Lionel Camus

PAH biomarker responses in polar cod (Boreogadus saida) exposed to benzo(a)pyrene

With expanding oil and gas activities into the Arctic region, there is a need to evaluate the induction capacity of polycyclic aromatic hydrocarbon (PAH) biomarkers on Arctic marine organisms and to test analytical methods that have been optimized for their temperate counterparts. Polar cod Boreogadus saida were injected intraperitoneally with cod liver oil (solvent control), 6.6+/-3.7, 85+/-48 or 378+/-190 microg kg(-1) wet weight of benzo(a)pyrene (B(a)P), or not injected (control), and liver and bile were sampled at 0 and 16 h and 1, 2, 4 and 7d. The mRNA expression of cytochrome P4501A1 (cyp1a1) and glutathione S-transferase (gst) genes showed a dose-dependent induction in the first 16 h following the injection and a return to basal levels after 4d. The aryl hydrocarbon receptor 2, however, showed no change in mRNA expression. The protein quantification of cytochrome P4501A (CYP1A), through Western blot analysis and the enzyme-linked immunosorbent assay (ELISA), presented similar but weaker and time-delayed responses (4-7d) compared to the gene (16 h to 2d). Ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) activities increased significantly at day 7 following the gene induction and increase in protein levels. Overall, these biomarkers showed dose-dependent but weak responses to B(a)P and low levels of bile metabolites. The mRNA expressions of oxidative stress genes, superoxide dismutases (sod(Cu/Zn) and sod(Mn)), catalase (cat) and glutathione peroxidase (gpx), were all up-regulated between 16 h and 2d of B(a)P exposure with cat (72-fold) and sod(Cu/Zn) (20-fold) giving the strongest responses in the highest dose. Finally, CAT protein level and enzyme activities showed less clear responses than the genes. The mRNA expression showed the earliest responses, followed by the protein levels. The enzymatic activities were the least sensitive and responded to the exposure after 7d. The study shows the induction capability of biomarkers in polar cod at very low bioavailable doses of B(a)P and provides new information on the selected biomarkers for use in oil monitoring in the Arctic.

Seasonal variation in biomarkers in blue mussel (Mytilus edulis), Icelandic scallop (Chlamys islandica) and Atlantic cod (Gadus morhua)—Implications for environmental monitoring in the Barents Sea

In the Barents Sea, the limited data on biological relevant indicators and their responses to various anthropogenic stressors have hindered the development of a consistent scientific basis for selecting indicator species and developing practical procedures for environmental monitoring. Accordingly, the main aim of the present study was to develop a common set of baseline values for contaminants and biomarkers in three species, and to identify their strengths and limitations in monitoring of the Barents Sea. Blue mussel (Mytilus edulis), Icelandic scallop (Chlamys islandica) and Atlantic cod (Gadus morhua) were sampled from a north Norwegian fjord in March, June, September and December 2010. Digestive glands from the bivalve species and liver from Atlantic cod were analysed for biomarkers of oxidative stress (catalase [CAT], glutathione peroxidase [GPX], glutathione-S-transferase activities [GST], lipid peroxidation as thiobarbituric reactive substances [TBARS] and total oxyradical scavenging capacity [TOSC]), biotransformation (ethoxyresorufine-O-deethylase activity [EROD]) and general stress (lysosomal membrane stability [LMS]). Concentrations of polycyclic aromatic hydrocarbons (PAHs) and metals in the bivalves and PAH metabolites in fish bile were quantified. Finally, energy reserves (total lipids, proteins and carbohydrates) and electron transport system (ETS) activity in the digestive gland of the bivalves and liver of Atlantic cod provided background information for reproductive cycle and general physiological status of the organisms. Blue mussel and Icelandic scallop showed very similar trends in biological cycle, biomarker expression and seasonality. Biomarker baselines in Atlantic cod showed weaker seasonal variability. However, important biological events may have been undetected due to the large time intervals between sampling occasions. Physiological biomarkers such as energy reserves and ETS activity were recommended as complementary parameters to the commonly used stress biomarkers, as they provided valuable information on the physiological status of the studied organisms. Interpretation of the seasonality in oxidative stress biomarkers was in general difficult but TOSC and lipid peroxidation were preferred over the antioxidant enzyme activities. This study is the first reporting seasonal baseline in these three species in a sub-Arctic location. Overall, the Icelandic scallop was considered the most adequate organism for environmental monitoring in the Barents Sea due to the interpretability of the biomarker data as well as its abundance, ease to handle and wide distribution from the southern Barents Sea to Svalbard.

Biomarker responses in polar cod (Boreogadus saida) exposed to the water soluble fraction of crude oil.

In order to mimic the biological effects of an oil spill in Arctic waters, we examined several types of biomarkers (genes, enzymes, metabolites, and DNA damage) in polar cod Boreogadus saida experimentally exposed to the water soluble fractions of crude oil. During 4 weeks of exposure, induction of the studied biomarkers exceeded baseline levels. The mRNA expression of the cytochrome P4501A1 (cyp1a1) gene was the most promising biomarker, with glutathione S-transferase (gst) as a suitable complement. The delayed ethoxyresorufin O-deethylase (EROD) and GST activities and their persistence following 2 weeks of depuration may allow detection of previous exposures in field samples. The composition of PAH metabolites in the bile indicated the bioavailability of different PAH size-classes. Although mRNA expressions of antioxidant defense genes were induced at start of the exposure, with the strongest responses from catalase and cytosolic superoxide dismutase, they were poor for oil monitoring purposes due to their very short response times. Significant DNA damage demonstrated genotoxicity even at low PAH concentrations (<15microgL(-1)) and was correlated with benzo(a)pyrene and pyrene metabolites in the bile.

Stability of lysosomal and cell membranes in haemocytes of the common mussel (Mytilus edulis): effect of low temperatures

Expanding industrial activities in the Arctic require an urgent assessment of the toxicity of chemicals at low temperatures. Organisms acclimatized to low temperature exhibit specific adaptations. For example, the amount of unsaturated lipids is increased to maintain the fluidity of the cell membranes. It has been hypothesized that such temperature-induced alterations in membrane lipid composition may affect the stability of lysosomal and cell membranes in the common mussel, Mytilus edulis, an organism exposed to seasonal temperature extremes. As mussels may be exposed to petroleum compounds along industrialized coastlines, we tested the combined effects of exposure to low temperature and the petroleum compound, phenanthrene, on haemocyte membrane stability. Test animals, acclimated to either 0 or 10 degrees C, were exposed to phenanthrene (0 = control or 500 micrograms l-1) and haemocytes were examined using the neutral red retention assay (lysosomal stability) and a fluorescence assay (cell membrane stability). At 0 degree C, lysosomal and cell membranes from uncontaminated mussels were destabilized compared with 10 degrees C (P = 0.0005). No significant effects (P > 0.05) of phenanthrene were detected at either temperature. Possible mechanisms underlying membrane destabilization include a weaker physical resistance of the membrane due to a higher amount of unsaturated lipids, a potentially higher level of reactive oxygen radicals at low temperature and the higher susceptibility of unsaturated lipids to oxidative stress. More work is required to better understand the consequences of this membrane destabilization at low temperature on the susceptibility of the organism to pollutants.

Unexpected Levels of Biological Activity during the Polar Night Offer New Perspectives on a Warming Arctic

The current understanding of Arctic ecosystems is deeply rooted in the classical view of a bottom-up controlled system with strong physical forcing and seasonality in primary-production regimes. Consequently, the Arctic polar night is commonly disregarded as a time of year when biological activities are reduced to a minimum due to a reduced food supply. Here, based upon a multidisciplinary ecosystem-scale study from the polar night at 79°N, we present an entirely different view. Instead of an ecosystem that has entered a resting state, we document a system with high activity levels and biological interactions across most trophic levels. In some habitats, biological diversity and presence of juvenile stages were elevated in winter months compared to the more productive and sunlit periods. Ultimately, our results suggest a different perspective regarding ecosystem function that will be of importance for future environmental management and decision making, especially at a time when Arctic regions are experiencing accelerated environmental change [1].

Biomarker responses in polar cod (Boreogadus saida) exposed to dietary crude oil.

Polar cod Boreogadus saida were exposed weekly to two doses of dietary crude oil for 4 weeks followed by 2 weeks of depuration. Administered doses corresponded on average to 4 and 9microgSigmaPAHsg(-1)fishweek(-1). Cytochrome P4501A1 (cyp1a1) and glutathione S-transferase (gst) mRNA expression, ethoxyresorufin O-deethylase (EROD) activity and metabolites in the bile showed strong and dose-dependent inductions at 2 and 4 weeks of exposure. Following 2 weeks depuration, mRNA expression of cyp1a1 and gst and PAH metabolites returned to basal levels while EROD activity and GST activity were still induced in the high oil treatment. The mRNA expressions of antioxidant defense genes (catalase, glutathione peroxidase and cytosolic and mitochondrial superoxide dismutase) did not change significantly during the experiment. Catalase activity was significantly depressed at week 2 in the high oil treatment. We conclude that the cyp1a1 mRNA expression, EROD activities and bile metabolites were the most reliable biomarkers of exposure while gst mRNA expression and GST activity were less sensitive and are considered only as complementary. Antioxidant defenses were poor biomarkers to assess effects of crude oil exposure in polar cod.

Seasonal baseline levels of physiological and biochemical parameters in polar cod (Boreogadus saida): Implications for environmental monitoring.

Seasonality of biomarker baseline levels were studied in polar cod (Boreogadus saida), caught in Kongsfjorden, Svalbard, in April, July, September and December, 2006-2007. Physiological parameters (condition factor, gonado- and hepato-somatic indexes, energy reserves, potential metabolic activity and antifreeze activity) in polar cod were used to interpret the seasonality of potential biomarkers. The highest levels of ethoxyresorufin-O-deethylase (EROD) activity occurred concomitantly with the highest potential metabolic activity in July due to e.g. intense feeding. During pre-spawning, EROD showed significant inhibition and gender differences. Hence, its potential use in environmental monitoring should imply gender differentiation at least during this period. Glutathione S-transferase and catalase activities were stable from April to September, but changed in December suggesting a link to low biological activity. Knowledge of the biomarker baseline levels and their seasonal trends in polar cod is essential for a trustworthy interpretation of forthcoming toxicity data and environmental monitoring in the Arctic.

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.

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.

Arctic versus temperate comparison of risk assessment metrics for 2-methyl-naphthalene.

Reliable risk assessment approaches for Arctic environments are requested to manage potential impacts associated with increased activities in Arctic regions. We performed toxicity tests on Arctic and temperate species exposed to the narcotic acting oil component, 2-methyl naphthalene. The experimental results were used to quantify concentration causing lethality to 50% of exposed individuals and no-effect concentration (individual level). For estimates at community level, the hazardous concentrations affecting 5% and 50% of the species were calculated from sensitivity distribution curves. These survival metrics were then used to elucidate whether temperate toxicity data used in risk assessment are sufficiently representative for the Arctic. Taking data uncertainty into consideration, we found no regional difference in tolerances to 2-methyl naphthalene either at the species level or at the community level. Hence these data support a conclusion that values of survival metrics for temperate regions are transferrable to the Arctic for the chemical 2-methyl naphthalene, as long as extrapolation techniques are properly applied and uncertainties are taken into consideration.