Responses of bay anchovy (Anchoa mitchilli) larvae under lethal and sublethal scenarios of crude oil exposure

Ecotoxicology and Environmental Safety 134 (2016) 264–272

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Responses of bay anchovy (Anchoa mitchilli) larvae under lethal and sublethal scenarios of crude oil exposure Tara A. Duffy a,n, William Childress a,b, Ralph Portier c, Edward J. Chesney a a

Louisiana Universities Marine Consortium, 8124 Hwy 56, Chauvin, LA 70344, USA Aquatic Germplasm and Genetic Resources Center, Department of Renewable Natural Resources, Louisiana State University Agricultural Center, 2288 Gourrier Ave, Baton Rouge, LA 70802, USA c Louisiana State University, Department of Environmental Sciences, Baton Rouge, LA, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 4 December 2015 Received in revised form 12 July 2016 Accepted 19 August 2016 Available online 16 September 2016

Bay anchovy (Anchoa mitchilli) is an ecologically important zooplanktivorous fish inhabiting estuaries of the Gulf of Mexico and eastern North America from Maine to Florida. Because they have a protracted spawning season (spring through fall) and are abundant at all life stages in coastal estuaries, their eggs and larvae likely encountered oil that reached the coast during the Deepwater Horizon oil spill. We compared responses to oil exposure at different life stages and at lethal and sublethal conditions using acute, 24 h exposures. In a series of experiments, bay anchovy larvae were exposed to high energy water accommodated fractions (HEWAF) and chemically-enhanced WAF (CEWAF) at two stages of larval development (5 and 21 days post hatch, dph). HEWAF oil exposures induced significantly greater life stage dependent sensitivity at 5 dph than at 21 dph but chemically dispersed (CEWAF) exposure mortality was more variable and LC50s were not significantly different between 5 and 21dph larvae. Acute exposure to two low-level concentrations of CEWAF did not result in significant mortality over 24 h, but resulted in a 25–77% reduction in larval survival and a 12–34% reduction in weight specific growth after six days of post-exposure growth following the initial 24 h exposure. These results show that younger (5 dph) bay anchovy larvae are more vulnerable to acute oil exposure than older (21 dph) larvae, and that acute responses do not accurately reflect potential population level mortality and impacts to growth and development. & 2016 Elsevier Inc. All rights reserved.

Keywords: Bay anchovy HEWAF CEWAF Mortality Growth Development

1. Introduction Bay anchovy, Anchoa mitchilli, is one of the most abundant species of fish in coastal bays and inlets from Maine to south Florida and the Gulf of Mexico (Hildebrand and Schroeder, 1928; Sheridan, 1978). Bay anchovy embryos and larvae typically dominate the ichthyoplankton catches in estuarine collections during the summer, while adults dominate trawl surveys in Gulf estuaries (Dovel, 1971; Flores-Coto et al., 1983; Olney, 1983; Houde and Alpern-Lovdal, 1984; Chesney and Baltz, 2001). Because of its small size at maturity (30–40 mm), serial spawning strategy, and rapid larval growth rate (0.5 mm d
1), bay anchovy have tremendous potential as an important secondary producer and trophic link as a forage fish in estuarine habitats (Luo and Brandt, 1993). Their planktivorous feeding strategy continues into the juvenile and adult stages, making bay anchovy and other closely related n Corresponding author. Current address: Northeastern University, Department of Marine and Environmental Sciences, 14 Holmes Hall, Boston, MA 02115, USA. E-mail address: [email protected] (T.A. Duffy).

http://dx.doi.org/10.1016/j.ecoenv.2016.08.010 0147-6513/& 2016 Elsevier Inc. All rights reserved.

engraulids important links for energy flow in estuarine systems of the Gulf of Mexico and southeastern Atlantic Ocean (Detwyler and Houde, 1970; Sheridan, 1978; Livingston, 1982; Houde and AlpernLovdal, 1984, 1985; Rakocinski et al., 1992). During the 2010 Deepwater Horizon (DWH) oil spill, significant amounts of oil reached coastal estuaries of the northern Gulf of Mexico (Silliman et al., 2012). Because bay anchovy is native to the Gulf of Mexico, and has abundant early life history stages in surface waters, it is likely that significant numbers of this species were exposed to crude oil at all life stages. Understanding the impacts of this oil spill on bay anchovy may help estimate potential damage to food webs and susceptibility of this ecologically important species to crude oil exposure during early life stages. Bay anchovy is also representative of the many other anchovy species that occur in the Gulf of Mexico (Robinette, 1983). Rapid growth and development in early stages of this species suggest it may be a sensitive sentinel for oil exposure in other fish species with rapid larval development (o48 h) and are native to the Gulf of Mexico. Evaluating oil sensitivity in an ecologically important species that, to our knowledge, had never previously been used for toxicological studies may provide new insight into how this

T.A. Duffy et al. / Ecotoxicology and Environmental Safety 134 (2016) 264–272

species may be impacted during an oil spill (Echols et al., 2015). In order to better predict the potential impacts of crude oil at the population level, it is important to establish how responses change during early development. In many cases, embryos and/or larvae are significantly more sensitive to oil exposure than juvenile and adult stages (Kristensen, 1994; Kazlauskiene et al., 2008; Brewton et al., 2013). Although there is a vast literature documenting impacts to fish in stages that encompass embryos to adults, few studies have compared sensitivities to crude oil at multiple points during the early life stages of a single species (Shen et al., 2012). Until recently there have been relatively few studies that documented responses to crude oil of tropical and subtropical species that are spawned at high temperature and hatch ( o24 h) and develop rapidly (Incardona et al., 2013, 2014). Because of the rapid developmental changes that occur early in bay anchovy development (Jones et al., 1978), we might expect differences in sensitivity to oil among larvae at different stages. Exposure of early life stages of nekton were likely to be especially important in areas where dispersed and weathered oil accumulated in surface waters or were mixed throughout the water column as small droplets (Fodrie and Heck, 2011; Redman et al., 2012). However, soluble oil constituents may persist in the water column at a range of concentrations that elicit sublethal responses. Short or low dose exposures can impair growth (Brewton et al., 2013; Vignet et al., 2014), swimming performance (Le Bihanic et al., 2014; Mager et al., 2014; Yu et al., 2015), cardiac development (Milinkovitch et al., 2013), and induce other physiological and genetic changes (Carls et al., 1999; Whitehead et al., 2011; Olsvik et al., 2012). These changes can undermine the fitness of early life stage fish and potentially contribute to reduced survival. Both survival and growth are demonstrably reduced when early life stages are exposed to oil constituents (Heintz et al., 2000; Brewton et al., 2013). Quantifying these sublethal impacts can help us to understand long-term impacts of oil exposure as well as population-level responses. The goal of this research was to assess both lethal and sublethal responses of bay anchovy larvae to chemically and mechanically dispersed crude oil in an ecologically important estuarine species of the Gulf of Mexico. Corexit9500™ (Nalco, Naperville, IL, USA) is one of the most widely used dispersants worldwide (Kleindienst et al., 2015) and was the primary dispersant used during the DWH spill (Kujawinski et al., 2011). Little is known about the susceptibility of bay anchovy to dispersed oil exposure, but several studies suggest that the dissolved, water accommodated fraction of crude oil (which includes polycyclic aromatic hydrocarbons (PAHs), and other components) is especially toxic to fish larvae (Paine et al., 1992; Carls et al., 2008; Mhadhbi et al., 2010). Short term (24 h) acute exposures were carried out to mimic realistic scenarios that likely occurred within coastal estuaries or within convergent zones (fronts) where bay anchovy embryos and larvae are commonly aggregated (Bowman and Iverson, 1978; Govoni, 1993). Specifically, 5 and 21 dph larvae were chosen for comparison because 5 dph larvae are newly feeding, competent larvae, while 21 dph larvae are approximately 10–12 mm TL (total length) and are on the cusp of metamorphosis (beginning around 15 mm TL, Jones et al., 1978). Because fast growth is considered to be important to the survival of early life stages of fish larvae (Ware, 1975; Houde, 1997), we also assessed the potential effects on growth following acute, low concentration doses of dispersed oil, then growth in oil free seawater, to estimate potential long-term impacts of acute oil exposure.

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2. Methods 2.1. Animal culture 2.1.1. Bay anchovy culture Bay anchovy embryos were collected from spawning adults held at the Louisiana Universities Marine Consortium. A spawning colony was established by collecting juvenile wild bay anchovies from estuarine waters of Louisiana and rearing them to the adult stage. Anchovy broodstock were held in a 400 gallon recirculating seawater system maintained under moderate summer spawning conditions (15 L: 9D light cycle; 25 psu; Temperature 27.5 °C). Adult bay anchovy were fed a mixture of brine shrimp, rotifers and a dry commercial fish feed (Aquaxcel, size 3, Cargill, Minneapolis, MN, USA). The holding system was fitted with a sump that contained an egg collection device. Embryos were removed from the egg collector daily, assessed for viability, and 500 embryos were moved to a 40 L aquarium filled with 25 psu seawater and housed in a temperature regulated water bath (  27 °C) for hatching. Hatching occurred in  18 h. Rotifers and live Nannochloropsis oculata and the Tahitian strain of Isochrysis galbana were introduced to the larval rearing aquaria to create 'green water' at approximately two days post hatch (dph). Rotifers were maintained in aquaria at 5–10 ml
1, and brine shrimp were introduced at approximately 10 dph as the larvae developed. 2.2. Experimental procedures 2.2.1. HEWAF and CEWAF preparation High energy water-accommodated fractions (HEWAFs) and chemically-enhanced water-accommodated fractions (CEWAFs) were made with source Macondo surrogate sweet Louisiana crude oil collected from the well head (BP Gulf Restoration Organization, Houston, TX). All HEWAF and CEWAF treatments were prepared in 25 psu seawater (5 mm filtered and UV sterilized) at 21 °C. A stock HEWAF solution was prepared according to Incardona et al. (2014) using 3.05 ml Macondo surrogate oil per 1 L of seawater. Briefly, HEWAF was prepared by adding 3.05 ml of oil to 1 L of water using a gas-tight syringe. The mixture was blended at low speed in a stainless steel commercial blender for 30 s and then poured into a separatory funnel, allowed to settle for one hour, and the liquid drained into a clean glass beaker. The stock solution was then diluted with clean 25 psu seawater to create a dilution series. Measurements of relative fluorescence were recorded for each of the diluted stock solutions using a Trilogy laboratory fluorometer (Turner Designs, Sunnyvale, CA USA) fitted with the crude oil module (7200-063). CEWAF dilutions were prepared according to Singer et al. (2000). CEWAFs were made in clean, 2 L aspirator jars with 1.2 L of seawater. The oil was chemically dispersed with Corexit9500™ (Nalco, Naperville, IL, USA) at a ratio of 20:1 (oil:dispersant, Singer et al., 2000). Briefly, between 0.15 and 1.5 ml crude oil and the appropriate volume of Corexit9500™ were added to seawater, vortexed with a stir bar to create a 20% vortex depth, stirred for 21 h, then allowed to settle for 3 h. CEWAF was then drained from aspirator jars, and stored in clean, covered glass beakers in the experiment incubation bath for  1 h to reach the experimental temperature. 2.2.2. LC50 Trials Originally, we attempted to establish bay anchovy LC50s with exposures to low energy WAF for comparison to dispersed WAF. However, in 24 h exposures negligible (o 10%) mortality was observed in both 5 and 21 dph bay anchovy larvae, even when exposed to 1:10 (oil:water) mixture (Duffy and Chesney, unpublished data using methods from Singer et al., 2000). Therefore, efforts

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were focused on dispersed WAF in this study. All oil exposures were carried out in 2 L glass beakers that contained a mixture of seawater, stock oil solutions, and plankton. Plankton was added to better mimic conditions to which wild larvae would be exposed to oil. Plankton consisted of rotifers at a density of 5/ml and a concentration of Nannochloropsis sufficient to create a turbidity of 5 NTU to maintain rotifer growth and reproduction. Approximately 150 brine shrimp were added to each beaker for any larvae 10 dph or older. Fifty larvae were randomly assigned to treatments by transferring them to 2 L beakers with a 50 ml glass beaker. After all larvae were transferred, additional clean seawater was added, then 400 ml of the oil solution was added to each beaker to reach 2 L and beakers were housed in a water bath held at 24.9 °C7 0.3 S.E. Beakers were lightly aerated using glass pipettes attached to plastic airline tubing and covered with food-grade plastic film. Each trial included treatments in duplicate, and trials were replicated several times so that all treatments were repeated in at least two trials for the range of oil concentrations. Results from replicated treatments were combined for statistical analyses. All exposures were conducted under 24 h light to promote photosynthesis by phytoplankton and to maintain dissolved oxygen levels. At the end of each HEWAF and CEWAF exposure, temperature (thermistor) and dissolved oxygen (YSI Pro ODO) were recorded and larvae were assessed as live, dead or moribund (abnormal or no swimming). Two, 450 ml water samples from each replicate were pooled for GC-MS analyses by combining samples. All treatments were filtered using a clean, 4.7 cm glass microfiber filter (VWR, Radnor, PA, USA) for pre-screening, followed by filtration with a 0.45 mm Whatmans cellulose nitrate membrane filter (GE Life Sciences, Pittsburg, PA, USA), added to a clean 1 L amber glass jar, and were immediately frozen at
20 °C for GC-MS analyses. One pooled sample was analyzed per nominal concentration in a given trial. 2.2.3. Low-level Oil Exposures and Growth Exposure to low concentrations of CEWAF in 5dph larvae were carried out as previously described, except that 100 individuals were used per duplicate treatments. Following low and moderate exposures of anchovy larvae to CEWAF (0.37 mg/L and 0.53 mg/L total PAHs (tPAH), respectively and 20 and 40 mg/L oil nominal dose, respectively). The remaining larvae were assessed for condition. Sixty five larvae that appeared to be healthy, (e.g. swimming and feeding normally) were moved to clean, 40 L static aquaria for an additional 6 days of growth. In order to transfer as little oiled water as possible, 75% of the oiled water was slowly exchanged with clean water at the appropriate temperature and salinity. Larvae were then transferred to aerated 40 L tanks in a small volume of water. This resulted in less than 1.2% of oiled water moved into the clean growout tanks. Water changes in the 40 L static tanks were carried out every other day to maintain low NH4 þ concentrations (o 0.25 mg/L) and to maintain optimal rotifer density (2–5 rotifers/ml). Total ammonia and pH were measured every other day using water quality kits (Mars Fish Care, API, Chalfont, PA, USA). Approximately 2 L of mixed algal culture (N. oculata and I. galbana.) was added twice daily to promote rotifer health and growth, and maintain high dissolved oxygen along with light aeration. After six days, surviving larvae were sacrificed and a randomly selected subset of anchovy larvae (16 per replicate) were measured (notochord length) and weighed (dry weight after 24 h at 38 °C and cooled in desiccant). This experiment was repeated for a total of four replicates per treatment.

2.3. Chemical Analyses 2.3.1. GC/MS Methods Extraction of PAHs and alkanes in water amended with oil follows methods outlined in EPA Method 8270D series (EPA, 2014). For analyses, flasks were rinsed with dichloromethane (DCM) to ensure the complete solubilization of all oil into the final, extractable liquid fraction. Approximately 200 ml of water was poured into a 250-ml separatory funnel and adjusted to a pH of 7. A 30-ml aliquot of dichloromethane (DCM) was added to the separatory funnel and spiked with a known amount of standard surrogate. The funnel was capped and shaken for approximately 3 min, venting occasionally to remove solvent pressure. The solvent and water were allowed to separate and the solvent was drained through an anhydrous sodium sulfate funnel into a 250-ml flat-bottom flask. The solvent addition and draining step were repeated twice. The sodium sulfate funnel is rinsed with DCM and allowed to drain completely. The flat-bottom flask was then placed on a rotary evaporation system and concentrated to a volume of 5– 10 ml DCM and placed in a calibrated extraction thimble. The extract volume of samples was concentrated by placing it under a nitrogen blow down concentrator that reduced the volume to 1.0 ml. The DCM extract was then exchanged to hexane using approximately 4–5 ml of hexane. A micro distillation column was added to the extraction thimble and placed in a hot water bath. The DCM was evaporated and the remaining hexane extract was reduced to a volume of 1–2 ml. The hexane extract was then placed beneath a nitrogen blow down device and reduced to a final volume of 1.0 ml hexane. 2.3.2. GC/MS Instrumental analyses After addition of internal standards, samples were analyzed using an Agilent 7890A GC fitted with a 0.25 mm ID  30 m HP5MS column and an Agilent 7683B autosampler. The injector was set to 250 °C and the detector to 280 °C. Detection of analytes involved the utilization of a HP 5975C Inert XL Series Mass Selective Detector operating in the Selected Ion Monitoring mode. The column was held at 60 °C for 1 min and then ramped at 25 °C/ min to 160 °C followed by 3 °C/min to 268 °C and 12 °C/min to 300 °C, where it was held for 8 min. Concentrations of parent PAHs were calculated based on calibrations using a five-point curve which was checked for each batch of samples analyzed. Concentrations were reported on a dry weight basis. Approximate alkylated PAH concentrations were calculated assuming the same response factors for each parent and corresponding alkylated analogues. For alkylated phenanthrene/anthracenes, the results were reported as pairs to incorporate the uncertainty of the measurements and quantification based on the average response factor of the individual parent PAHs (based on EPA Method 8270D (EPA, 2014)). Data from each category were combined into total PAHs and total alkanes, with total PAH concentration used for analysis as it usually represents the best estimate of toxicity (Barron et al., 2004; Wu et al., 2012; Vikebo et al., 2014). GCMS analyses were conducted on 70% of the samples. For the 30% of samples not analyzed by GCMS, total PAHs were estimated by fluorescence. A relationship was developed between tPAH concentration estimated by fluorescence measured for each HEWAF or CEWAF and the tPAHs as estimated by GCMS. At least one sample from every nominal concentration (CEWAF) or % HEWAF used in the experiments were analyzed by GCMS, with full coverage from the controls to the highest exposure doses. 2.4. Statistical Analysis LC50 values were estimated using R software (package¼‘drc’, R Core Team, 2015). Additional analyses were conducted using

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SigmaPlot™ (v. 11, Systat Software Inc., San Jose, CA, USA). Exposure mortality data were fit with a 3-parameter, sigmoidal model. LC50 values were compared with two-tailed T-tests. Mortality data was compared using a one-way ANOVA followed by post hoc Holm-Sidak pairwise comparisons and size data was compared using two-way ANOVAs with the same post hoc comparisons. Data from each trial were not significantly different, so both trials were combined for analyses, yielding 4 replicates per treatment. All data were normalized to total aromatic hydrocarbon (PAH) concentration because alkane concentrations showed little to no relationship with mortality.

3. Results 3.1. Water quality and oil chemistry In all experiments, dissolved oxygen (D.O.) was generally high, around 6.2 70.1 mg/L, but no low dissolved oxygen treatments o4.7 mg/L ( o60% D.O.) were used in the analyses. Additional water quality analyses indicated no significant changes in pH, and total ammonia was never above 0.25 mg/L. After 24 h, there were small differences in the composition of PAHs in the solutions of HEWAF and CEWAF, most notably in naphthalenic and Me-Phen./anthracenic fractions (Fig. 1). Composition of alkane and aromatic constituents for each treatment are available in the GRIIDC Database (https://data.gulfresearchinitiative.org; http://dx. doi.org/10.7266/N7416V3Z). Comparing initial RFU measurements of the stock oil mixture used in each treatment to the 24 h total PAH concentrations demonstrates that more tPAHs were maintained in CEWAF solutions than in HEWAF solutions (Fig. 2, ANCOVA, homogeneity of regression, p ¼0.13; mean, p ¼0.006). This demonstrates that for any given initial fluorescence measurement of WAF mixture, PAHs were more stable in CEWAF solutions during 24 h exposures than in HEWAF solutions. 16 14

Fig. 2. Initial fluorescence of stock used to make HEWAF (dark gray) and CEWAF (white) treatments compared to the total concentration of total PAHs after 24 h of exposure. HEWAF, y¼ 3.7  10
3x þ 0.18, R2 ¼ 0.52; CEWAF, y¼ 6.4  10
3x þ 0.97, R2 ¼ 0.84.

3.2. HEWAF and CEWAF LC50 s Bay anchovy at 5 dph were significantly more sensitive to HEWAF than 21 dph larvae, with LC50s of 0.69 mg/L ( 70.07 standard error, S.E., 95% confidence interval (95% C.I.)¼0.54– 0.84 mg/L) and 1.61 mg/L (70.09 S.E., 95% C.I. ¼1.43–1.80 mg/L) total PAHs (T-test, p o0.001, Fig. 3), respectively. CEWAF exposures yielded much higher LC50 values of 3.96 mg/L (70.09 S.E., 95% C. I.¼ 3.77–4.16 mg/L) and 4.75 mg/L (7 0.59 S.E., 95% C.I. ¼3.51– 5.99 mg/L, p ¼ 0.14, Fig. 4). However, there was no significant differences between 5 and 21 dph for CEWAF exposures. Comparisons of same-aged fish revealed significantly lower LC50 values for HEWAF relative to CEWAF (5 dph, T-test, p o0.001; 21 dph, p o0.001). Five dph larvae showed the steepest increases in mortality over a narrow range when exposed to both HEWAF and CEWAF, while mortality of 21 dph larvae increased more gradually with increasing PAH concentration (i.e. near-complete mortality was reached at a lower concentration in 5 dph larvae).

10

100

8

80 6

Mortality (%)

% Total PAHs

12

4 2

C Na 1- p h N C ap t h a 2- h l e N th n C ap a l e e 3- h N th n e C ap ale s 4- h N th nes ap al ht en ha es le C Flu nes 1- o Fl r e n u C D 2- ore e ib en F n z o C3 luo es t h -F ren i o lu e ph o s e n ren e es (D C BT 1D ) C BT 2- s C D 1P P C BT C h e h e 3-D s 2- ns n a B P T C he /A n n t h s 3- n t h r e Ph s/A ra n e c en nt e s/ hra ne An c s th en Be ra es nz ce Be o n n z [ a] P es a o Be [b nt yre h n z ] f lu ra n e o[ o c e k] ra n f lu n t e or he an ne th en e

0

Constituent Fig. 1. Percent of total PAHs of individual aromatic constituents as measured by GC-MS for HEWAF (black) and CEWAF (gray) samples to compare constituents that remain in each oil prep method after 24 h. Each bar represents the mean of representative samples (n ¼4) with a total PAH concentration between 0.5 and 1.5 μg/ L, error bars represent standard error. Only constituents that represent 41% of the total PAHs were included for clarity.

60

40

20

0 0

1

2

3

4

5

Fig. 3. Mortality following 24 h exposure to HEWAF for two ages of bay anchovy, 5 dph (gray) and 21 dph (black). Each data point represents mortality of a single replicate of n¼ 50 fish. Total PAH concentration represents the soluble fraction at the end of the 24 h exposure window.

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100

0.20

Dry weight (mg)

Mortality (%)

80

60

40

A a

0.15

b

0.10

b

0.05

20

0.00 0

0 2

4

6

8

10

20

40

12

Fig. 4. Mortality following 24 h exposure to CEWAF for two ages of bay anchovy, 5 dph (gray) and 21 dph (black). Each data point represents mortality of a single replicate of n ¼50 fish. Total PAH concentration represents the soluble fraction at the end of the 24 h exposure window.

3.3. Latent impacts of oil exposure Exposure of bay anchovy larvae to CEWAF at 0.37 μg/L (low dose) and 0.53 μg/L (moderate dose) total PAHs doses resulted in no statistically significant increased mortality relative to controls after 24 h (one-way ANOVA on ranks, p¼ 0.232). The remaining larvae were allowed to feed and grow for six additional days in clean seawater with plankton. Fish exposed to low and moderate CEWAF concentrations died at rates that ranged from 25 to 77% (Fig. 5). In comparison, control larval mortality was significantly lower than in treatments (9 72%, one way ANOVA, p ¼0.002). Additionally, larval growth was significantly slower in the surviving larvae exposed to low and moderate CEWAF doses (Fig. 6). Mean dry weight of anchovy larvae was significantly reduced in oil exposed larvae relative to controls by 27% and 34% (control¼ 0.137 mg ( 70.006 S.E.), low¼0.100 mg ( 70.005 S.E.), moderate¼0.090 mg ( 70.004 S.E.), n¼ 64 for all treatments) (2-way ANOVA, p o0.001, Fig. 6A). Dry weights of anchovy larvae exposed to low and moderate oil

Notochord length (mm)

0

8

B

a

b

6

b

4

2

0 0

0.24

1.23

Mean total PAHs (μg/L) Fig. 6. Mean dry weight (A) and notochord length (B) following acute (24h) exposure to CEWAF, and six days of post-exposure growth. Each bar represents n ¼ 64 fish from four replicates, 7 SE. Letters represent significant differences from the control.

concentrations did not differ significantly from each other (HolmSidak post hoc comparisons, p ¼0.160). Similarly, notochord length was reduced in exposed animals by 12% and 15%, (control ¼6.15 mm (70.06 S.E.), low¼ 5.40 mm (70.08 S.E.)), moderate¼ 5.25 mm ( 70.08 S.E.) (2-way ANOVA, p o0.001, Fig. 6B). Notochord lengths of larvae exposed to low and moderate oil concentrations also did not differ significantly from each other (Holm-Sidak post hoc comparisons, p ¼0.136).

4. Discussion

Fig. 5. Average mortality of 5 dph bay anchovy larvae at the end of 24h exposures to CEWAF (gray), and at the end of 6 days of post-exposure growth (black). Gray bars represents the mean mortality 7 SE of four replicate exposures with n ¼ 100 per replicate (24h exposure), black bars are n ¼ 65 per replicate (6 days of growth in clean water). Letters represent significant differences from the control, ns ¼ no significant differences compared to the controls.

Bay anchovy, Anchoa mitchilli, larvae responded to HEWAF and CEWAF exposures in a manner that demonstrated both sublethal and lethal consequences of exposure. Newly feeding (5 dph) larvae were significantly more sensitive to HEWAF than well-developed larvae that were beginning to metamorphose around 21 dph. However, the results were not statistically different for 5 and 21 dph larvae exposed to CEWAF, despite a higher LC50 estimate for 21 dph vs. 5 dph larvae. We also observed statistically significant latent mortality (up to 77%) in the 6 days following exposure to concentrations of CEWAF that resulted in no significant increase in mortality during the initial 24 h exposure. Weight was reduced in surviving larvae by up to one-third, and length was reduced up to 15% compared to control larvae over the same 6 day post exposure grow-out. These results suggest that acute exposure to dispersed crude oil may have disproportionate impacts on bay anchovy larvae of different ages, and that low-level exposures can have

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significant latent effects that may be significant at the population level if they can affect well developed larvae that would otherwise have a good probability of recruiting to the juvenile population. 4.1. PAH toxicity in mechanically and chemically dispersed crude oil Crude oil is a complex mixture of compounds, of which the polycyclic aromatic hydrocarbon (PAH) constituents are considered to be most toxic to aquatic animals (Barron et al., 2004; Carls and Meador, 2009; Vikebo et al., 2014) though they are not the only constituents that contribute to the toxic effects of oil (Gonzalez-Doncel et al., 2008). PAHs differ in water solubility (Pearlman et al., 1984), and at low total PAH concentrations (o 1– 2 mg/L) can cause severe impairment to fitness (Heintz et al., 2000; Carls et al., 2008) and acute mortality (Schein et al., 2009). Mixing energy in the form of waves creates droplets in suspension that can alter the solubility of PAH constituents within the water column (Li and Garrett, 1998). The addition of dispersants, including Corexit9500™, results in droplets of suspended oil that can be more easily biodegraded (Fiocco and Lewis, 1999). Dispersants may act to either bind or aid in the release of the soluble fraction, changing the availability of crude oil constituents and thus their toxicity (Kleindienst et al., 2015). Because we used different mixing energies to create both mechanically and chemically dispersed oil in this study, direct comparison of LC50 values between preparation methods is problematic (Singer et al., 2000; Brewton et al., 2013). Droplet size, distribution, and time in suspension change with each preparation method (Shiu et al., 1990; Singer et al., 1998, 2000; Olsvik et al., 2012; Forth et al., 2015), and CEWAF tends to create micelles of dispersant and oil that influence PAH availability (Singer et al., 1995). Therefore, it is difficult to make direct comparisons between results utilizing different preparation methods, as droplet size and retention may have varied dramatically between methods. Despite filtration of all analytical samples to 0.45 mm, small micelles (oil droplets surrounded by dispersant) may remain in solution (Forth et al., 2015) which can influence toxicity and analytical comparison (Singer et al., 1995; Mielbrecht et al., 2005). Initial fluorescence of oil mixtures, however, gave us an estimate of initial dissolved and suspended tPAHs (Brewton et al., 2013). For a given initial fluorescence measurement of WAF, dissolved tPAH values in CEWAF were significantly higher than in HEWAF after 24 h. This signifies that dissolved, but volatile, PAH constituents had stabilized in the CEWAF preparations due to 21 h of initial mixing and continued to be stable during the 24 h experiment (Forth et al., 2015). This is indicated by the clear differences in the greater percent composition of naphthalenes in the HEWAFs and higher relative percentages of phenanthrenes in the CEWAFs (Fig. 3). These data are some of the first to report responses of bay anchovy to oil exposure. Establishing LC50 s for comparison to other fish species is essential for understanding how estuarine vs. offshore species may have responded to oiling during the Deepwater Horizon oil spill (Echols et al., 2015). Data on numerous embryo and larvae of species from the Gulf of Mexico demonstrate LC50 values that are well within concentrations measured and expected during peak oiling of estuarine and open water systems within the Gulf of Mexico. Much of the oil exposure data for early life stages of fishes inhabiting the northern Gulf of Mexico comes from exposures of embryos that hatch during exposure. These results can be compared to larval exposure with caution since embryos tend to be less sensitive to PAHs than larvae (Kristensen, 1994; Carls et al., 1999). Mahi-mahi (Coryphaena hippurus) embryos exposed to both high energy and chemically enhanced water accommodated fraction demonstrate EC50 (effective concentration) values of 20.6–21.3 mg/L dissolved tPAH over 96 h (Esbaugh

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et al., 2016). Embryos of yellowfin tuna (Thunnus albacares) are potentially more sensitive than those of mahi-mahi, demonstrating EC50 values of 0.8–2.3 mg/L tPAH over 48 h, with greater amberjack (Seriola dumerili) demonstrating intermediate EC50 values of 13.5 mg/L (Incardona et al., 2014). These are relatively warm water species, which have embryos that develop quickly (o24 h to hatch) in upper surface waters of the Gulf of Mexico (Incardona et al., 2014) as do bay anchovy. Larval bay anchovy demonstrated LC50 values for different WAF preparations between 0.7 and 4.0 mg/ L which are comparable to the EC50 values for edema in mahimahi (Esbaugh et al., 2016). Oil research conducted with colder water species such as Atlantic herring (Clupea harengus) Atlantic cod (Gadus morhua), capelin (Mallotus villosus) and rainbow trout (Oncorhynchus mykiss) indicates relatively higher values at which approximately 50% mortality occurs to embryos or larvae (Nordtug et al., 2011; Frantzen et al., 2012; Ingvarsdottir et al., 2012; Martin et al., 2014). While it is difficult to make direct comparisons of LC50 values across these experiments due to different methodology, the general trend is that higher tPAH concentrations and longer exposure duration are required to elicit approximately 50% mortality (6–28 mg/L tPAH at treatment lengths from 4 to 32 d) in these cold water species. Bay anchovy appear to have comparable responses to dissolved PAHs to other pelagic larvae found in the Gulf of Mexico. This may indicate a higher sensitivity to crude oil exposure for rapidly developing life stages at high temperatures. 4.2. Early life stage comparisons Predicting and understanding the impacts of crude oil on the earliest life stages of ecologically and economically important, and native species is a necessary task to understand potential impacts to populations following an oil spill (Hilborn, 1996; Echols et al., 2015). Bay anchovy are serial spawners in the Gulf of Mexico with a protracted breeding season that occurs throughout spring and summer (Swingle, 1971; Houde and Alpern-Lovdal, 1985) indicating early life stages overlapped with DWH crude oil in Gulf of Mexico estuaries. Most significantly, bay anchovy 24 h LC50s averaged 0.7–1.6 mg/L tPAH for HEWAF and 4.0–4.8 mg/L tPAH for CEWAF. These concentrations are well within the range of values measured in the water column following the Deepwater Horizon Spill. For example, tPAH levels of 17.5–213 mg/L were measured in Barataria Bay, LA (May and June 2010; Whitehead et al., 2011) while levels between 3 and 3000 mg/L or greater were estimated at peak oiling in the same region (Pilcher et al., 2014). The greater sensitivity of 5 vs. 21 dph larvae to HEWAF suggests that contact with dissolved components of crude oil in the wild may have had disproportionate effects on survival depending upon the overlap between oil exposure and larvae of different ages. Bay anchovy embryos and yolk sac larvae often float in surface layers and have limited swimming ability (Flores-Coto, 1983; Morton, 1989). Consequently, these life stages may be particularly susceptible to direct oiling. Because bay anchovy showed significant differences in age dependent mortality as larvae, development may play a key role in susceptibility of early life history stages to oil exposure. Because bay anchovy are a small, highly abundant forage species that spawn daily, most fishes in the Gulf of Mexico had fewer early life stages present in the plankton at any given time during the summer when DWH occurred (Swingle, 1971; Houde and Alpern-Lovdal, 1985). Consequently the pool of bay anchovy larvae exposed was likely to be high compared to other estuarine species. Brewton et al. (2013) identified different responses in hepatic cytochrome P-4501A (cyp1A, biomarker of PAH exposure, see review in Lee and Anderson (2005)) gene expression and growth between larvae and juvenile spotted seatrout exposed to both HEWAF and CEWAF. Life stage differences in the response to oil in this species may be the result of ontogenetic shifts in physiology or behavior

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(Brewton et al., 2013), and may indicate that mortality is likely to be different between these stages. If we are to fully understand the potential impacts to nekton as a result of an oil spill, it is essential to study life stage-specific differences in response to oil (Fodrie and Heck, 2011; Vikebo et al., 2014). Numerous other fish species display life-stage dependent responses to crude oil ranging from differences in acute mortality to latent effects that include impacts to growth and behavior (Paine et al., 1992; Pollino and Holdway, 2002; McIntosh et al., 2010; Shen et al., 2012; Brewton et al., 2013). Most of these studies compared different developmental stages (embryos, larvae, juveniles) or focused on chronic exposures that span life stages (i.e. embryogenesis through larval hatch) rather than different ages within a developmental stage. For example, Pollino and Holdway (2002) found that crimson-spotted rainbowfish (Melanotaenia fluviatilis) larvae were more sensitive to crude oil than embryos during 96 h exposures, but that embryos showed significant developmental abnormalities at sublethal concentrations. Atlantic herring (Clupea harengus) displayed different sensitivities to oil and dispersed oil that depended on the timing of exposure (McIntosh et al., 2010). In herring, embryo sensitivity was greatest during the first 24 h, then declined rapidly as embryos developed, presumably due to a sharp decrease in permeability as the chorion water-hardened (McIntosh et al., 2010). These data underscore the need to know how sensitivity changes both within and among life stages, but given the paucity of life stage comparative data within a single species, more studies need to address these potential differences. There are several factors that differ between 5 and 21 dph bay anchovy that may underscore unique responses to HEWAF exposure depending upon size and stage of development. By 21 dph, larvae are approaching metamorphosis, developing pigmentation, and major organ systems (Jones et al., 1978). Detoxification mechanisms associated with liver development are likely by the time larvae reach 21 dph, which may partially explain the decrease in sensitivity to HEWAF relative to 5dph larvae. Exposure to dissolved PAHs is likely to be cutaneous in both life stages and smaller larvae will likely have a higher surface area to volume ratio (Jones et al., 1978), increasing the body burdens of fat-soluble PAHs. Younger bay anchovy larvae are much less efficient at prey capture (Chesney, 2008), so if foraging efficiency was reduced by exposure to PAHs then energy acquisition would be impacted more in 5 dph larvae. Despite non-significant differences in CEWAF LC50 values between different ages of larvae, the slope of the mortality response is strikingly different between 5 and 21 dph. This suggests a different mechanism in toxicity, further supporting the idea of ontogeny impacting the response to PAHs at different life history stages. 4.3. Latent impacts of oil exposure We observed significant sublethal and latent mortality effects by following growth and survival of fish larvae after they were exposed for 24 h. In a pioneering study designed to determine delayed impacts of oil exposure, pink salmon, Oncorhynchus gorbuscha, embryos were exposed to conditions that mimicked the oiling of substrate experienced by benthic organisms following the Exxon Valdez oil spill (Heintz et al., 2000). Researchers observed a 15% reduction in marine survival upon release of fry exposed to oil as embryos, as well as significant reductions in juvenile growth. Further, the authors extrapolate these small, but significant differences in growth to conclude that size at maturity would be reduced by approximately 33–50%, which has major implications for reproductive success (Heintz et al., 2000). Embryonic exposure to low levels of crude oil in zebrafish also demonstrate delayed effects. Acute (24 h) fuel oil exposure in common sole (Solea solea) juveniles resulted in low-levels of mortality within a day following

initial exposure and reduced survivorship (33% survival vs. 54% survival for controls) in those fish that survived initial exposure and were then reared in mesocosm tanks over three months (Claireaux et al., 2004). Hicken et al. (2011) found that reduced swimming performance in adults exposed as embryos for 48 h was likely tied to heart developmental malformations. These data demonstrate a potential mechanism that links impacts to individual fish with potential population-level consequences (Hicken et al., 2011). These studies highlight the potential population level impacts that are not easily observed in short-term studies. Relatively little work has been carried out to address delayed mortality in fishes, but several studies discuss the potential impact of delayed and population-level impacts (Carls et al., 1999; Incardona et al., 2014; Vignet et al., 2014). Crude oil and/or PAHs can directly inhibit growth across a range of exposures (Heintz et al., 2000; Claireaux et al., 2004; Frantzen et al., 2015). What is of equal or potentially greater value is understanding the potential impacts to growth (and other facets of early development) following exposure to short, but sublethal concentrations of PAHs. Short term exposures, such as those conducted by Heintz et al. (2000) and Mager et al. (2014), for example, demonstrate scenarios that are not only plausible, but are more likely to occur in the environment than in typical laboratory exposure protocols which emphasize endpoints like LC50. In an effort to understand potential impact to growth, Claireaux et al. (2004) found that juvenile common sole grew more slowly for several months following two exposures to fuel oil mixed with seawater, and that length, condition factor, and growth rate demonstrate reductions for fuel oil exposed fish. Growth can be reduced for months following exposure to crude oil, as shown by experiments conducted by Heintz et al. (2000). Spotted seatrout larvae grew more slowly in the presence of CEWAF and dispersant, and these delayed growth impacts were seen within three days of growth in clean water following exposure to HEWAF and Corexit™ (Brewton et al., 2013). These differences in growth were shortlived in that case, and no size differences were observed after four weeks of growth in clean water. This demonstrates that recovery via compensatory growth is possible in some cases. Delayed growth and mortality in zebrafish exposed to PCBs during embryonic development have been documented (DiPaolo et al., 2015), in addition to other environmental contaminants (Mirbahai and Chipman, 2014). Together, these data clearly demonstrate the latent and significant impacts of exposure from constituents of crude oil in early life stages of fishes. The mechanisms for latent effects due to low dose exposure to oil in bay anchovy are currently unknown, but additional studies are underway to better understand these. For example, morphological deformations such as spinal and cranial defects, and degradation of the developing finfold can reduce feeding capacity and swimming efficiency, resulting in larval starvation. These morphological changes are indicative of blue sac disease, which is a suite of developmental abnormalities that is common to PAH exposure in young fish (Incardona et al., 2013; Milinkovitch et al., 2013; Brette et al., 2014; Le Bihanic et al., 2014). Blue sac disease typically results in changes to cardiac function that may be tied to reduced metabolic rate (Mager et al., 2014; Klinger et al., 2015), impairing growth, development, and feeding behavior in a life stage when optimal growth is essential for survival. Mounting evidence suggests that cardiotoxicity of crude oil may be one of the most common mechanisms of impairment in developing fish exposed to PAHs (Brette et al., 2014). Bay anchovy are recently competent feeders at 5 dph, beginning feeding at around 3 dph. Bay anchovy larvae exhibit a distinct pause-travel search pattern, with pauses as they can for food becoming significantly shorter in duration leading up to metamorphosis (Chesney, 2008). Impacts to swimming ability, speed, and

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buoyancy regulation have been demonstrated in other species that have been exposed to PAHs as embryos and/or larvae (Le Bihanic et al., 2014; Mager et al., 2014; Klinger et al., 2015) which may be impairing the larvae's ability to successfully forage, leading to limitations in food acquisition and potentially starvation. HEWAF exposure at 1.2 mg/L tPAHs induced a significant decline in critical swimming velocity (Ucrit) in mahi-mahi juveniles that had been exposed for a brief 48 h spanning late embryo to early larval development (Mager et al., 2014). In this study, swimming efficiency was hypothesized to explain reductions in Ucrit without concurrent reductions in aerobic scope, as measured by respirometry. Further, this study also demonstrates latent impacts that appear to be characteristic of studies that follow fish larvae after short-term, sublethal exposure. While sustained swimming in mahi-mahi larvae is more relevant to feeding behavior in that species, impairment to swimming (especially burst swimming speed) could influence success of larval anchovies in successfully capturing food, which tends to increase dramatically over the first 5–6 days of feeding, concomitant with an increase in size and lipid stores (Tucker, 1988; Chesney, 2008). More work is needed to document reduced swimming and foraging success as the underlying mechanism in latent mortality and reduced growth following shortterm exposure to crude oil.

5. Conclusions This work establishes LC50 values for bay anchovy, an ecologically important species in Gulf of Mexico estuaries. We also demonstrated clear differences in acute mortality between early and late stage larvae in response to mechanically dispersed oil, and evaluated potential differences in toxicity between mechanically and chemically dispersed crude oil. Acute exposures, such as those performed in this study, induced significant latent impacts on both mortality and growth, indicating that assessing environmental effects based solely on acute (24 h) exposures that estimate LC50 values are not sufficient to predict impacts of oil exposure of bay anchovy larvae in the wild. These results suggest that bay anchovy larvae are similarly sensitive to dispersed crude oil to other fish larvae found in the Gulf of Mexico, and that they may a useful sentinel to understand population level impacts to fitness through assessment of delayed impacts.

Acknowledgments We gratefully acknowledge the help of Sarah Webb, Evan Kwityn, Joanna Griffiths, and Kathryn O'Shaughnessy. Erin Saal and Greg Olson conducted GC/MS analyses and analyzed data. This research was made possible by a grant from The Gulf of Mexico Research Initiative (project no. GoMRI-021). Data are publicly available through the Gulf of Mexico Research Initiative Information & Data Cooperative (GRIIDC) at https://data.gulfresearchinitiative.org. TAD and EJC designed the experiment, conducted the analyses, and prepared the manuscript, and TAD and BC conducted the experiments. The authors declare no conflict of interest. This work has not been previously published.

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