• Fish embryos;
  • Crude oil;
  • Toxicity;
  • Polynuclear aromatic hydrocarbons;
  • Dispersant


  1. Top of page
  2. Abstract
  8. Acknowledgements
  10. Supporting Information

The chronic toxicity of crude oil to fish embryos depends on the chemical constituents of the test oil and on factors that control the exposure of embryos to those constituents. The partitioning of chemicals from oil to water depends on the surface area of oil exposed to water and thus on the susceptibility of oil to be dispersed into droplets. The chronic toxicity of four different crude oils to embryos of rainbow trout (Oncorhynchus mykiss) was measured by exposure to the water-accommodated fraction (WAF; no droplet formation) and to the chemically enhanced WAF (CEWAF) of each oil. When effects were compared with the amount of WAF or CEWAF added to test solutions, chemical dispersion increased toxicity dramatically, by >35 to >300-fold, with the smallest difference measured for the lightest and least viscous oil. When effects were compared with measured concentrations of oil in test solutions, there were no differences in toxicity between WAF and CEWAF treatments, indicating that chemical dispersion promoted droplet formation and the partitioning of hydrocarbons from oil to water. On a dilution basis, the differences in toxicity among the four oils were correlated with the concentrations in oil of polynuclear aromatic hydrocarbons (PAH), particularly those with three to five rings, and with their viscosity, an index of dispersibility. However, when PAH concentrations were measured in solution, toxicity did not vary substantially among the four oils, suggesting that the PAH of each oil had equivalent toxicities and that differences in toxicity represented differences in dispersability. Environ. Toxicol. Chem. 2012;31:754–765. © 2011 SETAC


  1. Top of page
  2. Abstract
  8. Acknowledgements
  10. Supporting Information

Approximately five million tons of crude oil from a variety of sources enter the marine environment every year 1. The environmental impacts range from oiled shorelines and marine wildlife to intoxication of organisms that ingest oil or are exposed to the constituents that partition from oil into water. Crude oils from different sources vary widely in their chemical composition and physical properties. These properties influence the fate of oil following a spill, the types of spill response methods that might be effective for cleaning up the oil, and the risk of toxicity to aquatic organisms. Polynuclear aromatic hydrocarbons (PAH) constitute 0.5 to 3% of oil by weight 2 ( and are of particular concern; those with three or more aromatic rings are mutagenic or carcinogenic 3, 4. Human and environmental health concerns have focused traditionally on the U.S. Environmental Protection Agency's (U.S. EPA) 16 priority PAHs, most of which are unsubstituted. However, alkyl-substituted PAHs are toxic 5–7 and are abundant in mixtures of petroleum hydrocarbons, making up 80 to 90% of the total PAH in crude oils 2.

Alkyl PAHs with three or four rings have been implicated as the primary agents of oil toxicity to early developmental stages of fish and may be responsible for recruitment failure of species whose spawning coincides with an oil spill 8. The contamination of coastal spawning shoals by the 1989 Exxon Valdez oil spill demonstrated that embryonic pink salmon (Oncorhynchus kisutch) and Pacific herring (Clupea pallasi) were particularly vulnerable to toxicity 8, 9. Exposed fish showed a characteristic suite of pathologies defined as blue sac disease (BSD) as well as induction of mixed function oxygenase or cytochrome P4501A (CYP1A) proteins 10. These proteins catalyze the oxygenation of aromatic compounds and are inducible by many PAHs typical of crude oil 10, 11; induction following an oil spill is a clear sign of exposure to petroleum-derived PAH. Although low-molecular-weight (LMW) aromatics, such as naphthalenes, and C1–C10 aliphatics are acutely toxic by narcosis, they should contribute little to chronic toxicity because they are highly volatile, are readily diluted in water 12 (, and dissipate rapidly with mixing by wind and waves. The residual weathered crude oil is thereby enriched with medium-molecular-weight (MMW) compounds, which are associated with the chronic toxicity of oil to fish embryos 8, 9. High-molecular-weight (HMW) aliphatics and hydrocarbons such as waxes, resins, and asphaltenes are too large to dissolve readily in water or to be taken up by fish at toxic concentrations.

Crude oils are classified as light, medium, or heavy, depending on their relative proportions of LMW, MMW, and HMW compounds. Oils rich in LMW components should be more acutely toxic than those with a higher proportion of MMW and HMW components. Conversely, medium and heavy oils containing more three- to five-ringed alkyl PAHs should be more chronically toxic to fish embryos than lighter oils, which have proportionately more two-ringed naphthalenes 2. Therefore, the risk of spilled oil to fish embryos should be a function of chemical composition and might be estimated from models of chemical mixture toxicity. However, few of the thousands of alkyl PAHs in oil have been individually identified, analyzed, or tested, so direct measures of crude oil toxicity still provide the most useful way to assess relative risk among oils.

The risk of spilled oils also depends as much on the bioavailability of the toxic constituents as on their abundance and toxicity. Compared with lighter oils, heavy oils have physical properties that could reduce the rate of oil–water partitioning of larger hydrocarbons, including alkyl PAH. Partitioning could be diminished by poor dispersibility of heavy oils, reducing droplet formation and the surface-to-volume ratio of the oil–water interface. Thus, partitioning should vary with properties that affect dispersibility, such as viscosity, and the apparent toxicity of different oils may reflect differences in their physical characteristics rather than their chemistry.

One way to avoid this bias is to prepare test solutions with more uniform droplet sizes by using chemical dispersants. Oil dispersants are mixtures of surfactants in solvent, comprising anionic soaps and nonionic detergents that orient to the oil–water interface 13. They reduce surface tension and facilitate the formation of small oil–surfactant droplets in water, producing a chemically enhanced water-accommodated fraction (CEWAF) of oil. Compared with a water-accommodated fraction (WAF) of oil prepared by moderate stirring, CEWAF contains ten- to 100-fold more PAH and correspondingly elevates CYP1A induction and toxicity to fish embryos 14, 15.

The objective of the present study was to compare the toxicity of four different weathered crude oils to rainbow trout embryos, using chemical dispersants to reduce the influence of physical characteristics that affect oil–water interactions. Weathered oil avoided the confounding effects of acute lethality caused by LMW volatile compounds that would evaporate quickly following a spill. The bioavailability of alkyl PAHs to embryos exposed to WAF or CEWAF was demonstrated by CYP1A activity. Toxicity was assessed by percentage mortality, signs of BSD, percentage of surviving fish that appeared normal (no signs of BSD), and ratio of yolk sac to fish weight at swim-up, an index of growth and development. The concentrations of PAH and total petroleum hydrocarbons in test solutions of WAF and CEWAF were measured to understand the relationships among chemical composition, bioavailability of alkyl PAHs, and toxicity.


  1. Top of page
  2. Abstract
  8. Acknowledgements
  10. Supporting Information

Crude oil and dispersant

Four light-to-medium weathered crude oils were tested: Alaska North Slope crude (ANSC), Federated crude (FED; blend of several Alberta crude oils), medium South American crude (MESA), and Scotian light crude (SCOT; Table 1). All except MESA were from the Emergencies Science Division of Environment Canada in Ottawa, Ontario; MESA was provided by the Centre for Offshore Oil, Gas, and Energy Research (COOGER) at the Bedford Institute of Oceanography in Dartmouth, Nova Scotia, Canada. Corexit EC9500 (Nalco Energy Services) was used for dispersing the oils.

Table 1. Characteristics of each test oila
  • a

    ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude; NM = not measured; TPH = total petroleum hydrocarbons; TSH = total saturated hydrocarbons; TAH = total aromatic hydrocarbons; PAH = polynucelar aromatic hydrocarbons.

  • b

    API (American Petroleum Institute) gravity, developed by the API ( measures the relative specific gravity of petroleum liquids. A value >10 indicates a specific gravity less than that of water, and a value <10 indicates a specific gravity greater than that of water.

  • c

    CPI = carbon preference index, the ratio between the concentrations of odd and even alkanes. A predominance of odd alkanes (high CPI) indicates a relatively high contributions of terrigenous epicuticular plant waxes.

API gravityb31.838.929.753.2
Density at 15°C0.86630.82980.820.7655
Total alkanes (mg/g)60.610480.5172
Odd alkanes27.948.037.681.3
Even alkenes29.949.537.787.7
Petroleum hydrocarbons
 TPH (mg/g)504664639577
 TSH/TPH (%)82.188.481.895.3
 TAH/TPH (%)17.911.618.24.7
 Resolved peaks/TPH (%)19.828.720.547.7

Polynuclear aromatic hydrocarbons (µg/g; from Table 2

 Σ Non-alkyl PAH1,2821,0211,226325
 Σ Alkyl PAH11,64812,73414,3235,348
 Total PAH12,92913,75515,5495,673
Viscosity at 21°C (cP)11.55NM1

The four oils were weathered to remove most volatiles by placing aliquots of equal volume in two identical bowls held in a fume hood for 24 h. After 24 h, the two aliquots were mixed and stored at 4°C. The average weight loss was approximately 15 to 17%, except for MESA, which lost only 5% because it had been preweathered at COOGER by 24 h of aeration that caused a 13.8% weight loss; the total weight loss for MESA was approximately 19%.

Preparation of WAF and CEWAF

The WAF was prepared fresh daily by stirring crude oil with dechlorinated municipal water at a ratio of 1:9 in a beaker covered with aluminum foil. The vortex was adjusted to approximately one-third of the height of the mixture from the oil–water interface 16. After 18 h, the mixture was allowed to settle for 1 h to separate the water and oil phases, and the water phase (WAF) was aspirated via a disposable pipette for testing. Stock solutions of CEWAF were made in the same way as the WAF, except that after 18 h, Corexit 9500 was added to the surface of the oil–water mixture at a ratio of 1:10 dispersant:oil and stirred for 1 h. The CEWAF contained droplets of dispersed oil and was allowed to settle for 1 h before the cloudy bottom layer was aspirated for testing.

Experimental design

The embryonic toxicity of WAF and CEWAF from each oil and of Corexit 9500 was tested with a regression design, in which embryos were exposed to a graded series of oil concentrations with no replication. An exception was the Corexit controls, which were repeated because of unexpected mortality at the highest concentration. The endpoints of median lethal concentrations (LC50s) and median effective concentrations (EC50s) for a series of sublethal responses were estimated from the responses of fish to each concentration. The exposure followed a static, daily-renewal protocol, recognizing the need to create fresh solutions of WAF and CEWAF because of the loss of hydrocarbons from test solutions by volatility, absorption to test containers, and uptake by fish. Loadings increased in a logarithmic series from 1 to 10% v/v of WAF or from 0.01 to 0.1% v/v of CEWAF. Based on a preliminary experiment, an additional treatment of 18% v/v WAF was added for ANSC and a 0.18% v/v CEWAF treatment was added for SCOT to ensure that exposures were sufficient to calculate endpoints. Retene (7-isopropyl-1-methylphenanthrene, 100 µg/L; ICN Biomedical) served as a positive control; this concentration causes BSD in trout embryos without a significant amount of mortality 5. Corexit 9500 added directly to water at 1 to 10 µl/L served as dispersant controls, with the highest concentration typical of the highest CEWAF loading. The negative control and the diluent for all tests were dechlorinated municipal water from Lake Ontario (hardness 135 mg/L as CaCO3).

Chronic toxicity exposure protocol

All research was carried out under an approved Queen's University Animal Care Protocol (Hodson 2007 032), following the Guidelines of the Canadian Council on Animal Care ( Rainbow trout eyed eggs from the Rainbow Springs Trout Hatchery were acclimatized to 10°C for 1 h in their original container, divided among 12 stainless-steel bowls containing fresh dechlorinated water with aeration, and acclimated for 4 d. Water chemistry was measured daily, and the conditions were optimal for trout development (temperature 8.92 ± 0.18°C; dissolved oxygen 114 ± 4.66%; pH 8.11 ± 0.08; total ammonia 7.09 ± 4.36 µM). The chronic exposures to WAF and CEWAF began just prior to hatch, when 25 embryos were placed in stainless-steel bowls containing 1 L of test solution. Test solutions of WAF and CEWAF were prepared and renewed fresh daily until swim-up, mortality was recorded daily, and dead embryos were removed.

After 22 d, when control fish began to surface (swim-up), all fish were anesthetized in 1.0 g/L ethyl 3-aminobenzoate methanesulfonic acid (MS-222) and scored for signs of BSD following established protocols 14, 17. Signs were scored based on presence/absence (0–1) or severity (0–3, 3 as highest severity): pericardial edema (0–3), yolk sac edema (0–3), yolk/body hemorrhaging (0–1), ocular hemorrhaging (0–1), craniofacial deformity (0–1), spinal deformity (0–1), and fin rot (0–1). Edemas were given a greater weight than other signs because they were most closely associated with mortality, and the response range was sufficiently large that partial responses could be discriminated. The BSD score was the average of the summed scores for each fish for each sign of toxicity within each treatment, and the BSD index was the BSD score normalized to the maximum score (11) to give a range of 0 to 1. A severity index (SI) was also estimated, which combined pathology with mortality; fish that died were assigned the maximum BSD score plus 0.5. Survivors with no obvious signs of BSD (indexes of 0, equivalent to controls) were considered normal.

After scoring, the embryos were weighed to the nearest milligram before and after their yolk sacs were gently teased from the body with forceps. The ratio of the calculated yolk sac weight to fish weight (yolk-wt ratio) provided an index of growth and development. High ratios indicate a slow conversion of yolk to fish tissue and an inhibition of growth and development 14. After weighing, the embryos were placed in cryogenic vials (5/vial), flash-frozen in liquid nitrogen, and stored at −80°C for analysis of ethoxyresorufin-O-deethylase (EROD) activity, a measure of CYP1A induction and an indicator of the relative degree of exposure to PAH. Chronic toxicity was assessed from mortality rates, severity of BSD, percentage of fish considered normal, and yolk-weight ratios.

Ethoxyresorufin-O-deethylase assay

For each treatment, EROD activity was measured in four pools of five whole embryos. Fewer pools (zero to three) were available at the highest loadings of WAF or CEWAF because of mortality. Whole embryos were homogenized in buffer, the S9 fraction was recovered by centrifugation, and EROD activity was measured by a fluorescence method modified for microplate readers 18–20. The success of the enzyme assay was verified by analyzing an aliquot of archived liver S9 fractions from control and β-naphthoflavone (BNF; 10 µg/L)-exposed juvenile trout. These internal standards ensured that low values of EROD activity were not due to false negatives.

Characterization of oil exposures

Whole oil

The chemical characteristics of the whole crude oils were determined at Environment Canada laboratories in Ottawa, Ontario. Approximately 16 mg of each oil was loaded on preconditioned 3-g silica gel fractionation columns. Saturated and aromatic hydrocarbons were eluted with hexane (12 ml) and 50% DCM in hexane (v/v, 15 ml). One-half of each hexane fraction was analyzed for aliphatics, n-alkanes, and biomarker terpane and sterane compounds. One-half of the 50% DCM fraction was analyzed for alkyl homologous PAHs and other U.S. EPA priority unsubstituted PAHs. The remaining halves of the hexane and 50% DCM fractions were combined for analyses of the total petroleum hydrocarbons (TPH) and the unresolved complex mixture of hydrocarbons.

Analyses for TPH were performed on an Agilent 6890 gas chromatograph equipped with a flame-ionization detector. Analyses of the n-alkane distribution (n-C8 through n-C42, plus pristane and phytane), PAH compounds, and biomarkers were performed on an Agilent 6890 gas chromatograph with a 5973 mass selective detector. Details on quality control and quantification methods have been given by Wang et al. 21, 22.

Bioassays solutions

For each oil, water was sampled daily from the highest WAF or CEWAF treatments to assess the variation of exposure concentrations over time. The highest treatments were also sampled once at 0, 2, 4, 8, and 24 h after the daily renewal of test solution to describe the decline in hydrocarbon concentrations between renewals. Finally, every treatment was sampled once, immediately after the daily renewal of test solutions (time 0), to show the gradient of exposure concentrations. For each sample, a 1.8-ml water sample was added to 1.8 ml ethanol in a 7-ml scintillation vial and stored at 4°C until analysis. Waterborne total hydrocarbon concentrations were measured by spectrofluorometry 14, i.e., by recording fluorescence spectra with an excitation wavelength scan of 297 nm and emission wavelengths of 305 to 480 nm against standard curves of each crude oil in hexane diluted successively in a 50:50 ethanol:water solution. All standard curves were linear, with an r2 > 0.99. The linear regressions relating the logs of measured hydrocarbon concentrations to loadings of WAF and CEWAF of each oil were used to estimate LC50s and EC50s in units of micrograms per liter total hydrocarbons.

The concentrations of alkanes and PAHs in solution were also measured once at COOGER by gas chromatography–mass spectrometry (CG/MS) using modified U.S. EPA Methods 3510C and 8270 23, 24. For each crude oil, 1.0-L water samples were taken from the highest WAF (10% v/v) or CEWAF (0.1% v/v) treatments and from water and dispersant controls. The samples were extracted into DCM after spiking with a mixture of deuterated standards dried on silica gel. Each extract was concentrated by TurboVap, purified on silica gel, exchanged into isooctane, and spiked with internal standards. Extracts were analyzed with an Agilent 6890 GC coupled to a 5975 MS. The GC column was a Supelco MDN-5s 30 m × 250 µm × 0.25 µm (length × internal diameter × film thickness) with a 1-m retention gap of deactivated fused silica (Sigma Aldrich). The sample (1 µl) was injected by cool-on-column using oven track mode, with helium carrier gas at 1.0 ml/min. The oven temperature was held at 85°C for 2 min, ramped to 280°C at 4°C/min, and held for 20 min for a total run time of 70.75 min. The MS was operated in selected ion monitoring mode with specific ions and retention windows applied for each compound. Samples were calibrated against a mixture of aliphatic hydrocarbons and parent and alkyl PAH. When alkyl PAH standards were not available, the response of the nonalkyl congener was used for quantification with a detection limit of 100 ng/ml.

Statistical analysis

For each oil, EC50s values for EROD activity, BSD index, SI, and the ratio of yolk to fish weight were calculated by nonlinear regressions (GraphPad Prism 4.02; GraphPad Software) relating responses to the concentration of hydrocarbons measured by fluorescence. The EC50s were the concentrations of hydrocarbons associated with half-maximal responses relative to controls. For example, the maximal value for the normalized BSD index was 1.0, representing embryos with the highest degree of edema, spinal curvature, and so on. The lowest for control embryos was close to zero, and the EC50 was the exposure concentration at the midway point between the control indices and 1.0, as interpolated by the nonlinear regression. For the yolk-to-fish weight ratio, there was no method-defined maximal response, so the highest response of fish that showed little development over the test (highest loadings of WAF and CEWAF of MESA oil) was taken as the maximal response. The EROD activity for each pool of three or four fish was log transformed before regression to ensure homogeneity of variance 19. Median lethal concentrations and EC50s for percentage normal were calculated by probit analyses in SPSS 13.0 for Windows. When the greatest responses were less than 50% of maximal, EC50s and LC50s were reported as greater than the highest concentration tested.


  1. Top of page
  2. Abstract
  8. Acknowledgements
  10. Supporting Information

Test oils

Among the four test oils, SCOT had the highest concentrations of alkanes, the highest proportion of LMW alkanes (biomarker ratios), the highest concentrations of odd and even alkanes, the lowest concentrations of PAH, and the lowest viscosity (Table 1). There was much less variation in these characteristics among the remaining three oils. For the PAH, SCOT had the lowest concentrations of most classes of unsubstituted and alkyl PAHs (Table 2); among the remaining three oils, MESA had the highest and ANSC the lowest concentrations. The concentrations of total PAH relative to MESA oil were 88% (FED), 83% (ANSC), and 36% (SCOT). Across all four oils, the relative concentrations of each class of alkyl PAH were similar, with naphthalenes the predominant PAH class, increasing in proportion ANSC < FED < MESA < SCOT (Supplemental Data, Fig. S1). Scotian light crude also contained the highest proportion of fluorenes, but the other three oils contained two to six times higher proportions of all other classes of PAH. While there were similar concentrations of fluorenes and chrysenes in the three heavier oils (Table 2), ANSC had the highest concentrations of dibenzothiophenes and naphthobenzothiophenes, FED had the highest concentrations of phenanthrenes and pyrenes, and MESA was very similar to ANSC.

Table 2. Polynuclear aromatic hydrocarbon concentrations (µg/g oil) in weathered oils as testeda
  • a

    PAH = polynuclear aromatic hydrocarbons; ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude; C0 to C4 = the numbers of carbons in alkyl side chains.

  • b

    Blank spaces indicate data not available.

C0-naphthalene (N)36832747753
Σ alkyl (C1–C4)6,2687,4498,9664,330
C0-fluorene (F)11712814335
Σ alkyl (C1–C4)344371333274
C0-dibenzothiophene (DBT)210165163117
Σ Alkyl (C1–C4)1,5161,0211,500104
C0-phenanthrene (P)30526326851
Σ Alkyl (C1–C4)1,7912,3222,136412
C0-pyrene (PY)1217146
Σ Alkyl (C1–C4)458629383152
C0-naphthobenzothiophene (NBT)4122282
C4-NBT1111 4
Σ Alkyl (C1–C4)8104615977
C0-chrysene (C)102  3
Σ Alkyl (C1–C4)46048140769
Fluoranthene7  7
Benzo[k]fluoranthene   1
Benzo[a]pyrene3 32
Perylene   2
Indeno[1,2,3-cd]pyrene   3
Σ Nonalkyl PAH (including C0s)1,2821,0211,226325
Σ Alkyl PAH (excluding C0s)11,64812,73414,3235,348
Total PAH12,92913,75515,5495,673

Hydrocarbon concentrations in test solutions

The freshly mixed test dilutions of WAF and CEWAF included measurable amounts of alkyl and unsubstituted PAH (Fig. 1A and B) but almost no measurable PAH in samples taken at 24 h (data not shown). Overall, the proportion of total PAH represented by each family of alkyl PAH in 10% v/v WAF (Fig. 1A) was remarkably similar to that of whole oil (Supplemental Data, Fig. S1), except that C0 and C1 naphthalenes were enriched relative to C3 and C4 naphthalenes. Except for SCOT, the patterns of alkyl PAH distribution in 0.1% v/v CEWAF were similar to those of whole oil (Fig. 1B and Supplemental Data, Fig. S1). However, the proportions of three- or four-ringed alkyl homologs were higher than in whole oil, likely because the proportions of naphthalene homologs, the predominant PAH, were lower.

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Figure 1. Proportions of different classes of polynuclear aromatic hydrocarbons (PAHs) making up the total PAHs in a water-accommodated fraction (10% v/v; A) and a chemically enhanced water-accommodated fraction (0.1% v/v; B) of four weathered crude oils. The PAHs without alkyl substituents are referred to as C0 and C1 to C4 refer to the sum of all possible congeners containing one to four carbons in alkyl substituents. Other PAHs refer to the sum of all PAHs not included in the other categories; this comprises primarily five- or six-ringed nonalkyl-substituted PAHs. ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude.

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When the relative proportions of each component of total PAH in WAF or CEWAF were compared with those of whole oil, the scatterplots approximated a 1:1 line, with the highest correlations for FED and SCOT oils (Supplemental Data, Fig. S2). For ANSC and MESA oil, the less abundant PAHs (higher molecular weight) were somewhat underrepresented in WAF and overrepresented in CEWAF, the opposite being true for the more abundant low-molecular-weight naphthalenes (Supplemental Data, Fig. S2). For SCOT, the low-molecular-weight naphthalenes occurred in proportions in WAF and CEWAF similar to those in the parent oil, but the higher-molecular-weight, less abundant congeners were typically overrepresented in WAF and CEWAF.

The measured concentrations of alkanes and PAHs in WAF and CEWAF solutions of each crude oil were one to four orders of magnitude lower than concentrations in the corresponding whole oils (Supplemental Data, Fig. S3). Although the overall concentrations of PAH in 10% v/v WAF appeared higher than in 0.1% v/v CEWAF, these were not comparable concentrations. Total PAH concentrations in WAF and CEWAF varied by more than twofold among the four oils, with concentrations decreasing consistently in the order ANSC > FED > MESA; in contrast, SCOT WAF contained the highest concentrations total PAH concentrations, whereas SCOT CEWAF contained the lowest (Fig. 1A and B). During the 24-h daily-renewal interval, PAH concentrations declined exponentially (data not shown); by 24 h, concentrations were below detection in WAF solutions of each oil, but some alkyl PAH were still measurable in CEWAF. Concentrations of PAH in control solutions were not detectable.

In WAF and CEWAF treatments for each crude oil, the concentrations of total hydrocarbons estimated by fluorescence increased linearly with nominal loadings of WAF and CEWAF (Fig. 2). All regressions were statistically significant (p < 0.05) except for ANSC WAF, for which only three data points were above background fluorescence, and SCOT WAF, for which the slope was not significantly different from 0. For CEWAF, the slopes relating measured hydrocarbon concentrations to nominal CEWAF loadings of ANSC, MESA, and SCOT were similar (range 0.56–0.92; Table 3), but the slope for FED was distinctly higher (1.70). A similar trend was observed for WAF (Table 3). Thus, the treatments provided a graded series of test concentrations, but overall there were lower concentrations of hydrocarbons in WAF than in CEWAF solutions.

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Figure 2. Relationship between concentrations of total hydrocarbons (µg/L) measured by fluorescence spectrometry and the nominal amount of oil loaded to test solutions (% v/v) of water-accommodated fractions (WAF) and chemically enhanced water-accommodated fractions (CEWAF) of four weathered crude oils. ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude.

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Table 3. Regressions relating the log of measured concentrations (µg/L) of total hydrocarbons in test solutions to the log of nominal loadings (% v/v)a
Intercept (µg/L)Slope (µg/L)p For slopeIntercept (µg/L)Slope (µg/L)p For slope
  • a

    WAF = water-accommodated fraction; CEWAF = chemically enhanced water-accommodated fraction; ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude.



There was no mortality of negative control (water only) fish, and 8% mortality (two of 25 fish) of retene-positive control fish. Duplicate dispersant treatments, equivalent to the highest amount of dispersant (10 µl/L, equivalent to 0.001% v/v) in CEWAF dilutions, caused 48 and 4% mortality, respectively (average 26%).

For WAF treatments, mortality rates were low, increasing only at the highest loadings of each oil (Fig. 3A; equivalent to 10–18% v/v or higher). When expressed as measured concentrations of hydrocarbons, the apparent LC50s ranged from >362 (ANSC) to >1,744 µg/L (SCOT), because mortality did not exceed 50% at any test concentration (Table 4). Only MESA WAF was sufficiently toxic to generate more than 50% mortality and an estimate of the LC50 (880 µg/L), within the range of test loadings (Fig. 3A).

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Figure 3. Percentage mortality (A and B) and percentage normal (C and D) of rainbow trout embryos exposed from hatch to swim-up (22 d) to the water accommodated fraction (WAF; A and C) and the chemically enhanced WAF (CEWAF; B and D) of four weathered crude oils. Exposure was characterized as the measured concentrations of total hydrocarbons in test solutions. The dispersant controls were run in duplicate, and concentrations expressed are as nominal. ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude; DISP = dispersant.

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Table 4. Median lethal concentrations and median effective concentrations estimated using the measured concentrations of hydrocarbons in test solutions (µg/L)a
  22-Day LC50 (µg/L; 95% confidence limits)22-Day EC50 (µg/L; 95% confidence limits)
Percentage normalBSD indexSeverity indexRatio of yolk weight to fish weightEROD activity
  • a

    LC50 = median lethal concentration; EC50 = median effective concentration; BSD = blue sac disease; EROD = ethoxyresorufin-O-deethylase; WAF = water-accommodated fraction of oil; CEWAF = chemically enhanced WAF; ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude.

  • b

    EC50s marked with > indicate that the highest measured response was less than 50% of the maximum response.

  • c

    No CL = no confidence limits could be calculated because of the shape of the exposure–response relationship.

  • d

    — Indicates that ratios of EC50s could not be calculated because both EC50s were less than 50% of maximum responses.

WAFANSC>362133 (107–157)>362b>362>362>362
 FED>50872 (no CL)c>508506 (416–616)>508283 (115–692)
 MESA880 (830–987)657 (no CL)>895826 (780–875)823 (797–850)735 (681–793)
 SCOT>1,7441,440 (1,336–1,515)>1,744>1,744>1,744>1,744
CEWAFANSC764 (688–848)226 (134–363)>606663 (440–998)>1,015500 (353–710)
 FED714 (580–880)53 (35–75)>589619 (369–1,039)>1,218>589
 MESA614 (535–706)157 (136–179)>506560 (392–801)777 (630–957)517 (373–716)
 SCOT3,281 (2,569–5,094)1,168 (1,031–1,313)>2,3692,577 (2,299–2,890)>3,9962,415 (1,730–3,370)
Ratio (WAF/CEWAF)ANSC>0.50.6d>0.6> 0.7
 SCOT>0.51.2>0.7 >0.7

Mortality from CEWAF was much more clearly defined but was within the same range of hydrocarbon concentrations as WAF; LC50s ranged from 614 to 3,281 µg/L, equivalent to oil loadings of 0.06 to 0.16% v/v. There was little variation in LC50s among the three heavier oils (614–764 µg/L), but SCOT was approximately five times less toxic (Table 4). Compared with WAF, chemical dispersion had little effect on the toxicity of measured hydrocarbons, with estimated LC50 ratios of WAF/CEWAF ranging from >0.5 to 1.4. If these ratios were expressed as the nominal loadings of WAF and CEWAF, toxicity increased dramatically from WAF to CEWAF and to the greatest extent for ANSC, that is, by >260-fold, whereas the increase was 170- and >170-fold for MESA and FED oils and >35-fold for SCOT (Fig. 3B and Supplemental Data, Table S1).

Normal embryos at swim-up

For duplicate water control treatments, 100 and 88% (average 94%) of water control fish appeared normal. Although most fish exposed to 100 µg/L retene survived, none appeared normal. For dispersant controls, there was a gradient of toxicity, with the percentage normal declining progressively from 92 to 36% between 1 and 10 µl/L of dispersant, reflecting corresponding increases in mortality but few signs of toxicity typical of oil exposure.

The percentage of oil-exposed embryos that appeared normal declined with increasing loadings of WAF or CEWAF for all four crude oils (Fig. 3C and D). For WAF, the relative order of toxicity of the four oils was similar to that of mortality, that is, SCOT was clearly the least toxic, MESA and ANSC were intermediate in toxicity, and FED was most toxic, approximately 20 times more toxic than SCOT (Table 4). For SCOT and MESA, the percentage of embryos that were normal decreased sharply with increasing dose of WAF, in contrast to the lower slopes for ANSC and FED (Fig. 3C). With chemical dispersion, SCOT oil remained the least toxic, but there was less difference in toxicity among the other three (Fig. 3D). The slopes of toxicity curves were similar among all oils, in contrast to the WAF tests, and the effect of chemical dispersion on toxicity (ratio of WAF/CEWAF EC50s) was greatest for MESA (4.2-fold increase) and less for the other oils (0.6–1.4; Table 4). Overall, percentage normal was the only response metric for which EC50s could be calculated for every oil on the basis of oil loading or measured hydrocarbons.

Chronic toxicity: BSD index and SI

The mean BSD index and SI for duplicate retene treatments were higher than duplicate water controls (0.13 and 0.19 compared with 0.005 and 0.005). Dispersant caused no appreciable BSD; that is, the BSD index for dispersant-exposed trout ranged from 0 to 0.07, with no gradient related to dispersant concentration. In contrast, the SI, which includes a score for dead fish, increased with dispersant concentration from 0.04 to 0.48.

For WAF and CEWAF treatments, there was a progressive exposure-dependent increase in both BSD indices. At higher loadings, embryos showed significantly more severe BSD than water, dispersant, and retene controls, although exposure–response relationships for the BSD index were truncated by high mortality (Fig. 4). Thus, for most treatments, no EC50s could be calculated, because the highest responses did not exceed 50% of the maximum possible response (Table 4). For WAF, the toxicities of ANSC and FED oils based on both the BSD index and the SI were virtually the same (Fig. 4A and C and Table 4). The least toxic oil was SCOT, with MESA intermediate. Signs of BSD were more severe for CEWAF than for WAF, with little difference in EC50s among ANSC, FED, and MESA; SCOT was approximately fourfold less toxic than the other three oils (Table 4). However, the slope of exposure–response relationships for FED WAF and CEWAF was generally lower than that for any other oil. Although SCOT was least toxic, the slope of its exposure–response relationship was parallel to the slopes of ANSC and MESA. Based on measured hydrocarbons, there was little apparent increase in toxicity of oil with chemical dispersion, as indicated by the ratios of EC50s for the two indices, although most ratios were underestimates because responses to WAF did not exceed 50% of maximum for many treatments.

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Figure 4. Blue sac disease (BSD; BSD index in A and B; severity index in C and D) in rainbow trout embryos exposed from hatch to swim-up (22 d) to the water-accommodated fraction (WAF; A and C) and the chemically enhanced WAF (CEWAF; B and D) of four weathered crude oils. The BSD index includes only signs of pathology in living fish. The severity index includes both pathology and percentage mortality. Exposure was characterized as the measured concentrations of total hydrocarbons in test solutions. For SCOT WAF, the arrows pointing downward indicate that the BSD index and severity index were zero at the lower exposure concentrations. The dispersant controls were run in duplicate, and concentrations are expressed as nominal. ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude; DISP = dispersant.

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Ratio of yolk to fish weight

The yolk-weight ratio was least for water control fish (0.233, 0.286 for duplicates), reflecting their successful development to the feeding stage. For duplicate treatments of retene-exposed embryos, the ratios were higher (0.348, 0.50), as were the ratios for dispersant controls (0.388 and 0.302 for lowest and highest dispersant concentrations).

Yolk-weight ratios for WAF and CEWAF treatments increased with hydrocarbon concentrations for all four crude oils (Fig. 5), but the response for ANSC and FED was smaller within the range of test concentrations. Nevertheless, these two oils may be more toxic, because the response was first evident at lower concentrations of hydrocarbons than for the other two oils. The SCOT solutions were least toxic, with little effect on ratios until exposure concentrations exceeded those for all other oils; MESA toxicity was intermediate. The effect of chemical dispersion on yolk-weight ratios was difficult to estimate because reliable EC50s could not be calculated for WAF and CEWAF of most oils. However, SCOT oil was clearly the least toxic, and the other three had equivalent toxicities (Fig. 5B).

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Figure 5. Ratio of yolk to fish weight of rainbow trout embryos exposed from hatch to swim-up (22 d) to the water-accommodated fraction (WAF; A) and the chemically enhanced WAF (CEWAF; B) of four weathered crude oils. Fish weight was calculated from the weight of the whole embryo minus the weight of the yolk sac. Exposure was characterized as the measured concentrations of total hydrocarbons in test solutions. The dispersant controls were run in duplicate, and concentrations are expressed as nominal. ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude; DISP = dispersant.

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CYP1A induction

The whole-body EROD activity of water control embryos averaged 0.79 pmol resorufin/mg protein/min, approximately fourfold lower than retene-exposed positive control fish. Dispersant treatments increased EROD activity approximately threefold above controls, but with no exposure–response relationship from 1 to 10 µl/L dispersant.

In both WAF and CEWAF treatments, there was an exposure-dependent increase in EROD activity (Fig. 6), up to ninefold higher than the negative control. Exposure–response relationships for ANSC, FED, and MESA CEWAF were truncated by mortality at the highest loadings (0.1% v/v). The EC50 values indicated that potency of WAF for EROD induction increased in the order SCOT < MESA < ANSC < FED (Table 4). For CEWAF, the order of potency was SCOT < FED < ANSC < MESA, although the differences among ANSC, MESA, and FED were not large. The effect of chemical dispersion on potency was difficult to discern, because the EROD response was low for most treatments, precluding the calculation of ratios between EC50s for WAF and CEWAF. Nevertheless, the patterns appeared similar to those for other responses.

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Figure 6. Whole-body ethoxyresorufin-O-deethylase (EROD) activity of rainbow trout embryos exposed from hatch to swim-up (22 d) to the water accommodated fraction (WAF; A) and the chemically enhanced WAF (CEWAF; B) of four weathered crude oils. Test solutions were characterized by the measured concentrations of total hydrocarbons. The dispersant controls were run in duplicate, and concentrations are expressed as nominal. ANSC = Alaska North Slope crude; FED = Federated crude; MESA = medium South American crude; SCOT = Scotian light crude; DISP = dispersant.

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  1. Top of page
  2. Abstract
  8. Acknowledgements
  10. Supporting Information

Both WAF and CEWAF from all four crude oils were chronically toxic to trout embryos, causing an increased prevalence of BSD, a subsequent increase in mortality, and a lower proportion of fish that would be considered normal when the embryos made the transition to active feeding. The exposure-dependent induction of CYP1A proteins, as measured by increased EROD activity, indicated that the embryos had accumulated PAH, constituents of crude oil that are known to be toxic to fish. In fact, the signs of toxicity (BSD) were virtually the same as those of retene 5, an alkylphenanthrene used as the positive control. Retene causes cardiac toxicity, and the associated impairment of circulation retards development 25. The reduction in absorption of the yolk sac (increased yolk-wt ratios) for embryos exposed to the four oils was consistent with impaired embryonic development and typical of toxic effects observed in an array of fish species exposed to crude and refined oils 8, 14, 26; this suggests exposure to common toxic components, that is, alkyl PAHs.

There was remarkably little difference in toxicity between WAF and CEWAF when LC50s and EC50s were expressed as measured total hydrocarbon concentrations. The ratios of WAF LC50s or EC50s to CEWAF approximated 1.0, except for MESA oil, for which the ratio for percentage normal was 4.2, but ratios for most other responses to MESA were 1.5 or less. In contrast, the WAF to CEWAF ratios for LC50s and EC50s based on nominal loadings to test solutions (% v/v) ranged from >35 to >300 (and >360 for EROD induction; Supplemental Data, Table S1), although these ratios may be underestimates, because actual WAF EC50s were often greater than the highest concentration tested. Thus, more than 30 to 360 times less CEWAF than WAF was needed to achieve toxic concentrations of oil in water. The results are consistent with earlier studies showing that chemical dispersion increases the risk of oil toxicity by 10- to 100-fold when exposure is characterized as loading 14, 15. This suggests that dispersion, by suspending oil droplets in the water column, simply accelerates partitioning of hydrocarbons from oil to water and increases the exposure of embryos to hydrocarbons without changing hydrocarbon toxicity.

In terms of bioavailability, the fish likely responded to dissolved PAH and not to PAH in droplets 26. However, the method of sampling for analyses by GC-MS or by fluorescence prevents a distinction between PAH in droplets and dissolved PAH. By extracting samples with dichloromethane (GC-MS) and preserving samples with ethanol (fluorescence), oil in droplets is incorporated into the dissolved phase for analysis. Given the low solubility of most hydrocarbons in test solutions, they are likely present in small oil droplets, consistent with the strong correlation between the proportion of PAH in WAF and CEWAF test solutions and the proportion in the parent oil (e.g., ANSC, FED, Supplemental Data, Fig. S2). If most was in the dissolved phase, the highly soluble naphthalenes would be overrepresented in test solutions relative to the parent oil, and low-solubility, high-molecular-weight PAH would be underrepresented or less than detection. However, the opposite might have been the case for CEWAF of MESA oil and WAF and CEWAF of SCOT oil. In these cases, most high-molecular-weight PAHs (<10% of total PAHs) were overrepresented relative to whole oil, likely because the major constituents (>10% of the total PAHs) were underrepresented. The major constituents were the C0 to C4 naphthalenes, which are more water soluble, volatile, and biodegradable than PAHs with more than two rings. These characteristics, in addition to their absorption by fish and to the surfaces of test containers, likely contributed to their loss from test solutions over the 24 h between solution renewals.

Oil dispersion can also change the relative proportions of different PAHs in solution, although the effect is not consistent among oils. Except for SCOT, CEWAF solutions contained a higher proportion of MMW alkyl PAHs than did WAF or the parent crude oil (Fig. 1A and 1B), likely because of the greater entrainment of oil droplets into CEWAF relative to WAF. Couillard et al. 27 also reported that the HMW PAH constituted a higher proportion of total PAH in solution when the same MESA oil was chemically dispersed, consistent with the higher WAF to CEWAF ratios of EC50s for MESA observed in the present study.

Among the four test oils, SCOT had the lowest ratio of aromatic compounds to TPHs (4.7%, approximately one-third that of the other oils) and contained two to three times more alkanes than the other oils and correspondingly lower concentrations of total PAHs (0.57% by weight), less than half that of the other three oils (1.29–1.55%; Table 2). It would also be considered the lightest oil and was the least viscous. Chemical dispersion might have reduced differences in physical characteristics among oils. When the results are expressed as nominal loadings, the large spread among WAF LC50s and EC50s suggests that the toxicities of each oil were quite different, with ANSC being 2- to 10-fold less toxic than all other oils. However, when chemically dispersed, the amount of hydrocarbon measured in solution was much more uniform, and SCOT was clearly the least toxic oil, with ANSC toxicity similar to that of FED and MESA. Hence, the chemical dispersant reduced the influence of any unique physical interactions between each oil and water, likely by making the size of oil droplets in suspension more uniform among oils. Chemical dispersion makes all droplets the same size by reducing interfacial tension, and the droplet stability of WAF critically depends on the balance between interfacial tension and viscosity. The differing slopes of regressions relating fluorescence to dilution for WAF may be a viscosity effect. A comparison of droplet size distribution among the various WAFs might help to resolve the differences among oils. The reduction in the differences of toxicity among similar oils, along with the increased responses of fish embryos at overall lower rates of oil loading, demonstrates that chemical dispersion is a useful strategy for standardized testing to compare toxicity among oils.

Dispersant toxicity did not affect the measurement of oil toxicity. The primary response to dispersant alone was mortality (48%) at the highest concentration in only one of two replicates, which in turn influenced percentage normal and the BSD index. The dispersant-alone treatments caused only small increases in the signs of BSD at the highest test concentration, no change in the embryo development index, and no increase in EROD activity, suggesting that the dispersant was at least 10 times less toxic than oil itself and did not have the same mechanism of toxicity. Given that mortality was only 4% in the second replicate of the same dispersant concentration, it is likely that mortality in the first replicate was a false positive.

There was also the possibility of dispersant–oil interactions. If dispersant increased toxicity by acting independently or interactively with the constituents of oil, the ratios of WAF EC50s and LC50s to CEWAF EC50s and LC50s should consistently exceed 1.0; that is, the LC50s and EC50s of WAF expressed as measured hydrocarbon concentrations should be several-fold higher than those of CEWAF. However, for three of the four oils, the ratios were less than twofold greater or less than 1.0. Only for MESA did the ratio widely exceed 1.0, by 4.2 times for percentage normal (Table 4). These results are not sufficiently consistent to support the idea that the dispersant modified the toxicity of oil hydrocarbons.

The absence of dispersant–oil interactions might seem counterintuitive given that the dispersant might have been toxic at the highest concentration tested. The likely action of dispersant in the absence of oil would be a detergent-like effect on the external lipid membranes of embryos. Because the hydrophobic substituents of the dispersant would associate with membrane lipids, the hydrophilic substituents would solubilize the dispersant/lipid matrix, destroying the external membranes and hindering oxygen transfer and ion and osmotic regulation. Fish exposed to detergents develop a fuzzy appearance as damaged external membranes slough off; histologically, the gill epithelium is destroyed and oxygen uptake impaired 28. We did not observe this in fish surviving exposure to dispersant, and a fuzzy appearance of some dead fish was likely due to rapid decomposition. In contrast, when dispersant is mixed with oil to create CEWAF, the dispersant will associate first with oil droplets during preparation of CEWAF stock, and little dispersant, if any, will be free in solution to interact with fish tissues. As long as the oil remains dispersed, which was the case in the 24-h static daily-renewal protocol, interactive toxicity between dispersants and hydrocarbons would be unlikely.

Petroleum oils are complex mixtures of thousands of different compounds, and their toxicity varies as a function of their composition. Alkyl PAH are regarded as a major determinant of petroleum oil toxicity to fish embryos 6, and toxicity has been associated with oil fractions rich in alkyl phenanthrenes, fluorenes, naphthobenzothiophenes, chrysenes, and pyrenes 29. In single-compound exposures, alkyl phenanthrenes such as retene cause concentration-dependent increases in BSD of exposed embryos and CYP1A induction in juvenile trout 5, 7, 18, 30. These PAHs are generally more persistent and more water soluble than aliphatics 31 and are more likely to be bioavailable to fish. Therefore, PAHs and their alkyl homologs are likely the agents in crude oil causing the CYP1A induction, increased EROD activity and signs of BSD.

Compared with the other three oils, CEWAF of MESA had the lowest EC50s for inducing EROD activity, suggesting that it contained either higher potency CYP1A inducers or higher concentrations of inducers; for WAF, the ranking was ambiguous because firm EC50s were not available for FED and ANSC oils. The high potency of MESA CEWAF was consistent with high concentrations of alkyl PAHs in 0.1% v/v CEWAF, in 10% v/v WAF, and in the crude oil itself. In studies with mummichog embryos, chemical dispersion of MESA oil increased both toxicity and the concentrations of PAHs in solution 27. However, toxicity was most strongly correlated with the concentrations of total PAHs (>90% alkyl PAHs), whereas whole-body EROD induction was most strongly correlated with the HMW PAH fraction. Thus, the PAHs causing toxicity and induction might not be the same, and CYP1A induction may not be part of the primary mechanism of embryotoxicity 25.

If the four oils are ranked by CEWAF (Table 4) EC50s for percentage normal, a metric that integrates both lethal and sublethal responses, FED was clearly the most toxic. Scotian light crude was least toxic, likely because it had the lowest concentrations of alkyl PAHs, and the array of alkyl PAHs was dominated by the alkylnaphthalenes, which are least embryotoxic 29 and most rapidly lost during weathering (Fig. 1). The observed order of relative toxicity (FED > ANSC = MESA >> SCOT) seems intuitively to be correct, assuming that alkyl PAHs are the toxic components of each oil 6. Compared with SCOT, the three more toxic oils have six- to 110-fold higher proportions of the C1 to C4 alkyl derivatives of dibenzothiophenes, phenanthrenes, naphthobenzothiophenes, and chrysenes, the three- or four-ringed PAH most closely associated with embryotoxicity 29, and FED contained more alkylphenanthrenes that have been demonstrated to be embryotoxic 5, 7 than ANSC and MESA. Toxicities could also be compared by calculating the concentration of TPAHs from the total PAHs reported for 0.1% (v/v) CEWAF shown in Figure 1b and the EC50s expressed as dilutions (% v/v; Supplemental Data, Table S1), assuming a linear relationship between dilution and TPAH concentration. In this case, the percentage normal EC50s for all four oils were quite similar, ranging from 2.1 to 3.4 µg/L. With this perspective, the toxicity of TPAH in test solutions was relatively constant among oils, and the concentration of each oil in test solutions was the amount required to achieve toxic concentrations of TPAHs.

In summary, chemical dispersion of four crude oils caused dramatic increases in toxicity to trout embryos when effects were compared with the amount of WAF or CEWAF added to test solutions. However, the action of the dispersant was simply to increase the exposure of embryos to oil in water, because there was little difference in toxicity between WAF and CEWAF when effects were compared with measured concentrations of hydrocarbons. The differences in the chronic toxicity to trout embryos observed among four crude oils reflected their chemical composition, specifically the concentrations of three- or four-ringed alkyl-PAHs. The lightest oil was least toxic because it contained the lowest concentrations of these PAHs; thus more light oil was required in test solutions than heavy oil to achieve the same toxic concentration of TPAHs. Despite different sources, the remaining three crude oils varied little in toxicity, likely because there were only small differences in their concentrations of three- or four-ringed alkyl PAHs. These results will improve our capacity to assess the ecological risks of oil spills and the application of chemical dispersants.


  1. Top of page
  2. Abstract
  8. Acknowledgements
  10. Supporting Information

J. Scott, C. Khan, S. Wahoush, S. Kennedy, L. Lu, S. Fallatafti, B. Lemire, and the Analytical Services Unit of Queen's University provided technical support. The present study was funded by grants and contracts from the Natural Sciences and Engineering Research Council of Canada, the Petroleum Research Atlantic Canada, and the Department of Fisheries and Oceans Canada.


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  2. Abstract
  8. Acknowledgements
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  8. Acknowledgements
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

etc_1739_sm_SupplData.doc4634KSupplementary Data

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