Previous oil exposure alters Gulf Killifish
Fundulus grandis oil avoidance behavior
Charles W. Martin1 , Ashley M. McDonald1 , Guillaume Rieucau2 and
Brian J. Roberts2
1
2
UF/IFAS Nature Coast Biological Station, University of Florida, Cedar Key, FL, United States of America
Louisiana Universities Marine Consortium, Chauvin, LA, United States of America
ABSTRACT
Oil spills threaten the structure and function of ecological communities. The Deepwater
Horizon spill was predicted to have catastrophic consequences for nearshore fishes, but
field studies indicate resilience in populations and communities. Previous research
indicates many marsh fishes exhibit avoidance of oil contaminated areas, representing
one potential mechanism for this resilience. Here, we test whether prior oil exposure
of Gulf killifish Fundulus grandis alters this avoidance response. Using choice tests
between unoiled and oiled sediments at one of three randomized concentrations (low:
0.1 L oil m−2 , medium: 0.5 L oil m−2 , or high: 3.0 L oil m−2 ), we found that, even
at low prior exposure levels, killifish lose recognition of oiled sediments compared
to control, unexposed fish. Preference for unoiled sediments was absent across all oil
concentrations after oil exposure, and some evidence for preference of oiled sediments
at high exposure was demonstrated. These results highlight the lack of response to toxic
environments in exposed individuals, indicating altered behavior despite organism
survival. Future research should document additional sublethal consequences that
affect ecosystem and food web functioning.
Subjects Animal Behavior, Aquaculture, Fisheries and Fish Science, Marine Biology, Zoology,
Environmental Contamination and Remediation
Keywords Macondo, Deepwater Horizon, Fish, Gulf of Mexico, Salt marsh, Hydrocarbon
Submitted 22 October 2020
Accepted 25 November 2020
Published 18 December 2020
Corresponding author
Charles W. Martin,
martin.charles.w@gmail.com
Academic editor
Valsaraj KT
Additional Information and
Declarations can be found on
page 10
DOI 10.7717/peerj.10587
Copyright
2020 Martin et al.
Distributed under
Creative Commons CC-BY 4.0
OPEN ACCESS
INTRODUCTION
The 2010 Deepwater Horizon (DwH) oil spill in the Gulf of Mexico (USA) impacted
nearshore ecosystems from the Louisiana coast to the Florida Panhandle (Michel et al.,
2013). Over 87 days, approximately 4.9 million barrels of oil (McNutt et al., 2012) ultimately
covered an estimated 180,000 km2 of Gulf waters and over 1,100 km of coastal wetlands
with ∼95% of these being in Louisiana (Mitra et al., 2012; Michel et al., 2013; Nixon et al.,
2016). The spill predominantly impacted salt marshes in nearshore areas, with significant
economic implications given they serve as key habitats for the young of many commercial
and recreational fishery species (Peterson & Turner, 1994; Lellis-Dibble, McGlynn & Bigford,
2008; Rozas, Martin & Valentine, 2013; Baker et al., 2020). To date, field assessments of
coastal fish populations and communities have shown resistance and resilience to, and in
some cases rapid recovery from, the toxic effects of oil (Moody, Cebrian & Heck, 2013; Fodrie
et al., 2014; Able et al., 2015; Schaefer, Frazier & Barr, 2016). Several potential explanations
have been given for the unanticipated lack of severe impacts to populations/communities in
How to cite this article Martin CW, McDonald AM, Rieucau G, Roberts BJ. 2020. Previous oil exposure alters Gulf Killifish Fundulus
grandis oil avoidance behavior. PeerJ 8:e10587 http://doi.org/10.7717/peerj.10587
nearshore ecosystems (Fodrie et al., 2014), including behavioral emigration from oiled areas
(Martin, 2017), sublethal impacts to individuals (Whitehead et al., 2012; Dubansky et al.,
2013) that do not translate to higher levels of organization, indirect food web mechanisms
that provide predator release and/or stimulation of production (McCann et al., 2017),
and cessation of fishing (Fodrie & Heck, 2011; Schaefer, Frazier & Barr, 2016; Martin et al.,
2020).
Studies across a wide range of organisms, from zooplankton (Seuront, 2010) to
marine mammals (Smultea & Würsig, 1995; Ackleh et al., 2012), have indicated complex
recognition patterns and behavioral avoidance of oiled conditions. For example, calanoid
copepods alter swimming behavior to avoid water-soluble diesel oil to limit exposure
(Seuront, 2010). At larger spatial scales, sperm whales relocated from their historically
occupied areas due to the DwH oil spill (Ackleh et al., 2012). Dolphins exhibit similar
avoidance responses (Smultea & Würsig, 1995) and have been trained in the detection
of oil (Geraci, St Aubin & Reisman, 1983). Conversely, American lobsters have shown
attraction to hydrocarbons such as kerosene (Atema, 1976), which has been used as a bait
by commercial fishermen.
Fish behavior is also known to be affected by chemical pollutants (Saaristo et al., 2018;
Jacquin et al., 2020). Diverse taxonomic groups have demonstrated strong behavioral
responses to acute crude oil contamination (Weis et al., 2001; Martin, 2017; Schlenker et
al., 2019a). Freshwater fishes such as fathead minnows Pimephales promelas (Farr, Chabot
& Taylor, 1995), rainbow trout Oncorhynchus mykiss (Carr et al., 1990), pink salmon fry
Oncorhynchus gorbuscha (Rice, 1973), Caspian roach Rutilus caspicus (Lari et al., 2015), and
striped bass Morone saxatilis (Carr et al., 1990) avoid hydrocarbon contaminated areas,
albeit at different thresholds and concentrations. Estuarine and marine fishes such as
flatfishes (Moles, Rice & Norcross, 1994), juvenile spot Leiostomus xanthurus (Hinkle-Conn
et al., 1998), European seabass Dicentrarchus labrax (Claireaux et al., 2004), and mahi-mahi
Coryphaena hippurus (Schlenker et al., 2019a) exhibit an avoidance response to the toxic
chemical contaminants found in petroleum hydrocarbons.
In salt marshes, previous work (Martin, 2017) has demonstrated that ecologically
important fishes such as Gulf killifish Fundulus grandis, sailfin molly Poecilia latipinna,
and sheepshead minnow Cyprinodon variegatus avoid oil contaminated sediments, but
display a reduced response to weathered oil indicating they likely react to the volatile,
aromatic compounds that are lost as oil degrades and weathers due to a combination of
factors including UV exposure (Bacosa, Erdner & Liu, 2015), wave action (Daling et al.,
2014), microbial processing (Hazen et al., 2010), among others. Shallow, soft-sediment
areas dominate the inshore reaches of the northern Gulf of Mexico (Connor & Day,
1987) and benthic organisms living within and on these sediments serve as important
food sources for numerous species, including one of the most abundant Gulf coast
marsh species F. grandis. For example, Rozas & LaSalle (1990) reported that the major
dietary constituents of F. grandis are found associated with these sediments: fiddler crabs,
amphipods, and hydrobiid snails. As such, recognition of the quality and contamination
of these habitats is critical for F. grandis as these areas are linked with successful foraging
and fitness.
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Previous research indicates that oil exposure can alter fish detection of oil. For example,
exposure to the water-accommodated fraction of oil in Atlantic stingrays Hypanus sabinus
damaged olfactory function (Cave & Kajiura, 2018). After oil exposure, bicolor damselfish
Stegastes partitus showed a reduction in the response to conspecific alarm cues (Schlenker
et al., 2019b). This damage to olfactory mechanisms or central nervous processing may
further reduce an organism’s capacity to detect and respond to oil contamination (sensu
Schlenker et al., 2019a).
Here, we present the results of a series of experiments to test the effects of previous
oil exposure on oil-contaminated sediment avoidance in salt marsh fish. Using the
Gulf killifish F. grandis, a widely-used sentinel species for toxicological and ecological
studies (Able et al., 2015; Vastano et al., 2017; Jensen et al., 2019), we exposed animals to a
range of concentrations in experimentally oiled marsh conditions and then subsequently
tested behavioral avoidance patterns in simple choice tests. The overarching objective
of this project was to determine if prior exposure influences avoidance behavior and,
if so, to identify what exposure level influences these changes. Evidence that even the
lowest sublethal levels of hydrocarbon exposure result in non-avoidance of contaminated
sediments could suggest there are behaviorally-associated impacts to overall fitness or
survival of these ecologically important marsh fish.
MATERIALS & METHODS
Fish exposure
Fish were exposed to oil for 10–15 days in experimentally oiled marsh mesocosms at the
Louisiana Universities Marine Consortium in Cocodrie, LA during August/September 2019.
This exposure period is based on studies of known site fidelity in F. grandis (Nelson, Sutton
& DeVries, 2014; Jensen et al., 2019). Briefly, we utilized 12 hydrologically independent
Spartina alterniflora marsh mesocosms (3.05 m diameter, 1.83 m height) each with its
own paired tidal surge tank generating daily tidal cycles with range of 25 cm (flooding
marsh ∼10 cm at high tide) via a water control system of blowers and airlifts (Alt, 2019).
During flooded marsh conditions (∼40% of the time), fish had access to ∼7.3 m2 of
marsh platform (∼10 cm water depth at high tide) and the adjacent deeper water (∼40
cm deep at high tide) in the surrounding trough. When water was off the marsh, fish
were restricted to ∼1.4 m2 of surface area in the trough with water depths as low as
15 cm at low tide. Intact salt marsh plugs (30 cm diameter × 50 cm depth) at natural
densities from nearby unoiled marshes were established in mesocosms approximately
18 months prior to oiling. Light Louisiana Sweet (LLS) blended crude oil, API Gravity
40.1, similar to that which was released in the DwH oil spill, was acquired from Placid
Refining Company LLC in May 2018. This oil was evaporatively weathered by 30% of its
volatile components, as measured by gas chromatography, using a nitrogen gas sparging
system placed in the barrel of liquid oil as received from Placid over a period of 150 days.
The bubbling system not only expedited evaporation, but also mixed the contents of the
barrel to ensure conformity of its contents. A single application was applied to the water
at uniformly spaced locations at high tide to each mesocosm on July 8, 2019 at one of
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four concentrations, scaling roughly to Shoreline Cleanup and Assessment Team (SCAT)
categories observed on shorelines after the DwH spill (Michel et al., 2013; Nixon et al.,
2016): control/no oil (0.0 L oil m−2 ), low (0.1 L oil m−2 ), medium (0.5 L oil m−2 ), and
high (3.0 L oil m−2 ) oil concentrations. Additional details on the mesocosm setup can be
found at https://robertsresearchlab.weebly.com/mesocosms.html.
We captured F. grandis from the nearby salt marsh using baited minnow traps and
held them in a separate 450 liter aquarium for 2-4 days to minimize any mortality due to
handling before being introduced to the 15 cm wide trough surrounding plants within each
mesocosm. During low tide, fish were restricted to a 15 cm deep water column and at high
tide events fish gained access to oiled or unoiled experimental marsh platforms (at a water
depth of ∼10 cm) to forage. A total of 18 fish were added to each mesocosm between 22
August (12 fish added) and 27 August 2019 (6 additional fish added to increase available
fish for the avoidance experiment due to mortality observed after the first few days). A total
of 54 fish/treatment (18 fish in three mesocosm of each oil treatment) were introduced
to mesocosms and recaptured prior to experiments using dip nets. Fish were exposed
to oil treatments for 10 (27 August additions) to 15 (22 August additions) days with oil
having been further weathered in the mesocosms for 45 to 60 days prior to initiation of
the experiments described here. The mean ± standard error surface soil (0–5 cm) total
petroleum hydrocarbon (TPH) concentrations in the high oil treatments (419 ± 24 mg/g
soil) were ∼10 and ∼40 times higher than in the moderate (39 ± 5 mg/g soil) and low
(10 ± 0.4 mg/g soil) oil treatments (mean of 19 August and 9 September samplings;
E. Overton & B. J. Roberts, 2020, unpublished data). These concentrations are similar
to those found in Louisiana salt marsh field conditions (Lin et al., 2016). Only survivors
from these exposures were used in the subsequent experiment to measure sublethal effects,
and they were held for 48 h in separate aquaria containing unoiled seawater by treatment
prior to use in behavioral experiments to ensure no additional mortality. Seawater used
for holding fish and in the avoidance experiment (described below) was passed through
an ultraviolet filter then passed through a 0.2 µm filter to remove any background oil that
may confound results.
Avoidance experiment
To test the behavioral response of fish exposed to different oil concentrations, we used a
choice test following the design reported in Martin (2017). Thirty-eight-liter aquaria were
filled with 3 L total of clean, unoiled sediment to a depth of 18 cm. Each aquarium offered
a choice between unoiled and oiled sediment, randomized on each side of the aquaria to
reduce the chances of any unknown external cues affecting fish behavior. In addition, we
conducted trials with no oil on either side of the aquarium and found no preference; these
data were not used in further analyses. Concentrations followed previous experiments
(Martin, Hollis & Turner, 2015; Martin, 2017), and fish were given a choice between no oil
and low oil (10 mL oil per L sediment), no oil and medium oil (20 mL oil per L sediment),
or no oil and high oil (40 mL oil per L sediment) contamination. In these tests, we used 25%
weathered oil (from the same source barrel as used in the larger mesocosm experiment)
as this was representative of what came inshore (Reddy et al., 2012) and is the same degree
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of weathering used in previous experiments (Martin, 2017). Using gloves, we hand-mixed
sediment (both with or without oil) on the randomized side of the aquaria at the assigned
concentration and a thin layer (approximately 2 cm deep) of unoiled sand was placed
uniformly on top to prevent the oil/sand mixture from floating and affecting the adjacent,
unoiled side of the aquaria. Using only sediment in tanks prevented confounding effects
of vegetation due to structural refuge.
All F. grandis used in this study were adult individuals between 57 mm and 105 mm
and used only once in a trial. Mortality during the exposure duration in oiled mesocosms
limited the number of available fish, and as a result we replicated most comparisons 8
times, except for medium exposure fish (no oil vs medium oil was replicated 4 times) and
high exposure fish (only no vs low oil, the most conservative of the oil level options, was
tested with 8 replicates to avoid a lower sample size that would contribute to an inability
to statistically detect trends). We held salinity constant at 7.0 PSU and temperature ranged
from 26.8–28.6 ◦ C during trials (both comparable to the conditions in the mesocosms at
the time of collection).
For each trial, one fish from a randomized exposure was introduced to the aquarium,
allowed a 5-minute acclimation period, and its movements between the two sides of
the aquarium recorded using a GoPro camera over the course of 10 min. This trial period
mimics previous fish behavioral experiments (Gerlach et al., 2007; Paris et al., 2013; Martin,
2017). The side of the aquarium occupied by fish (no oil or assigned oil treatment) was
recorded by analyzing a frame systematically every 30 s over the trial period and noting
fish position within the aquarium. The proportion of time in each side was then calculated
as the number of observations taken on that side divided by the total observations.
Data were statistically analyzed in 2 ways, identical to analyses performed in Martin
(2017): (1) to determine whether fish deviated from an expected 1:1 occupancy pattern,
we conducted a paired t -test for each previous exposure level and oil vs no oil comparison
(Peterson & Renaud, 1989) and (2) to compare differences across treatments, we analyzed
proportion of time spent in oil using a general linear model (GLM) with factors of previous
exposure and oil vs. no oil comparison. Tukey’s post hoc test was used to determine
significant pairwise differences. Assumptions (normality and homogeneity of variance)
were tested for all comparisons and nonparametric alternatives (signed rank test) used if
transformations failed to meet assumptions and considered results significant at p < 0.05.
To graphically display data, a ratio of the number of times fish occupied each side of the
aquarium was generated and plotted, such that deviation below 1 indicates avoidance of
oil and above the line denotes preference for oil.
Ethics statement
All field collections were made under Louisiana Department of Wildlife and Fisheries
Scientific Collecting Permit # SCP 200. The use of vertebrate organisms was conducted with
IACUC approval and staff training from University of Florida under protocol 201710044.
As the goal of the study was to measure sublethal effects of oil on fish behavior, humane
endpoints were not used and were not possible during the 10–15 day exposure, as fish were
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Figure 1 Ratio of time spent in oil to time spent in control side of tank across previous exposures.
Dashed line indicates 1:1 (no preference), with values >1 indicating more time over oiled sediments and
<1 indicating less time over oiled sediments. Colors represent values for preference comparisons between
no oil and low (blue), medium (yellow), and high (red) oil concentrations. Asterisks indicate fish deviate from an expected 1:1 occupancy pattern (Table 1) and different letters indicate statistical differences
among treatments.
Full-size DOI: 10.7717/peerj.10587/fig-1
released into turbid mesocosms and unable to be monitored. Moreover, analgesics and
anaesthetics were not used because of the alterations to behavior that we were quantifying.
RESULTS
Previous exposure influenced fish preference patterns for oil contaminated sediments
(Fig. 1, Table 1). Unexposed (control) fish showed significant avoidance of the oiled side
of aquaria, regardless of the oil concentration choice given (Table 1). After exposure to
oil, even at low concentrations, this avoidance response disappeared. Fish unexposed to
oil spent on average 66% of time over uncontaminated sediments, a trend that decreased
with previous exposure to low (52%), medium (55%), and high (44%) concentrations. At
high previous exposure concentrations, we noted 7 of the 8 fish spent more time over oiled
sediments in the aquaria, although the time spent on the oiled side was not significant
(Table 1).
The oil concentration used in the choice test had a smaller influence on fish behavior
than previous oil exposure. Results of the GLM confirmed these results, which indicated
that previous exposure (F3,70 = 6.83, p < 0.001), not oil concentration in preference test
(F2,70 = 0.38, p = 0.684), drove behavioral patterns. Pairwise comparisons indicated that
control, unexposed fish had significantly stronger avoidance of oil than fish previously
exposed to low (p = 0.0034), medium (p = 0.0337), or high (p = 0.0029) oil concentrations,
with low, medium, and high not significantly different from each other (p > 0.05).
DISCUSSION
The 2010 DwH oil spill was an unprecedented stressor for northern Gulf ecosystems, with
oil impacting emergent and submerged plants (Lin & Mendelssohn, 2012; Silliman et al.,
2012), invertebrates (McCall & Pennings, 2012; Powers et al., 2017; Zengel et al., 2016), and
fishes (Fodrie & Heck, 2011; Able et al., 2015; Schaefer, Frazier & Barr, 2016). While the full
effect on the nearshore food web may not yet be fully realized because of the numerous and
complex indirect food web mechanisms (McCann et al., 2017; Barron et al., 2020), many
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Table 1 Statistical results (either paired t -test or signed rank test) for each previous exposure and
comparison. Control fish preferred no oil, a response which is lost even at low previous oil exposure. Significant p-values are shown in bold.
Previous Exposure
Control (No Oil)
Low Oil
Medium Oil
High Oil
N
Test Statistica
p
Comparison
Statistical Test
Low Oil vs No Oil
Paired t -test
8
−4.204
0.004
Medium Oil vs No Oil
Paired t -test
8
−7.105
<0.001
High Oil vs No Oil
Signed rank
8
2.388
0.016
Low Oil vs No Oil
Paired t -test
8
<0.001
1.000
Medium Oil vs No Oil
Paired t -test
8
0.235
0.821
High Oil vs No Oil
Signed rank
8
1.550
0.148
Low Oil vs No Oil
Paired t -test
8
−1.546
0.166
Medium Oil vs No Oil
Paired t -test
4
0.378
0.731
High Oil vs No Oil
Paired t -test
8
−1.080
0.316
Low Oil vs No Oil
Paired t -test
8
1.418
0.199
Notes.
a
Test statistic: T for Paired t -test, Z for Signed Rank test
studies have documented significant impacts to molecular, genomics, and development
of fishes (Whitehead et al., 2012; Dubansky et al., 2013) and resilience in populations and
communities to oil’s toxic effects (Fodrie et al., 2014; Martin et al., 2020). Among the
proposed explanations for this resilience, despite oil’s known toxicity, is the behavioral
emigration of organisms at small spatial scales to avoid exposure to contamination (Martin,
2017). Here, we demonstrate that exposure to oiled marshes, even at low concentrations
of weathered oil (0.1 L oil m−2 ), can impact a common marsh fish’s ability to avoid oil
contaminated sediments.
A further exploration of the current dataset indicated one anomalous trial in high
exposure fish. In this trial, the fish spent 65% of time over unoiled sediments. With the
exception of this trial, fish exposed to high oil concentrations spent 60% of time over oil
sediments (range: 50–75%), a significant preference (t (6) = 2.517; p = 0.045) for oiled
sediments at low concentration over unoiled sediments (the only test conducted on high
exposure fish as a conservative estimate due to lack of experimental organisms). We
hypothesize that this apparent preference for oiled sediments could be due to relaxation
of normal physiological functioning causing an anaesthetic effect (Barron et al., 2004).
Several studies have documented a narcotic effect on fishes due to chronic exposure to oil
(Barron et al., 2004; Incardona, Collier & Scholz, 2004). This can result in a nervous system
sedation (Lin & Tjeerdema, 2008), as well as increased respiratory rate (Brocksen & Bailey,
1973) and decreased swimming performance (Stieglitz et al., 2016). These effects can be
reversed, however, usually on the order of days after exposure has been removed (Brocksen
& Bailey, 1973). In this experiment, we only conducted choice trials at low concentrations
for the high exposure treatments, leaving open the possibility that a stronger response
could have been observed had more fish survived the exposure duration. As the goal
of this study was to document the alterations to F. grandis behavior and not to test the
physiological mechanisms driving the response to oil, we can only speculate that there could
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be acclimation or damage to olfactory or other organs (Cave & Kajiura, 2018; Schlenker et
al., 2019a) based on our experiments.
Previous exposure to oil is known to have many negative consequences for individuals.
For example, unexposed mahi-mahi avoided higher concentrations of water accommodated
fraction of oil, but exposed individuals demonstrated a lack of response (Schlenker et al.,
2019a). In this case, the authors explicitly tested for, and did not find, damage to olfactory
acuity from oil exposure. In other studies, damage to the higher order central nervous
system processing was implicated in this decreased oil avoidance behavior. Olfactory
damage is also known to occur with oil exposure in some fishes (such as Atlantic stingrays,
Cave & Kajiura, 2018) and reduced recognition of threats via these cues is also possible
(Schlenker et al., 2019b). The specific chemical constituents in weathered oil involved in
these detrimental physiological changes remain unclear. Given that weathered oil comprised
the bulk of the oil that came ashore (Reddy et al., 2012), it is likely that, based on field
abundance comparisons (Fodrie & Heck, 2011; Able et al., 2015; Martin et al., 2020) fish
survival may have been higher than expected and thus sublethal effects may constitute
the largest impact on marsh organisms. In our exposures, we noted mortality and lack of
recapture for some treatments, particularly in medium and high oil mesocosms, which
precluded the full range of preference tests in the avoidance experiment. Specifically,
out of the original 54 fish released we recaptured 54, 34, 24, and 10 fish in control, low,
medium, and high treatments, respectively. These differences in mortality corresponded
to survivorship of 100% (control), 62.0% (low), 44.4% (medium), and 18.5% (high) in
mesocosm treatments. Importantly, fish were held for 48 h post-exposure prior to use in
behavioral assays and no additional mortality was detected during this period or during
behavioral trials. We acknowledge this mortality and lack of recapture resulted in low
replication for some treatments (medium exposure and no oil vs. medium oil choice test)
and lack of comparisons for others (high exposure). As a result, findings from the lower
replication trials should be considered preliminary and serve as a template for future studies
to address this deficiency. Without a post-mortem examination, we cannot definitively state
whether killifish mortality during the mesocosm exposure duration was caused by toxicity
or because of behavioral responses to weathered oil exposure (e.g., foraging inefficiencies).
As such, these findings support the notion that even if fish survive oil exposure there are
significant behavioral responses that might influence their long-term survival.
The sublethal effects of the DwH oil spill have remained, for many organisms, largely
unexplored in the decade following the spill (but see Rozas, Minello & Miles, 2014; Cave
& Kajiura, 2018; Martin & Swenson, 2018; Schlenker et al., 2019a; Schlenker et al., 2019b).
Given the importance of physiological processes such as sensory mechanisms and olfaction
for many critical activities, such as foraging (Webster et al., 2007; Johannesen, Dunn &
Morrell, 2012), habitat recognition (Benfield & Aldrich, 1992; Forward et al., 2003), and
predator avoidance (Dixson, Munday & Jones, 2010; Martin et al., 2010), it is possible that
these and other sublethal effects resulting from oil exposure may have great consequences.
Previous studies have indicated that oil can have other important sublethal effects on fishes
and invertebrates. For example, oil presence triggered a 60% decrease in penaeid shrimp
Farfantepenaeus aztecus growth rate (Rozas, Minello & Miles, 2014) and foraging by darter
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gobies Gobionellus boleosoma can change 50–100% in sediments highly contaminated with
diesel fuel (Gregg, Fleeger & Carman, 1997). Spot L. xanthurus do not alter feeding behavior
at moderate-high concentrations of diesel oil suggesting continued exposure while feeding
on benthic organisms may occur (Hinkle-Conn et al., 1998). Given the known deleterious
impacts to other fishes, we anticipate that similar sublethal consequences were present
in marsh fishes after DwH, but have remained understudied. We propose that additional
research on the sublethal effects of oil (including impacts to top down control via predation
or predator release) need to be conducted to gain a broader understanding of the full scope
of DwH damages to northern Gulf of Mexico ecosystems.
Oil released from the DwH drilling rig was burned at the surface, collected on the water
or as it came ashore on wetlands and beaches, and chemically dispersed using Corexit R
dispersant (Peterson et al., 2012; Michel et al., 2013; Nixon et al., 2016). Wetlands accounted
for over half of the oiled shoreline (∼1,100 of ∼2,100 km), with >95% of oiled marshes
in Louisiana (Nixon et al., 2016). However, much of the oil remains unaccounted for
(McNutt et al., 2012) and is thought to reside in sediments throughout the region. Previous
spills such as the Exxon Valdez (Renner et al., 2006; Li & Boufadel, 2010), Florida barge in
Massachusetts (Culbertson et al., 2008), and Ixtoc-I (Schrope, 2010) all indicate that oil can
persist buried in the sediment where oil weathering rates are low (Boufadel et al., 2010).
Thus, marsh fishes may be vulnerable to sublethal oil exposure and the loss of avoidance
behaviors after exposure in marsh species may have more subtle, but still substantial,
implications for the marsh food web long after the oiling event. Unlike in pelagic species
where exposure is comparatively more limited because oil moves long distances across the
surface with currents and wind, weathers, biodegrades, or sinks to deep sediments, once oil
reaches marsh sediments exposure may be extended. Once exposed, these marsh fishes lose
recognition and remain vulnerable to oil contamination in the short or long term as oil gets
trapped by the plants and buried or slowly degraded over time. Enhancement of erosion
rates (Silliman et al., 2012; Martin, Hollis & Turner, 2015; Turner, McClenachan & Tweel,
2016) and sediment remobilization after large storm events, such as the frequent Gulf of
Mexico hurricanes (Khanna et al., 2013; Michel et al., 2013), may re-expose remaining oil
to saltmarsh flora and fauna, continuing to sublethally impact organisms for decades to
come. Many resident and transient species spend some part of their life cycles in these
contaminated areas and could be impacted for sustained periods, necessitating the need
for continued study of oil impacts and population trends in these vital ecosystems.
CONCLUSIONS
We tested whether prior exposure to oil alters the Gulf killifish’s avoidance response of
oil. Given the limitations and ethics of experimentally oiling field locations, we used an
ongoing mesocosm experiment to expose individuals to oil. After a short 48-hr holding
period to ensure no additional mortality due to the exposure duration, fish avoidance of
oil was then tested in simple choice tests. We found that, even at low oil exposure levels,
fish lost their response to oil compared to unexposed, control fish. This research suggests
that fish surviving short-term exposure durations may continue to incur sublethal effects.
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ACKNOWLEDGEMENTS
We thank the staff at Louisiana Universities Marine Consortium for the facilities and
logistical support needed to make this project possible, particularly members of the
Roberts lab for the initiation and maintenance of the mesocosm experiment (Charles
Schutte, Ryann Rossi, Ekaterina Bulygina, Stephanie Plaisance, Caitlin Bauer) and members
of the Education and Outreach team (Murt Conover, Aaron Bacala, and Tori Lambert) for
their assistance in organism collection and holding. We also thank Katie O’Shaughnessy
and two anonymous reviewers for their constructive criticism to a previous version of this
manuscript.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
This research was made possible by a grant from The Gulf of Mexico Research Initiative.
The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Grant Disclosures
The following grant information was disclosed by the authors:
The Gulf of Mexico Research Initiative.
Competing Interests
Guillaume Rieucau is an Academic Editor for PeerJ.
Author Contributions
• Charles W. Martin conceived and designed the experiments, performed the experiments,
analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the
paper, and approved the final draft.
• Ashley McDonald conceived and designed the experiments, performed the experiments,
authored or reviewed drafts of the paper, and approved the final draft.
• Guillaume Rieucau conceived and designed the experiments, authored or reviewed
drafts of the paper, and approved the final draft.
• Brian J. Roberts conceived and designed the experiments, prepared figures and/or tables,
authored or reviewed drafts of the paper, contributed resources in terms of equipment,
materials, and staff, and approved the final draft.
Animal Ethics
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
The use of vertebrate organisms was conducted with IACUC approval and staff training
from University of Florida under protocol 201710044.
Martin et al. (2020), PeerJ, DOI 10.7717/peerj.10587
10/18
Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving
body and any reference numbers):
All field collections were made under Louisiana Department of Wildlife and Fisheries
Scientific Collecting Permit # SCP 200.
Data Availability
The following information was supplied regarding data availability:
Data are publicly available through the Gulf of Mexico Research Initiative Information
& Data Cooperative (GRIIDC):
Martin, C.W., Roberts, B.J. 2020. Laboratory experiments to determine effects of
previous exposure to oil on Fundulus grandis avoidance behavior. Distributed by: Gulf of
Mexico Research Initiative Information and Data Cooperative (GRIIDC), Harte Research
Institute, Texas A&M University–Corpus Christi. doi:https://doi.org/10.7266/n7-3qjh2f11.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/10.7717/
peerj.10587#supplemental-information.
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