Partitioning of PAHs between Crude Oil Micro ... - ACS Publications

Article Cite This: Environ. Sci. Technol. 2018, 52, 14436−14444

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Partitioning of PAHs between Crude Oil Microdroplets, Water, and Copepod Biomass in Oil-in-Seawater Dispersions of Different Crude Oils Bjørn Henrik Hansen,*,† Anders J. Olsen,‡ Iurgi Salaberria,†,‡ Dag Altin,§ Ida Beathe Øverjordet,† Piero Gardinali,∥ Andy Booth,† and Trond Nordtug† †

Environment and New Resources, SINTEF Ocean, 7465 Trondheim, Norway Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway § BioTrix, 7022 Trondheim, Norway ∥ Department of Chemistry and Biochemistry, Southeast Environmental Research Center, Florida International University, North Miami, Florida 33199, United States

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‡

S Supporting Information *

ABSTRACT: 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 watersoluble 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.

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INTRODUCTION

dispersed oil and thus oil droplet bioavailability vary significantly between species. Previous studies with larvae of freshwater and marine fish indicate that they are not sensitive to oil droplets.2,5,6 In contrast, ingestion of oil droplets can contribute significantly to oil components associated with filter-feeding organisms.7−13 To predict the behavior of oil dispersions in oil spill modeling based on data generated by experimental exposures, observed effects must be related to parameters that can be predicted in time and space; this includes concentration and chemical composition of total and dissolved hydrocarbons and oil droplet size distribution. However, field studies commonly fail to provide sufficiently detailed data on the physicochemical composition of oil in time and space, meaning that laboratory study data are generally used in modeling and contingency

Crude oil released into the sea during blow-outs at depth will form dispersions of small oil droplets down to a few micrometers in size. These droplets contain constituents which will equilibrate between the oil matrix of each droplet and the surrounding water. The distribution of oil components between the two phases is largely determined by their water solubility and droplet surface-to-volume ratio.1 However, partitioning of oil components between droplets and water is also dependent on oil composition and the amount of oil present per volume of water, which varies with time, oil type, magnitude, and location/depth of release, and physical parameters such as temperature, pressure, light conditions, and sea/ocean currents.1 At high dispersion concentrations, oil droplets will contain the majority of oil components present in oil dispersions, with only a small fraction of these components dissolved in the aqueous phase. The toxicity of oil dispersions has been largely attributed to those components that are present in the dissolved phase due to their higher bioavailability.2−6 However, exposure and uptake routes of © 2018 American Chemical Society

Received: Revised: Accepted: Published: 14436

August 15, 2018 November 20, 2018 November 27, 2018 November 27, 2018 DOI: 10.1021/acs.est.8b04591 Environ. Sci. Technol. 2018, 52, 14436−14444

Article

Environmental Science & Technology planning.14−17 The role of dispersed oil droplets in crude oil toxicity remains as an important area of research. The main objective of the present study was to elucidate the partitioning of oil and oil components between oil, water, and biomass for different oil types. In particular, we wanted to study the fate of oil microdroplets when interacting with filter feeders and their impact on partitioning of polycyclic aromatic hydrocarbons (PAHs) between water and biota. The cold water marine copepod Calanus f inmarchicus (Gunnerus) was selected as a model species due to its key ecological role in the northeastern Atlantic.18,19 To identify potential oil-specific differences in partitioning dynamics, C. f inmarchicus were exposed to three moderately weathered crude oils with different physicochemical properties classified as paraffinic, naphthenic, and waxy oil.

copepods were counted and 20 live copepods (approximately 18 mg total wet weight) were sampled from each chamber and stored at −20 °C while awaiting body residue analysis. As controls, identical chamber design, feeding regime, and sampling regimes were used, but they were treated with seawater only. Exposure Characterization. Number, concentration, and size distribution of oil droplets and/or algae feed were measured daily in samples from the outlet of each chamber using a Multisizer 3 Coulter Counter (Beckman Coulter Inc., Brea CA, USA). To calculate the oil droplet concentration in each exposure chamber the algal mass was subtracted using a standard algal size distribution and baseline correction.11 Water was sampled from each exposure chamber after 24 and 72 h exposure in 1 L glass bottles for analysis of semivolatile organic components (SVOC; 800 mL) and in sealed glass vials without headspace for analysis of volatile organic components (VOC; 40 mL). A detailed summary of the sample preparation and analytical chemical methods is presented in the Supporting Information (S2). All analyzed components are listed in the Supporting Information (S3: Table S1) and are common target components used in postoil spill damage assessment.24,25 Body Residue Analysis. After weighing the copepod samples in conical, screw-capped sample vials (10 mL) with replaceable Teflon septa, 3 mL of KOH (6.5%) in methanol (80%) and surrogate internal standards (naphthalene-d8, phenanthrene-d10, chrysene-d12, phenol-d6, 4-methylphenold8) were added to each vial. The mixture was saponified for 2 h in an ultrasonic bath at 80 °C, followed by filtration and serial extraction with 4 mL of Milli-Q water/2 × 3 mL of hexane/4 mL of Milli-Q water and 0.5 mL of saturated NaCl. The combined organic extracts were dried with anhydrous Na2SO4 and concentrated to ∼0.5 mL using a Zymark Turbovap 500 Concentrator. Extract cleanup was performed by solid-phase extraction using 3 mL columns containing 0.5 g of normal phase silica packing (Superclean LC-Si, Supelco Bond Elut, SI, Agilent). The samples were eluted through the column with 3 × 2 mL of DCM:hexane (1:3). A list of the quantified target oil components is shown in the Supporting Information (Table S1), and a detailed description of body residue analysis including LOI/LOQ is given in Supporting Information S2−S4. It should be noted that this method does not distinguish between oil components that are taken up into the body and those adhered to the organism surface. Fluorescence Microscopy and Image Capture. After 4 days of exposure, surviving copepod feeding activity was assessed based on gut filling visualized by the autofluorescence of algal chlorophyll a and degradation products.7 To achieve simultaneous identification of oil droplets either ingested or adhered to the copepod surface, the B-2A (Nikon Corp., Tokyo, Japan) filter cube was replaced with a triband filter cube (D/F/Tr, Nikon Corp., Tokyo, Japan). To further improve color separation in the image, the signal in the blue channel from the camera was removed by postprocessing in Adobe PhotoShop v.12.1 (Adobe Corp., San Jose, CA, US). Data Analysis. Introducing dispersed oil to seawater in the presence of copepods will lead to the partitioning of oil and selected oil components between these environmental compartments until a dynamic equilibrium is reached. The following calculations assume this equilibrium is reached during the 96 h exposure, which is most likely not the case for high log Kow components.

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EXPERIMENTAL SECTION Copepods and Husbandry. Late copepodite stages (CV, the last stage before maturation) and adults of C. f inmarchicus were randomly selected from an in-house culture established in 2004 at the facilities of SINTEF/NTNU Sealab in Trondheim, Norway.20 During exposure, copepods were continuously fed 1.3 mg/L of the microalgae Rhodomonas baltica Karsten with a median cell diameter of 7.00 ± 0.035 μm (sphere equivalent based on volume) roughly corresponding to 150 μg carbon/L. Experiments were performed at dim light conditions at 10°C. Exposure. Three crude oils were selected to represent the three main oil types based on their physicochemical properties according to Daling et al.:21 paraffinic (Mississippi Canyon 252), naphthenic (Troll North Sea), and waxy (Alvheim North Sea). All oils were artificially weathered using a simple onestage distillation step (heating to 150 °C for 1 h) to reduce the content of volatiles (particularly BTEX) and increase the environmental relevancy of the tested oils,22 and the 150 °C residues were used in the experiments. Primary dispersions of 20 mg oil/L seawater were generated in-line as described by Nordtug et al.,23 creating a stabile and constant droplet size distribution with a mean volume based diameter of 10−14 μm. The primary dispersion was diluted in-line with filtered natural seawater at dispersion-to-water ratios of 1:1 (10 mg/L), 1:10 (2 mg/L), and 1:25 (0.4 mg/L) by computer-aided pulsing of 3-way solenoid valves. At each dilution step, 50% the dispersion was fed directly into an exposure chamber while the remaining 50% was filtered to remove the droplets and extract the water-soluble fraction (WSF) before being fed into a parallel exposure chamber. The filtration system used consisted of a custom-made in-line filtration unit (250 mL) containing loosely packed fine glass wool (15 g) above a Whatman GF/C and a GF/F Glass Microfiber Filter (Whatman Ltd., Maidstone, UK) with nominal particle retention of 1.2 and 0.7 μm, respectively. The design of the exposure chambers (5L) has been described previously.23 Dispersions and WSFs were continuously fed into each exposure chamber at a rate of 17 mL/min. Exposure solution volumes were maintained constant by a 300 μm mesh covered overflow outlet from each chamber. A detailed description of the exposure system can be found elsewhere.23 Experimental Design. Two hundred copepods were placed in each exposure chamber and exposed for 96 h to nominal concentrations of 0 (controls), 0.4, 2, or 10 mg/L of the dispersion and the corresponding WSFs of the paraffinic, naphthenic, or waxy oil; 4 replicates were included for each exposure (Supporting Information S1). After 96 h, surviving 14437

DOI: 10.1021/acs.est.8b04591 Environ. Sci. Technol. 2018, 52, 14436−14444

Article

Environmental Science & Technology

Statistical Analysis. Procedures for the evaluation of chemical data, detection limits, and procedures for removal of outliers are described in Supporting Information (S4). Control and exposed groups were compared by analysis of variance (ANOVA) and Dunnett’s posthoc test using GraphPad Prism statistic software, V4.00 (GraphPad Software, Inc.). Differences were considered significantly different when p < 0.05 unless stated otherwise.

The fraction of PAHs contained in the oil phase (Foil) was calculated as the difference in concentration between the dispersion (Cdisp) and the corresponding WSF (CWSF) related to the total concentration in the dispersion Foil = (Cdisp − C WSF)/Cdisp

(1)

The same equation was used for both for individual PAHs and for aggregated groups of PAHs. Bioconcentration factors (BCFs) for individual components (x) were calculated as BCFx = Cx bio/Cx WSF

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RESULTS AND DISCUSSION Exposure. The concentrations of both the dispersion and the WSF exposure series were maintained relatively constant throughout the experiment, proving that the exposure system worked acceptably. However, measured dispersion concentrations were lower than nominal values and also varied between the different oil qualities. The mean oil droplet concentrations of the highest exposure (measured by particle counting) were 7.0, 4.1, and 5.6 mg/L for the paraffinic, naphthenic, and waxy oil, respectively (Supporting Information S5 Table S3). This corresponds to measured concentrations of 70%, 41%, and 56% of the nominal concentration of 10 mg/L. Total hydrocarbon concentrations (THC; C5−C36) determined from GC/FID analyses revealed even lower values of 62%, 30%, and 33%, respectively. Discrepancies between measured and nominal values are possibly related to oil droplets surfacing in the settling chamber or exposure containers and adhesion to wetted surfaces of tanks and tubing in the system. Droplet size distributions were kept similar in all experiments, and differences between measured and nominal concentrations between the oil types are most likely related to their different physicochemical properties which will influence processes such as adhesion to surfaces (stickiness) and ability to coalesce. The difference between THC concentrations measured by GC/FID particle mass recorded by the particle counting of oil is assumed to indicate the fraction of the oil that is not extracted by DCM and therefore not present in the extracts analyzed by GC/FID. Survival of Copepods. At the highest dispersion concentration, most copepods displayed symptoms of narcosis observed as light discoloration and reduced swimming ability. For all oil types, a significantly (p < 0.05) higher mortality (17−24%) relative to the corresponding WSF (4−7%) and controls (2−5%) was only observed at the highest dispersion concentrations (Supporting Information 6 Figure S2). To test the short-term recovery of the surviving copepods, those copepods remaining after sampling from exposure to paraffinic oil were transferred to clean seawater for a subsequent 96 h. No further mortality was observed during the recovery period. Oil Component Partitioning between Oil and Water. A detailed summary of the concentrations of the different oil component fractions in each exposure is provided in the Supporting Information (S5 Table S3). As expected, the THC of all three oils studied was ∼30 times higher in the dispersion than the WSF for high-exposure concentrations. A 6−11-fold difference was observed for medium concentrations. The concentrations of the low-exposure concentration in most instances were too low to produce reliable data and are not included in the following sections unless otherwise stated. The total volatile component concentrations were low ( 5.9). We assume that these components are exclusively found in the oil phase. The AF for each of these components was calculated individually using eq 3. However, to estimate the oil-associated uptake it was assumed that the oil-associated body residue (BR) is the difference between the BR obtained in the dispersion exposure (CBRdisp) and that of the corresponding WSF (CBR WSF) C BR oil = C BRdisp − C BR WSF

(4)

The AF associated with oil droplets (AFoil) was then calculated according to eq 3, replacing Cx bio with CBR oil AF′x oil = C BR oil /Cx disp

(5)

where AF′x oil is the AF of a PAH with log Kow > 5.9. The oil volume associated with the biomass was then calculated based on the droplet concentrations in the water and the corresponding average AF′oil for each exposure group Voil bio = AF′x Voil disp

(6)

where Voil bio is the oil volume fraction associated with the biomass (ppm) and Voil disp the oil volume fraction in the dispersion (ppm). The relationship between AF and Kow for individual oil components was evaluated using an adapted version of the bilinear model,26 which is based on a hypothetical multicompartment biological system for chemical uptake and distribution log AF = a log Kow − b log(β log Kow + 1) + c

(7)

where a, b, and c are linear terms and β is the volume ratio between the lipid and the aqueous phases of the system. 14438


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Figure 1. Fraction (mean) of individual PAH’s associated with oil droplet fraction as a function of their log Kow in dispersions of paraffinic (A), naphthenic (B), and waxy (C) oil. Solid line and filled dots depict high concentrations, whereas medium concentrations are shown by broken line and open dots. Too few components were detected to allow a regression analysis for the low-dispersion exposures. Curve fit using a threeparameter nonlinear dose−response with restrictions on maximum and minimum value (1 and 0, respectively).

Since the aqueous solubility for a given component is constant at similar physical conditions, the equilibrium water concentration of a component according to eq 8 will remain constant as long as the molar fraction in the oil is maintained. As the oil:water ratio decreases, however, more of the total mass of individual components will be transferred to the water and thus the molar fraction in the oil matrix will ultimately be reduced. This will be associated with a decline in the water concentration. Since lipophilicity (e.g., measured as log Kow) is inversely related to solubility, the depletion of the mass fraction contained in the oil first declines for the components at in the lowest log Kow range (highest solubility). Further dilution causes successively more of the mass of high log Kow and low solubility components to be transferred to the water phase. This results in a higher mass fraction of the less soluble components (e.g., Kow > 5) in the aqueous phase at lower oil:water ratios (Figure 1). Similar observations of a relative increased dissolution of higher molecular weight oil components corresponding to an increase in their mole fraction have been reported in crude oil and coal−oil solubility studies31−33 but to our knowledge not in crude oil droplet dispersions. Thus, when dispersions are diluted, the composition and concentration of the WSF of crude oil dispersions will not simply follow the dilution factor. This is of importance in toxicity testing of oil dispersions, where the dissolved components are bioavailable and regarded as the main source of acute toxicity in marine organisms.2,6 High Kow components display higher acute toxicity per mass unit due to their higher potential for bioaccumulation. Since the water concentration of the heavier components may remain fairly unchanged while the lighter components become diluted, the relative contribution of heavier components to toxicity will increase with dilution. Although this causes the mass-based acute toxicity to increase during the dilution process as indicated, for instance, by a reduction in EC/LC50, the overall acute toxicity of the mixture is usually reduced or unchanged due to the decrease in total concentration of the WSF.34 PAH Bioconcentration and Accumulation. At medium and high concentrations of the three oils tested, uptake of PAHs in C. f inmarchicus biomass was significantly above control values after 96 h exposure (Supporting Information Tables S4−S6). A tendency toward accumulation at the lowest concentration was also apparent, although this could not be confirmed statistically due to high variation in the analysis results partly due to interference with biogenic material from the algae in the water. Copepods exposed to both dispersions

the volatiles constitute 50−93% of the characterized material in the WSFs and are expected to contribute significantly to the body residue. Unfortunately, we were not able to quantify the body residue of volatiles with the current methodology, and the partitioning studies were restricted to PAHs only. The volatiles (most with log Kow < 4.5) are expected to partition mainly to the water phase and have a similar BCF−Kow relationship as the PAHs. The highest PAH concentration in the dispersion exposures and WSF exposures was measured in the paraffinic oil and naphthenic oil, respectively. Individual concentrations of C0−C4 naphthalenes, 2−3 ring polycyclic aromatic hydrocarbons (PAHs), and 4−5 ring PAHs fractions were also low ( 97% for the 4−5 ring PAHs (Supporting Information Table S3). The mass distribution of individual PAHs between oil and water was calculated from the WSF concentrations and the difference in concentration between the dispersions and the corresponding WSFs (eq 1). The mass fraction of an individual component associated with the oil phase in the dispersion increased with log Kow. Components with log Kow > ∼6 have very low solubility in seawater, being almost exclusively associated with the oil droplets in the dispersion (Figure 1). Dilution of the dispersions caused a redistribution of the PAHs between the water and the oil droplet phases, increasing the mass fraction present in the water phase. This implies a net transfer of PAH mass from the oil to the water after dilution. This is visualized by the shift with approximately 1 log unit along the x axis between curve fits for medium and high concentrations in Figure 1. The shift in mass distributions shown is consistent with Raoult’s law27 and may be illustrated by using a simplified expression to describe the partitioning between an organic phase and water28−30 Cw = xoS l

(8)

where Cw is the chemical’s concentration in the aqueous phase (mol/L) in equilibrium with the organic phase, Sl is the aqueous solubility of the pure chemical (mol/L), and xo is the mole fraction of the chemical in the organic phase. 14439


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Figure 2. (A) Relationship between Kow and BCF calculated for individual PAHs in WSFs. Data compiled from three different oils and mediumand high-exposure concentrations (total N = 150). Curve fitted by bilinear model (R2 = 0.80). Bars indicate average ± STDEV. (B−D) Accumulation factors (AFs) for individual PAHs (mean values for n = 4) plotted as a function of log Kow. Dotted lines indicate the partitioning between water and biomass from 96 h exposure to WSF (from A), whereas the solid lines indicate partitioning between dispersions and biomass: (B) paraffinic oil; (C) naphthenic oil; (D) waxy oil.

solubility of the component in lipid and water. The lipid solubility of a component has traditionally been related to its Kow with the assumption that octanol is a suitable proxy for all relevant lipids in organisms. Deviation from a linear correlation between log BCF and log Kow (Figure 2A) demonstrates the shortcomings of this simple model and is supported by previous observations of deviations in the thermodynamic activities of large nonpolar molecules between fish lipids and octanol.44 It may be argued that steric hindrance of larger molecules may severely hamper their diffusion through biological membranes, resulting in low uptake rates and long equilibrium times. In line with this assumption, Connell and Hawker45 used a fugacity-related model to calculate the exposure time to equilibrium for a component with log Kow = 7 to be close to 325 days in fish. Correspondingly, Seto and Handoh46 calculated long exposure times to reach equilibrium for high-Kow components from a diffusion-related model and highlighted studies reporting equilibrium times of up to several weeks for high molecular weight pollutants in phytoplankton. In the present study, BCFs of components with Kow up to ∼6 were calculated, and at least some of the deviation from unity in the observed BCF−-Kow correlation may be related to insufficient time to reach equilibrium. Similar to the BCF, the accumulation of PAHs in the copepods exposed to dispersions may be related to the concentration of PAHs in the exposure solution including both water and oil droplets (Figures 2B−D) here denoted as the accumulation factor (AF, eq 3). It is evident that for the heavier PAHs the AFs of both the medium and the high exposures are lower in the dispersions compared to the BCF of the WSFs separated from the same dispersions. For components with relatively low log Kow values (5 lead to a leveling out of the BCF curve, a gradual decline is observed for AFs in all oil types. Heavier PAHs exhibit a lower dispersion AF than the WSF BCF due to the droplets retaining a large proportion of the oil components and preventing them from being readily bioavailable. It is also evident that a larger fraction of the heavier PAHs is associated with the copepods exposed to medium dispersion concentrations than for the high concentration. This is consistent with higher bioavailability

and their corresponding WSFs accumulated PAHs in an apparently concentration-dependent manner, reaching T-PAH body residues (BR T-PAH) of 193−288 μg g−1 wet weight at the highest dispersion concentrations for the three oil types tested. The uptake and toxicity of oil components in copepods in this study are consistent with results from earlier investigations of marine invertebrates exposed to single components35,36 and dispersed oil7−10,37,38 at similar concentration and time ranges, e.g., the T-PAH BRs converted to molar concentrations yield a range of 0.5−1.5 mmol/kg (wet weight), explaining the low mortality in our experiments as mortality typically occurs for nonpolar organic components for most invertebrates in the range 2−8 mmol/kg wet wt.35,39,40 Uptake of dissolved PAHs is generally concentration and time dependent through the process of passive diffusion,35,36 while uptake from dispersions may also be dependent on external adsorption (fouling),11 ingestion of contaminated algae,41 and/ or oil droplets.10−12,42 In a study by Berrojalbiz et al.,41 where the copepod Paracartia grani was exposed to either dissolved PAHs or PAHs in combination with PAH-contaminated algae (Rhodomonas salina), uptake of PAHs through contaminated algae was of limited importance for the overall PAH accumulation in copepods. Furthermore, oil fouling was also suggested to have a low contribution to oil accumulation in a study on C. f inmarchicus exposed to dispersed oil.11 On the basis of these findings, we will focus the rest of the paper primarily on passive uptake (bioconcentration of dissolved components) and active filtration of oil droplets (accumulation). For copepods exposed to WSFs of the three oils, bioconcentration factors (BCF) of individual PAHs could be calculated. After 96 h exposure to WSFs, BCFs (eq 1) there were no significant differences on the BCFs recorded from the different oil types. Compiled data (150 BCFs) from medium and high exposure of all three oils are shown in Figure 2A as a function of log Kow. An almost linear relationship is observed between the log values of BCF and Kow up to approximately log Kow = 5, beyond which log BCFs remain constant with increasing log Kow. For the whole Kow range, a nonlinear correlation (R2 = 0.800) by the bilinear model of Kubinyi et al.26 (eq 7) was found for all three oils. The falloff from a linear correlation between log BCF and log Kow at higher Kow values is consistent with studies on fish and several other aquatic organisms.43 Uptake of dissolved PAHs is generally considered a passive diffusion-driven process where the BCF of a component at equilibrium is proportional to the relative 14440


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Figure 3. (A) (Left) Section of copepod analyzed using fluorescence imaging. (Right) Image showing oil attached to the surface of the copepod (surface oil, A), oil stuck on the filtering apparatus (filtered oil, C), and oil located inside the digestive system of the copepod (ingested oil, B). (B) Representative fluorescence microscopy images of copepods exposed to dispersed oil (top three) and WSF (middle three) for the three different oils and controls (bottom three). Red color represents algae in the digestive system of copepods. Photo: Dag Altin, BioTrix.

suggesting that the observed relationships would also be valid for a broader range of crude oils. Oil Associated with Copepod BiomassFiltration and Fouling. Reduction in copepod filtration rates has been observed following exposure to dispersed oil7,11,13,37 and single-oil components.47 To address this issue in the context of oil droplet bioaccumulation, fluorescence microscopy was used to qualitatively assess the presence of algae in the copepod digestive system and the presence of oil droplets associated with copepods (Figure 3B). For copepods exposed to dispersions, oil droplets were observed as a green-yellow emission on the copepod surface, inside the digestive system, and on the filtering apparatus (Figure 3A). Algae present in the digestive system are colored orange-red (Figure 3B). Similar levels of algae were observed in the digestive system of copepods exposed to WSF and in control organisms. However,

of dissolved components and larger dissolved fraction in the medium concentration. No PAHs with log Kow > 6 were observed above the instrumental detection limit in the tissues of copepods exposed to WSF. However, components with log Kows up to 7 were present in detectable amounts in organisms exposed to dispersions (Figure 2B−D). The presence of droplets in the gut or adsorbed to the copepod surface (Figure 3A) is the most likely explanation for these observations. If the accumulation of components contained in oil is not dependent on Kow, the slope of the regression between Kow and AF should approach zero. Due to the variation in the data set this is not verifiable, but the slope especially for heavier PAHs appears to deviate from zero. This may indicate a transfer rate of components from the oil to the tissue that decreases with increasing log Kow. BCFs (for WSFs) and AFs (for dispersions) did not vary significantly between the three oil types, 14441


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associated with copepods and PAHs accumulated into copepod tissues. To a large extent, ingested oil droplets are probably excreted through fecal pellets.7,10 However, very slow PAH elimination in other studies17,48 suggests retainment of PAHs through partitioning to the lipid sac of copepods. When ovulating adult female copepods were exposed to dispersion or WSF, partitioning of heavy PAHs in their lipid sacs was displayed and elimination through transfer into copepod eggs was also evident.49 The potential transfer rates/kinetics of PAHs from droplets into copepod tissues, internal tissue partitioning, and elimination processes certainly deserves more attention.

in copepods exposed to high oil dispersions concentrations markedly lower intestinal algae contents were observed, indicating reduced filtration rates. This was also supported by a significant reduction (roughly estimated up to 90%) in fecal pellet production in dispersion-exposed copepods compared to controls and WSF-exposed copepods (data not shown). In fluorescence images, oil droplets were associated with the copepod surface as well as the gut (Figure 3A). Large PAHs with log Kow > 6 were mostly contained in the oil phase (Figure 1) and were below the detection limit in BR analyses of WSF-exposed copepods (Supporting Information Tables S4−S6). It is therefore likely that the accumulation of these components is related to the amount of oil associated with the copepod biomass. PAHs with log Kow > 5.9 were selected, and their average AFs (eq 5) at different dispersion concentrations are shown in Figure 4.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.8b04591.

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Description of the experimental design; sample preparation and analyses; target components; statistical treatment of outliers, including LOI/LOQ; data on analytical chemistry; copepod survival; algae concentrations in media (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +47 98283892; fax: +47 73595910; e-mail: bjorn.h. [email protected]. ORCID

Bjørn Henrik Hansen: 0000-0002-7599-4850 Ida Beathe Øverjordet: 0000-0001-5299-1756 Andy Booth: 0000-0002-4702-2210

Figure 4. Oil accumulation factors. Accumulation factor of heavy PAHs (log Kow > 5.9) in copepod (N = 8−10 except for the lowest concentration N = 3). (Mean and SD shown, N = 8−10 except N = 3 for the lowest concentration, no low exposure data for paraffinic).

Notes

The authors declare no competing financial interest.

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If these components are exclusively contained in the oil, the AF determined for heavy PAHs can be assigned to the presence of oil droplets in/on the biomass (eq 6). Assuming a density of the oil of 0.9, the estimated oil droplet concentration associated with copepods ranged between 2 and 10 g/kg biomass (wet weight), depending on exposure concentration and oil type. The highest associated oil concentration was found in the medium-exposure concentrations, suggesting a sustained filtration activity in copepods which is reduced in copepods exposed to high-dispersion concentrations. This is consistent with the observations done by fluorescence imaging as well as previous findings where an almost complete cessation of filtration activity was observed at dispersion concentrations > 4 mg/L.7,11 The average concentration of algae in the exposure solution was also higher for the highest exposure groups than for the control and low-exposure groups, indicating reduced filtration rates at high-exposure concentrations (Supporting Information S7, Figure S3). For all three oils the estimated average oil concentration associated with the biomass was higher in the medium exposure than in the high exposure. Potential differences in accumulation of oil components between oil types may result from varying degrees of stickiness between crude oils affecting the ability of the copepods to filter oil. Filtration may be an important driver for droplet uptake and subsequent PAH accumulation in filter feeders. On the basis of the present study, it is not possible to distinguish between PAHs still entrained within droplets

ACKNOWLEDGMENTS We thank Kjersti Almås and Marianne U. Rønsberg for their technical assistance with chemical analyses of water and biota samples. This work was supported by BP Exploration & Production Inc. and the BP Gulf Coast Restoration Organization.

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REFERENCES

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Partitioning of PAHs between Crude Oil Micro ... - ACS Publications

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