Oil droplet fouling on lesser sandeel (Ammodytes marinus) eggshells does not enhance the crude oil induced developmental toxicity.

The oil industry's expansion and increased operational activity at older installations, along with their demolition, contribute to rising cumulative pollution and a heightened risk of accidental oil spills. The lesser sandeel (Ammodytes marinus) is a keystone prey species in the North Sea and coastal systems. Their eggs adhere to the seabed substrate making them particularly vulnerable to oil exposure during embryonic development. We evaluated the sensitivity of sandeel embryos to crude oil in a laboratory by exposing them to dispersed oil at concentrations of 0, 15, 50, and 150 µg/L oil between 2 and 16 days post-fertilization. We assessed water and tissue concentrations of THC and tPAH, cyp1a expression, lipid distribution in the eyes, head and trunk, and morphological and functional deformities. Oil droplets accumulated on the eggshell in all oil treatment groups, to which the embryo responded by a dose-dependent rise in cyp1a expression. The oil exposure led to only minor sublethal deformities in the upper jaw and otic vesicle. The findings suggest that lesser sandeel embryos are resilient to crude oil exposure. The lowest observed effect level documented in this study was 36 µg THC/L and 3 µg tPAH/L. The inclusion of these species-specific data in risk assessment models will enhance the precision of risk evaluations for the North Atlantic ecosystems.

Use of TLM derived models to estimate toxicity of weathered MC252 oil based on conventional chemical data and the potential impact of unresolved polar components.

Target lipid model (TLM) and toxic unit (TU) approaches were applied to ecotoxicity and chemistry data from low-energy WAFs (LE-WAFs) of source and weathered crude oils originating from the Deepwater Horizon oil spill. The weathered oils included artificially weathered oils and naturally weathered samples collected in the Gulf of Mexico after the spill. Oil weathering greatly reduced the concentrations of identified LE-WAF components, however, the mass of uncharacterized polar material (UPC) in the LE-WAFs remained largely unchanged during the weathering process. While the TLM-derived calculations displayed a significant decrease in toxicity (TUs) for the heavily weathered oils, copepod toxicity, expressed as LC10-based TUs, were comparable between LE-WAFs of fresh and weathered oils. The discrepancy between observed and predicted toxicity for the LE-WAFs of artificially weathered oils may be related to limitations by the chemical analyses or increased toxicity due to generation of new unknown compounds during the weathering process.

A simple protocol for estimating the acute toxicity of unresolved polar compounds from field-weathered oils.

Crude oil spilled at sea is chemically altered through environmental processes such as dissolution, biodegradation, and photodegradation. Transformation of hydrocarbons to oxygenated species increases water-solubility. Metabolites and oxidation products largely remain uncharacterized by common analytical methods but may be more bioavailable to aquatic organisms. Studies have shown that unresolved (i.e. unidentified) polar compounds ('UPCs') may constitute > 90% of the water-accommodated fraction (WAF) of heavily weathered crude oils, but still there is a paucity of information characterizing their toxicological significance in relation to other oil-derived toxicants. In this study, low-energy WAFs (no droplets) were generated from two field-weathered oils (collected during the 2010 Deepwater Horizon incident) and their polar fractions were isolated through fractionation. To allow establishment of thresholds for acute toxicity (LC50) of the dissolved and polar fraction of field collected oils, we concentrated both WAFs and polar fractions to beyond field-documented concentrations, and the acute toxicity of both to the marine copepod Acartia tonsa was measured and compared to the toxicity of the native WAF (non-concentrated). The difference in toxic units (TUs) between the total of the mixture and of identified compounds of known toxicity (polycyclic aromatic hydrocarbons [PAHs] and alkyl phenols) in both WAF and polar fractions was used to estimate the contribution of the UPC to overall toxicity. This approach identified that UPC had a similar contribution to toxicity as identified compounds within the WAFs of the field-weathered oils. This signifies the relative importance of polar compounds when assessing environmental impacts of spilled and weathered oil.

Effects of mine tailing exposure on the development of early life stages of the marine copepod Calanus finmarchicus.

The demand for mineral resources is increasing mining activities worldwide. In Norway, marine tailing disposal (MTD) is practiced, introducing mineral particles into fjord ecosystems. We investigated the effects of two concentrations (high and low) of fine tailings from a CaCO3 processing plant on early life stages of the marine copepod Calanus finmarchicus. Results show that the exposure did not significantly impact hatching success or development in non- and early feeding life stages. However, feeding stage nauplii ingested tailings, which caused a significantly slower development in later nauplii stages in high exposure groups, with most individuals being two stages behind the control group. Further, high mortality occurred in late nauplii and early copepodite stages in low exposure groups, which could be caused by insufficient energy accumulation and depleted energy reserves during development. Individuals exposed to high exposure concentrations seemed to survive by arresting development and potentially by reduced activity, thereby conserving energy reserves. In nature, slower development could affect lipid storage buildup and reproduction.

Do oil droplets and chemical dispersants contribute to uptake of oil compounds and toxicity of crude oil dispersions in cold-water copepods?

Accidental crude oil spills to the marine environment cause dispersion of oil into the water column through the actions of breaking waves, a process that can be facilitated using chemical dispersants. Oil dispersions contain dispersed micron-sized oil droplets and dissolved oil components, and the toxicity of oil dispersions has been assumed to be associated primarily with the latter. However, most hydrophobic, bioaccumulative and toxic crude oil components are retained within the droplets which may interact with marine filter-feeders. We here summarize the findings of 15 years of research using a unique methodology to generate controlled concentrations and droplet size distributions of dispersed crude oil to study effects on the filter-feeding cold-water copepod Calanus finmarchicus. We focus primarily on the contribution of chemical dispersants and micron-sized oil droplets to uptake and toxicity of oil compounds. Oil dispersion exposures cause PAH uptake and oil droplet accumulation on copepod body surfaces and inside their gastrointestinal tract, and exposures to high exposure (mg/L range) reduce feeding activity, causes reproductive impairments and mortality. These effects were slightly higher in the presence of chemical dispersants, possibly due to higher filtration of chemically dispersed droplets. For C. finmarchicus, dispersions containing oil droplets caused more severe toxic effects than filtered dispersions, thus, oil droplets contribute to the observed toxicity. The methodology for generating crude oil dispersion is a valuable tool to isolate impacts of crude oil microdroplets and can facilitate future research on oil dispersion toxicity and produce data to improve oil spill models.

Effects of Exposure Timing on cyp1a Expression, PAH Elimination, and Lipid Utilization in Lumpfish Embryos Exposed to Produced Water.

Intentional discharges of produced water from oil production platforms to the marine environment contain a complex mixture of toxicants, including polycyclic aromatic hydrocarbons (PAHs). Early life stages of fish are highly sensitive to petrogenic exposure, and short-term exposure during critical periods of embryonic development may have detrimental effects on larvae health and survival. However, why different periods are more sensitive to exposure than others are not fully understood. Three identical exposure experiments (48 h, approx. 30 µg/L tPAH, sum 42 PAHs) on lumpfish (Cyclopterus lumpus) embryos were conducted where only exposure timing was varied: 0-48 h post fertilization (hpf, starting before chorion hardening), 36-84 hpf (starting after chorion hardening), and 240-288 hpf (during organogenesis). Total PAH (tPAH) uptake at the end of exposure was 5× higher when exposed during fertilization than when exposed late (during organogenesis). The first evidence of cyp1a induction in lumpfish during embryogenesis was observed after 84 hpf. Early exposure affected lipid droplet coagulation, indicating altered lipid utilization during embryogenesis. Although no significant impacts of exposure were observed on hatching success, hatching was delayed when exposed at the latest time point. This study shows that chorion properties, lipid content, biotransformation potential, and timing of produced water exposure during lumpfish embryogenesis affected PAH uptake and elimination.

Recommendations for advancing media preparation methods used to assess aquatic hazards of oils and spill response agents.

Laboratory preparation of aqueous test media is a critical step in developing toxicity information needed for oil spill response decision-making. Multiple methods have been used to prepare physically and chemically dispersed oils which influence test outcome, interpretation, and utility for hazard assessment and modeling. This paper aims to review media preparation strategies, highlight advantages and limitations, provide recommendations for improvement, and promote the standardization of methods to better inform assessment and modeling. A benefit of media preparation methods for oil that rely on low to moderate mixing energy coupled with a variable dilution design is that the dissolved oil composition of the water accommodation fraction (WAF) stock is consistent across diluted treatments.  Further, analyses that support exposure confirmation maybe reduced and reflect dissolved oil exposures that are bioavailable and amenable to toxicity modeling.  Variable loading tests provide a range of dissolved oil compositions that require analytical verification at each oil loading. Regardless of test design, a preliminary study is recommended to optimize WAF mixing and settling times to achieve equilibrium between oil and test media. Variable dilution tests involving chemical dispersants (CEWAF) or high energy mixing (HEWAF) can increase dissolved oil exposures in treatment dilutions due to droplet dissolution when compared to WAFs. In contrast, HEWAF/CEWAFs generated using variable oil loadings are expected to provide dissolved oil exposures more comparable to WAFs. Preparation methods that provide droplet oil exposures should be environmentally relevant and informed by oil droplet concentrations, compositions, sizes, and exposure durations characteristic of field spill scenarios. Oil droplet generators and passive dosing techniques offer advantages for delivering controlled constant or dynamic dissolved exposures and larger volumes of test media for toxicity testing. Adoption of proposed guidance for improving media preparation methods will provide greater comparability and utility of toxicity testing in oil spill response and assessment.

Crude oil exposure of early life stages of Atlantic haddock suggests threshold levels for developmental toxicity as low as 0.1 µg total polyaromatic hydrocarbon (TPAH)/L.

Atlantic haddock (Melanogrammus aeglefinus) embryos bind dispersed crude oil droplets to the eggshell and are consequently highly susceptible to toxicity from spilled oil. We established thresholds for developmental toxicity and identified any potential long-term or latent adverse effects that could impair the growth and survival of individuals. Embryos were exposed to oil for eight days (10, 80 and 300 µg oil/L, equivalent to 0.1, 0.8 and 3.0 µg TPAH/L). Acute and delayed mortality were observed at embryonic, larval, and juvenile stages with IC50 = 2.2, 0.39, and 0.27 µg TPAH/L, respectively. Exposure to 0.1 µg TPAH/L had no negative effect on growth or survival. However, yolk sac larvae showed significant reduction in the outgrowth (ballooning) of the cardiac ventricle in the absence of other extracardiac morphological defects. Due to this propensity for latent sublethal developmental toxicity, we recommend an effect threshold of 0.1 µg TPAH/L for risk assessment models.

Monitoring ocean water quality by deployment of lumpfish (Cyclopterus lumpus) eggs: In situ bioaccumulation and toxicity in embryos.

Fish embryos can bioaccumulate and are particularly sensitive to a wide range of contaminants, which makes them suitable sentinels for environmental biomonitoring. However, fish embryos are very rarely utilized in environmental monitoring surveys, possibly due to their fragility and seasonality. In the present work, we assessed the applicability of caged lumpfish (Cyclopterus lumpus) eggs for in situ biomonitoring of exposure and effects of organic contaminants focusing on polyaromatic hydrocarbons and phenolic compounds. Fertilized eggs (1 dpf) were transplanted for 17-19 days at different locations that differed in terms of contaminant load, depths and weather conditions, namely at three stations close to the city of Trondheim (two harbour areas and a one in the Fjord) and three stations at a coastal aquaculture facility. High survival upon retrieval after deployment showed that lumpfish eggs are relatively robust and survive encaging in different environments. Bioaccumulation of organic contaminants (PAHs and phenolic compounds) was measured and potential effects on hatching, development, survival and larvae morphometry were determined. Chemical analyses showed that especially PAHs were effectively accumulated in eggs in contaminated sites, with concentrations of Æ©PAHs being 15 - 25 times higher in harbour areas compared to those at the aquaculture facility. A higher incidence of embryonic deformations was observed in the most polluted deployment location, but larvae morphometry revealed no evidence of toxicity related to pollutant body burden. In conclusion, the in-situ exposure method was proven to work well, making it attractive for implementations in environmental monitoring programs.

A flow-through imaging system for automated measurement of ichthyoplankton.

Microscopic imaging and morphometric measurement of fish embryos and larvae is essential in environmental monitoring of fish populations and to evaluate larvae development in aquaculture. Traditional microscopy methods require time-consuming, repetitive work by human experts. We present a method for fast imaging and analysis of millimetre-scale ichthyoplankton suspended in seawater. Our system can be easily built from common and off-the-shelf components and uses open-source software for image capture and analysis. Our system obtains images of similar quality to traditional microscopy, and biological measurements comparable to those by human experts, with minimal human interaction. This saves time and effort, while increasing the size of data sets obtained. We demonstrate our approach with cod eggs and larvae, and present results showing biologically relevant endpoints including egg diameter, larval standard length, yolk volume and eye diameter, with comparison to similar measurements reported in the literature. • High throughput, microscope-scale imaging of fish eggs and larvae • Automated measurement of biologically relevant endpoints • Easily built from off-the-shelf components and open-source software.

Chemical and toxicological characterisation of residues from offshore in-situ burning of spilled fuel oils.

Burn residues collected after large scale experimental in situ burns performed in the North Sea were characterised with emphasis on chemistry and acute toxicity. Low-energy water accommodated fractions (WAFs) of three marine fuels (Ultra Low Sulphur Fuel Oil (ULSFO), bunker oil (IFO180), and marine gas oil (MGO) and their field-generated residues from in situ burning (ISB). were prepared to evaluate the potential impact of ISB residue to the environment. The toxicity effects on primary consumers were assessed by testing on early life stage (nauplii) of the marine copepod Calanus finmarchicus. Toxicity studies showed that ISB decreased the acute toxicity of the WAFs compared to the initial oils. WAF of MGO had highest toxicity, and ISB residue of MGO seems to be more toxic than WAFs of fresh ULSFO and IFO180. Additive toxicity expressed as toxic unit (TU) based on the chemical composition also indicated that the toxicity of WAFs from ISB residues were lower than for the initial oils. The 2-3 ring PAHs seem to contribute most to the TU. Overall, the three offshore burns reduced the total mass of PAHs in the water accommodated fractions by >90 % compared to the released unburned oils and caused a reduction of the acute toxicity to copepod nauplii.

Application of chemical herders do not increase acute crude oil toxicity to cold-water marine species.

Chemical herders may be used to sequester and thicken surface oil slicks to increase the time window for performing in situ burning of spilled oil on the sea surface. For herder use to be an environmentally safe oil spill response option, information regarding their potential ecotoxicity both alone and in combination with oil is needed. This study aimed at assessing if using herders can cause toxicity to cold-water marine organisms. Our objective was to test the two chemical herders Siltech OP-40 (OP-40) and ThickSlick-6535 (TS-6535) with and without oil for toxicity using sensitive life stages of cold-water marine copepod (Calanus finmarchicus) and fish (Gadus morhua). For herders alone, OP-40 was consistently more toxic than TS-6535. To test herders in combination with oil, low-energy water accommodated fractions (LE-WAFs, without vortex) with Alaskan North Slope crude oils were prepared with and without herders. Dissolution of oil components from surface oil was somewhat delayed following herder application, due to herder-induced reduction in contact area between water and oil. The LE-WAFs were also used for toxicity testing, and we observed no significant differences in toxicity thresholds between treatments to LE-WAFs generated with oil alone and oil treated with herders. The operational herder-to-oil ratio is very low (1:500), and the herders tested in the present work displayed acute toxicity at concentrations well above what would be expected following in situ application. Application of chemical herders to oil slicks is not expected to add significant effects to that of the oil for cold-water marine species exposed to herder-treated oil slicks.

The impact of exposure timing on embryo mortality and the partitioning of PAHs when cod eggs are exposed to dispersed and dissolved crude oil.

During sub-sea oil spills to the marine environment, oil droplets will rise towards the sea surface at a rate determined by their density and diameter as well as the vertical turbulence in the water. Micro-droplets (< 50 µm) are expected to have prolonged residence times in the water column. If present, pelagic fish eggs may thus be exposed to dispersed oil from subsurface oil spills for days, and the contribution of these micro-droplets to toxicity is not well known. The purpose of this work was to investigate to what extent timing of exposure and the presence of oil micro droplets affects PAH uptake and survival of pelagic Atlantic cod eggs. A single batch of eggs was separated in two groups and exposed to dispersions and corresponding water-soluble fraction at 3-7 days (Early exposure) and 9-13 days (Late exposure) post fertilization. Partitioning of PAHs between crude oil microdroplets, water and eggs was estimated as well as the contribution of oil droplets to PAH body residue and acute and delayed mortality. Timing of oil exposure clearly affects both the mortality rate and the timing of mortality. Even though the body residue of PAHs were lower when embryos were exposed in the later embryonic stage, mortality rate increased relative to the early exposure indicating that critical body residue threshold is stage specific. Although our results suggest that the dissolved fraction is the dominating driver for toxicity in cod embryos exposed to oil dispersions, crude oil micro droplets contribute to increased mortality as well.

Exposure to low environmental copper concentrations does not affect survival and development in Atlantic cod (Gadus morhua) early life stages.

In this study we investigated potential impacts of Cu exposure at low, environmentally relevant, concentrations on early live stages of Atlantic cod (Gadus morhua). Cod embryos and larvae were exposed to 0.5 µg/L (low), 2 µg/L (medium), and 6 µg/L (high) Cu from 4 to 17 days post fertilisation (dpf). Hatching success, mortality, oxygen consumption, biometric traits, and malformations were determined. A dynamic energy budget (DEB) model was applied to identify potential impacts on bioenergetics. A positive correlation was found between Cu exposure concentrations and Cu body burden in eggs, but not in larvae. The tested concentrations did not increase mortality in neither embryos nor larvae, or larvae deformations. Further, the DEB model did not indicate effects of the tested Cu concentrations.

Atlantic cod (Gadus morhua) embryos are highly sensitive to short-term 3,4-dichloroaniline exposure.

3,4-dichloroaniline (3,4-DCA) is one of the most widely produced anilines world-wide, used in plastic packaging, fabrics, pharmaceuticals, pesticides, dyes and paints as well as being a degradation product of several pesticides. 3,4-DCA has been detected in freshwater, brackish and marine environments. Although freshwater toxicity thresholds exist, very little toxicological information is available on marine and cold-water species. In this study, we exposed Atlantic cod (Gadus morhua) embryos (3-7 days post fertilization) to 3,4-DCA concentrations ranging from 8-747 µg/L for 4 days followed by a recovery period in clean sea water until 14 days post fertilization (dpf). The cod embryos were significantly more sensitive to acute 3,4-DCA exposure compared to other species tested and reported in the literature. At the highest concentration (747 µg/L), no embryos survived until hatch, and even at the lowest concentration (8 µg/L), a small, but significant increase in mortality was observed at 14 dpf. Delayed and concentration-dependent effects on surviving yolk-sac larvae, manifested as cardiac, developmental and morphometric alterations, more than a week after exposure suggest potential long-term effects of transient embryonic exposure to low concentrations of 3,4-DCA.

Sources, distribution and effects of rare earth elements in the marine environment: Current knowledge and research gaps.

Rare earth elements and yttrium (REY) are critical elements for a wide range of applications and consumer products. Their growing extraction and use can potentially lead to REY and anthropogenic-REY chemical complexes (ACC-REY) being released in the marine environment, causing concern regarding their potential effects on organisms and ecosystems. Here, we critically review the scientific knowledge on REY sources (geogenic and anthropogenic), factors affecting REY distribution and transfer in the marine environment, as well as accumulation in- and effects on marine biota. Further, we aim to draw the attention to research gaps that warrant further scientific attention to assess the potential risk posed by anthropogenic REY release. Geochemical processes affecting REY mobilisation from natural sources and factors affecting their distribution and transfer across marine compartments are well established, featuring a high variability dependent on local conditions. There is, however, a research gap with respect to evaluating the environmental distribution and fate of REY from anthropogenic sources, particularly regarding ACC-REY, which can have a high persistence in seawater. In addition, data on organismal uptake, accumulation, organ distribution and effects are scarce and at best fragmentary. Particularly, the effects of ACC-REY at organismal and community levels are, so far, not sufficiently studied. To assess the potential risks caused by anthropogenic REY release there is an urgent need to i) harmonise data reporting to promote comparability across studies and environmental matrices, ii) conduct research on transport, fate and behaviour of ACC-REY vs geogenic REY iii) deepen the knowledge on bioavailability, accumulation and effects of ACC-REY and REY mixtures at organismal and community level, which is essential for risk assessment of anthropogenic REY in marine ecosystems.

Effects of marine mine tailing exposure on the development, growth, and lipid accumulation in Calanus finmarchicus.

Marine tailing disposal (MTD) is sometimes practiced as an alternative to traditional mine tailing deposition on land. Environmental challenges connected to MTD include spreading of fine particulate matter in the water column and the potential release of metals and processing chemicals. This study investigated if tailing exposure affects the marine copepod Calanus finmarchicus, and whether effects are related to exposure to mineral particles or the presence of metals and/or processing chemicals in the tailings. We investigated the impacts of three different tailing compositions: calcium carbonate particles with and without processing chemicals and fine-grained tailings from a copper ore. Early life stages of C. finmarchicus were exposed over several developmental stages to low and high suspension concentrations for 15 days, and their development, oxygen consumption and biometry determined. The data was fitted in a dynamic energy budget (DEB) model to determine mechanisms underlying responses and to understand the primary modes of action related to mine tailing exposure. Results show that copepods exposed to tailings generally exhibited slower growth and accumulated less lipids. The presence of metals and processing chemicals did not influence these responses, suggesting that uptake of mineral particles was responsible for the observed effects. This was further supported by the applied DEB model, confirming that ingestion of tailing particles while feeding can result in less energy being available for growth and development.

Cellular level response of the bivalve Limecola balthica to seawater acidification due to potential CO2 leakage from a sub-seabed storage site in the southern Baltic Sea: TiTank experiment at representative hydrostatic pressure.

Understanding of biological responses of marine fauna to seawater acidification due to potential CO2 leakage from sub-seabed storage sites has improved recently, providing support to CCS environmental risk assessment. Physiological responses of benthic organisms to ambient hypercapnia have been previously investigated but rarely at the cellular level, particularly in areas of less common geochemical and ecological conditions such as brackish water and/or reduced oxygen levels. In this study, CO2-related responses of oxygen-dependent, antioxidant and detoxification systems as well as markers of neurotoxicity and acid-base balance in the Baltic clam Limecola balthica from the Baltic Sea were quantified in 50-day experiments. Experimental conditions included CO2 addition producing pH levels of 7.7, 7.0 and 6.3, respectively and hydrostatic pressure 900 kPa, simulating realistic seawater acidities following a CO2 seepage accident at the potential CO2-storage site in the Baltic. Reduced pH interfered with most biomarkers studied, and modifications to lactate dehydrogenase and malate dehydrogenase indicate that aerobiosis was a dominant energy production pathway. Hypercapnic stress was most evident in bivalves exposed to moderately acidic seawater environment (pH 7.0), showing a decrease of glutathione peroxidase activity, activation of catalase and suppression of glutathione S-transferase activity likely in response to enhanced free radical production. The clams subjected to pH 7.0 also demonstrated acetylcholinesterase activation that might be linked to prolonged impact of contaminants released from sediment. The most acidified conditions (pH 6.3) stimulated glutathione and malondialdehyde concentration in the bivalve tissue suggesting potential cell damage. Temporal variations of most biomarkers imply that after a 10-to-15-day initial phase of an acute disturbance, the metabolic and antioxidant defence systems recovered their capacities.

Effects of mine tailing exposure on early life stages of cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Mining and processing of minerals produce large quantities of tailings as waste. Some countries, including Norway, allow disposal of mine tailings in the sea. In this study we investigated the impacts of tailings from a calcium carbonate (CaCO3) processing plant on early live stages of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua). Fish eggs (3 days post fertilisation; dpf) were exposed for 48 h to three concentrations of tailings, nominally 1 mg L-1 (low, L); 10 mg L-1 (medium, M) and 100 mg L-1 (high, H); with L and M representing concentrations occurring at tailing release points. Results show that tailings rapidly adhered to eggs of both species, causing negative buoyancy (sinking of eggs) in M and H exposures. While tailings remained on egg surfaces in both species also after exposure termination, adhesion seemed more pronounced in cod, leading to larger impacts on buoyancy even after exposure. Tailing exposure further induced early hatching and significantly reduced survival in M and H exposed embryos in both fish species, and in cod from the L exposure group. Moreover, tailing exposure caused reduced survival and malformations in larvae, potentially related to premature hatching. This study shows that mineral particles adhere to haddock and cod eggs, affecting egg buoyancy, survival and development.

Toxicity and developmental effects of Arctic fuel oil types on early life stages of Atlantic cod (Gadus morhua).

Due to the heavy fuel oil (HFO) ban in Arctic maritime transport and new legislations restricting the sulphur content of fuel oils, new fuel oil types are continuously developed. However, the potential impacts of these new fuel oil types on marine ecosystems during accidental spills are largely unknown. In this study, we studied the toxicity of three marine fuel oils (two marine gas oils with low sulphur contents and a heavy fuel oil) in early life stages of cod (Gadus morhua). Embryos were exposed for 4 days to water-soluble fractions of fuel oils at concentrations ranging from 4.1 - 128.3 µg TPAH/L, followed by recovery in clean seawater until 17 days post fertilization. Exposure to all three fuel oils resulted in developmental toxicity, including severe morphological changes, deformations and cardiotoxicity. To assess underlying molecular mechanisms, we studied fuel oil-mediated activation of aryl hydrocarbon receptor (Ahr) gene battery and genes related to cardiovascular, angiogenesis and osteogenesis pathways. Overall, our results suggest comparable mechanisms of toxicity for the three fuel oils. All fuel oils caused concentration-dependant increases of cyp1a mRNA which paralleled ahrr, but not ahr1b transcript expression. On the angiogenesis and osteogenesis pathways, fuel oils produced concentration-specific transcriptional effects that were either increasing or decreasing, compared to control embryos. Based on the observed toxic responses, toxicity threshold values were estimated for individual endpoints to assess the most sensitive molecular and physiological effects, suggesting that unresolved petrogenic components may be significant contributors to the observed toxicity.