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The presence of genotoxic and potentially carcinogenic chemical contaminants in the estuarine and coastal marine environment is well documented. In this study, European flounder (Platichthys flesus) sampled from eight UK estuaries were analysed for hepatic DNA adducts, using the -postlabelling assay and liver histopathology as part of an on going survey to establish the health status of UK estuaries. Fish were collected from the estuaries Tyne, Mersey, Thames, Alde (reference site), Belfast, Forth, Clyde and Southampton. At the majority of contaminated sites (Southampton, Thames, Clyde, Tyne and Mersey) the predominant DNA adduct profile consisted of diagonal radioactive zones (DRZs). In contrast, flounder collected from the Forth, Alde and Belfast lacked DRZs with only background levels of DNA damage being observed. Statistically significant differences were observed between several of the sites sampled with the hepatic DNA adduct levels detected in flounder from Southampton, Thames and Clyde statistically elevated (P < 0.05) over those levels detected at the Tyne (Southampton and Thames only), Forth, Alde and Belfast. Histological analysis of these samples revealed a range of lesions including foci of cellular alteration, hepatocellular fibrillar inclusions, nuclear pleomorphisms along with non-toxicopathic changes/alterations, such as those associated with cell turnover (apoptosis, necrosis, regeneration) and immune-related functions (melanomacrophage aggregates, inflammation). Although it is difficult to associate higher prevalence of these lesion types with specific sites, generally, the lowest prevalence was seen in flounder captured from the Alde estuary, with higher prevalence (particularly of melanomacrophage aggregates, inflammation and necrotic foci) seen in fish from the contaminated sites.

Numerous studies focus on biological roles of the TP53 tumor suppressor gene in mammals but little is known about the actual function of TP53 in lower vertebrates. In this study, we used an in vivo functional assay in yeast to address the transactivation capacity of the flounder TP53 protein. We showed that the flounder TP53 acts as a sequence-specific transcription factor which is able to transactivate various human promoters containing a p53-responsive element (RE). This transcriptional activity was completely abrogated in the Val147Glu TP53 mutant previously identified in two flounder hepatic hyperplasia. In addition, we showed that the wild-type (wt) flounder TP53 but not the Val147Glu mutant inhibits cell growth when expressed in yeast. We finally reported that transcription regulation and growth inhibition by the wild-type flounder TP53 is temperature-dependent. The flounder TP53 optimal temperature appeared lower than those reported for the Xenopus and human homologues.

As a first stage in developing a DNA array-based approach to investigating the effects of pollutants on an environmentally relevant European fish species, we have constructed a 160-gene custom microarray for European flounder. Degenerate primers were used to amplify 110 different fragments of stress-related and other genes from European flounder cDNA and genomic DNA. Additionally, 22 fragments were obtained by suppressive subtractive hybridisation (SSH). These fragments were cloned and sequenced, then, with additional control genes, used to create a cDNA microarray for flounder. After optimisation of the arraying process, hepatic mRNA was isolated from flounder caught in the polluted Tyne and relatively unpolluted Alde estuaries. Fluorescent cDNA probes were synthesised from the mRNA and used in dual-colour hybridisations to the microarray. A number of transcripts were differentially expressed between Tyne and Alde female flounder but these changes were not significant, due to high inter-individual variation. However, in comparisons between Tyne and Alde male flounder, 11 transcripts were found to significantly differ in expression (P B/0.05). Seven transcripts were more highly expressed in the Tyne male fish (CYP1A, UDPGT, a-2HS-glycoprotein, dihydropyrimidine dehydrogenase, Cu/Zn SOD, aldehyde dehydrogenase and paraoxonase). Four transcripts (Elongation factor 1 (EF1), EF2, Int-6 and complement component C3) were found to be significantly less abundant in the Tyne male fish. Selected genes were assayed by real-time PCR, then normalised to a-tubulin. These assays confirmed the significance of the array results for CYP1A, UDPGT and EF1, but not for Cu/Zn SOD. This study provides a link between traditional single-gene biomarker studies and the emerging field of eco-toxicogenomics, demonstrating the utility of microarray studies on environmentally sampled, non-model organisms. # 2003 Elsevier B.V. All rights reserved.

From 1995 to 2000 biological effects were studied in liver of flounder (Platichtys flesus L.) from the German Bight. During the study period deleterious consequences of acute discharges of DDT and PCBs in early spring 1996 and after 1998 due to remobilization of contaminants from riverbed deepening of the River Elbe became evident. As core biomarker which reflects toxically induced liver pathologies and integrates effects of various classes of pollutants we measured the integrity of lysosomal membranes in individual flounder liver. During the study period, twice statistically significant disturbances of lysosomal function was detected in fish from the River Elbe: in summer 1996 and in spring 1999. Yet, the detrimental contaminant effects were not only restricted to individuals from the Elbe but expanded to those flounder inhabiting formerly less polluted reference areas. In contrast to flounder of the Elbe, their ability to recover from the lysosomal disorders were limited. While in autumn 2000 Elbe individuals showed clear signs of recovery, those fish caught in areas more distant to the source of toxicant input still maintained significantly decreased lysosomal membrane integrity. It can be speculated that fish populations which are not continuously exposed to chronic anthropogenic stress may have a lower potential or need a longer period to recover from the effects of pollution.

DNA from five tumors, three other-injured livers and two normal liver tissue samples from the European flounder were analyzed for mutations in exons 5-8 of the tumor suppressor gene p53 and at codons 12, 13 and 61 of Ha- and Ki-ras proto-oncogenes. No tumor-specific mutations were identified by direct sequencing and single-strand conformation polymorphism of these genes. A number of silent polymorphisms were noted in p53. In addition to a need for more extensive analyses of flounder liver tumor samples for ras or p53 mutations, other cancer-related genes should be investigated.

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.

We have applied a genotypic mutation detection system (the Restriction Site Mutation (RSM) assay) to detect mutations in the marine teleost flounder (Platichthys flesus). The aim of this study was to evaluate this species as an environmental indicator of genotoxic exposure. We have used the model genotoxin benzo[a]pyrene (B[a]P) to determine the limits of mutation detection in the p53 gene of flounder liver DNA. This study has revealed two important findings. Firstly, we were able to demonstrate that a polymorphism exists in the TaqI restriction site of exon 8 of the flounder p53 gene at codon 243. This polymorphic allele was present as a heterozygote at a mean frequency of 15%, whereas 85% carried the homozygous wild type sequence. Secondly, we established that B[a]P treatment resulted in specific mutational events at the adenine base of the same TaqI site, contrasting previous reports stating that there was a guanine preference for this chemical in mammalian DNA. This difference in mutation specificity may possibly be accounted for by sequence specific factors or by species differences in metabolic activation and/or DNA repair and are worthy of further study.

European flounder (Platichthys flesus) has shown an increased prevalence of liver tumors and lymphocystis disease (a viral infection) that correlated with pollution in field research in Dutch coastal and estuarine waters. Semi-field or mesocosm experiments confirmed the supposed causality. Although these types of research are highly relevant for the feral population, laboratory experiments are necessary to establish causal relationships between specific chemical pollutants and disease. Therefore, the effects on flounder of some of the potentially causative chemicals such as benzo[a]pyrene (BaP), dimethyl-benz[a]anthracene (DMBA), 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5 pentachlorobiphenyl (PCB-126), and bis(tri-n-butyltin)oxide (TBTO) were examined in several laboratory experiments. These effects were evaluated using general toxicological parameters and histopathology. For immune function assessment, attempts to develop an infection model with the lymphocystis virus were made, but appeared unsuccessful and immune function tests are not fully operational at the moment. Flounder has been successfully maintained and exposed to toxic substances in captivity in our laboratory. Short-term aqueous exposure to high levels of BaP or DMBA did not induce marked effects under our experimental conditions. Results of oral exposure of flounder to low levels of TCDD, PCB-126 or harbor sludge extract show significant induction of cytochrome P4501A (CYP1A) in hepatocytes. Oral exposure to high levels of TCDD or PCB-126 also significantly induced CYP1A immunoreactivity in epithelium in mesonephros and digestive tract and in endothelium in several organs. Remarkable was the induction of CYP1A in a distinct population of mononuclear cells in the mesonephros. Moreover, oral exposure to TCDD resulted in an increased mitotic activity and an increase of the hepatosomatic index in the 20 and 500 microgram TCDD/kg group respectively. Therefore, exposure to TCDD and related substances may promote the development of liver tumors in the field. Exposure to PCB-126 also significantly reduced the relative thymus volume, but other results indicate that flounder is relatively insensitive to this type of chemicals. Short-term aqueous exposure of flounder to TBTO, in concentrations that were in the same order of magnitude as upper TBT levels measured in the field, caused mortality after 7-12 days associated with gill lesions, and induced reduction of the non-specific resistance and decrease of the relative thymus volume. From these results we therefore conclude that TBTO might play a causal role in, for instance, increased prevalence of lymphocystis virus infections in the field

A number of flounders dwelling in highly contaminated coastal areas of Northern Europe develop liver tumours. In order to increase our understanding of the molecular pathogenesis of these sporadic tumours, we examined p53 mutations in eleven hyperplasia and six adenoma. p53 introns 4 to 8 were first sequenced to allow individual amplification of exons 5 to 8. DNA extracted from formalin-fixed livers was amplified and PCR products were directly sequenced. Two major results were obtained. (i) Flounders from different geographical areas displayed a high rate of sequence variation. Base substitutions were identified in both tumour and normal tissues and thus may be considered as polymorphic variations in individuals. (ii) One mutation was detected in two hyperplastic foci from the same flounder. This mutation was a T:A to A:T transversion at codon 147, resulting in the replacement of valine for glutamic acid. This residue took place in the L2 loop of the DNA binding surface. Its substitution by an hydrophilic and charged residue could thus impair p53 (protein) biological activity.

Information on occurrence and feeding ecology of cod (Gadus morhua), flounder (Platichtys Jesus), anadromous brown trout (Salmo trutta), and epipelagic fish in the Grenland fjords (SE Norway) is reviewed. Emphasis in previous studies have been on long-term trends in the abundance of juvenile demersal fish, and on the abundance of pelagic fishes, primarily sprat (Sprattus sprattus). The knowledge of distribution patterns and migrations of cod, flounder, and trout is incomplete, and only a single limited study of juvenile cod feeding habits have been carried out. New site-specific information is required as basis for the analyses of trophic transfer of contaminants in this heavily polluted fjord system. NORSK SAMMENDRAG: Informasjon om forekomst og fødevalg hos torsk, skrubbe, sjøørret og pelagisk fisk i Grenlandsfjordene i Telemark er oppsummert og vurdert. Hovedvekten i tidligere undersøkelser av fisk har vært lagt på analyser av langtidstrender i forekomst av fiskeyngel, samt mengdemåling av pelagisk fisk, spesielt brisling. Kunnskapen om fordeling av torsk, skrubbe og sjøørret er mangelfull, og kun ett begrenset arbeid dreier seg om fødevalg (til nylig bunnslått torskeyngel). For å analysere flyt av miljøgifter i næringsnettene i dette tungt forurensingsbelastede området, er det behov for betydelig ny kunnskap om både utbredelse, migrasjon og fødevalg til alle de nevnte artene.

The Tyne Estuary (North East England) is known to contain elevated levels of polycyclic aromatic hydrocarbons (PAH), compared with other less industrialised English waterways. Previous studies suggest that such contamination is responsible for the toxicity detected in invertebrate bioassays conducted on water and sediment samples collected from the Tyne. Here we present data from a biomonitoring study using hepatic DNA adducts (32P-postlabelling assay) and bile metabolites (synchronous fluorescence spectrometry) to investigate genotoxic exposure in flounder (Platichthys flesus) collected from three sites (Scotswood, Newcastle and Redheugh) along the Tyne Estuary. Flounder were also collected from a clean reference site, the Alde Estuary. Levels of bile metabolites (microgram kg-1 wet weight 1-OH pyrene equivalents) were elevated in flounder caught from the Tyne (Scotswood = 22,247 +/- 3408; Newcastle = 14,572 +/- 1888; Redheugh = 21,872 +/- 2935) compared with those collected from the Alde (632 +/- 56). The levels of DNA adducts (adducted nucleotides per 10(8) normal nucleotides) were also elevated in Tyne flounder (Scotswood = 24.6 +/- 3.2; Newcastle = 34.4 +/- 3.7; Redheugh = 27.6 +/- 6.3) compared with fish collected from the Alde (10.1 +/- 4.8), suggesting that a proportion of the bioavailable PAH was being converted into genotoxic metabolites. All DNA adduct profiles in flounder collected from the Tyne consisted of diagonal radioactive zones of radiolabelled adducts, which were not present in fish sampled from the Alde. The in vivo dosing of flounder with benzo[a]pyrene (BaP) to produced DNA adducts in similar chromatographic positions to the diagonal radioactive zones in the Tyne caught flounder are also described.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.

The integration of population genetics in ecotoxicology may be pertinent i) to detect possible differential physiological capacities or performances between individuals carrying particular alleles or genotypes, and ii) to explain their potential resistant or sensitive characters, sometimes resulting in increasing or decreasing frequencies in contaminated systems.1 Different studies were conducted on the genetic and physiological responses of the European flounder (Platichthys flesus) to pollution stress, especially in estuaries along the Atlantic coast of France.2,3 The authors highlighted that: i) particular alleles and genotypes (allozyme data) may be likely influenced (indirectly or directly) by chemical stress; ii) fish carrying these alleles or genotypes displayed higher DNA integrity over the whole contaminated populations. In addition, relationships between genotypes and phenotypes highlighted specific trends related to pollutant estuarine characteristics. In a more recent study,4 new DNA markers were explored in P. flesus; cDNA and genomic sequences of the genes encoding the glyceraldehyde-3-phosphate-deshydrogenase (GAPDH), the cytosolic creatine kinase(CK), the prostaglandin D synthase (PGDS) and the betaine homocysteine methyl-transferase (BHMT) were characterized.4 The tumor suppressor p53 gene was also considered in this study, the gene structure being already described. The exploration of the genetic polymorphism was carried out on several flounder populations along the French coast. The frequency of the p53-A allele was shown to be higher in contaminated populations (collected in the Gironde, Loire, Seine and Vilaine estuaries) compared to a reference site (Ster estuary), this increase ranging from 12.4% to 13.7%.4 In the Vilaine estuary, mean increases of 10% in BHMT-A and PGDS-A allele frequencies were also observed compared to the Ster estuary.4 Globally, the analysis of genetic diversity within populations and genetic differentiation suggested that the patterns of genetic structuration detected for flounders were probably more linked to local selection acting on the candidate genes than to the consequence of possible isolations by distance, or different histories of colonization.4 In the present report, new investigations were conducted on flounder populations stemming from environmentally contrasted estuaries. Our main objective was to confirm the possible selective effects of the contaminant on the candidate genes (GAPDH, CK, PGDS, BHMT, p53) by exploring the couplings between genotypes and phenotypes in the field. Journal of Xenobiotics 2013; volume 3(s1):e14

The inductive response of metallothionein (MT) synthesis to heavy-metal exposure in many Phyla is well documented; however, before this response can be used and interpreted as an environmental biomarker of exposure, the kinetics and dose responses must be known. We have carried out a series of experiments where we have studied these parameters of MT mRNA and MT protein induction in different tissues (liver, kidney, gills) of European flounder after intraperitoneal (ip) injection of Cd (1–320 μg/kg), Cu (25–200 μg/kg), Hg (235–100 μg/kg) and Zn (1–20 mg/kg). Basal hepatic levels of 320 μg MT/g wet tissue for the wild caught flounder used were five times higher than in fish from a pristine area and 10–20 times higher than in plaice or turbot obtained from pristine areas. Transient increases in renal MT mRNA levels were only observed 4–10 days post injection (p.i.) of Cu or Hg and there were no significant increases in MT protein levels in any tissue. Doses of >5 mg/kg Zn induced MT mRNA levels three- to four-fold in all tissues 2–4 days p.i. but only produced a transient elevation in MT levels at 4 days p.i. There was no measurable response to low doses of Cd, doses of >80 μg/kg Cd had no significant effect on hepatic MT levels but caused a significant elevation in gill and kidney levels 10 and 21 days p.i. MT mRNA levels in gill and kidney were induced four- to five-fold 24 h p.i. of Cd (and remained elevated for >10 days) whilst a response was only observed in liver (two-fold) 4 and 10 days p.i. These laboratory results, obtained with a wild population exhibiting moderately elevated basal MT levels indicate that the response to metal exposure is too small and too highly dependent upon the metal and exposure time to provide a robust biomarker of contaminant effect. This supports our previous observation of analyses of flounders from the southern North Sea and Dutch estuaries and would indicate that caging studies with ‘ultra-clean’ animals would be required for any monitoring studies.

In the small intestines of flounders adapted to sea-water or to fresh water, intracellular chloride activity is maintained above equilibrium activity. In sea-water adapted animals this accumulation is inhibited by piretanide whereas fresh-water adapted animals are insensitive to the drug. This indicates different chloride accumulation mechanisms.

The statistical procedure of analysing for trends over time in contaminant concentrations in fish provided by the International Council for the Exploration of the Sea (ICES) 1. is univariate and 2. analyses data on only one species at a time from a given sample area, even if data on more than one species are available. In this study temporal variations in contaminants in Atlantic cod ( Gadus morhua ) and European flounder ( Platichthys flesus ) from the Belgian coast are examined at the multispecies level rather than the single species level. The ICES univariate procedure 1. has been extended to the analysis of combined data for the two (or more) species and 2. is supplemented by the multivariate analysis of covariance procedure. Not only did the two species respond differently to variations in contaminant levels with time but even within a species contaminant levels did not vary in the same direction.

1. Everted intestinal sacs prepared from Platichthys flesus adapted to sea water (SW) have higher rates of salt and water transport than did sacs prepared from fresh water adapted (FW) fish. 2. Intestines mounted in Ussing chambers gave stable open-circuit voltages and short-circuit currents after pre-incubation for 20--30 min of --1.91 +/- 0.14 (14) mV and --45.0 +/- 5.1 (14) muA/cm2, SW fish, and --1.24 +/- 0.14 (11) mV and --18.2 +/- 3.6 (11) muA/cm2, FW fish. 3. Isotope flux measurements indicated a net Na transport of 21.5 +/- 4.1 (11) neq/cm2-min in SW fish intestines, but no significant Na transport in FW fish (7.6 +/- 7.6 (9) new/cm2-min). Both preparations showed an active Cl transport, 26.6 +/- 6.1 (12) neq/cm2-min for SW and 18.5 +/- 9.7 (17) neq/cm2-min for FW fish. 4. No significant difference in the uptake of Na or Cl across the mucosa was observed between FW and SW fish. The uptake of both ions showed some saturation at high concentrations. 5. Interactions between Na and Cl uptake were small, although Cl uptake was significantly higher in Na-free solutions. 6. It is concluded that electrogenic Cl transport may be the dominant mechanism for water and salt transport in flounder intestine, and that adaptation to a saline environment involves regulation of pumping rather than Na or Cl entry across the mucosal membrane.

Lymphocystis disease is a well-known viral infection of teleost fish species. It is characterized by conspicuous disease signs (Fig. 1A): small nodules which are located mostly on the body and fins. Occasionally, lymphocystis nodules occur in or on the surface of the body cavity and the internal organs as well as on the gills. Each nodule represents one single hypertrophied cell which may reach a size up to 2 mm in diameter and a volume of about a million-fold that of the normal cell, for example in European flounder (Platichthys flesus). There are infected fish with just one lymphocystis cell but in most cases several hypertrophied cells occur singly or grouped together in raspberry-like clusters with a tumorous appearance. Therefore, these signs are considered as “pseudo” tumors.

The spermatologic characteristics of European flounder (Platichthys flesus luscus) were determined. Flounder were collected during the spawning season and sperm of eight males was extracted by abdominal massage. Body weight and total length of the flounder were measured, volume, motility, duration of motility, spermatocrit, density, and pH of the sperm were determined, and correlations between the spermatologic characteristics and weight and length were investigated. Mean values were 0.7±0.16 ml for sperm volume, 87.5±3.66% for motility, 22.0±1.49 min for duration of motility, 94.0±1.22% for spermatocrit, 2.7±0.16 × 109/ml for density, and 6.9±0.05 for pH. Body length and sperm volume had positive correlations with body weight (p

Summary Instantaneous growth rates of 0-group European flounder, Platichthys flesus, obtained from two 9-day enclosure experiments were used to assess the relative quality of three eelgrass and three bare sand habitats in the Bay of Aarhus, Denmark. Measurements of temperature, dissolved oxygen, salinity, sediment structure and biomass of benthic macrofauna were made during the experiments. Gut content analysis was performed after retrieval of the fish. Growth rates of flounder at two bare sand sites were higher or equal to growth rates at the three eelgrass sites, and at a muddy bare sand site in both experiments. The variation in gut fullness of flounder between vegetated and bare sand sites followed the pattern observed in growth rates, but diverged from the spatial variation in prey abundance, which indicates that there were differences in the feeding efficiency between sites. Overall, habitat complexity, i.e. the presence or absence of vegetation seems to be the most important factor determining the growth rates and, by corollary, habitat quality for juvenile European flounder in the Bay of Aarhus.

The present study aimed at measuring the levels of legacy and emerging contaminants in fillet samples from four demersal fish caught in two fishing sites from Southern Norway, in order to assess possible implications for food safety. Levels of organochlorine compounds (OCs), organophosphate pesticides (OPs), polychlorinated biphenyls (PCBs), polybromodiphenyl ethers (PBDE), per- and polyfluoroalkyl substances (PFASs), and polycyclic aromatic hydrocarbons (PAHs) were measured in fillet from Atlantic cod (Gadus morhua), European plaice (Pleuronectes platessa), lemon sole (Microstomus kitt), and European flounder (Platichthys flesus) specimens. A negligible contamination by all the investigated chemicals was noted in both the fishing sites, as very low levels of OCs, PCBs, and PFASs were noted in a limited number of individuals for each species. Considering the levels of contaminants measured in fillets of the four demersal fish species, negligible risk for human health for Norwegian consumers can be supposed.