Prediction of Ecotoxicity of Heavy Crude Oil: Contribution ofMeasured ComponentsHyun-Joong Kang,† So-Young Lee,†,‡ Ji-Yeon Roh,† Un Hyuk Yim,§ Won Joon Shim,§

and Jung-Hwan Kwon†,*†Division of Environmental Science and Ecological Engineering, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 136-713,Republic of South Korea‡Department of Environmental Engineering, Ajou University, Woncheon-dong, Yeongtong-gu, Suwon 443-749, Republic of SouthKorea§Oil and POPs Research Group, Korea Institute of Ocean Science and Technology, 391 Jangmok-myon, Geoje 656-834, Republic ofSouth Korea

*S Supporting Information

ABSTRACT: A prediction model for estimating the ecotox-icity of the water-accommodated fraction (WAF) and water-soluble fraction (WSF) of heavy crude oil is proposed. Iranianheavy crude oil (IHC), one of the major components of theHebei Spirit oil spill in Korea in 2007, was used as a modelcrude oil for the preparation of the WAF and the WSF.Luminescence inhibition of Vibrio f ischeri was chosen as themodel ecotoxicity test for evaluating the baseline toxicity ofaromatic hydrocarbons in the IHC. The measured concen-tration of each chemical species in WAF and WSF agreed wellwith the predicted soluble concentration calculated usingRaoult’s law from the measured amount in the IHC. Thisindicates that the toxic potential of an oil mixture can be evaluated from the dissolved concentration of each species, which inturn, may be predicted from the composition of the crude or weathered oils. In addition, the contribution of each species in themixture to the apparent luminescence inhibition by the WAF and the WSF was assessed using a concentration-addition model.The relative contributions of benzene, toluene, ethylbenzene, xylenes (BTEX), polycyclic aromatic hydrocarbons (PAHs), andalkylated PAHs in luminescence inhibition were estimated to be 76%, 2%, and 21%, respectively. It was further identified that C3-and C4-naphthalenes were the most important aromatic hydrocarbons responsible for baseline toxicity. This indicates thatalkylated PAHs would be the major components of oil-spill residue. Further research is needed to evaluate the fate andecotoxicity of alkylated PAHs.

■ INTRODUCTION

Marine oil spills are some of the most serious environmentalproblems worldwide. The adverse ecological effects caused byoil-spill residues have been studied for decades.1−7 However,linking the observed environmental effects at many differentlevels to the components of oil-spill residue is still a verychallenging problem, because oil is a complex mixture ofnumerous chemicals, whose chemical composition changeswith time because of weathering processes.Among the various ecotoxicological end points applied to

assess the impact of oil spills on the ecosystem, narcotic effector baseline toxicity is regarded as one of the most importanttoxic modes of action. This effect has been intensively studiedto explain the effects of oil mixtures.8−13 Petroleum oils aremainly composed of many hydrophobic hydrocarbons such asstraight-, branched-, cyclic-aliphatic hydrocarbons, and variousmono- and poly cyclic aromatic hydrocarbons that accumulatein the lipid tissues of the bodies of living organisms. This lipid

accumulation is useful in explaining the results of laboratoryecotoxicity tests using either the critical body burden or targetlipid model, especially for single chemicals. It must be pointedout that none of these hydrocarbons, individually, is likely to bepresent at concentrations high enough to cause baseline toxicityafter an oil spill although chemical analyses of residual oilcomponents using bioassays revealed that the aromaticfractions containing many mono- and polycyclic aromatichydrocarbons are the most important fractions in terms ofecotoxicity of oil-spill residues.14,15 Furthermore, it is believedthat the mode of toxic action of each aromatic hydrocarbon issimilar and does not interfere with that of the otherhydrocarbons.12,13 The cumulative toxicity can help explain

Received: September 29, 2013Revised: January 24, 2014Accepted: February 3, 2014Published: February 3, 2014

Article

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© 2014 American Chemical Society 2962 dx.doi.org/10.1021/es404342k | Environ. Sci. Technol. 2014, 48, 2962−2970

the gaps between the chemical analysis results of oil-spillresidues and the overall observed toxicity.13,16−18 Thus, a fewmixture toxicity models have been built based on thedissolution of individual components in water and the additivityof toxic effects by each component.9,13 However, contributionof each chemicals in a mixture still need to be evaluated becauseidentified components are often not enough to explain theoverall ecotoxicological effects.7,19,20

In this study, we propose a predictive model forluminescence inhibition of Vibrio f ischeri as a modelecotoxicological end point resulting from the dissolved speciesof heavy crude oil and evaluated the mixture effects of heavycrude oil by taking into account the contribution of individualchemicals identified. This is possible because the V. f ischeri testis a well-established ecotoxicity test,21 the EC50 values forindividual hydrocarbons are well-documented, and quantitativestructure−activity relationships for them are available.22−25

Iranian heavy crude oil (IHC) was chosen as the model crudeoil because it was one of the main components of the HebeiSpirit oil spill, which occurred in 2007.26,27 The massconcentrations of chemical species such as benzene, toluene,ethylbenzene, xylenes (BTEX), polycyclic aromatic hydro-carbons (PAHs), and alkylated PAHs were measured in theIHC. The water-accommodated fraction (WAF) and water-soluble fraction (WSF) of IHC were prepared in the laboratoryand subjected to chemical analyses and luminescence inhibitiontests. The measured concentration of each species in the WAFand WSF was compared with the predicted dissolvedconcentration using Raoult’s law. The overall luminescenceinhibition caused by the WAF and WSF was compared with thesum of the contributions of the identified individual speciesusing a concentration-addition model. The relative contributionof the various chemical groups in IHC to the inhibition ofluminescence was compared in order to identify the majorspecies responsible for marine ecotoxicity after an oil spill.

■ THEORY

Dissolution of a chemical species i from a mixture is describedby Raoult’s law, which states that the maximum dissolvedconcentration (Ci) is the product of the mole fraction of thespecies in the mixture (xi) and the subcooled liquid solubility inseawater (Ssw,i

L ), assuming an ideal mixture.

=C x Si i sw,iL

(1)

Many organic chemicals in crude oil are solids in their pureform at ambient temperature and pressure. For substancesexisting as solids, Ssw,i

L is estimated from fusion entropy (ΔSm,i)and melting point (Tm,i) using the relation,28

=Δ

−⎡⎣⎢

⎛⎝⎜

⎞⎠⎟⎤⎦⎥S S

S

R

T

Texp 1i

Li

m,i m,i

(2)

where SiL and Si are the subcooled liquid solubility of species i

and the solubility of pure solid, respectively, R is the gasconstant (8.314 J mol−1K−1), and T is temperature in K.The increased chemical activity of hydrophobic solutes in an

electrolyte solution such as seawater (i.e., the “salting out”effect) is empirically modeled by the Setschenow equation.Including the Setschenow constant (KS,i), eq 2 is rewritten tocalculate the value of subcooled liquid solubility in seawater(Ssw,i

L ):

= −Δ

−⎡⎣⎢

⎛⎝⎜

⎞⎠⎟⎤⎦⎥S S K

S

R

T

Texp( [salt] )exp 1sw,i

Li S,i tot

m,i m,i

(3)

where [salt]tot is the total salt concentration in artificial seawater(0.5 mol L−1).For evaluating the overall effects of baseline toxicants using a

concentration-addition model, it is useful to express the toxicityof a chemical species using the concept of toxic units (TU) (eq4).

= CTU /EC50i i i (4)

Using this expression, the contribution of the toxicity of themixture can be calculated as the sum of the contributions of theindividual chemicals (eq 5).

∑=TU TUii (5)

Thus, we can estimate the overall toxic unit of a mixture by eqs1−5 once we know the mole fractions of each chemical species,their relevant physicochemical properties, and the toxicologicalend point values for the individual chemicals (EC50 in thisstudy).

■ EXPERIMENTAL SECTIONMaterials and Chemicals. Artificial seawater was prepared

at 3.5% (w/w) by dissolving the artificial sea salt obtained fromSigma-Aldrich (St. Louis, MO, U.S.). Polydimethylsiloxane(PDMS) tubing used for the preparation of the WSF waspurchased from Dong-Bang Silicone Inc. (Gimpo, Republic ofKorea). GC2-grade dichloromethane and hexane werepurchased from Burdick & Jackson (Morristown, NJ, U.S.).ACS-grade sodium sulfate and silica gel were purchased fromFisher Scientific (Seoul, Republic of Korea). PAH surrogatestandards (naphthalene-d8, acenaphthene-d10, phenanthrene-d12, chrysene-d12, and perylene-d12) and o-terphenyl used for asurrogate standard for total petroleum hydrocarbons (TPHs)were purchased from Supelco (Bellefonte, PA, U.S.). p-Terphenyl-d14 used as a GC internal standard for PAHs and5-α-androstane used as a GC internal standard for TPHs werealso purchased from Supelco. The IHC collected on-boardbefore harbor unloading to prevent mixing with other crude oilswas obtained from SGS Korea Co. (Seoul, Republic of Korea).

Preparation of WAF and WSF of IHC. The WAF of IHCwas prepared by gently stirring 1 L of artificial seawater for 24 husing a 2.54-cm stir bar at 200 rpm in the dark with 5, 10, 20,and 40 g of IHC, minimizing the vortex according to Singer etal.29 The WAF solution for chemical analysis and toxicity assaywas taken from the bottom of the vessel to separate it from thefloating oil film. Because we needed to evaluate the toxicity ofthe hydrophobic aromatic hydrocarbons, the WSF was alsoprepared by saturating the water with the hydrophobicchemical species of IHC, which are highly permeable throughPDMS. To prevent the direct contact of the oil phase withwater, a PDMS tube (ID = 2 mm, OD = 3 mm) containing 5 gof IHC in the lumen was submerged in 1 L of artificial seawater(Figure 1). The solution was stirred at 200 rpm for a designatedtime, in dark, again using a 2.54-cm stir bar. The contact surfacearea was 160 cm2, which is approximately twice the contact areaof the oil−water interface for the preparation of the WAF.Freshly prepared WAF and WSF solutions were immediatelysubjected to a luminescence inhibition test and liquid−liquidextraction for chemical analyses.

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Extraction of Chemicals from IHC, WAF, and WSF. Forthe analysis of chemical concentrations in IHC, the crude oilsample was first dissolved in n-hexane, followed by cleanupusing a silica-gel column and fractionation. The oil solution wasspiked with appropriate surrogates (100 μL of 200 μg mL−1 o-terphenyl and 100 μL of a mixture of naphthalene-d8,acenaphthene-d10, phenanthrene-d12, chrysene-d12, perylene-d12, 10 μg mL−1 each). The spiked oil samples were transferredto 3 g of activated silica gel column topped with ∼1 cmanhydrous granular sodium sulfate layer for sample cleanup andfractionation. The column was eluted with 15 mL of n-hexane(F1), followed by 30 mL of n-hexane/dichloromethane (50:50)solution (F2). Half of the F1 fraction was used for the analysisof saturates, and half of the F2 fraction was used for the analysisof alkylated PAH homologues and other target PAHs. Theremaining halves of F1 and F2 were combined to form F3 andused for the determination of total GC-detectable TPHs, GC-resolved peaks, and the GC-unresolved complex mixture(UCM) of hydrocarbons. The detailed procedure of thefractionation for the analysis of aromatic hydrocarbons isdescribed in literature.27,30

Freshly prepared WAF and WSF were extracted by liquid−liquid extraction using dichloromethane. First, 500 mL of theaqueous sample, spiked with 30 μL of 10 μg mL−1 of the TPHsurrogate standard (o-terphenyl) and 100 μL of 20 μg mL−1

PAHs surrogate standards (naphthalene-d8, acenaphthene-d10,phenanthrene-d12, chrysene-d12, perylene-d12) were extractedwith 50 mL dichloromethane three times by a modified EPAmethod 3510C.31 In short, the dichloromethane extract wasevaporated using a rotary evaporator and the solvent wasexchanged with approximately 20 mL of n-hexane. Afterconcentrating the n-hexane solution to ∼2 mL, the extractwas fractionated using the same fractionation method describedfor IHC for the instrumental analysis.Instrumental Analyses. Instrumental analyses were

performed according to target analytes. After fractionation,three fractions were concentrated to appropriate volumes,spiked with internal standards (5-α-androstane, p-terphenyl-d14,and 5-α-androstane for F1, F2, and F3, respectively), and then

adjusted to an accurate preinjection volume of 1.00 mL forGC/FID and GC/MS analyses. Analyses for n-alkanedistribution and TPH were performed on an Agilent 7890gas chromatograph equipped with a flame-ionization detector(FID) (Agilent Technologies, Inc., Santa Clara, CA, U.S.).Analyses of PAH compounds were performed on Agilent 5890GC equipped with Agilent 5972 mass selective detector (MS).In the case of BTEX, IHC was dissolved in pentane and directlyanalyzed using GC/MS.30 System control and data acquisitionwere achieved with HP G1034C MS ChemStation. Thedetailed analytical procedure, chromatographic conditions,analysis quality control, and quantification methodology havebeen described in literature.27,32

Luminescence Inhibition of Vibrio f ischeri. The acuteecotoxicity of WAF and WSF of IHC was assessed by theluminescence inhibition of V. f ischeri (strain NRRL B-11177)using Microtox Model 500 (Strategic Diagnostics Inc., Newark,DE, U.S.). Freeze-dried bacterium was purchased from thesupplier (Strategic Diagnostics Inc.). The TU of the sampleprepared after dilution of the WAF and WSF was calculatedafter processing the data using Microtox Omni software(Strategic Diagnostics Inc.), according to the test protocolprovided by the manufacturer. The results after 15 min ofexposure were used to quantitatively compare the toxicity.

Prediction of Dissolved Concentration and Lumines-cence Inhibition of Identified Species. As stated in theTheory section, the dissolved concentration of each species inthe solution was predicted using Raoult’s law. The molefraction of each species was calculated from the measuredcontent and the number average molecular weight (MWn).Because the MWn for the IHC used in this study has not beenreported, the MWn for West Texas crude oil estimated as 375 gmol−1 was used for calculating the mole fractions of thechemical species33 because MWn ranged from 295 to 400 gmol−1.33−35

The TU of the measured chemical species was calculated asdescribed earlier (eq 5). Because the sample solution wassubjected to the luminescence inhibition assay according to thestandard test protocol,21 dilution and partitioning of chemicalsbetween V. f ischeri and the solution lowers the freeconcentration of the chemicals from their concentrationsmeasured by chemical analysis. Thus, the free concentrationof chemical species i was calculated from the measured (orestimated) concentration in the WAF or WSF as follows:25

=+

CK f M V

C1

1 ( / ),V fischeri

free ilipw,i lip . w

i(6)

where Klipw,i is the lipid−water partition coefficient of i, f lip isthe lipid content of V. f ischeri (assumed as 0.01 kg kg−1), andMV.f ischeri is the mass of V. f ischeri in the test vessel. The values ofKlipw,i used are present in the literature (SupportingInformation, SI, Table S1); however, if literature values arenot available, they were estimated using the followingequation:36

= +K Klog 1.01 log 0.12lipw,i ow,i (7)

Because the baseline toxicity is related to the hydrophobicityof aromatic hydrocarbons, a quantitative structure−activityrelationship derived in our previous study25 was used to derivethe EC50 value of the individual chemicals without using themeasured values.

Figure 1. Schematic diagram of the preparation method for water-soluble fraction (WSF) of IHC.

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= −Klog(1/EC50) 1.36 log 2.75ow (8)

For all chemicals except for BTEX, the measuredconcentration in the WSF solution prepared for 24 h wasused to calculate TUi. Because BTEX are highly volatile inaqueous solution with high Henry’s law constants, thecorresponding TUi for BTEX was calculated using thepredicted aqueous concentration. In addition, the luminescenceinhibition of V. f ischeri showed a log Kow cutoff ofapproximately 5.0 due to the partitioning between the organismand the solution and a decreased fugacity ratio with increasing

log Kow.25 The TUi for aromatic hydrocarbons with log Kow

greater than 5.0 was not taken into account.

■ RESULTS AND DISCUSSION

Comparison of Methods for Preparing WAF and WSF.As shown in Table 1, concentrations of PAHs and alkylatedPAHs in the WAF and WSF solutions prepared at varying timesdid not significantly differ. This suggests that the method usedfor the preparation of the WAF did not result in a significantdifference in the dissolved concentration of hydrophobicaromatic hydrocarbons. In addition, the V. f ischeri luminescenceinhibition assay using the WSF solutions prepared at varying

Table 1. Comparison of the Measured Concentrations in WAF and WSF Solutions by Different Preparation Methods

chemical

measured concentration (μg L−1)

WAF (40 g L−1) WSF 3 h WSF 8 h WSF 24 h

naphthalene 19 ± 3 14 ± 1 12 ± 4 16 ± 1acenaphthylene ND ND ND NDacenaphthene 0.16 ± 0.01 0.15 ± 0.00 0.17 ± 0.01 0.16 ± 0.01fluorene 0.77 ± 0.03 0.81 ± 0.02 0.89 ± 0.02 0.83 ± 0.01phenanthrene 0.53 ± 0.02 0.55 ± 0.01 0.58 ± 0.01 0.60 ± 0.01anthracene 0.026 ± 0.002 0.013 ± 0.001 0.015 ± 0.001 0.016 ± 0.001fluoranthene 0.0019 ± 0.0002 0.0018 ± 0.0003 0.0025 ± 0.0002 0.0017 ± 0.0002pyrene 0.0066 ± 0.0003 0.0072 ± 0.0005 0.0068 ± 0.0004 0.0073 ± 0.0001benz[a]anthracene 0.0029 ± 0.0003 0.0029 ± 0.0001 0.0038 ± 0.0008 0.0025 ± 0.0003chrysene 0.0032 ± 0.0002 0.0035 ± 0.0003 0.0034 ± 0010 0.0041 ± 0.0001benzo[b]fluoranthene ND ND ND NDbenzo[k]fluoranthene ND ND ND NDbenzo[a]pyrene ND ND ND NDindeno[1,2,3-cd]pyrene ND ND ND NDdibenz[a,h]anthracene ND ND ND NDbenzo[ghi]perylene 0.00099 0.00050 0.00027 ND∑ EPA priority 16 PAHs 20 ± 3 16 ± 1 13 ± 4 18 ± 1biphenyl 0.67 ± 0.03 0.64 ± 0.01 0.70 ± 0.02 0.63 ± 0.02benzo[e]pyrene 0.00020 ND ND NDperylene ND ND ND NDdibenzothiophene 0.87 ± 0.02 0.89 ± 0.04 0.87 ± 0.15 0.79 ± 0.081-methylnaphthalene 17 ± 2 14 ± 0 14 ± 2 16 ± 12-methylnaphthalene 18 ± 2 16 ± 0 15 ± 2 17 ± 1C2-naphthalene 21 ± 1 21 ± 0 23 ± 1 22 ± 1C3-naphthalene 8.6 ± 0.3 8.6 ± 0.2 10 ± 0 9.0 ± 0.2C4-naphthalene 2.2 ± 0.1 2.0 ± 0.0 2.4 ± 0.0 2.2 ± 0.1C1-fluorene 0.49 ± 0.04 0.51 ± 0.01 0.60 ± 0.02 0.61 ± 0.01C2-fluorene 0.28 ± 0.00 0.32 ± 0.01 0.37 ± 0.01 0.34 ± 0.01C3-fluorene 0.13 ± 0.00 0.15 ± 0.00 0.16 ± 0.01 0.13 ± 0.011-methylphenanthrene 0.13 ± 0.01 0.14 ± 0.00 0.14 ± 0.00 0.15 ± 0.002-methylphenanthrene 0.12 ± 0.01 0.13 ± 0.00 0.14 ± 0.00 0.14 ± 0.003-methylphenanthrene 0.11 ± 0.00 0.12 ± 0.00 0.12 ± 0.00 0.12 ± 0.004/9-methylphenanthrene 0.18 ± 0.01 0.20 ± 0.00 0.20 ± 0.00 0.22 ± 0.00C2-phenanthrene 0.25 ± 0.01 0.26 ± 0.00 0.28 ± 0.01 0.29 ± 0.02C3-phenanthrene 0.093 ± 0.005 0.082 ± 0.012 0.097 ± 0.019 0.092 ± 0.033C4-phenanthrene 0.022 ± 0.003 0.027 ± 0.003 0.021 ± 0.004 0.019 ± 0.0021-methyldibenzothiophene 0.20 ± 0.00 0.22 ± 0.01 0.21 ± 0.04 0.18 ± 0.022-/3-methyldibenzothiophene 0.28 ± 0.00 0.30 ± 0.02 0.28 ± 0.06 0.23 ± 0.034-methyldibenzothiophene 0.36 ± 0.01 0.38 ± 0.02 0.37 ± 0.06 0.33 ± 0.03C2-dibenzothiophene 0.45 ± 0.02 0.50 ± 0.02 0.45 ± 0.09 0.39 ± 0.04C3-dibenzothiophene 0.13 ± 0.01 0.13 ± 0.00 0.13 ± 0.02 0.11 ± 0.01C1-chrysene 0.0031 ± 0.0006 0.0028 ± 0.0001 0.0033 ± 0.0006 0.0033 ± 0.0007C2-chrysene ND 0.00018 ND NDC3-chrysene ND ND ND ND∑ alkylated PAHs 71 ± 5 65 ± 1 68 ± 6 69 ± 2TPH 1397 ± 98 945 ± 10 720 ± 96 933 ± 56

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times up to 117 h showed consistent TU values ofapproximately 2.1 after 6 h (SI Figure S1). This indicatesthat almost all of the chemical species that exert toxic effects onV. f ischeri were, in fact, released from the IHC to the seawatersolution. Furthermore, 24-h duration appears to be sufficientfor most of the hydrophobic chemicals of the IHC that canpermeate the PDMS membrane to complete the process. Therewas no noticeable difference in the concentrations of aromatic

hydrocarbons between the WAF and WSF solutions. Thissuggests that most of the hydrophobic aromatic hydrocarbonsin the WAF solution are likely to be present in the dissolvedstate, i.e., in the solution prepared using the method thatminimized physical agitations.Unlike the case of aromatic hydrocarbons, TPH concen-

tration measured in the WAF solution prepared at the oil-loading rate of 40 g L−1 was approximately 40% greater than

Table 2. Comparison of Measured Chemical Concentrations in WAF and WSF, and Those Predicted by Eqs 1 and 2

chemicalmolar mass(g mol−1)

Tm(°C)a

ΔSm(J mol−1 K−1)b

KS

(L mol−1)c

aqueous solubility(mg L−1, at 25 °C)

concentration inIHC (μg g−1)

mole fraction(x) in IHC Cpre (μg L−1)S Ssw

L

benzene 78.11 5 35.4 0.20d 1789e 1429 1469 7.1 × 10−3 1.0 × 104

toluene 92.14 −95 37.2 0.21d 580e 455 3623 1.5 × 10−2 6.7 × 103

ethylbenzene 106.17 −95 51.4 0.23d 187e 143 1208 4.3 × 10−3 6.1 × 102

p-xylene 106.17 13 59.8 0.25d 214e 160 2468 8.7 × 10−3 1.4 × 103

m-xylene 106.17 −48 51.4 0.25d 160e 120 825 2.9 × 10−3 3.5 × 102

o-xylene 106.17 −25 54.9 0.23d 221e 170 1536 5.4 × 10−3 9.2 × 102

naphthalene 128.17 80 53.8 0.26d 30.60f 75.21 172 5.0 × 10−4 3.8 × 101

acenaphthylene 152.19 92 42.4 0.35 16.10g 33.75 0.28 6.8 × 10−7 2.3 × 10−2

acenaphthene 154.21 93 58.6 0.24d 4.16g 15.92 4.64 1.1 × 10−5 1.8 × 10−1

fluorine 166.22 115 50.5 0.27d 1.57f 7.19 27.3 6.2 × 10−5 4.4 × 10−1

phenanthrene 178.23 99 44.8 0.38 0.82f 2.028 50.9 1.1 × 10−4 2.2 × 10−1

anthracene 178.23 216 60.1 0.35 0.044f 3.013 3.38 7.1 × 10−6 2.1 × 10−2

fluoranthene 202.25 110 48.9 0.36 0.20g 0.70 0.56 1.0 × 10−6 7.3 × 10−4

pyrene 202.25 151 43.4 0.35 0.086f 0.52 3.64 6.7 × 10−6 3.5 × 10−3

benz[a]anthracene 228.29 161 49.2 0.36 0.017g 0.16 1.99 3.3 × 10−6 5.3 × 10−4

chrysene 228.29 256 55.5 0.37 0.00070f 0.080 8.85 1.5 × 10−5 1.2 × 10−3

benzo[b]fluoranthene 252.31 168 56.5h 0.35 0.0015i 0.026 1.17 1.7 × 10−6 4.5 × 10−5

benzo[k]fluoranthene 252.31 217 56.6h 0.35 0.00080i 0.043 0.15 2.2 × 10−7 9.3 × 10−6

benzo[a]pyrene 252.31 181 42.4 0.35 0.0015f 0.014 1.05 1.6 × 10−6 2.2 × 10−2

indeno[1,2,3-cd]pyrene 276.33 162 49.4 0.35 0.00019i 0.0020 0.22 3.0 × 10−7 5.9 × 10−7

dibenz[a,h]anthracene 278.35 270 58.3 0.34 0.0025j 0.53 0.49 6.6 × 10−7 3.5 × 10−4

benzo[ghi]perylene 276.33 273 31.3 0.29 0.00014i 0.0023 0.64 8.7 × 10−7 2.0 × 10−6

biphenyl 154.21 69 54.8 0.28d 6.71k 12.90 13.7 3.3 × 10−5 4.3 × 10−1

benzo[e]pyrene 252.31 181 36.5h 0.35 0.0050l 0.034 2.20 3.3 × 10−6 1.1 × 10−4

perylene 252.31 278 67.9 0.35 0.00040m 0.27 0.15 2.2 × 10−7 6.0 × 10−5

dibenzothiophene 184.26 98 56.4n 0.35 1.47j 5.20 88.8 1.8 × 10−4 9.4 × 10−1

1-methylnaphthalene 142.20 −30 49.3 0.44d 31.70e 6.34 451 1.2 × 10−3 7.53

2-methylnaphthalene 142.20 35 58.9 0.35 21.50f 18.05 540 1.4 × 10−3 25.7

C2-naphthalene 156.22 112 65.4 0.35 1.00o 6.62 1226 2.9 × 10−3 19.5

C3-naphthalene 170.25 63 56.5p 0.35 2.10m 3.34 1265 2.8 × 10−3 9.30

C4-naphthalene 184.28 72q 56.5p 0.35 1.39r 2.72 682 1.4 × 10−3 3.77

C1-fluorene 180.25 87 56.5p 0.35 1.092k 3.00 207 4.3 × 10−4 1.29

C2-fluorene 194.27 101q 56.5p 0.35 0.15r 0.56 114 2.2 × 10−4 1.2 × 10−1

C3-fluorene 208.30 94q 56.5p 0.35 0.10r 0.33 132 2.4 × 10−4 7.9 × 10−2

1-methylphenanthrene 192.26 123 56.5p 0.35 0.27s 1.68 41.8 8.2 × 10−5 1.4 × 10−1

2-methylphenanthrene 192.26 94q 56.5p 0.35 0.28t 0.89 44.2 8.6 × 10−5 7.7 × 10−2

3-methylphenanthrene 192.26 65 56.5p 0.35 0.28t 0.47 36.6 7.1 × 10−5 3.3 × 10−2

4/9-methylphenanthrene 192.26 54 56.5p 0.35 0.27u 0.34 66.0 1.3 × 10−4 4.4 × 10−2

C2-phenanthrene 206.28 109q 56.5p 0.35 0.071r 0.32 250 4.5 × 10−4 1.5 × 10−1

C3-phenanthrene 220.31 116q 56.5p 0.35 0.021r 0.11 167 2.8 × 10−4 3.1 × 10−2

C4-phenanthrene 234.34 128q 56.5p 0.35 0.0059r 0.041 82.4 1.3 × 10−4 5.4 × 10−3

1-methyldibenzothiophene 198.28 112q 56.5p 0.35 0.33r 1.60 76.5 1.4 × 10−4 2.3 × 10−1

2-/3-methyldibenzothiophene 198.28 112q 56.5p 0.35 0.33r 1.60 129 2.4 × 10−4 3.9 × 10−1

4-methyldibenzothiophene 198.28 112q 56.5p 0.35 0.33r 1.60 165 3.1 × 10−4 5.0 × 10−1

C2-dibenzothiophene 212.31 117q 56.5p 0.35 0.095r 0.52 553 9.8 × 10−4 5.0 × 10−1

C3-dibenzothiophene 226.34 128q 56.5p 0.35 0.027r 0.19 477 7.9 × 10−4 1.5 × 10−1

C1-chrysene 242.32 173 56.5p 0.35 0.013r 0.26 14.7 2.3 × 10−5 5.9 × 10−3

C2-chrysene 256.34 154q 56.5p 0.35 0.0037r 0.047 21.4 3.1 × 10−5 1.5 × 10−1

C3-chrysene 270.37 165q 56.5p 0.35 0.0011r 0.017 20.9 2.9 × 10−5 5.1 × 10−4

aRef 47. bRef 48. cRef 49. dRef 50. eRef 51. fRef 52. gRef 53. hRef 54. iRef 55. jRef 56. kRef 57. lRef 58. mRef 59. nRef 60. oRef 61. pEstimated valueusing Walden rule, ref 62. qEstimated value using MPBPVP v1.43.63 rEstimated value using WSKOW v1.42.63 sRef 64. tRef 65. uRef 66.

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that in the WSF solutions (Table 1). Mass fractions ofasphaltenes and resins of IHC were reported as 5.6 and 5.6%,repectively.27 These fractions may contain polar organicchemicals such as phenols that have much higher watersolubility than the hydrocarbons of similar size. Dissolution ofpolar organic chemicals may significantly decrease the molefraction in the oil mixture, as they dissolve in water due to theirrelatively high water solubility. This is in contrast to the highlyhydrophobic aromatic hydrocarbons, for which the initial molefraction changes only negligibly because of their limited watersolubility. It has been shown that the depletion of solublespecies in the nonaqueous phase liquid (NAPL) inducesdecreased concentration in water as the water/NAPL ratioincreases.37

Prediction of Dissolved Concentration of Chemicalsin Artificial Seawater. Table 2 shows the initial concentrationand the mole fraction of each chemical in the IHC. The tablealso shows the predicted concentration (Ci) in the artificialseawater and the physicochemical properties necessary tocalculate Ci (molar mass, Tm, ΔSm, KS, S, and Ssw

L ). For alkylatedPAHs, physicochemical properties were taken from representa-tive compounds listed in SI Table S2. The predicted values inTable 2 agree very well with the measured ones in Table 1, asshown in Figure 2. The differences between the measured and

predicted values were within a factor of 2 for more than 60% ofthe hydrocarbons evaluated. Relatively larger deviations werefound for chemicals with lower measured concentrations, suchas benzo[a]anthracene, probably because of the largerexperimental errors in quantification. Similarly, Sterling etal.34 showed that the measured soluble concentrations of PAHsagreed well with the predicted ones, especially for those withhigher mole fractions when a best-fit value of MWn was used.Because it is much easier to measure chemical composition incrude or weathered oils than in aqueous solutions, theestimation method proposed in this study could be used toevaluate the freely dissolved concentrations of each chemicalspecies in the WAF or WSF by measuring the mole fraction of

each chemical in the oil mixture. This information can then beused to evaluate the resulting ecotoxicological effects of eachchemical species. Because the MWn of a crude oil mixture is acritical parameter that determines the mole fraction of theindividual species, an accurate value of MWn is needed for agood prediction. In most studies, the MWn of crude oils wasestimated by comparing the measured vs predicted concen-tration of major species, and ranged from 295 to 400 gmol−1.33−35

Researchers have found that weathering of crude oils mayincrease their ecotoxicity, primarily because of the increase intoxic transformation products such as oxygenated hydro-carbons,6,7,38,39 in spite of the evaporation loss of smallerchemicals. These polar compounds are mostly not included inmonitoring of oil residues and need to be studied to fill gapsbetween observed ecotoxicity and chemically identifiedcomponents. Another possible reason in need of considerationis the increased mole fraction of heavier, and subsequently,more toxic components. Because chemicals with lowermolecular weights tend to have a higher vapor pressure, theMWn of spilled oil is likely to increase because of weatheringand the mole fraction of less volatile aromatic hydrocarbonsincreases. Although further validation of the toxic potentials forthese more persistent chemicals is needed, their increased molefraction may explain the increased ecotoxicity of weathered oils.

Prediction of Luminescence Inhibition and Compar-ison with Measured Data. The TU values of individualspecies were calculated as described above (detailed results arepresented in SI Table S3). As shown in Figure 3, the TU for the

WSF was the lowest and that for the WAF showed anincreasing trend with increasing oil-loading rate. These do notagree with the negligible changes in the measured concen-trations of hydrophobic aromatic hydrocarbons (Table 1).Because PDMS is a nonpolar polymeric phase, highly polarorganic compounds and inorganic species cannot easilypenetrate through the PDMS layer (Figure 1). Thus, the

Figure 2. Comparison of predicted and measured concentrations inwater-accommodated fraction (WAF) and water-soluble fraction(WSF). Open circles and open diamonds denote substances in WAFand WSF, respectively. Solid and dotted lines indicate 1:1 and 2:1 (or1:2) lines, respectively.

Figure 3. Toxic unit (TU) measured for WAF and WSF solutions.Error bars denote the standard errors (n = 3).

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differences in TU between the WSF and WAF solutions withvarying oil-loading rates may be attributed to the presence ofpolar organic compounds and/or inorganic chemicals. Asdiscussed above, the dissolved concentration of these chemicalsmay increase with increasing oil-loading rate if they are presentin a limited amount in the mixture. Crude oils are known tocontain a substantial fraction of inorganic substances such assulfur, vanadium, nickel, and copper compounds,40,41 whichmay be toxic to V. f ischeri. However, the extremely high oil-loading rates used for the preparation of WAF in this study (5−40 g L−1) are not likely to occur at oil-spill sites. Thus, thepotential contribution of these species to ecotoxicity fromspilled oil residues under field conditions should be limited.Figure 4 summarizes the comparison of TU measured for the

WAF using various oil-loading rates and for the WSF using theestimated contribution due to aromatic hydrocarbons. Thecontributions of the PAHs and alkylated PAHs were calculatedbased on the measured concentration in the 24-h WSF, whereasthat of the BTEX compounds was based on the predictedconcentration, because their actual concentrations were notmeasured in the WAF and WSF solutions. The relativecontribution of BTEX compounds, PAHs, and alkylated PAHsin luminescence inhibition was estimated at 76%, 2%, and 21%,respectively. Although the estimated contribution by BTEX isthe highest, the actual contribution might be lower thancalculated, because the BTEX compounds evaporate during theexperiment owing to their high Henry’s law constants.42

Interestingly, C3- and C4-naphthalenes and other alkylatedPAHs were identified as the most important aromatichydrocarbons responsible for baseline toxicity, with theexception of BTEX as suggested by earlier literature.8−12

Meanwhile, the contribution of the 16 priority PAHsdesignated by the US EPA was only approximately 2%. Thetoxic potentials of alkylated PAHs have been reported in a

limited number of studies and were comparable to, or evengreater than, that of the parent PAHs.43−46 This suggests thatfurther studies are required to study the fate and toxicity ofindividual alkylated PAHs and their mixtures in the environ-ment.

■ ASSOCIATED CONTENT*S Supporting InformationLiterature values of Klipw,i used in this study are summarized inTable S1, and TU values of each chemical species aresummarized in Tables S1 and S2. This material is availablefree of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +82 2 3290 3041; e-mail: junghwankwon@korea.ac.kr.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis research was a part of the project entitled “Oil SpillEnvironmental Impact Assessment and Environmental Restora-tion (PM57431)” funded by the Ministry of Oceans andFisheries, Korea, and the National Research Foundation ofKorea (NRF) grant (No. 2012R1A1B4000841).

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Figure 4. Contribution of the measured chemical species to the overall toxicity in toxic unit (TU). Values in parentheses represent the contributionof chemically identified components to TU using eqs 1−5.

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