Southeastern Naturalist R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 G82 2015 SOUTHEASTERN NATURALIST 14(4):G82–G92 Toxicity of Three Dispersants Alone and in Combination with Crude Oil on Blue Crab Callinectes sapidus Megalopae Rachel Fern1,*, Kim Withers1, Paul Zimba1, Tony Wood2, and Lee Schoech1 Abstract - During the Deepwater Horizon incident in 2010, ~1.8 million gallons of Corexit® dispersants were approved for use directly onto the released oil. Callinectes sapidus (Blue Crab) megalopae are pelagic and, therefore, likely to be one of the first organisms exposed to spilled oil and applied dispersants in open-ocean and nearshore waters. In this study, we examined acute toxicity of Corexit 9500, Corexit 9527, and MicroBlaze® (a microbial surfactant) alone and in combination with crude oil. We adapted methods from the established 48-h copepod toxicological assay and exposed Blue Crab megalopae for 48 h to varying dosages of each treatment. Oil treated with dispersant was more toxic than either oil or dispersant alone (48-h LC50 = 29.8 mg/L vs. 55.9 mg/L and 37.5–59.1 mg/L, respectively), and MicroBlaze was essentially non-toxic (48-h LC50: 7643 mg/L). Corexit 9527 was more toxic than Corexit 9500 both in solutions with oil and alone (48-h LC50 = 37.5 mg/L vs. 51.8 mg/L and 59.1 mg/L, respectively). Exposure to these toxicants not only induced mortality at certain dosage levels, but life-stage transitioning also seemed to be effected. The decreased ability to metamorphose, however, was not affected in a typical gradient manner, as with mortality; those that were exposed to a toxicant, overall, exhibited a decreased occurrence of metamorphosis (37% average decrease). This study provides essential baseline data needed for further investigations to determine optimal dosing of dispersants and balancing of dispersant use and dosage with anticipated crab-fishery impac ts. Introduction During the Deep Water Horizon incident in 2010 more than 2.1 million gallons of Corexit® dispersants were approved for use directly on the released oil; 1.4 million gallons were applied at the surface and 0.77 million gallons were applied at the wellhead (Kujawinski et al. 2011). The physical effects of this treatment were substantial (Bik et al. 2012, Dubansky et al. 2013, Zuijdgeest and Huettel 2012). However, the toxicity of oil and/or dispersants and bioremediation treatments, and the natural dilution and degradation processes of these substances are not well known. The Callinectes sapidus M.J. Rathbun (Blue Crab) life cycle begins with a “sponge” female, a name referring to the egg mass carried by a gravid female crab leaving the estuary or bay for oceanic waters. She releases her eggs in the open ocean, producing 750,000 to 8 million zoeae, the first larval stage. The planktonic zoeae remain in the oceanic waters until they transform into megalopae, the second larval stage. The megalopae often attach to floating debris and are tidally 1Center for Coastal Studies at Texas A&M University-Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX. 2National Oil-Spill Response School, 6300 Ocean Drive, Corpus Christi, TX. *Corresponding author - RachelRFern@gmail.com. Manuscript Editor: Joe Griffitt Southeastern Naturalist G83 R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 transported back into estuarine nursery areas where the final transformation into adulthood occurs (Churchill 1919). Blue Crab larvae are ideal test organisms to determine toxicities of oil, dispersants, and/or other bioremediation treatment combinations because the pelagic megalopae are generally planktonic and drift within the top 2 m of offshore waters (Shanks 1985, Sulkin and Van Heukelem 1982). Larval life-stages of crustaceans are often the organisms most sensitive to contaminants (Ahsanullah and Arnott 1978, Conner 1972); larvae of Crangon crangon L. (Sand Shrimp ), Carcinus maenus L. (Green Crab), and Homarus gammerus L. (European Lobster) were 1 to 3 orders of magnitude more sensitive to mercury, copper, and zinc than adults. Similarly, Ahsanhullah and Arnott (1978) found a 9-fold increase in sensitivity to copper, cadmium, and zinc for larval Paragrapsus quadridentatus (H. Milne Edwards) (Four-toothed Shore Crab) relative to adults. Early life-stages of Blue Crab are likely to be some of the first aquatic organisms exposed to oil spills in US waters, and the species’ commercial significance makes determining toxicity effects of oil and dispersants both ecologically and economically important. Average US landings of hard-shell Blue Crabs were 1.6 million pounds during 2006–2010, and the fishery was valued at more than $180 million in 2011 (NMFS 2012). Oil dispersants are mixtures of surfactants and hydrocarbon-based solvents that are used in response to oil spills to break oil into smaller, more dilutable molecular clusters (Kujawinski et al. 2011). Dispersants reduce the oil’s buoyancy, keeping it suspended in the water column and preventing oil slicks on the surface (George-Ares and Clark 2000). Due to the increase in surface-to-volume ratio, these smaller oil particles are more easily degraded and diluted by microbial communities, UV rays, and wave action (Atlas 1981, Brakstad 2008, NRC 2005). Two dispersants were used extensively during the Deepwater Horizon oil spill—Corexit 9527 and Corexit 9500. Corexit 9527 was only applied to the surface of the spill aerially and by small vessels depending on weather and oil conditions, which likely resulted in spatially and temporally heterogeneous surface-water concentrations (Kujawinski et al. 2011). Corexit 9500 was applied both at the surface of the spill and by a jet placed into the flow of oil and gas emerging from the 150-mdeep wellhead. However, due to wellhead-operation variability, jet application was inconsistent, the flow of Corexit 9500 was not constant, and the jet was not always inserted into the oil and gas stream. It was presumed that when the jet was properly inserted into the oil and gas flow and Corexit 9500 was being applied, the dispersant was mixed evenly with the oil as it rose up the water column to the surface (Kujawinski et al. 2011). When used appropriately, dispersants can help mitigate coastal impacts in the early stages of an oil spill (NRC 2005), particularly to habitats such as Rhizphora spp. (mangroves) and salt marshes (Duke et al. 2000, Getter et al. 1985). Dispersants alone may not be very toxic, but when added to oil, their toxicity and/or the toxicity of the oil is compounded (NRC 1989). These chemicals cause large increases in the number of oil particles dissolved and suspended in the water column, thus greatly increasing exposure of aquatic organisms (George-Ares and Southeastern Naturalist R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 G84 Clark 2000). Severe effects of dispersants on wildlife and microbial communities have been documented, particularly in coastal ecosystems (Couillard et al. 2005, Lindstrom and Braddock 2002, UnHyuk and GiMyeong 2009). Peak exposure of aquatic organisms likely occurs immediately following application of dispersants and wanes as the dissolved oil particles are diluted. However, repeated or heavy use of dispersants may damage populations of some pelagic organisms (Couillard et al. 2005, Perkins et al. 1973, Wells 1984). In our study, we determined the toxicity on Blue Crab megalopae of oil, dispersants, and a non-dispersant bioremediation treatment alone and in combination, and herein report our observations of the effects of the various treatments on subsequent metamorphosis. Methods Organism collection and lab structure On several occasions in 2012, we used a 500-mm plankton tow to collect Blue Crab megalopae directly off the ship channel in Port Aransas, TX. We used all organisms for experimental trials within 12 h of collection. We based our toxicological methods on the copepod 48-h toxicity assay (Gorbi et al. 1909). We aerated the seawater used in the trials for 3 h prior to use, manipulated the salinity with deionized water to match the salinity of the water from which we collected the organisms (usually 30–35 PSU), and vacuum-filtered it through a 48-mm filter to remove organisms and debris from experimental solutions to ensure homogenous exposure-conditions. In the 48-h copepod toxicity assay, 4–5 copepods were placed in each 25-mL vial; however, megalopae are larger than copepods, so we placed only 1 organism in each vial. Before beginning experimental treatments, we determined megalopae survival under test conditions to ensure oxygen and food availability would not affect exposure reactions of the megalopae. All megalopae metamorphosed within 48 hours in the vials and survived for 7 days, more than 3 times the time period for experimental trials. In experimental and control trials, we isolated the megalopae, introduced 1 into each 25-mL glass vial with 25-mL of treatment solution for a 48-h exposure period. Experimental design For treatments to test dispersant toxicity alone, each trial consisted of 15 individual megalopae tested at each of 6 concentrations: 0, 20, 50, 80, 110, and 140 mg/L of dispersant. For treatments testing toxicity of oil alone or in an oil and dispersant combination, trials consisted of 15 individual megalopae tested at each of 6 concentrations: 0, 15, 37.5, 60, 82.5, and 105 mg/L. Treatments testing MicroBlaze® (a microbial surfacant) also consisted of 15 individual megalopae tested at each of 6 concentrations: 0, 2000, 4000, 6000, 8000, and10000 mg/L. We performed preliminary trials for each treatment across a wide range of concentrations to determine appropriate concentration intervals for experimental trials. We performed a total of 4 trials for each treatment with a sample size at each concentration of 60 megalopae. Within each trial, we examined all concentration exposures simultaneously (including the control, or 0 mg/L concentration) Southeastern Naturalist G85 R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 in order to ensure homogenous testing conditions. Lab conditions remained constant throughout the 48-h testing periods—72 °F and fluorescent lighting remained on throughout the period. In this study, we used Bonny light-crude oil because it was easily accessible and has an American Petroluem Instutute gravity (API) similar to that of South Louisiana sweet-crude oil (33.4° and 35.9°, respectively), the oil that was spilled in the 2010 Macondo blowout. Bonny light-crude sulfur content (0.16%) is lower than that of South Louisiana sweet crude (0.33%); thus, the former is less corrosive and less environmentally threatening than the latter, making the results of this study conservative with regards to their application to the Gulf of Mexico. We dispersed crude oil with Corexit at the recommended 50:1 ratio. We placed all solutions on magnetic stir plates, using glass magnetic bullets, and mixed until we observed dissolution of the oil. MicroBlaze is packaged in concentrated form. We made a 6% MicroBlaze solution in seawater and mixed experimental concentrations using this stock solution. We chose a 48-h test period to mimic a realistic oil-spill exposure because water currents and turbulence generally dilute dispersed oil shortly after the dispersants are applied. At the termination of each experimental exposure period, we placed each surviving organism into a new vial containing clean seawater, and monitored it for an additional 48 hours to determine delayed mortality or delayed life-stage transitioning. Reference toxicant and data analysis Ours is the first study to establish toxicity of these dispersants and dispersed crude oil on Blue Crab megalopae; thus we used a reference toxicant, sodium dodecyl sulfate (SDS) to validate methodology and calculate 48-h LC50 values. Our megalopae SDS-exposure protocol was identical to that of the experimental trials. This surfactant has been employed in other studies as a reference toxicant and it has exhibited low variability and high reproducibility (Whiting et al. 1996). Thus, using SDS as the reference toxicant in this study helped ensure accuracy of our calculated 48-h LC50 values and validate our methodology. We recorded megalopae mortality and life-stage at the end of the exposure and post-exposure periods across all treatments. We present our data in a dose-response curve in order to illustrate relative toxicity of all combinations across increasing dosages. We made initial examinations of the data using a 2-factor design and evaluated time x treatment interactions. We conducted our analyses with SAS for Windows V9. Specifically, we used procedures PROBIT and ANOVA to establish 48-h LC50 values and determine within- and between-group differences, and Tukey’s HSD to test for significant differences between groups. In all cases, we set P = 0.05 to indicate significance. Results We were able to conduct a 1-way analysis of data because there was no significant time interaction. All treatments had significant differences among treatment levels (Table 1). Southeastern Naturalist R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 G86 Sodium dodecyl sulfate We used PROBIT analysis to examine mortality of megalopae on exposure to SDS to establish a standard 48-h LC50 value. Exposure to SDS resulted in a consistent mortality gradient across dosage levels, and we derived from the data a 48-h LC50 SDS value of 5.6 mg/L. This result is lower than the published SDS 48-h LC50 value of 9.8 mg/L for Blue Crab larvae (Whiting et al. 1996). However, the previously published value was calculated using newly hatched larvae (zoeae), which may have different physiological requirements. This finding implies that the megalopae life stage may be more sensitive to toxicants than other stages, most likely due to the increased physiological demands of metamorphosis that took place within the vials during exposure (Jacobi and Anger 1985, Mangum et al. 1985). Corexit 9500, 9527, and dispersed oil We monitored organisms exposed to Corexit 9500- and Corexit 9500-dispersed crude oil for acute and delayed mortality (mortality in the 48-h post-exposure period) and observed none. All control organisms survived and metamorphosed in the vials during the first 48-hour period. Oil dispersed with Corexit 9527 was significantly more toxic than any other solution or combination of crude oil and dispersant (Table 1). The dispersed oil was more toxic than the dispersant alone. The 48-h LC50 values for Corexit 9500 alone and Corexit 9500-dispersed oil were 59.1 mg/L and 51.8 mg/L, respectively. Although oil dispersed with Corexit 9500 showed a slight tendency to be more toxic than oil or the dispersant alone, the differences were not statistically significant. The 48-h LC50 values for Corexit 9527 and Corexit 9527-dispersed oil were 37.5 mg/L and 29.8 mg/L, respectively, which is significantly lower than the Corexit 9500 or Corexit 9500-dispersed oil (Table 1). All megalopae in the control group survived and metamorphosed in the vials during the 48-h experimental period. We did not observe delayed mortality in either the control or experimental treatments; organisms that survived the initial 48-h exposure period ultimately survived the experiment in its entirety. Crude oil and MicroBlaze In order to ensure exposure of the megalopae to oil, trials involving crude oil alone required the use of a dispersant to adequately suspend the crude oil in a seawater solution. We used low concentrations of SDS to disperse the oil into water Table 1. 48-hr LC50 values and ANOVA results comparing dosing level for each treatment compound. 48-hr LC50 Concentration mg/L Treatment mg/L F value P 140 110 80 50 20 0 C9527 + oil 29.8 263.1 less than 0.0001 - - - - - - C9500 59.1 408.0 less than 0.0001 - - - - - - C9527 37.5 772.4 less than 0.0001 - - - - - - C9500 + oil 51.8 414.4 less than 0.0001 105 83 60 38 15 0 MicroBlaze 7643.0 1065.4 less than 0.0001 10,000 8000 6000 4000 2000 0 Oil 55.9 - - 100 50 25 13 6 0 Southeastern Naturalist G87 R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 (SDS concentration less than 0.002 mg/L), substantially lower than the SDS no-observedeffect (NOEC) value of 0.59 mg/L (Whiting et al. 1996). We included an SDS plus seawater control at this concentration to ensure no toxicity at this dose. The 48-h LC50 value for crude oil was 55.9 mg/L (Table 1). MicroBlaze was essentially non-toxic—48-h LC50 = 7643 mg/L (Table 1). We attempted trials with MicroBlaze and oil combinations, but they resulted in abnormal dissolution behavior of the crude oil in seawater. When we used MicroBlaze as the sole dispersant in the solution, crude oil formed compact beads that did not dissolute until after 48 h. If we had used this solution that had already been exposed to the microbial surfactant for 48 h prior to the start of the experimental exposure, it would have yielded biased results because the added time allowed for oxygen and microbe-aided degradation of the oil. Thus, for the purposes of this study, which aimed to establish baseline acute toxicity of these chemicals, MicroBlaze-dispersed oil did not produce comparable or relevant results. However, the effects of this surfactant on crude oil and (other) dispersant solutions as a bioremediation agent are currently being investigated. Life-stage transitioning Exposure to these toxicants (crude oil and dispersants) not only induced mortality at certain dosage levels, but life-stage transitioning also seemed to be affected. The decreased ability to metamorphose, however, was not displayed in a typical gradient manner, as was mortality; rather, megalopae that were exposed to the toxicant exhibited a decreased likelihood of metamorphosis (Table 2). Transformation rates of organisms deceased at the end of the 48-h exposure period differed from those of organisms that survived the exposure. High transformation-ratios (alive:deceased) indicated more acute toxic effects of the solution on the organism. For instance, toxic effects of crude oil alone likely occurred sooner in the exposure period than those of the Corexit 9500 alone since a great proportion of those in the latter treatment transformed during exposure. Our analyses did not account for control organisms because all megalopae in the control treatments survived and metamorphosed within the initial 48-h exposure period. Thus, our null hypothesis was that all experimental megalopae would also Table 2. Metamorphosis frequencies across treatments of organisms that survived or died during the exposure period. Alive = Organisms that both metamorphosed and survived exposure, Dead = Organisms that had metamorphosed but died during the exposure period. Total = Cumulative proportion of organisms that transitioned, dead and alive, during the exposure period.. % Transitioned Treatment Alive Dead Total C9500 90.6 100.0 82.3 Oil/C9500 81.3 88.0 77.5 C9527 86.7 95.0 88.0 Oil/C9527 5.3 20.0 1.7 Oil 32.0 46.0 14.0 MicroBlaze 81.3 78.0 93.0 Southeastern Naturalist R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 G88 transform; this was not the case (Table 2). Megalopae exposed to oil dispersed with Corexit 9527 had the lowest transformation rate, especially among those organisms that survived the 48-h exposure period. Discussion Results from the trials involving dispersants (Corexit 9500 and Corexit 9527) and dispersed oil revealed higher toxicity of the dispersed oil relative to the dispersant alone. Dispersants allow easier uptake and ingestion of the crude oil via breakdown and dissolution of the oil into the water column (George-Ares and Clark 2000, Ramachandran et al. 2004). Dispersed oil also had a larger impact on life-stage transitioning than did the crude oil or dispersant alone. Both Corexit 9500 and 9527 were used during the 2010 Deepwater Horizon incident; Corexit 9500 was applied at both the wellhead and the surface, and Corexit 9527 was applied the surface only (Kujawinski et al. 2011). Corexit 9500 did not increase the toxicity of the oil as much as did Corexit 9527 (48-h LC50 51.83 mg/L vs 29.83 mg/L) and it did not impact life-stage transitioning as severely (77.5% vs 1.7% transformed). The 9500 formula was also less toxic alone than the 9527 formula (48-h LC50 = 59.09 mg/L vs 37.46 mg/L). Our results suggest that toxicity effects on Blue Crab larval populations might be reduced if Corexit 9500 rather than over Corexit 9527 is used as a dispersant for future oil releases in marine environments. However, other studies have found increased toxicity of crude oil treated with Corexit 9500 when compared to Corexit 9527 in Hydra viridissima Pallas (Green Hydra) and similar toxicity of 9500 and 9527 in Haliotis rufescens Swainson (Red Abalone) and Mysid holmesimysis Holmes (Kelp Forest Mysid) (Mitchell and Holdway 2000, Singer et al. 1996). Acute aquatic toxicity of selected dispersants must be considered in conjunction with targeted species, environmental conditions, and application concentration and treatment location because toxicity varies widely according to species, larval stage, and treatment methodology (George-Ares and Clark 2000) Life-stage transitioning Trials examining toxicity of dispersed oil can provide further insight by incorporating life-stage transitioning responses in the larvae. High transformation ratios (alive:deceased) indicate more-acute toxic effects of the solution on the organism. For instance, in trials involving of Corexit 9527-dispersed oil, only 1.7% of larvae metamorphosed during exposure (Table 2). More specifically, 20% of organisms that did not survive the exposure transformed before dying and only 5.3% of organisms that survived achieved metamorphosis. All larvae in control treatments transformed during the 48-h period. The treatment effects on life-stage transitioning and mortality occurrence before or after metamorphosis are outlined in Table 2. As previously mentioned, these were not dose-response effects. Rather, the effects suggest that exposure to any toxicant (however slight the dosage) influenced transformation of the megalopae in experimental trials. Southeastern Naturalist G89 R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 The megalopae that survived and did not metamorphose during the initial 48-h exposure period eventually transformed during the 48-h post-exposure period. While these organisms can delay transformation in light of unfavorable environmental conditions, they cannot delay it indefinitely (Forward et al. 1996). Metamorphosis delay is physiologically costly and may manifest in certain sublethal effects in later life stages, although further studies are needed to determine these long-term effects in Blue Crabs (Gebauer et al. 1999). Also, fewer organisms that died had metamorphosed during the 48-h exposure period than did the organisms that survived. This result implies that Blue Crabs may be most sensitive to the toxin during the actual metamorphosis. Additional investigations into the physiological effects of these dispersants and dispersed oil are needed. Although it is used often in smaller-scale terrestrial spills, MicroBlaze had not yet been tested in a laboratory setting so neither its performance as a dispersant, nor its relative toxicity to aquatic organisms was established prior to this study. Our results suggest that while it does not appear to be a viable option for immediate crude-oil dispersal, hydrocarbon-degradation effects occurring after 48 h of exposure may make this product a potential bioremediation additive for future oil-spill protocols; investigations into its utility are ongoing. Despite a slight depression of metamorphosis, this microbial surfactant also has the advantage of being essentially non-toxic. Adaptable methods We developed our method of assessing acute toxicity of a contaminant from a standard copepod 48–96-h LC50 assay. The success of the control-lifespan trials in the vials and the consistent mortality gradients of the reference toxicant and experimental doses indicate that this method is well suited for determining acute toxicity in megalopae. Our results also suggest that this method may be further adapted and used for toxicity assays of other larval or planktonic organisms. The SDS reference toxicant 48-h LC50 value found in this study (5.6 mg/L) differs from the published value (9.8 mg/L) for Blue Crab megalopae. As previously mentioned, the Whiting et al. (1996) study used larvae in an earlier life stage that may have different physiological requirements. The megalopae used in this study underwent an important and physiologically demanding transition in which the first carapace is developed and the telson undergoes fusion. The increase in physiological demands during metamorphosis may have caused the increase in sensitivity to the toxin. Additionally, the Whiting et al. (1996) study grouped the larvae together and exposed the organisms to the toxicant in open, 250-mL glass containers. In our experiments, we isolated each larva and exposed each organism in a separate, sealed 25-mL vial. The lack of dilution, evaporation, and interaction of other larvae with the toxicant may also have contributed to the higher toxicity. The lower value of 5.9 mg/L was consistent across all reference toxicant trials (S = 0.62). Study relevance and limitations The purposes of this study were to determine acute toxicity of dispersants commonly used in oil-spill response protocols, evaluate the potential of megalopae as Southeastern Naturalist R. Fern, K. Withers, P. Zimba, T. Wood, and L. Schoech 2015 Vol. 14, No. 4 G90 a model laboratory organism in toxicological studies, and establish any acute sublethal effects of these toxicants on Blue Crab megalopae, especially on life-stage metamorphosis. We did not address the physiological mechanism by which toxicity occurs. Future studies that describe those mechanisms could provide even further insight into the toxicological effects of oil and dispersants in the natural environment. Conclusions drawn from our data should only be applied to laboratory settings in which environmental variables are kept constant; toxicity of these chemicals can be affected by temperature and salinity (NRC 1989). After establishing the basic 48-h LC50 values for the dispersants and dispersed oil, experiments like this study should be performed under various temperature and salinity conditions to further understand how these dispersants interact with oil and aquatic organisms in natural settings. Additionally, in a natural landscape, water turbulence, UV-ray exposure and currents would all influence the exposure of the toxicants to organisms in the water column (Garrett et al 1998, Pace et al 1995). Concentrations of and exposure periods to dispersants, crude oil, and dispersed oil found in natural settings should be determined via water sampling or a larger scale mesocosm study capable of mimicking open-ocean forces on water-column chemistry. This study establishes baseline 48-h LC50 values for each of these dispersants alone and in combinations with crude oil that would facilitate future investigation into real-world exposure of these animals in various oil-spill scenarios. Acknowledgments We acknowledge the Center for Coastal Studies at Texas A&M University-Corpus Christi for the use of their laboratory space and equipment. 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