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.

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 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.

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.

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.

Comparison of artificially weathered Macondo oil with field samples and evidence that weathering does not increase environmental acute toxicity.

Macondo source oils and artificially weathered oil residues from 150 °C+ to 300 °C+, including artificially photo oxidized oils, were prepared and used for generating low energy water accommodated fractions (LE-WAFs) in order to assess the impact of oil weathering on WAF chemistry composition and toxicity to marine organisms. Two pelagic species representing primary producers (the marine algae Skeletonema pseudocostatum) and invertebrates (the marine copepod Acartia tonsa) were tested. Obtained acute toxicity levels, expressed as EC/LC50 values, were in the same range or above the obtained maximum WAF concentrations for WAFs from most weathering degrees. Based on % WAF dilutions, reduced toxicity was determined as a function of weathering. The chemical compositions of all WAFs were compared to compositions obtained from water samples reported in the GRIIDC database using multivariate analysis, indicating that WAFs of photo oxidized and two field weathered oils resembled the field data the most.

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.

Developmental effects in fish embryos exposed to oil dispersions - The impact of crude oil micro-droplets.

During accidental crude oil spills and permitted discharges of produced water into the marine environment, a large fraction of naturally occurring oil components will be contained in micron-sized oil droplets. Toxicity is assumed to be associated with the dissolved fraction of oil components, however the potential contribution of oil droplets to toxicity is currently not well known. In the present work we wanted to evaluate the contribution of oil droplets to effects on normal development of Atlantic cod (Gadus morhua) through exposing embryos for 96 h to un-filtered (dispersions containing droplets) and filtered (water soluble fractions) dispersions in a flow-through system at dispersion concentrations ranging from 0.14 to 4.34 mg oil/L. After exposure, the embryos were kept in clean seawater until hatch when survival, development and morphology were assessed. The experiment was performed at two different stages of embryonic development to cover two potentially sensitive stages (gastrulation and organogenesis). Exposure of cod embryos to crude oil dispersions caused acute and delayed toxicity, including manifestation of morphological deformations in hatched larvae. Oil droplets appear to contribute to some of the observed effects including mortality, larvae condition (standard length, body surface, and yolk sac size), spinal deformations as well as alterations in craniofacial and jaw development. The timing of exposure may be essential for the development of effects as higher acute mortality was observed when embryos were exposed from the start of gastrulation (Experiment 1) than when exposed during organogenesis (Experiment 2). Even though low mortality was observed when exposed during organogenesis, concentration-dependent mortality was observed during recovery.

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.

Modeling the toxicity of dissolved crude oil exposures to characterize the sensitivity of cod (Gadus morhua) larvae and role of individual and unresolved hydrocarbons.

Toxicity of weathered oil was investigated using Atlantic cod (Gadus morhua) larvae. A novel exposure system was applied to differentiate effects associated with dissolved and droplet oil with and without dispersant. After a 4-day exposure and subsequent 4-day recovery period, survival and growth were determined. Analytical data characterizing test oil composition included polyaromatic hydrocarbons (PAH) based on GC/MS and unresolved hydrocarbon classes obtained by two-dimensional chromatography coupled with flame ionization detection was used as input to an oil solubility model to calculate toxic units (TUs) of dissolved PAHs and whole oil, respectively. Critical target lipid body burdens derived from modeling characterizing the sensitivity of effect endpoints investigated were consistent across treatments and within the range previously reported for pelagic species. Individually measured PAHs captured only 3-11% of the TUs associated with the whole oil highlighting the limitations of traditional total PAH exposure metrics for expressing oil toxicity data.

Partitioning of PAHs between Crude Oil Microdroplets, Water, and Copepod Biomass in Oil-in-Seawater Dispersions of Different Crude Oils.

The impact of oil microdroplets on the partitioning of polycyclic aromatic hydrocarbons (PAHs) between water and marine zooplankton was evaluated. The experimental approach allowed direct comparison of crude oil dispersions (containing both micro-oil droplets and water-soluble fraction; WSF) with the corresponding WSF (without oil droplets). Dispersion concentration and oil type have an impact on the PAH composition of WSFs and therefore affect dispersion bioavailability. Higher T-PAH body residues were observed in copepods treated with dispersions compared to the corresponding WSFs. PAHs with log Kow 3-4.5 displayed comparable accumulation factors between treatments; however, accumulation factors for less soluble PAHs (log Kow = 4.5-6) were higher for the WSF than for the dispersions, suggesting low bioavailability for components contained in oil droplets. The higher PAH body residue in dispersion exposures is assumed to result mainly from copepods grazing on oil droplets, which offers an alternative uptake route to passive diffusion. To a large degree this route is controlled by the filtration rates of the copepods, which may be inversely related to droplet concentration.

Toxicokinetics of Crude Oil Components in Arctic Copepods.

The risk of accidental oil spills in the Arctic is on the rise due to increased shipping and oil exploration activities, making it essential to calibrate parameters for risk assessment of oil spills to Arctic conditions. The toxicokinetics of crude oil components were assessed by exposing one lipid-poor (CIII) and one lipid-rich (CV) stage of the Arctic copepod Calanus hyperboreus to crude oil WSF (water-soluble fraction). Water concentrations and total body residues (BR), as well as lipid volume fractions, were measured at regular intervals during exposure and recovery. Bioconcentration factors (BCFs) and elimination rates ( ke) for 26 petrogenic oil components were estimated from one-compartment models fitted to the BR data. Our parameters were compared to estimations made by the OMEGA bioaccumulation model, which uses the octanol-water partitioning coefficient ( KOW) in QSAR (quantitative structure-activity relationship) predictions. Our parameters for the lipid-poor CIIIs generally agreed with the OMEGA predictions, while neither the BCFs nor the kes for the lipid-rich CVs fitted within the realistic range of the OMEGA parameters. Both the uptake and elimination rates for the CVs were in general half an order of magnitude lower than the OMEGA predictions, showing an overestimation of these parameters by the OMEGA model.

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.

Does Microbial Biodegradation of Water-Soluble Components of Oil Reduce the Toxicity to Early Life Stages of Fish?

Microbial degradation following oil spills results in metabolites from the original oil. Metabolites are expected to display lower bioaccumulation potential and acute toxicity to marine organisms due to microbial-facilitated incorporation of chemical functional groups and a general decrease in lipophilicity. The toxicity and characterization of metabolites are poorly studied. The purpose of the present work was to evaluate the toxicity of degraded (0-21 days) water-soluble oil components. Low-energy water accommodated fraction (LE-WAF) of a weathered crude oil was prepared with nutrient amended seawater at 5 °C, kept in the dark, and sampled at 0, 10, 14, and 21 days. Samples were extracted with dichloromethane and toxicity experiments were conducted with reconstituted extracts. Toxicity experiments were conducted for 4 days on developing cod ( Gadus morhua) embryos during a critical period of their heart development. After exposure, embryos were kept in clean seawater and observed until 5 days post hatch. Survival, hatching, morphometric aberrations, and cardiac function was studied. The expected decrease in sublethal toxicity during the biodegradation period was not found, indicating that metabolites formed during biodegradation likely contributed to larvae toxicity.

Acute and physical effects of water-based drilling mud in the marine copepod Calanus finmarchicus.

The aim of this study was to investigate impacts of fine particulate fraction of a commonly used barite-containing drilling mud on the pelagic filter feeding copepod Calanus finmarchicus. The results show that the tested drilling mud had a low acute toxicity on C. finmarchicus (LC50 > 320 mg/L) and that the observed toxicity was likely caused by dissolved constituents in the mud and not the particle phase containing the weighting agent barite. Further, animals were exposed to drilling mud at a concentration of 10 mg/L for 168 hr followed by a 100 hr recovery phase. A rapid uptake of drilling mud particles was observed, while the excretion was slow and incomplete even after 100 hr recovery in clean seawater. The uptake of drilling mud particles caused a significant increase in sinking velocity of copepods, indicating that uptake of drilling mud particles affected their buoyancy. Long-term exposure to low concentrations of drilling mud could therefore cause physical effects such as impacts on the animal's buoyancy which may affect the energy budget of the copepods.

Maternal polycyclic aromatic hydrocarbon (PAH) transfer and effects on offspring of copepods exposed to dispersed oil with and without oil droplets.

Copepods of the genus Calanus have the potential for accumulating lipophilic oil components due to their high lipid content and found to filter and ingest oil droplets during exposure. As female copepods produce eggs at the expense of lipid storage, there is a concern for transfer of lipophilic contaminants to offspring. To assess the potential for maternal transfer of oil components, ovigerous female copepods (Calanus finmarchicus) were exposed to filtered and unfiltered oil dispersions for 4 days, collected and eggs maintained in clean seawater and hatching and gene expression examined in hatched nauplii. Oil droplet exposure contributed to polycyclic aromatic hydrocarbon (PAH) uptake in dispersion-treated adult copepods, as displayed through PAH body residue analyses and fluorescence microscopy. Applying the latter methodology, transfer of heavy PAH from copepod mothers to offspring were detected Subtle effects were observed in offspring as evidenced by a temporal reduction in hatching success appear to be occurring only when mothers were exposed to the unfiltered oil dispersions. Offspring reared in clean water through to late naupliar stages were collected for RNA extraction and preparation of libraries for high-throughput transcriptome sequencing. Differentially expressed genes were identified through pairwise comparisons between treatments. Among these, several expressed genes have known roles in responses to chemical stress including xenobiotic metabolism enzymes, antioxidants, chaperones, and components of the inflammatory response. While gene expression results suggest a transgenerational activation of stress responses, the increase in relatively small number of differentially expressed genes suggests a minor long-term effect on offspring following maternal exposure.

Exposure to crude oil micro-droplets causes reduced food uptake in copepods associated with alteration in their metabolic profiles.

Acute oil spills and produced water discharges may cause exposure of filter-feeding pelagic organisms to micron-sized dispersed oil droplets. The dissolved oil components are expected to be the main driver for oil dispersion toxicity; however, very few studies have investigated the specific contribution of oil droplets to toxicity. In the present work, the contribution of oil micro-droplet toxicity in dispersions was isolated by comparing exposures to oil dispersions (water soluble fraction with droplets) to concurrent exposure to filtered dispersions (water-soluble fractions without droplets). Physical (coloration) and behavioral (feeding activity) as well as molecular (metabolite profiling) responses to oil exposures in the copepod Calanus finmarchicus were studied. At high dispersion concentrations (4.1-5.6mg oil/L), copepods displayed carapace discoloration and reduced swimming activity. Reduced feeding activity, measured as algae uptake, gut filling and fecal pellet production, was evident also for lower concentrations (0.08mg oil/L). Alterations in metabolic profiles were also observed following exposure to oil dispersions. The pattern of responses were similar between two comparable experiments with different oil types, suggesting responses to be non-oil type specific. Furthermore, oil micro-droplets appear to contribute to some of the observed effects triggering a starvation-type response, manifested as a reduction in metabolite (homarine, acetylcholine, creatine and lactate) concentrations in copepods. Our work clearly displays a relationship between crude oil micro-droplet exposure and reduced uptake of algae in copepods.