Non-specific inflammatory lesions: Inflammatory lesions have previously been associated with non-specific pathogens and exposure to heavy metals and PCBs. Mussels from Blackpool pier exhibited the highest prevalence of inflammatory lesions (Figure 16).

Infectious Diseases: Mussels from the Church Scar exhibited the widest range and highest prevalence of infectious diseases compared to mussels from Blackpool Pier. However, prevalence in both mussel populations was low (Figure 17).

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Lytham St Annes 2.16 ± 0.25 * 18.85 ± 1.99 1.10 ± 1.06 *Black pool Pier 1.20 ± 0.13 * 16.43 ± 1.25 -2.09 ± 0.71 *

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Digestive gland pathology: Melanised bodies observed within the digestive epithelial cells are believed to be residual bodies or ‘post-lysosomes’ resulting from the lysosomal pathway used during normal digestion. These bodies contain undigested/indigestible material that may be endogenous or exogenous in origin. As such, elevated numbers of these residual bodies may indicate increased exposure to contaminants. Blackpool pier mussels displayed markedly higher prevalence of digestive gland pathology compared to the Ribble mussels (Figure 18). Degeneration/disintegration of the digestive gland epithelium has previously been correlated to acute toxicity exposure. The prevalence of this indicator was almost identical between both sites

Other pathology: Previous studies have shown that contaminated environments can result in the reduction of epithelial cell height of the digestive diverticula epithelium. Although a slight reduction of the epithelial cell height was found in Blackpool Pier mussels compared to those from Church Scar, there was no statistically significant difference found (Figure 19). The kidney is an important organ for the excretion of unmetabolised contaminants that manifest themselves histologically as melanised aggregates within the kidney epithelium. No statistically significant differences were found between the two mussel populations.

SedimentsSediments were sampled at six sites on the Ribble Estuary and analysed for the full suite of metal and organic contaminants. Organic contaminants, HBCD, OCPs, CBs and BDES were mostly undetected in 5 of 6 sites. PAHs and metals were detected in all sediment samples. Highest concentrations of all contaminants were found at site 2. This was particularly the case for PAHs. The difference in sediment type (muddy substrate) was likely to be responsible for the elevated concentrations at site 2.

ConclusionsOverall the environmental status of the Ribble estuary based on contaminant load, histopathology and fish and mussel biomarkers was good. EROD and bile metabolites in flounder were much lower at the mouth of the estuary compared to the “reference” site used. This was supported by the liver chemistry data with low levels of contaminants found in flounder collected from the Ribble. Histopathology of the Ribble flounder revealed an overall healthy population with low incidence of inflammatory lesions, infectious diseases and other histopathology markers.

The mussel populations measured were confirmed as M. edulis. The COMET assay revealed a higher level of DNA damage in the Ribble mussels compared to the other two populations (Blackpool and Brancaster). However, the scope for growth tests showed higher clearance rates and Scope for growth measurements in the Ribble mussels, which suggests that they were in an overall better condition than those collected from outside the estuary. The histopathological data appears to support this with an increased prevalence of inflammatory lesions and digestive gland pathology in the mussels outside the estuary. The contaminant concentrations in pooled whole mussel samples were low in all three populations.

This study was successful in applying an integrated mussel and flounder biological and chemical sampling program to a transitional water body. The results of the study suggest that the Ribble estuary is a relatively clean water body with low contaminant load and limited biological effect. However, it should be noted that both mussels and flounders were available only at the outer mouth of the Ribble estuary and therefore the data presented here may not reflect biological effects of contaminants that may occur further up the estuary. In addition, the hydrodynamics of the Ribble estuary dictates that the estuary virtually dries out at low tide (3-4hr of low water),

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Figure 19. Other mussel pathology

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and therefore, the use of this estuary as a model to demonstrate the integrated application of biological effects with chemical contaminants was limited.

l) Application of mussel biomarkers as an integrated suite of techniquesIntroductionThe Blue mussel, Mytilus edulis is an important biomonitoring species due to its ecological importance, geographical coverage and the range of biological effects techniques available. The biological effects techniques for M. edulis have been developed with the aim to further integrate chemical and biological monitoring tools and to apply the methods developed under this project to environmental monitoring scenarios. The biological response techniques used include whole organism responses (scope for growth), tissue responses (histopathology) and subcellular responses (NRR, comet assay and MXR). Application of these biomarker responses to selected estuarine and coastal water mussel populations around the UK has been carried out.

The main objective of this study was to apply mussel biological effects methodology, developed under this project, to a biological monitoring program. To integrate these biological effects methods with chemical analysis and histopathology in order to provide an overall assessment of mussel health, which can be used to determine environmental quality.

MethodSample collection: Mussels were collected from five sites around the British Isles. All mussels were collected on a falling tide within 1 m above of the water line. At least 200 mussels were collected at each site. They were transported back to the laboratory in cooler boxes containing damp paper towels and ice packs. The mussels collected for histopathology were placed in plastic bags containing their native seawater. Bags were sealed and placed in the cooler box for transport back to the lab. At two of the sites (Tees and Clyde) an overnight courier was used to transport the samples to the laboratory. For the other three sites, samples were brought to the laboratory on the same day in field cars. These samples were left overnight before being processed to ensure that all mussel populations received the same treatment prior to analysis.

Where possible the same mussels were used for several biomarker assays. For example, haemolymph was taken from mussels for the NRR and COMET assays. In addition, gill tissue and digestive gland was also taken from these mussels (snap frozen in liquid nitrogen). These tissues were stored for future speciation, metallothionein and gene microarray work.

Tissue chemistry: Whole mussel homogenates prepared from 50 mussels were used to determine contaminant tissue concentrations. Homogenated tissues were analysed for metals, organotins and organics (PAHs, PCBs, APEs).

Histopathology: Excised samples were placed into histological cassettes and immediately transferred to Davidson's seawater fixative. Fixation was allowed to proceed for 24 h before transfer of fixed samples to 70% industrial methylated spirit until further processing. Samples were processed in a Vision Bio-Systems Peloris vacuum infiltration processor followed by embedding in paraffin wax. Thin sections (3-5 m) were obtained using a rotary microtome and subsequently stained with haematoxylin and eosin. Sections were evaluated for numerous health index parameters in relation to site, season and species. All micrographs were captured using a Nikon DXM1200F digital video camera and the Lim Screen Measurement™ Lucia G image capture system (Nikon, UK).

Scope for Growth measurements were carried out in accordance with the manual ‘Practical Procedures for the measurement of scope for growth’ for marine mussels (Widdows and Staff, 1997).

MXR assay was carried out on M. edulis in line with the Cefas SOP. Briefly, mussels were opened with a fixed scalpel blade by cutting the posterior adductor muscle. The gills were rinsed in filtered seawater before being removed carefully from the mussel. Sections of gill tissue approx 4 x 4mm in size were prepared from the outer and inner lamellae next to the marginal groove of each demibranch. The prepared gill sections were placed in wells containing 0.5 ml of 0.5 µM calcein AM solution and 0.5 µM calcein AM plus 20 µM verapamil. The gills were incubated in the dark for 20 min. After the 20 min incubation the gills were removed from the calcein only solution and briefly washed in FSW. Gills exposed to calcein and verapamil were incubated again in the dark for 20 min after the addition of a further 0.25 µl of calcein. The fluorescence of the gill sections following exposure to calcein only and calcein plus verapamil was measured using image analysis software.

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NRR assay: The procedure used was that adapted from Lowe and Pipe (1994). In brief, approximately 0.1ml of haemolymph was removed from the adductor muscle of the mussel with a syringe containing approx 0.1ml of physiological saline. The haemolymph/ saline solution was placed in a microcentrifuge tube, from which a 40 µl sample was removed and pipetted onto the centre of a microscope slide. The slide was left in a dark humid chamber for 15 mins to allow the cells to adhere to the slide. Excess liquid was removed from the slide after this time and 40 µl of neutral red solution added. The neutral red solution was taken up inside the haemocytes and stored within the lysosome. The ability of the lysosome to retain the neutral red solution was checked every 15 min by light microscopy (x40). The test was terminated and the time recorded when greater than 50% of the haemocytes leaked the neutral red dye out of the lysosome into the cytosol.

Comet assay: DNA damage was assessed using the single cell gel electrophoresis (comet) assay previously described.

Results & DiscussionSpeciation: Speciation of 20 mussels from each site was carried out using PCR and gel electrophoresis techniques. Mytilus edulis was the most abundant making up 100% of the populations from the Thames and Lunderston and 95% from the Clyde and Tees (Table 6). Southampton showed the most well mixed population with an even split between M. edulis, M galloprovincialis and the hybrid.

Table 6. The percentage of each mussel species from the five sample sites (n = 20).Site Clyde Southampton Thames Tees LunderstonM. edulis 95% 33.33% 100% 95% 100%M. galloprovincialis 0% 33.33% 0% 0% 0%Hybrid 5% 33.33% 0% 5% 0%

Tissue chemistry: The organic contaminants (i.e. CBs and organochlorine pesticides) were either undetected or extremely low in all mussel populations. Highest concentrations of CBs were in the Clyde mussels, although the sum of all 28 CBs measured only amounted to 0.033 µg/ kg (ww). Low concentrations of PAHs were detected in mussels from all sites ranging between 0.2 and 126 µg/kg (ww). Slightly elevated concentrations of PAHs were found in the Tees mussels compared to all other sites (sum of 25 PAHs 823 µg/kg ww). Organotin concentrations were undetected in four of the five populations only present in the Southampton populations at low levels (0.045 mg/kg). Metal concentrations were low and almost identical in all five-mussel populations. Ranked metal concentrations were Fe> Zn> Mn> As>Cu.

Histopathology: A total of 33 health index parameters were recorded from each mussel from each of the sampling sites. Parameter groups were: reproductive markers, non-specific inflammatory lesions, infectious diseases and pathology.

Reproductive Markers: Physiological state in terms of reproductive markers was assessed with respect to adipogranular (ADG) tissue and reproductive status (Figure 20). ADG cells fuel gametogenesis and therefore these measures are inversely correlated. Disruption to the annual reproductive cycle dynamic may be experienced in mussels that reside in impacted environments. Southampton Water demonstrated similar levels of ADG rate and reproductive status. The Clyde demonstrated a relatively higher ADG rate compared to reproductive status. The gonadal status of mussels from the Thames was significantly lower than Southampton Water and the Clyde. This was considered abnormal because of the significant similarity of ADG rate between Southampton Water and the Thames. This may suggest that the Thames mussels were in a relatively compromised physiological state that impacted upon reproduction. This may be attributed to energy resources being directed elsewhere in relation to non-specific pathogens or exposure to contaminants.

Figure 20. Reproductive markers. Figure 21. Inflammatory lesions.

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Non-specific inflammatory lesions: Inflammatory lesions have previously been associated with non-specific pathogens and exposure to heavy metals and PCBs. Mussels from the Thames exhibited the highest prevalence of the five inflammatory lesions observed (Figure 21). Both Southampton Water and the Clyde exhibited similar prevalence with the exception of Southampton, which exhibited a higher prevalence of general inflammation.

Infectious Diseases: Mussels from the Thames exhibited the widest range of infectious diseases (Figure 22). In contrast, the Clyde exhibited the lowest range and prevalence. However, of the eight infectious diseases reported here, four were considered to be non-invasive commensals, which don’t cause detrimental harm to its host.

Figure 22. Infectious diseases. Figure 23. Digestive gland pathology.

Digestive gland pathology: Melanised bodies observed within the digestive epithelial cells are believed to be residual bodies or ‘post-lysosomes’ resulting from the lysosomal pathway used during normal digestion. These bodies contain undigested/indigestible material that may be endogenous or exogenous in origin. As such, elevated numbers of these residual bodies may indicate increased exposure to contaminants. Southampton and the Clyde exhibited similar levels with respect to prevalence. The Thames displayed the highest prevalence of 65% (Figure 23). Degeneration/disintegration of the digestive gland epithelium has previously been correlated to acute toxicity exposure. In descending order the highest prevalence was observed at the Thames, Southampton Water and the Clyde.

Other pathology: Previous studies show that contaminated environments can result in the reduction of epithelial cell height of the digestive diverticula epithelium. No significant differences were observed between sites with respect to epithelial cell height (Figure 24).

The kidney is an important organ for the excretion of unmetabolised contaminants that manifest themselves histologically as melanised aggregates within the kidney epithelium. Mussels from the Thames showed significantly elevated levels of these aggregates compared to the Clyde and Southampton Water.

Scope for Growth: Scope for growth data for mussels collected from four of the sites is shown in table 7. A higher clearance rate is often an indication of increased health. A significantly higher clearance rate was found in mussels collected from Southampton compared to the Clyde and Tees. Unfortunately, respiration and scope for growth measurements were not available for Southampton but from the clearance rate data it suggests that these mussels were in relatively good health. The Thames respiration data were significantly higher than that measured in the Clyde and Tees mussels (p<0.05), suggesting that the Thames mussels were in a relatively healthier condition than the other two mussel populations. However, despite this no significant differences in the overall scope for growth measurements were found.

Table 7. Scope for growth measurements of mussels collected from 4 sample sites. (mean ± SE, n=16).

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Figure 24. Other pathology

* Significant difference in clearance rate from Tees and Clyde mussels, p<0.05 (ANOVA, Tukey).† Significant difference in respiration from Tees and Clyde mussels, p<0.05 (ANOVA, Tukey).

MXR assayMussels exposed to elevated concentrations of environmental contaminants will have a tendency to produce MXR proteins as a detoxification process in order to reduce the intracellular concentration of contaminants. Therefore, mussel gill segments that display a reduced fluorescence are likely to have higher MXR protein expression as a result of exposure to environmental contaminants. For the five mussel populations in the present study the Lunderston Bay mussels were found to have a significantly higher fluorescence compared to all other populations when calcein only was added to the gill segments (Table 8, ANOVA, Tukey p<0.05). This result suggests that Lunderston Bay mussels were collected from a relatively ‘clean’ site with the mussels unlikely to be exposed to high contaminant concentrations. Thames and Tees mussels recorded the lowest fluorescent levels indicating an increased MXR protein expression through contaminant exposure.

Table 8. The relative fluorescence of mussel gill sections following exposure to 0.5 µM calcein only solution and 0.5 µM calcein plus 20 µM verapamil.

(* significant difference from all other mussel populations p<0.05 ANOVA, Tukey).

NRR assay: The results of the neutral red retention assay were inconclusive with no significant differences between the mean retention times of the five mussel populations (ANOVA, p>0.05). The mean retention times for the five populations ranged from approx 72 - 86 min.

Comet assay: For the comet assay the length of the tail moment is used as a measure of the genetic damage exhibited by the mussel as a result of contaminant exposure. With the length of tail moment dependent on the amount of DNA strand breaks within the sample, which is strongly influenced by contaminant exposure. For the mussels in this study, significant differences in mean tail moment were found between mussels collected from the Thames, Clyde and Lunderston compared to the Southampton and the Tees populations (Figure 25. p<0.05, ANOVA). Significantly less DNA damage was exhibited by the Southampton and Tees mussels when compared to the other 3 populations.

a) Southampton b) Tees c) Thames

d) Clyde e)Lunderston

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Site Clearance rate (l/h/g) Respiration (umol/h/g) Scope for Growth (J/h/g)Mean SE Mean SE Mean SE

Thames 3.09 0.17 17.13 † 1.76 6.07 1.05Clyde 2.16 0.07 8.93 0.72 5.61 0.48Tees 2.18 0.13 10.94 0.70 4.78 0.64

Southampton 3.89 * 0.52 - - - -

Site Relative Fluorescence (mean ± SE)  Calcein Calcein + VerapamilClyde 2.074 ± 0.380 10.569 ± 1.812Lunderston Bay 5.037 ± 1.164 * 21.866 ± 2.674Southampton 1.719 ± 0.277 7.681 ± 1.204Tees 1.541 ± 0.224 7.103 ± 1.007Thames 1.397 ± 0.194 8.335 ± 1.129

Figure 25. Mean tail moment of mussel haemocytes taken from the 5 mussel populations (mean ± SE, n=10). * significant difference from mean tail moment for Southampton and Tees mussels (p<0.05 ANOVA). Pictures above represent the actual tail moment of haemocytes from the 5 mussel populations.

ConclusionM. edulis made up at least 95 % of the population from 4 of the 5 sites with only the Southampton population containing an even division between M. edulis, M. galloprovincialis, and the hybrid. The Southampton population proved to be relatively healthier based on histopathology, scope for growth (respiration) and the COMET assay. Overall the worst conditioned mussels were those collected from the Thames. The Thames mussels showed the highest prevalence of inflammatory lesions, digestive gland pathology and kidney melanised aggregates as well as abnormalities in ADG rate, signifying adverse reproductive effects. This coincided with the lowest fluorescence reading in the MXR assay (indicating increased contaminant exposure) and significantly higher DNA damage from the COMET assay.

From the mussel chemistry data, both organic and inorganic contaminants were either low or undetected in all five-mussel populations measured. Therefore, no obvious differences in contaminant concentrations to explain the variations in biomarker response were found. However, this study has shown how biological effects techniques can be applied to determine the overall health status of a particular ecosystem. In this study the biological response methods proved to be more effective in determining the differences in health status between the mussel populations compared to analytical chemistry methods.

Table 9. Summary of histopathology, biomarker responses and contaminant concentrations in the five mussel populations.

m) Assessment of water quality; application of bioassays to offshore UK CSSEMP extracted water samples.With very few exceptions bulk seawater samples in UK coastal waters are not acutely toxic to marine organisms. Therefore, bioassay techniques such as those in general use for toxicity studies are not appropriate to assess water quality. An alternative approach for assessing water quality using such assays is to concentrate the contaminants from large volumes of seawater using solid phase extraction techniques and subsequently test

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Thames Clyde Lunderston Southampton TeesSpecies 100% edulis 95% edulis 100% edulis 33% edulis 95% edulis

5% hybrid 33% galloprovincialis 5% hybrid33% hybird

HistopathologyADG rate Abnormal repro effectsLesions Highest prevalenceDG Pathology Highest prevalenceKidney melanised aggregates Highest prevalenceScope for growthClearance rate sig higher (healthier)Respiration Sig higher (healthier)SFGMXR Lowest fluorescence Significantly higher

(i.e.high contam expo) fluorescence

------------No DNA damage------------

Organics ---------No marked differences between populations (low or undetected concentrations)-----------

these extracts with conventional bioassays. Water quality assessment can then be expressed as a “contaminant” concentration factor required to elicit toxicity. This approach has been used on offshore water samples and has allowed comparisons to be made of the relative toxicities of waters collected within the UK CSSEMP area. The aim of the work was to assess the suitability of new extraction techniques (C8 and ENV + solid phase extraction procedures) currently being developed within Europe (e.g. EU MODELKEY) and to assess the sensitivity and suitability of water quality bioassays using micro-scale methodology.

MethodSamples of water were collected from selected UK CSSEMP stations, aboard RV Cefas Endeavour, as part of the annual monitoring cruise programme between 2004 and 2006. Stations included those in the Irish Sea, English Channel and North Sea. A 50 L water sample was collected at each site by submerging a stainless steel churn to a depth of 1m using a weighted churn sampler. The water was immediately placed into a 50 L pressure vessel and extracted by C8 and ENV+ columns. The columns are a generic screen for non-polar compounds and polar compounds respectively and have been found in previous studies to remove the contaminants that are often responsible for the observed toxicity of waters. The columns were eluted in the laboratory by solvent extraction techniques and then reduced down to a final volume of 200µl. The concentrated extracts were re-diluted into seawater to obtain final concentrations of the original seawater of 1, 3.2, 10, 32, 100, 320 and 1000x. The dilution series were bioassayed using the Skeletonema costatum (algae), Tisbe battagliai (copepod) and oyster embryo bioassays following DTA guidelines. The results were analysed and EC50, LOEC and NOEC values derived using ToxCalc scientific statistical software. Toxicity concentration factors (i.e. concentration of contaminants by C8 and ENV+ required to elicit toxicity) were reported.

Results & DiscussionThe EC50 concentrations of the three bioassays for the extracted seawater samples are shown in figures 26-28. The position of the data points on each map indicate the location at which the seawater samples were taken, whilst the size indicates the EC50 value range. The larger the diameter of the data point the larger the EC50 concentration and therefore lower the toxicity.

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Thames Clyde Lunderston Southampton TeesSpecies 100% edulis 95% edulis 100% edulis 33% edulis 95% edulis

5% hybrid 33% galloprovincialis 5% hybrid33% hybird

HistopathologyADG rate Abnormal repro effectsLesions Highest prevalenceDG Pathology Highest prevalenceKidney melanised aggregates Highest prevalenceScope for growthClearance rate sig higher (healthier)Respiration Sig higher (healthier)SFGMXR Lowest fluorescence Significantly higher

(i.e.high contam expo) fluorescence

------------No DNA damage------------

Organics ---------No marked differences between populations (low or undetected concentrations)-----------

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Figure 27. The EC50 concentrations for Skeletonema costatum (microalgae) following 72 h exposure to offshore extracted seawater samples. Water samples collected from UK CSSEMP sites during the summer research cruise of 2004 to 2006.

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Figure 28. The EC50 concentrations for Tisbe battagliai (copepod)) following 48 h exposure to offshore extracted seawater samples. Water samples collected from UK CSSEMP sites during the summer research cruise of 2004 to 2006.

For all samples analysed, non-concentrated seawater did not cause any deleterious effects on Oyster, Skeletonema or Tisbe development. For the oyster bioassay in 2004 samples, lowest EC50 (3-17x concentrate.) were found in water samples located in the Belfast Lough, Tyne and Thames Warp areas of heavy industrialisation. Offshore samples tended to have an EC50 of > 24x concentrate. Similar differences in offshore and inshore EC50 concentrations were also found in 2005 and 2006 samples.

The results of the Skeletonema bioassay found a similar pattern to that reported for the oyster. In 2004, lowest EC50 concentrations were found in the Belfast Lough and Tyne (< 30x concentrate). In contrast, offshore in the North and Irish seas, the EC50 tended to be > 60x concentrate. In 2006, particularly low EC50s were recorded in Burbobight, Tees Bay and Carmarthen bay (< 8x concentrate). The results of the Tisbe bioassay coincided with the general findings of the two other bioassays. In 2004 Belfast Lough, Tyne and Liverpool Bay were found to have relatively low EC50s compared to offshore samples. The offshore samples for all three years tended to be > 50x concentrate.

This work program was successful in developing solid phase extraction procedure in combination with micro-scale bioassays for determining the water quality of inshore and offshore waters. All three bioassay species were sensitive enough to distinguish between the toxicity of inshore and offshore samples. The biological assessment of water quality in coastal and offshore waters using bioassays provides a measure of the bioavailability of contaminants, which has a clear advantage over chemical analysis alone, especially in areas where analytical techniques do not provide appropriate sensitivity.

In summary:The development of these techniques ensures that Cefas (Defra) can fulfil its obligations to the OSPAR JAMP CEMP programmes and to the UK CSSEMP. Results can be generated with appropriate AQC that are fit for purpose and as such can be used for national and international assessment of data. This work also keeps the UK at the forefront of methodology for biological effects techniques and enables the UK to influence and advise in an authoritative manner on the uptake of new techniques. Conversely, we are also able to advise if recommended techniques are not fit for purpose and should be removed from monitoring programmes such as the JAMP, thus ensuring that resources are not wasted on ineffective techniques. The current demand for integrated monitoring programmes and integrated assessment tools such as those being developed by OPSAR WKIMON, and recognised as being essential for the QSR 2010, and needed for the UK CSSEMP has been initiated in this work programme. The work using batteries of techniques on fish and mussels has addressed the practicalities of using multiple deployments of techniques at the same time on the same species. More research on this is needed to determine the base set of techniques that should be used and also to apply the integrated “health” assessment tools currently under development through OSPAR WKIMON. The integrated monitoring data generated to date in this programme will be fed into the OSPAR WKIMON initiatives to assist the process of defining and developing integrated assessment tools.

Cefas is an Executive Agency of Defra and can be called upon to respond to marine environmental emergencies such as the Sea Empress and more recently the Napoli grounding. The biological effects tools developed in this project and those already established in-house will allow Defra through Cefas to provide a rapid response to such scenarios. This will encompass a survey or monitoring strategy that is cost effective, uses tools that are fit for purpose and can be applied in a competent manner and generate data of the required standard.

3. Contribution to the development and application of biological effects techniques nationally and internationally.

a) MCERTS (Performance standards for laboratories undertaking direct toxicity assessments of effluents)Direct Toxicity Assessment (DTA) is the use of whole effluent ecotoxicity testing to assist in the assessment and control of complex industrial effluents discharged directly to controlled marine and fresh waters in the United

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Kingdom. Whole effluent ecotoxicity integrates the additive, synergistic and antagonistic effects of substances and their breakdown products within complex mixtures. The approach allows an integrated assessment of the combined biological effects of all constituents, including unknown substances and those for which EQSs, EALs or chemical analysis methods do not currently exist. The extension of MCERTS to include DTA is built on laboratories operating a suitable QMS based on recognised international standards and operating a level of AQC that is sufficient to ensure that the quality of test data is high. Cefas sits on the steering group of MCERTS to provide expert advice on methods, their application and AQC and interlinks with NMEAQC and BEQUALM.

b) ICES WGBEC The ICES WGBEC is an international group of expert scientists. Its purpose is to advise ICES and OSPAR on scientific aspects of biological effects techniques, which include the development of methods, the validation and uptake of methods into surveillance and monitoring programmes, issues of AQC, data assessment and specific questions raised by OSPAR. Cefas currently chairs this ICES working group. This project provides some of the support required for attending the meetings and inter-sessional work and allows Cefas (Defra) to have a strong influence in the use and uptake of biological effects work at an international level. This ensures that biological effects techniques advocated for marine monitoring programs (e.g. OSPAR JAMP CEMP) are robust, fit for purpose and cost effective and in turn this has knock-on effects for the UK CSEMP.

Contributions were also made to the OSPAR WKIMON; ICES REGNS data acquisition; liaison with the ICES data base for biological effects reporting, and presentations were made on bio effects techniques at a special session at the ICES Annual Science Conference in Vigo.

c) NMEAQC The NMEAQC is a UK-wide group of scientists concerned with the development and application of AQC aspects of methods used in the UK CSEMP. This project supports in a small way some of the related activities and advice to the work of this group. Cefas currently chair the NMEAQC group, which on average meets on five occasions during the year.

d) ConferencesIn 2003, contributions were made to SETAC conferences (e.g. Endocrine in York and Hamburg), SEB meeting in Edinburgh, an Offshore Oil and Gas Conference in Halifax and to The Rsc in Cambridge.

SETAC York: The Significance of Endocrine Disruption in Fish Offshore.SETAC Hamburg: The use of image analysis for improving data quality in aquatic bioassays.

SEB-Edinburgh: Use of Automated Image Analysis to determine endpoints in the Oyster Embryo Larval development test.Halifax: –offshore oil and gasRsc:– Cambridge

SETAC Lille:1. The use of early life stage bioassays of two marine macrophytes for measuring the toxicity of the antifouling biocides TBT, Irgarol 1051 and Diuron.2. Effects of chronic γ-irradiation upon germination success and growth of germlings of two species of marine macrophyte: Fucus vesiculosus and Ulva intestinalis.

In addition, work programmes conducted under this project were presented at the SETAC European annual meeting in Porto, Portugal in May 2007. These include:1. Biomarker responses in the Blue mussel, Mytilus edulis, an integrated approach to biological effects measurements.2. Comparison of the relative toxicities of UK offshore, solid-phase, seawater extracts using marine bioassay techniques.

ReferencesAlmeida, J. A., Diniz, Y. S., Marques, S. F. G., Faine, L. A., Ribas, B. O., Burneiko, R. C., and Novelli, E. L. B.

(2002). The use of the oxidative stress response as a biomarker in Nile tilapian (Oreochromis niloticus) exposed to in vivo cadmium contamination. Environmental International, 27: 673-679.

Brix, R., Noguerol, T-N, Piňa, B., Balaam, J., Nilsen, A.J., Tollefsen, K-E., Levy W., Schramm, K-W., Barceló, D. (2007). Inter-comparison of estrogenicity in water determined by yeast-based assays. Submitted for publication.

Chipman, J.K., George, S,G., Williams, T.D., Diab, A.M., Sabine, V., Godfrey, R.E., Minchin, S.D. (2005). Biomarkers fom (eco)toxicogenomics: the European flounder as a non-model organism and distinction between compensatory versus toxic responses. In: Proceedings of SETAC North America twenty-sixth annual meeting, Baltimore, USA, abstract 218.

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George, S. G., Wright, J., Bell, G., Geffen, A., and Taylor, S. (2000). Dietary effects of xenobiotic-induced oxidative damage in 'O' group plaice. Marine Environmental Research 50, 80-81.

Kirby, M.F., Allen, Y.T., Dyer, R.A., Feist, S.W. Katsiadaki, I., Matthiessen, P., Scott, A.P., Smith, A., Stentiford, G.D., Thain, J.E., Thomas, K.V., Tolhurst, L., & Waldock, M.J. 2004. Surveys of plasma vitellogenin and intersex in male flounder (Platichthys flesus) as measures of endocrine disruption by estrogenic contamination in United Kingdom: temporal trends. 1996–2001, Environ. Toxicol. Chem. 23, pp. 748–758.

Kleinkauf, A., Connor, L., Swarbreck, D., Levene, C., Walker, P., Johnson, P.J. and Leah, R.T. (2004). General condition biomarkers in relation to contaminant burden in European flounder (Platichthys flesus). Ecotoxicology and Environmental Safety, 58, 335-355.

Lowe, D.M., & Pipe, R.K. 1994. Contaminant induced lysosomal membrane damage in marine mussel digestive cells: an in vitro study. Aquatic toxicology. 30, 357-365.

Scott, A.P., Katsiadaki, I., Withames, P.R., Hylland, K., Davies, I.M., McIntosh, A.D., and Thain, J. (2006). Vitellogenin in the plasma of male cod (Gadhus morhua): A sign of estrogenic endocrine disruption in the open sea? Mar. Environ. Res. 61 (2) 149-170.

Sheader, D.L., Williams, T.D., Lyons, B.P., Chipman, J.K. (2006). Oxidative stress responses of European flounder (Platichthys flesus) to cadmium determined by a custom cDNA microarray.

Stagg, R.M., McIntosh, A., & Mackie, P. 1995. Elevation of hepatic monoxygenase activity in the dab (Limanda limanda L.) in relation to environmental contamination with petroleum hydrocarbons in the northern North Sea, Aquat. Toxicol. 33, pp. 245–264.

Widdow, J., & Staff, F., 1997. Practical procedures for the measurement of scope for growth. Produced for European ring-test by authors: Plymouth Marine Laboratory, Prospect Place, West Hoe, Plymouth, PL1 3DH, UK.

References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.

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1. BEQUALM: 14th Inter-laboratory comparison exercise for the luminescent bacteria toxicity assay. 2006. Ribo JM, Riva MC. Centre for research and innovation in toxicology UPC – Technical University of Catalonia.

2. QUASIMEME laboratory performance studies: BE-1 Imposex and intersex in Marine snails. Round 37 Exercise 614.

3. Gizzi G, Hoogenboom LAP, von Holst C, Anklam E. Determination of polychlorinated dibenzodioxins and polychlorinated dibenzofurans (PCDDs/PCDFs)in food and feed using a bioassay: Result of a validation study.

4. Besselink H, Schipper C, Klamer H, Leonards P, Verhaar H, Felzel E, Brouwer B, 2002. DR-CALUX interlaboratory validation study for sediments. Bioanalysis. Organohalogen compounds, 58, 417-420.

5. Rikke Brix, Tania-Noelia Noguerol, Benjamin Piña, Jan Balaam, Anja Julie Nilsen, Knut-Erik Tollefsen, Walkiria Levy, Karl-Werner Schramm, and Damià Barceló. 2007 Inter-comparison of estrogenicity in water determined by yeast-based assays. Submitted for publication.

6. Besselink H. DR-CALUX inter-laboratory validation study (RINCA).7. P. Leonards, M. Lamoree T. Hamers W. Brack G. Streck, J. Balaam, R. Brix, M. Machala, H. Klamer,

RIKZ, P. Korytar.2006. KD2.2 Validation report on sample pre-treatment and bioassay response of EDA method, including performance characteristics and evaluation of relative performance of applied EDA techniques at different laboratories for Interlaboratory study 1. Report for EU project SSPI-CT-2003-511237-2 MODELKEY (Models for Assessing and Forecasting the Impact of Environmental Key Pollutants on Marine and Freshwater Ecosystems and Biodiversity)

8. Multi-xenobiotic resistance in aquatic organisms: Mytilus edulis. A review. Cefas document.9. Girling JA. 2005. The potential use of macroalgal germlings to assess in-situ biocide concentrations’.

MSc Dissertation, University of Essex.10. Reynolds WJ, Goodsir F, Lyons BP, Knowles JK, Leonard KS, Gross-Sorokin M, Williams C

Copplestone D. 2006. Effects of chronic γ-radiation upon germination success and growth of two species of marine macrophyte: Fucus vesiculosus and Ulva intestinalis. SETAC poster.

11. Brooks SJ, Bolam T, Tolhurst L, Bassett J, La Roche J, Waldock M, Barry J, Thomas KV. Dissolved organic carbon reduces the toxicity of copper to germlings of the macroalgae, Fucus vesiculosus. Ecotoxicology and Environmental Safety (in press).

12. Sheader DL, Williams TD, Lyons BP, Chipman KJ (2006). Oxidative stress responses of European Flounder (Platichthys flesus) to cadmium determined by a custom cDNA microarray. Marine Environmental Research 62, 33-44.

13. Du Corbier FA, Rotchell JM, Stentiford G, Lyons BP (2005). Isolation of the Retinoblastoma cDNA from the Marine Flatfish Dab (Limanda limanda) and Evidence of Mutational Alterations in Liver Tumours. Environmental Science & Technology, (2005) vol 39, 4. pp 9785 - 9790.

14. Sheader DL, Gensberg K, Lyons BP, Chipman K (2004). Isolation of differentially expressed genes in contaminant exposed European flounder (Platichthys flesus) by suppressive subtractive hybridisation. Marine Environmental Research, 58, 553-558.

15. Thomas K.V., Balaam J., Hurst M.R., Thain J. (2004) Identification of in vitro estrogen and androgen receptor agonist in North Sea offshore produced water discharges, J Environ. Tox. And Chem Vol 23:5 pages 1156-1163.

16. Thain J.E., Hurst M.R. and Thomas K.V. (2006) Determination of dioxin-like activity in sediments from the East Shetland Basin. Dioxin 2006, Oslo.

17. Scott A.P., Katsiadaki I., Witthames P.R., Hylland K, Davies I.M., McIntosh A.D. and Thain J. (2006) Vitellogenin in the plasma of male cod (Gadus morhua): A sign of estrogenic endocrine disruption in the open sea? Mar. Environ. Res. 61 (2) 149-170.

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