Prosjektnr: /720 Bevilgningsperiode og finansiering fra Norges forskningsråd: : 1,500, : 1,000,000

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1 1 Effekter av produksjonsvann på egg og larveutvikling samt kjønnsdifferensiering hos torsk Havforskningsinstituttet Svardal, Asbjørn Forsker Prosjektnr: / : 1,000, : 1,000, : 1,000,000 Prosjektet er primært en undersøkelse av om eksponering av egg, plommesekklarver og yngel til produksjonsvann påvirker senere kjønnsdifferensiering. Et delmål er å studere effekten av produksjonsvann på egg- og larveutviklingen. Et annet delmål er å undersøke om eksponering av egg og larver til produksjonsvann kan påvirke otolittenes bilaterale symmetri på et senere utviklingstrinn. Dette har interesse siden det er blitt foreslått at graden av avvik fra denne symmetri kan reflektere ulike typer miljørelatert stress. Det finnes et stort behov for økt kunnskap om virkningene, spesielt langtidsvirkningene, av regulære utslipp av kjemikalier og oljeholdig produksjonvann fra petroleumsvirksomheten på norsk sokkel. Som et ledd i arbeidet med å framskaffe slik kunnskap skisseres et prosjekt hvor en vil studere bestemte bioeffekter av produsert vann. En vet at eksponering for visse stoffgrupper som finnes i produksjonsvann, f.eks. alkylfenoler, kan medføre endokrinologiske forstyrrelser både hos mennesker, andre mammalske organismer og fisk, med reproduksjonsproblemer som den endelige konsekvens. Problemet omfattes av den grunn med stor interesse både i vitenskapelige kretser og massemedia. Undersøkelsen gjøres ved å eksponere egg, larver og yngel direkte til produksjonsvann og drette yngel videre fram til kjønnsmodning for å se om produksjonsvann kan påvirke kjønnsdifferensieringen. Hormonell status vil bli undersøkt ved å ta blodprøver av fisken for å se om produksjonsvannet påvirker nivået av kjønnshormonene østradiol-17^x og testosteron. En vil også grundig studere egg og larveutvikling samt sammenligne vekst og overlevelse av de ulike larvegruppene gjennom startfôringsfasen og senere yngelstadium. 2 Contamination of fish in the North Sea by the offshore oil and gas industry Havforskningsinstituttet,Senter for marint miljø Klungsøyr, Jarle Forsker Prosjektnr: / : 1,500, : 1,000,000 Undersøke om kjemiske komponenter i operasjonelle utslipp fra oljeinstallasjoner bioakkumuleres og gir effekter på fisk, og om fiskens kvalitet som menneskeføde påvirkes negativt. Delmål: - Kartlegge nivåene av alkylfenoler og NPD/PAH i fisk fra Tampen- og Sleipnerregionen. Måle gallemetabolitter av NPD/PAH og bestemme sammenhengen med konsentrasjonene i lever/muskel. Måle ulike biomarkører for å bestemme biologiske effekter. Måle om kvaliteten på fiskemuskel forringes ved eksponering til oljekomponenter. - Modellere konsentrasjonsfeltene av alkylfenoler og NPD/PAH i utslippsområdene, og koble dette til forurensningsgraden i fisk. Prosjektet skal gi dokumentasjon på om nivåene av aromatiske hydrokarboner (NPD/PAH) og alkylfenoler er forhøyet i fisk fra havområder med store operasjonelle utslipp fra offshore olje- og gassindustri. Arbeidet vil gi informasjon om eksponering og belastningsgrad på fisk fra Tampen- og Sleipner-regionen, og vurdere dette opp mot ulike tilførselskilder og den generelle bakgrunnsbelastning av forurensning på fisk i Nordsjøen. Det skal gjennomføres målinger av biologiske effekter og faktorer som påvirker fiskens kvalitet som menneskeføde. Arbeidet skal kunne bidra til utforming av fremtidig overvåkning på forekomst og virkninger på fisk av oljeindustriens utslipp til sjø. 3 Hormonforstyrrende effekter av miljøgifter i produksjonsvann fra oljeinstallasjoner Havforskningsinstituttet,Senter for marint miljø Svardal, Asbjørn Forsker Prosjektnr: / : 1,000, : 1,000,000 Hovedmål: Utvikle og etablere metoder for å undersøke hvordan alkylfenoler i produksjonsvann virker på kjønnsutvikling og kjønnshormonsystemet i fisk. Delmål: 1.Utvikle metode for bestemmelse av alkylfenoler i sjøvann, plasma og vev

2 2.Etablere nødvendige metoder og undersøke normal kjønnsdifferensiering hos torsk 3.Etablere bestemmelse av enzymaktivitet for P450 aromatase 4.Undersøke opptak av alkylfenoler og PAH fra produksjonsvann i zooplankton og torskeyngel 5.Effekter av alkylfenoler på aromataseaktivitet i fiskeceller i kultur og i vev fra torskelarver Prosjektet fokuserer på langtidseffekter av alkylfenoler som forekommer i produksjonsvann fra oljeplattformer på fisk og følger dermed opp nylig gjennomførte undersøkelser på torsk ved Havforskningsinstituttet. Første del av prosjektet omfatter videreutvikling av metoder for analyse av alkylfenoler i sjøvann og biota, histologiske undersøkelser for å finne målbare kriterier for normal kjønnsutvikling hos torsk, og etablering av biokjemiske metoder for å studere mekanismer bak effektene alkylfenoler har på fisk. Metodene benyttes til å studere virkningsmekanismer i fiskeceller in vitro, og til undersøkelser av kjønnsutvikling og kjønnshormonmetabolisme hos torskeyngel som eksponeres for alkylfenoler i et eksperimentelt oppsett. Dette kan gi indikasjoner på tidskritiske vinduer der torsken er mer sårbar for påvirkning av kjemisk forurensning som kan forstyrre kjønnsutviklingen og dermed reproduksjonen. Metodene vil senere kunne anvendes for å gi detaljkunnskap om kjønnsutvikling og mekanismer og dermed et bedre grunnlag for å evaluere langtidseffekter av kjemisk forurensning på fisk. 4 GC/MS determination of produced water PAH and alkylphenol metabolites in marine fish Rogalandsforskning Beyer, Jonny Seniorforsker Prosjektnr: / : 74,059 Objectives To compile (and disseminate) an optimised protocol for GC/MS detection of bile metabolites of naphthalene and small PAHs that typically characterise PW discharges. To perform lab exposure of cod to PW related alkylphenols in order to produce suitable samples of fish bile for the GC/MS AP-metabolite feasibility study. To conduct the GC/MS AP-metabolite feasibility study aimed at developing and optimising a GC/MS protocol for detection of PW alkylphenol metabolites in fish bile. To use this GC/MS method in analyses of alkylphenol metabolites in bile from fish obtained at Tampen and at reference field locations. Atlantic cod is selected as preferred study species in the project but also other fish species (e.g. haddock) may be included depending on the outcome of the field sampling (described in NFR (Klungsøyr)). When fish are exposed to PAHs and APs originating from produced water (PW) discharges the PAH and AP parent compounds are metabolised and excreted in the gall bladder bile. Metabolites of PAH and alkylphenols (AP) in fish bile may thus be used as exposure markers of PW contamination at offshore oil fields in supplement to chemical analyses of PAH and AP parent compounds in fish tissues. The scope of the project is to develop GC/MS protocols optimised for detection of PAH and AP metabolitesin fish bile, and to validate the use of these measures as exposure-detection tools in assessment of PW contamination in fish. 5 Hydrocarbon release from oil droplets to seawater: experimental and computational verification of a model Rogalandsforskning Skadsheim, Arnfinn Seniorforsker Prosjektnr: / : 395,200 Verify predictions provided by the weathering model of Short & Heinz (1997) & Short (2002) for oil droplets. Improve the equations by correction terms if this improves the goodness of fit between the model and the data. Improve the understanding of relationships between hydrocarbon transfer between oil droplets, seawater and biota, as exemplified by mussels, semipermeable membrane devices and plastic strips that are frequently used in monitoring. Conduct a pre-project for studies on oil droplet behaviour, hydrocarbon transfer between compartments, and how toxic impact may be inflicted in marine waters. The actual impact of oil particle size on hydrocarbon release rates to seawater, bioavailability and bioconcentration of hydrocarbons has not yet been properly described. The model of Short & Heinz (1997) & Short (2002) has equations derived from PAH release from oil films to water. It may be used to describe release rates of various hydrocarbons from oil droplets to the surrounding seawater, but it needs to be verified and

3 possibly thereafter adjusted by goodness of fit calculations. The project proposes to expose mussels, semipermeable membrane devices and plastic strips to a North Sea and a Barents Sea crude. Each oil is dosed separately at 2 oil droplet sizes typical for produced water in a continuous flow system. Oil droplets will be separated from the water prior to analyses. The exposed "objects" serve as analyses and assessment of hydrocarbon availability for uptake in order to predict how hydrocarbons move between oil droplets, water and biota. The outcome will illustrate the relevance of bioavailability-assumptions in presently used fateexposure-effect models. It will improve toxicity identification and evaluation procedure for single substance and mixtures of substances, estimates of near and far field scenarios at discharge points, guide development of cleaning technologies as well as monitoring methods, and improve methods for ecotoxicological studies in the water column, sea-ice and sediments. 6 Impacts of metals from drill cuttings and mud to the marine water column Rogalandsforskning Westerlund, Stig Seniorforsker Prosjektnr: / : 667,759 Prosjektet utgjør et forprosjekt som skal generere metodiske resultater om metallers mobilitet i borekaks og slam. Det er foreslått bruk av sekvensiell ekstraksjon for å estimere mobilitet av metaller for å få et enkelt mål på biotilgjengelighet. Resultatene vil videre brukes til å utvikle mer relevante tester av biotilgengengligheten av metaller i borekaks og slam, samt måling av resuspensjon av slam og kaksmateriale og dertil estimering av utlekking fra denne type av prosesser, kombinert med giftighetstester. Baritt og illmenitt brukes hovedsaklig som vektstoffer i boreformuleringer, og slippes ut i store mengder med boreoperasjoner. Det er tidligere antatt at metallforurensinger og barium som er en hovedkomponent i baritt er meget sterkt bundet i partikler, og dermed lite biotilgjengelige, men dette er ikke godt dokumentert. Baritt- og illmenittpartikler er små, har lav synkehastighet, og spres derfor over store avstander med vannmassene. Pga. de store mengdene som slippes ut, og spredningsmønsteret, kan utlekking fra partiklene få relativt stor betydning for miljøet hvis det forekommer i tilstrekkelig grad. På denne bakgrunn tar prosjektet for seg utlekking til vannsøylen fra boreutslipp, spesielt utlekking av metaller (barium og andre metaller) fra baritt og illmenitt. Kjemisk sekvensiell ekstraksjon av deponert kaks og slam fra felt, fersk kaks og slam fra plattform, ubrukt slam samt baritt og ilmenitt partikler direkte vil bli gjennomført for å se om det er forskjeller i utlekking og dermed mulig biotilgjengelighet. Enkle eksperimentelle systemer for målinger av metaller ved suspendering og utløsning vil bli etablert. Effekter av suspendert og løst materiale vil bli målt ved enkle marine giftighetstester. 7 Identification of ecologically relevant toxic components in effluents from offshore activities (OffTiE) Norsk institutt for vannforskning Tollefsen, Knut-Erik Forsker Prosjektnr: / : 100,000 The objective of the project is to identify ecologically relevant group of toxic components in produced water by a bioassay-directed fractionation and identification procedure Effluents from oil and gas production activities (produced water) contain complex mixtures of organic and inorganic components. However, since environmental monitoring programs in the North Sea focus on a limited set of chemicals who's toxicological properties are either well documented or theoretically may be of concern, chemicals with less characterised toxic activity may not be properly addressed. This is of particular concern for chemicals that act through mechanisms involving perturbations of natural reproduction and development, and which may be more sensitive than the toxicological endpoints utilised in traditional chronic tests.the objective of the proposed project is to a) identify ecologically relevant groups of toxic components in produced water effluents by a bioassay-directed fractionation and identification procedure and b) assess the ecological risk these groups of chemicals pose to the marine environment. 8 Biodegradiation of oil in the seawater column with emphasis on Arctic conditions SINTEF Materialer og kjemi Brakstad, Odd Gunnar Forsker Prosjektnr: /720

4 : 588,000 Utvikling og etablering av eksperimentelle systemer for laboratorie-baserte studier av oljens biodegraderbarhet i den marine vannsøylen med vekt på simulering av arktiske betingelser. Delmål: -Etablering av systemer for anriking av mikrobielle populasjoner som degraderer olje i kaldt sjøvann (psykrofile mikroorganismer) -Etablering av et eksperimentelt to-fase system for simultane studier av oljeforbindelsers utløsning til vannfase og for bestemnielse av biodegraderingsprosesser i vann-og oljefaser i kaldt sjøvann. -Utvikling av et gjennomstrømmingssystem for biodegradering under arktiske betingelser -Sammenligning av oljeforbindelsers persistens i tempererte og kalde sjøvannssystemer Biodegradering av olje i den marine vannnsøyle er en funksjon av prosesser som skjer i både vann- og oljefaser. De fleste marine biodegraderingsstudier har blitt utført enten i vannfaser eller i dispersjoner og ved moderate til høye vanntemperaturer. I dette forprosjektet vil det bli utviklet og etablert systemer for simultane studier av viktige abiotiske (utløsning av oljekomponenter) og biotiske prosesser som bestemmer oljens skjebne i den marine vannsøyle. Systemer og prosedyrer vil bli etablert for anriking av mikrober som er viktige ved biodegradering av olje i arktiske miljøer (psykrofile mikroorganismer). Et statisk system som allerede er etablert ved SINTEF-gruppen sitt laboratoriet for målinger av biodegradering i et tofase system (basert på olje imobilisert til hydrofobe duker) vil bli tilpasset for kaldtvannsystemer (&61603; 0^U C). Dette systemet vil videre bli utviklet som et gjennomstrømmingssystem for å kunne studere nedbrytingshastigheter og - mekanismer under realistiske betingelser (simulerer naturlige feltsituasjoner). De eksperimentelle systemer vil bli benyttet for målinger av utløsning og nedbryting av komponenter fra to oljer med ulike egenskaper ved tempererte (13^U C) og kalde (&61603; 0^U C) sjøvannsbetingelser. 9 Chemical composition and toxicity of bioavailable polar crude oil fractions - a literature study SINTEF Materialer og kjemi Melbye, Alf Glein Forsker Prosjektnr: / : 200,000 By extensive literature search gain more knowledge on the toxicity and chemistry of the bioavailable polar components of crude oils. This project proposal is a revision of a the original project proposal, and has been revised according to the actual funding, and also with respect to the comments received from the Research Council of Norway. The project is limited to a literature stud y. The aim of the study is to gain more knowledge on the toxicity and chemistry of the bioavailable polar components of crude oils, which is poorly understood. The results will be used in later projects with focus on experimental work. 10 Long-term (chronic) effects of produced water effluents affecting reproduction in marine crustacean plankton. Introductory activities Institutt for biologi,norges teknisknaturvitenskapelige universitet Olsen, Anders J. Forsker Prosjektnr: / : 650,000 Principal objectives (covering introductory activities + main project):? establish novel insight into the susceptibility of pelagic crustacean plankton for chronic exposures to oil-related hydrocarbons, with special emphasis on C.finmarchicus. Reveal secondary impacts on other species or ecosystem structures induced by- or related to biological effects on the plankton level.? derive key input data to excising and future computerbased simulation tools? general competence build-up within the field of marine ecotoxicology. Sub-goals (covering introductory activities only. Shortened, for entire list see project description)? establishment of co-operation routines NTNU- Nordlandsforsning? a literature survey, contact with researchers in Norway and abroad? initial grazing tests with C.finmarchius, selection of feed algae? planning and decision on equipment for Calanus rearing? planning and decision on equipment and methodology for exposure and chemical addition? establishment of a doctoral program and announcement of the doctoral position This introductory project is primarily aimed to initiate and coordinate activities to be continued in the planned

5 follow-up (main) project. The main project primarily covers laboratory-based testing and evaluation of ecological significant chronic effects in marine zooplankton of long-range exposure to low-concentrated oil-related discharges. A number of issues are included in the introductory project, structurally, scientifically and methodologically. Structurally items include establishment of co-operation routines, assigning of different project tasks to the involved institutions and agreement and planning on student and doctoral supervision. Scientifically items include a literature survey, test cultivation of candidate feed algae, and initial grazing tests with C. finmarchicus. Methodological issues may also be regarded as part of the scientific issues, but is here treated separately. They include planning/construction of rearing and exposure equipment and chemical dosing technology. There is currently an increasing concern that emissions from the ongoing oil drilling activity on the Norwegian shelf shall induce detrimental effects on marine ecosystems, and reliable data from the project will therefore be beneficial both to the oil industry, environmental protection authorities and the fishing industry/seafood producers. 11 Hormone disruption and possible DNA damage on fish of alkylphenols in produced water from offshore oil installations Havforskningsinstituttet,Senter for marint miljø Svardal, Asbjørn Forsker Prosjektnr: / : 2,175, : 2,810, : 2,570,000 Investigate the effects of alkylated phenols from produced water from oil installations on sexual development and possible DNA damage in fish and study the mechanisms for these effects. A. Reproduction effects of alkylphenols and extracts of produced water on cod (Gadus morhua) 1. Development of analytical methods (vitellogenin, gene expression of FSH and LH) 2. Establishing protocol for extraction of produced water 3. Exposure and sampling of fish 4. Analysis of fish samples (steroids, aromatase, gonadotropins, vitellogenin, membrane lipids) 5. Spawning and sampling of eggs - determination of biological parameters 6. Studies of second generation effects - determination of biological parameters B. Mechanistic studies of effects of alkylphenols on fish cells in culture 1. Develop and verify assay for P450 aromatase 2. Develop method for determination of gene expression of P450 aromatase 3. Develop and evaluate Comet assay for analysis of single-stranded DNA breaks 4. Determine acute toxicity of C4-C7 alkylphenols on RTG-2 cells 5. Determine hormone disrupting effects on RTG-2 cells after exposure to C4-C7 alkylphenols 6.Determine DNA damage in RTG-2 cells after exposure to C4-C7 alkylphenols The project follows up a previous study on the effects of alkylphenols on cod. Sexually mature cod will be exposed through the feed to C 4 -C 7 alkylphenols and to extracts from produced water. The doses include similar and down to 1/100 of those used in the previous study. The exposed fish will be allowed to spawn naturally in enclosures to confirm the indications that alkylphenols postpone the onset of spawning in females and get exact information about important effects on reproduction. Secondgeneration effects will be determined in terms of malformations and hatching success of the offspring. Complementary to the in vivo studies the project will use in vitro systems to focus in more detail on steroid metabolism and possible DNA damage of alkylphenols. Rainbow trout gonadal cells are used as a convenient model system, and the knowledge obtained from these experiments will be transferred to the in vivo studies for analysis of gene expression and activity of the key enzyme P450 aromatase in brain and gonads of the exposed fish. The brain-pituitary-gonadal axis will be further investigated by analysis of gene expression of the gonadotropins in the pituitary and steroids level in plasma of the exposed fish. 12 Crude oil pollution measured in discharged processed water flows using optical polarisation Fysisk institutt,universitetet i Bergen Hammer, Erling A. Professor Prosjektnr: / : 386, : 137,000 Today the amount of discharged oil from oil platforms to the sea is measured by taking samples of the continuous discharges once a day with following laboratory analysis. Accidental discharges of oil between the samples can therefore occur without being monitored. Thus, there is a need for a robust instrument that can measure continuously the pollution of the discharged water. In the end of this project it should be possible to demonstrate a laboratory prototype of an instrument that can measure continuously the pollution in discharged water from oil production facilities down to 5 ppm. The optical polarisation plane is rotated if the medium is obtically active. Optical activity is caused by asymmetry of molecules that have a spiral shape. Different molecules

6 affect the optical rotation differently. Hydrocarbons, such as crude oils, contain complex organic molecules that contribute to rotation of the optical polarisasjon plane. Thus, by detecting the rotation of the polaritation angle of a polarised laser beam the concentration of crude in the water can be found down to 5 ppm. The main objective of the project is: 1. To map the specific polarisasjon plane rotation of optical active components in processed water from different oil/gas fields in order to evaluate the potential of this technology used in this or other industrial applications. 2. To develop a method for automatic detection of the angle of the polarisation plane. 13 Validation of methods and data for Environmental Risk Assessment off-shore Akvamiljø as Sanni, Steinar Direktør Prosjektnr: / : 2,330, : 2,330, : 2,330,000 Principal objective is to obtain more relevant and consistent systems for risk predictions and impact assessments that enable predictions of ecosystem health which can be monitored in the field. Sub-goals are to: - Establish links between Environmental Risk Assessment (ERA) and Biological Effect Monitoring. - Establish critical no harmful response limit values (acceptance criteria) for field biomarker signals. - Validate ERA model data by relevant laboratory experiments. - Extend ERA model to include biological effect parameters. - Establish a basis for interpretation of biomarker methods proposed for ERA related field monitoring. - Disseminate validated results. Environmental management of off-shore activities is based on sound methods of risk assessment, recently developed and in use by the industry, and being further developed in a related project ("ERMS"). To verify the reliability of the risk assessment, both input information (laboratory tests) and model output, require field validation. For extension to new areas with other environmental conditions (deep-sea, shallow near-shore and Arctic), and different discharge situations (produced water, drilling, irregular spills) the risk parameters must be validated for these particular conditions and situations. A basis for validation using biological methods will be established. Selection of methods with emphasis on "biomarkers" to serve as tools in field monitoring will be made. Relationships will be established between biomarkers and conventional end-point parameters that are used in current Environmental Risk Assessment (ERA). Critical "no harmful response" limit values will be established for the biomarker signals to serve as reference (PNEC) in the risk assessment. Field validation will be carried out to relate and verify exposure concentrations and biomarker responses for different discharges and areas. Linking biomarkers to the ERA creates a basis for field measurements of predicted biological effects. This "bridge" represents an important step forward in monitoring potential impacts that can be evaluated in relation to pre-assessed accepted criteria. 14 Pollutant exposure and effects in fish related to the discharge of produced water in the North Sea oil industry Rogalandsforskning Beyer, Jonny Seniorforsker Prosjektnr: /S : 1,945, : 1,954, : 697,000 1) To develop, use and field validate better tools for detection of PW related alkylphenol, metal and genotoxic exposure in marine fish based on analyses of fish bile samples. 2) To study the connection between PW exposure and relevant effect biomarkers in marine fish exposed to PW components in the lab and in the field, with emphasis on: - Biomarkers of genotoxic stress - Endocrine, reproduction and development effects - Effects on fish vitamin homeostasis - Proteomics (protein recognition) pattern in fish plasma The project addresses environmental issues related to produced water (PW) discharges from offshore installations. The question is whether fish populations in the water column in regions of the North Sea are in a condition of (sublethal) stress caused by a long-term, lowconcentration exposure to chemical constituents from the PW discharges. Atlantic cod is used as study organism, and a set of exposure and effect biomarkers is suggested in the project in order to evaluate the fish condition. The investigation is based on analyses of fish collected at the Tampen area, a region of the North Sea much affected by PW discharges. Studies in fish exposed to PW components under laboratory control are also carried out to describe the connection between exposure and effects. The project also includes development, refinement and validation of analytical tools for better detection of PW exposure and effects in marine fish.

7 15 Algorithms for automatic detection of oil spills in SAR images - ADOS - Institutt for informatikk,universitetet i Oslo Solberg, Anne Helene Schistad Førsteamanuensis Prosjektnr: /S : 610, : 585, : 604,000 Main objective: To fully exploit the potential for automatic detection of oil spills using new, advanced algorithms, and to demonstrate how well such a system will perform in terms of accuracy on a large set of SAR images both from the ERS satellite, ENVISAR and RADARSAT. Sub-goals: 1. To study SAR imaging of oil spills and similarly looking oceanographic phenomena 2. To develop improved algorithms for dark spot detection 3. To develop improved algorithms for dark spot feature extraction 4. To develop improved algorithms for spot classification 5. To verify and demonstrate the algorithms on a large set of images The quantity of marine pollution arising from illegal oily discharges from tank cleaning or bilge pumping is much larger than that following spectacular accidents like that of the Erika off the coast of the United Kingdom or the Exxon Valdez in Alaska. Remote sensing imagery may provide an effective tool for regular monitoring of large ocean areas. Given suitable meteorological conditions these oil spills can be seen from space using synthetic aperture radar imagery. There is a potential for automatic detection, either fully automatic systems or semiautomatic systems. The ADOS project aims at improving the previous attempts on automatic oil spill detection, and extending the algorithms to work for. both ENVISAT ASAR images and RADARSATimages, in addition to ERS SAR images (which the algorithms were designed for). 16 Long-term effects of offshore discharges on cold water zooplankton: establishing a test system for chronic exposure Akvaplan Niva AS Carroll, Jolynn Forskningskoordinator Prosjektnr: /S : 565, : 1,193, : 150,000 Objective: To study responses of Calanus spp. to longterm, sublethal exposure to selected offshore discharges and discharge components, by use of a laboratory based facility for culture through several generations. Sub-goals: To establish a multi-generation experimental system for studies of zooplankton responses to offshorerelated discharges, with emphasis on C. finnmarchicus. To assess the applicability of Calanus sp. as a test genus for effects studies. To conduct preliminary experiments addressing life history impacts to C. finnmarchicus from realistic exposure to produced water discharges, drilling discharges, and accidental oil spills. The project addresses the program call for the development of improved means to test long-term biological effects on the water column for keystone zooplankton species. The project will establish a laboratory system of multi-generation cultures of the copepod genus Calanus (with focus on C. finnmarchicus), and use this to begin testing a series of hypotheses on the response of Calanus to offshore discharges. The system will be based primarily on the technology and know-how of multi-generation culturing of this species that has been developed previously at the University of Tromsø and combined with the experience generated by the partners in studying the behaviour and ecophysiological aspects of zooplankton. This long-term test system with C. finnmarchicus will provide a supporting instrument for ecological risk assessment of offshore discharges, which is highly relevant both to the North Sea, the mid Norway shelf and the Barents Sea. 17 Establishment of the gill EROD assay as a biomarker of oil and produced water discharge Norges Fiskerihøgskole Jørgensen, Even Seniorforsker Prosjektnr: / : 100,000 Main objective: To establish the gill CYP1A bioassay as a biomarker of oil and produced water discharges

8 Partial objectives: - measure dose-dependent gill EROD responses to produced water in cod - investigate temporal characteristics of the gill EROD response during long term exposure of cod to produced water - measure gill EROD activity in cod caged at various distances from an oil platform - investigate possible relationships between gill CYP1A induction by produced water and hypo-osmoregulatory ability and gill histopathology Discharges and accidental spills from oil exploitation and exploration call for operational tools for monitoring and risk assessment purposes. A recently developed gill CYP1A assay appears to be an accurate, sensitive and straightforward way to measure the exposure of fish to waterborne, dioxin-like pollutants. The method implies that gill filament tips from fish is incubated in tissue culture well plates added buffer and 7-etoxyresorufin, after which the production of resorufin is measured (EROD activity). Recent experiments have shown that gills of a number of freshwater and marine fish species, including cod (Gadus morhua) respond with a times gill CYPlA induction to the AhR agonist &61538;-naphthoflavone, and to sediments and water containing a cocktail of pollutants, including polycyclic aromatic hydrocarbons. In the present project, the gill CYP1A response of cod to produced water and oil will be tested with the aim of implementing this biomarker as a tool in future monitoring and risk assessment. 18 Chemical Characterisation of Polar Components in Produced Water SINTEF Materialer og kjemi Daling, Per S. Seniorforsker Prosjektnr: /S : 640, : 980, : 940,000 The principal objective of this project is to obtain a thorough understanding of the chemical characteristics of polar components present in produced water and crude oil. The results from the project will form an essential basis for evaluation of possible long-term effects of produced water constituents in the marine environment. Sub-goals. Establish and adapt state-of-the art techniques for extraction of produced water. Establish and adapt state-of-the art techniques for adequate fractionation of extracts from produced water. Detailed and group-type chemical characterisation of polar fractions The project is designed to achieve a thorough understanding of the chemical characteristics of polar components in produced water. It is based on a literature review pre-project funded by the Research Council through the PROFO program in Present methodology for chemical analysis of organic constituents of produced water focus mainly on hydrocarbons (PAH) and to some extent phenols and naphtenic acids. The water soluble fraction of crude oils contain a substantial amount of unresolved complex material (UCM) which is poorly understood and not considered in regular analysis of produced water. The UCM is known to include polar components, like nitrogen-, sulphur- and oxygen-compounds (N,S,Ocompounds). The project will isolate polar fractions from produced water, or from laboratory prepared solutions representative for produced water, and use various analytical techniques to elucidate the chemical composition of these fractions. From what is known from preliminary investigations and literature, it is not reasonable to expect a full qualitative and quantitative characterisation on the single component level. However, a good group-type chemical characterisation in addition to determination of some selected single components is obtainable. Tailored extraction methods and state-of the art fractionation techniques will be applied. Method development will be done on laboratory prepared solutions of water soluble fraction (WSF) from crude oil. Standard mixtures of compounds proposed (in literature) to appear in polar fractions/ucm of crude oil will be prepared and used in method development and verification. Finally, established extraction, fractionation and analytical methods will be applied to investigate a few selected produced waters from Norwegian oil production facilities. The project will be conducted in co-operation with Norsk Hydro's research centre. Norsk Hydro has recently performed research activities on related areas, and efforts will be made to seek synergistic effects between this project and Norsk Hydro's research, and also to avoid possible duplication of work. 19 Weathering of marine oil spills under Arctic conditions Universitetssenteret på Svalbard AS Brandvik, Per Johan Førsteamanuensis Prosjektnr: /S : 1,404, : 1,800, : 1,505,000

9 The purpose of this project is to investigate the weathering processes in marine oil spills under Arctic conditions. The focus will be on the following processes: weathering of the bulk oil phase (evaporation, w/oemulsification and dispersion), dissolution of watersoluble components, photo-oxidation and biodegradation processes. Increased knowledge regarding these processes is important because they change the properties of an oil spill and increase the dissolution of components into the seawater. These processes may have a dual influence on the availability of potentially toxic hydrocarbons in the water column, by both increasing the bioavailability and eradicating them by e.g. evaporation or ultimate biodegradation which is complete biodegradation to inorganic compounds(h20 and CO2). The project will study the interactions between carefully selected oils and an Arctic marine environment with focus on important weathering processes (bulk oil properties, generation of water soluble components, photolysis and natural biodegradation) under real and simulated Arctic conditions. To obtain these ambiguous goals, the following topics will be studied as a function of oil type, temperature and presence of ice: - WP 1: Physical/chemical characteristics of oil spills in cold water and ice. - WP 2: Generation of water-soluble components from oil spills. Amount, composition and kinetics of the leaching process into the sea water. - WP 3: Radiation and photo induced degradation of oils under Arctic conditions and structural features of major degradation products. - WP 4: Natural biodegradation processes in oil spills under Arctic conditions. - WP 5: Verification of laboratory experiments with field studies on Svalbard. - WP 6: Reporting and dissemination of study results. 20 Long-term -chronic - effects of produced water effluents affecting reproduction in marine crustacean plankton Biologi, Institutt for,norges teknisknaturvitenskapelige universitet Olsen, Anders J. Forsker Prosjektnr: /S : 912, : 998, : 150,000 Principal objectives (total project) -establish novel insight into the susceptibility of pelagic crustacean plankton in northern seas for chronic exposures to oil-related hydrocarbons. Reveal secondary impacts on species or ecosystem structures related to biological effects on the plankton level -derive key input data to excisting and future computer-based simulation tools -general competence build-up within the field of marine ecotoxicology Main objective phase 1 -gain control with laboratory cultivation of C.finmarchicus under conditions comparable with future exposure regimes (shortened) Sub-goals phase 1 -gain control with parameters controlling growth, development and reproduction in C.finmarchicus (shortened) -select and evaluate biological quality indicators in laboratory cultures of C.finmarchicus (shortened) The overall theme for the project is development of appropriate experimental tools to evaluate long-term biological and ecological hazards of low-level produced water components on marine zoo-plankton. Experiments and data processing/deliveries will be tailored for implementation in contemporary and future data modelling tools for operationally and contingency planning. Due to its unique ecological significance as a main trophic link between micro alga and fish in the North Sea and Norwegian Sea the copepod Calanus finmarchicus has been selected as the main model plankton representative, and the majority of experimental work will be dedicated this species. Rearing and (exposure) test systems are principally laboratory based flow-through set-ups, allowing investigation of biological effects on all developmental stages. The present phase of the project (phase 1) is primarily devoted establishment of laboratory cultures of C.finmarchicus. Preferably multi-generation reproduction should be achieved, and physical and biological environmental variables will be strictly controlled in order to gain control with parameters controlling growth, development and reproduction. Health and function of the cultures/individuals will be routinely monitored by observation of specifically selected biological quality indicators. Indicator parameters which may serve as effect endpoints in future exposure studies will be preferred. 21 Experimental tests of petroleum-associated components on benthos at community, individual, and cellular levels - EXPAC - Akvaplan Niva AS Carroll, Jolynn Forskningskoordinator Prosjektnr: /S : 1,587, : 1,654, : 1,350,000

10 Identify effects to benthos from routine releases of drill cuttings and acute spills of petroleum-associated chemicals in diverse regions of Norway. 1. To develop hypotheses on impacts to the benthos from petroleum operations in higher latitudes. 2.To identify benthic community responses to routine discharges of drill cuttings and to evaluate effects on benthic organisms (organism- and cellular-level) from acute spills of petroleum-associated chemicals. 3. To propose improvements to the interpretation of benthic monitoring data from diverse environmental regions. The project will examine benthic processes in arctic and mid-latitude regions in order to derive specific conclusions on the sensitivity of benthic organisms and communities to acute spills of petroleum-related chemicals and routine releases of drill cuttings. The project will carry out a series of controlled experiments on whole sediment communities and individual benthic organisms with additions of drill cuttings and petroleumassociated contaminants, arriving at a set of hypotheses on the likely impacts on the benthos of petroleum production activities at higher latitudes. A series of testable hypotheses will be formulated based on an examination of real-world monitoring data sets collected under Norway's Petroleum Regional Monitoring Programme and results of mesocosm experiments performed previously at the Norwegian Institute for Water Research (NIVA) Station at Solbergstrand. These data sets will be examined in order to identify the geographic scope of responses to petroleum industrial activities. Through this work, the project intends to propose procedures to improve the interpretation of benthic monitoring data for diverse environmental regions in Norway. The project is linked to several on-going NFR projects within the Polarklima programme. By involving a Ph.D. student the project will advance the education and training of young scientists in the field of biological effects studies related to petroleum development and exploration activities. 22 Integrating monitoring methods for impacts of offshore discharges to the North Sea - IMONIT Norsk institutt for vannforskning Tollefsen, Knut-Erik Forsker Prosjektnr: /S : 1,130, : 1,324, : 1,259,000 The principle objective is to develop and validate a suite of bioassays as predictive (early warning) tools in assessment of long term effects of offshore discharges. Secondly, study spatial and temporal variation in effluent toxicity and identify toxic components. The objectives of the project will be achieved through laboratory-based development and a field-based validation of: 1. Robust, reliable, cost-effective and time-integrated sampling methodology 2. A suite of micro-scale and in vitro bioassays for assessment of the potential for sub-lethal long-term effects 3. Bioassay-directed fractionation and chemical identification procedure for resolving complex sample matrixes and to identify toxic compounds. One of the major obstacles for assessing the environmental impact of produced water discharges to the North Sea is to bridge the gap between the environmental risk determined by chemical discharge data (environmental impact models) and data from empirical effect assessment (water column monitoring programme). The gap in information from these conceptional different approaches increase the need for developing methods that link observed biological effects of offshore-related discharge to the presence of offshore related chemicals in the North Sea. One way to achieve this is to develop a testing system that combines the effect-integrative nature of micro-scale and in vitro biological assays (bioassays) with the highresolution of chemical analyses for use as predictive (early warning) tools for long-term effects. The methods will be used to study field- and time-specific variation in toxicity, causative chemicals and mixture toxicity in complex produced water effluents. The objectives will be accomplished on basis of the well functioning project group, recruitment of a Ph. D. scholar, as well as use of methods and results established in the NFR-funded pilot project "OffTiE". The results from this project is anticipated published and made available for evaluation of existing environmental impact models and the water column monitoring programme. 23 Effects of off-shore oil industry related discharges in the Arctic Rogalandsforskning Larsen, Bodil Katrine Forsker Prosjektnr: /S : 975, : 975, : 850,000 The primary objective is to determine whether UV irradiation enhances the toxicity of crude oil to the development of early life stages of Atlantic Cod larvae and Northern Shrimp larvae at lower temperatures (5-6 C). Two oil concentrations will be tested and UV

11 illumination will be applied. The importance of photomodification versus photosensitization in overall phototoxicity will be evaluated. The secondary objective is to further evaluate the two processes of toxicity by use of biological response parameters in early-life stages as well as adults organisms. Via synergy with other projects, we will evaluate whether photo-toxicity of oil to sub-arctic/arctic species represent a larger ecological problem than for temperate species Off-shore oil exploration and production industries are moving towards sub-arctic and arctic regions. These regions are characterized by specific ecological resources and are generally regarded as environmentally sensitive areas. Increased knowledge about the environmental impact of these industries is therefore requested. An important feature of the Arctic is the 6 months permanent solar illumination and consequent high levels of UV radiations, further enhanced by depletion of the ozone layer. UV irradiation has been proven to enhance toxicity of oil/pah's in temperate species, yet, photo-toxicity is often omitted in most of environmental studies. Phototoxicity is believed to have two different causes: Photomodification (activation of compound in water by UV) and photo-sensitization (activation of compounds by UV after accumulation). The project has designed the experiment to evaluate the importance of the two processes of photo-toxicity by using: 1. Effect/fitness parameters, studying mortality and developmental impairments in larvae of Atlantic cod and Northern shrimp. 2. Biological response parameters in cod and shrimp larvae as well as in adult Polar cod and Arctic amphipod. For this the project has chosen parameters of oxidative stress and DNA damage, as these are believed to be the primary causes of photo-toxicity. 24 Impacts of drilling mud discharges on water column organism and filter feeding bivalves Rogalandsforskning Westerlund, Stig Seniorforsker Prosjektnr: /S : 1,990, : 1,510,000 Primary objectives: 1.Study bioavailability of metals from suspended particles in the drilling mud discharge 2. Study effects of water based drilling mud on water column organisms (plantkton and fish) and filter feeding bivalves Secondary objective: Integrate test results on bioaccumulation and effects in an environmental risk based decision supporting framework (e.g. Environmental Impact Factor). The project will study the potential impact in the water column of discharges from ongoing drilling operations. The main focus will be on water column organisms, but filter-feeding bivalves are included. Three plankton species will be included; cod larvae, a model copepod (Acartia tonsa) and mussel larvae, in addition to juvenile cod (Gadus morhua). Tissue concentrations of metals in M. edulis, Placopecten magellanicus and cod exposed to used drilling mud with either barite or ilmenite as weighting materials, and the weighting materials alone, will be analysed and correlated to biomarker responses (lysosomal stability, oxidative stress, genetic damage, protein pattern, metallothionein, and gill histopathology) and biological effect parameters in bivalves (tissue growth and scope for growth). The planktonic species will be exposed to used water based mud with barite and barite alone. Ecologically relevant effect parameters will be studied in early life stages of cod, mussels and copepods (larval development and egg production of copepods, development and filtration rate of mussel larvae, growth and development of cod larvae). The results will be integrated in an evironmental risk assessment model. 25 Naturlige radionuklider i det marine miljø med vekt på Nordsjøen - oppsummering Norse Decom Varskog, Per Daglig leder Prosjektnr: / : 215,388 Fremskaffelse av eksisterende kunnskap om naturlige radionuklider i det marine miljø, antropogene og naturlige kilder, med vekt på Nordsjøen. 26 Radioactivity in produced water from Norwegian oil and gas installations - concentrations, bioavailability and doses to marine biota Institutt for energiteknikk - Kjeller Eriksen, Dag Øistein Seksjonsleder Prosjektnr: /S40

12 : 200, : 1,000, : 1,000, : 800,000 Main objective: To establish radiological safe discharge limits for Ra, Pb, and Po associated with other components in produced water from oil and gas installations. Sub-objectives: 1a. Establishing natural background levels of the relevant radionuclides in seawater, sediment and relevant organisms. b. Assess natural sources of Ra to seawater on the Norwegian continental shelf. 2a. Determine levels and distribution of relevant radionuclides in produced water. b. Study mobility and sedimentation mechanisms of the radionuclides given different discharge scenarios. 3. Study bioavailability and bioaccumulation of Ra, Pb, and Po in biota and food chain with discharge scenarios as 2b. 4. Assess effects of ionizing radiation on marine organisms. 5. Model the concentration of the radionuclides in seawater and biota using the DREAM model. Include adsorption and sedimentation. 6a. Risk assessment based on dose from radiation to biota and man. b. Recommendation of relevant PNEC values. Large amount of produced water containing elevated levels of radionuclides is discharged to the sea in connection with oil and gas production on the Norwegian Continental Shelf. So far no study has been performed to assess the potential radiological effects on marine biota in connection with radionuclide discharges to the North Sea. The produced water contains elevated levels of 226Ra and 228Ra. It is normally assumed that when this water, which is poor in sulphate, gets in contact with seawater rich in sulphate, RaSO4 precipitates. The precipitated salts can then attach to other organic or inorganic particles and sequentially settle to the bottom close to the installation. Data on the speciation of radium, and the effect of major components in produced water on the speciation of radium, is however sparse. In today s oil production, several chemicals are added, i.e. scale and corrosion inhibitors, emulsion breakers and surfactants, sulphide removers, etc. These chemicals are usually organic compounds comprising functional groups, which may also interact with cations such as Ra2+. Thus, radium may exist in compounds more easily accessible for uptake in biota than the inorganic aqueous or foodborne form. An understanding of how different compounds in produced water affect the mobility, bioavailability and sedimentation of radium is essential to determine the fate and effects of radium discharges. For evaluation purposes the behaviour of produced water radium must be compared to radium already present in seawater. An assessment of existing data on natural radioactivity in the North Sea has revealed a lack of data in this area. This project aims at assessing this problem thorough an interdisiplinary study comprising survey of background levels, mobility studies of relevant radioactive isotopes, bioavailability studied at controlled environments, studies on effects on biota, modelling of the discharge, and calculation of doses to biota and humans. 27 Effects on development, sex differentiation and reproduction of cod (Gadus morhua) exposed to produced water during early life stages Havforskningsinstituttet Klungsøyr, Jarle Forsker Prosjektnr: /S : 3,021, : 947, : 1,989,000 To investigate the effects of produced water on cod from early life stages to the next generation by following the growth rate, frequency of malformations, sexual development and spawning success. This main objective can be subdivided into: - Develop methods and describe criteria for identification of inter-sexual conditions in cod during early life stages. - Study the influence of produced water on the sexual development of larvae, and find the time windows where cod are most sensitive. - Determine frequency of malformations in exposed larvae compared to control. - Identify long-term effects of exposure to produced water in early life on the reproduction potential of fist-time spawning cod, and study second generation effects in the offspring. The objective of the project is to obtain improved knowledge about the effects of produced water from offshore petroleum industry on the development and reproduction of coastal cod. Long time exposure studies will be carried out on eggs, larvae and juvenile cod in all temporal windows where developmental processes are most likely affected. The fish exposed continuously during early life will be held in enclosures until it matures and spawn for the first time 2 years after hatching, and the reproduction success closely followed. The analyses span from chemical analysis and molecular aspects (protein and gene expression), through histological analyses and oocyte counts to whole animal responses like sex distribution, frequency of malformations and

13 measurements of spawning success. Effects on egg quality; malformation and hatching success for secondgeneration cod will be monitored. 28 The use of passive sampling devices in monitoring of potential impact of offshore discharges and accidental oil spills (PASSIMPACT) Norsk institutt for vannforskning Tollefsen, Knut-Erik Forsker Prosjektnr: /S : 1,000, : 1,000, : 1,000,000 The objective of the present project is to optimize and validate a suite of passive sampling devices for chemical (and biological) monitoring of organic chemicals (polar and non-polar) from oil related discharges (regular offshore discharges and accidental spills).the objective will be achieved through the following sub-objectives: 1) Characterize the sampling kinetics of the passive sampling devices POCIS, Emporedisk and SPMD for classical pollutants and newly emerging polar components of particular toxic concern, and compare the sampling kinetics with that of a typical sentinel monitoring species like blue mussels 2) Determine the effect of biofouling on the sampling kinetics of POCIS, Emporedisks, SPMD and blue mussels to determine the susceptibility to variations in environmental conditions 3) Validate the use of a combined sampling strategy using a suite of passive sampling devices (POCIS, SPMD, and Emporedisks) and blue mussels in a field study and a simulated spill situation 4) Concept validation by comparing results obtained in field studies and those emerging from the DREAM model for a selection of compounds studied above. Currently, there is a need to combine chemical characterization with toxicological screening of extracts to address chemicals that are of environmental concern. This applies in particular to potential use of passive samplers as pre-concentrating devices for bioassayassisted fractionation and chemical identification procedures which previously has shown that the acutely toxic organic chemicals in produced water are mainly found in fraction with log Kow< 3, whereas chemicals with the potential for more long term effects are found in fractions with a log Kow> 3. As a consequence, there is a need to develop a combined passive sampling strategy using time-integrative samplers that discriminate between bio-available fractions and that are able to be used for both chemical characterization and toxicity testing. The present project is directly related to research needs identified in the PROOF program background document and application guidelines for This applies in particular to the time-integrated measurement of PW components in seawater, and a comparison with similar data in iological tissues. The measurements of the components will be combined with toxicological screening of resulting extracts, thus linking chemical data to effect/biomarker responses. The practical work will be divided into four work packages (WPs): 1) laboratory studies with a controlled exposure situation (i.e. with and without bio-fouling); 2) controlled laboratory study of the water accommodated fraction (WAF) of oil; 3) field study at an oil field; 4) data and concept validation using the DREAM models. 29 Identification of Estrogen-Like Compounds in Produced Water from Offshore Oil Installations. Havforskningsinstituttet Boitsov, Stepan Forsker Prosjektnr: /S : 400,000 Main goal: Determine the presence and identify clusters of unidentified alkylphenol isomers and possibly other compounds in produced water exhibiting toxic effect on marine biota. Subgoals: - Synthesise mixtures of longchain alkylphenols to be used as standards for identification. - Extract large volumes of produced water and analyse chromatograms of produced water extracts for alkylphenol presence. Resolve some of the chromatogram peaks by means of mathematical analysis to elucidate the maximum possible number of unidentified alkylphenol isomers. - Identify parasubstituted long-chain alkylphenol isomers being present in produced water which have not been identified before. - Make an attempt at identification of thiols present in produced water. This project focuses on identification of the yet unidentified compounds, long-chain para-substituted alkylphenols and possibly thiols, in produced water. Chemical syntheses will be carried out to provide alkylphenol standards unavailable commercially. Statistical analysis will allow to separate the overlapping chromatogram peaks and resolve the chromatograms.

14 30 Environmental effects of oil and gas exploration on the benthic fauna of the Norwegian continental shelf: an analysis using the OLF database Biologisk institutt,universitetet i Oslo Gray, John Stuart Professor Prosjektnr: /S : 600, : 600, : 600, Can the OLF database be used to generate predictions of effects of the industry in other areas of the world? - by means of a comparison of tradtional and new multivariate methods for making predictions of effects. 2.The development of field-based species-sensitivity distributions (SSDs) - For sets of species that show declines in abundance with contaminant concentration, prepare EC50 values and then derive SSDs for ca. 200 species. 3.Rationalizing and improving the monitoring programme - test scenarios of reduced sampling effort on precision and accuracy of monnitoring both chemical an biological variables. We will use traditional and new multivariate statistical methods to analyse effects of using oil-based and non-olibased drilling fluids on the benthos of the Norwegian continental shelf. These methods allow one to test hypotheses of cause and effect and thus may move from a descriptive to a predictive tool. In order to do such analyses datasets will have to be selected which comply with set criteria. Most SSDs are based on laboratory data and often from non-marine species. We have started development of field-based SSDs which show the combined effects of a variety of chemicals. plots for Ba, Cd, Cu and PAHs show encouraging results and we will plot a wide variety of SSDs for combined data and data split into sub-sets varying sediment properties and depth.such SSDs will be of great benefit as management tools for minimising effects on the marine environment The present monitoring programme is probably not efficient for the stated purposes. We will erect scenarios and test whether a reduced sampling programme can give similar accuracy and precision in monitoring effects of the industry on the marine environment. A series of simulations will be done on selected data chosen to illustrate temporal and spatial changes. The results should be of interest to both environmental authorities and oil companies. 31 Long term effects on Arctic ecosystem from accidental discharges Akvamiljø as Sanni, Steinar Direktør Prosjektnr: /S : 1,200, : 1,200, : 1,200,000 Prosjektets hovedmål og delmål The principal objective is to investigate if there is significant reason for concern about long term effects on Arctic ecosystem from accidental discharges. A pollution scenario which may occur as a result of a large oil spill in open sea with subsequent drift into the ice margin has been selected for the studies. It is thought that drift into the ice may cause oil to be trapped and stored and re-released, exposing the fauna of the ice margin repeatedly over a long time period. The goals of the project are to investigate the potential for such effects through exposure of the ice amphipod Gammarus wilkitzkii, an organism with a key position in the ice margin ecosystem. Effects on the reproduction cycle of G. wilkitzkii with links to short/medium term effects will be studied. The effects of repeated, sub-lethal exposures of oil as well as dietary uptake of oil components via plankton will also be investigated. The overall null hypothesis for the studies is that no long term effects can be pointed out. Off-shore oil exploration and production is moving towards Arctic regions. A strict regulation according to a precautionary principle has been invoked for the oil and gas industry. There is a strong official requirement to avoid long term environmental impacts or effects that may be in conflict with the fisheries. The knowledge about the environmental impact of such activities in these regions has to be expanded. Since regular discharges will practically be prohibited, the possible environmental spill scenarios on the marine environment from the oil industry are mainly focused on accidental spills. These may include large as well as small repeated discharges related to oil spill events in the course of production, processing or transport and to subsequent re-releases of temporarily stored oil, e.g. trapped in sea ice. At present it is difficult

15 to express the probability for the occurrence of such events or its associated environmental risk, so a scenario based approach is used to identify relevant research targets. The selected scenario is related to oil drift into ice margin, and the project work is focusing on potentially long term effects on ice margin community of trapped oil. This may occur as oil trapped underneath the sea ice or as frozen within the sea ice. Potential long term effects are determined by results of experiments. From the results it will be attempted to express their influence on expected restitution time. The data on uptake and effects will be relevant for validation of hazard and risk estimates as well as a support for environmentally related decision making at accidents as well as industrial operations in Arctic waters. The work will be divided into three work packages, high-lighting different aspects of potential effects on the ice amphipod Gammarus wilkitzkii. WP1: Uptake and effects of water dissolved and food-bound hydrocarbons. WP2: Effects on reproduction cycle and population with links to short term/medium term effects. WP3: Long term effects by repeated exposures of released oil. The presence of UV-light will be included as an experimental factor due to the combination of large amounts of sunlight in the Arctic summer and its influence on oil (PAH) toxicity. Two different doses of oil will be tested in order to assess the difference in effects in relation to oil load (exposure concentrations). The project will be carried out in close co-operation with a doctorate study related to UV effects on Arctic amphipods funded by the German Research Council. 32 Parameterisation of the Environmental Impacts on Bottom Fauna of Water-based Drilling Fluids and Cuttings Norsk institutt for vannforskning Olsgard, Frode Forsker Prosjektnr: /S : 600, : 600, : 600, : 600,000 The principal objective : Through existing field data study effects of WBM discharges on bottom fauna structure and function. Through literature studies investigate and quantify the impact of benthic fauna on sedimentary processes in order to better parameterise the Par Track Bottom Fauna Module of the ERMS-model subgoal: Quantify relationships between WBM discharges and faunal effects Quantify relations between benthic local fauna composition and sedimentary processes like sediment trapping, sediment de-stabilisation, bioturbation, bioirrigation and sediment resuspension in order to optimize the ERMS-model Discharges of contaminated drill-cuttings have caused appreciable ecological changes in the bottom fauna around many Norwegian oil and gas platforms. Drillinginduced disturbances reduce the diversity and complexity of pristine bottom fauna and restricts their functions. The effects may be due to physical disturbance (sedimentation and smothering), chemical toxicity or heavy organic loading (and subsequent oxygen problems in the sediments). In order to aid the prediction and evaluation of harmful biological effects and the environmental risk of using various chemicals in the Norwegian offshore drilling activities, modelling has been introduced as an important tool. The models have till now mainly been applied for processes in the water column, but recently transport of sediments and chemicals to the seabed has been included in the modelling work. The seabed part of the model has till now been based on physical/chemical processes, without incorporating the impacts of the bottom-dwellin. We will also quantitatively describe biological effects of drilling discharges on the bottom fauna. This project aims to provide important information needed to better evaluate and predict risk of effects of drill-cuttings disposal and also predict the restitution time needed for a new bottom-dwelling fauna to establish on seabed receiving larger amounts of drilling discharges. 33 Proteome Expression Signatures (PES) in fish as a diagnostic tool to evaluate the environmental impacts of offshore oil and gas exploration Rogalandsforskning Andersen, Odd Ketil Forskningsleder Prosjektnr: /S : 1,000, : 1,000, : 1,000,000 Principal objective and sub-goals The principal objective of the study is to develop a proteome based diagnostic system for cod in Environmental Risk Assessment (ERA) and Environmental Monitoring of produced water. The secondary objectives are: - To identify PES in cod exposed to PW and it's components in controlled exposure studies - Identification of the structure of key diagnostic proteins that express the effects of the different PW components to develop suites of biomarkers

16 - To identify PES in caged and wild cod from offshore water column monitoring and regional fish sampling cruises - Correlate proteome changes with functional disorder where possible to provide a basis for including proteome analysis within the ERA model. Project summary: Recent studies on fish examining the impact of produced water released offshore have revealed an extremely complex response to oil micro droplets and oil spiked with relevant doses of poly-aromatic hydrocarbons (PAHs) and alkylated phenols (AP). Protein expression signatures (PES) obtained from exposed and control fish show both up and down regulation of between proteins, with the majority of responses observed being sex dependent, even in juvenile fish. Produced water is an unstable mix of many components which may be unique for individual oil fields. Components in produced water, PAHs, APs and a variety of polar organic compounds (POCs), are over represented in comparison to concentrations found in the oil. The POCs are the least studied components of the produced water both with respect to acute toxicity and long-term effects. In this study we will seek to sample from cod exposure experiments financed through the PROOF, especially "Comparative oral and water based exposures of cod to produced water components", and other programs. Based on these findings individual proteins will be sought to be identified for further development as biomarkers of chronic exposure to low doses of oil, synthetic mixes of PAHs, APs and POCs at concentrations similar to those found in produced water. The proteome response in both wild and caged cod from the North-sea will be compared against results from the laboratory exposures with the aim of identifying and validating diagnostically important proteins. To perform the identification of diagnostically important proteins a cooperation with national and international research groups within the field of proteomics will be established. If possible some genomic analyses will be performed on the Cod as cooperation with other research groups to increase the mechanistic interpretation of the proteom responses. 34 Predicting chronic effects in Fish from sublethal markers Norsk institutt for vannforskning Hylland, Ketil Professor Prosjektnr: /S : 1,500, : 2,000, : 1,500,000 This main objective can be divided into the following sub-goals: -to establish tools to detect biochemical and physiological changes in selected fish species -to identify specific early markers that are predictive of adverse effects of contaminants in fish -to quantify adaptation processes that may invalidate the methods currently in use in environmental monitoring -to develop a toolbox of methods for environmental assessment of offshore effluents Hypotheses to be tested by the project are: -will effects observed at early life stages accurately predict later-life effects in the two model species Atlantic cod and zebrafish? -can adaptation be accounted for in an identification of markers for later effects in the models? -is it valid to compare responses in two fish species with such different life history traits? -to which extent does existing monitoring methods detect effects that may lead to population-relevant impacts? Offshore activities input large volumes of moderately contaminated effluents into the North Sea. There is uncertainty as to the biological effects of the effluents, although there are signs that some biological systems are affected. The current methods to assess effects in the pelagic zone mainly depend on health assessments of fish or mussels, i.e. biomarkers. The methods currently available are not optimal for offshore-relevant contaminants and there is limited knowledge of whether responses change over time (adaptation). This project aims to use state-of-the-art methods both to identify effects at an early point in a deleterious process and to identify changes in responses over time. The project will use zebrafish as a model to identify early responses using microarray technology. Results from the zebrafish studies will be carried over to a one-year study in which Atlantic cod will be exposed through water to selected offshore effluent (produced water) components, i.e. alkylphenols and PAHs. In addition to screening by microarray analyses, fish will be analysed for a range of contaminantspecific, contaminant-related and health related endpoints at regular intervals during long-term exposures. At the end of the exposures of both fish, hatching success will be determined. The project will produce new knowledge of responses to long-term low level and intermittent exposure to contaminants. It will further identify endpoints that are suitable for monitoring and enable an improved assessment of methods currently in use. 35 Effects of produced water to Atlantic cod: Mechanistic studies and biomarker development with proteomics based methods. Molekylærbiologisk institutt,universitetet i Bergen

17 Goksøyr, Anders Professor Prosjektnr: /S : 1,015, : 1,008, : 1,043, Screen already collected tissue materials from Atlantic cod exposed for produced water for changes in protein pattern by 2-D gels and identify changes in protein pattern by MALDI-TOF and MS/MS. 2. Continue collaboration with IMR and Akvamiljø/Rogaland Research to obtain material from exposed cod, also including studies over more than one generation. 3. Search for post translational modifications, like phosphorylation, glutathionylation etc. Either by antibodies, fluorescent probes or directly with mass spectrometric methods. 4. Develop other proteomic methods for such studies which may help to speed up identification of protein with differential expression pattern, as with LC-MS/MS. 5. Make probes and synthetic peptides from de novo sequences to produce antibodies to identified proteins. 6. Validate such antibodies on already obtained materials in addition to use them on new lab and field studies. Effects of contaminants on an organism start at the molecular level, and are transferred to higher levels of biological organization if the exposure is persistent and toxicological repair is overwhelmed. Long-term effects of produced water on the reproduction of Atlantic cod can therefore be studied at the molecular level. This project aims at utilising proteomic based techniques to study and identify possible changes in protein expression due to such exposure. We believe that this approach may detect possible changes with an unbiased eye. We want to further develop proteomics based technology, i.e. 2- dimensional gel electrophoresis (2-DE) combined with different mass spectrometry techniques and database search to identify mechanisms involved due to exposure of Atlantic cod to produced water from oil and gas production. An important aim is to develop new biomarkers to be used in future biomonitoring programmes. This will be applied to laboratory experiments and field samplings. As one of the concerns of these discharges are endocrine disruptive effects from alkylated phenols, we will focus on changes in plasma, but also study other organs like gonads and liver in addition to whole larvae extracts. 36 Comparative oral and water based exposures of cod to produced water components. Akvamiljø as Sanni, Steinar Direktør Prosjektnr: /S : 350,000 The principal objective is to provide sample material of cod that makes it possible to compare and assess oral and water exposures. This is a highly relevant interpretation aspect in all the on-going effect projects in PROOF. The sampled material from this additional exposure will be analysed and the results integrated in the respective projects. These are 3 on-going PROOF projects where comparison of chemical uptake and biological responses in fish is a common sub-goal in all. Based on this additional exposure, the sub-goals will be to provide samples and supporting data on: a mixture of four selected dissolved alkyl phenols compared to oral exposure administered in feed, artificial produced water based on crude oil dispersions (incl. dispersions spiked with alkyl phenols and PAH components), a real produced water sampled from an oil field installation. The data will be discussed in a common context which will improve the interpretation basis considerably in all 3 projects. There are presently three on-going PROOF projects at RF-Akvamiljø and Institute of Marine Research (IMR) concerning effects of produced water components in cod. The projects have different objectives, and they focus on different aspects of effects in fish. Consequently, the studies vary in the way the fish are exposed to contaminants, as well as in the measurements of contaminant uptake, responses in the fish, and effect endpoints. The projects have, however, a close thematic relationship, and the information value of the results will be strongly augmented by making it possible to compare the levels of exposure and effects between them. The additional experiment applied for will establish a basis to relate oral (dietary) and waterborne uptake of produced water components. This is an essential aspect that will improve the basis for interpreting all the three "parent" projects, and also to relate more precisely the results of other studies to them. This will increase the usefulness of the results for regulatory purposes, such as determination of critical discharge limits, monitoring threshold levels, etc. The on-going projects will contribute to the common study within their existing budget frames. The data will be issued in a common report and integrated in the separate parent projects. In particular, the results will be used to clarify and quantify differences in uptake and effect responses to different modes of dietary and waterborne exposure in cod. These aspects of the results will be considered published in collaboration by the involved participants.

18 37 The unresolved complex mixture (UCM) of petrogenic oils: Impacts in the seawater column SINTEF Materialer og kjemi - Trondheim Brakstad, Odd Gunnar Forsker Prosjektnr: /S : 1,000,000 representing one pseudo-oil compound group, or separated in several toxic fractions partially characterized by chemical analysis. The main objective of this project is to determine the toxicities of the unresolved complex mixture (UCM) and UCM-fractions of petrogenic oils with selected marine bioassays. The objectives are separated as follows: -Determination of bioassay-assisted toxicities of UCMrich fractions from in-reservoir biodegraded oils -Determination of the contribution of essential UCMfractions in crude, evaporated and photo-transformed oils with selected bioassays It is intended that data from this project can be used to include the UCM, or several UCM fractions, as compound group(s) in the exposure models to assess the environmental impacts of the UCM fractions in relation to existing compound groups included in the models. The unresolved complex mixtures (UCMs) of petrogenic oils represent recalcitrant fractions of the oil after discharges to the marine water column. UCMs may resist both evaporative and biodegradation processes in the water column, and photo-transformation processes may result in toxic UCM-related compounds. Extensive parts of the UCM consist of water-soluble polar compounds which are bioavailable, and the UCM has been associated with toxicity to marine organisms. This project will in 2005 focus on the effects of the UCM found in petrogenic oils and the water-soluble fractions (WSFs) of the oils. Bioassay-assisted fractionation procedures will be initiated with a number of rapid screening bioassays to compare the toxicities of different UCM-fractions separated by established chemical techniques. Chemical characterization of the most toxic fractions will be performed mainly as a part of another NFR-sponsored project (NFR project ). The sub-acute effects of selected UCM-fractions on marine phytoplankton photosystem II will also be examined. Relevant data may be used in existing exposure models. Two in-reservoir biodegraded oils (Heidrun Åre and Troll) will be used as UCM-rich oils, while a paraffinic Statfjord oil (low UCM) will be used as reference oil. The impacts of some oil weathering processes (sea surface evaporation and photo-transformation) will be used to increase the contribution of the UCM in the oil WSFs. Toxicityrelated data will be available for exposure model tools (Oscar, Dream), either for the whole UCM-fraction