Endre Aas

PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure : a laboratory experiment with Atlantic cod

In order to perform environmental risk assessments with regard to oil contamination in the sea, it is important to obtain knowledge about threshold levels for possible adverse effects in marine organisms. With this objective in mind, selected biomarkers were studied in Atlantic cod (Gadus morhua) chronically exposed to mechanically dispersed crude oil. The fish were exposed for 30 days in a continuous flow system to nominal concentrations of 0.06, 0.25 and 1 ppm North Sea crude oil. Fish were sampled five times during the exposure period. In addition, the 1 ppm group and the control group were sampled 1 week after the end of exposure. Polyaromatic hydrocarbon (PAH) concentrations in the seawater were analysed regularly by direct fluorescence and, at one occasion, by gas chromatography with mass spectrographic detection (GC/MS) measurements. Liver samples were analysed for parent PAH levels by means of GC/MS measurements, and PAH metabolites in bile were analysed by means of fixed wavelength fluorescence. Cytochrome P450 induction in liver was estimated by ethoxyresorufin-O-deethylase (EROD) activity, and hepatic DNA adducts were analysed by the 32P-postlabelling assay. The parent PAH concentrations in liver showed peak levels 3 days after the start of exposure, followed by a reduction towards the end of the experiment. In contrast, the PAH metabolites in bile and EROD activity showed generally increasing levels throughout the whole exposure period, indicating an increased biotransformation efficiency. The level of DNA adducts in the 1 ppm group showed a stable increase during the entire exposure period. Only a slight, non-significant decrease in DNA adduct levels was observed after 7 days of recovery in clean water. Exposure-dependent responses were observed for all three biomarkers. The lowest nominal concentration of dispersed oil in water, 0.06 ppm, corresponded to a measured total PAH concentration in the water of 0.3 ppb. Atlantic cod exposed to this concentration showed increased levels of PAH metabolites in bile and a slight induction of CYP1A, as well as formation of DNA adducts when compared with control fish. Particularly noteworthy is the detection of DNA adducts at such a low exposure concentration of oil in water, which, to our knowledge, is a novel finding. These dose-response data may serve as useful contributions when assessing environmental risk with regard to marine oil pollution.

Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) exposed by caging to polluted sediments in Sørfjorden, Norway

Flounder (Platichthys flesus L.) and Atlantic cod (Gadus morhua L.) were subjected to caging at polluted sediments in a Norwegian fjord (Sorfjorden) for a period of 3 months. Three caging sites were located close to metal smelters, whereas a fourth site was located 30 km away as a reference. In sediment samples, polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and heavy metals were elevated at the innermost sites (1, 2 and 3) compared with the reference location (site 4). In fish, the biliary levels of fluorescent aromatic compounds (FACs) were elevated 5–20 fold in both species at the polluted sites. A two-fold difference in heavy metal levels was observed in cod (site 2 vs. 4), whereas no differences were seen in flounder. Pesticides bioaccumulated in a diffuse manner at all sites. In flounder at the innermost sites, plasma aspartate aminotransferase (AST) and hepatic cytochrome P450 1A (CYP1A) dependent 7-ethoxyresorufin O-deethylase (EROD) activity were elevated 4–5 and 5–10 fold, respectively, compared with the reference site. Both of these biomarkers were significantly correlated with FACs levels. For other biomarkers, the site effect was more marginal. The biomarkers seemed in general more responsive in flounder than in cod. The present study demonstrates biomarker measurements in caged fish as a promising approach for evaluating accumulation and effects of pollutants in marine teleosts.

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

Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.Fixed wavelength fluorescence (FF) of bile has been evaluated as a monitoring tool for the screening of polyaromatic hydrocarbon (PAH) contamination in fish. The methodology was studied through laboratory and field experiments with Atlantic cod (Gadus morhua L.) and flounder (Platichthys flesus L.) exposed to various forms of PAH contamination. The present study demonstrates the ability of FF screening to discriminate between 2-, 4- and 5-ring PAH metabolites by using the wavelength pairs 290/335 nm, 341/383 nm and 380/430 nm, respectively. In general, the degree of fluorescence interference between these metabolite groups appears to be low. Dose- and time-response patterns of the FF signals were shown to give a good reflection of the PAH exposure. Further, the necessity of an appropriate dilution of bile samples prior to fluorescence measurements is demonstrated by a study of inner filter effect. Normally a dilution of 1000-2000-fold is necessary. Individual differences in the bile density, e.g. measured as the concentration of the bile pigment biliverdin, have to be allowed for when applying the FF method. However, it is shown that normalizing the FF signals to biliverdin concentrations on an individual basis added extra error to the data set. The simple, rapid and cost-effective FF method is found to be well suited for screening fish for PAH contamination.

Evidence of uptake, biotransformation and DNA binding of polyaromatic hydrocarbons in Atlantic cod and corkwing wrasse caught in the vicinity of an aluminium works.

Feral Atlantic cod (Gadus morhua) and corkwing wrasse (Symphodus melops) were investigated for polyaromatic hydrocarbon (PAH) contamination in the Karmsund strait, western Norway. This strait is highly contaminated with PAHs, and a main source is the chronic release of gas-scrubbing effluents from a local aluminium works. In both species, the level of biliary PAH metabolites and hepatic DNA adducts were higher in fish collected near the aluminium works. Interestingly, a significantly higher level of both biliary PAH metabolites and hepatic DNA adducts was found in corkwing wrasse as compared to cod, indicating a higher potential for genotoxic effects in this species. Hepatic cytochrome P4501A (CYP1A) in cod estimated by ethoxyresorufin-O-deethylase and an immunoassay technique (ELISA), seemed to be weakly induced at the contaminated sites. At the most contaminated site, skin ulcers and fin erosion were detected in about 70 and 45% of the cods, respectively. The data demonstrated that both cod and corkwing wrasse may be suitable target species for PAH pollution monitoring.

PAH in fish bile detected by fixed wavelength fluorescence

Abstract Fixed wavelength fluorescence (FF) for detection of polyaromatic hydrocarbon (PAH) metabolites in fish bile has been studied, and is presented through a laboratory and a field experiment. The method, which can be regarded as a biomarker of exposure to PAH, has proved to be a simple, rapid and cost effective method for monitoring PAH contamination.

PAH metabolites in bile and EROD activity in North Sea fish

Abstract Atlantic cod ( Gadus morhua ), haddock ( Melanogrammus aeglefinus ), and long rough dab ( Hippoglossoides platessoides ) were sampled from areas with oil production in the North Sea. Egersundbanken, an area without oil production, was used as a reference site. Ethoxyresorufin- O -deethylase (EROD) activity was measured in liver to estimate cytochrome P4501A levels. Bile was analysed by fixed wavelength fluorescence (FF) detection for polyaromatic hydrocarbon (PAH) metabolites. No increased levels of EROD activity were detected in fish caught in the vicinity of oil production fields, when compared to the reference site, Egersundbanken. No differences in levels of PAH metabolites in bile were detected between the stations for any of the species. Therefore, contamination from 2–5 ring PAH compounds in the vicinity of oil fields in the North Sea may not be of great concern for these fish species.

Ethoxyresorufin-O-deethylase activity and fixed wavelength fluorescence detection of PAHs metabolites in bile in turbot (Scophthalmus maximus L.) exposed to a dispersed topped crude oil in a continuous flow system

Long term effects of sublethal concentrations of oil on the marine environment have become of general concern. Cytochrome P4501A activity (EROD) in liver and fixed wavelength fluorescence detection of PAHs metabolites (FF) have in this study been used as biomarkers for dispersed oil exposure on a long term period of juvenile turbot (Scophthalmus maximus L.). A Continuous Flow System was used to carry out the study. The fish were continuously exposed to 0.125, 0.5 or 2.0 mg litre−1 dispersed topped crude oil for 6, 15, 24 h, 4 and 21 days followed by a 9 days recovery period in clean seawater. No induction of the cytochrome P4501A was measured. A maximum level in bile metabolites (4- to 5-fold) was recorded after 24 h of exposure revealing thereby a detoxification process, but a decline occurred from day 4 to day 21. This study demonstrated that FF detection of PAHs metabolites in bile could be a more sensitive biomarker than EROD activity in a long term exposure to sublethal concentration of oil.

Bioavailability of PAH in effluent water from an aluminium works evaluated by transplant caging and biliary fluorescence measurements of Atlantic cod (Gadus morhua L.)

Abstract Fixed wavelength fluorescence (FF) of bile was used as a biomarker for PAH exposure in fish caged adjacent to an aluminium works. After 30 days of caging, a 20–50 fold increase of pyrene fluorescence was found in groups caged adjacent to the works as compared to two control groups. The PAH uptake occurred mainly from suspended particles rather than from PAH contaminated sediments at the caging sites. Our results demonstrates the combined use of fish caging and bile-fluorescence measurements as a powerful field model for the monitoring of coastal waters subjected to PAH pollution.

DNA adduct levels in fish from pristine areas are not detectable or low when analysed using the nuclease P1 version of the 32P-postlabelling technique.

In order to understand and apply DNA adduct formation in fish liver as a biomarker for aquatic pollution, information concerning the natural background levels in non-contaminated organisms, caused by endogenous compounds, is of fundamental importance. In this study, DNA adducts were analysed in liver of 11 fish species from arctic and sub-arctic areas in the northern Atlantic using the nuclease P1 version of the 32P-postlabelling technique. The collected fish were assumed not to have been influenced by anthropogenic pollution apart from possible long-range transported pollutants. As polycyclic aromatic hydrocarbons (PAHs) are thought to be fundamental in forming the type of DNA adducts detected by the method used, biliary PAH metabolite levels were measured in a selection of the investigated species. In all investigated individuals, the levels of PAH metabolites were undetectable. Controlled on-site exposure experiments with benzo[a]pyrene (polar cod) and laboratory experiments with crude oil (polar cod and Atlantic cod) were conducted. DNA adducts were formed in both these species. The field-sampled fish showed undetectable levels of DNA adducts or levels just above the detection limit. The present study supports the assumption that when DNA adducts are detected by the nuclease P1 version of the 32P-postlabelling method in fish liver, it can be interpreted as DNA damage caused by pollutants.

The Arctic is no longer put on ice: evaluation of Polar cod (Boreogadus saida) as a monitoring species of oil pollution in cold waters.

The withdrawing Arctic ice edge will facilitate future sea transport and exploration activities in the area, which calls for the establishment of relevant cold water monitoring species. The present study presents first results of field baseline levels for core oil pollution biomarkers in Polar cod (Boreogadussaida) sampled from pristine, Arctic waters. Furthermore, biomarker response levels were characterized in controlled laboratory exposure experiments running over 2 weeks. Fish exposed to a simulated petrogenic spill (1ppm dispersed, crude oil) exhibited elevated hepatic EROD activity, bile PAH-metabolites, and hepatic DNA-adducts, whereas male individuals exposed to simulated produced water (30ppb nonylphenol) exhibited a strong induction of plasma vitellogenin. In conclusion, the results demonstrated low and robust biomarker baseline levels that were clearly different from exposure responses. In combination with its high abundance and circumpolar distribution, the Polar cod seems well qualified for oil pollution monitoring in Arctic waters.