Corrosion and disintegration of munition shells from the World Wars increase the risk that explosives are released into the marine environment, exposing a variety of organisms. Only few studies investigated contamination of fish with explosives in the field under environmental conditions. Here we present a comprehensive study on the contamination status of dab (Limanda limanda) from a munition dumpsite and from reference sites in the Baltic Sea. Bile of 236 dab from four different study sites, including a dumpsite for conventional munitions, was investigated and explosive compounds were detected by high performance liquid chromatography-mass spectrometry. Five explosive compounds were identified, including 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrolouene, and hexahydro-1,3,5-trinitro-1,3,5-triazine. 48% of the samples from the dumpsite contained at least one explosive compound. The results prove that toxic explosive compounds from a dumpsite in the Baltic Sea are accumulated by flatfish and may therefore pose a risk to fish health and human food safety.

Liver tumours in flatfish have been diagnosed using histopathology for decades in order to monitor the impacts of marine pollution in coastal and estuarine environments. This traditional method has been coupled with molecular analyses of tumours in the liver of the dab, Limanda limanda, in order to elucidate underpinning molecular level aetiology of such disease. A laser capture microdissection technique for molecular investigation of cancer has been applied in fish. The present study provides optimized steps for environmental sample utilisation: a procedure for field sample collection and handling; a method allowing reliable identification of lesions on frozen tissue sections; and, downstream molecular analyses of tumourigenesis markers (retinoblastoma gene) in laser microdissected samples. This approach facilitates the use of wild flatfish as a model of environmentally-induced tumourigenesis, and has wide applicability to any disease pathology for which the underpinning molecular aetiology is required.

In the framework of the ICON project, environmental genotoxicity and cytotoxicity levels were assessed in blood erythrocytes of dab (Limanda limanda) and haddock (Melanogrammus aeglefinus) collected at 25 stations in the North Sea and near the coast of Iceland in August–October 2008. Micronuclei, nuclear buds and bi-nucleated cells with nucleoplasmic bridges were assessed as environmental genotoxicity biomarkers, and the frequency of fragmented-apoptotic and bi-nucleated erythrocytes were assessed as environmental cytotoxicity biomarkers. The lowest frequencies of genotoxic and cytotoxic abnormalities were detected in fish from the Icelandic study stations. The highest frequencies of abnormalities were recorded in dab from the Dogger Bank and the German Bight, in haddock from the Egersund Bank and from an area off the Firth of Forth (North Sea). In fish from the Icelandic reference area, frequencies of genotoxicity and cytotoxicity responses were significantly lower than in fish from most areas of the North Sea.

Liver tumours in flatfish have been diagnosed using histopathology for decades to monitor the impacts of marine pollution. Here we describe the application of specific gene (retinoblastoma, Rb) profiling in laser capture micro-dissected samples, and a suppression subtractive hybridization (SSH) approach to isolate differentially expressed genes in hepatocellular adenoma (HCA) samples from dab, Limanda limanda.The Rb profiles from apparently normal and HCA micro-dissected samples of fish from the North Sea showed no significant difference, and genotypic heterogeneity within defined histological phenotypes was observed. In the SSH, sequences associated with cell signalling, cell cycle, gene expression regulation, protein transport and protein degradation were isolated. These included up-regulation of arrestin domain containing 3 (arrdc3), Rac-1 and tribbles, and down-regulation of ankyrin repeat/sterile alpha-motif domain-containing protein 1B-like (ANKS1B-like), c-fos, CDKN1B and RhoA-like sequences, previously implicated in mammalian HCA. This study offers new candidates involved in fish liver tumour development.

The metals cadmium, mercury and lead, and the ICES7 CB levels were analysed in the common dab Limanda limanda to investigate whether concentrations of these determinants are at levels above established OSPAR assessment criteria around England and Wales. The results indicate that CB118 is above the OSPAR derived EAC at 22 out of 29 sampled stations, with 15 showing significant reductions. Cadmium is increasing significantly at six stations with two sites in the North Sea breaching corresponding OSPAR EAC. Mean results for all other PCBs and metals stayed below the EAC thresholds, with Hg concentrations even falling below BAC levels at South Eddystone located in the western English Channel. This study shows that PCBs banned over 20years ago are still found at concentrations in fish liver tissues which may pose a health risk to both the individual fish and their predators.

The Seine Estuary is well known to be widely contaminated by organic pollutants and especially by polycyclic aromatic hydrocarbons (PAHs). Fish are known to metabolize PAHs, leading to different toxic effects at both cellular and sub-cellular levels. In this work, we studied the relationships between the 7-ethoxyresorufin-O-deethylase (EROD) activity in the liver, the level of DNA strand breaks in blood cells and the concentration of PAH metabolites in the bile of the sentinel flatfish species Limanda limanda. Muscle and liver samples were analysed for parent PAH levels. Female and male dabs of two size classes (juveniles and adults) were collected by trawling in two sites with different degrees of pollution during March and September 2005 and 2006. Significant effects of sex, age, site and season were demonstrated on EROD activity and the level of strand breaks. Parent PAH concentrations in dabs did not allow discriminating of the two sampling sites. However, for PAH metabolites, significant differences were observed with sites and seasons. Dabs collected at the mouth of the estuary appeared to be the most impacted when looking at the results obtained with the three selected markers. The significant correlations observed between the level of PAH metabolites and the level of DNA lesions showed the importance of a combined analysis of chemical and biochemical markers to correctly assess the contribution of chemical contamination to the toxic effects measured in situ in fish.

Background, Aim and Scope Polycyclic aromatic hydrocarbons (PAH) are important environmental contaminants which may lead to increased levels of neoplastic aberrations or tumours in fish liver. Therefore, monitoring of PAH and their effects are part of several international environmental programmes. The aim of the present field study was to investigate the concentrations of the PAH metabolites in fish bile, to elucidate spatial, seasonal and species differences as well as to discuss different strategies of normalisation with regard to environmental monitoring. Materials and Methods: PAH metabolites were determined in the bile fluid of dab (Limanda limanda) and flounder (Platichthys flesus) caught in the North Sea and Baltic Sea between 1997 and 2004. After enzymatic deconjugation, two metabolites were determined by means of HPLC. The limit of detection and the limit of quantification were calculated. The accuracy of the method was tested with a standard reference material. Results were referred to bile volume as well as to biliverdin. Results: The main metabolite, 1-hydroxypyrene, was determined in concentrations from <0.7 to 838 ng/ml in bile of dab (Limanda limanda) and flounder (Platichthys flesus) caught between 1997 and 2004. The values for 1-hydroxyphenanthrene in fish bile were considerably lower (<0.4 - 87 ng/ml). Significant differences in the 1-hydroxypyrene levels were found between summer and winter surveys as well as between the sampling sites in the data set from 2004 (383 dabs and 62 flounders): Highest levels of PAH contamination were found in dab from the German Bight and in flounder from the Baltic Sea. Discussion: Spatial differences in 1-hydroxypyrene concentrations between North Sea and Baltic Sea were discussed, as well as differences in relation to season, sex and species. Three parameters of normalisation (biliary protein, biliverdin and bile pigments) were discussed. Biliverdin was identified as a suitable parameter for the normalisation of PAH metabolites in field samples. Conclusions: Spatial differences in 1-hydroxypyrene concentrations of dab demonstrate the usefulness of PAH metabolites in fish bile as a monitoring parameter in marine regions. Significant differences in 1-hydroxypyrene concentrations were found between summer and winter sampling campaigns. This may be linked to an annual cycle of 1-hydroxyprene in dab. It is also possible that bile synthesis/release in dab differs between the seasons. There is no indication for a time trend from 1997 to 2004. Recommendations and Perspectives: It is recommended to relate PAH metabolites in fish bile to biliverdin concentrations. Although the concentrations are low in offshore regions and bile volumes are small, the method presented here allows one to measure PAH metabolites on an individual level which is a crucial prerequisite for meaningful monitoring studies.