Tor Fredrik Holth

Protein carbonyls and antioxidant defenses in corkwing wrasse (Symphodus melops) from a heavy metal polluted and a PAH polluted site.

The use of fish in environmental monitoring has become increasingly important in recent years as anthropogenic substances, many of which function as prooxidants, are accumulating in aquatic environments. We have measured a battery of antioxidant defenses as a measure of oxidative status, as well as protein carbonylation as a measure of oxidative damage, in corkwing wrasse (Symphodus melops) captured near a disused copper mine, where water and sediment are contaminated with heavy metals, and an aluminum smelter, a site contaminated with PAHs. Results were compared to two different reference sites. Fish at the heavy metal site had lower glucose-6-phosphate dehydrogenase activity and elevated protein carbonyls (1.8 times) compared to fish from the reference site. At the PAH site, EROD was increased 2-fold, while total glutathione and methemoglobin reductase concentration, were decreased. No differences were seen in protein carbonyl levels at the PAH site. Measures of both antioxidant defenses and oxidative damage should be used when assessing effects of xenobiotics on oxidative stress in fish species.

Polycyclic Aromatic Hydrocarbon (PAH) Metabolites in Atlantic Cod Exposed via Water or Diet to a Synthetic Produced Water

Bile metabolites are widely accepted as measures of exposure to polycyclic aromatic hydrocarbons (PAH) and have also been used to assess exposure to alkyl phenols (AP). The aim of this study was to clarify relationships between exposure (through water or diet) and subsequent accumulation of specific PAH and AP metabolites in Atlantic cod (Gadus morhua). Atlantic cod were exposed through water or diet to a synthetic mixture of PAH, alkylated PAH, and AP, simulating the composition of North Sea produced water (formation water; separated from oil or gas on the platform). Fish were exposed through water for 11 mo and the results reported here are from 2 and 8 mo. Fish were subjected to one of four exposures: “control,” “low,” “high,” or “pulsed high.” Bile samples were analyzed by gas chromatography/time-of-flight mass spectroscopy (GC/MS-ToF) for identification of PAH and AP metabolites. Cod exposed through diet were divided into six groups receiving different groups of compounds (AP, PAH, or alkylated PAH), a mixture of all compounds, a low-concentration mixture, and controls. A dose-dependent relationship was found for metabolites for most of the PAH and AP, although results were less clear for the more volatile substances such as phenol and naphthalene. The concentration of bile metabolites from fish exposed through water rose with increasing lipophilicity, but this relationship was less clear for fish exposed through diet. Overall, data indicated that bile metabolites of AP and PAH in fish are reliable markers of both water exposure and dietary exposure to such substances, although with the possible exception of the more volatile species such as phenol and naphthalene.

Effects of water accommodated fractions of crude oils and diesel on a suite of biomarkers in Atlantic cod (Gadus morhua)

The aim of this study was to characterize concentration- and time-dependent responses in juvenile Atlantic cod (Gadus morhua) following exposure for one and three weeks to the water-soluble fraction (WAF) of three weathered oils: Arabian Light crude oil (ALC), North Sea crude oil (NSC) and ship-diesel. The sum of polycyclic aromatic hydrocarbons (PAH) in water was highest after one week of exposure and within environmentally relevant concentrations. PAH metabolites in bile confirmed exposure to and uptake of PAHs. Hepatic cytochrome P450 1A (CYP1A) gene expression (mRNA quantification) increased dramatically following exposure to all three oil types (fold-change up to 165) and there was a time lag between gene and protein expression. Hepatic CYP1A protein concentration and ethoxyresorufin-O-deethylase (EROD) activity were more variable among individuals and treatments than gene expression. EROD activity in liver and gills increased in fish exposed to WAF from the two crude oils, but not in fish exposed to WAF from diesel. Exposure to diesel appeared to induce oxidative stress to a greater extent than exposure to crude oils. Other biomarkers (glutathione S-transferases, acetylcholine esterase, vitellogenin) did not appear to respond to the exposure and hence did not discriminate among oils. Biomarker responses in cod after exposure to weathered crude oils and diesel suggested that the CYP1A system and oxidative stress markers have the highest potential for discriminating among different oil types and to monitor the environmental consequences of spills.

Species-dependent sensitivity to contaminants: An approach using primary hepatocyte cultures with three marine fish species

There is limited knowledge about the sensitivity of different fish species to environmental pollutants. Such information is pivotal in risk assessment and to understand why some species appear to be more tolerant to contaminants than others. The aim of the current study was to evaluate whether primary hepatocyte cultures of three marine fish species could be established in the field and whether their sensitivity to selected contaminants would differ. Primary hepatocyte cultures of three marine fish species (plaice, long rough dab, Atlantic cod) were established and exposed for 24 h to copper (20-2500 mg L⁻¹) and statins (1-200 mg L⁻¹). Endpoints were esterase activity, metabolic activity and reduced glutathione (GSH) content, all using fluorescent probes. Flatfish hepatocytes were more susceptible to copper and statin exposure than hepatocytes from cod. This study has shown that species-dependent differences in contaminant sensitivity can be investigated using primary hepatocyte cultures.

Relationship Between Polycyclic Aromatic Hydrocarbon (PAH) Accumulation in Semipermeable Membrane Devices and PAH Bile Metabolite Levels in Atlantic Cod (Gadus morhua)

The extent to which accumulations of polycyclic aromatic hydrocarbons (PAH) by semipermeable membrane devices (SPMD) may be correlated to the concentration of PAH metabolites in fish was examined. Atlantic cod (Gadus morhua) and SPMD were exposed over a 4-wk period to a mixture that was designed to simulate the composition of PAH in produced water discharges in the North Sea. Fish bile and whole SPMD were analyzed after exposure in a high, low, and a control treatment tank. PAH metabolites were detected and quantified by means of high-performance liquid chromatography (HPLC) or gas chromatography–time-of-flight mass spectroscopy (GC-ToF-MS) and PAH in SPMD by gas chromatography–mass spectroscopy (GC-MS). The level and duration of exposure were generally reflected in both matrices. Exposure water concentrations, calculated from SPMD accumulations, showed some considerable differences from nominal concentrations for volatile compounds. Concentration factors (CF) for fish based on SPMD-derived water concentrations ranged from 2.9 to 354.3 L/g. CF increased with hydrophobicity with more variation at higher log K ow. Regression analysis of accumulations of individual compounds in fish and SPMDs showed reasonable but compound-specific correlation, r 2 = .54−.85 (C1-phenanthrenes/anthracenes and pyrene, respectively). Comparison of the two exposures suggests that fish CF may not be entirely independent of water concentrations. Overall the results show that accumulation of up to four-ring PAH in SPMD may be related to internal exposure of fish to these substances when exposed through water, as evidenced by metabolite concentrations. The usefulness of using SPMD to measure exposure concentrations in a large-scale flow-through study is also examined.

Acetylcholine esterase inhibitors in effluents from oil production platforms in the North Sea.

Inhibition of acetylcholine esterase (AChE) activity is a biomarker for the exposure to neurotoxic compounds such as organophosphates and is intimately associated with the toxicity of several pesticides. In the present study, the AChE inhibiting potential of organic extracts of production water (produced water) from oil and gas production platforms in the Norwegian sector of the North Sea was determined in an in vitro bioassay based on commercially available purified AChE from the electric organ of Electrophorus electricus (L.). The results from the studies show that produced water contains a combination of AChE inhibiting compounds and compounds stimulating AChE enzymatic activity. The AChE inhibition was predominantly caused by unidentified aromatic compounds in the oil/particulate fraction of produced water, whereas polar compounds in both the water soluble and oil/particulate fraction of produced water caused an apparent stimulation of AChE activity. Substrate saturation studies with fixed concentrations of produced water extracts confirmed that the inhibition occurred in a non-destructive and competitive manner. The concentrations of AChE inhibitors (7.9-453 ng paraoxon-equivalents L⁻¹, 2.2-178 μg dichlorvos-equivalents L⁻¹) were in many cases found to be several orders of magnitude higher than background levels. The findings demonstrate that produced water contains potentially neurotoxic compounds and suggest that further laboratory studies with fish or field studies in the vicinity of oil production facilities are highly warranted.

Pristine Arctic: background mapping of PAHs, PAH metabolites and inorganic trace elements in the North-Atlantic Arctic and sub-Arctic coastal environment.

As the ice cap of the Arctic diminishes due to global warming, the polar sailing route will be open larger parts of the year. These changes are likely to increase the pollution load on the pristine Arctic due to large vessel traffic from specific contaminant groups, such as polycyclic aromatic hydrocarbons (PAHs). A well-documented baseline for PAH concentrations in the biota in the remote regions of the Nordic Seas and the sub-Arctic is currently limited, but will be vital in order to assess future changes in PAH contamination in the region. Blue mussels (Mytilus edulis) were collected from remote sites in Greenland, Iceland, the Faroe Islands, Norway and Sweden as well as from urban sites in the same countries for comparison. Cod (Gadus morhua) was caught north of Iceland and along the Norwegian coast. Sixteen priority PAH congeners and the inorganic trace elements arsenic, cadmium, mercury and lead were analysed in the blue mussel samples as well as PAH metabolites in cod bile. Σ16PAHs ranged from 28 ng/g dry weight (d.w.) (Álftafjörður, NW Iceland) to 480 ng/g d.w. (Ísafjörður, NW Iceland). Mussel samples from Mjóifjörður, East Iceland and Maarmorilik, West Greenland, contained elevated levels of Σ16PAHs, 370 and 280 ng/g d.w., respectively. Levels of inorganic trace elements varied with highest levels of arsenic in mussels from Ísafjörður, Iceland (79 ng/g d.w.), cadmium in mussels from Mjóifjörður, Iceland (4.3 ng/g d.w.), mercury in mussels from Sørenfjorden, Norway (0.23 ng/g d.w.) and lead in mussels from Maarmorilik, Greenland (21 ng/g d.w.). 1-OH-pyrene was only found above limits of quantification (0.5 ng/mL) in samples from the Norwegian coast, ranging between 44 and 140 ng/ml bile. Generally, PAH levels were low in mussels from the remote sites investigated in the study, which indicates limited current effect on the environment.

Assessment of lysosomal membrane stability and peroxisome proliferation in the head kidney of Atlantic cod (Gadus morhua) following long-term exposure to produced water components.

There is a need for sensitive biological effect methods by which to detect impacts of chronic exposure to low concentrations of contaminants. Two methods shown to be potentially useful for monitoring purposes in fish include lysosomal membrane stability and peroxisome proliferation. These biological endpoints were assessed in Atlantic cod (Gadus morhua) head kidney following exposure to a mixture of produced water components including polycyclic aromatic hydrocarbons, phenol, and alkylphenols. Lysosomal damage of head kidney cells occurred within the first two weeks and did not recover during the entire exposure period (32 weeks). Lysosomal membrane stability was not affected by gender and was responsive at low concentrations of contamination, indicating that lysosomal membrane stability measured in the head kidney could be a useful biomarker for effects of offshore pollution. Peroxisome proliferation, measured as acyl-CoA oxidase activity in the head kidney, appeared to be a potential biomarker in male cod exposed less than 16 weeks.

Bioaccumulation and lack of oxidative stress response in the ragworm H. diversicolor following exposure to 226Ra in sediment

The main effluent from oil and gas production, produced water, from some platforms in the North Sea contains elevated concentrations of (226)Ra. The aim of this study was to investigate whether (226)Ra in sediment would accumulate in and affect sediment-dwelling organisms. In addition, we wanted to determine if the bioavailability would be modulated by the presence of a scale inhibitor which is used during oil and gas production. Hediste diversicolor was therefore exposed to different levels of (226)Ra (30-6600 Bq kg(-1)) in combination with scale inhibitor in the sediments in a flow through system. The levels of radioactivity in the exposures were close to levels that can be measured in proximity to oil/gas production facilities. (226)Ra spiked to natural sediment partitioned into pore water and accumulated in the sediment-dwelling polychaete following a four-week exposure period. The results suggest that (226)Ra did not bind strongly to sediment (low sediment:water partitioning coefficient), but it was not shown to bioaccumulate in any great extent (bioaccumulation factors of 0.019-0.022). Exposure of H. diversicolor in sediments with up to 6600 Bq kg(-1)(226)Ra had no measurable effect on the total oxyradical scavenging capacity of the organisms compared to control. So although they accumulated the alpha-emitter, the treatments did not appear to cause oxidative stress in polychaete tissues.

Accumulation of lead (Pb) in brown trout (Salmo trutta) from a lake downstream a former shooting range

An environmental survey was performed in Lake Kyrtjønn, a small lake within an abandoned shooting range in the south of Norway. In Lake Kyrtjønn the total water concentrations of Pb (14µg/L), Cu (6.1µg/L) and Sb (1.3µg/L) were elevated compared to the nearby reference Lake Stitjønn, where the total concentrations of Pb, Cu and Sb were 0.76, 1.8 and 0.12µg/L, respectively. Brown trout (Salmo trutta) from Lake Kyrtjønn had very high levels of Pb in bone (104mg/kg w.w.), kidney (161mg/kg w.w.) and the gills (137mg/kg d.w), and a strong inhibition of the ALA-D enzyme activity were observed in the blood (24% of control). Dry fertilized brown trout eggs were placed in the small outlet streams from Lake Kyrtjønn and the reference lake for 6 months, and the concentrations of Pb and Cu in eggs from the Lake Kyrtjønn stream were significantly higher than in eggs from the reference. More than 90% of Pb accumulated in the egg shell, whereas more than 80% of the Cu and Zn accumulated in the egg interior. Pb in the lake sediments was elevated in the upper 2-5cm layer (410-2700mg/kg d.w), and was predominantly associated with redox sensitive fractions (e.g., organic materials, hydroxides) indicating low potential mobility and bioavailability of the deposited Pb. Only minor amounts of Cu and Sb were deposited in the sediments. The present work showed that the adult brown trout, as well as fertilized eggs and alevins, may be subjected to increased stress due to chronic exposure to Pb, whereas exposure to Cu, Zn and Sb were of less importance.