Science of the Total Environment 543 (2016) 644–651
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Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
The effects of weathering and chemical dispersion on Deepwater Horizon
crude oil toxicity to mahi-mahi (Coryphaena hippurus) early life stages
Andrew J. Esbaugh a,⁎, Edward M. Mager b, John D. Stieglitz b, Ronald Hoenig b, Tanya L. Brown c,
Barbara L. French c, Tiffany L. Linbo c, Claire Lay d, Heather Forth d, Nathaniel L. Scholz c, John P. Incardona c,
Jeffrey M. Morris d, Daniel D. Benetti b, Martin Grosell b
a
Department of Marine Science, University of Texas, Marine Science Institute, 750 Channel View Dr., Port Aransas, TX 78373, United States
Department of Marine Biology and Ecology, University of Miami, Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Cswy., Miami, FL 33149, United States
c
Environmental and Fisheries Science Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E.,
Seattle, WA 98112, United States
d
Stratus Consulting/Abt Associates, 1881 Ninth Street, Suite 201, Boulder, CO 80302, United States
b
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Weathering of crude oil increases the
toxicity of water accommodated fractions.
• Dispersant does not change lethal or
sub-lethal oil toxicity in larval fish.
• Pelagic larvae are sensitive to weathered oil at low μg/l ΣPAH concentrations.
• There is a close relationship between
survival and cardiotoxicity between oil
types.
a r t i c l e
i n f o
Article history:
Received 24 September 2015
Received in revised form 11 November 2015
Accepted 13 November 2015
Available online xxxx
Editor: D. Barcelo
Keywords:
Polycyclic aromatic hydrocarbon
PAH
a b s t r a c t
To better understand the impact of the Deepwater Horizon (DWH) incident on commercially and ecologically important pelagic fish species, a mahi-mahi spawning program was developed to assess the effect of embryonic exposure to DWH crude oil with particular emphasis on the effects of weathering and dispersant on the magnitude
of toxicity. Acute lethality (96 h LC50) ranged from 45.8 (28.4–63.1) μg l−1 ΣPAH for wellhead (source) oil to 8.8
(7.4–10.3) μg l−1 ΣPAH for samples collected from the surface slick, reinforcing previous work that weathered oil
is more toxic on a ΣPAH basis. Differences in toxicity appear related to the amount of dissolved 3 ringed PAHs. The
dispersant Corexit 9500 did not influence acute lethality of oil preparations. Embryonic oil exposure resulted in
cardiotoxicity after 48 h, as evident from pericardial edema and reduced atrial contractility. Whereas pericardial
edema appeared to correlate well with acute lethality at 96 h, atrial contractility did not. However, sub-lethal
cardiotoxicity may impact long-term performance and survival. Dispersant did not affect the occurrence of peri-
⁎ Corresponding author.
E-mail address: [email protected] (A.J. Esbaugh).
http://dx.doi.org/10.1016/j.scitotenv.2015.11.068
0048-9697/© 2016 Elsevier B.V. All rights reserved.
A.J. Esbaugh et al. / Science of the Total Environment 543 (2016) 644–651
Dispersant
Corexit 9500
Pericardial edema
Atrial contractility
645
cardial edema; however, there was an apparent reduction in atrial contractility at 48 h of exposure. Pericardial
edema at 48 h and lethality at 96 h were equally sensitive endpoints in mahi-mahi.
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
During the spring and summer of 2010 the Deepwater Horizon
(DWH) incident released millions of barrels of crude oil into the northern Gulf of Mexico (GOM), making it the largest marine oil spill in
United States history (Crone & Tolstoy, 2010; Ryerson et al., 2012;
Camilli et al., 2012; McNutt et al., 2012). This resulted in extensive oiling
of the pelagic zone and fouling of shoreline habitats. The timing of the
DWH spill also coincided with the spawning seasons for many commercially important fish species (Rooker et al., 2012; Rooker et al., 2013;
Block et al., 2005; Brown-Peterson et al., 2001; Gibbs & Collette, 1959)
and likely resulted in exposure to both breeding and early life stage
fish. The embryos of marine fish are particularly susceptible to developmental toxicity from crude oil-derived polycyclic aromatic hydrocarbons (PAHs), which cause a suite of sub-lethal morphological and
cardiovascular defects (Incardona et al., 2004; Incardona et al., 2009;
Incardona et al., 2011a; Incardona et al., 2012; Dubansky et al., 2013;
Zhang et al., 2012; Huang et al., 2012). In aquatic environments crude
oil releases a complex mixture (Carls & Meador, 2009) of PAHs into
the dissolved phase, many of which have their own unique toxicity
characteristics (Incardona et al., 2004; Incardona et al., 2006;
Incardona et al., 2011b). The composition of individual PAHs in the
water column results from a combination of solubility and natural
weathering processes. Weathering typically removes low molecular
weight hydrocarbons from oil water mixtures through evaporation,
subsequently producing oil slicks with proportionally higher molecular
weight and low solubility PAHs (Carls & Meador, 2009). However, the
DWH spill was distinct from past surface spills in that the crude oil
was released from the seafloor, facilitating greater dissolution of low
molecular weight hydrocarbons into the water column during ascent
(Reddy et al., 2012). This was further complicated by the unprecedented
1.8 million gallons of chemical dispersants that were applied to the surface and sub-surface in an effort to solubilize crude oil and minimize
slick formation (Lehr et al., 2010). Dispersants such as the heavily
used Corexit 9500 influence oil solubility and PAH bioavailability. Importantly, these dispersants have also been shown to be toxic to fish,
such as the Atlantic herring and inland silverside (Hemmer et al.,
2011; Adams et al., 2014a).
Historically, oil exposure was thought to exhibit toxic effects primarily through narcotic mechanisms, which led to the target lipid model of
toxicity that related oil constituent toxicity to the respective octanol/
water partitioning coefficient (Di Toro et al., 2000). The solubility of individual constituents was subsequently integrated with the model to
estimate the toxic potential of complex oil water mixtures (Di Toro
et al., 2007). Under this scenario low molecular weight PAHs and volatile BTEXs were estimated to be the more harmful components in complex mixtures regardless of the organism (Di Toro et al., 2007).
However, over the last 15 years a range of complementary studies
have established that cardiotoxicity in early life stage fish following
crude oil exposure is specifically associated with water soluble PAHs
(Incardona et al., 2009; Marty et al., 1997; Carls et al., 1999; Incardona
et al., 2005; Adams et al., 2014b). The primary injury phenotype was extensively characterized in boreal species following the 1989 Exxon
Valdez oil spill in Alaska (Peterson et al., 2003), and is marked by fluid
accumulation around the heart and other secondary malformations
(Incardona et al., 2011a). Interestingly, studies indicated that 3 ring
PAHs were the primary cause of embryonic heart failure by disrupting
cardiac function (Incardona et al., 2004; Incardona et al., 2009;
Incardona et al., 2005), and furthermore that the relative proportion of
3 ring PAHs is predictive of embryonic cardiotoxicity to a complex oil
mixture (Incardona et al., 2004). This has raised the important question
of whether natural weathering reduces toxicity by eliminating the
higher solubility toxic compounds, or increases toxicity by concentrating those components with greater individual toxicity.
Despite the rich literature on PAH toxicity to larval and embryonic
fish, relatively little is known about species native to the pelagic zone
of the GOM. We recently demonstrated that four representative pelagic
species – bluefin tuna, yellowfin tuna, amberjack and mahi-mahi –
show characteristic embryonic cardiotoxicity when exposed to high energy water accommodated fractions (WAFs) of naturally or artificially
weathered MC252 crude oil at low μg l−1 total (∑) PAH concentrations
(Incardona et al., 2014; Mager et al., 2014). However, a systematic assessment of acute lethality and cardiotoxicity that incorporates the
state of weathering and degree of chemical dispersion has yet to be performed on GOM pelagic species. This information is crucial when
attempting to assess the damage to fish and fisheries resulting from
the DWH oil spill. To this end, the current study established a mahimahi (Coryphaena hippurus) spawning brood stock culture specifically
to obtain newly fertilized embryos for toxicity testing with a variety of
DWH-relevant PAH mixtures. The main objective was to assess the differential toxicity of source, artificially weathered and naturally weathered oil, as well as the influence of chemical dispersion on the toxicity
of these field-collected samples. A secondary objective was to directly
compare standardized lethal concentration 50 (LC50) tests to sublethal measures of cardiotoxicity to the different oil types with the intent of defining the proposed link between the two endpoints.
2. Experimental methods
2.1. Animals
Mahi-mahi broodstock (C. hippurus) were captured off the coast of
Miami, FL using hook and line angling. The fish were subsequently
transferred to the University of Miami Experimental Hatchery
(UMEH), where they were acclimated in 80 m3 fiberglass maturation
tanks equipped with re-circulated and temperature controlled water
(IACUC protocol # 12–064). All embryos used in the experiments described herein were collected within 2–10 h following a volitional
(non-induced) spawn using standard UMEH methods (Stieglitz et al.,
2012). A prophylactic formalin treatment (37% formaldehyde solution
at 100 μl/l for 1 h) was administered to the embryos, followed by
30 min of flushing with a minimum of 300% water volume in the treatment vessel using filtered, UV-sterilized seawater. A small sample of
eggs was collected from each spawn to microscopically assess fertilization rate and embryo quality. Spawns demonstrating low fertilization
rate (b 85%) or frequent developmental abnormalities (N5%) were not
used.
2.2. Preparation of water accommodated fractions
Three distinct sources of MC252 crude oil were used to generate
WAFs. A field sample was collected on July 29, 2010 from the hold of
barge number CTC02404, which was receiving slick oil from various
skimmer vessels. This oil type had a DOSS concentration below detection limit (b1 μg g−1). This sample is referred to as slick A throughout.
An artificially weathered source sample was generated by slow heating
at 70 °C until the total volume was reduced by 20%. The third sample
was unweathered source oil that was obtained from the riser pipe.
Corexit 9500 was used as dispersant. In all cases, samples were delivered to the University of Miami under chain of custody. High energy
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water accommodated fractions (HEWAFs) were prepared as described
previously (Incardona et al., 2014; Incardona et al., 2013) at a loading
rate of 1 g of oil per liter of seawater. Chemically enhanced water accommodated fractions (CEWAFs) were prepared in 2 l or 5 l aspirator
bottles at a loading rate of 1 g of oil per liter of filtered (1 μm), UVsterilized seawater. Corexit 9500 was added at a rate of 100 mg per g
of oil and mixed for 18–24 h at an approximately 10% vortex using a
stir plate and Teflon coated magnetic stir bar. After mixing the CEWAF
was allowed to settle for 3–6 h, after which the lower 90% was collected
and used for exposures. All WAFs were prepared the day of use. Chemical composition of the respective WAF types is shown in supplemental
Figs. S1–3.
Due to logistical constraints, only unfiltered samples were measured
for toxicity assessments. The dissolved fraction of each PAH analyte was
subsequently estimated for each WAF preparation type. Because Deepwater Horizon (DWH) oil is a complex mixture with many unknown
components, we used empirical data for these estimates. Our participation in the DWH NRDA provided access to chemical analyses of many
samples of WAF preparations using the same oil types and methods as
the exposure treatments. These samples included paired filtered and
unfiltered samples taken from dilutions of each of the six WAF preparations. To produce filtered samples, WAF dilutions were passed through
two stacked 0.3-μm glass fiber filters under low suction (b 5 cm–10 cm
Hg); this filtered out the oil droplets, leaving only the dissolved analytes
in the filtered sample. Regressions of the filtered concentration versus
unfiltered concentration were performed for each of 50 PAH analytes
within each WAF (see Fig. S4). For each regression, we fitted an equation of the form:
½dissolved ¼ a 1 eb½unfiltered
where “a” is the modeled maximum found in the solution, and “b” is
proportional to the slope of the relationship approaching the maximum.
These models were fitted using the nls function in the stats package in
(R Core Team, 2015). When this equation fit poorly for insoluble
analytes (e.g., Kow N 5.3) (Redman et al., 2012) or analytes with low concentrations in the oils used to produce the WAFs, we assumed that those
analytes did not dissolve, and estimated the dissolved concentration as
zero. For a few highly soluble analytes (naphthalene, C1-naphthalene,
and biphenyl), measurements of filtered concentrations were often
greater than the corresponding unfiltered concentrations. For these
analytes, we estimated the dissolved concentrations as 100% of the unfiltered concentrations. The regression results and solubility assessments described above were applied to measured unfiltered
concentrations to estimate the dissolved fraction of each analyte (see
Supplemental Table S1).
2.3. Toxicity testing
All tests were performed in a temperature controlled environmental
chamber (26 °C) with a 16:8 light/dark cycle. Treatments were generated by spiking 5 l of filtered and sterile seawater with varying levels
of WAF using a glass syringe. The treatment was mixed on a stir plate
for 5 min and then aliquoted into 4 1 l replicates. A single replicate
consisted of 20 embryos in 1 l of test solution held in a 1 l glass beaker,
with four replicates per treatment. Survival tests consisted of five test
concentrations and a control, while sub-lethal tests consisted of four
concentrations and a control. This difference was due to the length of
time needed to process sublethal endpoints relative to survival. Tests
were monitored daily for survival. Mortality was assessed visually and
by lack of response to prodding. All dead animals were removed from
the test daily using glass transfer pipettes. Survival tests were deemed
unreliable if control survival at hatch was less than 70%, or if the subsequent survival of hatched larvae was less than 80%. The defined hatch
point for the purposes of test reliability was 48 h as mahi-mahi typically
hatch at 35–40 hpf. No water changes were performed.
Survival tests were concluded after 96 h while cardiotoxicity endpoints were assessed after 48 h. For all imaging, 2–3 larvae were captured at a time and transferred to a petri dish, where they were
individually mounted atop 2% methylcellulose in seawater. This ensured rapid imaging of specimens, avoiding potential temperature elevation on the microscope stage. Two stereoscope stations allowed
sequential processing of 2 replicates at a time; larvae were imaged continuously until all replicates were completed. Crude oil exposure replicates were processed in random order with unexposed control fish
evenly spaced throughout. Images (640 × 480) were collected using
FireI-400 industrial digital video cameras (Unibrain, San Ramon, CA)
mounted on Nikon SMZ800 stereomicroscopes, using MacBook laptops
and BTV Carbon Pro software. Images were calibrated using a stage
micrometer.
2.4. Image analysis
For scoring the presence or absence of edema, still frames and videos
were assessed for the shape of the yolk mass. Larvae were scored as normal if the anterior portion of the yolk sac was smooth and rounded with
a bullet-shaped tip and if there were are no obvious indentations on the
yolk sac due to pressure from fluid buildup in the pericardial area.
Edema was scored positive if the anterior portion of the yolk sac was
concave or pushed to a sharp point, and/or if indentations indicated
by dark, angular lines were seen pushing on the yolk sac due to pressure
from fluid buildup in the pericardial area. Atrial diameter was measured
in digital video frames stopped at diastole (maximal relaxation) and
systole (maximal contraction), and used to measure contractility calculated as fractional shortening using the formula (diastolic diameter −
systolic diameter) / diastolic diameter × 100. All scoring of sublethal
endpoints was performed blind, whereby the oil dosage information
was withheld from the scoring individual.
2.5. Water chemistry analysis
Samples for total sum PAH (ΣPAH) analysis were collected from
each WAF preparation immediately after generation, as well as from
each treatment concentration immediately after dilution. Each sample
was collected into a 250 ml amber glass bottle with no head space and
immediately stored at 4 °C. The ΣPAH measurements comprised a
panel of 50 different PAH analytes. This is more than the 33–40 PAHs
measured in past studies, with the consequence of shifting the ΣPAH
metric upwards. For CEWAF samples, four 10 ml samples were collected
for dioctylsulfosuccinate sodium salt (DOSS) analysis and stored at 4 °C.
For source oil WAFs, three additional 20 ml samples were collected in
glass vials with no head space for volatile analysis, including benzene,
toluene, ethylbenzene and xylenes (BTEX). Preliminary analyses
showed artificially weathered source and slick A oil WAF preparations
contained negligible BTEX concentrations. All samples were then
shipped overnight on ice to ALS Environmental (Kelso, WA) for analysis
by gas chromatography and mass spectrometry – selective ion monitoring (GC/MS-SIM; based on EPA method 8270D).
To complement GC/MS-SIM quantitation of PAHs, a previously
established fluorescence method was also used to calculate ΣPAH for
each treatment (Greer et al., 2012). Briefly, a percent WAF standard
curve was generated for each prepared WAF in a 50% ethanol solution.
A 10 ml sample from each treatment concentration was diluted in
10 ml of ethanol. All standards and samples were sonicated for 3 min
and centrifuged at 10,000 rpm for 10 min to precipitate salts. The supernatant was then transferred to a quartz cuvette and measured for fluorescence using a LS-45 spectrophotometer (PerkinElmer). The exact
wavelengths were determined for each individual WAF using an
excitation-emission scan ranging from 200 to 600 nm. Two distinct
peaks were generally observed at excitation wavelengths of
A.J. Esbaugh et al. / Science of the Total Environment 543 (2016) 644–651
approximately 224 and 258 nm. Instrument software was used to calculate the peak area (relative fluorescence units; rfu), which was used to
calculate a standard curve (R2 ≥ 0.99) of log[rfu] versus log[% WAF].
The commercially measured PAH concentrations in the stock WAF
were then used to convert all fluorescence values to ΣPAH. An independent clean seawater sample from the UMEH facility was used to account
for background fluorescence. This analysis provided ΣPAH values for
one concentration of the 96 h LC50 source HEWAF test, as well as four
concentrations for the slick A CEWAF test.
The following water quality parameters were also monitored for all
tests: temperature, pH, dissolved oxygen (DO), salinity and total ammonia. Temperature and DO were measured daily in all replicates using a
ProODO handheld optical DO probe and meter (YSI, Inc., Yellow Springs,
OH) and pH was measured daily using a PHM201 meter (Radiometer,
Copenhagen, Denmark) fitted with a combination glass electrode.
Both pH and DO probes were calibrated daily. Salinity was measured
daily using a refractometer and total ammonia was measured in final
water samples using the colorimetric assay (Verdouw et al., 1978).
2.6. Statistics
Exposure-response curves were fit using the U. S. Environmental
Protection Agency's TRAP software package. All data were first fit to a
tolerance type Gaussian model with 3 parameters using logtransformed data. In some instances this method yielded a poor fit
with no convergence; therefore, a non-linear regression method was
used (see Table S2 and S3). Comparison of LC50 and cardiotoxicity effective concentration 50 (EC50) values was based on overlapping 95% confidence intervals, with non-overlapping intervals signifying a significant
difference between estimates. Hypothesis testing of cardiac contractility
was performed using a one-way analysis of variance and Dunnett posthoc test (P ≤ 0.05) using the SigmaPlot 12.5 software package. When
data did not conform to test assumptions an analysis of variance on
ranks with a Dunn's post-hoc test was used. The effect of a single concentration of dispersant on cardiac endpoints was tested using a
student's t-test. Note that hypothesis testing was used in lieu of EC50
because the upper bounds of the endpoint (maximal response) were
not apparent in the data set. All raw data from toxicity tests are presented in supplemental Tables S4–6.
3. Results
3.1. Chemical composition of WAF preparations
Of the three oil types, slick A was the most enriched in 3 ringed PAHs,
with tricyclics accounting for approximately 60% of the total ΣPAH in
both HEWAF and CEWAFs (Figs. S1–3). Source and artificially weathered source HEWAFs had similar profiles that were approximately 50%
2-ring PAHs. The weathered source CEWAF profile was similar to the
HEWAF; however, the source CEWAF contained 80% 2-ring PAHs. The
estimated dissolved fraction of all WAF preparations was more enriched
647
in 2-ringed PAHs, and accounted for 84–94% of ΣPAH in weathered and
source WAFs, but only 27–33% in slick A WAFs. The measured BTEX
were generally similar in total concentration between source HEWAF
and CEWAF (Fig. S4), but when calculated relative to the total ΣPAH
the CEWAF contained approximately 17 BTEX per unit PAH while the
HEWAF contained only 0.7 BTEX per unit PAH.
3.2. Lethal concentration toxicity tests
96 h LC50 estimates for the six WAF preparations are shown in
Table 1. There were marked differences in toxicity between oil samples
related to weathering state. When expressed on the basis of either total
or dissolved ΣPAH, source oil was significantly less toxic than the slick A
oil in both HEWAF and CEWAF preparations. Toxicity of the artificially
weathered source oil was not significantly different from either source
or slick A when expressed as dissolved ΣPAH; however, when
expressed as total ΣPAH weathered source HEWAF was also significantly more toxic than source oil. When expressed based on the dissolved 3 ring PAH composition, LC50s were nearly indistinguishable
between all three oil samples and WAF types. The only notable exception was a marginally, but significantly, higher estimate for slick A
HEWAF and CEWAF relative to the source oil CEWAF. No differences between CEWAFs and HEWAFs were apparent within an oil type when
expressed as dissolved ΣPAH or 3 ring PAH; however, the CEWAF was
more toxic than the HEWAF for source oil based on total ΣPAH.
To assess the influence of chemical dispersion on crude oil toxicity,
survival data were analyzed based on the measured DOSS concentrations in the respective CEWAFs. The toxicity of all three oil types was
significantly more severe than the Corexit 9500 alone treatment (Fig.
1). Corexit 9500 alone resulted in a DOSS LC50 of 3.9 mg l−1, but ranged
between 22 and 918 μg l−1 in the presence of oil.
3.3. Sub-lethal cardiotoxicity tests
The occurrence of pericardial edema and reduced cardiac contractility were used as endpoints to assess sub-lethal toxicity after 48 h of exposure to the various WAF types. No differences in the EC50 for the
occurrence of pericardial edema were apparent between oils or preparation methods when expressed as total or dissolved ΣPAH (Table 2);
although, a suitable pericardial edema EC50 could not be calculated
for slick A HEWAF. When expressed as dissolved 3 ring PAHs, the slick
A CEWAF had a significantly higher EC50 than other CEWAFs, but this
value was within a similar range to the HEWAF preparation. The range
of test concentrations was also not sufficient to define a maximum response in cardiac contractility, and therefore LOEC values were used in
place of EC50s to define toxicity. This precluded quantitative comparisons of toxicity between oil types and WAF preparation methods. For
HEWAF assays the LOEC values for reduction of contractility (Table 3)
followed a qualitatively similar trend to that described for survival and
pericardial edema; however, HEWAF LOEC values are 4 to 7 times
higher than the respective CEWAF LOECs (Table 3). Overall, the different
Table 1
Acute lethality estimates (96 h LC50s) for mahi-mahi exposed to different Deepwater Horizon oil types using both high energy and chemically dispersed preparations. Water-accommodated fractions (WAFs) are expressed as total ΣPAH, estimated dissolved ΣPAH and estimated dissolved 3 ring polycyclic aromatic hydrocarbon (PAH) concentrations. Bracketed values
represent 95% confidence intervals.
WAF preparation
High energy
Chemically enhanced
Corexit 9500 only
96 h LC50
Source
Weathered
Slick A
Source
Weathered
Slick A
DOSS (mg l−1)
Total (μg l−1)
Dissolved (μg l−1)
3 ring (μg l−1)
45.8 (28.4–63.1)
12.3 (9.5–16.1)
8.8 (7.4–10.3)
25.3 (23.2–27.7)
8.7 (0–59.8)
9.5 (8.5–10.5)
3.9 (3.7–4.1)
21.3 (5.4–37.3)
5.4 (2.1–13.6)
3.5 (3.1–4.1)
20.6 (18.9–22.5)
3.7 (0–19.9)
3.4 (3.2–3.7)
–
1.3 (0.3–2.3)
0.8 (0.3–2.1)
2.1 (1.8–2.4)
1.3 (1.2–1.4)
0.4 (0–2.4)
2.1 (2–2.3)
–
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Fig. 1. The effects of the dispersant Corexit 9500 on PAH toxicity to larval mahi-mahi. Left panel: Percent survival plotted against dispersant derived dioctyl sodium sulfosuccinate (DOSS)
in various exposure preparations (mean ± SEM). Note that the artificially weathered oil type is not included due to a steep dose response. Right panel: the calculated DOSS 96 h LC50
values (±95% confidence interval) for chemically enhanced WAF exposures.
WAFs showed a close relationship between toxicity manifested as either
mortality or pericardial edema (Fig. 2). Importantly, effective concentrations for mortality (96 h) were in a similar range as the concentrations producing edema (measured at 48 h); the latter occurred below
the LC50 in only one test (Source CEWAF).
4. Discussion
In the wake of the DWH oil spill many challenges arose for injury assessment related to fish and fisheries in the GOM. These included a lack
of relevant toxicity information, as little was known about the effects of
PAH exposure on pelagic larvae native to the GOM. A second challenge
was incorporating natural weathering and chemical dispersion processes into toxicity estimates for potentially affected species. To our
knowledge this is the first study to describe acute mortality for embryonic PAH exposure in a species native to the GOM pelagic zone. We also
provide additional information to a now expanding body of literature
(Incardona et al., 2014; Mager et al., 2014) describing the cardiotoxicity
of PAHs on pelagic embryos from the GOM. Finally, by using a variety of
oil types and WAF preparation methods we demonstrate the effects
of natural weathering processes and chemical dispersion on PAH
toxicity as determined through both acute lethality and sub-lethal
cardiotoxicity.
Acute lethality for mahi-mahi, as defined by LC50, varied from 8.8 to
45.8 μg l−1 total ∑PAH depending on the state of weathering. The naturally weathered slick A WAFs were more acutely lethal than nonweathered source oil; a trend unaffected by chemical dispersion from
Corexit 9500. These results reinforce the importance of accounting for
the chemical composition of oil as part of the injury assessment process
for the DWH spill. For example, available field data from the DWH oil
spill report ΣPAH concentrations ranging from b 0.01–77 μg l−1
(Bejarano et al., 2013) and as high as 85 μg l−1 (Diercks et al., 2010).
While the upper range of the reported concentrations is above the
mahi-mahi LC50 regardless of oil type, if these reported concentrations
are chemically similar to the slick A oil then the upper range exceeds the
LC50 by almost an order of magnitude. These data support previous
conclusions drawn for early life stage PAH toxicity, including studies
that have shown oil becomes more toxic to embryos and larvae of Pacific herring (Carls et al., 1999) and pink salmon (Heintz et al., 1999)
as it weathers. However, these earlier findings have recently been challenged (Neff et al., 2013; Page et al., 2012). As described earlier, the high
solubility and purported narcosis mechanism of toxicity should mean a
higher toxic potential of low molecular weight PAHs and BTEX (Di Toro
et al., 2007), which are proportionately and progressively lost during
weathering. Nonetheless, the current results clearly demonstrate that
weathered oil WAFs are more toxic than non-weathered source oil
preparations on a ΣPAH basis. It is well established that sub-lethal
cardiotoxicity to complex oil mixtures is attributable to high molecular
weight PAHs (Incardona et al., 2004; Incardona et al., 2006; Incardona
et al., 2011b), most notably the 3 ring compounds (Incardona et al.,
2004). As expected, HEWAF preparations of the slick A oil were
enriched in high molecular weight PAHs relative to the source oil and
artificially weathered source oils; a trend that was magnified in the dissolved fraction (Fig. S4). In fact, expressing acute lethality in terms of
dissolved 3 ring PAH concentration largely eliminated the difference between oil types. This may suggest that different toxicity expressed in
terms of total ΣPAH between oil types was the result of the relative
abundance of 3 ring PAHs; however, the complex nature of oil mixtures
Table 2
Incidence of pericardial edema (48 h EC50) for mahi-mahi exposed to different Deepwater Horizon oil types using both high energy and chemically dispersed preparations. Water-accommodated fractions (WAFs) are expressed as total ΣPAH, estimated dissolved ΣPAH and estimated dissolved 3 ring PAH concentrations. Bracketed values represent the 95% confidence
intervals.
WAF preparation
High energy
Chemically enhanced
Corexit 9500 only
48 h EC50
Source
Weathered
Slick A
Source
Weathered
Slick A
DOSS
Total (μg l−1)
Dissolved (μg l−1)
3 ring (μg l−1)
7.3 (1.4–36.7)
5.7 (1.4–24.0)
N5.1
11.5 (7.4–17.9)
11.3 (7.6–16.8)
13.0 (8.1–20.7)
NA (1400)
6.0 (1.7–21.9)
2.6 (0.7–9.7)
N2.6
9.9 (6.4–15.4)
5.2 (3.5–7.7)
3.6 (1.6–7.9)
0.4 (0.1–1.3)
0.4 (0.1–1.5)
N1.5
0.6 (0.4–0.9)
0.6 (0.4–0.9)
2.7 (1.2–5.9)
A.J. Esbaugh et al. / Science of the Total Environment 543 (2016) 644–651
Table 3
Reduction in atrial contractility (48 h LOEC) for mahi-mahi exposed to different Deepwater
Horizon oil types using both high energy and chemically dispersed preparations. Wateraccommodated fractions (WAFs) are expressed as total ΣPAH, estimated dissolved ΣPAH
and estimated dissolved 3 ring PAH concentrations. Bracketed values represent the NOEC.
PAH 48 h LOEC — contractility
WAF preparation
High energy
Chemically enhanced
Corexit 9500 only
Source
Weathered
Slick A
Source
Weathered
Slick A
DOSS
Total (μg
l−1)
Dissolved (μg
l−1)
3 ring (μg
l−1)
21.6 (10.3)
15.9 (7.9)
4.8 (1.2)
2.4 (1.2)
5.4 (2.7)
3.5 (0.3)
NA (1400)
15.2 (8)
6.9 (3.6)
2.6 (0.6)
2.1 (1.1)
1.3 (2.4)
0.4 (0.04)
–
0.88 (0.47)
1.05 (0.54)
1.49 (0.37)
0.13 (0.07)
0.28 (0.15)
0.29 (0.03)
–
makes this conclusion somewhat difficult. At the very least, acute lethality is similar to cardiotoxicity whereby high molecular weight PAH concentration (≥ 3 ring) determines toxicity to complex PAH mixtures
(Incardona et al., 2004).
ΣPAH measurements represent the sum of two distinct PAH fractions: the dissolved fraction and the micro-droplet fraction (Redman
et al., 2012). A dissolution model was used to apply dissolved PAH correction factors to the total ΣPAH measures of toxicity and thereby assess
toxicity with respect to the dissolved fractions. Both CEWAF and
HEWAF dissolved toxicity patterns were consistent with those for
total ΣPAH. More importantly, the significant difference in total ΣPAH
acute lethality observed between HEWAF and CEWAF source oil preparations was no longer present. The impact of Corexit 9500 on acute lethality was further addressed by expressing toxicity based on
measured DOSS concentrations — a principle ingredient of Corexit
9500. Acute lethality based on DOSS concentration was between 4 and
177 times more severe in the presence of oil than in the presence
Corexit 9500 alone. In combination, these data show that the effects of
Corexit 9500 on acute lethality are limited to the chemical solubility of
PAHs as no additive effects at the biological level were observed when
considering the dissolved ΣPAH LC50s. This is generally consistent
with survival endpoints using a variety of oil types and fish species,
with either dispersant and oil showing no difference or in some cases
reduced toxicity relative to oil alone (Hemmer et al., 2011; Fuller
et al., 2004). Furthermore, the effect of dispersant and oil was no different than oil alone in seabass, Dicentrarchus labrax, exposed to a series of
649
environmental challenge tests (Claireaux et al., 2013). However, dispersant and oil did result in an increased CYP1a response relative to oil
alone using three different oil types in rainbow trout, Oncorhynchus
mykiss (Ramachandran et al., 2004). In the context of developing fish
embryos, the toxicity of Corexit 9500 would be expected to arise from
its surfactant action on biological membranes, and thus occur at concentrations above which surfactants form membrane-active micelles, or its
critical micellar concentration (CMC). The CMC of DOSS in seawater is
1.25 mg l−1 (Steffy et al., 2011), and consistent with toxicity based on
micellar membrane action, the DOSS LC50 for mahi-mahi larvae following embryonic exposure was above this at 3.9 mg l−1 (Table 1).
As anticipated from previous studies on mahi-mahi (Mager et al.,
2014) and other species (Incardona et al., 2004; Incardona et al., 2009;
Incardona et al., 2011a; Incardona et al., 2012), mahi-mahi showed
sub-lethal cardiotoxicity after 48 h of exposure to WAF preparations.
This was most consistently evident as reduced atrial contractility and
pericardial edema, which were both observed in all tests; no secondary
malformations (e.g., jaw or body axis defects) were observed. It has generally been assumed that larval cardiotoxicity results in reduced survival and the data provided here support that viewpoint. A qualitative
assessment of LC50 and pericardial edema EC50 across oil types supports the premise that acute lethality occurs from complications stemming from circulatory failure. Interestingly, cardiotoxicity after 48 h of
exposure was not found to be a more sensitive endpoint than acute lethality following 96 h of exposure. It seems likely that fast developing
species – a trait common for many pelagic GOM species – may suffer
the effects of cardiac impairment earlier than slower developing species.
The clear link between cardiotoxicity and acute lethality in the current
study also allows for the extrapolation of acute survival thresholds of
other commercially important GOM species with similar early life histories, ontogenetic timing, and defined pericardial edema sensitivity
(Incardona et al., 2014). In particular, bluefin and yellowfin tuna have
48 h EC50s of 0.9 and 2.5 μg l−1, respectively, for pericardial edema
when exposed to artificially or naturally weathered oil HEWAF
(Incardona et al., 2014). The current study would therefore predict
96 h LC50 values in a similar range, which in the case of bluefin tuna
is nearly 100-times lower than the upper range of reported field
concentrations.
The relationship between acute lethality and atrial contractility is
less robust than that for pericardial edema; however, the difference in
endpoint metric makes comparisons more difficult. Significant effects
on atrial contractility did not arise until concentrations exceeded the
pericardial edema EC50 in two of three HEWAF preparations. In fact,
Fig. 2. Comparison of the effects of dissolved ΣPAH exposure on acute lethality, the occurrence of pericardial edema and reduced atrial contractility. Left panel: no dispersant high energy
WAF. Right panel: chemically enhanced WAF. NOEC = no observed effect concentration for the reduced atrial contractility, LOEC = lowest observed effect concentration for reduced atrial
contractility. Error bars denote the 95% confidence interval for LC50 estimates. A suitable pericardial edema EC50 estimate could not be generated for the slick A HEWAF.
650
A.J. Esbaugh et al. / Science of the Total Environment 543 (2016) 644–651
the 48 h LOEC for the weathered HEWAF exceeded the 96 h LC50. Interestingly, the atrial contractility LOECs for all CEWAF preparations were
lower than the respective HEWAF NOECs. This difference persisted
even when accounting for the dissolved only fraction and 3 ring composition of the respective WAF types, which suggests that some interactive
effect is present for this particular endpoint. The different responses between pericardial edema and atrial contractility also suggest different
performance consequences. The occurrence of pericardial edema correlates with acute lethality, reflecting more severe cardiac dysfunction
(Brette et al., 2013) with clear population level significance. In contrast,
inhibited atrial contraction will reduce performance through reduced
cardiac output and may alter heart development due to a transient
phase of reduced contractility, potentially leading to reduced cardiorespiratory function or even delayed mortality. However, at present it is
unclear how persistent such cardiac effects are in surviving fish. Both
mahi-mahi and zebrafish show reduced swim performance in recovery
and grow-out studies following embryonic PAH exposure (Mager et al.,
2014; Hicken et al., 2011); however, no differences were observed in
aerobic scope in mahi-mahi (Mager et al., 2014) suggesting that the effects on aerobic swim performance were not due to reduced cardiorespiratory capacity. In contrast, zebrafish showed persistent effects on
cardiac morphology a year after embryonic exposure, which likely contributed to reduced swim performance in this species; however, aerobic
scope was not evaluated (Hicken et al., 2011). Clearly future work is
necessary to further elucidate the functional significance and fitness
consequences of sub-lethal cardiotoxicity in larval fish species, especially with respect to the persistence of compromised cardiorespiratory
phenotypes.
Acknowledgments
This work was supported by funds provided as part of the Natural
Resource Damage Assessment (NRDA) for the DWH oil spill. Data presented here are a subset of a larger toxicological database that is being
generated as part of the Deepwater Horizon Natural Resource Damage
Assessment. Therefore, these data will be subject to additional analysis
and interpretation which may include interpretation in the context of
additional data not presented here. We thank Cathy Laetz for assistance
with data collection, and Jana Labenia and David Baldwin for assistance
with data analyses. M.G. is a Maytag professor of ichthyology.
Appendix A. Supplementary data
Supplementary material.
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Supplemental Information: Table S1: Measured total concentration for 50 PAHs from unfiltered samples compared to estimated dissolved concentrations (µg/L) in representative stock solutions for each oil type and preparation. PAH Analyte
Acenaphthene
Acenaphthylene
Anthracene
Benz(a)anthracene
Benzo(a)fluoranthene
Benzo(a)pyrene
Benzo(b)fluoranthene
Benzo(b)fluorene
Benzo(e)pyrene
Benzo(g,h,i)perylene
Benzo(j+k)fluoranthene
Biphenyl
C1-Chrysenes
C1-Dibenzothiophenes
C1-Fluoranthenes/Pyrenes
C1-Fluorenes
C1-Naphthalenes
C1-Naphthobenzothiophenes
C1-Phenanthrenes/Anthracenes
C2-Chrysenes
C2-Dibenzothiophenes
C2-Fluoranthenes/Pyrenes
C2-Fluorenes
C2-Naphthalenes
C2-Naphthobenzothiophenes
C2-Phenanthrenes/Anthracenes
C3-Chrysenes
C3-Dibenzothiophenes
C3-Fluoranthenes/Pyrenes
C3-Fluorenes
C3-Naphthalenes
C3-Naphthobenzothiophenes
C3-Phenanthrenes/Anthracenes
C4-Chrysenes
C4-Dibenzothiophenes
C4-Fluoranthenes/Pyrenes
C4-Naphthalenes
C4-Naphthobenzothiophenes
C4-Phenanthrenes/Anthracenes
Chrysene+Triphenylene
Dibenz(a,h)anthracene
Source
HEWAF
Tot. Dis.
7.01 0.295
0
0
3.91 0.1
3.04 0.011
0
0
1.15 0
2.23 0.006
7.24 0.019
4.13 0.006
1
0
0
0
80.3 5.38
39.6 0.019
61.5 0.505
31.5 0.061
110
1.72
733
61.96
24.7 0
239
1.673
55.8 0
84.9 0.286
51.7 0.075
148
0.77
858
22.9
30.3 0
249
0.691
39
0
68.6 0.152
61
0.071
137
0.281
529
4.86
24.6 0
166
0.212
29.4 0
35.1 0
46.8 0
286
1.02
13
0
113
0.156
17.3 0.022
1.02 0
Source
CEWAF
Tot. Dis.
0.649 0.251
0
0
0.274 0.096
0.097 0.012
0
0
0.023 0
0.073 0.006
0.328 0.026
0.14 0.004
0.029 0.007
0
0
9.84 4.79
1.49 0.027
2.86 0.565
1.18 0.088
6.19 2.13
119
71.6
0.852 0
11.5 1.98
1.8
0
3.64 0.333
2.11 0.085
7.05 1.15
65
22.1
1.04 0
11
0.762
1.37 0
3.09 0.157
2.55 0.082
6.14 0.388
27.8 4.616
0.807 0
6.9
0.264
1.11 0
2.69 0
1.7
0
12.9 1.21
0.479 0
4.13 0.028
0.655 0.031
0.036 0.01
Weathered
HEWAF
Tot. Dis.
6.95 0.399
0
0
8.75 0.223
6.75 0.035
0
0
1.9 0
4.75 0.121
14.2 0.04
10.2 0
1.58 0
0
0
123 7.5
106 0
133 0.879
70.9 0.137
222 2.97
940 81.1
63.6 0
503 2.7
147 0
197 0.681
128 0.269
340 2.29
1660 35.34
80
0
563 1.28
109 0
162 0.362
149 0.198
303 1.39
1250 8.91
66.7 0
385 0.335
88.7 0
92.9 0
115 0
747 1.89
36.6 0
265 0
39.6 0.468
2.06 0
Weathered
CEWAF
Tot. Dis.
5.95 0.324
0
0
3.89 0.184
2.39 0.011
0
0
0.888 0
2.11 0
6.49 0.043
3.87 0
0.906 0
0.272 0
52.9 5.35
39.4 0.024
55.4 0.712
25.8 0.14
95.8 2.5
446
54.7
21.2 0
220
2.15
48.5 0
79.9 0.505
46.1 0.224
139
2.38
684
25.1
27.4 0
225
0.75
36.5 0
58.1 0.341
54.4 0.193
115
1.37
474
6.596
22.8 0
151
0.251
28.6 0
33
0
41.6 0.12
266
1.54
11.3 0
103
0.247
16.5 0.026
1.15 0
Slick A
HEWAF
Tot. Dis.
1.45 0.063
1.17 0
2.38 0.039
2.16 0.011
0
0
0.532 0
2.34 0.003
4.63 0.021
3.73 0
0.646 0
0
0
3.32 0.273
39.9 0.026
48.1 0.643
24
0.138
68.6 1.7
2.76 0.406
24.6 0
208 1.99
45.4 0
83.2 0.483
43.4 0.27
113 1.88
77.6 3.93
28.2 0
242 0.762
28.9 0
62.7 0.239
46.7 0.226
108 1.452
181 2.961
22.8 0
132 0.224
19.5 0
34
0
38.8 0.134
133 0.895
9.99 0
103 0
20.6 0.033
0.744 0
Slick A
CEWAF
Tot. Dis.
0
0
0
0
0.145 0
0.109 0.013
0
0
0
0
0.125 0.006
0.323 0.014
0.213 0
0.027 0.005
0
0
0
0
2.21 0.021
3.74 0.518
1.34 0.108
6.08 1.69
0
0
1.23 0
15.8 1.131
2.18 0
5.33 0.317
2.55 0.212
9.39 3.34
10.6 2.31
1.38 0
15.3 0.314
1.23 0
4.2
0.057
2.92 0.157
8.52 2.612
16.6 0.975
0.701 0
9.08 0.203
0.725 0
7.64 0
2.2
0.141
10.8 0.402
0
0
5.72 0
1.19 0.017
0.025 0.009
PAH Analyte
Dibenzofuran
Dibenzothiophene
Fluoranthene
Fluorene
Indeno(1,2,3-cd)pyrene
Naphthalene
Naphthobenzothiophene
Phenanthrene
Pyrene
Source
HEWAF
Tot. Dis.
0
0
17.5 0.497
2.27 0.008
48.7 2.771
0.422 0
389
107
6.29 0.005
105
2.442
7.97 0.026
Source
CEWAF
Tot. Dis.
0
0
1.24 0.535
0.096 0
5.05 2.81
0
0
143
108
0.226 0.006
7.3
2.73
0.323 0.032
Weathered
HEWAF
Tot. Dis.
0
0
38.3 0.836
4.11 0.028
94.4 4.28
0
0
208 110
14.9 0.007
218 3.97
15.6 0.041
Weathered
CEWAF
Tot. Dis.
0
0
16.4 0.623
2.17 0.011
44.3 3.26
0.386 0
125
41.2
6.02 0.006
98.6 3.02
7.08 0.03
Slick A
HEWAF
Tot. Dis.
2.28 0.184
11.9 0.436
1.5 0.011
18.8 1.35
0.17 0
0
0
6.53 0.007
72.3 2.32
4.55 0.03
Slick A
CEWAF
Tot. Dis.
0
0
1.16 0.359
0.128 0
2.33 1.12
0
0
0
0
0.407 0.006
7.19 1.8
0.441 0.022
Table S2: The statistical models employed using TRAP software for 96 h lethal concentration 50 estimates. All estimates were performed using 3 parameter models. Source HEWAF Source CEWAF Weathered HEWAF Weathered CEWAF
Slick A HEWAF Slick A CEWAF Corexit only Analysis Type Non‐linear Tolerance Tolerance Non‐linear
Tolerance Non‐linear Tolerance Model Logistic Gaussian Gaussian Logistic
Gaussian Logistic Gaussian Transformation None Log Log None Log None Log Table S3: The statistical models employed using TRAP software for 48 h effective concentration 50 estimates for the occurrence of pericardial edema. All estimates were performed using 3 parameter models. Source HEWAF Source CEWAF Weathered HEWAF Weathered CEWAF Slick A HEWAF Slick A CEWAF Corexit only Analysis Type Tolerance
Tolerance Tolerance Tolerance Tolerance
Tolerance t‐test only Model Gaussian
Gaussian Gaussian Gaussian Gaussian
Gaussian NA Transformation Log Log Log Log Log Log None Table S4: Raw data for all 96 h acute lethality toxicity tests. Dose is the total ∑PAH or DOSS concentration (µg l‐1) and # is the mean number of survivors after 96 h of exposure in the four replicates. All tests contained 20 individuals at beginning of the test. Source HEWAF Dose # Source CEWAF Dose # 0.01 14.75 16.9 11.5 30.7 13.25 41.4 9.25 118.7 0 213.1 0 0.17 4.5 8.2 16.7 27.5 41.6 Weathered HEWAF Dose # 14.75 0.16 12.75
19.25 4.7 11
17.5 12.9 5.25
16.5 37.3 0
6.25 122.5 0
0.75 393.7 0
Weathered CEWAF Dose # Slick A HEWAF Dose # Slick A CEWAF Dose
# Dose 0.31
1.0
6.5
29.6
145.9
596
0.08
2.2
6.5
12.2
23.3
53.2
0.02
1.6
4.4
9.8
19.2
33.1
.75 14.25
680 17
1400 14.75
4000 10.75
4400 2
5100 0
12.5
14.75
11
0
0
0
18.5
17.5
11
4.75
0.5
0
16 17.5 16.5 7.25 0 0 DOSS # Table S5: Raw data for all 48 h pericardial edema toxicity tests. Dose is the total ∑PAH or DOSS concentration (µg l‐1) and % is the mean percent of individuals exhibiting edema after 48 h of exposure in the four replicates. All tests contained 20 individuals at beginning of the test. Source HEWAF Dose % 0 3.5 6.4 10.3 21.5 18 52 61 81 91 Source CEWAF Dose % 0 1.2 2.4 4.0 8.5 13 8 3 10 32 Weathered HEWAF Dose % Weathered CEWAF Dose
% 0 2.4 4.1 7.9 15.9 0
1.5
2.7
5.4
10.9
9
19
37
69
77
20
5
15
29
49
Slick A HEWAF Dose % 0
0.4
1.2
4.8
5.1
3
1
7
15
26
Slick A CEWAF Dose % Dose % 0
0
0.35
3.49
16.37
0 1400 9
10
8 5 8 13 69 DOSS Table S6: Raw data for all 48 h atrial contractility tests. Dose is the total ∑PAH or DOSS concentration (µg l‐1). Cont is the mean measured contractility after 48 h of exposure in the four replicates. All tests contained 20 individuals at beginning of the test. Source Source Weathered Weathered Slick A Slick A DOSS HEWAF CEWAF HEWAF CEWAF HEWAF CEWAF Dose Cont Dose Cont Dose Cont Dose Cont Dose Cont Dose Cont Dose Cont 0 3.5 6.4 10.3 21.5 30.3 28.1 27.5 23.8 24.9 0 1.2 2.4 4.0 8.5 30.3 29.8 28.4 28.7 26.4 0 2.4 4.1 7.9 15.9 27.5
26.0
27.5
26.7
21.3
0
1.5
2.7
5.4
10.9
27.5
23.7
26.6
22.8
23.1
0
0.4
1.2
4.8
5.1
28.7
28.5
27.3
25.7
25.0
0
0
0.35
3.49
16.37
30.7 30.3 29.9 26.6 23.7 0 1400 30.7
32.2
Figure Captions: Figure S1: Individual PAH composition of high energy water‐accommodated preparations employed for 96 h LC50 toxicity testing. Figure S2: Individual PAH composition of chemically enhanced water‐accommodated preparations employed for 96 h LC50 toxicity testing with a 10:1 oil to dispersant ratio. Figure S3: Left panel: The concentration of volatile BTEXs in source oil high energy and chemically enhanced water‐accommodated fractions employed for 96 h LC50 toxicity testing. Right panel: The BTEX composition in high energy and chemically enhanced water‐accommodated fractions expressed relative to the total PAH concentration. Note that no significant BTEXs were observed in artificially weathered or slick A oil preparations. Figure S4: Example of dissolved to unfiltered relationships for a single analyte, acenapthene, in each of the six WAF preparations studied. Each dot shows the relationship of the filtered to unfiltered concentration for a particular dilution within each combination. Lines show the fitted equations estimating dissolved concentrations (solid), 1:1 relationship (dotted), and modeled maximum dissolved concentrations (dashed; its y‐intercept is “a” in the equation). Outliers (hollow triangles) were removed when a filtered measurement was 110% or more of the unfiltered measurement. Figure S5: The relative composition of 2, 3 and 4 ring polycyclic aromatic hydrocarbons in the total and dissolved fractions of high energy and chemically enhanced water accommodated fractions from three different oil types. Biphenyl
Naphthalene
C1-Naphthalenes
C2-Naphthalenes
C3-Naphthalenes
C4-Naphthalenes
Dibenzofuran
Acenaphthylene
Acenaphthene
Fluorene
C1 - Fluorenes
C2 - Fluorenes
C3 - Fluorenes
Anthracene
Phenanthrene
C1-Phenanthrenes/Anthracenes
C2-Phenanthrenes/Anthracenes
C3-Phenanthrenes/Anthracenes
C4-Phenanthrenes/Anthracenes
Dibenzothiophene
C1 - Dibenzothiophenes
C2 - Dibenzothiophenes
C3 - Dibenzothiophenes
C4 - Dibenzothiophenes
Benzo(b)fluorene
Fluoranthene
Pyrene
C1 - Fluoranthenes/Pyrenes
C2 - Fluoranthenes/Pyrenes
C3 - Fluoranthenes/Pyrenes
C4-Fluoranthenes/Pyrenes
Naphthobenzothiophene
C1-Naphthobenzothiophenes
C2-Naphthobenzothiophenes
C3-Naphthobenzothiophenes
C4-Naphthobenzothiophenes
Benz(a)anthracene
Chrysene+Triphenylene
C1 - Chrysenes
C2 - Chrysenes
C3 - Chrysenes
C4 - Chrysenes
Benzo(a)fluoranthene
Benzo(b)fluoranthene
Benzo(j+k)fluoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Indeno(1,2,3-cd)pyrene
Dibenz(a,h)anthracene
Benzo(g,h,i)perylene
% Total PAH
% Total PAH
% Total PAH
Figure S1 20
2 ring
3 ring
4 ring
10
10
10
>4 ring
15
Slick
5
0
20
15
Weathered
5
0
20
15
Source
5
0
Biphenyl
Naphthalene
C1-Naphthalenes
C2-Naphthalenes
C3-Naphthalenes
C4-Naphthalenes
Dibenzofuran
Acenaphthylene
Acenaphthene
Fluorene
C1 - Fluorenes
C2 - Fluorenes
C3 - Fluorenes
Anthracene
Phenanthrene
C1-Phenanthrenes/Anthracenes
C2-Phenanthrenes/Anthracenes
C3-Phenanthrenes/Anthracenes
C4-Phenanthrenes/Anthracenes
Dibenzothiophene
C1 - Dibenzothiophenes
C2 - Dibenzothiophenes
C3 - Dibenzothiophenes
C4 - Dibenzothiophenes
Benzo(b)fluorene
Fluoranthene
Pyrene
C1 - Fluoranthenes/Pyrenes
C2 - Fluoranthenes/Pyrenes
C3 - Fluoranthenes/Pyrenes
C4-Fluoranthenes/Pyrenes
Naphthobenzothiophene
C1-Naphthobenzothiophenes
C2-Naphthobenzothiophenes
C3-Naphthobenzothiophenes
C4-Naphthobenzothiophenes
Benz(a)anthracene
Chrysene+Triphenylene
C1 - Chrysenes
C2 - Chrysenes
C3 - Chrysenes
C4 - Chrysenes
Benzo(a)fluoranthene
Benzo(b)fluoranthene
Benzo(j+k)fluoranthene
Benzo(e)pyrene
Benzo(a)pyrene
Indeno(1,2,3-cd)pyrene
Dibenz(a,h)anthracene
Benzo(g,h,i)perylene
% Total PAH
% Total PAH
% Total PAH
Figure S2 40
2 ring
3 ring
4 ring
10
20
>4 ring
30
20
Slick
0
40
30
20
10
Weathered
0
40
30
10
Source
0
1000
500
0
o-Xylene
1500
m,p-Xylenes
2000
Ethylbenzene
2500
BTEX to Total PAH Ratio
HEWAF
CEWAF
Toluene
Benzene
3000
o-Xylene
3500
m,p-Xylenes
Ethylbenzene
Toluene
Benzene
BTEX (μg/l)
Figure S3 10
8
6
4
2
0
Figure S4 Figure S5 HEWAF
Percent Total PAH
100
80
CEWAF
Slick A
Weathered
Source
100
80
60
60
40
40
20
20
0
0
Percent Dissolved PAH
2 Ring 3 Ring 4 Ring
100
100
80
80
60
60
40
40
20
20
0
0
2 Ring 3 Ring 4 Ring
2 Ring 3 Ring 4 Ring
2 Ring 3 Ring 4 Ring