Bjørn Henrik Hansen

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.

Effects of atrazine on hepatic metabolism and endocrine homeostasis in rainbow trout (Oncorhynchus mykiss).

The herbicide atrazine (ATZ) is one of the most widely used pesticides in the world and is now under scrutiny for its alleged capacity to disrupt the endocrine system. Exhibiting negligible interaction with the estrogen receptor (ER), ATZs mode of action remains to be elucidated. ATZ may act as an inducer of the enzyme aromatase, which converts androgens to estrogens, although other mechanisms should also be taken into consideration such as impairment of hepatic metabolism. Therefore we administered juvenile rainbow trout (Oncorhynchus mykiss) a dose of either 2 or 200 microg ATZ/kg, or of carrier control phosphate buffered saline (PBS) and we measured plasma concentrations of testosterone (T), 17beta-estradiol (E2) and vitellogenin (Vtg) 6 days after exposure. Simultaneously we analyzed hepatic gene expression of cytochrome P450 (CYP) 1A and pi-class glutathione S-transferase (GST-P), and catalase (CAT) activity. Although sex steroid levels showed no significant alterations, we found a dose-dependent increase in Vtg and a concomitant decrease in CYP1A. There was no effect of ATZ on GST-P mRNA levels but GST-P was positively correlated with CYP1A. Also, CYP1A was negatively correlated with liver CAT and E2, and varied with T concentrations in a hormetic manner. The results showed that ATZ can alter hepatic metabolism, induce estrogenic effects and oxidative stress in vivo, and that these effects are linked.

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.

Chemical and toxicological characterization of an unresolved complex mixture‐rich biodegraded crude oil

Chemical and toxicological characterization of unresolved complex mixtures in the water-soluble fraction of an artificially weathered Norwegian Sea crude oil was determined by a combination of chemical analysis and toxicity testing in fish in vitro bioassays. The water-soluble fraction of the crude oil was separated into 14 increasingly polar fractions by preparative high-pressure liquid chromatography. The in vitro toxicity (7-ethoxyresorufin O-deethylase activity, estrogenicity, and metabolic inhibition) of these fractions was characterized in a primary culture of liver cells (hepatocytes) from rainbow trout (Oncorhynchus mykiss). The main contributor to toxicity was one of the most polar fractions, accounting gravimetrically for more than 70% of the organic material in the water-soluble fraction and dominated by an unresolved complex mixture. Chemical analysis by gas chromatography-mass spectrometry and comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry identified a large number of cyclic and aromatic sulfoxide compounds and low amounts of benzothiophenes (<0.1% of total mass) in this fraction. Commonly monitored toxic components of crude oil (e.g., naphthalenes, polycyclic aromatic hydrocarbons, and alkylated phenols) eluted in less polar fractions, characterized by somewhat lower toxicity. Normalization of in vitro responses to the mass in each fraction demonstrated a more even distribution of toxicity, indicating that toxicity in the individual fractions was related to the amount of material present. Although polar and nonpolar compounds contribute additively to crude oil toxicity, the water-soluble fraction was dominated by polar compounds because of their high aqueous solubility and the high oil-water loading. Under these conditions, the polar unresolved complex mixture-rich fraction might account for a large portion of crude oil toxicity because of its high abundance in the water-soluble fraction.

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.

Molecular effects of diethanolamine exposure on Calanus finmarchicus (Crustacea: Copepoda).

Alkanolamines are surface-active chemicals used in a wide range of industrial, agricultural and pharmaceutical applications and products. Of particular interest is the use of alkanolamines such as diethanolamine (DEA) in the removal of CO(2) from natural gas and for CO(2) capture following fossil fuel combustion. Despite this widespread use, relatively little is known about the ecotoxicological impacts of these compounds. In an attempt to assess the potential effects of alkanolamines in the marine environment, a key species in the North Atlantic, the planktonic copepod Calanus finmarchicus, was studied for molecular effects following sublethal exposure to DEA. DEA-induced alterations in transcriptome and metabolome profiling were assessed using a suppression subtractive hybridization (SSH) gene library method and high resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR), respectively. Effects were observed on transcription of genes reportedly involved in lipid metabolism, antioxidant systems, metal binding, and amino acid and protein catabolism. These effects were accompanied by altered expression of fatty acid derivates, amino acids (threonine, methionine, glutamine, arginine, alanine and leucine) and cholines (choline, phosphocholine and glycerophosphocholine). Together, SSH and HR-MAS NMR offer complementary screening tools for the assessment of molecular responses of C. finmarchicus to DEA and can be used in the study of other chemicals and organisms. Concentration-response and time-response relationships between DEA exposure and single gene transcription were investigated using quantitative PCR. Specific relationships were found between DEA exposure and the transcription of genes involved in protein catabolism (ubiquitin-specific protease-7), metal ion homeostasis (ferritin) and defence against oxidative stress (gamma-glutamylcysteine synthase, glutathione synthase and Cu/Zn-superoxide dismutase). At the lowest alkanolamine concentration used in these experiments, which corresponded to 0.5% of the LC(50) concentration, no transcriptional effects were observed, giving information regarding the lower molecular effect level. Finally, similar transcription patterns were observed for a number of different genes following exposure to DEA, which indicates analogous mechanisms of toxicity and response.

Gross CO2 and CH4 emissions from the Nam Ngum and Nam Leuk sub-tropical reservoirs in Lao PDR.

Gross CO2 and CH4 emissions (degassing and diffusion from the reservoir) and the carbon balance were assessed in 2009-2010 in two Southeast Asian sub-tropical reservoirs: the Nam Ngum and Nam Leuk Reservoirs (Lao PDR). These two reservoirs are within the same climatic area but differ mainly in age, size, residence time and initial biomass stock. The Nam Leuk Reservoir was impounded in 1999 after partial vegetation clearance and burning. However, GHG emissions are still significant 10 years after impoundment. CH4 diffusive flux ranged from 0.8 (January 2010) to 11.9 mmol m(-2) d(-1) (April 2009) and CO2 diffusive flux ranged from -10.6 (October 2009) to 38.2 mmol m(-2) d(-1) (April 2009). These values are comparable to other tropical reservoirs. Moreover, degassing fluxes at the outlet of the powerhouse downstream of the turbines were very low. The tentative annual carbon balance calculation indicates that this reservoir was a carbon source with an annual carbon export (atmosphere+downstream river) of about 2.2±1.0 GgC yr(-1). The Nam Ngum Reservoir was impounded in 1971 without any significant biomass removal. Diffusive and degassing CO2 and CH4 fluxes were lower than for other tropical reservoirs. Particularly, CO2 diffusive fluxes were always negative with values ranging from -21.2 (April 2009) to -2.7 mmol m(-2) d(-1) (January 2010). CH4 diffusive flux ranged from 0.1 (October 2009) to 0.6 mmol m(-2) d(-1) (April 2009) and no degassing downstream of the turbines was measured. As a consequence of these low values, the reservoir was a carbon sink with an estimated annual uptake of - 53±35 GgC yr(-1).

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.

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.