Jan Fredrik Børseth

Biomarker responses and PAH uptake in Mya truncata following exposure to oil-contaminated sediment in an Arctic fjord (Svalbard).

Expanding industrial activity (notably oil and gas exploration) in the Arctic requires assessment of the potential impact of chemicals on marine organisms living in seawater at low temperature. The bivalve Mya truncata is common in Svalbard fjord (Norway) where it experiences low temperature throughout the year. To measure the impact of polycyclic aromatic hydrocarbons (PAH) on M. truncata, the responses of three biomarkers [total oxyradical scavenging capacity-assay (TOSC), plasma membrane stability of haemocytes and respiration rates] were investigated from bivalves exposed to sediment contaminated with a PAH mixture (crude oil). After two weeks of exposure to the contaminated sediment, TOSC showed no change. The high TOSC value (4010+/-1339 unit mg(-1) protein) of Mya truncata (control group) is thought to protect biomolecules with a low turnover rate efficiently in a low food availability environment. In the exposed bivalves, the haemocyte cellular membranes were significantly destabilised compared with controls (P<0.05). Respiration rate of control and PAH-exposed individuals (0.055+/-0.020 mg O(2) dw(-1) h(-1)) was similar and relatively low as is typical for polar bivalves, reflecting a strategy to minimise energy expenditure to cope with 9 months of starvation. Bioaccumulation of PAH by M. truncata was also low, due probably to a combination of low metabolic rate and reduced solubility of the oil compounds at low temperature. Data indicated an uptake of mainly low molecular weight compounds (two and three ring molecules). A good correlation of logBAF(lipid) (bioaccumulation factor) and logK(ow) (octanol/water partitioning coefficient) was shown (r(2)=0.87). Tissue sensitivity and/or functional differences (digestive gland vs. haemocytes), PAH uptake route (dietary vs. gills), the low metabolic rate of M. truncata and the low environmental temperature (reducing the bioavailability of PAH) are factors that help explain these findings.

Total oxyradical scavenging capacity and cell membrane stability of haemocytes of the Arctic scallop, Chlamys islandicus, following benzo(a)pyrene exposure.

Industrial activities, notably oil and gas industries, are expanding in the Arctic. Most of the biomarkers were developed using temperate organisms living at temperatures above 10 degrees C. Little is known about the biomarker responses of organisms living between -1.88 and 5 degrees C. Therefore, assessment of the toxicity of chemicals to cold-water adapted species is required. In this study, the Arctic scallop, Chlamys islandicus, was selected as a key species for bio-monitoring because of wide distribution in Arctic waters and its commercial value. Test animals, stored in seawater at 2 degrees C, were injected with benzo(a)pyrene (diluted in cod liver oil 5 mg ml(-1)) in the adductor muscle every 24 h for four days giving a final dose of 0, 74 and 90.6 mg kg(-1) wet weight for control, low and high dose, respectively. The biomarkers used were total oxyradical scavenging capacity (TOSC) in the digestive gland and cell membrane stability of haemocytes. TOSC values were significantly reduced (ca. 30%) in exposed groups (P < 0.05), indicating a depletion in oxyradical molecular scavengers. The antioxidant defences appeared to be overwhelmed by the reactive oxygen species as the plasma membranes of haemocytes were destabilised (P < 0.05) probably due to lipid peroxidation. These data indicate that reactive oxygen species (ROS) were produced by Arctic scallops via the metabolisation of benzo(a)pyrene at 2 degrees C.

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.

Biomarkers in Natural Fish Populations Indicate Adverse Biological Effects of Offshore Oil Production

Background Despite the growing awareness of the necessity of a sustainable development, the global economy continues to depend largely on the consumption of non-renewable energy resources. One such energy resource is fossil oil extracted from the seabed at offshore oil platforms. This type of oil production causes continuous environmental pollution from drilling waste, discharge of large amounts of produced water, and accidental spills. Methods and principal findings Samples from natural populations of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua) in two North Sea areas with extensive oil production were investigated. Exposure to and uptake of polycyclic aromatic hydrocarbons (PAHs) were demonstrated, and biomarker analyses revealed adverse biological effects, including induction of biotransformation enzymes, oxidative stress, altered fatty acid composition, and genotoxicity. Genotoxicity was reflected by a hepatic DNA adduct pattern typical for exposure to a mixture of PAHs. Control material was collected from a North Sea area without oil production and from remote Icelandic waters. The difference between the two control areas indicates significant background pollution in the North Sea. Conclusion It is most remarkable to obtain biomarker responses in natural fish populations in the open sea that are similar to the biomarker responses in fish from highly polluted areas close to a point source. Risk assessment of various threats to the marine fish populations in the North Sea, such as overfishing, global warming, and eutrophication, should also take into account the ecologically relevant impact of offshore oil production.

Heart rate, respiration and total oxyradical scavenging capacity of the Arctic spider crab, Hyas araneus, following exposure to polycyclic aromatic compounds via sediment and injection.

Increasing industrial activity in the European Arctic has raised concerns of the potential anthropogenic impact of chemicals on this polar marine ecosystem. For the past 20 years or so, biomarkers have been developed to provide early-warning signals of detrimental impacts of chemicals on the marine ecosystem, however, most biomarker methods have been established for organisms living in temperate rather than polar waters. Little is known about biomarker responses in organisms living within the temperature range of -1.88 to +5 degrees C. In this study, established biomarkers from temperate studies were tested on the Arctic spider crab Hyas araneus to validate their use in polar ecosystems. H. araneus is common in Svalbard fjord (Norway), although it is a temperate water species occurring from northern Spain to Svalbard at depths from 10 to 1200 m. In this paper, the effects of oil were investigated at 2 degrees C via two routes: (i) injection and (ii) contaminated sediment. After 2 weeks of exposure, heart rate, oxygen consumption and total oxyradical scavenging capacity (TOSC) were measured in the same individuals. In both methods of contaminant exposure, heart rate showed a significant increase compared with the control (P < 0.0001, n = 7); mean heart rate values (+/- S.D.) of H. araneus were 49.06 (+/- 13.72), 57.56 (+/- 7.28) and 63.30 (+/- 6.57) beats per minute in control, injected and sediment-treated groups, respectively. Respiration of H. araneus was not affected significantly by either oil treatment (P > 0.05), but two individuals (n = 8) showed a marked increase in oxygen uptake in the sediment-exposed group. The basal oxygen consumption of control H. araneus was lower (0.025 mg O(2) g wet wt.(-1) h(-1)) than reported for H. araneus living in temperate water. Although TOSC of H. araneus was not affected significantly by either exposure treatment (P > 0.05) the mean TOSC value in the sediment-exposed group was lower than the control, indicating some saturation of the oxyradical scavenging system. Results indicate that although low temperature appears to be the main factor reducing the bioavailability of polycyclic-aromatic hydrocarbons, the relatively low metabolic rate of Arctic H. araneus is also implicated in decreased uptake and metabolism of oil compounds into reactive oxygen species (ROS).

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.

Environmental harm assessment of a wastewater discharge from Hammerfest LNG: a study with biomarkers in mussels (Mytilus sp.) and Atlantic cod (Gadus morhua).

Biologically treated wastewater (WW) from the Hammerfest LNG (liquefied natural gas) plant is discharged to the sea. A study using biomarkers in mussels and Atlantic cod was performed to examine whether this discharge meets a zero harmful emission requirement. Caging of mussels close to the outfall and exposure of mussels and fish to WW in the laboratory were conducted, and a suite of contaminant responsive markers was assessed in exposed animals. In mussels the markers included chemical contaminant levels, haemocyte lysosomal instability and nucleus integrity, cellular energy allocation, digestive gland and gonad histopathology and shell-opening behaviour. In fish, biliary PAH metabolites and gill histopathology biomarkers were measured. A consistent cause-effect relationship between WW treatments and markers measured in test animals was not found. The results therefore indicate that the WW emission is unlikely to represent a significant stress factor for the local marine environment under the conditions studied.

Membrane destabilisation of haemocytes measured by uptake of fluorescent probes

Abstract Lysosomal stability measured as neutral red retention (NRR) in haemocytes of mussels has been used to measure impact of environmental pollution. In this study we have tested whether membrane destabilisation also may be measured in haemocytes by a fluorescent plate reader after addition of the fluorescent probes BODIPY FL verapamil (BFLV) and ethidium homodimer-1 (EthD-1). Blue mussels (Mytilus edulis) have been exposed to pyrene, Cu and Zn, and the ratio of fluorescence calculated after different time intervals. Healthy granulated haemocytes efficiently take up BFLV to their lysosomes, while uptake is decreased in haemocytes of exposed mussels. EthD-1 is taken up generally with time and the fluorescence will increase upon binding to nucleic acids; however, damaged cells take up EthD-1 to a greater extent. Thereby, the ratio of the fluorescent probes enhances the signal. The effect was verified by fluorescence microscope studies. The results with pyrene demonstrated significant reduction in ratio of fluorescence after treatment with 0.4, 1, and 2.5 mg/l for 1 week, as did 2 mg/l Cu and Zn. The results have been compared with the NRR assay, demonstrating similar sensitivity. A comparison of the methods has also been performed with mussels collected from a metal-polluted field site. The measured fluorescence ratio may be an alternative to the NRR assay, providing automated reading.