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.

Comparison of two field systems for determination of crude oil biodegradation in cold seawater.

Marine oil spills have devastating environmental impacts and extrapolation of experimental fate and impact data from the lab to the field remains challenging due to the lack of comparable field data. In this work we compared two field systems used to study in situ oil depletion with emphasis on biodegradation and associated microbial communities. The systems were based on (i) oil impregnated clay beads and (ii) hydrophobic Fluortex adsorbents coated with thin oil films. The bacterial communities associated with the two systems displayed similar compositions of dominant bacterial taxa. Initial abundances of Oceanospirillales were observed in both systems with later emergences of Flavobacteriales, Alteromonadales and Rhodobacterales. Depletion of oil compounds was significantly faster in the Fluortex system and most likely related to the greater bioavailability of oil compounds as compared to the clay bead system.

Comprehensive Two-Dimensional Gas Chromatography with Peak Tracking for Screening of Constituent Biodegradation in Petroleum UVCB Substances.

Petroleum substances, as archetypical UVCBs (substances of unknown or variable composition, complex reaction products, or biological substances), pose a challenge for chemical risk assessment as they contain hundreds to thousands of individual constituents. It is particularly challenging to determine the biodegradability of petroleum substances since each constituent behaves differently. Testing the whole substance provides an average biodegradation, but it would be effectively impossible to obtain all constituents and test them individually. To overcome this challenge, comprehensive two-dimensional gas chromatography (GC × GC) in combination with advanced data-handling algorithms was applied to track and calculate degradation half-times (DT50s) of individual constituents in two dispersed middle distillate gas oils in seawater. By tracking >1000 peaks (representing ∼53-54% of the total mass across the entire chromatographic area), known biodegradation patterns of oil constituents were confirmed and extended to include many hundreds not currently investigated by traditional one-dimensional GC methods. Approximately 95% of the total tracked peak mass biodegraded after 64 days. By tracking the microbial community evolution, a correlation between the presence of functional microbial communities and the observed progression of DT50s between chemical classes was demonstrated. This approach could be used to screen the persistence of GC × GC-amenable constituents of petroleum substance UVCBs.

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.

No observed developmental effects in early life stages of capelin (Mallotus villosus) exposed to a water-soluble fraction of crude oil during embryonic development.

The rise in offshore oil and gas operations, maritime shipping, and tourism in northern latitudes enhances the risk of oil spills to sub-Arctic and Arctic coastal environments. Therefore, there is a need to understand the potential adverse effects of petroleum on key species in these areas. Here, we investigated the effects of oil exposure on the early life stages of capelin (Mallotus villosus), an ecologically and commercially important Barents Sea forage fish species that spawns along the coast of Northern Norway. Capelin embryos were exposed to five different concentrations (corresponding to 0.5-19 µg/L total PAHs) of water-soluble fraction (WSF) of crude oil from 6 days post fertilization (dpf) until hatch (25 dpf), and development of larvae in clean seawater was monitored until 52 dpf. None of the investigated endpoints (embryo development, larval length, heart rate, arrhythmia, and larval mortality) showed any effects. Our results suggest that the early life stages of capelin may be more robust to crude oil exposure than similar life stages of other fish species.

Physicochemical characterization and quantification of nanoplastics: applicability, limitations and complementarity of batch and fractionation methods.

A comprehensive physicochemical characterization of heterogeneous nanoplastic (NPL) samples remains an analytical challenge requiring a combination of orthogonal measurement techniques to improve the accuracy and robustness of the results. Here, batch methods, including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM), as well as separation/fractionation methods such as centrifugal liquid sedimentation (CLS) and field-flow fractionation (FFF)-multi-angle light scattering (MALS) combined with pyrolysis gas chromatography mass spectrometry (pyGC-MS) or Raman microspectroscopy (RM) were evaluated for NPL size, shape, and chemical composition measurements and for quantification. A set of representative/test particles of different chemical natures, including (i) polydisperse polyethylene (PE), (ii) (doped) polystyrene (PS) NPLs, (iii) titanium dioxide, and (iv) iron oxide nanoparticles (spherical and elongated), was used to assess the applicability and limitations of the selected methodologies. Particle sizes and number-based concentrations obtained by orthogonal batch methods (DLS, NTA, TRPS) were comparable for monodisperse spherical samples, while higher deviations were observed for polydisperse, agglomerated samples and for non-spherical particles, especially for light scattering methods. CLS and TRPS offer further insight with increased size resolution, while detailed morphological information can be derived by electron microscopy (EM)-based approaches. Combined techniques such as FFF coupled to MALS and RM can provide complementary information on physical and chemical properties by online measurements, while pyGC-MS analysis of FFF fractions can be used for the identification of polymer particles (vs. inorganic particles) and for their offline (semi)quantification. However, NPL analysis in complex samples will continue to present a serious challenge for the evaluated techniques without significant improvements in sample preparation.

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.

Combined effects of crude oil exposure and warming on eggs and larvae of an arctic forage fish.

Climate change, along with environmental pollution, can act synergistically on an organism to amplify adverse effects of exposure. The Arctic is undergoing profound climatic change and an increase in human activity, resulting in a heightened risk of accidental oil spills. Embryos and larvae of polar cod (Boreogadus saida), a key Arctic forage fish species, were exposed to low levels of crude oil concurrently with a 2.3 °C increase in water temperature. Here we show synergistic adverse effects of increased temperature and crude oil exposure on early life stages documented by an increased prevalence of malformations and mortality in exposed larvae. The combined effects of these stressors were most prevalent in the first feeding larval stages despite embryonic exposure, highlighting potential long-term consequences of exposure for survival, growth, and reproduction. Our findings suggest that a warmer Arctic with greater human activity will adversely impact early life stages of this circumpolar forage fish.

DNA damage and health effects in juvenile haddock (Melanogrammus aeglefinus) exposed to PAHs associated with oil-polluted sediment or produced water.

The research objective was to study the presence of DNA damages in haddock exposed to petrogenic or pyrogenic polyaromatic hydrocarbons (PAHs) from different sources: 1) extracts of oil produced water (PW), dominated by 2-ring PAHs; 2) distillation fractions of crude oil (representing oil-based drilling mud), dominated by 3-ring PAHs; 3) heavy pyrogenic PAHs, mixture of 4/5/6-ring PAHs. The biological effect of the different PAH sources was studied by feeding juvenile haddock with low doses of PAHs (0.3-0.7 mg PAH/kg fish/day) for two months, followed by a two-months recovery. In addition to the oral exposure, a group of fish was exposed to 12 single compounds of PAHs (4/5/6-ring) via intraperitoneal injection. The main endpoint was the analysis of hepatic and intestinal DNA adducts. In addition, PAH burden in liver, bile metabolites, gene and protein expression of CYP1A, GST activity, lipid peroxidation, skeletal deformities and histopathology of livers were evaluated. Juvenile haddock responded quickly to both intraperitoneal injection and oral exposure of 4/5/6-ring PAHs. High levels of DNA adducts were detected in livers three days after the dose of the single compound exposure. Fish had also high levels of DNA adducts in liver after being fed with extracts dominated by 2-ring PAHs (a PW exposure scenario) and 3-ring PAHs (simulating an oil exposure scenario). Elevated levels of DNA adducts were observed in the liver of all exposed groups after the 2 months of recovery. High levels of DNA adduct were found also in the intestines of individuals exposed to oil or heavy PAHs, but not in the PW or control groups. This suggests that the intestinal barrier is very important for detoxification of orally exposures of PAHs.

The use of PAH, metabolite and lipid profiling to assess exposure and effects of produced water discharges on pelagic copepods.

Several laboratory studies have demonstrated that exposure to oil components cause toxicity to copepods, however, this has never been shown in natural populations of copepods. In the present study, we sampled copepods in an area of the North Sea with high density of oil production platforms discharging produced water. Environmental modelling was used to predict produced water and copepod trajectories prior to copepod sampling in situ. To maximise output from a minimal number of field samples, a novel and combined methodology was developed to allow exploitation of the same extract for several purposes; contaminant body burden, lipidomics, and metabolomics analysis. PAH body burdens were low compared to laboratory experiments where correlations between PAH body burden and acute toxicity, reproduction and molecular endpoints had been established. Still, station-specific PAH profiles strongly indicated copepod exposure to PW. NMR metabolomics, focusing on water-soluble metabolites, suggested no correlation between metabolites and stations. Interestingly, lipidomics analyses suggested site-specific fingerprints and profiles displayed for acyl-glycerols and wax esters. Potential effects of produced water exposure on lipid metabolism in copepods cannot be ruled out and deserves more attention. Our study exemplifies the importance of incorporating novel and improved analytical methodologies in environmental monitoring.

Establishing a link between composition and toxicity of offshore produced waters using comprehensive analysis techniques - A way forward for discharge monitoring?

Extracts of produced waters from five mature Norwegian Sea oil fields were examined as total organic extracts (TOEs) and after fractionation into operationally-defined 'polar' and 'apolar' fractions. The TOEs and fractions were examined by gas chromatography (GC), GC-mass spectrometry (GC-MS), two dimensional GC-MS (GC × GC-MS) and liquid chromatography with high-resolution spectrometry (LC-HRMS) techniques. Low molecular weight aromatics, phenols and other common petroleum-derived hydrocarbons were characterized and quantified in the TOEs and fractions. In addition, a range of more uncommon polar and apolar constituents, including those likely derived from production chemicals, such as trithiolane, imidazolines and quaternary amine compounds (so-called 'quats'), were tentatively identified, using GC × GC-MS and LC-HRMS. The acute toxicity of the TOEs and subfractions was investigated using early life stages of the marine copepod Acartia tonsa. Toxicity varied significantly for different PW TOEs and subfractions. For some PWs, the toxicity was attributed mainly to the 'polar' components, while that of other PWs was associated mainly with the 'apolar' components. Importantly, the observed toxicity could not be explained by the presence of the commonly reported compounds only. Although, due to the vast chemical complexity even of the sub-fractions of the PW extracts, specific compounds driving the observed toxicity could be not be elucidated in this study, the proposed approach may suggest a way forward for future revisions of monitoring regimes for PW discharges.

Embryonic exposure to produced water can cause cardiac toxicity and deformations in Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) larvae.

Regular discharges of produced water from the oil and gas industry represents the largest direct discharge of effluent into the marine environment worldwide. Organic compound classes typically reported in produced water include saturated hydrocarbons, monoaromatic and polyaromatic hydrocarbons (MAHs, PAHs) as well as oxygenated compounds, such as phenols, acids and ketones. This forms a cocktail of known and suspect toxicants, but limited knowledge is yet available on the sub-lethal toxicity of produced water to cold-water marine fish species. In the present work, we conducted a 4-day exposure of embryos of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) to produced water extracts equivalent to 1:50, 1:500 and 1:5000 times dilutions of raw effluent. No significant reduction in survival or hatching success was observed, however, for cod, hatching was initiated earlier for exposed embryos in a concentration-dependent manner. During recovery, significantly reduced embryonic heart rate was observed for both species. After hatch, larvae subjected to embryonic exposure to produced water extracts were smaller, and displayed signs of cardiotoxicity, jaw and craniofacial deformations. In order to improve risk assessment and regulation of produced water discharges, it is important to identify which produced water components contribute to these effects.

Accumulation and toxicity of monoaromatic petroleum hydrocarbons in early life stages of cod and haddock.

A multitude of recent studies have documented the detrimental effects of crude oil exposure on early life stages of fish, including larvae and embryos. While polycyclic aromatic hydrocarbons (PAHs), particularly alkyl PAHs, are often considered the main cause of observed toxic effects, other crude oil derived organic compounds are usually overlooked. In the current study, comprehensive two-dimensional gas chromatography coupled to mass spectrometry was applied to investigate the body burden of a wide range of petrogenic compounds in Atlantic haddock (Melanogrammus aeglefinus) and cod (Gadus morhua) embryos that had been exposed to sublethal doses of dispersed crude oil. Several groups of alkylated monoaromatic compounds (e.g. alkyl tetralins, indanes and alkyl benzenes), as well as highly alkylated PAHs, were found to accumulate in the fish embryos upon crude oil exposure. To investigate the toxicity of the monoaromatic compounds, two models (1-isopropyl-4-methyltetralin and 1-isopropyl-4-methylindane) were synthesized and shown to bioaccumulate and cause delayed hatching in developing embryos. Minor developmental effects, including craniofacial and jaw deformations and pericardial edemas, were also observed at the highest studied concentrations of the alkylindane.

Adhesion of mechanically and chemically dispersed crude oil droplets to eggs of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus).

Crude oil accidentally spilled into the marine environment undergoes natural weathering processes that result in oil components being dissolved into the water column or present in particulate form as dispersed oil droplets. Oil components dissolved in seawater are typically considered as more bioavailable to pelagic marine organisms and the main driver of crude oil toxicity, however, recent studies indicate that oil droplets may also contribute. The adhesion of crude oil droplets onto the eggs of pelagic fish species may cause enhanced transfer of oil components via the egg surface causing toxicity during the sensitive embryonic developmental stage. In the current study, we utilized an oil droplet dispersion generator to generate defined oil droplets sizes/concentrations and exposed Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) to investigate if the potential for dispersed oil droplets to adhere onto the surface of eggs was species-dependent. The influence of a commercial chemical dispersant on the adhesion process was also studied. A key finding was that the adhesion of oil droplets was significantly higher for haddock than cod, highlighting key differences and exposure risks between the two species. Scanning electron microscopy indicates that the differences in oil droplet adhesion may be driven by the surface morphology of the eggs. Another important finding was that the adhesion capacity of oil droplets to fish eggs is significantly reduced (cod 37.3%, haddock 41.7%) in the presence of the chemical dispersant.

Crude oil exposures reveal roles for intracellular calcium cycling in haddock craniofacial and cardiac development.

Recent studies have shown that crude oil exposure affects cardiac development in fish by disrupting excitation-contraction (EC) coupling. We previously found that eggs of Atlantic haddock (Melanogrammus aeglefinus) bind dispersed oil droplets, potentially leading to more profound toxic effects from uptake of polycyclic aromatic hydrocarbons (PAHs). Using lower concentrations of dispersed crude oil (0.7-7 µg/L ∑PAH), here we exposed a broader range of developmental stages over both short and prolonged durations. We quantified effects on cardiac function and morphogenesis, characterized novel craniofacial defects, and examined the expression of genes encoding potential targets underlying cardiac and craniofacial defects. Because of oil droplet binding, a 24-hr exposure was sufficient to create severe cardiac and craniofacial abnormalities. The specific nature of the craniofacial abnormalities suggests that crude oil may target common craniofacial and cardiac precursor cells either directly or indirectly by affecting ion channels and intracellular calcium in particular. Furthermore, down-regulation of genes encoding specific components of the EC coupling machinery suggests that crude oil disrupts excitation-transcription coupling or normal feedback regulation of ion channels blocked by PAHs. These data support a unifying hypothesis whereby depletion of intracellular calcium pools by crude oil-derived PAHs disrupts several pathways critical for organogenesis in fish.

Application of gas chromatography/tandem mass spectrometry to determine a wide range of petrogenic alkylated polycyclic aromatic hydrocarbons in biotic samples.

RATIONALE: Due to the inherent toxicity of these compounds, analysis of petrogenic alkylated polycyclic aromatic hydrocarbons (PAHs) and heteroaromatic compounds in environmental samples is crucial. However, most existing methods are not sensitive enough when the analyte concentrations are low. Gas chromatography/tandem mass spectrometry (GC/MS/MS) is well suited to tackle this challenge, but methods are yet to be fully developed. METHODS: A gas chromatograph coupled with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode was applied for the analysis. MRM transitions and analytical conditions were optimized for the cluster analysis of 24 different compound classes of alkylated PAHs and heteroaromatics present in crude oils. Further, the method was verified and applied to analyze the body burden of these compounds in crude oil-exposed haddock eggs. RESULTS: A comprehensive and sensitive method for the determination of C1-C4 alkylated naphthalenes, benzothiophenes, fluorenes, phenanthrenes, dibenzothiophenes, chrysenes, pyrenes and fluoranthenes was developed. The method showed good precision (RSD <15%), and the obtained concentrations were similar to those obtained by traditional GC/MS analysis. CONCLUSIONS: We demonstrate the successful application of this method towards crude oil-exposed fish eggs, where we are able to precisely measure total PAH body burden below 0.5 µg/g. The demonstrated method facilitates further miniaturization in sample preparation. Copyright © 2016 John Wiley & Sons, Ltd.

Optimization and comparison of miniaturized extraction techniques for PAHs from crude oil exposed Atlantic cod and haddock eggs.

Two miniaturized extraction methods for a wide range of 2-6 ring polycyclic aromatic hydrocarbons (PAHs) and their alkylated homologues in small lipid-rich biota samples (≤100 mg) have been developed. Both methods utilize liquid extraction (LE) prior to a clean-up step using either normal phase solid phase extraction (SPE) or mixed-phase dispersive SPE (dSPE). Optimization of the methods was achieved by comparing the type and amount of sorbents, drying agents, and solvents used. In order to improve the limits of detection (LOD) of target PAHs under high sensitivity gas chromatography-tandem mass spectrometry analysis, specific emphasis was given to minimizing lipid co-extraction. The optimized LE-SPE method comprised extraction with dichloromethane/n-hexane (1:1, v/v) and clean-up by silica SPE, whereas the optimized LE-dSPE method comprised extraction with acetonitrile and clean-up with PSA and C18 sorbents. The methods were validated and directly compared through the analysis of Atlantic cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) eggs exposed to oil. The LE-SPE method resulted in lower levels of co-extracted lipids (14.1 ± 1.7 ng/µL) than the LE-dSPE method (60 ± 14 ng/µL). Achieved PAH LODs for the LE-SPE method were typically an order of magnitude lower (<5 ng/g) than for the LE-dSPE method (<125 ng/g). The LE-SPE method offers the possibility for PAH analysis of small samples of fish eggs (~100 mg) exposed to small quantities of crude oil (~1-10 µg/L total PAHs).

Oil droplet interaction with suspended sediment in the seawater column: influence of physical parameters and chemical dispersants.

The interaction of dispersed oil droplets with large diameter suspended particulate materials (SPM) has been little studied. In the current study, particle size, oil characteristics and chemical dispersant significantly influence the adsorption of oil droplets to SPM in seawater. Sediments with a smaller particulate size (clay) approaching that of the oil droplets (2-20 µm) adsorbed more oil per gram than sediments with large particle size (sand). Heavier, more polar oils with a high asphaltene content adsorbed more efficiently to SPM than lighter, less polar oils. A decrease in the smaller, more water soluble oil components in the sediment adsorbed oil was observed for all oil types. Addition of chemical dispersant decreased the adsorption of oil droplets to suspended carbonate sand in an exponential-like manner. No change in the relative distribution of compounds adsorbed to the sediment was observed, indicating dispersants do not alter the dissolution of compounds from oil droplets.