Trond Nordtug

Biodegradation of dispersed Macondo oil in seawater at low temperature and different oil droplet sizes

During the Deepwater Horizon (DWH) accident in 2010 a dispersant (Corexit 9500) was applied at the wellhead to disperse the Macondo oil and reduce the formation of surface slicks. A subsurface plume of small oil droplets was generated near the leaking well at 900-1300 m depth. A novel laboratory system was established to investigate biodegradation of small droplet oil dispersions (10 μm or 30 μm droplet sizes) of the Macondo oil premixed with Corexit 9500, using coastal Norwegian seawater at a temperature similar to the DWH plume (4-5°C). Biotransformation of volatile and semivolatile hydrocarbons and oil compound groups was generally faster in the 10 μm than in the 30 μm dispersions, showing the importance of oil droplet size for biodegradation. These data therefore indicated that dispersant treatment to reduce the oil droplet size may increase the biodegradation rates of oil compounds in the deepwater oil droplets.

Modulation of steroidogenesis and xenobiotic biotransformation responses in zebrafish (Danio rerio) exposed to water-soluble fraction of crude oil

The induction of CYP enzyme activities, particularly CYP1A1, through the aryl hydrocarbon receptor (AhR) in most vertebrate species is among the most studied biochemical response to planar and aromatic organic contaminant exposure. Since P450 families play central roles in the oxidative metabolism of a wide range of exogenous and endogenous compounds, interactions between the biotransformation processes and reproductive physiological responses are inevitable. Steroidogenesis is the process by which specialized cells in specific tissues, such as the gonad, brain (neurosteroids) and kidney, synthesize steroid hormones. In the present study, we evaluated the effects of water-soluble fraction (WSF) of crude oil on the xenobiotic biotransformation and steroidogenic processes in the head (brain) and whole-body tissue of a model species by transcript analysis using quantitative (real-time) polymerase chain reaction (qPCR), enzyme activities and steroid hormone (testosterone: T and 17beta-estradiol: E2) levels using enzyme immune assay (EIA). Our data showed that exposure of fish to WSF produced an apparent concentration-specific increase of AhR1, CYP1A1 and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) mRNA levels, and decrease of AhR2. On the activity level, WSF produced concentration-specific increase of ethoxyresorufin O-deethylase (EROD), benzyloxyresorufin (BROD) methoxyresorufin (MROD) and pentoxyresorufin (PROD) activities in whole-body tissue. In the steroidogenic pathway, WSF exposure produced apparent concentration-specific decrease of ER* and ERbeta, steroidogenic acute regulatory (StAR) protein, cytochrome P450 side-chain cleavage (P450scc), P450aromA and P450aromB mRNA expression. For steroid hormones, while T levels decreased, E2 levels increased in an apparent WSF concentration-specific manner. In general, the xenobiotic biotransformation and estrogenic responses showed negative relationship after exposure of zebrafish to WSF, suggesting an interaction between these physiological pathways. The relationship between WSF mediated changes in brain StAR, P450scc, 3beta-HSD, ER*alpha, ERbeta, P450aromA, P450aromB and whole-body steroid hormone levels suggests that the experimental animals might be experiencing altered neurosteroidogenesis probably through increased activity level of the biotransformation system. Thus, these responses might represent sensitive diagnostic tools for short-term and acute exposure of fish or other aquatic organisms to WSF.

Comparative study on acute effects of water accommodated fractions of an artificially weathered crude oil on Calanus finmarchicus and Calanus glacialis (Crustacea: Copepoda)

Extrapolation of ecotoxicological data from temperate species for use in risk assessment in the polar environments may be difficult since polar organisms as a rule differ from temperate species in terms of life span length, developmental time, surface-to-volume ratios, metabolic rates, total energy usage and lipid content for energy storage. In the current work we performed a comparative study where two closely related and morphologically similar copepod species, Calanus finmarchicus (temperate-boreal) and Calanus glacialis (arctic), were exposed to water accommodated fractions (WAF) of oil in a series of parallel experiments. The two species, adapted to 10°C and 2°C, respectively, were compared on the basis of acute ecotoxicity (LC(50)) and the WAF-mediated induction of the gene encoding glutathione S-transferase (GST). In addition, an experiment was conducted in order to reveal relationships between lipid content and acute toxicity. LC(50) values differed between the two species, and the Arctic copepod appeared less sensitive than the temperate-boreal species. The lipid contents of the two species, measured biometrically, were comparable, and the relationships between lipid content and response (reduced survival) to acute WAF exposure followed the same trend: Lipid-rich copepods survived longer than lipid-poor copepods at the same exposure concentration. In terms of GST expression, both species showed concentration-dependent and exposure time-dependent trends. However, as for the acute toxicity data, the Arctic copepod appeared to respond slower and with a lower intensity. From the study it can be concluded that temperature and lipid content are important factors for assessing differences between temperate and Arctic species, and that a delayed response in organisms adapted to low temperatures needs to be corrected for when extrapolating toxicity data from species with other temperature optimums for use in Arctic environments.

Suppression subtractive hybridization library prepared from the copepod Calanus finmarchicus exposed to a sublethal mixture of environmental stressors

A library of expressed sequence tags (ESTs) was constructed by the use of suppression subtractive hybridization polymerase chain reaction (SSH PCR) technique from the marine copepod Calanus finmarchicus. Samples used were from controls (seawater, 10 degrees C) and exposed (sublethal mixture) individuals. The sublethal exposure regime consisted of a mixture of mono ethanol amine (MEA), water-soluble fractions of oil (WSFs), copper (Cu) and elevated temperature (17 degrees C). The resulting 189 unique ESTs consisted of 127 putatively up-regulated genes and 54 putatively down-regulated genes. Annotation analyses revealed altered expression of a wide variety of genes, among these putative heat shock protein 90 (HSP-90), antioxidants (thioredoxin reductase, glutathione peroxidase) and cytochrome P450 enzymes. In addition, sequences showing high similarity to enzymes involved in fatty acid metabolism, energy metabolism and amine handling were found further confirming the effects of the exposure. The annotated sequences are discussed in relation to the present exposure as well as known physiological mechanisms known in C. finmarchicus and related copepod species. The sequenced ESTs from our C. finmarchicus library will provide an excellent tool for future studies on this species, both from a toxicogenomic and systems biology point of view.

Gene Expression of GST and CYP330A1 in Lipid-Rich and Lipid-Poor Female Calanus finmarchicus (Copepoda: Crustacea) Exposed to Dispersed Oil

The copepod Calanus finmarchicus is a marine ecological key species in the Northern Atlantic food web. This species was exposed to an artificially weathered North Sea oil dispersion (oil droplets and water-soluble fractions [WSF]) and a filtered dispersion (containing only WSF) in serial dilution. Female copepods were divided into lipid-rich and lipid-poor for each exposure followed by gene expression analyses of glutathione S-transferase (GST) and cytochrome P-450 330A1 (CYP330A1). Lipid-rich copepods exhibited elevated transcription of GST and reduced transcription of CYP330A1 after exposure to both dispersed oil and WSF. In contrast, lipid-poor copepods exhibited increased transcription of CYP330A1 following exposure to WSF but not the dispersion. Data suggested that small lipid storage promotes increased bioavailability of accumulated oil compounds. Variations in response in CYP330A1 gene expression indicate that oil constituents may exert different modes of toxic action in copepods depending on their reproductive stages. The contribution of oil droplets to the observed effects seemed to be low as GST gene expression was similar after exposure to both dispersed oil and WSF. However, feeding rate in copepods exposed to dispersed oil was reduced, and this may have decreased the uptake of oil constituents via the diet. Although quantitatively higher mortality was observed in copepods exposed to the highest dispersion levels, this may result from smothering of animals by oil droplets. Furthermore, increasing dilution of both the dispersions and the WSF altered their distributions and chemical composition, which may influence the bioavailability of spilled crude oil to pelagic marine organisms.

Acute toxicity of naturally and chemically dispersed oil on the filter-feeding copepod Calanus finmarchicus

Following oil spills in the marine environment, natural dispersion (by breaking waves) will form micron-sized oil droplets that disperse into the pelagic environment. Enhancing the dispersion process chemically will increase the oil concentration temporarily and result in higher bioavailability for pelagic organisms exposed to oil-dispersant plume. The toxicity of dispersed oil to pelagic organisms is a critical component in evaluating the net environmental consequences of dispersant use or non-use in open waters. To assess the potential for environmental effects, numerical models are being used, and for these to reliably predict the toxicity of chemically dispersed oil, it is essential to know if the dispersant affects the specific toxicity of the oil itself. In order to test the potential changes in specific toxicity of the oil due to the presence of chemical dispersant, copepods (Calanus finmarchicus) were subjected to a continuous exposure of chemically (4 percent Dasic w/w dispersant) and naturally dispersed oil (same droplet size range and composition) for four days. On average the addition of dispersant decreased 96h LC(50)-values by a factor of 1.6, while for LC(10) and LC(90) these factors were 2.9 and 0.9, respectively. This indicates that after 96h of exposure the dispersant slightly increased the specific toxicity of the oil at median and low effect levels, but reduced the toxicity at high effect levels. Decreased filtrations for the exposed groups were confirmed using particle counting and fluorescence microscopy. However, no differences in these endpoints were found between chemically and naturally dispersed oil. The ultimate goal was to evaluate if models used for risk and damage assessment can use similar specific toxicity for both chemically and naturally dispersed oil. The slight differences in toxicity between chemically and naturally dispersed oil suggest that risk assessment should be based on the whole concentration response curve to ensure survival of C. finmarchicus.

Method for generating parameterized ecotoxicity data of dispersed oil for use in environmental modelling

The aim of the work was to establish methodology for realistic laboratory-based test exposures of organisms to oil dispersions, specifically designed to generate parameterized toxicity data. Such data are needed to improve the value of numerical models used to predict fate and effects of oil spills and different oil spill responses. A method for continuous and predictable in-line production of oil dispersions with defined size distribution of different oil qualities was successfully established. The system enables simultaneous comparison between the effects of different concentrations of dispersion and their corresponding equilibrium water soluble fractions. Thus, net effects of the oil droplet fraction may be estimated. The method provides data for comparing the toxicity of oil dispersions generated both mechanically and with the use of chemical dispersions, incorporating the toxicity of both dissolved oil and droplets of oil.

Expression of ecdysteroids and cytochrome P450 enzymes during lipid turnover and reproduction in Calanus finmarchicus (Crustacea: Copepoda)

The marine copepod Calanus finmarchicus is the most abundant zooplankton species in the northern regions of the Atlantic Ocean and the Barents Sea. Very little is known about molecular regulation of hormone metabolism, moulting and reproduction in copepods. To investigate these processes, we sampled adult male and female copepods (females at three distinct reproductive stages) and copepodites stage five (CV) from the culture at SINTEF/NTNU Sealab. Copepods were individually photographed, analyzed biometrically (body size, length and lipid storage size) and for ecdysteroid concentrations. In addition, we analyzed copepods for gene expression of three putative cytochrome P450 enzymes possibly involved in ecdysteroid regulation: CYP301A1, CYP305A1 and CYP330A1. The CV group exhibited the highest ecdysteroid concentrations and the largest lipid storage size, and a significant positive correlation was found between these parameters. Also, two of the P450 enzymes (CYP305A1 and CYP330A1) were more highly expressed at CV than at the adult stage, suggesting that these P450 enzymes are involved in ecdysteroid synthesis and lipid storage regulation. The expression of CYP330A1 was higher in newly moulted females than in females that had produced eggs. In addition, we observed that ecdysteroid concentrations were higher in females with large egg sacs, suggesting that ecdysteroids may be involved in egg maturation and reproduction. The CYP301A1 was more highly expressed in males and post-spawning females, and may be involved in ecdysteroid degradation since these groups also exhibited the lowest ecdysteroid concentrations.

Biotransformation and Dissolution of Petroleum Hydrocarbons in Natural Flowing Seawater at Low Temperature

The objective of this study was to establish methods for controlled studies of hydrocarbon depletion from thin oil films in cold natural seawater, and to determine biotransformation in relation to other essential depletion processes. Mineral oil was immobilized on the surface of hydrophobic Fluortex fabrics and used for studies of microbial biodegradation in an experimental seawater flow-through system at low temperatures (5.9–7.4°C) during a test period of 42 days. The seawater was collected from a depth of 90 m, and microbial characterization by epifluorescence microscopy, fluorescence in situ hybridization, and most-probable number analysis showed relatively larger fractions of archaea and oil-degrading microbes than in the corresponding surface water. Chemical analysis of hydrocarbons attached to the fabrics during the test perweree re(H)-hopane showed that the oil remained adsorbed to the fabrics during the test period.Comparison of depletion analysis with calculation of hydrocarbon dissolution in a flow-through system indicated that naphthalenes and smaller PAH compounds (alkylated 2-ring and 3-ring compounds) were removed from the fabrics by dissolution. The data further implied that depletion of n-alkanes and 4–5 ring PAH hydrocarbons were the result of biotransformation processes. PCR amplification of bacterial 16S rRNA genes from microbes adhering on the immobilized oil surfaces showed the dominance of a few bands when analysed in denaturing gradient gel electrophoresis (DGGE). Sequence analysis of DGGE bands revealed phylogenetic affiliation to the α- and γ-subdivisions of proteobacteria and to the Chloroflexus–Flavobacterium–Bacteroides group.

Oil droplets do not affect assimilation and survival probability of first feeding larvae of North-East Arctic cod

Oil exploration and production in the Atlantic moves northwards towards spawning and nursery areas of fish species that sustain some of the worlds largest fisheries. Models are therefore needed that can simulate the effects of accidental oil spills on early life stages of these fish. In this study, we combined an individual based model and a microcosm approach to infer effects of the water soluble fraction (WSF) and of an oil dispersion (WSF and droplets) on two key endpoints of North East Arctic cod (Gadus morhua) larvae: food assimilation rate and survival probability. Both exposure types (WSF and dispersion) decreased assimilation rate (control: 0.4 d(-1)) and survival probability (control: 0.96) in a concentration-dependent fashion, with EC(50)s of about 2 (feeding) and 40 μg/L ∑PAH in the WSF (survival probability). No consistent differences were found between the ECs from the two exposure types indicating no additional oil droplet effects in the oil dispersion. During post exposure, effects on the two endpoints disappeared, which was confirmed by an image analyses we performed of gut content fluorescence. Our results also show that the larvae model fitted the experimental data from the two exposure types equally well, indicating that the presence of oil droplets did not affect model performance. More complex models that explicitly consider possible mechanisms of oil droplet toxicity - in addition to the toxicity of the WSF - on the two examined endpoints during a 17 day time frame do therefore not have a higher accuracy than simpler models that neglect oil droplet toxicity.