A baseline study of Greenland halibut off the Faroe Islands

Transcription

1 A baseline study of Greenland halibut off the Faroe Islands November 2000

2 A baseline study of Greenland halibut off the Faroe Islands RF-2000/221 AM-2000/008 This document may only be reproduced with the permission of RF or the client.

3 Our reference: No. of pages: 85 Authors Bjørn Einar Grøsvik, Anne Bjørnstad, Atle Nævdal, Stig Westerlund and Endre Aas Project Quality Assurance. Odd Ketil Andersen Client(s): GEM Project title: Hydrocarbon baseline study Version No. / date: Vers. 2 / Distribution restriction: Open Scope: The objective of this study has been to establish baseline levels of metals, polycyclic aromatic hydrocarbons and selected biological effect parameters in Greenland halibut (Reinhardtius hippoglossoides) as well as characterisation of sediment, from the area opened for oil exploration after 1 st licence round, south east of the Faroe Islands. The results of this investigation form the baseline to which future results will be compared. Key-words: Faroe islands; Baseline; Greenland halibut; Sediment; Hydrocarbon; Metal; Biomarkers RF - Rogaland Research has a certified Quality System in compliance with the standard NS - EN ISO 9001 Project Manager for Akvamiljø for RF - Rogaland Research Bjørn Einar Grøsvik Steinar Sanni Troels Gyde Jacobsen This document may only be reproduced with the permission of RF or the client.

4 Summary RF-Rogaland Research has in collaboration with Akvamiljø and Biosense performed a baseline study of metals, polycyclic aromatic hydrocarbons (PAH) and selected biological effect parameters in Greenland halibut (Reinhardtius hippoglossoides) as well as characterisation of sediment, from the area opened for oil exploration after 1 st licence round, south east of the Faroe Islands. The results of this investigation form the baseline to which future results will be compared. The aim is to be able to assess, in future monitoring programmes, whether oil activities may lead to environmental changes. The task was given by Fiskirannsoknarstovan on behalf of the GEM programme. The GEM programme is a consortium of oil companies with interest in exploring the Faroes area. Greenland halibut (Reinhardtius hippoglossoides) was chosen as indicator fish species, as this is a commercially important species in this area. Two sampling cruises were performed, the first from 27 th May 3 rd June and the second from 11 th 15 November 1999, both with the research vessel Magnus Heinason. This allowed for covering both spring and autumn conditions. Sediment was sampled during the cruise in November. The analyses of the sediment samples demonstrated low levels of metals, and not detectable levels of total hydrocarbons or single compounds of PAH. There was some variation in grain size distribution between different areas. The total organic matter in the sediment samples was relatively low with some variation between stations. All data supported the characterisation of the sediment to be uncontaminated. 50 Greenland halibut were sampled per cruise, 25 females and 25 males. The fish were of homogenous size and age. Levels of metals in liver and muscle were found to be generally low. Metals were also analysed in bile. Single components of PAH were analysed in fish muscle and no detectable levels were found. Neither were detectable levels of PAH metabolites found in bile. Activities of the detoxifying enzyme cytochrome P450 1A (CYP1A) measured in liver were low. DNA adducts were also analysed in livers and found to be below the detection limit. Levels of vitellogenin (Vtg) and zona radiata proteins (Zrp) were measured in blood, as these proteins are shown to be induced by oestrogen mimicking compounds. Crossreactive bands were found in both males and females. Further work is required to investigate whether this is a normal pattern in Greenland halibut. Liver and gills were fixed and embedded in paraffin as possible reference material for future studies. There is still limited knowledge about natural variations in some of the biological parameters analysed, like liver CYP 1A activity and levels of Vtg/Zrp in blood. The data obtained through this study confirms that the area studied, south and south east of the Faroe Islands, to be a clean area with regard to the chemical and biological parameters studied and the data are reflecting natural conditions in the area. - ii -

5 Preface This work was performed by RF-Rogaland Research (RF), Akvamiljø and Biosense as part of a programme to establish baseline conditions in Faeroes waters prior exploration drilling for hydrocarbons. Fiskirannsoknarstovan, Faroes gave the commission on the behalf of the GEM program. Field surveys were performed with Research Vessel Magnus Heinason. Karina Nattestad (Fiskirannsoknarstovan) and Anne Bjørnstad (RF) performed the field sampling. Randi Mikalsen (RF) performed the analyses of grain size distribution and total organic matter in sediments. Metal analyses were performed by Stig Westerlund (RF). PAH and THC analyses were performed by Kjell Birger Øysæd (RF), Sigfryd Torgrimsen (RF), Atle Nævdal (RF) and Grete Jonsson (RF). Bile metabolite analyses performed by Endre Aas (RF) EROD measurements by Endre Aas and Bjørn Einar Grøsvik. DNA adduct by Birgitta Liewenborg and Lennart Balk (University of Stockholm), Vtg and Zrp analyses by Sven Inge Kristiansen (Biosense) and Histology embedding and first characterisation by Kjell I. Flesjå (Veterinærinstituttet i Sandnes). Project leader in RF has been Bjørn Einar Grøsvik. Front cover: View from R/V Magnus Heinason, the North Sea, November Photo: Anne Bjørnstad. - iii -

6 Contents 1 INTRODUCTION Chemical analyses Biological response parameters PAH metabolites in bile Cytochrome P4501A DNA adducts Vitellogenin and zona radiata proteins in blood Histology MATERIAL AND METHODS Field work Sampling locations Sediment Fish Grain size distribution and total organic material in sediment Metals Sediment Bile and liver Extraction and analyses of hydrocarbons Sample treatment GC/FID analysis for total hydrocarbon (THC) quantification PAH analysis Calculation and Quality assurance Fluorescence detection of PAH metabolites in bile Cytochrome P4501A activity in liver Samples Analysis Enzyme-linked immunosorbent assay (ELISA) DNA adducts Paraffin embedding of liver and gill samples RESULTS AND DISCUSSION Sediment Grain size distribution and total organic material Metal analyses of sediment iv -

7 3.1.3 Total hydrocarbon (THC) analyses PAH analyses of sediment Greenland halibut Metals in liver and muscle Metals in bile PAH analyses of fish samples PAH metabolites in bile Cytochrome P4501A activity DNA adducts Levels of vitellogenin and zona radiata proteins in blood Histology CONCLUSION REFERENCES APPENDIX Data from field surveys Position of stations for sediment sampling Raw data Fish sampling Trawl data Sampling overview Particle distribution and total organic matter Metal analyses Metals in liver and muscle of fish sampled in May Metals in liver and muscle of fish sampled in Novenber Metals in bile of fish sampled in May Metals in bile of fish sampled in November Analyses of hydrocarbons in sediment and fish samples Recovery, fish samples and sediment samples Printout of a representative chromatogram of THC in sediment GC/MS printouts of a representative analysis of fish muscle GC/MS printout of a representative sediment analysis GC/MS printouts of a calibration standard Example of GC/MS printout of an oil sample Analyses of PAH metabolites in bile Raw data EROD, Vtg and Zrp measurements v -

8 Abbreviations CYP1A ELISA GC/MS-SIM ICP-MS LOQ EROD MDL NPD PAH QIS RIS SFT THC TIC TLC TOM Vtg Zrp Cytochrome P4501A Enzyme linked immunosorbent assay Gas chromatography/mass spectrometry in single ion mode Inductively coupled plasma mass spectrometry Limit of quantification Ethoxy-resorufin-o-deethylase Method detection limit Naphtalene, phenanthrene, dibenzothiophene and their C 1 -C 3 alcyl substituted homologues. NPD compounds are included in the term PAH Polycyclic aromatic hydrocarbons Internal standards for quantification Internal standards for recovery calculation State Pollution Authority in Norway Total hydrocarbon Total ion chromatogram Thin liquid chromatography Total organic matter Vitellogenin Zona radiata proteins - vi -

9 1 Introduction A study to establish baseline levels of chemical components in sediment and fish from the Faroes waters as well as baseline levels of selected biological effect parameters in fish have been performed by RF-Rogaland Research (RF) in collaboration with Akvamiljø and Biosense. The task was given by Fiskirannsoknarstovan, Faroe Islands, spring 1999 on behalf of the GEM programme. The GEM programme is a consortium established in 1997 by oil companies with interest in exploring the Faeroes area. The objectives were to establish a database of baseline levels prior to exploration for petroleum and to be able to compare results in future monitoring programmes to assess impact of offshore industry activities on the environment. The area opened for oil exploration after 1 st license round (August 2000) is shown in Figure 1. Greenland halibut (Reinhardtius hippoglossoides) was chosen as the monitoring species, as this is an economically important species in this area. Two cruises have been performed, one in spring (27 May-3 June 1999), and one in autumn (11-15 November 1999). 100 Greenland halibut were sampled totally, 25 from each sex per cruise. Sediment samples were collected at the cruise in November. Generally, monitoring studies of offshore activities have been limited to chemical studies of sediment and species characterisation in soft bottom fauna, as required in monitoring guidelines from the Pollution Authorities, e.g. (SFT 1999). The presented work focus on selected biological effect parameters (or biomarkers) in tissue of Greenland halibut in addition to chemical levels in fish and sediment. Bottom fauna analyses will be covered in separate report, by other investigators. The reason for including biomarkers is to try to detect effects of chemicals as early warning signals before irreversible damage has occurred. This is to our knowledge the first study that implies biomarker measurements in a reference study

10 7 W 6 W 5 W 4 W Figure 1. Area opened for oil exploration after 1 st license round, August Chemical analyses Sediment samples were analysed for particle distribution, total organic matter (TOM), metals, total hydrocarbons (THC), and polycyclic aromatic hydrocarbons (PAH). The two- and three- rings PAH which are the major PAH compounds in oil are usually named NPD. NPD includes naphtalene, phenanthrene and dibenzothiophene and their C 1 -C 3 alcyl substituted homologues. NPD is included in the term PAH. The following chemical parameters were measured in Greenland halibut: Metals in liver and bile, and mercury levels in muscle. PAH and were measured in muscle. 1.2 Biological response parameters When using biological parameters in baseline studies, it is important to obtain knowledge of the range of natural variation, for example due to age, sex and seasonal changes. The selected response parameters applied in this study are listed below: PAH metabolites in bile After PAH compounds are taken up by fish they get biotransformed into polar metabolites, which enhances the efficiency of excretion. The bile is thought to be the - 2 -

11 dominant excretion route of metabolites of especially larger PAH molecules in fish (Meador, et al. 1995). The fact that PAH metabolites are stored for some time and thereby concentrated in the gall bladder makes the gall bladder bile a suited matrix for sensitive measures of PAH metabolites. The liquid nature of bile also makes an extraction step redundant, which is necessary for tissue analyses. Chemical measurements of parent PAH do not provide a good assessment of the PAH exposure in fish due to the high metabolic rate, transforming the parent PAH into metabolites. A characteristic feature of PAH compounds is their fluorescing properties. All PAH molecules absorb ultraviolet light followed by emission of light of a longer wavelength. The fluorescence properties, i.e. optimal excitation and emission wavelengths and signal intensity, varies between PAH compounds and is dependent on size, structure and eventual substituents on the molecule. Generally, the optimal excitation wavelength increases with increasing size of the PAH molecule (Vo-Dinh 1978), i.e. smaller PAHs need more energy (shorter wavelength of the excitation light) than the larger molecules. This variability can be utilised in simple detection methods for PAHs. By synchronous fluorescence analyses it is possible to obtain measures of level of groups of PAHs (Aas et al. 2000) Cytochrome P4501A Cytochrome P4501A (CYP1A) belongs to a group of membrane bound enzymes, as well as a superfamily of genes, essential in the metabolism of both endogenous and exogenous compounds in cells. PAH compounds from petrogenic origin have also shown to be potent inducers of CYP1A in several fish species. CYP1A induction, estimated for instance by ethoxy-resorufin-o-deethylase (EROD) activity, has been widely used as a biomarker for planar organic contaminants, and among them PAHs (Goksøyr and Førlin 1992; Bucheli and Fent 1995). Ethoxyresorufin is a substrate for CYP1A and the formation of resorufin is thereby an indirect measure of CYP1A activity. In addition to possible pollutants, natural factors like sex, age and temperature also play important roles in regulating the CYP1A level (Førlin 1980). When applying CYP1A as a biomarker, it is therefore of prime importance to include possible natural variations when interpreting the results DNA adducts PAH metabolites have been shown to form covalent adducts with the DNA molecule (Varanasi et al. 1989). DNA-PAH adducts are found to be a crucial factor in the aetiology of cancer development, and their presence has for this reason been suggested as a highly relevant biomarker of PAH exposure in e.g. fish (Sikka, et al. 1990; Shugart and Theodorakis 1998). Hepatic adducts with benzo[a]pyrene from PAH contamination of pyrolytic origin are most often reported, but adducts can also be formed with lighter PAH compounds, like chrysene which is a constituent in most mineral oils (Kurelec et al. 1992; Noaksson et al. 1998). Recently, Harvey et al. (1999) reported hepatic DNA adducts in three teleost species exposed to the Sea Empress oil spill in Wales, UK. The 32 P postlabelling assay of liver and blood cells is presently the most sensitive and frequently applied technique for detecting DNA adducts in fish (Reichert, et al. 1998)

12 With this method it is possible to detect one adduct in about nucleotides (Reddy and Randerath 1986). The method involves enzymatic hydrolysation of DNA to 3 monophosphates, followed by enrichment of DNA adducts and labelling with [ 32 P] phosphate. The labelled adducts are then separated by two-dimensional, thin-layer chromatography (TCL) on polyethyleneimine-modified cellulose sheets. Finally, radiolabelled adducts are detected and quantified by autoradiography and liquid scintillation counting or imaging analysis Vitellogenin and zona radiata proteins in blood Vitellogenin and zona radiata proteins are important constituent parts of the egg. Vitellogenin is the precursor of egg yolk, while the zona radiata proteins constitute the dominant part of the eggshell. They are produced in the liver of maturing females, transported by the blood, and taken up by the growing oocyte. The processes are regulated by oestrogens and male and juvenile fish are supposed to have low levels of these proteins. However, induced levels of these proteins in male and juvenile fish have been reported after exposure to environmental pollutants that have the possibility to mimic natural oestrogens (xenoestrogens) and thereby inducing unscheduled oogenesis (White et al. 1994; Sumpter and Jobling 1995; Arukwe, et al. 1997). Induced levels of Vtg in male and juvenile fish have also been reported in wild population of flounder (Platichthys flesus L.) and carp (Cyprinus carpio) living in polluted estuaries or polluted rivers, respectively, probably mainly due to discharges from sewage treatment plants, but also due to industrial outlets (Lye et al. 1997; (Folmar et al. 1996). Some of the chemicals used in oil production can act as xenoestrogens, e.g. some alcylated phenols like octyl- and nonylphenol (Nimrod and Benson 1996); (Gronen et al. 1999) Histology Cellular alterations detected by histological methods are changes on a higher biological level compared to changes in enzyme activities and may represent a more severe effect. Samples of liver and gill tissue were fixed and embedded in paraffin. A subset was briefly characterised, while the main objective was to store it as a possible reference material for future studies. Liver is the main organ for metabolism and used in studies of cellular alterations (Myers et al. 1987). Gills are the first barrier for the organism towards water carried contaminants, and are also used to study histopathological lesions (Spies et al. 1996; Teh et al. 1997)

13 RF Rogaland Research, Norway. 2 Material and methods 2.1 Field work Fieldwork was performed by Fiskirannsoknarsstovan s research vessel Magnus Heinason. Two sampling cruises were performed, 27 th May 3 rd June, and 11 th 15 th November 1999, to cover both spring and autumn conditions in case of seasonal variances in the biological response parameters. For each cruise 50 Greenland halibut were sampled, 25 females and 25 males. In November sediment samples were also collected Sampling locations The sampling area for fish and sediments are shown in Figure 2. The red lines indicate the trawl tracks. The area south of the Faroe Islands was selected as a reference region, while the area south east of the Faroes is covering the area for possible oil exploration. Raw data from both surveys are shown in Chapter 6.1. FAROE ISLANDS 62 N d e 10-d 10-c 10-b 10-a 10-f a b 8 9-c 61 N a 1-b 1-c 1-d 2 SHETLAND 60 N 2 W 3 W 4 W 5 W 6 W 7 W 8 W Sediment stations Trawling Nov Trawling May 1999 ORKNEY 59 N Figure 2. Sampling area. Locations for sediment stations are indicated. Red lines indicate trawl tracks

14 2.1.2 Sediment Handling of sediment samples and parameters measured were done according to SFT Guidelines (SFT 99:01). Sediments were collected with a Van Veen grab at the locations given in Figure 2 and Chapter A total of 20 grab samples were collected, and each sample was divided into sub samples for analyse of: 1. Grain size distribution and amount of total organic matter (TOM). 2. Metals (Li, Cr, Fe, Ni, Cu, Zn, As, Sr, Ag, Cd, Ba, Pb and Hg). 3. Total hydrocarbon content (THC) 4. Polycyclic aromatic hydrocarbons (PAH) and C 1 -C 3 alcyl substituted compounds of naphtalene, phenanthrene and dibenzothiophene (NPD). Sediments for hydrocarbon analysis were collected with a metal spoon, and then packed in pre cleaned aluminum foil. Finally the package was put in a plastic bag and frozen at 20 o C prior to analysis. Sediments for heavy metal analysis, grain size distribution and TOM were collected with a teflon spoon, packed in plastic bags, and stored at 20 o C prior to analysis. Upon retrieval each grab sample was described with reference to visible characteristics and features including type, color, odour, presence of biological material Fish Greenland halibut was collected by trawl and transferred into storage tanks with running seawater, to keep them alive prior to sampling. Dead fish were not included in the sampling. From each fish, samples from otoliths, blood, gills, bile, liver and muscle were taken as described in Chapter In addition biological data of individual fish such as total length, total weight, liver weight, gonad weight, sex and maturation are presented in Chapter Maturation stages range from 0 to 3, where stage 0 is juvenile fish, and stage 3 is pre-spawning fish. 2.2 Grain size distribution and total organic material in sediment Grain size distribution of sediment samples was determined according to (Buchanan 1984). Total organic material was determined as loss on ignition according to Norsk Standard NS Metals Sediment Prior to the chemical analysis, sediments were digested according to Norwegian Standard NS This digestion procedure together with the pre-treatment method described below, are recommended by SFT (The State Pollution Authority in Norway)

15 The sediment samples were, after arrival at the laboratory, dried at 50 o C to a weight that remained constant. After drying, the samples were crushed and homogenised in an agate mil and sieved with a plastic sieve 0.5 mm. The fraction smaller than 0.5 mm were analysed. For digestion of the sediment, approximately 1 g of sediment was placed in a vessel, and 10 ml 7 M HNO 3 is added. The vessel was sealed and placed in an autoclave. Temperature in the autoclave was maintained at 120 C, for 30 min. The autoclave was then allowed to cool and the samples were diluted with water to 50 ml. Reference material, MESS-1 and BEST-1, were similarly digested, for quality control of the analytical procedure Bile and liver Bile and liver samples were frozen at 20 C and stored until analysis. In the laboratory 100 µl bile were transferred to a screw-capped polypropylene test-tube and 100 µl conc. HNO 3 and 100 µl H 2 O 2 were added. Digestion was performed by placing the test tubes in water bath for 3 hours. The samples were then diluted to 5 ml. The wet liver samples were digested with HNO 3 in a microwave oven. The procedure was verified with certified material. This verification showed between 5-10 % deviation from the certified values. Analyses. Analyses of all metals were performed by inductively coupled plasma mass spectrometry (ICP-MS), using a VG-PQ2+ system with standard mienard nebulizer and Ni sampling and Skimmer cones. In addition to the usual standard curves, Indium was used as an internal standard. The Hg analysis was performed by cold vapour atomic absorption spectroscopy (CV-AAS) in an automated flow injection system (FIMS) from Perkin-Elmer (Westerlund et al. 1998). 2.4 Extraction and analyses of hydrocarbons Sample treatment Sediment samples were stored at 20 C until homogenisation, saponification and extraction. Before saponification and extraction, n-eicosan-d42 (n-c 20 D 42 ) was added as internal standard for the THC analysis. Fish samples were stored at 20 C until sample preparation started. The samples were thawed and homogenised by cutting them into small pieces, and stored at 20 C, in heat-treated glass containers, until extraction. All samples. Internal standards for quantification, QIS, were added to the homogenised samples (sample weight approximately 5 g) and the samples were boiled for two hours in a 10% (w/v) solution of potassium hydroxide in methanol, to achieve saponification. The digests were filtered and extracted three times with cyclohexane. The extracts were combined. To dry the samples, an excess of anhydrous sodium sulphate (pest. grade) was added. Finally the samples were filtered through glass sinter filters (pore size 2) and - 7 -

16 concentrated to 1 ml by use of TurboVap 500 (Zymark Corporation, Hopkinton, MA, USA). Cleanup of the extracts was performed by solid phase extraction using 3 ml tubes containing 0.5 g of normal phase packing (Supelclean LC-Si, Supelco INC, Bellefonte, PA, USA). The compounds of interest were eluted with a mixture of dichloromethane and cyclohexane (1+3). The purified extracts were concentrated to 0.5 ml. Internal standards for recovery calculation (RIS) was added to the extract and, finally, the sample extracts were transferred to autosampler vials and stored refrigerated until GC/MS-analysis GC/FID analysis for total hydrocarbon (THC) quantification. THC analysis of the sediment samples was performed by Gas Chromatography (HP5890 Series II, Hewlett Packard, USA) equipped with HP7673 auto injector and Flame Ionisation detector (FID). The GC was equipped with a HP ULTRA 1 (Crosslinked-Methyl-Siloxane 50mx0.2mmx0.33µm). Injector temperature was set to 290 C and detector temperature to 320 C. The column oven temperature was started at 50 C and ramped at 25 C/min to 120 C, 3 C/min to 320 C and held for 20 min at 320 C. Helium was used as carrier gas with a flow rate at 1 ml/min. Sample injection volume was 1µl PAH analysis GC/MS-SIM analysis of samples PAH analysis of all samples was performed by Gas Chromatography (HP5890, Hewlett Packard, USA) connected to a Mass Spectrometer (Finnigan SSQ7000, USA) and analysed in selected ion mode (GC/MS-SIM). The GC was equipped with a CP-SIL 8CB fused silica column (Chrompack, 50 m x 0.25 mm i.d., film thickness 0.25 µm). Injector and detector temperatures were both 300 C. The column was held at 50 C for 1 min, ramped at 25 C/min to 120 C, 3 C/min to 320 C and held for 17 min at 320 C. Helium was used as carrier gas with a flow rate of 0.6 ml/min at 50 C. The selected masses (m/z) for both PAH and deuterated internal standards are shown in Table 1. Expected recovery is 30% for naphthalenes (2-rings), 50% for larger ring systems. Table 1 Selected masses (m/z) of PAH and standards PAH compounds Masses m/z Naphthalene 128,2 C1-naphthalenes 142,2 Acenaphthylene 152,2 Acenaphthene 154,2 C2-naphthalenes 156,2 fluoranthene 166,2 C3-naphthalenes 170,2 phenanthrene 178,2 anthracene 178,2 dibenzothiophene 184,2-8 -

17 C1-phenanthrenes/anthracenes 192,2 C1-dibenzothiophenes 198,2 fluoranthene, pyrene 202,2 C2-phenanthrenes/anthracenes 206,2 C2-dibenzothiophenes 212,2 benzo(a)anthracene 228,2 Chrysene 228,2 C1-chrysene 242,2 benzo(b)fluoranthene 252,3 benzo(k)fluoranthene 252,3 benzo(a)pyrene 252,3 C2-chrysene 256,3 indeno(1,2,3-cd)pyrene 276,3 benzo(g,h,i)perylene 276,3 dibenzo(a,h)anthracene 278,3 naphthalene-d8, QIS 136,2 phenanthrene-d10, QIS 188,2 Dibenzothiophene-d8, QIS 192,2 fluoranthene-d10, QIS 212,2 pyrene-d10, QIS 212,2 chrysene-d12, QIS 240,2 benzo(a)pyrene-d12, QIS 264,3 Dibenzo(a,h)anthracene-d14, QIS 292,3 1-methylnaphthalene-d10, RIS 152,2 anthracene-d10, RIS 188,2 perylene-d12, RIS 264, Calculation and Quality assurance Calibration standards for the various PAHs were prepared in seven different concentrations. Response factor curves were calculated for the individual PAHs and used for calculation of sample concentrations (calculation program used was SI-QUAN, Kjell Urdal, SINTEF, Oslo). The reproducibility of the response factors for three different standards, approximately covering the expected concentration range, was checked for each series of samples analysed. Recovery of 2, 3 and 5 ring PAHs was found by calculating the QIS/RIS area ratio of selected compounds. A certified reference material (SRM2974, NIST, Gaithersburg, MD, USA) or a blank sample spiked with an appropriate amount of a certified mixture of PAHs (Dr Ehrenstorfer Reference Materials, Augsburg, Germany) was used as control samples. Internal standards. To perform accurate quantitative analyses, we use several different internal standards. These deuterated compounds are chosen to eliminate differences in behaviour and response for different compounds when the concentration varies. Internal control. To confirm that our analytical results are reliable, standards, spiked samples, certified reference material and, if possible, blank samples were analysed - 9 -

18 along with the rest of the samples. Recovery was also calculated for 2-ring, 3-ring, and 5-ring compounds. Interlaboratory studies. Akvamiljø participates regularly in international round Robin tests for sediment and biota. Sources of error Traces of many compounds may be present in a sample because of the nature of the sample, and are visible as sample matrix. Some compounds in the matrix that are not PAHs, can be seen in the chromatograms (matrix peaks) and may influence the analysis. Matrix peaks can indicate a false positive value or cover small peaks that should have been detected (false negative). These facts are taken into consideration when the results are evaluated. To perform a quantitative analysis, several different internal standards are added. These deuterated compounds are about 99% pure, and the remaining nondeuterated PAHs are present in standards and samples. They can therefore give rise to small background peaks in the chromatogram (detection of false positive). When handling volatile compounds there is always a risk of loosing part of the compounds. To eliminate the error that could occur, internal standards (QIS) are added before the samples are extracted, standards that behaves similarly to the compounds of interest. In this way we eliminate the effect of the loss. Recovery data tells us how many per cent are lost, and whether or not this is acceptable analytically. Recovery internal standards (RIS) are added to the extracted samples before they are analysed on GC/MS. PAHs are light sensitive. The samples are therefore kept covered and away from sunlight throughout the whole procedure to avoid degradation. Representative chromatograms of sediment and fish samples, standards, and also the typical naphthalene pattern from oil are give in Chapter Fluorescence detection of PAH metabolites in bile Synchronous fluorescence spectrometry (SFS) were performed on a Perkin Elmer LS50B luminescence spectrometer. Samples were diluted 1:1000 in methanol:water (1:1), and a difference between excitation and emission wavelengths of 42 nm was used. This λ is found to be suitable for the identification of PAH metabolites (Aas et al. 2000). Slit widths were set at 2.5 nm for both excitation and emission wavelengths. Bile samples were analysed in quartz cuvettes. Detection limits correspond to a naphthalene equivalents concentration of 180 ng/ml bile, a pyrene equivalent concentration of 14 ng/ml bile and for benzo[a]pyrene equivalents, 0.7 ng/ml bile. The detection limit was defined as the concentration corresponding to a fluorescence signal three times the fluorescence signal of the solvent

19 2.6 Cytochrome P4501A activity in liver Samples All liver samples were homogenised onboard in 0.1 M Na-phosphate, 0.15 M KCl, 1 MM ethylene diamine tetra acetic acid (EDTA), 1 mm dithiothreitol (DDT) and 10 % glycerol (ph 7.4), centrifuged for 20 min at g. The supernatant was frozen on liquid nitrogen, and stored at 80 C until analysis. For samples analysed in November, the S12 fraction seemed to have coagulated. For that reason extra parallels of frozen liver samples were homogenised and centrifuged at the laboratory before being analysed for EROD activity Analysis Specific EROD activity in liver samples was measured by a fluorescence plate reader technique, according to the method described in (Eggens and Galgani 1992). A Fluoroscan II plate reader (Labsystems OY, Finland) was used. The described method was modified by measuring activity in S12 fraction (12 000g centrifugation of liver homogenate), and excitation/emission wavelengths of 544/584 nm were applied. Protein concentrations in the liver samples were measured according to the method described in (Bradford 1976). Hepatic S-12 fraction of cod exposed intraperitoneally to 5 mg kg -1 benzo[a]pyrene for 3 days served as a positive control in all analytical series. A nonexposed cod was included as a negative control, also in all analytical series. The samples from the November cruise were analysed using a Perkin Elmer LS50B fluorometer. However, the method followed were identical to the samples from the May cruise. 2.7 Enzyme-linked immunosorbent assay (ELISA) ELISA measurements were performed by Biosense Laboratories AS, Bergen, Norway. Antibodies used for Western and ELISA measurements were polyclonal anti-turbot Vitellogenin (CS-2, Biosense) and polyclonal anti-salmon zona radiata proteins (O-173, Biosense). SDS-PAGE was performed using 7.5 or 12 % acrylamide gel (Laemmli 1970). Western blotting was performed using SDS-PAGE and electrophoretic transfer onto nitrocellulose (Towbin et al. 1979). Detection of peroxidase staining of Western blots and enzyme-linked immunosorbent assay (ELISA) was performed as described in (Nilsen et al. 1998). Antibody dilution for ELISA; CS-2: 1:1000, O-173: 1: DNA adducts A subset from each cruise (a total of 20 samples) were analysed for occurrence of DNA adducts in liver. DNA adduct formation in was analysed according to the 32 P postlabelling method, described in (Ericson et al. 1998). Adducts were resolved in two dimensions by chromatography, and located and quantitated by storage phosphor

20 imaging technology (PhosphorImager SI and ImageQuant, 5.0 software, Molecular Dynamics). 2.9 Paraffin embedding of liver and gill samples Liver and gill from all of the fish were fixed in buffered formalin (4 % formaldehyde, 1.7 % methanol and phosphate, ph 7.4) (Norsk Medisinaldepot), dehydrated and embedded in paraffin. Sliced sections were stained with hematoxylin and eosin. A subset (10 livers and gills of each cruise) was also briefly characterised

21 3 Results and discussion 3.1 Sediment 20 sediment samples were collected during the cruise in November Sampling sites are shown in Figure 2 and Chapter 6.1. Stations 1-9 are from the licensed area south east of the Faroe Islands, while Station 10 is from a possible future reference area south of the Faroe Islands. All the sediment samples had a grey/brown colour. They had no characteristic odour and contained some biological material as sea stars, brittle stars, sea mice, crustaceans, however, species characterisation of the sediment samples were not within the scope of this investigation Grain size distribution and total organic material Grain size distribution analyses of the 20 sediment stations are shown in Figure 3 and Chapter 6.2. The % weight of silt and clay (particles < 63 µm) was varying from 0.3 % to 20 %. When Station 9 were subtracted the mean weight % of silt and clay in this area was 5.0 ± 0.9 %. Mean of Station 9 was 12.9 ± 5.2 %. Mean of Station 10 was 1.3 ± 0.8 %. Total organic material (TOM) measured as ignition loss is shown in Figure 4. The TOM varied between %. The sediment stations were taken from approx. the same depth, m (Chapter ). Values for skewness and kurtosis for the sediment samples are given in Chapter % Particle distribution (%) 80 % 60 % 40 % 20 % 0 % 1-a 1-b 1-c 1-d a 9-b 9-c 9-d 10-a 10-b 10-c 10-d 10-e 10-f > 4 mm 2-4 mm 1-2 mm mm mm µm µm < 63 µm Station Figure 3. Grain size distribution (% weight) in the surface sediment

22 4.5 4 Loss on ignition (%) a 1-b 1-c 1-d a 9-b 9-c 9-d 10-a 10-b 10-c 10-d 10-e 10-f Station Figure 4. Content of TOM measured as loss on ignition in the sediment samples Metal analyses of sediment Levels of metals found in the sediment samples are given in Table 2. Metal data characterise the sediment as uncontaminated with chemical levels according to sediments of class I (SFT 1997). However, this is a guideline of fjords and coastal waters in Norway, and could be misleading for a reference area in open sea. Some variation between stations is observed, probably effected by differences in grain size distribution. Levels of barium were very low compared with those found elsewhere on the margin (AFEN 2000). As barium sulphate has been used to increase the density of drilling mud, the content of barium in the sediment has been used as an indicator of the spread of drilling cuttings on the sea floor. However, it is important to be aware that there are also biogenic sources of barium in the deep-sea and particularly at continental margins

23 Table 2. Metal analyses of sediment samples. Data are given as mg/kg dry weight. % dry matter in samples reported as DM. Mean and std. dev. of all stations is given below. Li Cr Fe Ni Cu Zn As Station mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 1-a b c d a b c d a b c d e f Mean std dev Sr Ag Cd Ba Pb Hg DM Station mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg % 1-a b c d a b < c d a b c d e < f M ean std dev

24 3.1.3 Total hydrocarbon (THC) analyses The 20 sediment samples were extracted and analysed for THC. None of the sediment samples show hydrocarbons above levels of detection (LOD) which was 2 mg/kg sediment, while limit of quantification (LOQ) was 5 mg/kg sediment in these analyses. In Chapter is given a representative chromatogram of the sediment samples. Low levels were also found further east in the same region, in this case quantified to means of 2.2 and 3.1 mg/kg (AFEN 2000) PAH analyses of sediment The 20 sediment samples were analysed for individual PAH and NPD components. The analyses did not show detectable amount of any PAH or NPD in the samples (Table 3). For single compounds in sediment samples the method detection limit (MDL) was 1.5 µg/kg, and the limit of quantification (LOQ) 6 µg/kg. For the compounds naphthalene, phenanthrene, anthracene, fluoranthene and pyrene a peak was found in the chromatograms, however, this is believed to be caused by addition of deuterated internal standards and notified as below method detection limit. C 1 and C 2 phenanthrenes/anthracenes were also not detected. The peaks that are visible in the respective time windows of the chromatograms do not match the pattern that is typical for the respective C 1, and C 2 compounds, and are interpreted as matrix peaks (Table 3). For details of the chromatograms, see Chapter The term PAH is including all aromatic compounds and their alcyl substituted homologues that consists of two or more condensed benzene rings. The PAH analysed in this project are shown in Table 3. Another frequently used term is NPD. NPD is included in PAH, but is often reported separately because these 2- and 3- rings PAH are of particular interest in work related to oil activities. Generally, PAH and NPD are reported from baseline surveys from other regions in the North Sea. Means of 28 and 41 µg/kg NPD and means of 74 and 107 µg/kg of 2-6 rings PAH are reported in sediments from the Scottish continental slope (AFEN 2000). Baseline investigations at Njord (west of Trondheim, Norway) reports mean NPD levels to be 73 µg/kg and 3-6 ring PAH levels to be 98 µg/kg (Botnen et al. 1997). At Norne (Trænabanken, Norway) levels of NPD and PAH were reported to be 63 and 89 µg/kg, respectively (Myhrvold et al. 1996)

25 Table 3. PAH levels in sediment samples (µg/kg dry weight) PAH compounds Sediment (µg/kg) Comments Naphthalene < 1.5 C1-naphthalenes not detected C2-naphthalenes not detected C3-naphthalenes not detected Acenaphthylene not detected Acenaphthene not detected Fluorene not detected Phenanthrene < 1.5 Anthracene < 1.5 C1-phenanthrenes/anthracenes not detected matrix peaks; noise C2-phenanthrenes/anthracenes not detected matrix peaks; noise Dibenzothiophene not detected C1-dibenzothiophenes not detected C2-dibenzothiophenes not detected Fluoranthene < 1.5 Pyrene < 1.5 benzo(a)anthracene not detected chrysene not detected C1-chrysene not detected C2-chrysene not detected benzo(b)fluoranthene not detected benzo(k)fluoranthene not detected benzo(a)pyrene not detected indeno(1,2,3-cd)pyrene not detected benzo(g,h,i)perylene not detected dibenzo(a,h)anthracene not detected 3.2 Greenland halibut Biological data of Greenland halibut are summarised in Table 4 while individual data are given in Chapter Otoliths for age determination were taken only from fish sampled in November. The age ranged from 7 to 12 years. Mean age of males was 8.0 ± 0.9 years, while for females it was 9.1 ± 0.3 years. Table 4. Length, weight and age of Greenland halibut (n.a. = not analysed). Sex Sampled n Length (cm) Weight (Kg) Age (year) Male May ± ± 0.6 n.a. Female May ± ± 0.5 n.a. Male Nov ± ± ± 0.9 Female Nov ± ± ± Metals in liver and muscle Chemical levels of metals in liver and muscle are presented in Tables 5 and 6. The levels of heavy metals were generally low and within the range reported from other fish species from the Arctic (AMAP 1998). However, levels of Hg in muscle was approx

26 0.1 mg/kg which is slightly higher than similar data reported for Greenland halibut caught at Greenland, reporting Hg levels from mg/kg (AMAP 1998). Limits of Hg levels in cod muscle to be classified as uncontaminated are set to <0.1 mg/kg Hg (SFT 1997). Table 5. Levels of metal (mg/kg wet weight) in liver and muscle of Greenland halibut. Samples of male fish were analysed individually and presented as mean ± std. dev., while samples of female fish were analysed as pooled samples (n.a. = not analysed). Liver Liver Liver Liver Muscle Muscle May-99 May-99 Nov-99 Nov-99 May-99 Nov-99 Mean (n=23) Pooled Mean (n=25) Pooled Pooled Pooled Element mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg V 0.20 ± ± Cr 0.37 ± <0.4 < <0.4 Mn 1.07 ± n.a. n.a n.a. Fe 61 ± ± Co 0.03 ± ± Ni 0.04 ± ± Cu 12.1 ± ± Zn 21.8 ± ± As 19.0 ± ± Se <1 <1 <1 <1 2.9 <1 Mo 0.07 ± ± <0.02 Ag 0.11 ± ± Cd 0.79 ± ± Sn <1 <1 <0.2 < <0.02 Ba <1 <1 <0.2 < <0.2 Pb <0.03 <0.03 <0.03 <0.03 <0.03 <0.03 Table 6. Levels of Hg (mg/kg wet weight) in muscle and liver tissue of Greenland halibut. Tissue Sampling time n Hg (mg/kg) Muscle May ± Muscle May-99 pooled sample (25) 0,053 Muscle Nov ± Muscle Nov-99 pooled sample (25) Liver May-99 pooled sample (25) Metals in bile The metal components of the bile reflects the recent metal exposure or physiological active elements, as the bile is regularly evacuated into the gut upon digestion. The advantage of using the bile is that it is a concentrate, which do not need extraction prior to analysis. It may not give an equal representative picture of all metals presently metabolised/excreted by the organism, as some metals are directly excreted through gills and kidney. The secretion of metals in bile is not necessarily an excretion of trace

27 metals, since much might be reabsorbed in the intestine. The amount and concentration of the bile content should be considered when interpreting the data. The presented data have not been normalised to e.g. biliverdin or protein content of the bile as it may also add noise to the data set. The presented data indicates the range in variation in fish sampled in spring and autumn and between sex. Some metals have higher levels in fish sampled in May compared to fish sampled in November e.g. Cr, Fe and Ni, while levels of Zn, Ag, Cd and Pb were higher in fish sampled in November. Higher levels of Cr and Fe were observed in females compared to males sampled in November (Table 7). In a study of trace metals in bile and liver in fish from the North Sea, it was possible to group the fish from the different areas based on element composition. The bile analysis results produced a stronger multivariate statistical grouping than the liver samples (Westerlund et al. 1998). Levels of metals in bile are not a validated biomarker, but it has a potential as a simple and cost effective biomarker of exposure. However, more background data is needed to evaluate the method, and its use is being evaluated in ongoing programmes. Table 7. Levels of metal in bile of Greenland halibut (mean ± std.dev.) (n.a. = not analysed). May-99 Nov-99 Nov-99 males (n = 25) males (n = 20) females (n=17) Element µg/l µg/l µg/l Al 64 ± 61 n.a. n.a. V 35 ± 14 n.a. n.a. Cr 392 ± ± ± 59 Mn 15 ± 9 18 ± ± 10 Fe 3287 ± ± ± 940 Co 2.4 ± ± ± 1.3 Ni 13.9 ± ± ± 4.3 Cu 2093 ± ± ± 2453 Zn 188 ± ± ± 541 As 946 ± ± ± 904 Se n.a. 642 ± ± 379 Sr 2496 ± ± ± 572 Mo n.a. 2.0 ± ± 1.2 Ag 13.3 ± ± ± 19.6 Cd 2.2 ± ± ± 6.3 Sn n.a ± ± 59.9 Pb 1.57 ± ± ±

28 3.2.3 PAH analyses of fish samples A total of 52 fish muscle samples were analysed for individual PAH and NPD components, males were analysed individually (n= 50) and females as pooled samples. The analyses did not show any detectable amount of any PAH or NPD in the samples (Table 8). For single compounds in sediment and fish muscle samples the method detection limit (MDL) was approximately 1 µg/kg, and limit of quantification (LOQ) 4 µg/kg. For the compounds naphthalene, phenanthrene, anthracene, fluoranthene and pyrene peaks were found in the chromatograms, however, this is believed to be caused by non deuterated impurities in the internal standards and notified as below method detection limit. C 1, C 2, and C 3 naphthalenes were also notified as not detected. The peaks that are visible in the respective time windows of the chromatograms do not match the pattern that is typical for naphthalenes from oil, and are interpreted as matrix peaks (Table 8). Representative chromatograms for the analysed samples are given in Chapter The recovery was satisfactory. Only one sample (FGH 99014) had a recovery lower than expected. For details about recovery of the samples, see attached Chapter Table 8. PAH levels in fish muscle samples (µg/kg wet weight) PAH compounds Fish, µg/kg Comments Naphthalene < 1 C1-naphthalenes not detected matrix peaks C2-naphthalenes not detected matrix peaks C3-naphthalenes not detected matrix peaks Acenaphthylene not detected Acenaphthene not detected Fluorene not detected Phenanthrene < 1 Anthracene < 1 C1-phenanthrenes/anthracenes not detected C2-phenanthrenes/anthracenes not detected Dibenzothiophene not detected C1-dibenzothiophenes not detected C2-dibenzothiophenes not detected Fluoranthene < 1 Pyrene < 1 Benzo(a)anthracene not detected Chrysene not detected

29 C1-chrysene C2-chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(a)pyrene Indeno(1,2,3-cd)pyrene Benzo(g,h,i)perylene Dibenzo(a,h)anthracene not detected not detected not detected not detected not detected not detected not detected not detected PAH metabolites in bile PAH metabolites were measured in bile from all individuals. All analysed bile samples exhibited a fluorescence signal corresponding to PAH levels below detection limit. Detection limits corresponded to naphthalene equivalents concentration of 180 ng/ml bile, a pyrene equivalents concentration of 14 ng/ml bile and benzo[a]pyrene equivalents of 0.7 ng/ml bile. The results are illustrated by synchronous fluorescence spectrography ( λ=42nm) (Figure 5). Biliverdin analyses are used as an aid in the interpretation of the fluorescence results. Since no detectable levels were found, biliverdin levels were not included. Data of bile analyses are given in Chapter

30 Figure 5. Synchronous fluorescence spectrography ( λ=42nm) of representative bile samples compared to bile from cod exposed to crude oil. The figure also shows that precipitated bile samples should be avoided as they show interfering fluorescence signals with possible PAH metabolites. Bile from cod exposed to dispersed crude oil Precipitated bile Solvent Representative bile samples Cytochrome P4501A activity Low levels were observed for cytochrome P4501A (CYP1A) activities measured as EROD activity in males and females caught in both cruises (May and November) (Table 9). Even though, slightly higher levels were observed in fish sampled in May, compared to the fish sampled in November, and in males compared to females, the CYP1A enzyme did not seem to be induced. To our knowledge these are the first measurements of CYP1A activity in Greenland halibut. The levels observed seem comparable to basal levels in cod (Aas et al. In press). Variation due to season and sex should be expected and are found for other species like salmon (Salmo salar L.) and turbot (Larsen et al. 1992; Arukwe and Goksøyr 1997). We tried to obtain more information on the CYP1A response in Greenland halibut by trying to keep them in tanks for exposing them to CYP1A inducers onboard, but they seem extremely sensitive to stress after trawling, and all died within the first day. Table 9. EROD activities in liver of Greenland halibut given as mean ± standard deviation. Sex N Sampling time EROD (pmol/min/mg protein) Male 24 May ± 0.8 Female 25 May ± 0.7 Male 20 November ± 0.4 Females 21 November ±

31 3.2.6 DNA adducts A subset of 20 liver samples was analysed for DNA adducts by 32 P-postlabelling. DNA adducts were not detected, except for one fish which had DNA adducts just above background levels (Table 10). In order to establish a background for each sample, 4 different areas on each TLC sheet (representing about 80 % of the TLC surface) was investigated for the background value by the use of Phosphor Imager and the program Image Quant. A representative TLC is demonstrated in Figure 6 together with the TLC of fish no and of a cod exposed to oil in the laboratory. DNA adducts have been analysed in wild caught fish from other areas in the arctic also without finding detectable levels (Lennart Balk, pers. comm.). Table 10. DNA adducts measured in liver of Greenland halibut. (*) DNA adducts were considered to occur when background level were surpassed 1.5 times. Fish no. Sex Sampled Adducts (nmol/mol) FGH99001 F Jun FGH99002 F Jun FGH99004 F Jun FGH99005 F Jun FGH99006 F Jun FGH99037 M Jun FGH99038 M Jun FGH99039 M Jun FGH99040 M Jun FGH99041 M Jun FGH99054 F Nov FGH99058 F Nov FGH99070 F Nov FGH99074 M Nov FGH99078 M Nov FGH99081 M Nov FGH99083 F Nov FGH99085 F Nov FGH99090 M Nov FGH99091 M Nov A B C Figure 6. Autoradiogram of TLC plates after 32 P postlabelling. Shown is (A) a representative autoradiogram of Greenland halibut showing no adducts. (B) Autoradiogram of fish showing adducts just above background. (C) An example of adducts taken from cod exposed to 0.06 ppm oil for 30 days (Aas et al. In press)

32 3.2.7 Levels of vitellogenin and zona radiata proteins in blood Vitellogenin and zona radiata proteins are important constituent parts of the egg. They are produced in the liver of maturing females, transported by the blood, and taken up by the growing oocyte. The processes are regulated by oestrogens and male and juvenile fish are supposed to have low levels of these proteins. However, induced levels of these proteins in male and juvenile fish have been reported after exposure to environmental pollutants that have the possibility to mimic natural oestrogens (xenoestrogens) and thereby inducing unscheduled oogenesis (White et al. 1994; Sumpter and Jobling 1995; Arukwe et al. 1997). As some of the chemicals used in oil production can act as xenoestrogens, e.g. some alcylated phenols like octyl- and nonylphenol (Nimrod and Benson 1996; Gronen et al. 1999), measurements of these biomarkers were included. There are no species specific Vtg or Zrp antibodies purified from Greenland halibut. For that reason detection of these proteins were performed by polyclonal anti-turbot vitellogenin (CS-2) and polyclonal anti-salmon zona radiata proteins (O-173). Western blots with polyclonal anti-turbot vitellogenin (CS-2) demonstrated crossreaction with vitellogenin in both female and male samples in addition to some unspecific cross reactivity in males. Whether occurrence of vitellogenin in male Greenland halibut is a natural pattern warrants further investigation. In ELISA measurements, we did see an increase in absorbance for females according to maturation status, but due to the interference of unspecific cross-reactivity in males, we have chosen not to present the data with vitellogenin. A test of new available antibodies, or most preferably developments of specific antibodies to Greenland halibut would be required. This could also allow for quantitative determination of vitellogenin. Results with polyclonal anti-salmon zona radiata proteins (O-173) demonstrated also cross-reactivity with the antibodies in both males and females. However, results with ELISA demonstrated as expected low levels in males. For females the results demonstrated high levels in females with maturation status 2 and 3, while females with maturation status 1 had levels of Zrp protein in blood at background levels (Table 11 and Figure 7). For females with maturation status 2, Zrp levels were approximately similar either sampled in May or November (n=7 and 9 respectively) (Table 11). Individual data are given in Chapter

33 Table 11. Levels of Zrp cross reacting proteins in blood of Greenland halibut expressed as absorbance at 492 nm given as mean ± std. dev. Fish were divided according to sex, maturation (0-3, where stage 0 is juvenile fish and stage 3 is pre spawning fish) and sampling time. Sex Maturation Sampling time n Zrp (absorbance at 492 nm) Female 1 May ± Female 1 November ± Female 2 May ± Female 2 November ± Female 3 November ± Male 1 May ± Male 1 November ± Male 2 May ± Male 2 November ± Male 3 May Male 3 November ± Absorbance at 492 nm F1 F2 F3 M1 M2 M3 Sex and maturation Zrp Figure 7. Levels of Zrp cross reacting proteins in blood of Greenland halibut expressed as absorbance at 492 nm given as mean + std. dev. Fish were divided according to sex (female (F), male (M), maturation (0-3, where stage 0 is juvenile fish and stage 3 is pre spawning fish). Values for sampling times in May and November were pooled Histology Liver and gills from all fish were embedded in paraffin and stored for possible histological analyses in future studies. A subset, liver and gills of 10 fish per cruise, were sliced, stained and briefly characterised. In the liver samples, one necrosis and one granuloma was noted, but was explained most probably due to parasites. In the rest of the material, no pathological changes were found. Secondary lamellas of the gills were found to be cudgel-shaped, probably a normal pattern in Greenland halibut. Some variations in the cytoplasm in the livers were noted, probably due to variation of depositing of fat and glycogen. Status of nutrition, sex, maturation and age would effect such observations

34 4 Conclusion RF Rogaland Research, Norway. The results from the analyses of the sediment samples demonstrated low levels of metals and not detectable levels of total hydrocarbon or single compounds of PAH. There was some variation in grain size distribution between different areas. The total organic matter in the sediment samples was relatively low with some variations between stations. All data supported the characterisation of the sediment to be uncontaminated. Levels of metals in fish liver and muscle were found to be generally low. Levels of metals were also analysed in bile. Single components of PAH were analysed in muscle and no detectable levels were found. Neither were detectable levels of PAH metabolites found in bile. Activities of the detoxifying enzyme cytochrome P450 1A (CYP1A) measured in liver were generally low. Levels of DNA adducts were also measured and found to be below levels of detection. Levels of vitellogenin and zona radiata proteins were measured in blood as these proteins are shown to be induced by oestrogen mimicking compounds. Cross-reactive bands were found in both males and females. Further work is required to investigate whether this is a normal pattern in Greenland halibut. There is still limited knowledge about natural variations in some of the biological parameters analysed, like CYP1A activity and levels of vitellogenin/zona radiata proteins in blood. The data obtained through this study confirms the area studied, south and south east of the Faroe Islands, to be a clean area with regard to the chemical and biological parameters studied and the data are documenting natural conditions in the area. Greenland halibut migrates between the Faroe Islands, Iceland and Greenland. In future monitoring programmes, additional monitoring of a more stationary deep-sea species should be considered

35 5 References AFEN (2000). Atlantic margin environmental surveys of the sea floor 1996 & 1998, Atlantic Frontier Environmental Network: CD-ROM. AMAP (1998). AMAP Assessment Report: Arctic Pollution Issues. Oslo, Norway, pp 859. Arukwe, A. and A. Goksøyr (1997). Changes in three hepatic cytochrome P450 subfamilies during a reproductive cycle in turbot (Scophthalmus maximus L). Journal of Experimental Zoology 277(4): Arukwe, A., F. R. Knudsen and A. Goksøyr (1997). Fish zona radiata (eggshell) protein: A sensitive biomarker for environmental estrogens. Environmental Health Perspectives 105(4): Botnen, H. B., Ø. F. Tvedten, O. Mjaavatten and O. Grahl-Nielsen (1997). Grunnlagsundersøkelse av miljøforholdene på havbunnen ved Njord, UNIFOB. pp 185. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: Buchanan, J. B. (1984). Sediment analysis. methods for the study of marine benthos. N. S. Holme and A. D. McIntyre. Oxford, UK, Blackwell Scientific Publications. IBP Handbook 16: Bucheli, T. D. and K. Fent (1995). Induction of cytochrome P450 as a biomarker for environmental contamination in aquatic ecosystems. Crit. Rev. in Env. Sci. Technol. 25(3): Eggens, M. L. and F. Galgani (1992). Ethoxyresorufin-O-deethylase (EROD) activity in flatfish: Fast determination with a fluorescence plate-reader. Marine Environmental Research 33: Ericson, G., E. Lindesjöö and L. Balk (1998). DNA adducts and histopathological lesions in perch (Perca fluviatilis) and northern pike (Esox lucius) along a polycyclic aromatic hydrocarbon gradient on the Swedish coastline of the Baltic Sea. Canadian Journal of Fisheries and Aquatic Sciences 55(4): Folmar, L. C., N. D. Denslow, V. Rao, M. Chow, D. A. Crain, J. Enblom, J. Marcino and L. J. Guillette (1996). Vitellogenin induction and reduced serum testosterone concentrations in feral male carp (Cyprinus carpio) captured near a major metropolitan sewage treatment plant. Environmental Health Perspectives 104(10): Førlin (1980). Effects of clophen A50, 3-methylcholanthrene, pregnenolone-16 alpha-carbonitrile, and phenobarbital on the hepatic microsomal cytochrome P-450-dependent monooxygenase system in rainbow trout, Salmo gairdneri, of different age and sex. Toxicology and Applied Pharmacology 54(3): Goksøyr, A. and L. Førlin (1992). The cytochrome P-450 system in fish, aquatic toxicology and environmental monitoring. Aquatic Toxicology 22: Gronen, S., N. Denslow, S. Manning, S. Barnes, D. Barnes and M. Brouwer (1999). Serum vitellogenin levels and reproductive impairment of male Japanese medaka (Oryzias latipes) exposed to 4-tertoctylphenol. Environmental Health Perspectives 107(5):

36 Harvey, J. S., B. P. Lyons, T. S. Page, C. Stewart and J. M. Parry (1999). An assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in selected invertebrate and vertebrate species. Mutation Research 441(1): Kurelec, B., A. Garg, S. Krca, S. Britvic, D. Lucic and R. C. Gupta (1992). DNA adducts in carp exposed to artificial diesel-2 oil slicks. European Journal of Phamacology, Environmental Toxicology and Pharmacology 1: Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: Larsen, H. E., M. Celander and A. Goks yr (1992). The Cytochrome P450 System of Atlantic Salmon (Salmo salar): II Variations in Hepatic Catalytic Activities and Isozyme Patterns during an Annual Reproductive Cycle. Fish Physiology and Biochemistry 10(4): Lye, C. M., C. L. J. Frid, M. E. Gill and D. McCormick (1997). Abnormalities in the Reproductive Health of Flounder Platichthys flesus Exposed to Effluent from a Sewage Treatment Works. Marine Pollution Bulletin 34(1): Meador, J. P., J. E. Stein, W. L. Reichert and U. Varanasi (1995). Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms. Reviews of Environmental Contamination and Toxicology 143: Myers, M. S., L. D. Rhodes and B. B. McCain (1987). Pathologic anatomy and patterns of occurrence of hepatic neoplasms, putative preneoplastic lesions, and other idiophatic hepatic conditions in English sole (Parophrys vetulus) from Puget Sound, Washington. J. Natl. Cancer Inst. 78: Myhrvold, A., S. Westerlund, J. P. Aabel and O. H. J. Christie (1996). Grunnlagsundersøkelse på Nornefeltet pp 127. Nilsen, B. M., K. Berg and A. Goksøyr (1998). Induction of Cytochrome P450 1A (CYP1A) in Fish: A biomarker for Environmental Pollution. Methods in Molecular Biology. I. R. Phillips and E. A. Shephard. Totowa, NJ, Humana Press Inc. 107: Nimrod, A. C. and W. H. Benson (1996). Environmental estrogenic effects of alkylphenol ethoxylates. Critical Reviews in Toxicology 26(3): Noaksson, E., U. Tjärnlund, G. Ericson and L. Balk (1998). Biological effects on viviparous blenny exposed to chrysene and held in synthetic as well as in natural brackish water. Marine Environmental Research 46(1-5): Norsk Standard NS Vannundersøkelse. Tørrstoff og gløderest i vannslam og sedimenter. Norsk Standardiseringsforbund. Reddy, M. V. and K. Randerath (1986). Nuclease Pl-mediated enhancement of sensitivity of 32 P- postlabeling test for structurally diverse DNA adducts. Carcinogenesis 7: Regoli, F., P. GB, B. E, N. M and O. E (1997). Biochemical characterization of the antioxidant system in the scallop Adamussium colbecki, a sentinel organism for monitoring the Antarctic environment. vol

37 Reichert, W. L., M. S. Myers, K. Peck-Miller, B. French, B. F. Anulacion, T. K. Collier, J. E. Stein and U. Varanasi (1998). Molecular epizootiology of genotoxic events in marine fish: Linking contaminant exposure, DNA damage, and tissue-level alterations. Mutation Research 411: SFT (1997). Classification of environmental quality in fjords and coastal waters. A guide. SFT 97:03. pp 36. SFT (1999). Environmental monitoring of petroleum activities on the Norwegian shelf; Guidelines, SFT. pp 123. Shugart, L. and C. Theodorakis (1998). New trends in biological monitoring: application of biomarkers to genetic ecotoxicology. Biotherapy 11(2-3): Sikka, H. C., J. P. Rutkowski, C. Kandaswami, S. Kumar, K. Earley and R. C. Gupta (1990). Formation and persistence of DNA adducts in the liver of brown bullheads exposed to benzo[a]pyrene. Cancer Letters 49: Spies, R. B., J. J. Stegeman, D. E. Hinton, B. Woodin, R. Smolowitz, M. Okihiro and D. Shea (1996). Biomarkers of hydrocarbon exposure and sublethal effects in embiotocid fishes from a natural petroleum seep in the Santa Barbara Channel. Aquatic Toxicology 34(3): Sumpter, J. P. and S. Jobling (1995). Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environmental Health Perspectives 103: Teh, S. J., S. M. Adams and D. E. Hinton (1997). Histopathologic biomarkers in feral freshwater fish populations exposed to different types of contaminant stress. Aquatic Toxicology 37(1): Towbin, H., T. Staehelin and J. Gordon (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci., USA 76: Varanasi, U., W. L. Reichert and J. E. Stein (1989). 32P-postlabeling analysis of DNA adducts in liver of wild English sole (Parophrys vetulus) and winter flounder (Pseudopleuronectes americanus). Cancer Research 49: Vo-Dinh, T. (1978). Multicomponent analysis by synchronous luminescence spectrometry. Analytical Chemistry 50(3): Westerlund, S., E. Aas and O. K. Andersen (1998). The use of bile in fish in a screening method for trace metal exposure. Marine Environmental Research 46(1-5): White, R., S. Jobling, S. A. Hoare, J. P. Sumpter and M. G. Parker (1994). Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135(1): Aas, E., T. Baussant, L. Balk, B. Liewenborg and O. K. Andersen (In press). PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure; A laboratory experiment with Atlantic cod. Aquatic Toxicology. Aas, E., J. Beyer and A. Goksoyr (2000). Fixed wavelength fluorescence (FF) of bile as a monitoring tool for polyaromatic hydrocarbon exposure in fish: an evaluation of compound specificity, inner filter effect and signal interpretation. Biomarkers 5(1):

38 6 Appendix

39 6.1 Data from field surveys Position of stations for sediment sampling. Station Position Trawl no. Depth 1-a N W b N W c N W d N W N W N W N W N W N W N W a N W b N W c N W d N W a N W b N W c N W d N W e N W f N W * There were taken sub samples (by Karina Nattestad, Fiskirannsoknarsstovan) to evaluate the biological content in the grab samples

40 6.1.2 Raw data Fish sampling Raw data Fish sampling May 1999 Fish nr. Length Fish Liver Gonad Sex-F/M & Trawl nr [cm] weight weight weight maturation [g] [g] [g] FGH , ,5 5,0 F FGH , ,5 8,5 F FGH , ,0 1,0 M FGH , ,0 9,5 F FGH , ,0 11,5 F FGH , ,0 1,5 F FGH , ,5 5,0 F FGH , ,5 1,0 M FGH , ,0 2,5 M FGH , ,5 4,5 F FGH , ,0 2,0 F FGH , ,5 13,0 F FGH , ,5 3,0 F FGH , ,0 5,5 M FGH , ,0 3,0 F FGH , ,5 2,5 M FGH , ,0 6,5 F FGH , ,5 2,0 F FGH , ,0 5,5 F FGH , ,5 15,5 M-2(3) FGH , ,0 1,5 M FGH , ,0 6,0 F FGH , ,5 18,0 M-2(3) FGH , ,0 2,5 F FGH , ,0 1,0 F

41 Raw data Fish sampling May 1999 (continued) Fish nr. Length Fish Liver Gonad Sex-M/F & Trawl nr [cm] weight [g] weight [g] weight [g] maturation stage FGH , ,5 4,5 F FGH , ,5 2,0 F FGH , ,0 2,0 F FGH , ,5 7,0 M FGH , ,5 1,0 F FGH , ,0 4,0 F FGH , ,0 2,5 F FGH , ,0 6,0 F FGH , ,0 8,5 M FGH , ,0 2,0 F FGH , ,5 1,0 M FGH , ,0 10,5 M FGH , ,0 4,5 M FGH , ,0 1,5 M FGH , ,5 3,5 M FGH , ,5 1,0 M FGH , ,5 8,5 M FGH , ,0 2,5 M FGH , ,5 1,0 M FGH , ,0 4,0 M FGH , ,0 11,0 M FGH , ,0 9,5 M FGH , ,7 13,0 M FGH , ,0 2,0 M FGH , ,0 1,0 M

42 Raw data Fish sampling November 1999 Fish nr. Length Fish Liver Gonad Sex-M/F & Age Trawl no. [cm] weigh weight weight Maturation stage (years) [g] [g] [g] FGH , ,9 F FGH , ,0 M FGH , F-2 9* FGH , F FGH , F-3?** FGH , F-1?** FGH , M FGH , F FGH , F FGH , F FGH , F FGH , M FGH , F FGH , F FGH , F FGH , F FGH , F FGH , F FGH , F FGH , F FGH , M FGH , M FGH , M FGH , M * Unsure, only 1 otholite ** Could not be determined

43 Raw data Fish sampling November 1999 (continued) Fish nr. Length Fish Liver Gonade Sex-M/F & Age Trawl nr (cm) weight (g) weight (g) weight (g) Maturation stage (years) FGH , M FGH , M FGH , M FGH , M FGH , M FGH , F FGH , M FGH , M FGH , F FGH , F FGH , F FGH , M-1 -* FGH , M FGH , M FGH , M FGH , M FGH , M FGH , M FGH , M FGH , F FGH , F FGH , M FGH , M FGH , M FGH , F-2 11** FGH , F * Unsure, only 1 otholite ** Could not be determined

44 6.1.3 Trawl data Trawl data May 1999 Sampling locations & trawl data from the May-survey. Trawl nr Position Depth Temp Date Trawl- Comments (m) [ C] time [min] o 51 N 7 o 06 W 60 o 56 N 7 o 08 W o 55 N 7 o 09 W 61 o 07 N 7 o 15 W , mm 2, mm o 10 N 7 o 18 W 509 3, mm 61 o 02 N 7 o 11 W 501 1, o 04 N 7 o 13 W 572 2, mm 61 o 14 N 7 o 22 W 560 1, o 13 N 7 o 21 W 61 o 03 N 7 o 13 W , mm o 52 N 7 o 09 W 576 0, mm 61 o 02 N 7 o 13 W 577 2, o 28 N 5 o 58 W 476 0, mm 60 o 38 N 6 o 04 W 452 1, o 49 N 5 o 55 W 441 4, mm 60 o 52 N 5 o 35 W 464 1, o 53 N 5 o 30 W 457 0, mm 61 o 01 N 5 o 17 W 459 2, o 24 N 4 o 47 W 455 2, mm 61 o 37 N 4 o 39 W 452 2,0-36 -

45 Trawl data November Sampling locations & trawl data from the November-survey. Trawl nr Position Depth (m) Temp Date Trawl- Trawl -size ( C) time (min) o 49 N 5 o 52 W 60 o 50 N 5 o 44 W o 51 N 5 o 43 W 60 o 53 N 5 o 30 W o 02 N 5 o 15 W 61 o 10 N 5 o 08 W o 09 N 5 o 08 W 61 o 03 N 5 o 15 W o 38 N 6 o 04 W 60 o 34 N 6 o 01 W o 33 N 6 o 01 W 60 o 29 N 5 o 58 W o 28 N 5 o 58 W 60 o 33 N 6 o 00 W o 05 N 7 o 13 W 61 o 07 N 7 o 14 W , mm 3, mm 3, mm 2, mm 4, mm 4, mm 2, mm 4, mm

46 6.1.4 Sampling overview. Sample Table 1. Description of samples, treatment of samples and analysis. Sample volume Treatment of sample Analysis Biological data Total length of the fish, total weight of the fish, liver weight, gonad weight (and maturation) were measured Condition index, LSI, GSI Blood 1 ml The blood samples were centrifuged at 2000g for 4 min (at 4 o C)., and the supernatant transferred to a new cryo tube and put on liquid nitrogen. Vitellogenin (Vtg) To verify cross-reactions with commercial available antibodies to fish Vtg, and to determine levels. Liver a). 1 g a) 4ml cold homogenising buffer a) To measure induction of per g liver was added to the liver, cytochrom P450 1A and the mixture was (CYP1A) (as EROD homogenised (in a Potter- activity). Elvehjem type of homogeniser) for 20 sec at 800 rpm. The homogenate was then transferred to centrifuge tubes and centrifuged at G for 20 min (at 4 o C). The supernatant (cytosol fraction) was carefully b) If positive controls i.e. Greenland halibut exposed to PAH are obtained, immunodetection methods (ELISA) may also be performed. transferred to cryo tubes and finally put on liquid nitrogen b). 0.5 g. b) Transferred directly to cryo tubes and put on liquid nitrogen. c) segment of 4 mm c) Preserved in 4% Formaldehyd (added phosphate buffer). Stored in fridge. c) Indication of genetic DNA damage (a subset will be d) 2 g d) Transferred directly to cryo tubes analysed). and put in freezer (- 20 o C). e) the rest of the liver e) Wrapped in Al foil and put on liquid nitrogen. d) Embedded in paraffin for Histopathology analysis (a subset will be characterised). e) Metals (not Hg) in liver

47 f) PAH concentrations in liver Sample Sample volume Treatment of sample Analysis Bile 0,5 1,8 ml (depending on the size of the bladder) Transferred directly to cryo tubes and put on liquid nitrogen. a) Bile metabolites from PAH degradation (measured by fixed wavelength - fluorescence. b) Metals in bile (ICP/MS) Gills Segment of 1 cm Preserved in 4 % Formaldehyd (added phosphate buffer). Stored in fridge. Embedded in paraffin for Histopathology analysis (a subset will be characterised). Muscle a) 5 gram b) 10 gram a) Transferred to a plastic sample bag and put in freezer (- 20 o C). b) Wrapped in Al foil and put on liquid nitrogen. a) Metals (Hg) b) PAH concentrations in muscle (optional). Otoliths * - Stored in paper bags at room temperature. Determination of age * Otoliths were only sampled at an individual level in November, however Petur Steingrund (Fiskirannsoknarstovan) collected otoliths from a number of Greenland halibut on the survey in May

48 6.2 Particle distribution and total organic matter S tation: 1-a. Lab re f.no.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Size Phi Weight Weight Cumulative Grain size distribution Station 1-a (m m ) (g ) (% ) w e ig h t (% ) > Silt/Clay Sand Granule < W e ig ht e xclude d org. m a tte W e ig ht include d org. m a tte S ke wne ss 1.16 Loss on ignitio 4.0 % Size (mm) Ku rto s is S tation: 1-b, Lab.re f.no.: Pe riod of analys is: RF-Miljølab. Grain size distribution by sieving Grain size distribution Station 1-b Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig ht (% ) > Silt/Clay Sand Granule < W e ight e xclude d org. m a tte W e ight include d org. m a tte Skewness 1.14 Loss on ignitio 3.5 % Ku rto s is Size (mm) S tasjon: 1-c, Lab.re f.nr.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Grain siz e distribution Station 1-c Size Phi Weight Weight Cumulative (m m ) (g ) (% ) w e ig h t (% ) > < W e ight e xclude d org. m a tte W e ight include d org. m a tte S ke wne ss 1.40 Loss on ignition 2.6 % Kurtosis Silt/Clay Sand Size (mm) Granule S tation: 1-d, Lab.re f.no.: Period of analysis: RF-Miljølab. G ra in s iz e d is trib u tio n b y s ie v in g Size Phi Weight Weight Cumulative (m m ) (g ) (% ) w e ig h t (% ) > < W e ight e xclude d org. m a W e ight include d org. m a t S k e w n e s s Lo s s o n ig n itio n 3.3 % Kurtos is 2.04 Silt/Clay Grain size distribution Station 1-d Sand Size (mm) Granule

49 S tation: 2, La b.re f.no.: Pe riod of ana lysis: RF-Miljøla b. Grain size distribution by sieving Grain size distribution Station 2 Size Phi Weight Weight Cumulative (m m ) (g ) (% ) w e ig h t (% ) > < W e ig ht e xclud e d org. m a W e ig ht include d o rg. m a t S ke wne ss 0.63 Loss on ignition 3.3 % Ku rto s is Silt/Clay Sand Size (mm) Granule S tation: 4, Lab.re f.no.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Grain size distribution Station 4 Size Phi Weight Weight Cumulative (m m ) (g ) (% ) w e ig h t (% ) > < S ilt/c la y Sand Granule W e ig h t e xc lu d e d o rg. m a tte r W e ig h t in c lu d e d o rg. m a tte r S ke wne ss 2.62 Loss on ignition 2.7 % Kurtosis 7.12 Size (mm) Stasjon: 5.re f.nr.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig ht (% ) Grain size distribution Station 5 > < We ight e xclude d org. ma tte We ight include d org. ma tte r S ilt/cla y Sand Granule S ke wne ss 0.81 Loss on ignition 3.8 % Kurto s is Size (mm) S tation: 6, Lab.re f.nr.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Grain siz e distribution Station 6 Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig ht (% ) > < We ight e xclude d org. ma tte We ight include d org. ma tte r Silt/Clay Sand Granule S ke wne ss 2.15 Loss on ignition 2.7 % Kurtosis 4.85 Size (mm)

50 S tasjon: 7, Lab.re f.nr.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig h t (% ) > < Weight excluded org. matte W e ig h t in c lu d e d o rg. m a tte S ke wne ss 1.33 Loss on ignitio 3.3 % Kurtosis Grain size distribution Station 7 Silt/Clay Sand Granule Size (mm) S tation: 8, re f.no.: Pe riod of analysis: RF-Miljølab. G ra in s ize d is trib u tio n b y s ie v in g Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig h t (% ) > < Silt/Clay Grain siz e distribution Station 8 Sand Granule W e ight e xclude d org. ma tte W e ight include d org. ma tte S ke wne s s 1.67 Loss on ignitio 3.4 % Kurtosis 2.64 Size (mm) S tation: 9-a, Lab.re f.no.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig ht (% ) > < Weight excluded org. matte We ight include d org. ma tte S ke wne ss 1.19 Loss on ignitio 4.2 % Ku rto s is Grain size distribution Station 9-a Silt/Clay Sand Granule Size (mm) S tation: 9-b, Lab.re f.no.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Size Phi Weight Weight Cumulative Grain size distribution Station 9-b (m m ) (g ) (% ) we ig h t (% ) > < Weight excluded org. matte Silt/Clay Sand Granule W e ig h t in c lu d e d o rg. m a tte S ke wne ss 0.88 Loss on ignition 4.0 % Ku rto s is Size (mm)

51 S tation: 9-c, re f.no.: Pe riod of analysis: RF-Miljølab. G ra in s iz e d is trib u tio n b y s ie v in g Size Phi Weight Weight Cumulative (m m ) (g ) (% ) w e ig h t (% ) Grain siz e distribution Station 9-c > < W e ig h t e xc lu d e d o rg. m a tte W e ight include d org. m a tte S k e w n e s s Lo s s o n ig n itio 4.1 % Ku rto s is Silt/Cla y Sand Size (mm) Granule S tation: 9-d, Lab.re f.no.: Pe riod of analys is: RF-Miljølab. Grain size distribution by sieving Grain size distribution Station 9-d Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig ht (% ) > Silt/Clay Sand Granule < W e ight e xclude d org. ma tte W e ig h t in c lu d e d o rg. m a tte S k e w n e s s Lo s s o n ig n itio 2.9 % Kurtosis 3.52 Size (mm) S tasjon: 10-a, Lab.re f.no.: Pe riod of analysis: RF-Miljølab. G ra in s iz e d is trib u tio n b y s ie v in g Grain size distribution Station 10-a Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig h t (% ) > Silt/Clay Sand Granule < Weight excluded org. matte W e ight include d org. ma tte S k e w n e s s Lo s s o n ig n itio 3.1 % Size (mm) Ku rto s is S tation: 10-b.re f.nr.: Pe riod of analysis : RF-Miljølab. Grain size distribution by sieving Grain size distribution Station 10-b Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig h t (% ) > < Silt/Clay Sand Granule W e ight e xclude d org. ma tte W e ight include d org. ma tte S ke wne ss 0.65 Loss on ignitio 3.3 % Ku rto s is Size (mm)

52 S tation: 10-c, Lab.re f.no.: Pe riod of ana lys is : R F-Miljøla b. Grain size distribution by sieving Grain size distribution Station 10-c Size Phi Weight Weight Cumulative (m m ) (g ) (% ) w e ig h t (% ) > < Silt/Clay Sand Granule W e ight e xclude d org. m a tte W e ight include d org. m a tte S ke wne ss 0.87 Loss on ig nition 3.1 % Kurtosis 0.43 Size (mm) S tas jon: 10-d, re f.nr.: Pe riod of analys is: RF-Miljølab. G ra in s iz e d is trib u tio n b y s ie v in g Grain size distribution Station 10-d Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig h t (% ) > < Silt/Clay Sand Granule W e ight e xclude d org. ma tte W e ig h t in c lu d e d o rg. m a tte S k e wn e s s Lo s s o n ig n itio 3.3 % Kurtos is 0.22 Size (mm) S tation: 10-e, Lab.re f.no.: Pe riod of analysis: RF-Miljølab. G ra in s iz e d is trib u tio n b y s ie v in g Grain siz e distribution Station 10-e Size Phi Weight Weight Cumulative (mm) (g) (%) weight (%) > < W e ight e xclude d org. ma tte W e ig h t in c lu d e d o rg. m a tte S k e w n e s s Lo s s o n ig n itio 2.5 % Kurto s is Silt/Clay Sand Size (mm) G ranule S tation: 10-f, re f.no.: Pe riod of analysis: RF-Miljølab. Grain size distribution by sieving Grain size distribution Station 10-f Size Phi Weight Weight Cumulative (m m ) (g ) (% ) we ig h t (% ) > < W e ight e xclude d org. m a tte W e ight include d org. m a tte Silt/Clay Sand Granule S ke wne ss 1.36 Loss on ignitio 2.4 % Kurtosis 1.18 Size (mm)

53 6.3 Metal analyses Metals in liver and muscle of fish sampled in May

54 - 46 -

55 - 47 -

56 6.3.2 Metals in liver and muscle of fish sampled in Novenber

57 - 49 -

58 - 50 -

59 6.3.3 Metals in bile of fish sampled in May

60 - 52 -

61 6.3.4 Metals in bile of fish sampled in November

62 - 54 -

63 6.4 Analyses of hydrocarbons in sediment and fish samples Recovery, fish samples and sediment samples Recovery, fish samples Sample name: FGH99003 FGH99008 FGH99009 FGH99014 FGH020 FGH99021 FGH99023 FGH99029 FGH99034 FGH99036 FGH99037 Sampling date: Recovery (ratio) 2-ring PAHs 0,42 0,46 0,40 0,21 0,39 0,40 0,42 0,42 0,40 0,44 0,42 3-ring PAHs 0,77 0,75 0,80 0,31 0,82 0,74 0,77 0,78 0,78 0,81 0,78 5-ring PAHs 0,80 0,81 0,79 0,23 0,79 0,77 0,78 0,79 0,78 0,81 0,78 Sample name: FGH99038 FGH99040 FGH99041 FGH99042 FGH99043 FGH99044 FGH99046 FGH99047 FGH99048 FGH99050 FGH99052 Sampling date: Recovery (ratio) 2-ring PAHs 0,45 0,43 0,41 0,48 0,45 0,50 0,38 0,42 0,39 0,40 0,37 3-ring PAHs 0,81 0,8 0,86 0,84 0,77 0,87 0,77 0,81 0,91 0,92 0,79 5-ring PAHs 0,80 0,8 0,78 0,79 0,84 0,80 0,79 0,82 0,62 0,69 0,56 Sample name: FGH99057 FGH99062 FGH99071 FGH99039 FGH99045 FGH99049 FGH99073 FGH99073 FGH99074 FGH99075 FGH99076 Sampling date: Recovery (ratio) 2-ring PAHs 0,41 0,42 0,41 0,45 0,42 0,49 0,39 0,37 0,41 0,37 0,35 3-ring PAHs 0,83 0,78 0,9 0,87 0,88 0,84 0,82 0,81 0,90 0,87 0,72 5-ring PAHs 0,74 0,71 0,74 0,82 0,77 0,78 0,77 0,71 0,60 0,71 0,73 Sample name: FGH99077 FGH99078 FGH99079 FGH99081 FGH99082 FGH99086 FGH99087 FGH99088 FGH99089 FGH99090 FGH99091 Sampling date: Recovery (ratio) 2-ring PAHs 0,51 0,34 0,36 0,35 0,40 0,37 0,40 0,26 0,35 0,35 0,36 3-ring PAHs 0,84 0,82 0,88 0,85 0,88 0,91 0,91 0,69 0,82 0,8 0,81 5-ring PAHs 0,70 0,64 0,74 0,82 0,81 0,74 0,73 0,72 0,66 0,65 0,68 Sample name: FGH99092 FGH99093 FGH99096 FGH99097 FGH FGH99016 Pooled, mai Pooled, nov Sampling date: Recovery (ratio) 2-ring PAHs 0,36 0,39 0,39 0,39 0,40 0,43 0,50 0,41 3-ring PAHs 0,78 0,85 0,86 0,87 0,79 0,81 0,89 0,76 5-ring PAHs 0,73 0,68 0,83 0,76 0,71 0,59 0,71 0,61 Recovery, sediment samples Sample name: St 1 start St 1 slutt St1 slutt re2 St1 slutt re3 St 2 slutt St 4 st St 5 start St 6 start St 7 start St 8 start Sampling date: Recovery (ratio) 2-ring PAHs 0,39 0,41 0,31 0,32 0,38 0,39 0,35 0,39 0,32 0,40 3-ring PAHs 0,75 0,75 0,70 0,74 0,74 0,78 0,74 0,79 0,76 0,80 5-ring PAHs 0,71 0,72 0,74 0,73 0,69 0,78 0,74 0,76 0,73 0,73 Sample name: St 9 st r1 St 9 st r2 St 9 st r3 ST 9 slutt St 10 st re1 ST 10 st r2 St 10 st r3 St 10 st r4 St 10 st r5 St 10 slutt Sampling date: Recovery (ratio) 2-ring PAHs 0,38 0,39 0,37 0,39 0,38 0,38 0,37 0,37 0,34 0,35 3-ring PAHs 0,75 0,73 0,73 0,77 0,74 0,76 0,70 0,76 0,69 0,69 5-ring PAHs 0,74 0,75 0,71 0,78 0,72 0,72 0,68 0,75 0,69 0,

64 6.4.2 Printout of a representative chromatogram of THC in sediment

65 6.4.3 GC/MS printouts of a representative analysis of fish muscle 26apr21ep is sample FGH99020, a typical example of analysis from fish. Total ion chromatogram (TIC):

66 m/z 128, naphthalene m/z 142, C1 naphthalenes m/z 156 C2 naphthalenes m/z 170, C3 naphthalenes

67 m/z 152, acenaphthylene m/z 154, acenaphthene m/z 166, fluorene RF Rogaland Research, Norway

68 m/z 178, phenanthrene and anthracene m/z 192, C1 phenanthrenes/anthracenes m/z 206, C2 phenanthrenes/anthracenes RF Rogaland Research, Norway

69 m/z 184, dibenzothiophene m/z 198, C1 dibenzothiophenes m/z 212, C2 dibenzothiophenes RF Rogaland Research, Norway

70 m/z 202, fluoranthene, pyrene RF Rogaland Research, Norway. m/z 212, deuterated fluoranthene and pyrene (internal standards)

71 m/z 228, benzo(a)anthracene, chrysene m/z 242, C1 chrysene m/z 256, C2 chrysenes RF Rogaland Research, Norway

72 m/z 252, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene m/z 276, indeno(1,2,3-c,d)pyrene, benzo(g,h,i)perylene m/z 278, dibenzo(a,h)anthracene

73 6.4.4 GC/MS printout of a representative sediment analysis 22mar19.ep ia sample Station 8-start, a typical example of sediment analysis Total ion chromatogram (TIC):

74 m/z 128, naphthalene m/z 142, C1 naphthalenes m/z 156 C2 naphthalenes m/z 170, C3 naphthalenes

75 m/z 152, acenaphthylene m/z 154, acenaphthene m/z 166, fluorene RF Rogaland Research, Norway

76 m/z 178, phenanthrene and anthracene m/z 192, C1 phenanthrenes/anthracenes m/z 206, C2 phenanthrenes/anthracenes RF Rogaland Research, Norway

77 m/z 184, dibenzothiophene m/z 198, C1 dibenzothiophenes m/z 212, C2 dibenzothiophenes RF Rogaland Research, Norway

78 m/z 202, fluoranthene, pyrene RF Rogaland Research, Norway. m/z 212, deuterated fluoranthene and pyrene (internal standards)

79 m/z 228, benzo(a)anthracene, chrysene m/z 242, C1 chrysene m/z 256, C2 chrysenes RF Rogaland Research, Norway

80 m/z 252, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene m/z 276, indeno(1,2,3-c,d)pyrene, benzo(g,h,i)perylene m/z 278, dibenzo(a,h)anthracene