ICES WGBEC REPORT 2006

ICES MARINE HABITAT COMMITTEE ICES CM 2006/MHC:04 Ref. ACME

REPORT OF THE WORKING GROUP ON BIOLOGICAL EFFECTS OF CONTAMINANTS (WGBEC)

27-31 MARCH 2006

ICES HEADQUARTERS, COPENHAGEN, DENMARK

International Council for the Exploration of the Sea Conseil International pour l’Exploration de la Mer H.C. Andersens Boulevard 44-46 DK-1553 Copenhagen V Denmark Telephone (+45) 33 38 67 00 Telefax (+45) 33 93 42 15 www.ices.dk [email protected]

Recommended format for purposes of citation: ICES. 2006. Report of the Working Group on Biological Effects of Contaminants (WGBEC), 27–31 March 2006, ICES Headquarters, Copenhagen, Denmark. ICES CM 2006/MHC:04. 79 pp.

For permission to reproduce material from this publication, please apply to the General Secretary.

The document is a report of an Expert Group under the auspices of the International Council for the Exploration of the Sea and does not necessarily represent the views of the Council. © 2006 International Council for the Exploration of the Sea.

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Contents

Executive Summary ...... 1

1 Opening of the meeting ...... 4

2 Adoption of the agenda ...... 4

3 Appointment of Rapporteurs...... 4

4 REGNS integrated assessment of the North Sea...... 4

5 Assess the amount of biological effects data on the ICES database and develop a means to encourage further submissions as well as the use of the data ...... 5

6 Review progress with the TIMES...... 7

7 Consider progress with activities: i) BEQUALM, ii) EU FIRE Project iii) Prestige oil spill, and iv) BSRP/Baltic activities ...... 10 7.1 BEQUALM ...... 10 7.2 EU FIRE Project...... 12 7.3 Consider progress with national activities pertaining to the Prestige oil spill ...... 13 7.4 Review BSRP/Baltic activities ...... 13

8 Review the use of background responses in biological effects ...... 14

9 WGBEC to consider and assess the long term impact of oil spills on marine and coastal life as requested from OSPAR and provide a guidance document on the use of biological effects techniques for oil spill situations ..... 15 9.1 Background...... 16 9.2 Strategy for using biological effects methods...... 17 9.3 Selection of appropriate target species ...... 19 9.4 Confounding factors ...... 20 9.5 Selection of appropriate biological effects techniques ...... 20

10 Provide expert knowledge to the ICES Data Centre as appropriate ...... 23

11 Evaluate the WKIMON 2006 workshop and feedback from SIME (Feb 2006 meeting) ...... 24

12 Assess the development of the CEMP Guidelines...... 28 12.1 Respond to WKIMON/SIME: Are biological effects methods appropriate to meet the requirements of the OSPAR JAMP?; and...... 28 12.2 What methods need to be developed?...... 28 12.3 Respond to WKIMON: review integrated methods to assess effects on biota from lindane and BFRs...... 33 12.4 Respond to WKIMON: Review of comet assay and is application...... 34 12.5 Respond to WKIMON: Compare across species (12e) and...... 35 12.6 Selection of species, gender and size range (12f)...... 35 12.7 Respond to WKIMON: Review background levels for ALA-D...... 36

13 Assess the development of the CEMP...... 36 ii | ICES WGBEC Report 2006

14 Any other business; Review progress with 1) genomics and proteomics 2) nanotechnology, 3) 2-tier approach to monitoring, and 4) amphipod decline in the Baltic ...... 36 14.1 Review progress with genomics and proteomics...... 36 14.2 Review progress with nanotechnology...... 38 14.3 2-tier biomarker approach to monitoring...... 39 14.4 Amphipod decline and crash in the Baltic ...... 40

15 Joint discussions with WGSAEM and WGMS ...... 42 15.1 WGBEC terms of reference item j: Development of assessment criteria and integrated assessment...... 42 15.2 WGBEC used the opportunity to meet with WGSAEM to initiate an assessment of imposex data on the ICES database...... 42 15.3 WGBEC terms of reference k. (WGMS Agenda item 4): Passive samplers: review the response of the WGBEC to WGMS suggestions for areas of cooperative work on (bio)availability and related issues to report on opportunities for cooperative work...... 44 15.4 WGBEC terms of reference l; discuss and report back to ACME on potential contributions for the ecosystem overview of the advisory reports ie WGRED reports of 2005 and 2006...... 46

16 Recommendations and action list...... 47 16.1 Recommendations ...... 47

17 Adoption of the report and closure of the meeting ...... 49

Annex 1: List of participants ...... 52

Annex 2: WGBEC Terms of reference 2005 ...... 54

Annex 3: WGBEC draft agenda...... 55

Annex 4: List of Rapporteurs ...... 57

Annex 5: List of documents ...... 58

Annex 6: WGBEC draft resolutions ...... 60

Annex 7: Progress with national activities pertaining to the Prestige oil spill (Agenda Item 7.3) ...... 62

Annex 8: Provide expert knowledge to the ICES data centre as appropriate (Agenda item 10)...... 68

Annex 9: Assessment of temporal trends in imposex (Agenda Item 15)...... 71

Annex 10: A regional assessment of VDSI in dogwhelks from Sullom Voe and Yell Sound (Agenda item 15) ...... 75

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Executive Summary

The Working Group on the Biological Effects of Contaminants [WGBEC] (Chair, John Thain, United Kingdom) met in Copenhagen, from 27–31 March 2006. A summary of the key outcomes of the meeting in respect of the Terms of Reference is described below.

REGNS integrated assessment of the North Sea

The Chair of REGNS had provided WGBEC with documents outlining the current progress with the REGNS programme. The group reviewed the overall assessment and some comments were made regarding the assessment scope and methodology employed. It was noted that although many parameters had been included in the assessment, contaminants and their effects were not represented. Rather the assessment had been restricted to biological and hydrographic parameters and the overall conclusions of the assessment were related to these. Some concern was expressed regarding the level of data aggregation, annual averaging and the potential for correlating parameters not causally related in such an assessment.

It was considered by the group that biological effects of contaminants should form an important component of an integrated ecosystem assessment; however the data-sets available to WGBEC do not match the spatial and temporal scale that seems to be required for REGNS.

Biological effects data will be relevant to the forthcoming thematic assessment on ‘chemical pollution’. In this respect WGBEC would like more information from REGNS on the proposed structure of the thematic assessment on ‘chemical pollution’ to determine the value of submitting data for assessment.

Assess the amount of biological effects data on the ICES data base and develop a means to encourage further data submissions as well as the use of the data

WGBEC reviewed the amount of biological effects data on the ICES database. It was noted that the amount of biological effects data on the ICES database has increased since the last review by WGBEC in April 2005, however overall, the amount of data remains limited and still mostly consists of imposex parameters. The amount of biological effects work undertaken by OSPAR contracting parties is not reflected by the amount of data on the ICES database. It would appear that lack of transformation programmes for national monitoring data remains a major problem for ICES submissions. In this respect it was recommended that moving to free formats would help overcome this problem and it was agreed that an example of a data submission file be uploaded onto the ICES web site.

Review progress with ICES TIMES

At each meeting WGBEC reviews progress with the publication of biological effects methods appropriate for use in monitoring programmes. Publication of these methods is important as the method documents are registered with the ICES database to allow data submitted for these techniques to be associated with a standard protocol. Progress on the drafting of the documents was discussed and a plan of action agreed for taking forward the production of these documents over the next two years.

Consider progress with activities of BEQUALM, EU FIRE and the Prestige oil spill

BEQUALM provides important AQC for biological effects methods used within the JAMP CEMP. An overview of progress and current activities was presented to the group. It was noted that for some components such as some biomarkers and benthic community analysis that uptake remains poor. Progress with the EU FIRE project was presented and it was noted

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that the final report will be available in 2007. Presentations were given of current work within programmes associated with the Prestige oil spill along with several background documents. The programme is extensive and uses many biological effects techniques and therefore of considerable use and importance to WGBEC in understanding how techniques should be deployed and how data may be interpreted in oil spill scenarios.

Review the use of background responses in biological effects

This item was first discussed at the WGBEC 2005 meeting. The philosophy developed then was discussed and also jointly with a presentation from the WGSAEM. This is an important issue for the interpretation and assessment of biological effects parameters. It was agreed that relevant effect data now be compiled by an intersessional WGBEC subgroup, with the aim of proposing baseline/background responses for the relevant biological effects techniques so that the proposal can be assessed at the meetings by WKIMON III and WGBEC in 2007. The proposals to be forwarded to WGSAEM for consultation and further development as appropriate at their meeting in 2007.

Assess the long-term impact of oil spills on marine and coastal life based on a request from OSPAR and develop guidelines for use of biological effects techniques in oil spill situations

The group reviewed a report that had been produced intersessionally and developed this further in response to the OSPAR request. The report includes a review of recent oil spills, a strategy for using biological effects techniques, selection of appropriate target species, confounding factors and selection of appropriate biological effect techniques. The above guidelines are recommended for use in oil spill situations and need to be used as appropriate for the type of oil spill, the conditions under which the oil spill occurred and the variable locations (i.e. type of coastline, hydrography, etc.). It is emphasised that the methods need to be used in a fit for purpose manner and these are described within the guideline.

Provide expert knowledge to the ICES data centre as appropriate

WGBEC liaised with the ICES data centre and had discussions on several issues relating to minimum fields and standard parameters, and how to ensure quality and comparability of biological effects data submitted to the ICES database. Separate lists for recommended fields were compiled but further work in several areas (e.g. checking of standard parameter combinations and specifications for data screening) need to be taken forward intersessionally.

Evaluate the report from the ICES OSPAR WKIMON II workshop and feedback from SIME

The group spent much time on this subject, reviewing the report and documents from WKIMON II and the report and requests from SIME. Ketil Hylland, the Co-Chair of WKIMON, provided much useful background information. The group felt that it could provide guidance on which biological effects methods are appropriate for inclusion in an integrated monitoring programme, how the methods could be used in an integrated manner and advice on assessment criteria. In this respect the group produced a strategy on the core methods that should be used, which included fish and mussels and this was based on the ICES WGBEC recommended list and requirements of the JAMP CEMP. A strategy for integrating data was also suggested along with a process for updating the JAMP guidelines. WGBEC emphasized their willingness to actively support the development of background documents with assessment criteria for methods in time for WKIMON III and were prepared to work on an integration strategy document. The group was also prepared to develop a demonstration programme for the integrated guideline and could prepare this for 2008.

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Assess the development of the CEMP guidelines

The current state of biological effects techniques within the CEMP was reviewed. It was agreed that WGBEC could recommended the removal of sediment sea water elutriate bioassays and oxidative stress from the CEMP and include VTG in cod and flounder. Oxidative stress (two new methods), cellular energy allocation and aromatase are to be considered as appropriate methods for inclusion for the OSPAR JAMP/CEMP at the next ICES WGBEC meeting in 2007.

A review of integrated methods to assess effects on biota from lindane and BFRs and a review of the comet assay was undertaken but the group felt that further work on these topics needs to be undertaken intersessionally for the WGBEC meeting in 2007.

Reviewing progress with biological effects techniques - new developments

Progress with gene expression (transcriptomic) profiling was presented to the meeting. DNA microarrays have been constructed for four estuarine/marine species, using flounder, Atlantic salmon, striped sea bream and mussel and their use in monitoring programmes was elaborated with data from a study under GENIPOL and a study using blue mussel in Italy. The microarrays are now widely available for use and WGBEC were of the opinion that this is a rapidly advancing technology that is exciting and needs further take up and evaluation.

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1 Opening of the meeting

The Chair, John Thain (UK), opened the meeting at 10:00 hr on Monday 27 March 2006. It was noted that WGBEC had not met at the ICES headquarters for some considerable time and that it was a privilege to be invited to see the new facilities and to meet back to back with other ICES groups, WGSAEM, MCWG and WGMS. WGBEC were warmly welcomed to ICES by Vivian Piil and formerly in the latter part of the morning by the ICES General Secretary, Gerd Hubold. The Chair thanked ICES for hosting the meeting and also welcomed WGBEC participants to Copenhagen. The participants introduced themselves and their affiliations and described their area of interest and field of expertise. The list of attendees is given in Annex 1.

2 Adoption of the agenda

The Terms of Reference for the meeting can be found in Annex 2. The draft agenda (Annex 3) was adopted by the meeting and a tentative agenda timetable agreed. It was noted that Agenda Items 8 and 12 were direct requests from OSPAR and Agenda Item 4 was a request from ICES REGNS Study Group. Items 15 J1-3, were items that could possibly require input from other ICES WG meeting simultaneously at ICES.

3 Appointment of Rapporteurs

Principle Rapporteurs were appointed for the Agenda Items and are given in Annex 4.

A list of documents used at the meeting is given in Annex 5.

4 REGNS integrated assessment of the North Sea

Background documents available for this item included the request letter from the ICES REGNS Chair and a presentation of the results of the overall North Sea assessment conducted during Phase I of REGNS.

The group went over the presentation of the overall assessment and some comments were made regarding the assessment scope and methodology employed. It was noted that although many parameters had been included in the assessment, contaminants and their effects were not represented. Rather the assessment had been restricted to biological and hydrographic parameters and the overall conclusions of the assessment were related to these. Some concern was expressed regarding the level of data aggregation, annual averaging and the potential for correlating parameters not causally related in such an assessment.

It was noted that the sub-regional data tables provided by REGNS prior to the WG meeting did not currently contain any biological effects data.

It was considered by the group that biological effects of contaminants should form an important component of an integrated ecosystem assessment; however the data-sets available to WGBEC do not match the spatial and temporal scale that seems to be required for REGNS. The spatial distribution of the available data is concentrated near the coast or around localised offshore contaminant sources, rather than evenly distributed across the region. Long term biological effects time-series of the scale considered in the overall assessment are also lacking. Imposex/intersex and fish disease data-sets were highlighted as having the greatest spatial and temporal coverage.

WGBEC would be willing to work with REGNS on an assessment of the data-sets collated during 2005 and presented to the REGNS workshop in 2005. The spatial extent of these data

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types can be seen in the WGBEC 2005 report (Section 4.3). These data are available partly on the ICES database, but some are held nationally. None appear on the sub-regional data tables.

Biological effects data will be relevant to the forthcoming thematic assessment on ‘chemical pollution’. Although much data are available, they are largely related to local or specific issues and an assessment of data aggregated by the sub-regional ‘eco-hydrodynamic units’ proposed from the overall assessment would not be appropriate. WGBEC need more information on the likely structure of the thematic assessment on ‘chemical pollution’ before further data-sets for assessment could be identified. Local data-sets concerning biological effects along contaminant gradients in coastal and offshore (near oil and gas installations) for example may be appropriate if the thematic assessment was structured to accommodate it. Much of these data are held nationally rather than centrally and there would be a lot of effort associated with collating it for REGNS.

Action

The Chair of WGBEC will discuss with the REGNS Chair whether there would be value in a member of WGBEC attending the REGNS workshop in May.

Recommendation

WGBEC would like more information from REGNS on the proposed structure of the thematic assessment on ‘chemical pollution’ to determine the value of submitting data for assessment.

5 Assess the amount of biological effects data on the ICES database and develop a means to encourage further submissions as well as the use of the data

A summary of the biological effects data held on the ICES database was provided by ICES and presented as Table 5.1 below.

It was noted that the amount of biological effects data on the ICES database has increased since the last review by WGBEC in April 2005, however overall, the amount of data remains limited and is still mostly consists of imposex parameters. Additional data received in the past year consisted of imposex data from Norway (for 2004), Denmark (for 1998–2005) and the United Kingdom (for 1987–1991, 1993 and 1995), intersex data from Netherlands and United Kingdom (for 2002–2004 and 1997–2000, respectively) and bile metabolite data from Norway for 2000–2004. Whilst this is an improvement on the situation last year it still does not reflect the amount of biological effects work undertaken by OSPAR contracting parties and will continue to hamper effective data assessments for the OSPAR area as a whole. Lack of transformation programmes for national monitoring data remains a major problem for ICES submissions. Moving to free formats would help overcome this problem.

Contracting parties were encouraged to input available imposex data by 25 August 2006, in time for the next MON assessment.

The following issues were noted with the current datasets and where appropriate amendments will need to be addressed and data resubmitted where required.

• The use of invalid parameter codes, e.g. IMPOSEX_SH; • A reduction in the VDSI and RPSI data for Denmark (due to corrected re- submissions of data); • Miss-interpretation of record hierarchy for version 3.2 resulting in additional station counts.

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Table 5.1: Summary of the biological effects data held on the ICES database.

NUMBER OF STATIONS IN DATA SUBMITTED TO ICES COUNTRY PARAMETERS 1987 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Imposex/intersex Denmark IMPF% 11 9 11 34 18 11 INTSI 3 LNFPR 3 PCI 11 7 7 19 17 10 RPLI 14 % STERF 3 VDSI 11 8 11 37 18 11 Netherlands INTSI 2 3 2 7 Norway IMPS 5 6 6 13 8 9 9 20 IMPOSEX_SH 5 6 6 13 8 9 9 20 LNFPE 5 6 6 13 8 9 9 20 LNMPE 5 6 6 13 8 9 9 20 United Kingdom IMPF% X X X X X X INTSI X X X X PCI X RPSI X X X X X X X X X X X X X X % STERF X X X X X X X X X X VDSI X X X X X X X X X X X X X X others Norway ALAD 7 9 11 13 14 4 4 4 ABS380 9 11 13 14 4 4 4 BAP3OH 13 4 4 4 4 EROD 7 9 11 13 14 4 4 4 MT 7 9 11 13 14 4 NAP2OH 12 PA1OH 12 14 4 4 4 PYR1OH 9 11 13 14 4 4 4 United Kingdom EROD X X X X X X X * Note: Number of stations not given for UK as submissions include duplicate station records.

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ICES requested that WGBEC produce an example data submission file for biological effects data to aid data submitters. This was undertaken jointly with ICES using a UK v3.2 data submission as the basis and will be uploaded to the ICES website on completion.

Action

In order to assist data submitters, an example of a data submission file for biological effects data will be produced and uploaded to the ICES website. This will be undertaken by Jacqueline Jones and Marilynn Sørensen.

Recommendation

In order to encourage submission of data to the ICES database WGBEC recommends that ICES move to free formats as soon as practicable

6 Review progress with the TIMES

WGBEC commissions the preparation and publication of biological effects methods, appropriate for use in monitoring programmes, particularly in the OSPAR maritime area. Methods are also registered with the ICES database to allow data submitted for these techniques to be associated with a standard protocol. To date ICES WGBEC has commissioned and published 16 methods, available via the ICES website.

One method was published in 2004 (Freek Ariese, Jonny Beyer, Grete Jonsson, Cinta Porte Visa and Margaret M Krahn. “Review of analytical methods for determining metabolites of polycyclic aromatic hydrocarbons (PAHs) in fish bile”).

In 2005 there were five methods outstanding that had been identified as requiring publication but had not yet been commissioned or completed. These were: 1 ) Scope for growth (commissioned, peer reviewed); 2 ) Antioxidant enzyme induction (commissioned, not completed); 3 ) Blue mussel histopathology (authors identified); 4 ) Gonadal histology of flounder (authors identified); 5 ) Extraction techniques and in vitro testing (authors identified).

At WGBEC 2005 Rob Roy (Canada) stepped down as biological effects editor of ICES TIMES and Matt Gubbins (UK) took over this task. Rob Roy would complete the Scope for Growth review and Matt Gubbins was to assume responsibility for new manuscripts.

Due to delays in finding an appropriate reviewer Rob Roy was unable to complete the review process for the Scope for Growth method and passed this task to Matt Gubbins in September 2005. Reviewers’ comments were returned to the author, John Widdows in January and amendments to the manuscript / responses to reviewers comments are expected back from the author by end of March 2006. The final document will be forwarded to WGBEC members for approval and then sent to ICES for publication.

The author for method 2 above on antioxidant enzyme induction, agreed to author the method, but has not produced a manuscript. Since it is proposed to remove oxidative stress from the OSPAR CEMP, pending further review of these methods by WGBEC, it is suggested to prioritise other methods for publication.

At WGBEC 2005, two further methods were put forward for commission. These were: a ) MDR/MXR in blue mussel (Angela Kohler et al.); b ) Histopathology in invertebrates (Brita Sundelin et al.).

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And two further methods were identified as requiring revision: c ) Oyster embryo bioassay (John Thain); d ) EROD determination (Matt Gubbins).

The method documents a) and b) above are under preparation. Angela Kohler indicated that she has identified co-authors and intends to write a review of methods for MDR/MXR, to include activity assays and potentially protein and mRNA analysis. Following discussion it was decided that a single standardised method, rather than a review of available methods would be most useful for ICES TIMES. Therefore the method document will concentrate on the Calcein-AM dye efflux method of MXR activity determination. This document is expected to be completed in early 2007. Brita Sundelin and co-authors are nearing completion of a document describing methods for the determination of embryo aberrations in amphipods under method b) above. Method c) concerning OEB is currently under review in the UK and the TIMES document will be updated on the basis of this review for next year (mid-2007). Method d) concerning EROD activity requires revision to take account of the option to analyse microsomal fractions. This will be addressed by Matt Gubbins this year.

The requirement for new TIMES method documents were considered by reviewing the table at 6.1 below (adapted from WKIMON II, annex 19). Sediment pore water bioassays and reproductive success in fish stand out as methods recommended by WGBEC and adopted under OSPAR JAMP but do not yet have TIMES methods identified. Jakob Strand was identified as a possible author for the method on reproductive success in fish.

Table 6.1: Status of Biological Effects methods in OSPAR/ICES and availability of TIMES methods.

TECHNIQUE JAMP CEMP WGBEC TIMES CAT/STATUS RECOMMENDED General Biological Effects INVERTEBRATES Whole sediment bioassays Yes II Yes 28, 29 Sediment pore water bioassays Yes II Yes No Sediment sea water elutriates No Water bioassays OEB / Tisbe Yes II Yes 11 Lysosomal integrity NRR mussel Yes 36 MXR/MDR in mussels Yes No SFG in mussels Yes No AChE in mussels Yes 22 MT in mussels Yes 26 Histopathology in mussels Yes No

Benthic community analysis Yes Yes ?

FISH AChE Yes 22 Lysosomal stability Yes II ?? 36 CYP1A Yes II Yes 13, 23 Liver neoplasia/ hyperplasia Yes I – V Yes 38 Liver nodules Yes I – V Yes 38 Externally visible fish diseases Yes I - V Yes 19 Reproductive success Yes II Yes No

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TECHNIQUE JAMP CEMP WGBEC TIMES CAT/STATUS RECOMMENDED Metal-specific biological effects Metallothionein Yes II Yes 26 ALA-D Yes II Yes 34 Oxidative stress No

PAH-specific biological effects CYP1A- EROD Yes II Yes 13, 23 DNA adducts Yes II Yes 25 PAH metabolites Yes II Yes 39 Liver pathology Yes I - V Yes 38

TBT- specific biological effects Imposex/intersex in gastropods Yes I - M Yes 24, 37

Endocrine disruption Vitellogenin in cod Yes 31 Vitellogenin in flounder Yes 31 Intersex in male flounder Yes No

It was decided that the previously identified method concerning extraction techniques and in vitro testing (Vethaak, et al.) should be separated to cover standardised extraction methodologies, DR-CALUX and YES / YAS yeast screening methodologies. It will be necessary to consider the implications of publishing the patented DR CALUX assay before requesting a draft resolution for publication from ICES.

To summarise, the following methods in Table 6.2 are required as TIMES manuscripts. The methods concerning embryo aberrations in amphipods, MXR, EROD activity and extraction procedures for bioassays will require draft resolutions for publication in 2006.

Table 6.2: Manuscripts identified by WGBEC for publications in the TIMES series.

METHOD LEAD AUTHOR ICES DRAFT RESOLUTION? DEADLINE TO ICES Scope for growth John Widdows Already 1 June 2006 Oyster embryo bioassay John Thain Already September 2007 Embryo aberrations in Brita Sundelin et al. Needed 2006 October 2006 amphipods MDR / MXR Calcein – Angela Kohler et al. Needed 2006 Early 2007 am efflux EROD assay amendment Matt Gubbins Needed 2006 December 2006 Extraction techniques for Dick Vethaak / John Needed 2006 Early 2007 bioassays Thain Blue mussel Miren Cajaraville Needed 2007 discuss with author histopathology YES / YAS screen John Thain No Late 2007 DR-CALUX Dick Vethaak / John No needs consideration Needs consideration Thain Reproductive success in Jakob Strand No Late 2007 eelpout Gonadal histology of Steve Feist No Post 2007 flounder

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Recommendation

Permission is requested from ICES to commission the list of methods in Table 6.2 above. Requests for publication of method documents concerning embryo aberrations in amphipods, MXR, EROD activity and extraction procedures for bioassays will be put forward as Draft Resolutions in 2006.

7 Consider progress with activities: i) BEQUALM, ii) EU FIRE Project iii) Prestige oil spill, and iv) BSRP/Baltic activities

7.1 BEQUALM

Background

A presentation on the Biological Effects Quality Assurance in Monitoring Programmes (BEQUALM) scheme was given on behalf of the BEQUALM Project Office, Centre Environment, Fisheries and Aquaculture Science (Cefas), UK. The self-funded scheme was launched in September 2004 following a three-year research phase funded by the European Commission’s Standards, Measurements and Testing programme. The costs of running the self-funded scheme are recouped entirely from registration fees paid by participants.

BEQUALM was set up to develop protocols and quality standards for a range of biological effects techniques and devise an AQC framework for monitoring compliance of laboratories generating data from these techniques for national and international monitoring programmes. The primary driver is the OSPAR JAMP CEMP, but the techniques used are also applicable to other monitoring programmes run by HELCOM and MEDPOL. All OSPAR JAMP CEMP biological effects data submitted to the ICES database by OSPAR contracting parties should have accompanying QA provided by BEQUALM, and in the case of imposex this is provided by QUASIMEME. This is necessary to allow assessment of data across the whole OSPAR area. Data submitted to the ICES database without appropriate QA may be down graded or not used in OSPAR assessments. External QA/QC is also required for biological effects techniques used for regulatory purposes, and to this end these types of assays have been included as part of the BEQUALM repertoire. The Water Framework Directive will also require QA/QC for much of the data generated as part of the quality assessments for water bodies and subsequent monitoring, and BEQUALM is in a good position to accommodate this need. Indeed, with the introduction of the EU Marine Strategy it would seem sensible that OSPAR and WFD have consistent AQC procedures for biological effects measurements.

The BEQUALM scheme comprises three components:

• Whole Organism (bioassays and fish disease) - led by the Centre for Environment, Fisheries and Aquaculture Science (Cefas); • Biomarkers – led by the Norwegian Institute for Water Research (NIVA); • Community Analysis - led by the UK National Marine Biological Analytical Quality Control Scheme (NMBAQC).

The BEQUALM Project Office (Cefas) acts as the overall administrative and co-ordinating centre for the whole scheme. A website (www.bequalm.org) has been set up providing information on the activities of all components. Training CD-ROMs, developed for some assays as part of the EU funded programme are given to participants as part of the registration fee. These are updated as required to include e.g. changes in methodology and QC criteria as the scheme progresses.

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Whole organism component activities

The bioassays component has successfully conducted two rounds of intercalibrations in the first two years of the self-funded scheme. Four assays (Tisbe battagliai, Daphnia magna, Corophium volutator, Arenicola marina) were offered in Year 1 and a further two assays (Skeletonema costatum and Acartia tonsa) added to the portfolio in Year 2. For each bioassay a single intercalibration was conducted, using zinc sulphate as the reference material for the water column assays and Ivermectin-spiked sediment for the sediment assays. For fish disease assessment, two intercalibrations have taken place; one using a series of images sent electronically and another using a set of slides. This will be followed up in March 2006 by a five-day workshop to provide feedback for existing participants whilst simultaneously providing training for fish histopathologists, with the view to encouraging additional uptake to the BEQUALM fish disease programme.

The number of registrations for each assay in each year is presented in Table 7.1.1 Organisations from France, Germany, Denmark, England, Scotland, Northern Ireland, Eire, Netherlands, and Tazmania took part. In addition, the fish disease workshop is attracting participants from Canada, Spain, Norway, the Russian Federation and Switzerland. The Acartia was not taken forward due to lack of uptake. Acartia, together with Corophium and Skeletonema, is the PARCOM regulatory species for the Harmonised Offshore Chemical Notification Scheme. At the moment there is no official requirement from PARCOM for external quality control/QA for the toxicity tests that are being conducted as part of the risk assessment of chemicals used in the offshore oil and gas industry. BEQUALM has offered to give a presentation at the next meeting of EOSCA (European Offshore Speciality Chemicals Association) to promote the scheme and highlight the importance of external QA/QC for toxicity tests used within this regulatory context.

Table 7.1.1: Number of participants for each assay under the Whole Organism Component.

YEAR TISBE DAPHNIA COROPHIUM ARENICOLA SKELETONEMA FISH TOTAL ASSAY ASSAY ASSAY ASSAY ASSAY DISEASE 1 7 4 10 7 -- 6 34 2 5 8 7 5 8 (25) 33 (58)

(Numbers in brackets indicate participant numbers for the forthcoming fish disease workshop).

In 2005 the whole organism component was expanded further to include QA/QC for the Luminescent Bacteria Assay, with the University of Catalonia as the Lead Laboratory. An intercalibration is currently taking place with 27 participants from Spain, Netherlands, Belgium and the UK.

Biomarker Component Activities

NIVA successfully organised and conducted intercalibrations for EROD, CYP1A, VTG and protein analysis during November 2004 to November 2005. The number of participants for each assay is presented in Table 7.1.2. Organisations from South Korea, UK, Denmark, Germany, France, Croatia, Netherlands and Finland took part.

A call for expression of interest to participate in QA/QC exercises for additional assays will be made shortly. These assays will include Ache (acetyl cholinesterase), neutral red retention (lysosomal stability), the yeast oestrogen screen (YES assay) and DNA adducts.

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Table 7.1.2: Number of participants for each assay under the Biomarker Component

YEAR EROD CYP1A VTG PROTEIN TOTAL 1 13 5 5 9 32

Community analysis component activities

Benthos

In 2004, the NMBAQC extended the UK scheme into Europe by inviting organisations to participate in two of the five components offered – the “Own Sample” and “Ring-Test” exercises. Uptake from Europe has been very disappointing, with only one laboratory, from Germany, participating in the first year and one from Eire in 2005. (It should be noted that labs from several countries in Europe took part in the EU BEQUALM development programme). Each year, around 20 organisations from the UK participate in this scheme. Despite the OSPAR requirement for AQC for benthic analyses across the convention, laboratories are not signing up to the scheme. The reasons for this are not clear and the lack of uptake needs to be urgently addressed through the ICES working group and SIME. Despite being unable to generate interest in benthos AQC outside the UK, the NMBAQC is continuing to extend its remit, taking forward AQC requirements under the WFD by holding a workshop on fish sampling and is also about to embark on the first fish ring test. In addition, a workshop on epibiota is planned.

Phytoplankton

The Marine Institute in Dublin, under the auspices of NMBAQC/BEQUALM has recently conducted a Phytoplankton Enumeration and Identification intercalibration. The purpose of this is to compare the performance of laboratories engaged in national official or non-official phytoplankton monitoring programmes throughout Ireland and the UK. Six organizations, with a total of 16 analysts have taken part. Data is being collated and a report was sent out to participants in February 2006. A wash-up meeting/workshop is planned in March 2006 where the results of the intercalibration will be discussed. It is anticipated that in the next intercalibration, organizations throughout Europe will be invited to participate.

WGBEC welcomed the report and progress made by BEQUALM in its first year as a self- funding scheme. Although progress has been made WGBEC would also like to highlight to ICES/OSPAR that in certain areas, notably the benthic community component and for some biomarkers that the scheme has had very poor participation. Clearly such a position is not financially sustainable and it should be noted that if circumstances do not change then BEQUALM will not be able to provide the QA component for some biological effects measurements required by OSPAR (JAMP CEMP) for data submission to ICES.

Action

The Chair will contact QUASIMEME to clarify what is needed for the current state for PAH metabolite analyses.

7.2 EU FIRE Project

Dick Vethaak (Netherlands) presented the results on the flounder exposure studies carried out within the EU-FIRE project and recently published in the Credo cluster newsletter (www.credocluster.info/news.html). So far results for 3 studies were flounders were exposed for three months to hexabromocyclododecane (HBCD), tetrabromobisphenol-A (TBBPA), and for one month to 2,4,6-tribromophenol (2,4,6-TBP in environmentally relevant test setup have been reported. The following parameters were included in the risk analysis: general health and toxicity parameters (behaviour, survival, growth rate, and relative liver and gonad weight) EROD) and CYP 19 (aromatase, responsible for oestrogen synthesis) activities, and levels of

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thyroid hormones and vitellogenin (VTG) in plasma. All animals were examined histologically with emphasis on tissues of major endocrine importance (liver, kidney, gonads, and thyroid). Chemical analysis was performed on individual muscle samples from all fish for exposure assessment. In general no major endocrine effects of BFRs tested in fish were found. Subtle effects observed that related to TBBPA exposure were a mild increase in male gonads CYP 19 activity but without any evidence for estrogenic effects. Levels of unbound thyroid hormone (T4) in plasma increased linearly with internal TBBPA concentration, however, T3 levels were not affected and histology showed no signs of altered thyroid gland activity. A fourth exposure experiment using a pentaBDE mix is currently underway.

Another study within the EU-FIRE project showed that some PBDE congeners have the capacity to inhibit TCDD-induced EROD activity in primary cultured carp hepatocytes (Kuiper et al., 2004). PBDE-47 exhibited the strongest inhibition (down to 6% of the TCDD control value). This finding stresses the need for caution when interpreting EROD data on environmental samples.

Other Dutch work within and outside EU projects is in progress to develop an in vitro thyroid receptor assay, or thyroid related biomarkers.

7.3 Consider progress with national activities pertaining to the Prestige oil spill

Concepción Martínez-Gómez made a presentation explaining the preliminary mid-term results of the first two years (2003-2004) obtained by the Oceanographic Centre of Murcia (Spanish Institute of Oceanography, IEO) within the frame work of the DEEP project (Distribution, fate and effects of the fuel oil in the coastal zone affected by the Prestige oil spill. VEM 2003- 20068). Estibaliz Díaz made a presentation with the results of the IMPRES and Prestepse (VEM 2003-20082 CO&, 2003-2006) projects obtained by AZTI regarding of the Effects of the Prestige oil spill on commercially exploited fishes in the Basque Country.

During this year a special number of the Marine Pollution Bulletin will be published containing different studies and approaches for the assessments of the Prestige oil spill.

In addition, in 2005, the research groups involved in the Prestige Oil Spill (POS) research met at the “Symposium on marine accidental oil spill” in Vigo (Spain 13-16 July; Vertimar 2005: Strategic Action Symposium (MET)). During this meeting, all the groups involved in projects presented the current status of their work and this can be found as follows:

Oral presentations: http://otvm.uvigo.es/vertimar2005/orales.html

Posters: http://otvm.uvigo.es/vertimar2005/poster.html

The major findings concerning biological techniques that were presented in this Symposium are described in Annex 7.

WGBEC welcomed the reports and progress with the ongoing biological effects monitoring programme following the Prestige oil spill. The group felt that this was important work in terms of developing strategies for the use of biological effects techniques in oil spill scenarios and in understanding and interpreting this type of data.

WGBEC would like to be kept informed of strategies and results with this extensive monitoring programme in 2007.

7.4 Review BSRP/Baltic activities

Several background papers were tabled prior to the meeting in Copenhagen, predominantly on the output of the EU BEEP programme. However, presenters of the work were at the last minute unable to attend the meeting. The biological effects work previously undertaken in the

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Baltic and the current programme is of great interest to WGBEC and the group would welcome input and an overview of results and activities at its meeting in 2007.

8 Review the use of background responses in biological effects

The discussions in this agenda item is a continuation of the WGBEC-meeting in Reykjavik 2005, where it was recognised that settings of baselines/background response levels for relevant biological effect techniques are an important issue. At this meeting some basic principles for setting the baseline levels was proposed.

It is recognised that settings baseline/background response levels have an important role in integrating biological effect parameters into assessments of the environmental conditions in the marine environment. The general philosophy is that elevated levels compared to a background response indicate that unintended/unacceptable levels of biological effects caused by hazardous substances can occur.

At the meeting WGSAEM was invited to make comments to the discussion on the procedures for setting background response levels using the settings of background levels for contaminants and reference conditions for fish disease data as examples.

The approach for setting background levels for contaminants in the marine environment includes firstly that the background concentrations have to be set, secondly the criteria to assess levels close to background concentration has to be derived.

WGSAEM explained that expert judgement has been used for setting the background concentration levels for contaminants in the marine environment within the OSPAR region, and it was also stressed that setting these values is an evolving process, since it is based on a pragmatic approach, which includes the present knowledge and it involves some scientifically based compromises.

It was noted that the background response levels should be values, which seem reasonable for the entire OSPAR region, if possible. However, the values have also to be biological and chemical meaningful, and that the assessment criteria should be useful for interpretation of data from monitoring programmes.

In the example with the prevalence of fish diseases in the North Sea, and regional differences in levels and trends are observed in different ICES rectangles in the period 1980-2005. It was by WGPDMO suggested to use regional reference values to assess temporal changes since the disease prevalence differ between regions.

WGSAEM also stated that when compiling and analysing available data one has to be aware of that data can be biased by data from a single country/region, and therefore not reflecting the ideal reference conditions. The analyses of background levels should include some randomness in the selection of data used. It was recommended to weight stations equally in situations more data was available from one station than another.

WGBEC recognises that there is a further need to look more in to the data available for the different biological effect techniques. And in the selection and use of data it will be important to reduce the influence of confounding factors like species differences, seasonality etc.

It is important that the available data represent appropriate “reference sites” and that the criteria to include/exclude data from so-called “reference stations” at present must be based on expert judgments.

It is recognised that there is a need for absolute baseline values for the main monitoring species as there are variations also between reference sites. Such values may be identified

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through sampling the appropriate species in Icelandic waters. It was suggested that data from controlled exposure studies, for instance from mesocosms experiments, described in the open literature also can be included the analyses.

Because of interlaboratory variability some older data should not be included, because identical techniques and QA procedures have not been in place.

At the moment only assessment criteria for TBT-specific effects, i.e. imposex and intersex, in five gastropod species used in monitoring within the OSPAR region are in place.

UK and Norway presented first proposals for background response levels for whole organism techniques with Oyster Embryo, Amphipod, Polychaete, SFG mussel, External fish disease, Histopathology, Liver nodules, Hepato. fib. incl. and FCA as well as the sub-cellular techniques; EROD, Bile metabolites, DNA adducts, Ache, VTG, Intersex, Lysosomal latency, Metalothionein and ALA-D related to specific species. Denmark has proposed background levels for reproductive success in eelpout; see also WKI-MON report 2006 in agenda item 12.

Action

Form a subgroup (John Thain, Ketil Hylland, Jakob Strand and Dick Vethaak), compile the required data with support as available from other WGBEC members, start the process of developing baseline responses and draft findings for WKIMON and WGBEC in 2007. This will be communicated to the WGBEC members preparing documents on assessment criteria for specific methods.

Recommendation

WGBEC recommends that the relevant effect data now be compiled by an intersessional WGBEC subgroup with the aim of proposing baseline/background responses for the relevant biological effects techniques so that the proposal can be assessed at the meetings by WKIMON III and WGBEC in 2007.

The proposals be forwarded to WGSAEM for consultation and further development as appropriate at their meeting in 2007.

9 WGBEC to consider and assess the long term impact of oil spills on marine and coastal life as requested from OSPAR and provide a guidance document on the use of biological effects techniques for oil spill situations

OSPAR were requesting information about what is known about the long-term impacts of oil spills, taking account of the work that has been done following a number of recent incidents (Braer, Sea Empress, Erika, Prestige, Tricolor). Are there particular issues that we should be worried about? Or need to take action about at a generic level?

WGBEC formed a sub group to take forward this request. This group was led by Concha Martinez who had provided background documents on this item prior to the meeting.

A guidance document is presented below on how to use biological techniques in oil spill situations.

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9.1 Background

The degree and duration of damage from oil spills is a function of several factors: 1 ) the type of oil spilled (The greatest toxic damage has been produced by lighter oils in more enclosed areas); 2 ) the quantity and duration of the spill; 3 ) seasonal, oceanographic, and meteorological conditions; 4 ) nature of exposed biota; 5 ) habitat and substratum; 6 ) geographic location; and 7 ) type of spill cleanup used (Clark and Finley 1977).

Therefore, some adaptations to this guidance may be required to take into account the differences of each particular oil spill situation / scenario. Table 9.1 below shows the location of recent major oil spills, the type and amount of oil spilled and the area affected.

Table 9.1: A review of recent major oil spills re: nature of oil spill, type of oil and area affected.

DATE COUNTRY OIL SPILL TYPE OF OIL OIL SPILL AREA AFFECTED AFFECTED VOLUME 1969 EEUU Florida Nº 2 650–700 t West Falmouth area of Buzzards Bay, Massachusetts

1969 UK Hamilton Trader Nº 6 640 m3 Liverpool Bay

1970 Canada Arrow Nº 6 12 000 m3 New Scotland 1972 EEUU Argo Merchant Nº 6 5700 m3

1972 EEUU Tamano Nº 6 400 m3 Maine 1976 France Bohlen Petroleo crudo 6500 t Iroise Sea (Sein island) pesado 1976 EEUU Mobiloil Nº 6 640 m3 Columbia River 1978 UK Amocco Cadiz Light Arabian 22 3000 t Northern shores of Britanny (375 km France and Light Libian 40% of total shoreline) cargo was lost 40 000–50 000 sunk into tidal to the air sediments 1980 France Tanio Nº 2 6500 t Batz island 1984 Finland M/S Eira POR 180 250 t Gulf of Bothnia, Vassa archipielago1,500 km2

1984 EEUU Nestuca Nº 6 11 000 m3 Washington 1989 Alaska Exxon Valdez 38 800 t 1 200 miles of coastal area 470 miles away from the accident point 1991 Italy Haven Heavy Iranian 14 4000 t Ligurian sea, NW Mediterranean

10 000 tonnes sank to the bottom 1992 Spain Aegean sea Brent crude oil 80 000 t Galician coast 1993 UK Braer Gulflfaks 85 000 t Shetland Norwegian crude

1996 UK Sea Empress “Forties Blend” 72 360 t South Wales, 200 km shoreline crude oil and heavy fuel oil

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DATE COUNTRY OIL SPILL TYPE OF OIL OIL SPILL AREA AFFECTED AFFECTED VOLUME 1997 France Katja Nº 6 183 m3 Seine beach

Maritime beach

Calvados beach

1997 Japan Nakhodka Nº 6; Bunker C 5000 t 1200 km shoreline (linear km) heavy oil

1999 France Erika Nº 2 20 000 t North Atlantic ocean French coastal area 400 km

2001 Denmark Baltic Carrier Nº 6 2700 t

2002 Spain Prestige oil spill Nº 2 60 000 t Galician-Cantabrian Continental 2 shelf (30 000 km ) France (M-100) Spanish coastal area 2600 km

9.2 Strategy for using biological effects methods

Operational guidance to apply biological techniques before oil spill situations.

It was noted from the literature that assessments of short and long-term impacts are frequently hampered by a lack of pre-spill baseline data on resources, hydrocarbon levels in the marine compartments and background levels of biomarker responses or reference values of biological measurements. With the experience obtained in previous catastrophes, the assessment of the impact produced by an oil spill situation using biological techniques can be successfully established in those areas where previous background reference data sets exist. When these data sets do not exist, the only possibility to perform an impact analysis is through the selection of post spill reference/control areas, if this is possible.

Therefore, it is highly desirable for biomonitoring programmes in areas of greatest risk and threat from oil spills that baseline biological, chemical and biomarker data is obtained i.e. before any oil spill occurs. Typically these would be in maritime areas with a high risk of shipping accidents, major oil tanker traffic routes, major oil ports, and coastlines with economically important fisheries and ecologically important ecosystems.

In order to establish a baseline biomonitoring programme for an area it would be essential also to take into account oceanographic and climatic information (e.g. marine currents and wind predominance, nature of the bottom substrate, littoral geomorphology etc.).

Operational guidance in applying biological techniques in oil spill situations

In an oil spill situation there are generally three main aims: 1 ) Measurement of toxicity; to estimate the potential toxicity of the fuel oil and this is usually undertaken by laboratory experiments e.g. bioassays which are rapid and cheap. 2 ) Spatial effects; a study undertaken to estimate the extent of the magnitude of the damage on the marine ecosystems affected by the oil spill (a field sampling programme using biomarkers, bioassays in conjunction with analytical chemistry).

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3 ) Temporal effects; a programme of work to evaluate the course of time to recovery after the oil spill and the effectiveness of any policy measures taken (a field sampling programme using biomarkers, bioassays in conjunction with analytical chemistry).

It is imperative that after an oil spill situation, a monitoring programme using biological techniques must is initiated as soon as possible; otherwise information and assessment on the spatial and temporal effects resulting from the spill will be compromised.

Identification of spatial scale where organisms have been affected by the oil spill

In any oil spill situation it is important to assess the spatial scale of impact of the oil. This is made much easier if a baseline and background survey has been undertaken as recommended above. Exploratory sampling must take place as soon as possible after the oil spill to include areas where suspicions of pollution exit and also in assumed control areas. It is highly recommendable that this exploratory sampling is performed in a coordinated way with chemical and fisheries monitoring.

Data obtained during exploratory spatial monitoring (chemical and biological data) can then be used to design a strategic sampling programme. In general a detailed field annual sampling programme with rigorous statistical analysis is necessary to be able to demonstrate the environmental persistence or long-term recovery (perhaps up to 12 years). As an example a sampling programme of not less than four years has been recommended following the Prestige oil spill as this time frame is required to take account of medium-term fluctuations of biological responses using biomarkers.

In the programme design field samples must be collected from stations were biological response have been detected, but also in control stations (if they exist) and in those stations where though any initial responses have not been observed they are susceptible to be affected because the simulation of the spillage dispersion indicate that may be affected “a posteriori”.

The validity of a sampling programme will be determined by the degree of accuracy with which it represents temporal and spatial variability in “environmental quality” for the duration of the monitoring programme. In this respect relevant factors that must be considered include the following:

• Chemical composition of the oil; • Sampling location (degree of system homogeneity and hence the need for sample stratification, number of sampling locations, accessibility, and safety precautions); • Sampling time and frequency (system homogeneity over time, random and cyclic variations); • Estimated nature and magnitude of natural variation in the biological components to be measured; • Estimated nature and magnitude of the man-made impact under investigation; • Duration of sampling period—discrete or composite samples; • Economic and practical considerations; • Quality control.

After each sampling cycle, all results should be evaluated and assessed to identify weak points and inconsistencies that can be corrected to increase the quality and statistical robustness of the programmes.

The number of stations in a sampling programme will be governed by the spatial scale of the impacted coastline, the cost of conducting the programme and the predicted economic,

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ecological and environmental impact and importance. In any case, considerations of replicability in the sampling design must not be obviated, especially when the spatial scale were at regional scale. For every oil spill situation the sampling design will be unique and it must be statistically robust with specific aims, see Peterson et al, (2001) for further information.

9.3 Selection of appropriate target species

It is generally the case that biological techniques are applied to target organisms representative of the marine environment (coastal areas, inner shelf, middle and outer shelf) where the oil spill has occurred. There is also a tendency to use recognised monitoring species such as the blue mussel or commercial fish or species for which biological effects techniques are well documented.

The following organisms are proposed as useful target species in biomonitoring programmes in oil spill situations. However, particular situations may require the use of different species and in these cases their selection will be based on site specific monitoring criteria.

TARGET ORGANISMS BIOLOGICAL TECHNIQUE SPECIES

Molluscs Mussel (Mytilus edulis, M. Embryo toxicity Bioassays galloprovincialis) Biomarkers Clams (Crassostrea gigas) Flounder (Platichthys flesus) Fish exposure assays Fish Dab (Limanda limanda) Biomarkers Plaice (Pleuronectes platessa) Four-spotted megrim (Lepidorhombus boscii) Dragonet (Callyonimus lyra) Common sole (Solea solea) Hake (Merluccius merluccius) Mugil (Chelon labrosus) Grey mullet (Mullus barbatus)

Pelagic fish: anchovy (Engraulis encrasicholus) Whiting (Micromessitius poutassou)

Sea urchins Paracentrotus lividus Embryo toxicity bioassays

Amphipods Gammarus aequicauda Bioassays Gammarus locusta Corophium votulator Copépodos Bioassays

Algae Isochrysis galbana Bioassays

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9.4 Confounding factors

Consideration was given to confounding factors that need to be taken into account when designing a biological effects / chemical monitoring programme and in assessing data from a post spill monitoring programme. These confounding factors were:

• Migratory processes: behaviour of fish permanently residing in the affected area or passing through the affected area; • Absence of pre-oil spill data: i.e. it is quite difficult to judge whether the biomarker activities are on basal or induced levels; • Seasonal variations in temperature affect the biological activities in fish and may also affect the behaviour of the oil; • Temperature of the habitat also influences the time course of biological effect induction; • The maturation and reproductive cycle of fish, which may affect the enzymatic activities, has to be taken into account in both field and laboratory experiments.

9.5 Selection of appropriate biological effects techniques

Biomarkers

Some of the biomarkers proposed below are being used successfully as tools to identify biological responses after an oil spill situation. Others still require additional development before they can give a complete diagnosis of pollution effects in coastal environments. It is important to differentiate between those biomarkers which reflect acute response to acute exposure directly after the spill and those reflecting long-term effects of month and years.

Biomarkers of acute exposure are induction of biotransformation, glutathione- conjugating and antioxidant enzymes. Lysosomal membrane stability reflects the whole range of time scale in its response from very early to long term effects. Biomarkers measuring long-term effects putatively resulting in carcinogenesis and reproductive failure indicate prolonged oxidative stress, DNA damage, reduction of contaminant efflux and enzymes of steroid metabolism.

It is essential that a set of biomarkers should be used in conjunction with supporting analytical water, sediment and tissue chemistry and other biological measurements.

Bioassays

Bioassays are usually performed in oil spill situations in order to evaluate the toxicity of the water, sediments, the elutriates and the water-accommodated fraction (WAF). It should be noted that some studies have demonstrated the impact of natural light (UV) on the toxicity of elutriates and WAF (Allred and Giesy, 1985) and this may need to be taken into account in the assessment of field collected water samples.

The table below (Table 9.5.1) provides guidance on the current biological effects techniques that can be used in oil spill situations with special reference to; the type of measured response, the timescale of response of each method, the timescale of expected effect in the field, the organism used, the target tissue, the purpose of using the method and the citation for the prescribed method.

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Table 9.5.1: Application of biological effects techniques after an oil spill situation.

ACUTE EFFECTS BIOMARKERS TIMESCALE OF TIMESCALE OF ORGANISM TARGET TISSUE PURPOSE METHODS RESPONSE BY EXPECTED EFFECT METHOD IN FIELD PAH bile metabolites Hours Days to months Fish Bile Indicates exposure to Ariese et al., 2005. depending on PAH conditions Lysosomal stability Hours Days to months Mussel Blood cells Sub-cellular damage Moore and Lowe, 2004. depending on fish liver BEEP conditions Köhler et al., 2002 EROD activity Days Days to months Fish Liver Induction of Galgani et al., 1991 Induction CP4501 depending on detoxification conditions mechanism GST activity Days Days to months Fish Liver Induction of Habig and Jakoby, 1981 depending on Mussels Digestive gland detoxification conditions mechanism Antioxidant enzymes: SOD, , Days Days to months Mussels Digestive gland Induction of oxidative Karacoh et al., 1998 CAT, GPX and GR DT- depending on stress response Greenwald, 1985 diaphorase conditions Fish Liver enzymes Long-term effects Organism Target tissue Methods biomarkers Lipid peroxidation/oxidative Days Months Molluscs Digestive gland Cellular response to To be identified. stress fish liver oxidative stress Lysosomal stability Hours Months Mussels Digestive gland Interference with Moore and Lowe, 2004. Haemolynpha steroid metabolism / BEEP effects of reproductive performance / Fish Liver endocrine disruption Köhler et al., 2002 ED effect Hours / days Months Imposex in mussels Gonads To be identified Aromatase inhibition Fish Vitellogenin in males serum MXR activity inhibition Days Months Mussel Gill Inhibition of To be identified. fish Whole animals contaminant efflux at sub-cellular level Micronuclei Days Months Mussels Digestive gland Genotoxic damage Olive D.L. 1988; Akcha et al., 2002; Reichert et al., Fish Liver 1999; Singh et al., 1988; Aas et al., 2000; Sánchez et

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ACUTE EFFECTS BIOMARKERS TIMESCALE OF TIMESCALE OF ORGANISM TARGET TISSUE PURPOSE METHODS RESPONSE BY EXPECTED EFFECT METHOD IN FIELD al., 2000; Taban et al., 2004; Nishikawa et al., 2005; Baršienė, 2002; Bagni et al., 2005; Baršienė et al., 2006b). Enzyme altered foci 1+ years 1+ years Fish Liver Initiation of To be identified. carcinogenesis Histopathology 1+ years 1+ Years Fish Target organs: including Tissue damage / Feist et al., 2004; Köhler, Mussels liver, gills and gonads neoplastic disease 2004.

Bioassays Organism Toxicity of fuel SFG Days Years Whole organism Physiological To be identified Mussels (M. edulis) impairment

Embryo-larval test Days Days to weeks Sea urchin Whole organism Mortality and Thain, 1991. (on field samples) depending on (Paracentrotus deformity in embryos conditions lividus) Mussels (M. galloprovincialis) Oyster (Crassostrea gigas) Algal growth inhibition test Days Days to weeks Isochrysis galbana Whole organism Mortality To be identified depending on conditions DR CALUX Hours Days to months GMO In vitro Measure PAHs To be identified (on field samples: water, depending on sediment, tissue extracts) conditions Sediment bioassays: Weeks Days to months Whole organism Mortality and Corophium spp. depending on behaviour Arenicola spp. conditions

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Action

The above review and guidelines suggested by WGBEC will be forwarded to the OSPAR Secretariat (Richard Emmerson).

Recommendations 1 ) The above guidelines are recommended for use in oil spill situations and need to be used as appropriate for the type of oil spill, the conditions under which the oil spill occurred and the variable locations (i.e. type of coastline, hydrography, etc.). Therefore the methods need to be used in a fit for purpose manner and due consideration should be given to: • Type of oil; • Duration of spill; • Geographic extent of spill; • Type of habitats (rocky shore / mud / ice); • Weather and hydrographic conditions; • Type of clean up operations employed; • Socio-economic considerations e.g. aquaculture or sensitive ecological areas vs. offshore; • Statistical robustness to sampling design; • Appropriate reference sites need to be identified; • In areas of high risk to oil spills it is essential to establish baseline responses for the methods to be used; • Appropriate integration of sampling with chemistry should be conducted according to the integrated guidelines under development (WKIMON); • Toxicity profiling using specific biomarkers / bioassays of the major oil types transported in the ICES / OSPAR area should be conducted. 2 ) For the major oil types transported in a given region, toxicological profiling / hazard identification using bioassays should be employed to determine the responses to the given oil types. The importance of undertaking this research is to establish the applicability of biological effects techniques (bioassays and biomarkers) for risk assessment, management and policy purposes after oil spill accidents.

10 Provide expert knowledge to the ICES Data Centre as appropriate

Jacqueline Jones liaised with the ICES Data Centre and co-ordinated this agenda item.

ICES informed WGBEC that its recommendation at WGBEC 2005 for a sub-group to be created within B-MBA for PAH-related parameters was not possible.

The additional parameter codes requested for the cytochemical method for lysosomal stability were agreed with ICES and added to the RECODE database.

The group reviewed the minimum fields and standard parameters recommended at WGBEC 2005, to ensure quality and comparability of biological effects data submitted to the ICES database. It was agreed that separate lists for recommended fields should be compiled for biological effects in biota, sediment and water (Tables 10.1–10.3).

These tables are found in Annex 8:

Table 10.1 Recommended data fields for biological effects in biota

Table 10.2 Recommended data fields for biological effects in sediment

Table 10.3 Recommended data fields for biological effects in water

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Recommended parameters and cofactors

The group reviewed the parameters and co-factors recommended for biological effects data at WGBEC 2005. These were revised for each technique. The details will be provided in a table and be supplemented with explanatory text intersessionally.

Specifications for data screening

There is a need to check for standard parameter combinations.

Action

John Thain, Matt Gubbins and Jacqueline Jones to check for standard parameter combinations and specifications for data screening; to be carried out intersessionally.

11 Evaluate the WKIMON 2006 workshop and feedback from SIME (Feb 2006 meeting)

An introduction to the WKIMON process was given to the group by Ketil Hylland (Norway, WKIMON I/II Co-Chair). He highlighted the need for WGBEC to give guidance to WKIMON on:

• which biological effects methods are appropriate for inclusion in such a programme; • integration (both design and assessment); • assessment criteria.

WGBEC discussed the different aspects of methodology. Aldo Viarengo (Italy) described a strategy under development in MEDPOL countries, in which a tiered approach was used with a single method (lysosomal stability) as a primary screening tool. In that strategy, further analyses were performed if there was a response for lysosomal stability (see Agenda Item 14.3 for more details).

Selection of methods

A range of desirable properties for relevant methods were identified:

• It is essential that methods have an ability to separate contaminant-related effects from influence by other factors (e.g. natural variability, starvation); • The methods used should ideally have an ability to predict effects on “ecosystem health”; • They should be sensitive to contaminants, i.e. provide “early warning”; • “general health” of the organism should be reflected in at least one of the methods selected; • The range of methods used should include a range of mechanisms of toxic action, e.g. estrogenicity/androgenicity, carcinogenicity, genotoxicity and mutagenicity.

Using the list of methods recommended by WGBEC, a general design could be established for both fish (Figure 11.1) and blue mussel (Figure 11.2). The design includes biological effect parameters, chemical determinands and support parameters. Some of the methods for fish are already included in the CEMP (indicated in the figure). All methods are recommended, but a more limited set is seen as required to achieve the aim of an integrated understanding of effects. For biological effects such core methods include the concentration of PAH-metabolites, CYP1A/EROD induction, lysosomal stability, liver histopathology, acetyl cholinesterase inhibition (Ache), the Comet assay and plasma vitellogenin concentration (indicated in Figure 11.1). In addition, some physiological characteristics of individual fish would be required, e.g. GSI, LSI and condition factor. The remaining biological effects methods may be included as appropriate and may at a later point in time be included in the core programme. See section below for a discussion on mechanisms for keeping a guideline continuously updated.

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Figure 11.1: Overview of methods to be included in a programme for selected fish species. (Blue: included in CEMP; solid-lined boxes: prioritised components (only applies to tissue and subcellular responses); italics: ICES WGBEC promising methods).

Blue mussels are viewed by WGBEC as an important monitoring species, not least because of its ecological importance and its geographical coverage (basically the entire OSPAR, MEDPOL and HELCOM areas). A range of biological effects techniques for blue mussels have been recommended by WGBEC, but there are also methods viewed as appropriate for integrated chemical and biological monitoring on the list of promising methods. The core methods suggested for blue mussel are scope for growth, histopathology, metallothionein concentration, lysosomal stability, Ache inhibition and the Comet assay.

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Figure 11.2: Overview of methods to be included in a programme for blue mussel; (Blue: included in CEMP; solid-lined boxes: prioritised components (only applies to tissue and subcellular responses); italics: ICES WGBEC promising methods).

Integration

Discussion on integration within WKIMON was initiated by a presentation by the Chair. As was indicated in the WKIMON I report, there are different levels of integration in such a programme. Above all, it is important that methods are chosen to achieve some extent of ecosystem integration. This would be achieved by including basic components describing the state for water, sediment and biota (Figure 11.3). A second level of integration is ensuring that sampling is co-ordinated. This would mean:

• samples for chemical analyses and biological effects should be taken from the same individual; • hydrography should be done at the same sites and preferably same time as other types of sampling; • sampling of sediment, blue mussel and fish should be done in the same area whenever appropriate.

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Figure 11.3: Overview of components in an integrated monitoring programme; solid-lined boxes – prioritised components.

Assessment

Results may be assessed either (i) using assessment criteria for single methods or (ii) using assessment criteria for sets of methods. In both cases results would have to be integrated at a higher level to provide an ecosystem health assessment, much as that developed for the UK Fullmonti programme (see SIME 2006 summary record).

Ideally, biological effects methods that are associated with similar mechanisms of action should be assessed together. Our knowledge of responses is not sufficiently advanced at this time to facilitate this approach however so methods will need to be assessed individually. This will mean that results for individual biological effects methods will be scored in three categories: unaffected, moderately affected and strongly affected. As used in Fullmonti, results from each will then need to be weighted according to ecological relevance and combined to yield a total score. This process will not necessarily entail simple addition, but will more probably be of the form “at least two strongly affected” or “not more than three moderately affected”. Guidance on how this process should be run needs to be developed.

A full integration for an area, i.e. assessment of ecosystem health, will need results for sediment toxicity, fish biological effects, mussel biological effects and hydrography. Hydrography data (temperature, salinity, stratification) will be used in the interpretation of biological effects data. Chemistry data (for selected components), sediment toxicity, fish biological effects and mussel biological effects will each contribute a score from a process as outlined above. The final output from this process will thus be four scores (numbers). A mechanism needs to be established for the adjustment of limits between the different categories following some years of application in monitoring programmes.

Short background documents with assessment criteria are needed for the methods included in the guideline, but it is most urgent for the core set of methods. WGBEC members indicated their willingness to contribute to this process with a deadline of September 2006.

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Updating guidelines

WGBEC noted that some existing guidelines were outdated (as commented on by WKIMON II). It is seen as vital that a mechanism for continuous update of methodology and guidelines is established. One option would be regular (e.g. every second year) updates of available methods from the WGBEC recommended methods list.

A demonstration programme

As indicated by SIME (Summary record 2006), earlier international workshops (e.g. Oslo, Bermuda, Bremerhaven and BECPELAG) have been found to be very useful in improving our knowledge of biological effects methods. Following the establishment of integrated guidelines it would be useful to test the appropriateness of the guideline in a demonstration programme. This programme would need to co-ordinated by e.g. WGBEC and should include at least one station in each country. A full programme, including fish, mussels and sediment, should be included for at least four stations within the demonstration programme. Following adoption of a draft guideline in 2007, such a programme could be run in 2008.

Recommendations 1 ) Brief background documents with assessment criteria need to be developed for selected methods in time for WKIMON III. The deadline for documents is September 2006. 2 ) A document needs to be prepared on an integration strategy before September 2006. 3 ) A demonstration programme for the integrated guideline should be prepared for 2008.

12 Assess the development of the CEMP Guidelines

12.1 Respond to WKIMON/SIME: Are biological effects methods appropriate to meet the requirements of the OSPAR JAMP?; and

12.2 What methods need to be developed?

The issue of how to define the role of biological effects methods under the CEMP was considered at WKIMON and OSPAR SIME, 2006. It was agreed by SIME that: e ) biological effects techniques are included under the CEMP to address the following JAMP Hazardous Substances questions: i ) What are the concentrations in the marine environment, and the effects, of the substances on the OSPAR List of chemicals for priority action (“priority chemicals”)? Are they at, or approaching, background levels for naturally occurring substances and close to zero for man made substances? ii ) Are there any problems emerging related to the presence of hazardous substances in the marine environment? In particular, are any unintended/unacceptable biological responses, or unintended/unacceptable levels of such responses, being caused by exposure to hazardous substances? f ) in order to address these questions the techniques should be deployed: i ) for investigative monitoring or screening; ii ) as an exploratory tool for identifying both the effects of contaminants not included in the monitoring programme and combined effects of contaminants; iii ) as part of an integrated chemical and biological effects monitoring programme.

WGBEC noted the request from WKIMON / SIME and addressed this issue by reviewing the tabled CEMP methods ‘Biological effects technique review and status’ in Annex 8 of the OSPAR SIME 2006 summary record (and Annex 20 of the WKIMON II report). Methods were considered by the group for their suitability for inclusion under the CEMP and the table of methods was updated with respect to

ICES WGBEC Report 2006 | 29 whether techniques were proven / widely available, of value across the OSPAR area, and whether monitoring data, AQC and assessment criteria were available.

As a result of this discussion, sediment sea water elutriate bioassays and oxidative stress are recommended to be removed from the CEMP. In the case of sediment elutriate bioassays this was primarily because pore water bioassays are preferable techniques and now available and in use. Oxidative stress was removed because there is a lack of methodology, QA and clear demonstration of a response to contaminants in field monitoring situations. Members of the group pointed out that two methods for determining oxidative stress (Lipofuscin and Glutathione Reductase) were particularly robust and well tested and should be considered again for inclusion in the OSPAR JAMP next year. Steven George (UK) and Angela Kohler (Germany) agreed to put forward the case for inclusion of these methods at WGBEC 2007.

It was also noted that VTG (in male cod / flounder) is a proven, widely available method, of value across the OSPAR area and much monitoring data is already available. It was proposed to include this method in the OSPAR CEMP.

Cellular Energy Allocation will be reviewed and considered for inclusion at WGBEC 2007. Ketil Hylland (Norway) agreed to present the case for this method to the group at the next meeting. Recent developments with the use of aromatase in fish were raised and this method will also be considered for adoption as both an ICES recommended technique and for inclusion in the OSPAR JAMP at the next meeting. Dick Vethaak (The Netherlands) agreed to present the case for Aromatase at WGBEC 2007.

During the revision of the CEMP tables it was noted that the ‘scope’ column was misleading as several methods were listed as metal or PAH-specific, when they are known not to be. Methods listed in Appendix 7 of the CEMP under metal and PAH-specific methods were adopted to answer specific JAMP questions regarding these two groups of contaminants. It should be noted that although these methods are appropriate to monitor for the effects of metals / PAH, with the exception of ALA-D and PAH bile metabolites, none of the methods are, in fact, contaminant-specific.

The proposed revisions to the CEMP tables are given below (Table 12.1).

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Table 12.1. Recommended revision to the status of biological effects methods in the CEMP.

TECHNIQUE SCOPE WGBEC PROVEN AND WELL APPLICABLE CONSIDERED TO DATA FOR THE GUIDELINES/ AQC ASSESSMENT CURRENT CONCLUSIONS RECOMMEND WIDELY DOCUMENTED ACROSS BE OF VALUE TECHNIQUE TECHNICAL ANNEX CRITERIA CEMP FURTHER WORK AVAILABLE OSPAR MA ACROSS THE AVAILABLE UPDATING REQUIRED CAT/STAT NEEDED OSPAR MA US INVERTEBRATES Whole sediment General Yes Yes SIME 6/4/8 Assumed Yes Yes Update by B To be II bioassays Spain but test Secretariat developed by TIMES 28 organisms WGBEC and 29 not indigenous across region Sediment pore General Yes Yes SIME 6/4/8 Yes Yes Data Update by B Further II Extraction water bioassays Spain availability Secretariat development guidelines TIME 11 poor needed needed Water bioassays General Yes Yes SIME 6/4/9 Yes Yes Yes Update by B Further II Extraction OEB / Tisbe NL and UK Secretariat evaluation guidelines needed needed; species used needs to be clearly defined Scope for Growth General Yes Yes TIMES Yes Yes Data No TA No Need not AQC and imminent availability development currently assessment [John Thain poor in criteria need (UK) has CEMP development offered to develop a BD FISH CYP1A General Yes Yes [in prep] Yes – but Value Yes Update by B-a Need to be II Requires Belgium? no single questionable Secretariat developed review TIMES 23 species Lysosomal stability General Yes Yes No BD ? Yes Yes but as Update by [lead B-a Proposed at II AQC under TIMES 38 research country] WGBEC 05, BEQUALM programmes- background needs to be no external QA document to actioned. be prepared Background doc on proposed AC needed

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TECHNIQUE SCOPE WGBEC PROVEN AND WELL APPLICABLE CONSIDERED TO DATA FOR THE GUIDELINES/ AQC ASSESSMENT CURRENT CONCLUSIONS RECOMMEND WIDELY DOCUMENTED ACROSS BE OF VALUE TECHNIQUE TECHNICAL ANNEX CRITERIA CEMP FURTHER WORK AVAILABLE OSPAR MA ACROSS THE AVAILABLE UPDATING REQUIRED CAT/STAT NEEDED OSPAR MA US Liver neoplasia/ General Yes Yes SIME 6/4/5 Depends on Yes Yes Update by UK and B Further I – Vol. Species hyperplasia UK and DE availability DE development differences and TIMES 19 of target by AC need species WGPDMO 2 attention 006 Liver nodules General Yes Yes SIME 6/4/5 Depends on Yes Yes Update by UK and B Further I – Vol. Species UK and DE availability DE development differences and TIMES 19 of target by AC need species WGPDMO 2 attention 006 Externally visible General Yes Yes SIME 6/4/5 Depends on Yes Yes Update by UK and Yes Further I – Vol. Species fish diseases UK and DE availability DE development differences and TIMES 19 of target by AC need species WGPDMO 2 attention 006 Reproductive General Yes Yes SIME 6/4/2 No – No – relevant to Yes Update by lead B-a Further II Consider success DK dependent the Baltic area country evaluation adoption in on single only needed alternative species species outside Baltic Metallothionein Metal- Yes Yes SIME 6/4/4 Not clear Of limited use in Yes Update by Norway B-a Further II Evaluation of specific N fish to site to reflect updated evaluation genomic TIMES 26 specific issues method needed approaches to MT needed ALA-D Metal- Yes Yes SIME 6/4/5 Further Yes – where Pb Yes but limited Update by Norway B-a Further II Assessment specific N studies is considered an to Norway to reflect updated evaluation of criteria need needed issue method proposals by further work, Norway limited scope needed for successful external QA CYP1A- EROD PAH- Yes Yes [in prep] Yes – but Value Yes Update by B-a Need to be II Requires specific Belgium? no single questionable Secretariat developed review TIMES 23 species DNA adducts PAH- Yes Proven but [in prep] Yes –but Yes Yes but limited yes B-a Need to be II Cheaper specific not widely Belgium no single availability developed alternative available TIMES 25 species methods need development AC and AQC needed

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TECHNIQUE SCOPE WGBEC PROVEN AND WELL APPLICABLE CONSIDERED TO DATA FOR THE GUIDELINES/ AQC ASSESSMENT CURRENT CONCLUSIONS RECOMMEND WIDELY DOCUMENTED ACROSS BE OF VALUE TECHNIQUE TECHNICAL ANNEX CRITERIA CEMP FURTHER WORK AVAILABLE OSPAR MA ACROSS THE AVAILABLE UPDATING REQUIRED CAT/STAT NEEDED OSPAR MA US PAH metabolites PAH- Yes Yes TIMES 39, Yes – but Yes Yes yes Q-a Need to be II Assessment specific Background no single developed criteria require doc [in prep] species development Belgium? Liver pathology PAH- Yes Yes SIME 6/4/5 Depends on Yes Yes yes B Further I – Vol. Species specific UK and DE availability development differences of target by need to be species WGPDMO 2 addressed 006 Imposex/intersex in TBT- Yes Yes TIMES 24 Yes – Yes Yes yes Q Provisional I – M gastropods specific and 37 methods 2004-15 OSPAR and AC guidelines available for 5 species in OSPAR MA Vitellogenin in cod Endocri Yes Yes SIME 6/4/6 dependant Yes Yes [TA needs to be B-a for Proposals not Could be / flounder ne UK on species developed] cod from the UK currently applied to disrupto TIMES availability need in eelpout and rs document deveopment CEMP other species should be

investigated. AQC and AC need development

WGBEC recommended technique – of sufficient standing for OSPAR-wide deployment; SIME XX/XX/XX - Background document provided for SIME; TA – Technical Annex; CEMP Category rated I or II; V = Voluntary, M = Mandatory; B = Bequalm, B-a = available under BEQUALM, Q = Quasimeme, Q-a = available under Quasimeme

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Action

Oxidative stress (two new methods), Cellular energy allocation and aromatase are to be considered as appropriate methods for inclusion for the OSPAR JAMP/CEMP at the next ICES WGBEC meeting in 2007.

Recommendations 1 ) OSPAR is recommended to remove sediment sea water elutriate bioassays and oxidative stress from the CEMP and include VTG in cod and flounder. 2 ) OSPAR is invited to take account of the proposed revisions to the CEMP biological effects tables at 12.1. above.

12.3 Respond to WKIMON: review integrated methods to assess effects on biota from lindane and BFRs

Lindane

At present, there is only very limited information available on biological effects measurements which are specific for lindane (γ-hexachlorocyclohexane). It is possible that effects might be detectable by non-specific methods (e.g. fish liver histopathology). It might also be possible to exploit other known properties, such as the endocrine disrupting properties of lindane.

There is a paucity of information in mechanistic effects of lindane therefore an initial experiment using trancriptomic analysis of lindane treated flounder has been conducted. In common with effects of a pcb-mixture (arochlor 1254) there was an immediate very strong transitory induction of fibrinogen gene expression, however, there was no CYP1A response and contrasting responses of other genes. Lindane induced changes in expression of genes associated with Fe and haem metabolism, decreases in 5-ALA synthase and biliverdin reductase expression (induced by arochlor) and of cytochrome C and ferritin expression were accompanied by increases of the oxidative stress genes, CAT, THRXN, PRXN which may be a consequence of Fe2+ release. Interestingly, expression of the phase II xenobiotic metabolising enzymes ST and UDPGT were decreased and of most significant interest was the finding that TTR precursor expression was decreased. A diagnostic set of gene responses has been compiled and this preliminary study may provide useful clues for follow up mechanistic studies.

BFRs

As regards the applicability of biological effects methods for BFRs in field research/monitoring, a range of classical biomarkers such as CYP1A, Ah receptor, oestrogen/androgen receptors and DR-CALUX have been found not to be applicable. Most of the biological effects of flame retardants appear to be related to interference with thyroid hormones and possibly interference with the enzyme aromatase. The development of in vitro thyroid receptor assays, or thyroid related biomarkers is currently underway and require further validation.

In conclusion, it is still too early to suggest what biological effects methods should be included in integrated assessments for lindane and BFRs. To facilitate effect studies on lindane it is requested that lindane should be included in the list of chemical determinands.

Action

It was suggested that WGBEC will prepare a state-of-the-art paper on this issue including the open literature and the final results of the EU-project FIRE. Dick Vethaak kindly volunteered to provide such a review paper for next years meeting, Rolf Schneider would prepare a review paper for lindane and Ketil Hylland one on perfluorinated substances.

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12.4 Respond to WKIMON: Review of comet assay and is application

The comet assay is a technique to quantify DNA strand breaks and alkali labile sites on a single cell level. The extent of DNA damage is regarded as a measure for genotoxicity. The method is well known and is on the WGBECs list of promising methods. The method itself is quick and easy, however the evaluation of the comets can take some time. The collection of appropriate samples (e.g. blood) from marine fish may be problematic, because DNA damage can be easily induced in the samples during catch of the fish or storage of the cells. Therefore experienced people are needed to avoid incorrect sampling. It is also possible to perform the assay directly on board of a ship to avoid any storage of the samples at all (WGBEC 2006 background document 12d i).

Thierry Burgeot presented the results of a project on the relationship between the chemical contamination and the occurrence of DNA lesions in flatfish species from the eastern English Channel. During two cruises samples were collected by trawling in the Seine and in the Somme Bay in March and September 2001. At each site, juvenile and adult dabs of both sexes were sampled. The erythrocytes of the fish were investigated for DNA lesions using the comet assay. Gonad, muscle and liver tissue samples were also collected for chemical analyses of PAHs, PCB and other compounds. In the dab, significant effects of sex and age have been demonstrated in the number of DNA strand breaks. Whereas in March significant differences in the number of DNA strand breaks were observed between the sampling sites, no difference was shown in September. This difference in results could be explained by seasonal variations in the formation of these DNA lesions that could be attributed to variations in tissue chemical concentration. In fish, this latter parameter has already been shown to evolve in time with both lipid contents and/or spawning cycle. From a technical point of view, comet assay sensitivity was affected by high intra- and inter-individual variability that accounted for nearly 70% of the total variance. The site factor represented no more than 30%. This point was improved with a more elevated number of cells selected (75 cells per slide). The presented results have been published by Akcha et al. (2003, 2004). Comet assay as a rapid and simple method can be applied in field for monitoring. The main constraint is the time consuming slide scoring. Basal level and an automatically detection method with optical fluorescence microscope should be developed.

Matt Gubbins presented work conducted at FRS which demonstrated that the comet assay can be used to identify statistically elevated levels of damage in plaice erythrocytes from two areas with contrasting pollution levels (background document 12 d i). The recorded median percentage Tail DNA levels had an elevated level 2.2 % higher at the site with higher level of environmental contamination.

The causative agent(s) of the elevated damage have not been identified. Routine monitoring of sediment and plaice liver from both areas (in the sea off Colonsay (reference site) and at the Garroch Head dump site) informed this study of the general levels of PAH and CB contaminants which were considerably higher at the Garroch Head (up to 16263 µg/kg PAH and 2187 µg/kg PCB; Webster et al., 2005). Due to the skewed distribution of the tail intensity data, the suitability of a number of summary statistics proposed for the analysis of comet data was investigated. No significant difference was observed in the upper 90th and 95th percentiles. The median tail intensity of pooled data from Colonsay was 1.1% (n = 1550, range 0 to 94%) while a significantly (p<0.014) elevated level of damage of 3.3% was found at Garroch Head (n = 1350, range 0 to 89%). Variation between fish within sites was significant (p<0.001), irrespective of summary statistic. No relationship was observed between Comet tail intensity and fish length. Fish to fish variation within site was observed and it would be useful to record further fish parameters such as gender, disease and infestation status etc.

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Variability of comet data

Various laboratories use different methods to acquire data from the comet assay. Comets can be scored visually giving a measure of DNA damage or image analysis software can be used which enables various parameters to be simultaneously recorded. Published information provides guidance on scoring to ensure accurate results such as randomised scoring, not to take data from areas close to bubbles with the gels which could interfere with the results.

At BTS/UKEMS Spring congress 2006 it was discussed that when slides contain ‘hedgehogs’ that their numbers should be noted but not scored. A recent addition to the market place, is an automatic scoring system in which slides are loaded into a machine which automatically scores all comets on gels, enabling high throughput. Having seen this demonstrated the system may not be able to distinguish cells that are close together or overlapping and therefore generate erroneous results. Depth of field of the camera and the ability to focus comets at different depths in the gels is under consideration.

Choice of protocol

There are many variations in the comet assay protocols and some differences are necessary in terms of pH, electrophoresis conditions and buffers etc depending on the choice of cells being investigated and the information on genetic damage required. Some investigate work may be necessary to determine the suitability of individual buffers for the cell of interest.

Choice of parameters

The International Working Group on Genotoxicty Testing met in September 2005 and recommends reporting percentage DNA in the comet tail. Other parameter including tail length and tail moment are traditionally found in the literature and are also still currently reported. Tail length is influenced by electrophoresis conditions and non standardised conditions hinders inter laboratory comparison of data. There is still debate on the use of olive tail moment versus percentage DNA damage in the tail.

Confounding Affects on the assay

Cell preparation has to be consistent. Preparation should be under subdued/darkroom or safe lighting conditions to prevent UV induced damage. The assay is useful in determining DNA damage. In vivo and field work it may be difficult to state the causative agent for this damage.

Action

Ulrike Kammann, Matt Gubbins and Thierry Burgeot agreed to review the method before WGBEC 2007.

12.5 Respond to WKIMON: Compare across species (12e) and

12.6 Selection of species, gender and size range (12f)

A number of fish and molluscan species are used for environmental biomonitoring of biological effects of contaminants across the OSPAR area of interest. Comparability of response characteristics between species is clearly an issue that needs to be addressed in species selected for monitoring. Responses do vary between species as a consequence of differences in regulation of biochemical and metabolic functions resulting in differences in sensitivity. In some cases this will also reflect genomic differences as well. This argues strongly for limiting the numbers of species chosen for monitoring purposes; and also selection of representative species for which there is a good database.

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Methods used for assessing biological effects need to be generic and this is an especially important criterion for any new tests being developed, particularly in the development of DNA micro-arrays. Not only should tests be transferable between species as far as possible, but the informational capacity for interpretation of responses should also be generic. Interpretation of biological effects will also be facilitated by rigorous standardisation of animal size, gender and time of year for sampling the selected species.

Recommendation

The recommendation of WGBEC is that a limited number of specific fish and molluscs should be identified for monitoring purposes in the different OSPAR sub-regions and that generic testing be applied where possible in order to generate data that can be interpreted globally rather than locally.

12.7 Respond to WKIMON: Review background levels for ALA-D

Ketil Hylland had prepared a background document on ALA-D for the WKIMON II meeting. The group felt that this document covered the topic adequately.

13 Assess the development of the CEMP

This agenda item was not discussed as it had been covered in agenda items 12.1. and 12.2.

14 Any other business; Review progress with 1) genomics and proteomics 2) nanotechnology, 3) 2-tier approach to monitoring, and 4) amphipod decline in the Baltic

14.1 Review progress with genomics and proteomics

Presented by Steve George and Aldo Viarengo

The “omic” technologies for simultaneous profiling changes in expression of several thousands of genes, proteins and hundreds of metabolites in cells and tissues developed over the past decade have been applied to environmental toxicology in aquatic organisms over the past four years.

Genomics

Gene expression (transcriptomic) profiling has been carried out in 4 estuarine/marine species:-

European flounder (Platichthys flesus). Low density (120 gene) and high density (>7000 gene) liver DNA microarrays have been constructed and characterized (www.genipol.stir.ac.uk). One field study was performed with the low density array (Williams, T.D., Gensberg, K., Minchin, S.D, Chipman J.K. (2003) A DNA expression array to detect toxic stress in European flounder (Platichthys flesus). Aquat. Toxicol (65) 141–157)

With the high density (GENIPOL) array, gene expression profiles have been determined for test exposures to nine compounds (two PAHs, a PCB mixture, Cd, Cr, EE2, E2, lindane, PFOA, tBHP) and diagnostic gene sets have been computed for biological effects (www.genipol.stir.ac.uk). Analyses of immature male fish from six differentially polluted sites have demonstrated site differentiation (see Figure 14.1.1.).

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1.10 1.00 Elbe H 0.90 0.80 0.70 0.60 Helgolan Alde 0.50 0.40 0.30 0.20 Elbe

0.10 0 Ald -0.10 Tyne T -0.20 Elbe -0.30

-0.40

-0.50 Tyne H -0.60 -0.70 Tyne Team, -0.80

-0.90 PCA component 1 (54.71% variance) Helgo... -0.90 -0.70 -0.50 -0.30 -0.10 0 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10

Figure 14.1.1. PCA of microarray data from individual flounder.

Computational analysis of these gene expression profiles on the basis of the diagnostic gene responses for the test compounds has enabled classification of sites on the basis of the biological responses to these pollutants. For the UK sites where supporting chemistry and biomarker data are available, the conventional biomarker and genomic data are in agreement with the contaminant profiles. For practical use it is recommended that a simple classification scheme based upon statistical significances of responses is used (see Table 14.1.1).

Table 14.1.1: Classification scheme for comparing genomic data with contaminant profiles.

CLASS COMPOUND ALDE TYNE TYNE ELBE Reference HOWDEN TEAM HELGOLAND CUXHAVEN BRUNSBUTTEL PAH 3MC 0.06 0.06 0.25 0.06 PCB AROCHLOR 0.39 0.67 0.13 0.13 0.007 CH LINDANE 0.14 0.67 0.13 Perox.proliferator PFOA 0.09 0.06 0.005 heavy metal Cd 0.005 0.32 0.13 0.2 estrogenic E2 0.38 0.07 0.38 0.07 viral/bacterial infection furunc vaccine 0.19 0.5 0.01 0.05 0.00004 oxidative stress tBHP 0.15 0.0003

DIAGNOSIS no effect not significantly different from controls concern p>0.1 cf. test treatment severe impact p<0.1 cf. test treatment

Quantitative RTPCR methods for CYP1A, MT, SOD and VTG were developed for validation of the flounder microarray. These are suitable for long term status and trends monitoring are and an intercomparison with current TIMES methods for these biomarkers has been performed under the UK NMMP programme (George, S., Gubbins, M., MacIntosh, A., Reynolds, R., Sabine, V. Scott, A., Thain, J. (2004) A comparison of pollutant biomarker responses with transcriptional responses in European flounders (Platicthys flesus) subjected to estuarine pollution. Mar. Environ. Res. (58) 571–575). SOPs are available for deployment. Cross species applicability if the flounder array to other flatfish … Dab, Halibut, Plaice, Amercian Winter and Japanese Flounder has been established. The array is available for deployment in experimental and field conditions and take up is encouraged. Primary contact for use Prof. S. George (U. Stirling, UK)

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Striped sea Bream (Lithognathus mormaratus). A high density (5000 gene) liver DNA microarray has been constructed, characterised and tested with 4 prototypical compounds (PAH, PCB, Cd, E2). A limited field study has been carried out and cross species reactivity with a has been confirmed. The primary contact is Dr. M. Tom (IOLR, Haifa, Il). A higher density microarray is currently being developed for a related Sparid, Sparus aurata.

Atlantic Salmon (Salmo salar). A high density (17000) EST multi tissue DNA microarray has been constructed and is currently under test. For progress the primary contact is Dr. J. Taggart (U. Stirling, UK).

Blue mussel (Mytilus galloprovincialis). A low density (25 gene) DNA microarray has been constructed and responses to heavy metals (Cd, Cu. Hg), oil, H2O2 and field exposure (4 sites) evaluated. A medium density 1850 gene DNA microarray has been constructed (50% of spots are characterized) and responses to heavy metals (Cd, Cu, Hg mix), organic compounds (PAH, PCB, dioxin mix) have been tested. Preliminary field testing has been undertaken and test applications are encouraged. The primary contact is Prof. A. Viarengo (Alessandria, It). You

Proteomics

Studies using 2D PAGE analysis have been carried out on two marine (Atlantic salmon, cod) and one estuarine (flounder) fish species and responses to EE2, a PAH, a PCB mixture, Cd, PFOA , tBHP, EE2 and crude oil have been evaluated. Analyses of flounder from 2 field sites have been carried out. Whilst promising results have been obtained, due to the limited analytical capability (ca 1500 proteins) and necessity for de novo sequence identifications (MS) of proteins in animals with uncharacterised genomes (i.e. Our monitoring species), at the moment it is considered to be a research tool only and the group concludes that it should not be used in routine out biological effects monitoring.

WGBEC noted that the GENIPOL flounder DNA microarray and accompanying q-RTPCR procedures have been validated for field use and are now available for community use.

Recommendations 1 ) The the use of the above procedures in pilot studies and intecomparison with existing procedures is encouraged. 2 ) Similarly pilot studies should be performed with the blue mussel arrays.

14.2 Review progress with nanotechnology

Nanotechnology is a major innovative scientific and economic growth area, which may present a variety of hazards for environmental and human health. The surface properties and very small size of nanoparticles and nanotubes provides surfaces that may bind and transport toxic chemical pollutants, as well as possibly being toxic in their own right by generating reactive radicals. There is a wealth of evidence for the harmful effects of nanoscale combustion-derived particulates (ultrafines), which when inhaled can cause a number of pulmonary pathologies in mammals and humans. However, release of manufactured nanoparticles into the aquatic environment is largely an unknown. There are potential hazards associated with nanomaterials and possible harmful effects that may result from exposure of aquatic animals to nanoparticles. Possible nanoparticle association with naturally occurring colloids and particles must be considered together with how this could affect their bioavailability and uptake into cells and organisms. Uptake by endocytotic routes are identified as probable major mechanisms of entry into cells; potentially leading to various types of toxic cell injury. Uptake of C60 fullerene (“Buckyball” - carbon nanoparticle) has been demonstrated in hemocytes (phagocytic blood cells) of blue mussels (Mytilus edulis). These cells are the major component of the molluscan immune system and there were significant reductions in lysosomal stability (NRR – neutral red retention) after 60 minutes exposure of the cells at 1 and 10 µg.ml−1 of carbon nanoparticles. The

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higher level consequences for damage to animal health, ecological risk and possible food chain risks for humans also need to be considered in the light of known behaviours and toxicities for inhaled and ingested nanoparticles in the terrestrial environment. It is concluded that a precautionary approach is required with individual evaluation of new nanomaterials for risk to the health of the environment. Although current toxicity testing protocols should be generally applicable to identify harmful effects associated with nanoparticles, research into new methods is required to address the special properties of nanomaterials.

14.3 2-tier biomarker approach to monitoring

Aldo Viarengo gave a presentation on the 2-tier approach to monitoring using the blue mussel (see agenda item 11 above) and also outlined a proposal for taking this work forward (Proposal for the UNEP MAP biomonitoring program – Palermo Meeting, December 2006).

The proposal uses in situ deployments of caged organisms since it is believed that this is more effective in describing the relationship between biological effects and tissue concentration of chemical pollutants (i.e. in terms of the stress response in the organisms). The programme should mainly utilize caged molluscs (Mytilus sp. when possible). This choice is due to the biological characteristics of these organisms (filter feeding, lamellibranch, sedentary molluscs), and the fact that these organisms may be caged without feeding problems and may be cheaply transported to the laboratory in cold, humid boxes that guarantee animals in perfect biological conditions for the biomarker analysis

The proposal for the biomonitoring programme is based on a 2-tier approach and was described and presented to the group under agenda item 11 above.

Molluscs should remain in cages for 3-4 weeks, a period of time during which, despite the possible slight differences in temperature, food availability, etc. occurring at each site, it is possible to sample animals showing no changes in gonad development. Under these conditions, any observed changes in organism physiology can be related to the effects of toxic chemicals present in the water and accumulated in the tissue of these filter feeding molluscs.

The use of caged organisms in field studies allows a higher standardisation of the results and a simpler comparison of control organisms with animals collected at potentially polluted sites.

This strategy also permits a correct evaluation of the concentration of different chemicals accumulated in the organisms during the caging period. In fact a relationship between biological effects and pollutant concentration cannot be inferred utilising wild animals since harmful compounds may have a very different biological half life in these mussels, e.g. from days (copper) to months and years (cadmium, POPs respectively). In addition, it has to be taken into consideration that animals from wild populations may be at different stages of gonad maturity, therefore showing different biological responses and pollutant accumulation, for example PAHs and PCBs in the egg lipid envelop.

A second important point with regard to the planning of a “biological mussel watch” programme for any national or regional monitoring purpose may be the consideration of a two-tier approach which comprises:-

Tier 1. The possibility of using a highly sensitive and low cost biomarker, such as lysosomal membrane stability or lipofuscin accumulation, as a screening tool. The more polluted site will be evident by using also the end point of mortality during 30 days of exposure and survival in air (stress on stress response): 2 simple, low cost, well established end points.

Tier 2. Where lysosomal membrane stability (or another screening biomarker) has shown significant (up to 20% variation with respect of control values) changes that may be related to the effects of pollution, the set of biomarkers will be enlarged to a full battery of 6-8 biological effects techniques. Data generated in this manner may then be evaluated and integrated using

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tools such as an expert system (Narbonne et al., 2005) developed in the EU BEEP programme. This will also permit effects to be related to tissue residue levels in the mussels. The selection of the biomarkers is crucial in the utilization of the data, the development of an integrated stress index and the application of the expert system. In this respect a “limited set” of biomarkers can be used and may permit the expert system to rank the stress syndrome (from A = control to E = pathological status), but some expert judgement may be needed for selection of the most appropriate techniques. However, it is important to note that the expert sytem developed for the blue mussel permits the use of all the known blue mussel biomarkers (Dagnino, in press).

In addition a single microarray containing 25 genes related to stress response in the blue mussel has recently been developed and could be easily used as a single molecular analysis tool to rank pollutant-related stress. Interestingly, it is very simple and a low cost technique and may be of great value for deployment in biomonitoring programmes.

Recommendation

WGBEC could see value in the use of expert systems for multi-biomaker monitoring and would support such an approach. In addition, the development and application of mussel microarrays as a basis for future use in monitoring programmes should be encouraged, supported and data using these tools needs to be generated.

14.4 Amphipod decline and crash in the Baltic

Brita Sundelin gave a presentation on the decline of amphipod populations in the Baltic.

In the Baltic proper a decline of M. affinis populations has been observed since the 1970s and today abundance is only 1/10 of what it was 25 years ago. Crustaceans are the invertebrate group most susceptible to hypoxia [1] and hypoxia seems to have influenced the populations in the Baltic proper [2]. Besides the decline of both the freshwater amphipod M. affinis and the marine species P. femorata, the bivalve Macoma balthica and the priapulid Halicryptus spinulosus have increased, while Bylgides sarsi (former Harmothoe sarsi), sensitive to lower salinity has decreased. The reason to the change species composition is not verified but it is likely that both oxygen deficiency as well as lower salinity have affected the bottom fauna.

During this time span the Bothnian Sea amphipod populations have shown high abundances until 1998 when populations dramatically declined on Swedish coastal and offshore stations in Kvarken and Bothnian Bay. Southern Baltic Sea stations were affected in 2002 while a central station in the Bothnian Sea showed normal abundance until 2003. Sampling at this central sea station in January 2004 resulted in very few specimens, indicating that the collapse has extended to central Bothnian Sea stations. Finnish coastal stations do not show this dramatic decline so far (pers comm. Ari Laine). In 2004 offshore areas show some degree of recovery while coastal stations did not.

Normally the M. affinis populations show cyclic fluctuations in the Gulf of Bothnia covering 6– 7 years resulting in low abundance on individual stations during separate years, but this pattern of simultaneously low abundances on all coastal and offshore stations has never been seen before as far as we know. Today abundances may have declined to an extent where effects on the ecosystem are difficult to avoid. A similar decline of the closely related species Diporeia spp. has occurred in the North America Great Lakes. This decline coincides with the introduction and spread of the mussels Dreissena polymorpha and Dreissena bugensis but the mechanisms for the negative response have not yet been clarified. The dramatic population decline has been discussed during different ICES working group meetings e.g. working group for biological effects of contaminants (WGBEC) in March 2003 and ICES Baltic Sea Region Project, study group on Baltic Ecosystem Health (SEGH) in Nov 2003. Both the M. affinis and the Diporeia spp decline were discussed on specific session at the international limnology conference held in Finland 2004 (SIL 2004).

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In May 2003 a workshop with participation of Swedish EPA and Finnish and Swedish researchers was arranged in order to discuss possible hypotheses to observed decline. The following hypotheses were considered less plausible.

• New anthropogenic substances: The contaminant-sensitive variables in the national monitoring programme “embryonic development of M. affinis” show a decreasing trend in the Bothnian Sea until 2002 which during the last years this trend has stopped but frequencies are within background levels. • Hypoxia: Even if the bottom water oxygen concentration in the Bothnian Sea have decreased slightly during the last few years [3] the oxygen concentrations are way beyond concentrations negatively affecting M. affinis [4]. • Competition: In the Gulf of Bothnia the newly invaded polychaete Marenzelleria viridis is excluded, as increasing abundance of M. viridis at Swedish monitoring stations does not correlate to decreasing abundance of M. affinis and despite a very high abundances of M. viridis on the Finnish coast a similar amphipod decline has not been observed. • Predation. The density of M. affinis main predator Saduria entomon has decreased simultaneously during the corresponding period.

Until recently we hypothesised that food deficiency might be a plausible explanation since an increased outflow of fresh water to the Gulf of Bothnia were recorded during the last years. An annual average increase of 10-40 % above mean value between 1961-1990 was recorded from 1998 to 2001 and in 2000 the outflow was more than 200 % above the average during several months to the Bothnian Sea. Large river outflow resulted in increased deposition of terrestrial carbon rich in humus into the Gulf of Bothnia. The terrestrial carbon is essential in many aquatic food webs [5], but humus carbon is recalcitrant and not readily assimilated [6]. In addition increased outflow of brown-coloured humic particles may decrease the secci depth resulting in insufficient light for photosynthesising algae. In the Bothnian Sea an increased ratio between production of bacteria/autotrophic algae has been observed after the increased river outflow (Agneta Andersson, UMF Umeå University, monitoring data). Compensatory feeding is a response to such low quality food among gammaridean amphipods but despite increased feeding, declining growth and female gonad size was recorded [7].

Therefore we hypothesised that:

• Decreased amounts of high quality food i.e. diatoms in the spring phytoplankton bloom. • Increased percentage low quality terrestrial carbon from river outflow has diluted the high quality carbon in the sediment

M. affinis is a semelparous deposit feeder with sedimentation of phytoplankton as the main carbon source. M. affinis was suggested to feed on detritus and bacteria responding to phytoplankton [8, 9]. Recent studies reported that both Diporeia spp and M. affinis preferably feeds directly on fresh newly settled phytoplankton [10, 11]. Both amphipods feed intermittently and gut fullness is primarily observed after the spring phytoplankton bloom [12, 13]. Later studies on Diporeia spp. have shown that gut fullness declines after the spring bloom but increases later in the autumn [10]. Between periods of phytoplankton blooms the amphipods can cope without food or subsist on low quality food by using energy stored as lipids, facilitated herein by the low temperatures in their soft bottom habitat. The very low pre-spring-bloom ammonia excretion rate in M. affinis [14] further underlined the importance of stored lipids during periods of starvation.

However, sediment samples between 1996 to 2003 were stored in the freeze and to examine whether quality of sediment resources had changed during this period the sediment was analysed for the content of bioavailable amino acids, closely correlated to growth of deposit feeding bottom fauna, according to Mayer [15]. Nitrogen in the form of polymerized amino acids is

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generally the principal food source for deposit feeders. Mayers’ biomimetic approach is a recent technique that allows measuring nutritional quality of the sedimentary organic matter. The technique mimics the natural digestion that would take place in a deposit feeder by incubating sediments in vitro with the bacterial proteolytic enzymes. After the digestion, the non-digested nitrogeneous compounds are eliminated and the dissolved amino acids are stained and measured with HPLC or with spectro-fluorometry. However, these analyses failed to show any correlation between time for amphipod crash and bioavailable amino acids. In addition the content of the fatty acid 16:1n-7, which is a biomarker for diatom food item, increased after the crash. Thus, we suggest that food deficiency is not the main or only reason to the dramatic decline.

In January 2005 we observed high densities of white opaque amphipods. They had a similar appearance as the microsporidian infested Northamerican species Diporeia sp. In a seasonal survey carried out in 2005 we recorded very high frequencies of these presumed infested amphipods. Histological examination verified that a great percentage of the adult populations were infested by an unknown parasite located in the muscle tissue. Males were more seriously affected than females. Infested females show lower fertilization success than healthy females and they produce higher degree of degenerated gonads and embryos of females with parasites have a comparatively high percentage of membrane dysfunctions.

We have no information about distribution of this parasite in other areas but the white amphipods occur on all investigated stations in the Bothnian Sea. However, whether this is a new parasite for M. affinis or if it has increased due to e.g. decreased condition in amphipods is not clear. Possibly the dramatic decline is caused by a combination of factors, short periods of decreased food resources made the amphipods more sensitive to old or newly transmitted parasites from invaded species (Marenzelleria). It is also possible that unknown contaminants have increased in the Bothnian Sea. Higher abundance of parasite infested amphipods has been recorded in contaminated sites.

15 Joint discussions with WGSAEM and WGMS

15.1 WGBEC terms of reference item j: Development of assessment criteria and integrated assessment

It was noted that in the WGBEC terms of reference that the group should prepare with WGSAEM documents on assessment criteria and discuss the development of assessment criteria. The group felt that this request had been superceeded by the outputs from the WKIMON 11 workshop and the requests to WGBEC contained within the OSPAR SIME summary record report of February 2006. In this respect WGBEC addressed the main issues of concern and imitated a way forward and this is reported in agenda item 11 above. The Chair agreed to forward WGBECs comments to the OSPAR secretariat (Richard Emmerson) in time for consideration if appropriate at the OSPAR ASMO meeting in late April 2006.

15.2 WGBEC used the opportunity to meet with WGSAEM to initiate an assessment of imposex data on the ICES database.

This assessment was imitated by Matt Gubbins (WGBEC) and Rob Fryer (WGSAEM).

Imposex data submitted by OSPAR Contracting Parties were due to be assessed by OSPAR MON during their 2005 meeting. However, problems with data extraction from the ICES database prior to and during the meeting prevented this assessment process. Following the meeting these issues were resolved and an assessment is now possible. It was decided that a preliminary assessment was an appropriate task for ICES WGBEC / WGSAEM to take on, as a means of developing appropriate methodology for OSPAR MON.

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Two background documents from Fisheries Research Services, UK were made available prior to the meetings of the two working groups (Fryer and Gubbins, WGBEC 15 - J1 a and b). These described a temporal trend assessment of Vas Deferens Sequence Index (VDSI) and Penis Classification Index (PCI) data in the ICES database for all sites with long time-series (J1a) and a preliminary regional assessment of VDSI data in dogwhelks, Nucella lapillus from the Sullom Voe and Yell Sound region of Shetland (J1b). These documents are available as Annexes (9 and 10) to this report.

Presentations of these preliminary assessments were made to WGSAEM and, in a joint session, to WGBEC.

Temporal trend assessment (Background document: Annex 9)

Temporal trend assessment was restricted to sites with 5 or more years of data on the ICES database and to parameters relevant to the OSPAR assessment criteria. Of the data available in the ICES database, 34 time-series from sites in Denmark, Norway and the UK covering 3 species (Nucella lapillus, Neptunea antiqua, Buccinum undatum) were appropriate for assessment. For Nucella and Neptunea, VDSI was assessed and for Buccinum PCI was used.

The imposex data was scaled to lie between 0-1, a linear logistic function of time was fitted to the time-series assuming quasi-binomial errors, and the upper one-sided 95% confidence limit on the fitted line in the final year of the time-series was used to make a precautionary classification of the data using the assessment criteria developed for OSPAR.

Thirty of the estimated trends were downwards over time, four upwards. Of these, 10 downward trends and one upward trend (probably caused by a known local point source) were significant at the 5% level. All sites were assigned a precautionary OSPAR classification (from A – D) using the 95% upper limit from the final year in the series.

Regional assessment – Sullom Voe / Yell Sound (Background document: Annex 10)

As a preliminary demonstration of how a regional assessment could be conducted for imposex data, the case study of Sullom Voe oil terminal in Shetland (UK) was used. There is heavy shipping activity in the area, centred around the oil terminal and imposex in dogwhelk populations in the Voe and surrounding yell Sound area has been monitored at 20 sites since 1987.

Fitted temporal trends at all sites were used to post-stratify the data into two groups (heavily impacted Voe sites and less affected Sound sites further from the oil terminal). The data were then modelled using a generalised linear mixed model assuming quasi-binomial errors. The fixed effects allowed for a separate trend in each stratum, whilst the random effects allowed for variation between stations within strata and between-year variation common to all stations within strata. The fitted model showed a significant downward trend in VDSI in the Sound stratum and a non-significant downward trend in the Voe stratum. The precautionary classification of each stratum according to the OSPAR assessment criteria depends on whether one uses the upper one-sided 95% confidence limit on the mean level in the stratum or the upper one-sided 95% prediction limit on the level in a station sampled at random in the stratum.

Adapting this approach to make a regional assessment of e.g. an OSPAR region might be possible, but the spatial correlation in imposex levels between stations would also have to be considered. For large areas of the ICES / OSPAR area, long time-series are not available and in particular for most of the North Sea, only three years of monitoring data spanning 10–12 years are available. With such data, a regional assessment could only make limited statements about temporal trends and might have to restrict attention to statements about levels. In addition, care

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will have to be taken to separate OSPAR ‘hotspot’ sampling stations around ports and other point sources from background stations representative of a wider regional area.

Several modifications to the statistical approaches used for both temporal trend and regional assessments were proposed in the background documents and suggested by the working groups. These modifications addressed such issues as the use of mean values of indices compared to individual index data and the heterogeneity of variance associated with vas deferens stages. These specific statistical recommendations are outlined in the WGSAEM report.

Action

The assessment of imposex data should be revised for OSPAR MON, taking into account any new imposex data submitted to the ICES database and the modifications to the statistical approach suggested in the background documents and raised during the WG meetings.

Matt Gubbins will present current status on imposex assessments to WGBEC 2007.

Recommendations 1 ) Only a small proportion of the ICES/OSPAR areas are represented by the data submitted to the ICES database. All ICES members and Contracting Parties to OSPAR in particular are strongly encouraged to submit their imposex data to the ICES database to inform the OSPAR assessment process. 2 ) For temporal trend assessment of sites with heavily affected populations of dogwhelks, additional parameters of imposex which may be more sensitive to change than VDSI at these levels should also be assessed, for example RPSI and % sterile females.

15.3 WGBEC terms of reference k. (WGMS Agenda item 4): Passive samplers: review the response of the WGBEC to WGMS suggestions for areas of cooperative work on (bio)availability and related issues to report on opportunities for cooperative work

A position paper on this topic was presented at the WGBEC 2005 meeting. The paper described the current state of water and sediment extraction procedures and subsequent in vitro / in vivo bioassay techniques. Although extraction techniques are powerful methods to pre-concentrate samples for bioassay testing, it is recognised that these methods may produce a biased extraction profile of pollutants and in some cases also introduce toxicity that is not directly related to the environmental sample. For instance, it has been documented that trace levels of solvent impurities in solvents or sampling device residues has caused toxicity or bioassay incompatibility. When deemed necessary, the extract may be further treated to remove interferences (a procedure called cleanup), and/or exchanged into a water-miscible, non-toxic solvent suited for addition to the aqueous bio-assay (solvent-exchange). With these uncertainties in mind it was proposed to consult on these issues with the back to back meeting with WGSAEM and MCWG in Copenhagen 2006.

In this respect the WGMS met with WGBEC in plenary to discuss recent developments in, and opportunities presented by, passive sampling. WGMS members gave three short presentations, concerning passive sampling of water, of sediment, and the possible application of the samples obtained to subsequent bioassays.

Water

Passive sampling of water provides a powerful tool to measure the free dissolved concentrations of lipophilic organic contaminants in water. Results were shown of an experiment carried out in the Netherlands in which silicone rubber samplers were deployed on eight diverse locations, on

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eight occasions over four years, in association with transplanted mussels. After sic weeks exposure, the concentrations in the mussels were found to be closely related to the very low free concentrations in the water determined by the passive samplers. The calculated bioconcentration factors were closely related to the relevant octanol-water partition coefficients (Kow), and were consistent over time and locations. Such relationships provide validation that the free dissolved concentrations in the water measured by passive samplers are biologically relevant, and suggest that, broadly speaking, concentrations in mussels are in equilibrium with the free concentrations.

Sediment

Passive sampling in sediment also gives access to the free dissolved concentrations of lipophilic contaminants, but this time in pore water. The sampling process described involved exposing silicone rubber to sediment, in different ratios, shaking for periods of up to three weeks to allow equilibration to occur prior to analysis of the samplers. Bioconcentration factors applied to concentrations in bulk sediment greatly predict much higher concentrations in biota than are actually found, In contrast, such partitioning applied to concentrations estimated from passive samplers give much better predictions of the observed accumulations in the biota. It therefore seems likely that passive samplers also address the activity of contaminants in sediments, and should be relevant to bioaccumulation and effects. WGMS is in the process of designing a more thorough validation exercise.

Bioassay

Foppe Smedes responded to WGBEC comments about their interest in using contaminants collected by passive samplers as sources of exposure material for use in bioassays. He explained that, because of the range of Kow values shown by organic contaminants, analysis of classical water samples gave a rather poor indication of the contaminants that would accumulated by organisms. Passive samplers in water gave a better indication, but because of the kinetics of equilibrations of passive samplers, representation could be reasonable up to log Kow of about 4, but became progressively less good at higher Kow.

Passive samplers in sediment could achieve equilibrium, within reasonable periods of time, with compounds of log Kow up to about 8. Therefore, passive sampling of sediments currently appeared to offer greater potential for application to bioassays than passive sampling of water.

WGBEC noted the progress made over the past three years in silicone rubber passive samplers and their value in accumulating lipophilic compounds from water and sediments. Indeed there is good evidence to show that these samplers can be used as surrogates for mussels. However, where biological effects techniques are measured in mussels it is imperative that the effects measurements are related to contaminant tissue concentrations and therefore it would be hard to see in such situations where silicone rubber passive samplers would be beneficial. In addition WGBEC felt that there was no direct applicability to bioassay extraction procedures as described in the position paper. However group members felt that some investigative work would be worthwhile to: a ) deploy silicone rubber passive samplers in field situations (aqueous environment) for extraction and subsequent in vivo bioassay and compare this with bulk water extraction b ) deploy silicone rubber passive samplers in field situations (aqueous environment) for extraction and subsequent in vitro bioassay and compare this with mussel tissue extracts from mussels deployed at the same site.

Foppe Smedes indicated that he could be contacted by WGBEC members to provide addresses for purchase of silicon materials.

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Action

The above investigations were proposed and would be taken forward intersessionally by WGBEC members. The Chair, John Thain would liaise with Foppe Smedes and Ian Davies from WGMS.

15.4 WGBEC terms of reference l; discuss and report back to ACME on potential contributions for the ecosystem overview of the advisory reports ie WGRED reports of 2005 and 2006.

WGBEC was invited by ACME “to discuss and report on potential contributions to the ecosystem overview of the advisory reports describing the quantity and quality of marine habitat and/or the health of the marine ecosystem, and to consider and report on potential indicators of significant change in these ecosystem attributes.”

The partner commissions have asked ICES to find a clearer way of providing its advice on how to pursue an ecosystem-based approach to the management of human activities that impact marine waters - and ICES wants feedback from the ICES working groups.

The basis for the WGBEC discussions were the WGRED reports for regional ecosystem description 2005 and 2006 (draft) and the REGNS document (agenda item 4), which include ‘ecosystem overviews’ or short descriptions of the regional ecosystems. In these, the focus was on information required for the stock assessment and fisheries management advice. Very typically, the conclusive chapter of WGRED 2006 is titled “Advancing the use of environmental information in ICES fisheries (and other) advice”, and following examples are addressed:

• Long-term climate changes and Global warming • Regime shifts and its implications to fisheries management • Recovery strategies and ecological niche theory

However, the request from the partner commissions goes beyond the advice for fisheries management; it includes advice on the management of all human activities that impact marine ecosystems.

WGBEC has some comments and suggestions on information that could be useful for supporting management decisions on the broader integrated management initiative.

Advice in a framework for the protection and preservation of the marine environment requires the assessment of more indicators (physical, chemical, biological) if the intention is to include all important environmental variables and factors affecting the marine ecosystem.

In the WGRED and REGNS reports and documents, contaminants and their effects on the status of ecosystems are only mentioned briefly for the Baltic, not at all for the other eight regions. WGBEC believe that these clearly anthropogenic factors are also very important for descriptions of ecosystem status and for prediction and monitoring of impact of management actions and decisions.

Information on spatial and temporal trends of contaminants and their effects on organisms is available to some extent and should be included in regional assessments. Various monitoring programmes provide information on this. How to include contaminant-related factors in integrated assessments on a larger scale is presently under discussion and development, for instance within WKIMON.

Recommendation

WGBEC noted work within WGRED with interest. Since contaminant effects are important ecosystem descriptors, the group would welcome further communication with WGRE

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16 Recommendations and action list

16.1 Recommendations

Several recommendations were made by the WGBEC, see also Annex 6. 1 ) WGBEC would like more information from REGNS on the proposed structure of the thematic assessment on ‘chemical pollution’ to determine the value of submitting data for assessment. 2 ) In order to encourage submission of data to the ICES database WGBEC recommends that ICES move to free formats as soon as practicable 3 ) Permission is requested from ICES to commission over the next two years the list of eleven methods in Table 6.2 of the WGBEC report. Requests for publication of method documents concerning embryo aberrations in amphipods, MXR, EROD activity and extraction procedures for bioassays will be put forward as Draft Resolutions in 2006. 4 ) WGBEC recommends that the relevant effect data now be compiled by an intersessional WGBEC subgroup with the aim of proposing baseline/background responses for the relevant biological effects techniques so that the proposal can be assessed at the meetings by WKIMON III and WGBEC in 2007. The proposals should also be forwarded to WGSAEM for consultation and further development as appropriate at their meeting in 2007. 5 ) WGBEC recommends that brief background documents with assessment criteria need to be developed for selected methods in time for WKIMON III. The deadline for documents is September / October 2006. In addition WGBEC recommends a document needs to be prepared on an integration strategy before September / October 2006 and a demonstration programme for the integrated guideline should be prepared for 2008. 6 ) OSPAR is recommended to remove sediment sea water elutriate bioassays and oxidative stress from the OSPAR JAMP CEMP and include VTG in cod and flounder. 7 ) OSPAR is invited to take account of the proposed revisions by WGBEC to the CEMP biological effects tables outlined in the WGBEC report at agenda item 12a. 8 ) The recommendation of WGBEC is that a limited number of specific fish and molluscs should be identified for monitoring purposes in the different OSPAR sub- regions and that generic testing be applied where possible in order to generate data that can be interpreted globally rather than locally. 9 ) WGBEC recommends and encourages the use of the GENIPOL flounder DNA microarray and accompanying q-RTPCR procedures for use in pilot studies and intecomparison with existing monitoring biological effects procedures. Similarly pilot studies should be performed with the blue mussel arrays. 10 ) Only a small proportion of the ICES/OSPAR areas are represented by the data submitted to the ICES database. All ICES members and Contracting Parties to OSPAR in particular are strongly encouraged to submit their imposex data to the ICES database to inform the OSPAR assessment process. For temporal trend assessment of sites with heavily affected populations of dogwhelks, additional parameters of imposex which may be more sensitive to change than VDSI at these levels should also be assessed, for example RPSI and % sterile females. 11 ) WGBEC noted work within WGRED with interest. Since contaminant effects are important ecosystem descriptors, the WGBEC group would welcome and recommend further communication with WGRED.

Action list 1 ) The Chair (John Thain) of WGBEC will discuss with the REGNS chair whether there would be value in a member of WGBEC attending the REGNS workshop in May 2006.

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2 ) In order to encourage data submissions to the ICES database a submission file for biological effects data will be produced and uploaded to the ICES web site. This will be undertaken by Jacqueline Jones, Marilynn Sorenson. 3 ) The Chair (John Thain) of WGBEC will contact QUASIMEME to clarify what is needed for the current state for PAH metabolite analyses for AQC purposes . 4 ) To take forward the development of background responses for biological effects measurements WGBEC will form a subgroup (John Thain, Ketil Hylland, Jakob Strand and Dick Vethaak), compile the required data with support as available from other WGBEC members, and start the process of developing baseline responses and draft findings for WKIMON III and WGBEC in 2007. This will be communicated to the WGBEC members preparing documents on assessment criteria for specific methods.

In response to an OSPAR request for information on the measurement of long term biological effects of oil spills WGBEC have undertaken a review of the current status and application of techniques and suggested guidelines. The above review and guidelines will be forwarded to the OSPAR secretariat (Richard Emmerson).

1 ) In relation to the ICES database John Thain, Matt Gubbins and Jacqueline Jones will check for standard parameter combinations and specifications for biological effects data screening; To be carried out intersessionally. 2 ) In response to the WKIMON / SIME question as to whether biological effects techniques are appropriate to meet the requirements of the OSPAR JAMP. WGBEC identified oxidative stress, cellular energy allocation and aromatase for evaluation and will review these in full intersessionally and at the next ICES WGBEC meeting in 2007. 3 ) Brief background documents with assessment criteria need to be developed for selected methods in time for WKIMON III. The deadline for documents is September / October 2006. A document needs to be prepared on an integration strategy before September / October 2006. A demonstration programme for the integrated guideline should be prepared for discussion at the ICES WGBEC meeting in 2007 for implementation in 2008. These initiatives will be relayed to the OSPAR secretariat and if appropriate be taken forward by John Thain, Ketil Hylland and other WGBEC group members. 4 ) In response to the request from WKIMON for WGBEC to review integrated methods to assess effects on biota from lindane and BFRs. It was suggested that WGBEC will prepare a state-of-the-art paper on this issue including the open literature and the final results of the EU-project FIRE. Dick Vethaak kindly volunteered to provide such an review paper for next years meeting, Rolf Schneider would prepare a review paper for lindane and Ketil Hylland one on perfluorinated substances. 5 ) In response to WKIMON for WGBEC to review the comet assay and is application. WGBEC (Ulrike Kammann, Matt Gubbins and Thierry Burgeot) agreed to review the method before WGBEC 2007. 6 ) The assessment of imposex data should be revised for OSPAR MON, taking into account any new imposex data submitted to the ICES database and the modifications to the statistical approach suggested in the background documents and raised during the WG meetings. Matt Gubbins will present current status on imposex assessments to WGBEC 2007. 7 ) WGBEC with WGMS proposed some on some practical investigations that would be taken forward intersessionally by WGBEC members. The Chair, John Thain would liaise with Foppe Smedes and Ian Davies from WGMS.

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17 Adoption of the report and closure of the meeting

A draft report of the meeting was prepared and circulated for comment by the WG and corrections and amendments made as appropriate. The Chair agreed to incorporate these in a second draft which would be circulated by e-mail at a later date.

The Chair thanked members of the WG for their support and contribution to the meeting. The Chair also thanked ICES Headquarters for their hospitality and for hosting the meeting and secretariat support

The Chair closed the meeting at 15:00 hrs on 31 March 2006.

References

Agenda Item 7.2. Kuiper, R.V., Bergman, A., Vos, J.G., van den Berg, M. 2004. Some polybrominated diphenyl ether (PBDE) flame retardants with wide environmental distribution inhibit TCDD- induced EROD activity in primary cultured carp (Cyprinus carpio) hepatocytes. Aquatic Toxicology, 10;68(2): 129–39. Vethaak, D., Fernandez, R., Wester, P., Leonards, P., Munro Jensen, B., Dubbeldam, M., Anselm, H., and Kuiper, R. 2005. No major endocrine effects of BFRs tested in fish in environmentally relevant test setup. Credo cluster news 5—FIRE WP4.

Agenda Item 9

Ariese F., Beyer J., Jonsson G., Visa, C.P., and Krahn M.M. 2005. Review of analytical methods for determining metabolites of polycyclic aromatic compounds (PACs) in fish bile. ICES Techniques in Marine Enviromental Series. Nº 39. 41 pp.

Akcha, F., Vincent-Hurbert, F., and Pfhol-Leszkowicz, A. 2003. Potential value of the content assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds. Mutat. Res. Genet. Toxicol. Envrin. Mutagen, 534, 21–32.

Bucke, D., Vethaak, D., Lang, T. and Mellergaard, S. 1996. Common diseases and parasites of fish in the North Atlantic: Training guide for identification. ICES Techniques in Marine Enviromental Series. Nº 19. 27 pp.

Feist, S.W., Lang, T., Stentiford, G.D., and Köhler A. 2004. Biological effects of contaminants: Use of liver pathology of the European flatfish dab (Limanda limanda) and flounder (Platichthys flesus L.) for monitoring. ICES Techniques in Marine Enviromental Series, 38. 42 pp.

Moore, M.N., and Lowe, D. 2004. Biological effects of contaminants: Measurements of Lysosomal membrane stability. ICES Techniques in Marine Enviromental Series, 36. 31 pp.

Stagg, R., and McInstonsh, A. 1998. Biological effects of contaminants: Determination of CYP1A-dependent mono-oxygenase activity in dab by fluorimetric measurement of EROD activity. ICES Techniques in Marine Enviromental Series, 23. 16 pp.

Singh, N.P., Mc Coy, M.T., Tice, R.R., Schneider, E.L. 1988. A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell. Res., 175: 184–191.

Thain J.E. 1991. Biological effects of contaminants: oyster (Crassostrea gigas) embryo assay. ICES Techniques in Marine Enviromental Series, 11. 12 pp.

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Agenda Item 12.4. Akcha, F., Vincent Hubert, F., and Pfhol-Leszkowicz, A. 2003. Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds. Mutation Research, 534: 21–32. Akcha, F., Leday, G., Pfohl-Leszkowicz, A. 2004. Measurement of DNA adducts and strand breaks in dab (Limanda limanda) collected in the field: effects of biotic (age, sex) and abiotic (sampling site and period) factors on the extent of DNA damage Mutation Research, 552: 197–207. Webster, L., Phillips, L., Russel, M., Dalgarno, E., and Moffat, C. 2005 J. Environ. Monit., 7, 1–10.

Agenda Item 14.3. Narbonne, J.F., Aarab, N., Clerandeau C., Daubeze M., Narbonne J., Champeau O., and Garrigues P. 2005. Scale of classification based on biochemical markers in mussels: application to pollution monitoring in Mediterranean coasts and temporal trends. Biomarkers, 10(1): 58–71. Dagnino, A., Allen, I., Moore, M.N., Canesi, L., and Viarengo, A. Integration of biomarker data into an organism health index: development of an expert system and its validation with field and laboratory data in mussels”. Marine Ecology Progress Series (submitted).

Agenda item 14.4. 1. Gray, J.S., Wu, R.S.S., and Or, Y.Y. 2002. Effects of hypoxia and organic enrichment on the coastal marine environment. Mar Ecol Prog Ser., 238: 249–279. 2. Eriksson, Wiklund, A.-K., and Sundelin, B. 2004. Biomarker sensitivity to temperature and hypoxia - a seven-year field study. Mar. Ecol. Prog. Ser. In press. 3. Bjerkebæk-Lindberg, A.E., Gorringe, P. 2003. In Bottniska viken 2002. Ed. by K. Wiklund. Umeå Marine Research Center. pp 7–9. 4. Eriksson-Wiklund, A-K., and Sundelin, B. 2001. Impaired reproduction of the amphipods Monoporeia affinis and Pontoporeia femorata as a result of moderate hypoxia and increased temperature Mar. Ecol. Prog. Ser., 222: 131–141. 5. Pace, M.L., Cole, J.J., Carpenter, S.R., Kitchell, J.F., Hodgson, J.R., Van de Bogert, M.C., Bade, D.L., Kritzberg, E.S., and Bastviken, D. 2004. Whole-lake carbon-13 additions reveal terrestrial support of aquatic food webs. Nature, 427: 240–243. 6. Wetzel, R.G. 1995. Death detritus and energy flow in aquatic ecosystems. Freshwater Biol., 33: 83–89. 7. Cruz-Rivera, E., and Hay, M.E. 2000. Can quantity replace quality? Food choice, compensatory feeding, and fitness of marine mesograzers. Ecology, 81(1): 201–219. 8 Uitto, A., and Sarvala, J. 1991. Seasonal growth of the benthic amphipods Pontoporeia affinis and P. femorata in a Baltic archipelago in relation to environmental factors. Mar Biol., 111: 237–246. 9. Lehtonen, K.K., and Andersin, A.B. 1998. Population dynamics, response to sedimentation and role in benthic metabolism of the amphipod Monoporeia affinis in an open-sea area in the the northern Baltic Sea. Mar. Ecol. Prog. Ser., 168: 71–85. 10. Aljetlawi, A.A., Albertsson, J., Leonardsson, K. 2000. Effect of food and sediment pre- treatment in experiments with a deposit-feeding amphipod Monoporeia affinis. J. Exp. Mar. Biol. Ecol., 249: 263–280. 11. Guiguer, K.R.A., and Barton, D.R. 2002. The trophic role of Diporeia (Amphipoda) in Colpoys Bay (Georgian Bay) Benthic food web: A stable isotope approach. J Great Lakes Res., 28: 228–239.

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12. Dermott, R.M., and Corning, K. 1988. Seasonal ingestion rates of Pontoporeia hoyi (Amphipoda) populations in Lake Ontario. Can J Fish Aquat Sci., 45: 1886–1895. 13. Quigley, M.A. 1988. Gut fullness of deposit-feeding amphipod, Pontoporeia hoyi in southeastern Lake Michigan. J Great Lakes Res, 14: 178–187. 14. Lehtonen, K.K. 1994. Metabolic effects of short-term starvation on the benthic amphipod Pontoporeia affinis (Lindström) from the northern Baltic Sea. J Exp Mar Biol Ecol., 176: 269–283. 15. Mayer, L.M., Schick, L.L., Sawyer, T., Plante, C.J., Jumars, P.A., and Self, RL. 1995. Bioavailable amino acids in sediments: A biomimetic kinetic-based approach. Limnol Oceanogr., 40: 511–520.

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Annex 1: List of participants

NAME ADDRESS PHONE/FAX EMAIL Maija Balode Institute of Aquatic Ecology TEL: +37 19471203 [email protected] University of Latvia FAX: +37 17601995 8 Dougovgrivas St LV-1048,Riga Latvia Thierry Burgeot IFREMER TEL: +33 240374051 [email protected] rue de l’Ile d’Yeu FAX: +33 240374075 B.P. 21105 F-44311 Nantes Cédex 03 France Estibaliz Diaz AZTI TEL: +34 946029100 [email protected] Txatxarramendi Irla FAX: +34 94687006 48395 Sukarrieta/Pedernales Spain Stephen George Institute of Aquaculture TEL: +44 (0) [email protected] University of Stirling 1786467922 [email protected] Stirling FK9 4LA Matt J. Gubbins Fisheries Research Services TEL: +44 [email protected] Marine Laboratory (0)1224295681 P.O. Box 101 375 Victoria Road Aberdeen AB11 9DB United Kingdom Ake Granmo Goteborg University TEL: +46 52318534 [email protected] Kristineberg Marine FAX: +46 52318502 Research Station 45034 Fiskebackskil Sweden Ketil Hylland Norwegian Institute for TEL: +47 22185170 [email protected] Water Research (NIVA) FAX: +47 22185200 PB 173 Kjelsås N-0411 Oslo Norway Jacqueline Jones CEFAS TEL: +44 1621787293 [email protected] Burham-on-Crouch FAX: +44 1621784989 Laboratory Remembrance Avenue Burnham-on-Crouch UK-Burnham-on-Crouch CM0 8AH United Kingdom Ulrike Kammann Institut f. Fischereiökologie TEL: +49 4038905198 [email protected] BFA f. Fischerei fisch.de Palmaille 9 22767 Hamburg Germany Angela Köhler Alfred-Wegener-Institut TEL: +49 47148311407 akoehler@awi- Foundation for Polar and FAX: +49 47148311425 bremerhaven.de Marine Research Postfach 12 0161, D-27515 Bremerhaven

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NAME ADDRESS PHONE/FAX EMAIL Concepción Martínez Instituto Español de TEL: +34 968180500 [email protected] Oceanografía FAX: +34 968184441 Centro Oceanográfico de Murcia Varadero 1, Lo Pagán 30740 San Pedro del Pinatar (Murcia) Spain M. Moore Plymouth Marine TEL: +44 1752633120 [email protected] Laboratory FAX: +44 1752633120 Prospect Place, The Hoe Plymouth PL1 3DH UK Rolf Schneider Institut für Ostseeforschung TEL: +49 3815197213 rolf.schneider@io- Seestrasse 15 warnemuende.de D-18119 Rostock Germany Jakob Strand NERI TEL: +45 46301865 [email protected] Department of Marine Ecology Frederiksborgvej 399 PO Box 358 DK-4000 Roskilde Denmark Britta Sundelin Department of Applied TEL: +46 86747235 [email protected] Environmental Science, ITM Stockholms Universitet 106 91 Stockholm Sweden John Thain CEFAS TEL: +44 [email protected] (Chair) Burham-on-Crouch (0)1621787239 Laboratory FAX: +44 Remembrance Avenue (0)1621784989 Burnham-on-Crouch UK-Burnham-on-Crouch CM0 8AH United Kingdom Dick Vethaak RIKZ TEL: +31 703114219 a.d.vethaak@ P.O. Box 20907 FAX: +31 703114200 rikz.rws.minvenw.nl 2500 EX The Hague The Netherlands Aldo Viarengo Via Bellini N 25 TEL: +39 0131360370 [email protected] 15100 Alexandria FAX: + 39 9357182439 Italy Pekka Vuorinen Finnish Game and Fish. Res. TEL : +35 8205751 277 [email protected] Institute FAX : +35 8205751 202 P.O. Box 2 FIN-00791 Helsinki Finland

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Annex 2: WGBEC Terms of reference 2005

2005/2/MHC04 The Working Group on Biological Effects of Contaminants [WGBEC] (Chair: John Thain, CEFAS, UK) will meet jointly with WGMS, MCWG, and WGSAEM at ICES Headquarters, Copenhagen, Denmark with in Copenhagen from 27–31 March 2006 to: a ) review progress with publication and electronic dissemination of biological effects techniques in the ICES TIMES series; b ) critically evaluate the progress with BEQUALM, ICES/BSRP SGEH and other relevant national (s.a. PRESTIGE oil spill) and international projects and where relevant the extent to which they meet the requirements of OSPAR and HELCOM; c ) assess and report on the amount of biological effects data submitted to the new ICES database; d ) evaluate the report from WKIMON second workshop; e ) review and update sub-regional data tables and where necessary include new data (parameters) and/or existing data (parameters) updated where relevant. The data tables will be subject to thematic assessment to be undertaken at a REGNS thematic assessment workshop; f ) produce a guidance document on the use biological effects techniques for oil spill situations; g ) evaluate documents prepared intersessionally for “background” biological effects responses; h ) provide expert knowledge and guidance to ICES Data Centre on a continuous basis. i ) respond together with MCWG to request from WKIMON on the following issues: i ) Review the selection of available and developing biological effects methods and the degree to which they are/may be appropriate to meet the requirements of the OSPAR Joint Assessment and Monitoring Programme (JAMP), as detailed to meet the needs of the OSPAR Hazardous Substances Strategy, and the need for integrated monitoring. ii ) Where suitable contaminant-specific biological effects methods do not exist, investigate the possibility for their development. iii ) Review appropriate integrated methods to assess effects on biota and the health of the marine environment from lindane and BFRs. iv ) Review the specificity of the COMET assay for DNA damage in relation to the detection of the effects of hazardous substances and potential confounding effects of other factors affecting the assay. v ) Review available data for each biological effects method to clarify whether data can be compared across the range of recommended fish species: vi ) Selection of species, gender and size ranges: vii ) Review background levels for ALA-D in relevant species (where information is available).

Joint discussions on: j ) preparing the documents on assessment criteria and discuss the development of integrated assessment with WGSAEM in 2006. See g) above. i ) passive samplers and their use and application for chemical contaminant and biological effects monitoring. ii ) Discuss and report on potential contributions for the ecosystem overview of the advisory reports describing the quantity and quality of marine habitat and/or the health of the marine ecosystem, and to consider and report on potential indicators of significant change in these ecosystem attributes.

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Annex 3: WGBEC draft agenda

ICES Working Group on Biological Effects of Contaminants (WGBEC).

ICES Headquarters, Copenhagen, 27–31 March 2006.

1. Opening of the meeting; 2. Adoption of the agenda; 3. Appointment of Rapporteurs; 4. Review and update sub regional data tables for submission to ICES REGNS; 5. Assess the amount of biological effects data on the ICES database and develop means to encourage further submissions as well as the use of the data; 6. Review progress with ICES TIMES series; 7. Consider progress with national/international activities: i) BEQUALM ii) ICES/BSRP SGEH and PRESTIGE oil spill and any others; 8. Review the use of “background” responses in biological effects; 9. Produce guidance document on the use of biological effects techniques in oil spill situations; 10. Provide expert knowledge to ICES Data Centre as appropriate: 11. Evaluate the report from the ICES OSPAR WKIMON II workshop and feedback fro OSPAR SIME; 12. Respond on request from ICES OSPAR WKIMON on a number of issues; a) are biological effects methods appropriate to meet the requirements of the OSPAR JAMP. b) where suitable methods do not exist can they be developed. c) review integrated methods to assess effects on biota from lindane and BFRs. d) review of COMET assay and its application. e) review data for each biological effect method and assess whether bio effects data can be compared across species. f) selection of species, gender and size range. g) review background levels for ALA-D 13. Assess the development of the CEMP guidelines; 14. Any other business; a) review progress with genomics and proteomics b) progress with nanotechnology c) development of a 2-tier biomarker approach for monitoring

d) amphipod decline in the Baltic

15. Joint discussion with other ICES working groups meeting at ICES – WGSAEM, WGMS and MCWG. The mechanism on how this will take place is still under discussion.

Topics for discussion; ƒ Development of assessment criteria for biological effects techniques and integrated assessments.

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ƒ Passive samplers and their use and application for biological effects monitoring ƒ Provide information to ACME on the quantity and quality of habitat and the health of marine ecosystems.

16. Recommendations and action list;

17. Adoption of the report and closure of the meeting.

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Annex 4: List of Rapporteurs

AGENDA TOPIC LEAD, COLLATOR, RAPPORTEUR ITEM 1 Opening of meeting John T. 2 Adoption agenda – additions, AOB John T. 3 Appointment of rapporteurs John T 4 Review regional data tables for ICES Matt G REGNS 5 Assess amount of bio effects data on ICES John T Jacqueline J database 6 Review progress with TIMES Matt G 7 Progress with BEQUALM John T / Ketil H Progress on PRESTIGE oil spill Concha M / Estibaliz D 8 Review the use of “background” responses in Jakob S biological effects 9 Produce guidance document on bio effects Concha M et al. relating to oil spills 10 ICES database issues Jacqueline J / John T / Matt G 11 Review OSPAR WKIMON II and issues….. Ketil H / John T 12 Requests from WKIMON John T i) are bio effects methods appropriate to meet Matt G requirements of OSPAR JAMP ii) where suitable methods do not exist can Matt G they be developed iii) review integrated methods to assess Dick V / Rolf S effects for lindane and BFRs iv)review COMET assay and its application Ulrike K v) review data for each biological effect Mike M method and assess whether bio effects data can be compared across species vi) selection of species, gender and size range Mike M vii) review background levels for ALA-D Ketil H 13 Assess development of the CEMP John T 14 Any other business a) review progress with genomics and Steve G proteomics Aldo V b) progress with nanotechnology Mike M c) development of 2-tier biomarker approach Aldo V for monitoring d) amphipod decline in the Baltic Brita S 15 Joint discussions as appropriate J1) Development of assessment criteria for Mat G with WGSAEM biological effects and integrated assessment J2) Passive samplers and their use and John T / Ketil H / Dick V with WGSM application for biological effects monitoring J3) Provide information to ACME on the Rolf S / Jakob S quantity and quality of habitat and health of marine ecosystems 16 Recommendations and Action list John T 17 Adoption of the report John T

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Annex 5: List of documents

AGENDA DOCUMENT ITEM 1 a ICES information and housekeeping 1 b List of attendees

2 a Draft agenda 2 b Tentative timetable 2 c List of background documents 2 d WGBEC Terms of Reference 2 e Terms of Reference for all Working Groups meeting back-to-back at ICES

3 a List of rapporteurs

4 a Request from REGNS – Letter from AJ Kenny 4 b REGNS overview of assessment so far – Report from A J Kenny 4 c REGNS ICES report from last year – Report fro AJ Kenny

5 a ICES database – letter from ICES Data Centre

7 i a Bequalm progress review – report from BEQUALM project office 7iii a Effects of Prestige oil spill on mussels…. - Orbea et al. Mar. Ecol. Prog. Ser., 306: 177–198. 2006 7iii b PAHs, biomarker responses ……hake, anchovy. Prestige oil spill Diez et al. Pwt Pt presentation 7iii c Cell and tissue biomarkers in mussels. Prestige oil spill – Margomes et al. MPB in press 2005(6) 7iii d Signs of recovery of mussel health. Prestige oil spill – Cajaraville et al., departmental report 7iii e-h Four published papers on birds relating to the Prestige oil spill 7 iv a Baltic Sea BEEP project. Lehtonen et al., MPB xxx(2006) in press 7iv b Monitoring biological effects of pollut … in the Baltic. Lehtonen et al. MPB xxx 2006 in press 7iv c Biomarker responses in flounder …in Baltic Sea. - Barsiene et al. MPB xxx 2006 in press 7iv d Biomaker responses as indicators of effect in Baltic …- Schiedek et al. MPB xxx 2005(6) in press 7iv e Indices of environmental pollution of the Baltic … Broeg et al. MPB 3088 in press 7iv f Liver pathology in Baltic flounder… -Lang et al. MPB in press

8 a Impaired larval development in eelpout……Strand et al. Fish Phys. and Biochem 30: 37-46 2005 8 b Development disorders of eelpout… Gerchen et al. MPB xxx 2005(6) 8 c Background responses in bio effects methods review – report by John T

9 a Case study info: Braer oil spill – report by Mat G. 9 b Case study info: Prestige oil spill – report by Concha M 9 c Biological effects techniques in oil spill zones – report by Janine B 9 d Guidelines document in oil spill situations – report by Concha M 9 e EA UK guide to procedures following oil spill – EA UK report 9 f DNA damage measured by single cell gel. Erika – Lemiere et al. Mutation Res., 581: 11–21 2005 9 g Sub lethal iological responses. UK Blue Book extract 9 h Case study Sea Empress – report by John T 9 i Chronic impacts of oil pollution in the sea. Peterson et al. Mar. Ecol. Prog. Ser., 241: 235–236 2004 9 j Sea otter populations. Exxon Valdez – Bodkin et al. Mar. Ecol. Prog. Ser., 241: 237–253, 2002 9 k Harlequin duck pop.. re Exxon Valdez - Esler et al. Mar. Ecol. Prog. Ser., 241: 271–286. 2002 9 l Sampling design .. programme …Exxon Valdez..Peterson et al. Mar Ecol Prog Ser 210: 265-283 2001 11 a WKIMON report from meeting 2005 11 b WKIMON report from meeting 2006

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AGENDA DOCUMENT ITEM 11 c SIME reports from meeting of Feb 2006

12 a (i) ICES WGBEC last revised methods list 2004 12 b (i) Review of current state of OSPAR JAMP techniques from WKIMON II – SIME report

12 d (i) Comet assay poster, DNA damage in plaice – poster by Matt G

13 a CEMP review – SIME document

14 a Peroxisomal proteomics a new tool – Jia Mi et al. Proteomics 5:3954-3965 2005 14 b Nanotechnology. Defra UK scoping study report

J 1(a) Imposex assessment – trends. Report by Rob Fryer and Matt Gubbins J 1 (b) Imposex assessment – Sullom Voe. Report by Rob Fryer and Matt Gubbins J 3 (a) ToRs for Chairs of WG – joint meeting J 3 (b and c) WGRED reports of 2005 and 2006

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Annex 6: WGBEC draft resolutions

Category 1

Recommendation for the publication of ICES TIMES papers

Permission is sought to continue with publications of the following documents 1 ) Revision of the bivalve embryo bioassay, (J. Thain) 2 ) Revision of the determination of EROD, (M. Gubbins to lead) 3 ) MDR/MXR in blue mussel (Angela Köhler to lead) 4 ) Histopatholgy in invertebrates (Brita Sundelin) 5 ) Extraction techniques and in vitro testing (D. Vethaak, J. Thain, K.-E. Tollrfesen 6 ) Reproductive success in the eel pout (J Strand) 7 ) YES and YAS screens for endocrine disruption (J Thain and D Vethaak) 8 ) Blue mussel histopathology (I. Marigomez, D. Lowe) 9 ) Gonadal histology of flounder (S. Feist)

WGBEC would like to proceed with all topics and prioritised these as 1, 2 and 4 to finalise in Oct/ Nov 2006, 3, 4, 5 and 6 to elicit in 2007 and 7, 8 and 9 the following year.

Justification

Protocols are needed for national and international programmes as well as the OSPAR programmes.

Category 2

The Working Group on Biological Effects of Contaminants [WGBEC] (Chair: John Thain, CEFAS, UK) will meet in Alessandria from 15-19 March 2007 to: a) Review progress with publication and electronic dissemination of biological effects techniques in the ICES TIMES series; b) Consider progress with activities such as BEQUALM, HELCOM area/BSRP project, biological effects monitoring programmes in MEDPOL, USA, Canada and other relevant national and international projects; c) Assess the amount of biological effects data submitted to the new ICES database; d) Receive and evaluate further reports on the Prestige oil spill; e) Evaluate the report from WKIMON third workshop and the intersessional work undertaken by WGBEC members, including the development of assessment criteria, integrated monitoring and the development of a field demonstration programme; f) Evaluate documents prepared intersessionally for “background” biological effects responses; g) Review the “recommended list” of biological effect techniques; h) Receive and evaluate reports prepared intersessionally by WGBEC on oxidative stress, cellular energy allocation and aromatase; i) Review integrated methods for assessment of effects on biota from lindane, BFRs and methodology of the COMET assay; j) Report on progress with the assessment of imposex data; k) Report on progress with intersessional activities using passive samplers: l) Review the use of in vitro and in vivo biological effects techniques for monitoring purposes and WFD activities;

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Supporting information

Priority: The activities of this group will enable ICES to advise on issues relating to the design, implementation and execution of regional research and monitoring programmes pertaining to hazardous substances in the marine environment. To develop procedure for quality assurance of biological effects data and to improve assessments of data relating to the biological effects of contaminants in the marine environment. Scientific Justification and relation a) It is important for WGBEC to keep track of publication progress with to Action Plan: biological effects methods it has sponsored. Protocols are needed for national and international programmes as well as the OSPAR programmes. b) AQC for biological effects methods has been identified as critical by international organisations; following commercialisation of BEQUALM; it is important that WGBEC keeps track of developments with this programme. In addition it is important that WGBEC keeps track and evaluates other national and international programmes that can influence the uptake of biological effect techniques, e.g., BSRP. c) Biological effects data can be entered into the revamped ICES database and WGBEC needs to monitor the data reporting acitivities. d) There is an ongoing biological effects monitoring programme following the Prestige oil spill; as in previous years WGBEC wishes to be kept informed of strategies and results. e) The output from WKIMON III will influence the uptake of biological effects techniques across the OSPAR area and WGBEC should be allowed to evaluate the resulting guidelines. f) “Background” biological responses were discussed at WGBEC 2006. However, further work needs to be undertaken on this topic. g) WGBEC keeps a watching brief on the progress and development of biological effect techniques and reviews its list every three years for reporting back to ICES and OSPAR. h) WKIMON have requested that WGBEC assess the applicability of these techniques for inclusion in the nOSPAR JAMP CEMP. i) In response to a request from WKIMON a review will be carried out intersessionally for WGBEC to evaluate in 2007. j) Continue the work already undertaken at WGBEC 2006 (with WGSAEM) on the assessment of imposex data and any activities in this respect undertaken by OSPAR MON. k) Investigations were initiated on the use of passive samplers during the joint meeting with WGSMin 2006 and these activities need to be reported back to WGBEC. l) in vitro and in vivo methods methods are now being more widely used in monitoring programmes and WGBEC needs to evaluate progress and application of these techniques.

Resource requirement: The main input to this group is from National experts. Each attendee is self- funded from their own / organisation / institute resources. Participants: The Group is normally attended by ca. 16 members and guests. Secretariat Facilities: None required. Financial: No financial implications. Linkages to Advisory There are no obvious linkages with other advisory committees. Committees: Linkages to other Committees or There are linkages with WGSAEM, MCWG, WGMS and WGPDMO. Groups: Linkages to other Organisations: None identified. Secretarial Marginal Cost Share: None identified.

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Annex 7: Progress with national activities pertaining to the Prestige oil spill (Agenda Item 7.3)

The major findings concerning biological techniques in relation to the Prestige oil spill that were presented at the the “Symposium on marine accidental oil spill” in Vigo (Spain 13–16 July; Vertimar 2005: Strategic Action Symposium (MET)) were as follows:

Biological effects related to the Prestige oil spill in demersal fish from the northern Iberian shelf.

C. Martínez-Gómez, J.A. Campillo, J. Benedicto, B. Fernández, J. Valdés, I. García, and F. Sánchez.

Liver EROD, GST, Catalase and GR activities as well as DNA integrity were determined in two demersal fish species in seven geographical areas along the Galician and Cantabrian shelf five months after the Prestige oil spill (POS) (November 2002). The simultaneous use of both species allowed to obtain information from different shelf environments since C. lyra can be considered representative for the northern Iberian inner shelf whereas L. boscii represents the northern Iberian middle and outer shelf. The results show differences in biomarker responses between the species. EROD activity showed significant differences between specimens collected from different areas. Significantly higher values were observed in specimens from Cantabria and eastern Asturias (eastern Cantabrian shelf). For GST, GR and Catalase activities, significant differences between areas were also found. Higher values of these activities were found in the closest geographical area to the accident (Finisterre). One of the key findings in this study is the significant positive correlation between GR activities (measured in L. boscii) and tar aggregate data deriving from the POS (R = 0.869; p = 0.011) and between CAT activities (measured in L. boscii) and tar aggregate data (R = 0.814; p = 0.026). Differences in EROD activities in C. lyra were found when data from Finisterre, the most impacted area, were compared with those from eastern Asturias (the less impacted area). However, these differences were not statistically significant. The highest mean GST activity was found in Galicia N, which was considered as the second most impacted area by the POS, and the lowest were also found in Asturias W, although they were not statistically significant. CAT and GR activities in C. lyra did not present any differences which can be associated with the POS. Though clear differences in DNA integrity between areas were found, the low number of individual fish analysed for most stations does not permit a useful comparison between areas

Biomonitoring of the Galician and Cantabrian coast six-months after the Prestige oil spill

R. Martin-Skilton, R. Lavado, and C. Porte

Hydroxilated naphtalenes in bile, liver EROD, GST and catalase activities were determined in two demersal fish species (Micromessitius poutassou and Lepidorhombus boscii) in 7 sampling sites along the Galician and Cantabrian shelf six months after the Prestige oil spill. Hydroxilated naphtalenes and alkylated naphthols were detected in L. boscii collected in the Galician coast, indicating high exposure to hydrocarbons in the sampling site closest to the accident. The results shown differences in biomarker responses between the species. No significant differences among stations were observed for GST activity in any of the species tested. Significant differences among sampling sites were observed for EROD activity only in M. poutassou, values in some sampling sites located at eastern Cantabrian continental shelf being higher. Catalase activities were also significantly different among stations but only in L. boscii.

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Endocrine alteration and other biochemical responses in juvenile turbot exposed to the Prestige fuel oil

R. Martin-Skilton, F. Saborido-Rey, and Cinta Porte

Juvenile turbot (Scophthalmus maximus) were exposed to different concentrations of the Prestige fuel oil through the diet, and the effects on hepatic biotransformation enzymes and steroid levels monitored after six weeks exposure. The work demonstrated that acute exposure to Prestige fuel oil elicits alterations in some hepatic biotransformation enzymes (EROD and UDPG) with different sensitivities, and leads to decreased levels of testosterone and estradiol in plasma of juvenile turbot.

PAHs, biomarker responses, histopathology and reproductive parameters in hake and anchovy after the prestige oil spill

E. Díaz, L. Arregi, P. Alvarez, U. Cotano, H. Murua, M. Santos, A. Alday, E. Saitua, A. Orbea, R. Werding, U. Izagirre, I. Cancio, I. Marigómez, M. Cajaraville, and G. Díez

This study presents an assessment of biological effects derived from the exposure to the POS on European hake (Merluccius merluccius) and anchovy (Engraulis encrasicholus). Preliminary histopathological examination of hake liver from spring sampling revealed high prevalence of parasitic infestation by nematodes (Anisakis) and variable prevalence of other alterations or lesions such as accumulation of melanomacrophage centers, immflamatory responses with lymphocyte accumulation, hepatocellular nuclear polymorphism and necrotic foci. Anchovy larval growth studies showed that anchovy larvae younger than 15 days presented significant differences in the Somatic Index between 2000 and 2003 (p< 0.05) and larvae from 10 to 15 days in the case of 2003 and 2004.

Field application of biochemical markers and physiological index (SFG) in the mussel Mytilus galloprovincialis from the coast affected by the Prestige oil spill

J.A. Campillo and M. Albentosa.

Antioxidant enzymatic activities (SOD, CAT, GPX and GR) and SFG in Mytilus galloprovincialis were determined on the Galician-Cantabrian coast 15 months after the Prestige oils spill. The results showed increased SOD levels and reduced SFG in mussels collected from two sites affected by the POS, and reached the levels shown by mussels from areas suffering chronic pollution. Multiple regression analyses revealed significant negative relationships between two antioxidant activities (SOD and GR) and the Physiological stress detected from the SFG.

Biological effects of the Prestige oil spill in mussels from Galicia and the bay of Biscay using biomarkers, histopathology and genomic and proteomic approaches

L. Garmendia, A. Orbea, R.l Werding, A. Gómez-Mendikute, U. Izagirre, D.Raingeard, E. Bilbao, I. Apraiz, M. Ortiz-Zarragoitia, S. Cristóbal, I. Cancio, M. Soto, I. Marigómez and M. P. Cajaraville.

An evaluation of the possible effects of the POS on Mytilus galloprovincialis populations was performed by applaying several cell and tissue-level biomarkers: peroxisome proliferation (acyl- Coa oxidase, AOX activity); lysosomal response as change in the estructure (volume density, Vv, surface-to volume ratio, S/V, and numerical density Nv) and membrane stability (labilisation period, LP); cell type replacement (volume density of basophilic cells, VvBAS) in digestive gland epithelium; and changes in the morphology of digestive alveoli and gonad development stages were assessed as supporting parameters. Possible genotoxic damages were

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assessed by measuring the frequency of micronuclei in isolated gill cells of mussels from selected localities. Mussel samples were collected in April, July and September 2003 and in February, April, July and October 2004. Employed biomarkers evidenced exposure to toxic chemicals and disturbed health in mussels from Galicia and Basque Country. Highest degree of disturbance was detected in areas most impacted by the oil spill (Galicia). Biomarkers responses were more marked in 2003, a certain recovery trend being envisaged during 2004.

Acute toxicity of individual polycyclic aromatic hydrocarbons (PAH), and Prestige-fuel water accomodated fractions (WAF) to early life stages of commercial marine organisms, zooplankton and phytoplankton

R. Beiras, J. Bellas, L. Saco-Álvarez, J.C. Mariño-Balsa, P. Pérez, and N. Fernández.

Acute toxicity of phenanthrene, fluoranthene and pyrene was assessed by using early life stages of commercial marine organism (larvae of decapod crustacean Palaemon serratus, larvae of sea urchin Paracentrotus lividus and embryos of Gadus morhua), zooplankton (copepodod Acartia tonsa) and natural phytoplankton assemblages. The three compounds tested were toxic at concentrations ranging from 1/4 to 1/8 saturation in seawater. Crustacean seemed particularly sensitive to PAHs. The experiment support that planktonic organisms were affected during the first weeks after the Prestige oil spill, when concentrations of intermediate molecular weight aromatics in the water column remain close to saturation. Natural phytoplankton assemblages showed higher sensitivity to PAHs than aquaculture reared microalgal species. In majority of all bioassays, toxicity was enhanced by light.

Ecotoxicity of the water-soluble fraction of the Prestige fuel-oil

J. M. Navas, M. Babín, P. García, P. Heranz, C. Fernández, and J. V. Tarazona.

An evaluation of the toxicity of the water soluble fraction (WSF) of the Prestige fuel oil was performed using fish cell line RTG-2. Fish cell lines were exposed to different dilutions of the WSF but not cytotoxic effects were detected. Similarly, no acute toxicity was evidenced in Daphnia Magna. However, strong toxic effects were observed in algae (Chlorella vulgaris) growth inhibition tests, even for very high dilutions of the WSF. Results obtained indicate that toxicity of WSF shows a strong variation among taxa and that acute toxicity bioassays following established protocols can not evidence all the implications of these toxic processes. In addition, this WSF was able to provoke a strong induction of EROD activities in the RTG-2 cells in vitro, indicating that this WSF could be causing long-term deleterious effects.

Prestige's fuel soluble phase activates the aryl hydrocarbon receptor

S. Casado, M. Babín, J. V. Tarazona, and J. M. Navas.

A study about the specific mechanism mediated by the aryl hydrocarbon receptor (AhR) was conducted using two cells lines (RT-LW1, RTG-2) exposed to several dilutions of the WSF of the Prestige fuel oil. In both cells lines EROD activity and CYP1A transcription levels, studied by means of RT-PCR, increased with increasing concentrations of the WSF. The use of permanently transfected cell systems and of immunoassays evidenced the activation of AhR by ligand-binding mechanisms. Since AhR activation may cause a disruption of the estrogen receptor pathways, and hence a reduction of reproductive performance, the results suggest the possibility of long term deleterious effects of fuel oil.

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Immune parameters of mussels treated with Prestige fuel oil

M. C. Ordás, J. Albaiges, J.M. Bayona, A. Ordás, and A. Figueras.

A study was conducted to asses the effects of the Prestige fuel oil on the immune system of the mussel (Mytilus galloprovincialis). Different amounts of fuel were added to the tanks in which mussels were maintained, and a number of individuals were monthly sampled to measure several immune parameters, cellular and humoral. The statistical analysis showed that only the measured humoral parameters protein concentration and lysozyme activity, were significantly different between fuel-oil-exposed and control mussels.

Effect of Prestige oil spill on the lipid composition of common octopus (Octopus vulgaris) in Galician coast

M.P. Sieiro, J.M. Antonio, S. Aubourg, C. Piñeiro, A. Guerra, F. Rocha, and J.M. Bayona.

A study was initiated to check if the octopus-coccidian system could be used for biomonitoring the environmental health in relation to the contamination discharge event from Prestige. The severity of infection (estimated from infrapopulation counts of coccidian sporocysts) was higher in sampling stations more affected by the spill from Prestige, suggesting that either the host or the parasite (or both) populations size have been changed from 2002 to 2004. Unfortunately there was not possibility to sample just prior and after the spill. Further studies are being carried out to ascertain the role of the contamination from Prestige in the above found epidemiological differences.

Evaluation of the toxicity of seawater and sandy sediments affected by the Prestige fuel-oil spill by using the marine invertebrate embryogenesis bioassay

L. Saco-Álvarez and R. Beiras.

An evaluation of the toxicity of seawater and sand samples from an area of the Galician coast and shelf after the Prestige oil spill was attempted by using bivalves (Crassostrea gigas and Venerupis pollastra) and sea-urchin (Paracentrotus lividus) embryogenesis bioassay. Coastal sand elutriates were in general non toxic for embryos, not even during the first weeks after the fuel oil spill. In contrast, high toxicity was found in the seawater during the first days after the fuel oil spill in the most impacted areas, with complete inhibition of embryogenesis even after four-fold dilution with control water. In a lower degree toxicity persisted for two months in light-exposed coastal waters-accommodated fraction, rather than the more visible oil slick, in a oil spill event. In shelf samples the embryogenesis was successful, indicating a lack of toxicity in the sediments.

Evaluation of the effects of Prestige oil on Mytilus galloprovincialis from the coast of A Coruña

T. Rábade, B. Laffon, E. Pásaro and J. Méndez.

A study to asses DNA damage in Mytilus galloprovincialis was performed by applying the comet assay technique. Mussels and seawater samples were collected from two different locations intensely affected by the Prestige oil spill, in 10 different samplings from November 2003 to June 2004. DNA damage in mussels gills and PAH content in mussel tissues were analysed and then maintained under laboratory conditions for seven days and then PAH content and DNA damage were evaluated again. No significant differences were obtained in comet tall length between the two locations analysed at the arrival to the laboratory. Significant differences in DNA damage were observed between the arrival and after the recovery for mussels from both locations, indicating that DNA repair took place. A high correlation coefficient was found

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between total PAHs content in sea water and tail length, supporting the utility of the comet assay in biomonitoring programmes.

Experimental study on the effects of Prestige oil on the blue mussel Mytilus galloprovincialis

I. Aldao, B. Laffon, E. Pásaro, and J. Méndez.

In this study mussels were exposed to Prestige oil (1.5 to 3 ppm). Significant increases in DNA damage using comet assay technique were observed in oil-exposed individuals with regard to their corresponding controls, being these increases constant during the exposure period (14 days). DNA damage evaluated after the recovery phase decreased in control individuals, but surprisingly increased in oil-exposed mussels. Authors concluded that recovery time assayed (seven days) was insufficient. There was a good association between tail length and total PAHs in mussel tissues.

Ecotoxicity of sediments contaminated by the oil spill using juveniles of the fish

N. Jimenez-Tenorio, M. J. Salamanca-Marin, C.Morales-Caselles, M. L. Gonzáles De Canales, C. Sarasquete and A. Delvalls .

Two sublethal bioassays with different species of juveniles of the fish (Sparus aurata and Solea senegalensis) were carried out to determine the effects produced in these organisms associated with the contaminants bound to sediments impacted by the oil spill. The results were obtained by comparing an area that suffered an acute impact caused by the Prestige oil spill (Galicia) and other with low or inexistent rate of oil spill. The sediments were subjected to chemical analysis and tested using a battery of chronic sediment toxicity and evaluating histopathological damage in different target tissues (liver and gills), metallothionein content and EROD activity. The results obtained from both bioassays shown clear and similar information. High levels of EROD activity in organisms exposed to sediments with high concentrations of PAHs in comparison with the control sites. Histopathological results shown different histomorphological alterations. Bioassays using juveniles of the fish shown results sensitive enough to determine the hazardousness associated with this contaminated sediment, demonstrating the importance to use chronic bioassays as a useful tool to determine the quality of the sediments and completing the limitations that offer the data of acute bioassays.

Determination of chronic toxicity in sediments impacted by the Prestige oil spill and in sediments affected by continuous spills in the bay of Algeciras using C. maenas and R. philippinarum

C. Morales-Caselles, M. L. Martín-Díaz, M. C. Sarasquete, I.Riba, and Ángel Delvalls

This study, which is a part of an integrated sediment assessment attempted to determine sediment toxicity caused by contaminants from the spills using a battery of acute toxicity test. Three bioassays in solid phase were performed: the microbial bioassay microtox, a bioassay with the polychaeta Arenicola marina, a test with the amphipod Corophium volutator and a bioassay using embryos of the sea urchin Paracentrotus lividus (conducted in sediment elutriates). Microtox test showed similar responses in sediments affected by chronic input of contaminants and those affected by acute impact caused by the Prestige oil spill. The amphipods bioassay and the sea urchin test agree that sediments affected by chronic contamination are more toxic than those impacted acutely by the oil spill. Polychaeta experiment detected some of the samples of sediments acutely impacted and were so toxic as those from chronically impacted sites. Conclusion obtained were that acute toxicity tests seem to be a good tool to assess the effects associated with the sediment contamination caused by oil spill. However, given that the acute toxicity do not give all the information necessary for evaluation of the quality of the sediments impacted by oil spill, sublethal bioassays should be performed to determine chronic effects.

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Bioassays with the amphipods Gammarus aequicauda and Gammarus locusta for oil pollution assesment on European Coasts.

C. Sanz-Lázaro, A. Marín, M. Borredat, J. Lloret, and L. Marín-Guirao

Study to validate the use of two amphipod species Gammarus locusta and Gammarus aequicauda in bioassays for assessing oil toxicity in European coasts. These species were chosen because they are equivalent and have a broad distribution along European coasts. First results showed that these amphipods are suitable to assess oil derivated products sediment toxicty. Due to the fact that there is no European Directive related with oil spills pollution assessment, authors proposed the use of bioassays with these species as a reliable tool for assessing pollution on European coasts.

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Annex 8: Provide expert knowledge to the ICES data centre as appropriate (Agenda item 10)

Lists for recommended fields for biological effects in biota, sediment and water (Tables 10.1– 10.3).

• Table 10.1 Recommended data fields for biological effects in biota • Table 10.2 Recommended data fields for biological effects in sediment • Table 10.3 Recommended data fields for biological effects in water

Table 10.1: Recommended data fields for biological effects in biota.

MONITORING PROGRAMME INFORMATION ERF 3.2 RECOMMENDED FIELDS INPUT CODE DESCRIPTION REFERENCE CODE DESCRIPTION WGBEC recommendations for biota STATN Station name data - biological effects WGBEC recommendations for biota POSYS Positioning system data - biological effects WGBEC recommendations for biota SEXCO Sex code data - biological effects WGBEC recommendations for biota STAGE Stage of development data - biological effects WGBEC recommendations for biota BULKID Bulk identification data - biological effects WGBEC recommendations for biota METOA Method of analysis data - biological effects WGBEC recommendations for biota REFSK Reference or source key data - biological effects WGBEC recommendations for biota FINFL Factors potentially data - biological effects influencing guideline compliance WGBEC recommendations for biota PURPM Purpose of monitoring data - biological effects WGBEC recommendations for biota SMTYP Sampler type data - biological effects WGBEC recommendations for biota ORGSP Origin of specimen data - biological effects WGBEC recommendations for biota METPT Method of pre-treatment data - biological effects

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Table 10.2: Recommended data fields for biological effects in sediment.

MONITORING PROGRAMME INFORMATION ERF 3.2 RECOMMENDED FIELDS INPUT CODE DESCRIPTION REFERENCE CODE DESCRIPTION WGBEC recommendations for STATN Station name sediment data - biological effects WGBEC recommendations for POSYS Positioning system sediment data - biological effects WGBEC recommendations for METOA Method of analysis sediment data - biological effects WGBEC recommendations for REFSK Reference or source key sediment data - biological effects WGBEC recommendations for FINFL Factors potentially sediment data - biological influencing guideline effects compliance WGBEC recommendations for SREFW Source of reference sea sediment data - biological water effects WGBEC recommendations for PURPM Purpose of monitoring sediment data - biological effects WGBEC recommendations for SMTYP Sampler type sediment data - biological effects WGBEC recommendations for ORGSP Origin of specimen sediment data - biological effects WGBEC recommendations for SPECI Species sediment data - biological effects

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Table 10.3: Recommended data fields for biological effects in water.

MONITORING PROGRAMME INFORMATION ERF 3.2 RECOMMENDED FIELDS INPUT CODE DESCRIPTION REFERENCE CODE DESCRIPTION WGBEC recommendations STATN Station name for water data - biological effects WGBEC recommendations POSYS Positioning system for water data - biological effects WGBEC recommendations STIME Sampling start time (UTC) for water data - biological effects WGBEC recommendations DEPHU Pressure/depth where for water data - biological sample is taken (sediment effects depth upper) WGBEC recommendations METOA Method of analysis for water data - biological effects WGBEC recommendations REFSK Reference or source key for water data - biological effects WGBEC recommendations FINFL Factors potentially for water data - biological influencing guideline effects compliance WGBEC recommendations SREFW Source of reference sea for water data - biological water effects WGBEC recommendations PURPM Purpose of monitoring for water data - biological effects WGBEC recommendations SMTYP Sampler type for water data - biological effects WGBEC recommendations ORGSP Origin of specimen for water data - biological effects WGBEC recommendations SPECI Species for water data - biological effects

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Annex 9: Assessment of temporal trends in imposex (Agenda Item 15)

WGBEC met with WGSAEM to initiate an assessment of imposex data on the ICES database – an assessment of temporal trends in imposex

Rob Fryer and Matt Gubbins, Fisheries Research Services, Aberdeen, UK

Introduction

Here, we present an assessment of temporal trends in imposex levels using data held in the ICES database. We use a simple extension of the methodology used by OSPAR MON to assess trends in contaminant levels. For simplicity, we restrict attention to time-series with at least five years of data and consider Vas Deferens Sequence Index (VDSI) as our measure of imposex (Penis Classification Index, PCI, for Buccinum undatum). This gives us 34 time-series from three countries (Denmark, Norway, and UK) in three species (Buccinum undatum, Neptunea antiqua, and Nucella lapillus).

Methodology

VDSI is constrained to lie between 0 and 6, so the distributional assumption of (log-)normal errors used in contaminant temporal trend assessments is not appropriate. However, a simple alternative is to transform the data to lie between 0 and 1 (i.e. by taking VDSI / 6) and to model the data as if they were under-dispersed binomial proportions. We

• fitted a linear logistic function of time to each time-series; • assessed the evidence of any trend over time; • used the upper one-sided 95% confidence limit on the fitted line in the final year of the time-series to make a precautionary classification of the data using the assessment criteria developed for OSPAR (OSPAR, 2004) (Table A9.1).

Table A9.1: OSPAR assessment criteria for TBT, prepared during a technical TBT workshop, 6–7 November 2003, The Hague. The levels of imposex in the 5 key gastropod species monitored in the OSPAR/ICES area are related to a six-class assessment scheme A-F. Although VDSI in Nucella can reach 6, VDSI in Neptunea can not exceed 4.0 and PCI in Buccinum can not exceed 3.5.

ASSESSMENT NUCELLA NASSARIUS BUCCINUM NEPTUNEA LITTORINA CLASS VDSI VDSI PCI VDSI ISI A < 0.3 < 0.3 < 0.3 < 0.3 < 0.3 B 0.3 – <2.0 0.3 – <2.0 C 2.0 – < 4.0 0.3 – <2.0 0.3 – <2.0 2.0 – <4.0 D 4.0 -–5.0 2.0 – 3.5 2.0 – <3.5 4 0.3 – < 0.5 E >5.0 > 3.5 3.5 0.5 – 1.2 F > 1.2

Results

The data and fitted models are summarised in Figure A9.1 and Table A9.2. Nearly all the estimated trends are downwards. There are 10 significant downward trends (at the 5% level) and only 1 significant upwards trend, at Scarf Stane in Shetland, UK, which may be due to a large pelagic fishing vessel that often moors close to the monitoring station. The classification ranges from A/B (for some Buccinum time-series) through to D.

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Thoughts • It would be easy to extend the methodology to use smoothers to describe non-linear trends, but there is no real evidence that they are needed here. • VDSI in Neptunea antiqua can not exceed 4 and PCI in Buccinum undatum can not exceed 3.5, so we should have scaled the data for these species differently (i.e. by taking VDSI / 4 and PCI / 3.5 respectively). • Imposex indices are obtained by sampling 40 individuals for Nucella lapillus or Neptunea antiqua and 100 individuals for Buccinum undatum (OSPAR 2002). However, only the females are used to determine the VDSI or PCI indices. The number of females in the sample might be used to weight each observation. • The assumption of quasi-binomial errors is pragmatic and appears reasonable for these data. However, VDSI is obtained by scaling each female (between 0 and 6) and taking the mean scale. These scales are ordinal – they have no sense of distance between them – and it is ‘harder’ to move between some scales than others. For example, it is particularly ‘hard’ to progress to VDSI >4.0 in Nucella lapillus. This is apparent from dose response curves for Nucella VDSI. Davies (2000) has also shown that the half life of stage 4 is much longer than the half lives of stages 1-3 during the development of imposex in transplanted adult Nucella populations. Alternative weighting schemes need to be investigated which allow for greater variability in VDSI when the mean level is between 1 and 3. A more realistic, but more complex, statistical model would be a proportional-odds or proportional- hazards model (McCullogh and Nelder, 1989) fitted to the individual observations.

References Davies, I.M., 2000. Kinetics of the development of imposex in transplanted adult dogwhelks, Nucella lapillus. Environmental pollution, 107: 445–449. McCullagh, P., Nelder, J.A. 1989. Generalized Linear Models (second edition). Chapman and Hall, London. OSPAR. 2002. Revised technical annex 3 of the OSPAR guidelines for contaminant-specific biological effects monitoring (TBT-specific biological effects monitoring). Annex 10, summary record, ASMO, 2002. 18 pp. OSPAR. 2004. Proposal for Assessment Criteria for TBT – Specific Biological Effects. ASMO 04/3/3. OSPAR Environmental Assessment and Monitoring Committee, Stockholm, 29 March–2 April 2004.

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VDSI or PCI

Denmark schultz´grund gilleleje ven s ven n anholt Buccinum undatum 4

2

0

skagen halskov

4

2

0

Denmark hirtshals n gilleleje halskov ven n ven s Neptunea antiqua 4

2

0

Norway færder melandholmen Nucella lapillus 4

2

0

UK norther geo burgo taing easterwick samphrey the brough Nucella lapillus 4

2

0

uynarey orfasay billia skerry east of ollaberry moss bank

4

2

0

little roe grunn taing voxter ness tivaka taing skaw taing

4

2

0

the kames northward mavis grind noust of burraland scarf stane

4

2

0

1990 2000 1990 2000 1990 2000 1990 2000 1990 2000

Figure A9.1: The data (circles) and fitted models (solid lines) with pointwise (two-sided) 90% confidence bands (grey shaded areas). The thin dashed horizontal lines are the boundaries between the assessment classes. The time-series lies in a particular assessment class (or better) with 95% confidence if the upper confidence limit in the final year lies below the corresponding assessment boundary.

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Table A9.2. Results for each time-series: trend indicates whether the estimated trend is positive of negative; p trend gives the significance of the trend with values significant at the 5% level highlighted in bold; fit is the fitted value in the last monitoring year; ucl is the upper one-sided 95% confidence limit in the last monitoring year; class is the classification.

COUNTRY SPECIES SITE TREND P TREND FIT UCL CLASS Denmark Buccinum undatum Schultz´grund - 0.001 0.04 0.06 A/B Gilleleje - 0.046 0.07 0.18 A/B Ven s - 0.024 0.13 0.26 A/B Ven n - 0.110 0.12 0.46 C Anholt - 0.454 0.29 0.60 C Skagen - 0.727 0.22 0.72 C Halskov - 0.148 0.92 1.27 C Neptunea antiqua Hirtshals n - 0.975 1.41 2.24 C Gilleleje - 0.159 2.61 3.35 C Halskov - 0.278 3.88 4.04 D Ven n + 0.953 3.83 4.09 D Ven s - 0.379 3.65 4.09 D Norway Nucella lapillus Færder - 0.078 3.83 3.96 C Melandholmen + 0.905 4.21 4.49 D UK Nucella lapillus Norther geo - 0.029 0.07 0.36 B Burgo taing - 0.013 0.35 0.68 B Easterwick + 0.292 0.30 0.74 B Samphrey - 0.047 0.30 0.83 B The Brough - 0.068 0.48 1.16 B Uynarey - 0.012 0.82 1.32 B Orfasay - 0.079 0.68 1.53 B Billia Skerry - 0.107 1.32 1.88 B East of Ollaberry - 0.003 1.60 2.09 C Moss Bank - 0.096 1.53 2.51 C Little Roe - 0.114 3.23 3.90 C Grunn Taing - 0.206 3.77 4.01 D Voxter Ness - 0.002 3.90 4.14 D Tivaka Taing - 0.058 3.91 4.22 D SKAW Taing - 0.272 4.10 4.41 D The Kames - 0.002 4.04 4.44 D Northward - 0.065 4.06 4.48 D Mavis Grind - 0.201 4.24 4.48 D Noust of Burraland - 0.316 4.15 4.52 D Scarf Stane + 0.006 4.30 4.54 D

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Annex 10: A regional assessment of VDSI in dogwhelks from Sullom Voe and Yell Sound (Agenda item 15)

WGBEC used the opportunity to meet with WGSAEM to initiate an assessment of imposex data on the ICES database – a regional assessment of VDSI in dogwhelks

Rob Fryer and Matt Gubbins, Fisheries Research Services

Introduction

The oil terminal at Sullom Voe, Shetland, has been in operation for more than 20 years. Tankers enter the Voe via the northern or south-eastern entrances to Yell Sound and berth at the terminal loading jetties. Imposex levels have been monitored in the Sullom Voe region of Shetland since 1987. Twenty stations are monitored, mostly bi-annually (Figure A10.1).

Figure A10.1: Imposex sampling sites in Sullom Voe and Yell Sound. The colour indicates the strata used for assessment purposes (see Figure A10.3).

The Vas Deferens Sequence Index (VDSI) data from these stations were analysed for temporal trends and compared with OSPAR assessment criteria (OSPAR, 2004) as part of an assessment of imposex data held in the ICES database (Fryer and Gubbins, 2006). The results are reproduced below (Figure A10.2).

The results clear indicate the patterns of change at individual stations. However, it can be difficult to combine the results of many individual assessments and convert them into

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management advice. For example, although most of the fitted lines suggest decreasing VDSI levels, only eight are significant (at the 5% level) and one of these is an increasing trend at station 4 (Scarf Stane). VDSI levels also range widely between stations.

Here, we present a regional assessment of the data that attempts to make more general statements about VDSI levels and trends in Sullom Voe and Yell Sound.

VDSI 20 norther geo 2 burgo taing 1 easterwick 16 samphrey 19 the brough

4

2

0

17 uynarey 15 orfasay 3 billia skerry 5 east of ollaberry 14 moss bank

4

2

0

18 little roe 6 grunn taing 10 voxter ness 7 tivaka taing 13 skaw taing

4

2

0

12 the kames 11 northward 9 mavis grind 8 noust of burraland 4 scarf stane

4

2

0

1990 1995 2000 1990 1995 2000 1990 1995 2000 1990 1995 2000 1990 1995 2000

Figure A10.2: VDSI data from Sullom Voe and Yell Sound (open circles) with fitted trends (solid lines) and pointwise 90% confidence bands (grey shaded areas). The thin horizontal lines are the boundaries between the assessment classes (OSPAR, 2004). Each time-series is labelled by its number and name.

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Methodology

Superimposing the fitted trends for each time-series reveals several groupings of stations (Figure A10.3).

VDSI

6

4 9 1211138 4 107 6

18

2

14 5 3

17 15 19 16 2 1 0 20

1990 1995 2000 Figure A10.3: Fitted trends for each time-series in Sullom Voe and Yell Sound. Each line is labelled by its station number. The colour indicates the post-stratification.

• All the stations in the Voe itself (6–13, 18) cluster together. The VDSI at Station 18 appears slightly less than the others due to the unusually low observation in 2001, which we omit in later analysis. • The remaining stations (except for station 4) also cluster together. VDSI levels appear to decrease moving north up Yell Sound (i.e. away from the oil terminal), but a similar effect is not evident moving south east (see stations 14 and 17). Some hydrographic information may give more insight here. • Station 4 is clearly unusual. It is the only station outside the Voe that has high levels of VDSI, and is the only station with a significant increasing trend (Fryer and Gubbins, 2006). The high levels of VDSI may be due to very localised activities; for example, one of the largest Scottish pelagic fishing vessels often moors to a pier close to the monitoring station. Since Station 4 may be monitoring a local point source, we omit it from the regional assessment.

For further analysis, we post-stratify Sullom Voe into two: the Voe (stations 6–13, 18) and the Sound (stations 1–3, 5, 14–17, 19, 20). The categorisation of stations 5 and 18 is data driven, rather than geographically driven, and it would be desirable to reinforce this decision with hydrographical arguments.

We modelled the data using a generalised linear mixed model, assuming quasi-binomial errors and fixed and random effects:

• fixed ~ time + strata + time.strata • random ~ station + time.station + year + year.strata

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where time denotes a continuous variable and year the same data but expressed as a categorical variable. The fixed effects allow for

• a trend over time; • a different level for each stratum; • an interaction between time and strata (i.e. a different trend in each stratum).

The random effects allow for

• variation in levels between stations (within strata); • variation in trends between stations (within strata); • between-year variation in VDSI common to all stations; • between-year variation in VDSI common to all stations within a stratum (but possibly different between strata).

Results

The fitted model is shown in Figure A10.4. The slopes differ between strata (p = 0.02). There is a significant downwards trend in the Sound (p = 0.0001) whilst the estimated trend in the Voe is less steep and not significant at the 5% level (p = 0.09).

VDSI

6

4 Voe

2

Sound

0

1988 1992 1996 2000 2004 Figure A10.4: The fitted model showing the mean VDSI level in both the Voe and the Sound (solid lines) with pointwise 90% confidence bands (grey shaded areas). The thin grey lines represent the boundaries between OSPAR classes (OSPAR, 2004).

The upper (one-sided) 95% confidence limits on the fitted lines in 2004 are 0.9 and 4.5 for the Sound and the Voe respectively. Thus, a classification for the Sound and the Voe based on mean VDSI levels (averaged across stations) might be C and D respectively (c.f. OSPAR assessment criteria, OSPAR, 2004; Fryer and Gubbins, 2006).

However, an upper (one-sided) 95% prediction limit for the VDSI level in a station sampled at random from the Sound or the Voe is 1.6 and 5.2 respectively, leading to precautionary classifications of C and E.

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Thoughts

• We have assumed that the between-station and within-station variances are common to both strata. However, inspection of the raw data (Figure A10.2) and the superimposed trends (Figure A10.3) suggests that both types of variation are greater in the Sound than the Voe. This means that the upper 95% confidence and prediction limits are probably too low for the Sound and too high for the Voe. It also means that that the trend is probably ‘too significant’ in the Sound and ‘not significant enough’ in the Voe. We might need to model the data for each stratum separately. This would be defensible since the between-year variation common to both stratum was negligible (see below). • The larger within-station variation in the Sound is probably because VDSI measurements between 0 and 3 evident in this group of stations are more inherently variable than VDSI measurements between 4 and 5 which are more prevalent in the Voe. This is also discussed in Fryer and Gubbins (2006). • Some of the between-site variation in the Sound might be structural rather than random (e.g. the decline in VDSI moving north). We might explain some of this variation by using additional fixed effects. • We have assumed that the station and time.station random effects are uncorrelated. This needs to be checked. • The between-year variation common to both strata was negligible, but the between- year variation within strata was significant, as was the variation in levels and trends between stations. The variance components could be used to say how many stations are needed within each stratum to detect trends with a specified power, or to classify the stratum with a certain precision. • Although there is no significant trend in VDSI in the Voe (at the 5% level), it would be interesting to compare this assessment with one for % sterile females, an alternative measure of imposex recommended for Nucella in the OSPAR guidelines (OSPAR, 2002). Although OSPAR assessment criteria have not been developed for % sterile females, it may be a more sensitive index than VDSI for temporal trend assessments of highly affected populations. It is also closely related to the reproductive potential of the population.

References Fryer, R.J., and Gubbins, M.J. 2006. An assessment of temporal trends in VDSI. Working document to WGBEC and WGSAEM. OSPAR. 2002. Revised technical annex 3 of the OSPAR guidelines for contaminant-specific biological effects monitoring (TBT-specific biological effects monitoring). Annex 10, summary record, ASMO, 2002. 18 pp. OSPAR, 2004. Proposal for Assessment Criteria for TBT – Specific Biological Effects. ASMO 04/3/3. OSPAR Environmental Assessment and Monitoring Committee, Stockholm, 29 March–2 April 2004.