Chemosphere 120 (2015) 131–137
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Acute aquatic toxicity studies of Gulf of Mexico water samples collected following the Deepwater Horizon incident (May 12, 2010 to December 11, 2010) B.S. Echols ⇑, A.J. Smith, P.R. Gardinali, G.M. Rand Southeast Environmental Research Center, Florida International University, North Miami, FL 33181, United States
h i g h l i g h t s Water samples were taken at 44 sites following the Deepwater Horizon incident. Water accommodated fractions (WAFs) and oil-in-water dispersions (OWDs) were tested. Mysids were generally more sensitive than inland silversides. Most mortality observed occurred in samples collected before the well was capped.
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Article history: Received 5 February 2014 Received in revised form 9 May 2014 Accepted 14 June 2014
Handling Editor: S. Jobling Keywords: Oil spill Acute toxicity Monitoring Gulf of Mexico Deepwater Horizon
a b s t r a c t The potential for the Deepwater Horizon MC-252 oil incident to affect ecosystems in the Gulf of Mexico (GOM) was evaluated using Americamysis bahia, Menidia beryllina and Vibrio fischeri (MicrotoxÒ assay). Organisms were exposed to GOM water samples collected in May–December 2010. Samples were collected where oil was visibly present on the water surface or the presence of hydrocarbons at depth was indicated by fluorescence data or reduced dissolved oxygen. Toxicity tests were conducted using water-accommodated fractions (WAFs), and oil-in-water dispersions (OWDs). Water samples collected from May to June 2010 were used for screening tests, with OWD samples slightly more acutely toxic than WAFs. Water samples collected in July through December 2010 were subjected to definitive acute testing with both species. In A. bahia tests, total PAH concentrations for OWD exposures ranged from non-detect to 23.0 lg L 1, while WAF exposures ranged from non-detect to 1.88 lg L 1. Mortality was >20% in five OWD exposures with A. bahia and three of the WAF definitive tests. Total PAH concentrations were lower for M. beryllina tests, ranging from non-detect to 0.64 lg L 1 and non-detect to 0.17 lg L 1 for OWD and WAF exposures, respectively. Only tests from two water samples in both the WAFs and OWDs exhibited >20% mortality to M. beryllina. MicrotoxÒ assays showed stimulatory and inhibitory responses with no relationship with PAH exposure concentrations. Most mortality in A. bahia and M. beryllina occurred in water samples collected before the well was capped in July 2010 with a clear decline in mortality associated with a decline in total PAH water concentrations. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Abbreviations: DWH, Deepwater Horizon; GOM, Gulf of Mexico; BTEX, Benzene, toluene, ethylbenzene, and xylenes; COC, chain of custody; GC/MS-SIM, gas chromatograph/mass spectrometry-selected ion monitoring; IC50, median concentration inhibiting 50% of a sample population; MC-252, Mississippi Canyon Block 252; NOAA, National Oceanic and Atmospheric Administration; NRDA, Natural Resource Damage Assessment; PAH, polycyclic aromatic hydrocarbons; QA/QC, quality assurance/quality control; ROV, remotely operated vehicle; USEPA, U.S. Environmental Protection Agency; WET, whole effluent toxicity. ⇑ Corresponding author. Address: 3000 NE 151st St., North Miami, FL 33181, United States. Tel.: +1 (540) 808 5395. E-mail address: [email protected] (B.S. Echols). http://dx.doi.org/10.1016/j.chemosphere.2014.06.048 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
Crude oil was released into the Gulf of Mexico (GOM) from a depth of approximately 5,000 feet in the Mississippi Canyon 252 (MC-252) lease block (28° 44.20´N, 88°22.23´W) for 87 days, starting on April 20, 2010, until the well was successfully capped on July 15, 2010 (OSAT, 2010). Multiple ecosystems in the GOM may have been exposed and the extent to which each ecosystem may be impacted depends on a host of physical, chemical, and biological factors as well as the inherent changes that can occur in the crude oil as a result of dissolution, weathering, degradation and the
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addition of dispersant. Therefore, during and after the discharge period, different types and stages of oil (e.g., weathered oil, dispersed weathered oil) present in water and/or sediment may have potentially posed a hazard to aquatic life in the GOM. A crucial first step toward understanding potential crude oil impacts to biota is to quantitatively determine the potential aquatic toxicity of MC252 oil and how the toxicity may be altered as a result of the changes in crude oil characteristics and composition (Neff, 1999). Aquatic toxicity tests are a useful assessment tool that can be used to estimate the presence and the intensity of an effect(s), as a result of chemical exposures in water and/or sediment (Neff, 1999; Fingas, 2011). The potential for the DWH MC252 oil incident to affect different marine ecosystems in the GOM is presently being evaluated in an ongoing aquatic toxicity program to characterize the potential effects that might result from exposure to fresh- and weatheredMC-252 crude oil (Rand et al., 2011, 2012). As part of this program, an initial step was to apply the USEPA whole effluent toxicity testing (WET) approach for the protection of aquatic life (USEPA, 1991), to evaluate the toxicity of field-collected water samples from the GOM, during release of oil and after the well was capped. WET testing is a useful initial step for assessing effects because it employs the use of standard procedures and standard marine test organisms and it evaluates potential acute and chronic responses as a result of exposures to an aggregate field-collected sample (USEPA, 2002). This study presents laboratory results from part of this program, in which standard acute WET toxicity tests were conducted with mysid shrimp (Americamysis bahia) and silverside minnow (Menidia beryllina) exposed up to 96 h to field-collected water samples from the GOM prior to and after the MC-252 well was capped along with the MicrotoxÒ bacterial assays. Exposure media used field-collected water samples prepared as a variable dilution of both water-accommodated fractions (WAFs), where samples were stirred and the aqueous phase collected for exposure, and oil-in-water dispersions (OWDs), where the whole sample was used. Although WAF preparations may not be typical of field-collected conditions they do contain the dissolved fraction of hydrocarbons which are more bioavailable and toxic (Neff and Anderson, 1981; Pruell et al., 1986). OWDs more accurately simulate field conditions, but they are multiphase solutions complicating chemical analyses and replication (Anderson et al., 1974; Neff, 1999). WAF solution exposure media are typically preferred over OWD exposures in aquatic toxicity testing because they produce more replicable conditions. For testing in this study, the combination of both exposure media were used to determine potential differential toxicity between samples (in WAFs) containing dissolved oil constituents and samples (OWDs) containing both dissolved and insoluble (e.g., droplets) constituents. Mortality was the biological endpoint used for both single species acute toxicity tests and the median concentration value that inhibited light production in 50% of the test population (IC50) of bacteria was used for the MicrotoxÒ assay. Total PAH concentrations were measured for all WAFs and OWDs. There is much criticism and serious debate on the use and uncertainties of the standard descriptive summary statistics (LC50, EC50, NOECs, etc.) using both hypothesis testing and regression-based approaches, which form the basis of almost every effects assessment (Warne and Van Dam, 2008; Landis and Chapman, 2011; Jaeger, 2012). Therefore, greater than 20% mortality for an exposure treatment in an acute test with the standard test species was the measurement endpoint considered environmentally relevant in this study and is supported by regulatory precedent for aqueous effluent discharges under the National Pollutant Discharge Elimination System (NPDES) for protection of aquatic life under the Clean Water Act (Suter et al., 1995). The investigators understand that the latter
does not apply when considering endangered, threatened and keystone species. 2. Materials and methods 2.1. Study area Water samples were collected for aquatic toxicity testing from May 12 to December 11, 2010, within 2 km of the MC-252 wellhead as well as far-field (6250 km). Details of sampling locations are included in Fig. 1. 2.2. Sample collection Sampling transects were selected based on the extent of surface oil identified in aerial and satellite imagery and from water column profiling data obtained from response vessels. At each sampling location, real-time, in situ UV fluorescence and dissolved oxygen (DO) were measured using sensors packaged with a conductivitytemperature-depth profiler. These parameters, as well as visual observations of oil, were used to guide decisions regarding the depths at which water samples were collected. Fluorescence and DO anomalies have been used in previous work to assess the presence of hydrocarbons in the water column or the consumption of oxygen by increased microbial activity (Hazen et al., 2010). If a fluorescence anomaly and/or a distinct decrease in DO were observed relative to background, or if oil was visible on the water surface, then whole water samples were collected for laboratory toxicity testing. Water samples were collected at multiple depths using a conventional hydrowire with 5-L, 10-L, or 20-L GO-FLO sampling bottles on a rosette multi-bottle array or attached to an ROV-tethered management system. Standard decontamination procedures were used between sample collection events. Surface water samples were collected via bucket grab or peristaltic pump. Samples were aliquoted from the surface grabs or GO-FLO into 1-L, 2.5-L, or 3.8-L certified clean amber glass containers with PTFE-on lids. Minimal headspace was maintained in each bottle. Water samples were stored in the dark at 4 °C until prepared for shipment. Samples were received at FIU within seven days of collection. All original samples were then stored in the dark in a refrigerated unit at 4 °C until test preparation. 2.3. Test species Three species were selected for toxicity testing and include the mysid shrimp (A. bahia), the inland silverside (M. beryllina) and the light producing bacteria used in MicrotoxÒ assays, V. fisheri. Mysid shrimp and inland silversides were selected as test species as they are recommended marine test species by the USEPA (USEPA, 2002), naturally inhabit coastal waters in the Gulf of Mexico; and both species can tolerate higher salinities (15–30 ppt), similar to ranges common in the GOM which should preclude effects of changes in salinity alone. The MicrotoxÒ assay was selected because it was being used in the field as part of the analysis onboard research vessels deployed during the Response efforts during the DWH incident. Mysids and inland silversides were purchased from a commercial supplier and upon delivery animals were acclimated in a temperature-controlled incubator for at least 48 h prior to toxicity testing. Organisms were acclimated to the appropriate test salinity, matching that of field-collected samples, with gradual additions of laboratory saltwater. Laboratory saltwater was obtained from a natural saltwater well that was air-sparged, and passed through a particle filter, carbon filtration and UV sterilized. This water
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Fig. 1. Locations of screening and definitive level toxicity samples collected from 5/12/2010 to 12/11/2010.
ranged from 29–32 ppt and was used as dilution water for all definitive tests. During holding, organisms were fed daily with 0.99 for all analytes was used to demonstrate the linear range of the detector. Analyte concentrations were calculated based on surrogate standards naphthalene-d8, phenanthrene-d10, chrysene-d12, acenapthene-d10, and perylene-d12. Surrogate standard recoveries were calculated based on internal standards benzo(a)pyrene-d12 and fluorene-d10. Alkylated homologues were quantitated based on the response factors of the parent compounds. For simplicity and to provide the reader with a reference point for the toxicological assessment interpretation, analytical chemistry results are summarized as total PAH concentration in the water samples expressed in parts per billion (lg L 1). 2.7. Quality assurance/quality control All of the biological and chemistry data presented in this paper were subjected to formal data verification and validation prior to use. All laboratory data were reviewed by the laboratory’s internal Quality Assurance Unit and external (independent) auditors to
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assure that each study was performed in accordance with the protocol and laboratory standard operating procedures (SOPs). Sample processing activities, analytical procedures, and storage and holding times for the chemical analyses were generally consistent with the Analytical Quality Assurance Plan. Mississippi Canyon 252 (Deepwater Horizon) Natural Resource Damage Assessment. Version 2.2 (NOAA, 2010). Batch QA/QC was used to assess method precision and accuracy by analyzing laboratory blank(s), fortified blank(s), sample duplicates (when available), and standard reference materials. Both the ecotoxicology and analytical chemistry laboratories at Florida International University are NELACaccredited. 3. Results and discussion Water was collected from 44 sites in the GOM between May 12 and December 11, 2010 at multiple depths within 9 km of the well head and at far-field distances (6250 km) from the wellhead (Fig. 1). The first 14 water samples underwent acute toxicity tests with 100% WAF and 100% OWD (screening tests). The remaining 30 water samples underwent acute toxicity tests using a variable dilution series (definitive tests). Screening and definitive single species acute toxicity tests were conducted with mysids (96 h) and, silversides (96 h) along with the MicrotoxÒ bacterial assay. Control mortality throughout all testing was 0% for A. bahia and less than 5% for M. beryllina and thus well within standard guideline requirements. During testing, salinities were slightly higher (5–30 ppt ± 10%) than the recommended values in the USEPA guidelines due to the use of field-collected water samples. In general, water quality parameters remained within acceptable ranges (USEPA, 2002). Control salinities were within recommended guidelines and were not adjusted to salinities of field-collected samples. The pH measurements were also within standard ranges and no fluctuations were observed during the duration of any toxicity test. Although DO levels fell below 4 mg L 1 in some of the treatments by test termination, test chambers were not aerated to eliminate any potential effects on PAH concentrations in the exposure media. Dissolved oxygen did not appear to contribute to toxicity in those treatments with low DO because toxicity occurred more rapidly declining DO. Note that collection of the 44 water samples in the GOM were based on where oil was visibly present on the water surface or where the presence of hydrocarbons at depth was indicated by fluorescence data or was suggested by reduced dissolved DO concentrations. 3.1. Single-species screening toxicity tests The results presented in Fig. 2 show the concentrations of total PAHs for the full-strength (100% sample) OWD and WAF exposure media prepared at test initiation in relation to their collection date in the GOM. As noted by the different magnitudes of the concentration scales total PAH concentrations in OWD samples were generally higher than WAFs. The maximum total PAH concentrations for OWD samples was 18 175 lg L 1 while WAF samples reached total PAH concentrations of up to 895 lg L 1. Most of the samples collected during the earlier stages of the response were restricted to the top of the water column (61 m depth) and are likely representative of ‘‘worst case’’ surface conditions (e.g. within or directly under an oil slick). Samples collected May 12 to June 16, 2010 for screening toxicity tests contained visible floating oil while those collected later in the year were identified by the fluorescence anomaly or the DO sag rather than by visual evidence of oil, sheen or oil droplets. Results also indicate that samples collected at or below 1000 m contained lower maximum total PAH concentrations at 0.170 lg L 1 and 0.076 lg L 1 for
Fig. 2. Total PAH concentrations (lg L 1) measured in OWDs and WAFs prepared from field-collected samples in relationship to the field collection date. Grey symbols represent samples where no PAHs were detected.
the OWDs and WAFs, respectively. The levels of total PAHs in the sample seems related to the amount of visible oil rather than the collection location. OWD preparations were slightly more acutely toxic than WAFs for both A. bahia and M. beryllina. Mysids were generally more sensitive than M. beryllina. Ten of the 14 water samples resulted in >20% mortality for A. bahia, following the 96 h static-renewal exposures to both undiluted WAF and OWD preparations. In tests with M. beryllina, eight of the 14 water samples caused >20% mortality following the 96 h static-renewal exposures to both undiluted WAF and OWD preparations. WAFs were slightly less toxic than OWDs for both species and A. bahia was more sensitive than M. beryllina. The maximum total PAH concentration for mysid OWD tests associated with 620% mortality was 59 lg L 1, while the maximum concentration in tests with >20% mortality was 2962 lg L 1. For A. bahia WAF tests, total PAH concentrations ranged from 0.02 to 15 lg L 1 in exposures with 620% mortality. In exposures with >20% mortality, up to 895 lg L 1 total PAH was observed. In silverside OWD tests, the maximum total PAH concentration associated with 620% mortality was 0.11 lg L 1, while the highest concentration observed in samples with >20% mortality was 11 175 lg L 1. For WAF tests using M. beryllina, 620% mortality was observed at total PAH concentrations up to 64 lg L 1. In samples with >20% mortality, the maximum total PAH concentration observed was 259 lg L 1. 3.2. Single-species definitive toxicity tests A variable dilution method was used to prepare both OWD and WAF exposure test media of 6.25–100% of the original sample. Use of variable dilution is the preferred method to prepare treatment exposure media for oil since it creates reproducible conditions and concentrations of PAHs in exposure test media compared to
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Table 2 Total PAH concentrations (lg L
1
) using a variable dilution series of a 100% sample.
Dilution (%)
Measured WAF concentration (lg L 1 TPAH)
Measured OWD concentration (lg L 1 TPAH)
100 50 25 12.5 6.25
45.61 19.34 10.68 4.64 2.60
1124.02 437.91 212.69 64.33 26.93
serial dilution (Barron and Ka’aihue, 2003; Neff, 1999). In support of this method dilutions of a single field-collected sample (GOM Field Sample JF2-2 km-surf-TOX-20100524-E125) were prepared in Table 2. Results indicate the use of this method as concentrations of WAF TPAH (lg L 1) were within 25% of expected nominal concentrations (Table 2). Thirty (collected July 10 through December 11, 2010) of the 44 GOM collected water samples were subjected to definitive acute toxicity testing with both species. Samples collected after June 16, 2010 contained total PAHs below 23.1 lg L 1, while samples collected after the well was capped (July 15, 2010) contained total PAHs below 0.22 lg L 1. For each species, a total of 60 definitive tests were conducted; 30 were with OWD and 30 with WAF preparation using variable dilutions. Total PAH concentrations for mysid OWD tests ranged from non-detect to 23.03 lg L 1 while WAF tests ranged from non-detect to 1.88 lg L 1. Total PAH concentrations for M. beryllina OWD tests ranged from non-detect to 0.64 lg L 1 while WAF tests ranged from non-detect to 0.17 lg L 1. Of the 30 OWD definitive tests with A. bahia, only five OWD preparations exhibited >20% mortality. Only three of the WAF definitive tests out of 30 exhibited >20% mortality. One OWD and two WAF exposure media showed a concentration-response in mysids. For M. beryllina, only tests from two water samples in both the WAFs and OWDs exhibited >20% mortality. However, since mortality in controls in all four tests exceeded the quality criterion of 10% and the total PAH concentrations were low or undetected in exposure media mortality in treatments was not attributed to oil. 3.3. MicrotoxÒ assays MicrotoxÒ assays with Vibrio fischeri showed both stimulatory (negative) and inhibitory effects on light production for both the Table 3 Responses of Vibrio fischeri (Microtox) to OWDs and WAFs of Field Collected Samples collected from May 12 to June 16, 2010 for Screening Toxicity Studies. Samples selected for Screening Studies Contained Visible Floating Oil. Sample No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
OWDs % Inhibition of lighta,b 29.2 22.0 27.0 29.6 42.3 27.2 32.4 25.9 17.8 36.3 49.2 40.4 19.8 70.4
WAFs Total PAHs lg L 1 (T0)
% Inhibition of lighta,b
Total PAHs lg L 1 (T0)
14.7 0.92 0.26 0.69 65.4 0.11 2245 353 2824 1402 0.10 616 4152 18174
24.5 27.6 24.7 18.7 11.7 100 23.5 0.33 33.6 23.6 34.3 62.5 37.1 15.5
2.21 1.20 0.28 0.003 62.9 0.65 46.3 48.1 49.0 48.9 2.75 21.2 51.6 259.3
a Percent inhibition of light production at 15 min exposure. Based on Microtox protocol, these numbers correspond to an 18.1% dilution of original sample (81.9% of original sample). b Negative effect is a stimulatory effect.
Fig. 3. Total PAH concentrations in field samples and toxicity responses of mysid shrimp and silverside minnows in test samples collected over time (May 2010 to December 2010). Gray circles in the background indicate actual measurements in samples collected from the water column during the DWH incident. Green and blue circles correspond to OWD and WAF sample concentrations while the color of the edge shows toxicity in the reference species above the 20% threshold. Black circles are samples tested for toxicity that had TPAHs below the detection limits. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5- and 15-minute exposures. Results of Screening Sample studies are provided in Table 3. Inhibition of normal metabolism, as indicated by decreased rate (%) luminescence did not appear to have a direct relationship with either PAH exposure concentrations, location of sample, or depth. Overall, the results were not consistent with the results from single species acute toxicity tests. This assay was not useful for evaluating the toxicity of field collected water samples from the GOM, especially when visible oil was present. 4. Conclusions Total PAH exposure media concentrations from WAFs and OWDs used for toxicity tests were compared to the 10,828 total PAH measurements from water samples collected in the Gulf of Mexico during the Deepwater Horizon incident in 2010 based on the empirical data presented in the Summary Report for Sub-Sea and Sub-Surface Oil and Dispersant Detection: Sampling and Monitoring (OSAT, 2010), as well as the field data collected as part of NRDA activities (Fig. 3). WAF and OWD exposure test media PAH concentrations used for toxicity testing were generally consistent with the total PAH concentrations measured in the field from May to July 2010 as well as following successful capping of the MC252 wellhead. The highest WAF and OWD exposure media total PAH concentrations used in single species toxicity tests mirror the highest (top 5%) total PAH concentrations out of the 10,828 water samples analyzed from the GOM (Fig. 3) from May to mid-July, 2010. Mysids were more sensitive to the field-collected samples than M. beryllina based on total PAH concentrations. Gala et al. (2001) also found that in the laboratory with spiked preparation exposures of light and heavy crude oils, mysids were also more sensitive than M. beryllina, when expressed as total PAHs and
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LC50s expressed as total PAHs (sum of 41 parent and alkylated PAHs) showed the least variability. Note since the aquatic organisms were exposed in this study to a mixture of hydrocarbons in WAFs and OWDs, acute toxicity expressed as the concentration of one subset of components (e.g., total PAHs) is not independent of the presence of other constituent component types in the mixture (Gala et al., 2001). For silversides, OWD studies were generally more toxic than WAFs in the screening tests (field samples collected May to June 2010) when there was observable oil in the sample, but were consistent for the remaining samples evaluated in the definitive tests. Mysid results were typically similar for the WAF and OWD studies. For OWD and WAF exposures with mysids, most of the mortality occurred in the first 48 h of the 96 h test. In nearly all samples analyzed, OWDs contained more PAHs than WAFs. Most mortality to mysids and silversides occurred initially (surface samples collected May to July 2010), with a clear decline in mortality associated with a decline in total PAH water concentrations following capping of the well in July 2010. It should be noted that most samples with measurable toxicity in the laboratory studies had visible oil, droplets or a surface sheen when collected in the field. It can be concluded from this study that there was minimal acute toxicity (mortality) to two standard toxicity test species during oil release and prior to closure of the well from the DWH incident. Following well closure, PAH concentrations decreased with little to no mortality. This is supported by results from the Operational Science Advisory Team (OSAT) assessment which is an interagency team selected to assess real-time data collected by the response of the DWH incident relative to specific indicators (OSAT, 2010). In the OSAT report from water samples collected (May and June 2010) during response, it was indicated that only six near-surface water samples (1 m) in the offshore sampling zone exceeded the EPA chronic aquatic life benchmark and only one exceeded the acute aquatic life benchmark for PAHs (OSAT, 2011). Furthermore, no significant mortality was observed in silversides and limited effects were observed in mysid tests (3 out of 99). Although sampling and acute toxicity testing are extensive but yet limited compared to the size of the GOM, if the USEPA standard toxicity test species are representative and accepted as a sound biological indicator, it can be concluded that the potential for acute toxicity from water exposures to MC252 were also limited. Acknowledgments This work was supported by BP Exploration and Production Inc. and the BP Gulf Coast Restoration Organization. References Anderson, J.W., Neff, J.M., Cox, B.A., Tatem, H.E., Hightower, G.M., 1974. Characteristics of dispersions and water-soluble extracts of crude and refined oils and their toxicity to estuarine crustaceans and fish. Mar. Biol. 27, 75–88. Barron, M.G., Káaihue, L., 2003. Critical evaluation of CROSERF test methods for oil dispersant toxicity testing under subarctic conditions. Mar. Poll. Bull. 46, 1191– 1199. Clark, J.R., Bragin, G.E., Febbo, E.J, Letinski, D.J., 2001. Toxicity of Physically and Chemically Dispersed Oils Under Continuous and Environmentally Realistic Exposure Conditions: Applicability to Dispersant Use Decisions in Spill Response Planning. In: Proceedings, 2001 International Oil Spill Conference. American Petroleum Institute, Washington, DC. pp. 1249–1255.
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Acute aquatic toxicity studies of Gulf of Mexico water samples collected following the Deepwater Horizon incident (May 12, 2010 to December 11, 2010) B.S. Echols ⇑, A.J. Smith, P.R. Gardinali, G.M. Rand Southeast Environmental Research Center, Florida International University, North Miami, FL 33181, United States
h i g h l i g h t s Water samples were taken at 44 sites following the Deepwater Horizon incident. Water accommodated fractions (WAFs) and oil-in-water dispersions (OWDs) were tested. Mysids were generally more sensitive than inland silversides. Most mortality observed occurred in samples collected before the well was capped.
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Article history: Received 5 February 2014 Received in revised form 9 May 2014 Accepted 14 June 2014
Handling Editor: S. Jobling Keywords: Oil spill Acute toxicity Monitoring Gulf of Mexico Deepwater Horizon
a b s t r a c t The potential for the Deepwater Horizon MC-252 oil incident to affect ecosystems in the Gulf of Mexico (GOM) was evaluated using Americamysis bahia, Menidia beryllina and Vibrio fischeri (MicrotoxÒ assay). Organisms were exposed to GOM water samples collected in May–December 2010. Samples were collected where oil was visibly present on the water surface or the presence of hydrocarbons at depth was indicated by fluorescence data or reduced dissolved oxygen. Toxicity tests were conducted using water-accommodated fractions (WAFs), and oil-in-water dispersions (OWDs). Water samples collected from May to June 2010 were used for screening tests, with OWD samples slightly more acutely toxic than WAFs. Water samples collected in July through December 2010 were subjected to definitive acute testing with both species. In A. bahia tests, total PAH concentrations for OWD exposures ranged from non-detect to 23.0 lg L 1, while WAF exposures ranged from non-detect to 1.88 lg L 1. Mortality was >20% in five OWD exposures with A. bahia and three of the WAF definitive tests. Total PAH concentrations were lower for M. beryllina tests, ranging from non-detect to 0.64 lg L 1 and non-detect to 0.17 lg L 1 for OWD and WAF exposures, respectively. Only tests from two water samples in both the WAFs and OWDs exhibited >20% mortality to M. beryllina. MicrotoxÒ assays showed stimulatory and inhibitory responses with no relationship with PAH exposure concentrations. Most mortality in A. bahia and M. beryllina occurred in water samples collected before the well was capped in July 2010 with a clear decline in mortality associated with a decline in total PAH water concentrations. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Abbreviations: DWH, Deepwater Horizon; GOM, Gulf of Mexico; BTEX, Benzene, toluene, ethylbenzene, and xylenes; COC, chain of custody; GC/MS-SIM, gas chromatograph/mass spectrometry-selected ion monitoring; IC50, median concentration inhibiting 50% of a sample population; MC-252, Mississippi Canyon Block 252; NOAA, National Oceanic and Atmospheric Administration; NRDA, Natural Resource Damage Assessment; PAH, polycyclic aromatic hydrocarbons; QA/QC, quality assurance/quality control; ROV, remotely operated vehicle; USEPA, U.S. Environmental Protection Agency; WET, whole effluent toxicity. ⇑ Corresponding author. Address: 3000 NE 151st St., North Miami, FL 33181, United States. Tel.: +1 (540) 808 5395. E-mail address: [email protected] (B.S. Echols). http://dx.doi.org/10.1016/j.chemosphere.2014.06.048 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
Crude oil was released into the Gulf of Mexico (GOM) from a depth of approximately 5,000 feet in the Mississippi Canyon 252 (MC-252) lease block (28° 44.20´N, 88°22.23´W) for 87 days, starting on April 20, 2010, until the well was successfully capped on July 15, 2010 (OSAT, 2010). Multiple ecosystems in the GOM may have been exposed and the extent to which each ecosystem may be impacted depends on a host of physical, chemical, and biological factors as well as the inherent changes that can occur in the crude oil as a result of dissolution, weathering, degradation and the
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addition of dispersant. Therefore, during and after the discharge period, different types and stages of oil (e.g., weathered oil, dispersed weathered oil) present in water and/or sediment may have potentially posed a hazard to aquatic life in the GOM. A crucial first step toward understanding potential crude oil impacts to biota is to quantitatively determine the potential aquatic toxicity of MC252 oil and how the toxicity may be altered as a result of the changes in crude oil characteristics and composition (Neff, 1999). Aquatic toxicity tests are a useful assessment tool that can be used to estimate the presence and the intensity of an effect(s), as a result of chemical exposures in water and/or sediment (Neff, 1999; Fingas, 2011). The potential for the DWH MC252 oil incident to affect different marine ecosystems in the GOM is presently being evaluated in an ongoing aquatic toxicity program to characterize the potential effects that might result from exposure to fresh- and weatheredMC-252 crude oil (Rand et al., 2011, 2012). As part of this program, an initial step was to apply the USEPA whole effluent toxicity testing (WET) approach for the protection of aquatic life (USEPA, 1991), to evaluate the toxicity of field-collected water samples from the GOM, during release of oil and after the well was capped. WET testing is a useful initial step for assessing effects because it employs the use of standard procedures and standard marine test organisms and it evaluates potential acute and chronic responses as a result of exposures to an aggregate field-collected sample (USEPA, 2002). This study presents laboratory results from part of this program, in which standard acute WET toxicity tests were conducted with mysid shrimp (Americamysis bahia) and silverside minnow (Menidia beryllina) exposed up to 96 h to field-collected water samples from the GOM prior to and after the MC-252 well was capped along with the MicrotoxÒ bacterial assays. Exposure media used field-collected water samples prepared as a variable dilution of both water-accommodated fractions (WAFs), where samples were stirred and the aqueous phase collected for exposure, and oil-in-water dispersions (OWDs), where the whole sample was used. Although WAF preparations may not be typical of field-collected conditions they do contain the dissolved fraction of hydrocarbons which are more bioavailable and toxic (Neff and Anderson, 1981; Pruell et al., 1986). OWDs more accurately simulate field conditions, but they are multiphase solutions complicating chemical analyses and replication (Anderson et al., 1974; Neff, 1999). WAF solution exposure media are typically preferred over OWD exposures in aquatic toxicity testing because they produce more replicable conditions. For testing in this study, the combination of both exposure media were used to determine potential differential toxicity between samples (in WAFs) containing dissolved oil constituents and samples (OWDs) containing both dissolved and insoluble (e.g., droplets) constituents. Mortality was the biological endpoint used for both single species acute toxicity tests and the median concentration value that inhibited light production in 50% of the test population (IC50) of bacteria was used for the MicrotoxÒ assay. Total PAH concentrations were measured for all WAFs and OWDs. There is much criticism and serious debate on the use and uncertainties of the standard descriptive summary statistics (LC50, EC50, NOECs, etc.) using both hypothesis testing and regression-based approaches, which form the basis of almost every effects assessment (Warne and Van Dam, 2008; Landis and Chapman, 2011; Jaeger, 2012). Therefore, greater than 20% mortality for an exposure treatment in an acute test with the standard test species was the measurement endpoint considered environmentally relevant in this study and is supported by regulatory precedent for aqueous effluent discharges under the National Pollutant Discharge Elimination System (NPDES) for protection of aquatic life under the Clean Water Act (Suter et al., 1995). The investigators understand that the latter
does not apply when considering endangered, threatened and keystone species. 2. Materials and methods 2.1. Study area Water samples were collected for aquatic toxicity testing from May 12 to December 11, 2010, within 2 km of the MC-252 wellhead as well as far-field (6250 km). Details of sampling locations are included in Fig. 1. 2.2. Sample collection Sampling transects were selected based on the extent of surface oil identified in aerial and satellite imagery and from water column profiling data obtained from response vessels. At each sampling location, real-time, in situ UV fluorescence and dissolved oxygen (DO) were measured using sensors packaged with a conductivitytemperature-depth profiler. These parameters, as well as visual observations of oil, were used to guide decisions regarding the depths at which water samples were collected. Fluorescence and DO anomalies have been used in previous work to assess the presence of hydrocarbons in the water column or the consumption of oxygen by increased microbial activity (Hazen et al., 2010). If a fluorescence anomaly and/or a distinct decrease in DO were observed relative to background, or if oil was visible on the water surface, then whole water samples were collected for laboratory toxicity testing. Water samples were collected at multiple depths using a conventional hydrowire with 5-L, 10-L, or 20-L GO-FLO sampling bottles on a rosette multi-bottle array or attached to an ROV-tethered management system. Standard decontamination procedures were used between sample collection events. Surface water samples were collected via bucket grab or peristaltic pump. Samples were aliquoted from the surface grabs or GO-FLO into 1-L, 2.5-L, or 3.8-L certified clean amber glass containers with PTFE-on lids. Minimal headspace was maintained in each bottle. Water samples were stored in the dark at 4 °C until prepared for shipment. Samples were received at FIU within seven days of collection. All original samples were then stored in the dark in a refrigerated unit at 4 °C until test preparation. 2.3. Test species Three species were selected for toxicity testing and include the mysid shrimp (A. bahia), the inland silverside (M. beryllina) and the light producing bacteria used in MicrotoxÒ assays, V. fisheri. Mysid shrimp and inland silversides were selected as test species as they are recommended marine test species by the USEPA (USEPA, 2002), naturally inhabit coastal waters in the Gulf of Mexico; and both species can tolerate higher salinities (15–30 ppt), similar to ranges common in the GOM which should preclude effects of changes in salinity alone. The MicrotoxÒ assay was selected because it was being used in the field as part of the analysis onboard research vessels deployed during the Response efforts during the DWH incident. Mysids and inland silversides were purchased from a commercial supplier and upon delivery animals were acclimated in a temperature-controlled incubator for at least 48 h prior to toxicity testing. Organisms were acclimated to the appropriate test salinity, matching that of field-collected samples, with gradual additions of laboratory saltwater. Laboratory saltwater was obtained from a natural saltwater well that was air-sparged, and passed through a particle filter, carbon filtration and UV sterilized. This water
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Fig. 1. Locations of screening and definitive level toxicity samples collected from 5/12/2010 to 12/11/2010.
ranged from 29–32 ppt and was used as dilution water for all definitive tests. During holding, organisms were fed daily with 0.99 for all analytes was used to demonstrate the linear range of the detector. Analyte concentrations were calculated based on surrogate standards naphthalene-d8, phenanthrene-d10, chrysene-d12, acenapthene-d10, and perylene-d12. Surrogate standard recoveries were calculated based on internal standards benzo(a)pyrene-d12 and fluorene-d10. Alkylated homologues were quantitated based on the response factors of the parent compounds. For simplicity and to provide the reader with a reference point for the toxicological assessment interpretation, analytical chemistry results are summarized as total PAH concentration in the water samples expressed in parts per billion (lg L 1). 2.7. Quality assurance/quality control All of the biological and chemistry data presented in this paper were subjected to formal data verification and validation prior to use. All laboratory data were reviewed by the laboratory’s internal Quality Assurance Unit and external (independent) auditors to
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assure that each study was performed in accordance with the protocol and laboratory standard operating procedures (SOPs). Sample processing activities, analytical procedures, and storage and holding times for the chemical analyses were generally consistent with the Analytical Quality Assurance Plan. Mississippi Canyon 252 (Deepwater Horizon) Natural Resource Damage Assessment. Version 2.2 (NOAA, 2010). Batch QA/QC was used to assess method precision and accuracy by analyzing laboratory blank(s), fortified blank(s), sample duplicates (when available), and standard reference materials. Both the ecotoxicology and analytical chemistry laboratories at Florida International University are NELACaccredited. 3. Results and discussion Water was collected from 44 sites in the GOM between May 12 and December 11, 2010 at multiple depths within 9 km of the well head and at far-field distances (6250 km) from the wellhead (Fig. 1). The first 14 water samples underwent acute toxicity tests with 100% WAF and 100% OWD (screening tests). The remaining 30 water samples underwent acute toxicity tests using a variable dilution series (definitive tests). Screening and definitive single species acute toxicity tests were conducted with mysids (96 h) and, silversides (96 h) along with the MicrotoxÒ bacterial assay. Control mortality throughout all testing was 0% for A. bahia and less than 5% for M. beryllina and thus well within standard guideline requirements. During testing, salinities were slightly higher (5–30 ppt ± 10%) than the recommended values in the USEPA guidelines due to the use of field-collected water samples. In general, water quality parameters remained within acceptable ranges (USEPA, 2002). Control salinities were within recommended guidelines and were not adjusted to salinities of field-collected samples. The pH measurements were also within standard ranges and no fluctuations were observed during the duration of any toxicity test. Although DO levels fell below 4 mg L 1 in some of the treatments by test termination, test chambers were not aerated to eliminate any potential effects on PAH concentrations in the exposure media. Dissolved oxygen did not appear to contribute to toxicity in those treatments with low DO because toxicity occurred more rapidly declining DO. Note that collection of the 44 water samples in the GOM were based on where oil was visibly present on the water surface or where the presence of hydrocarbons at depth was indicated by fluorescence data or was suggested by reduced dissolved DO concentrations. 3.1. Single-species screening toxicity tests The results presented in Fig. 2 show the concentrations of total PAHs for the full-strength (100% sample) OWD and WAF exposure media prepared at test initiation in relation to their collection date in the GOM. As noted by the different magnitudes of the concentration scales total PAH concentrations in OWD samples were generally higher than WAFs. The maximum total PAH concentrations for OWD samples was 18 175 lg L 1 while WAF samples reached total PAH concentrations of up to 895 lg L 1. Most of the samples collected during the earlier stages of the response were restricted to the top of the water column (61 m depth) and are likely representative of ‘‘worst case’’ surface conditions (e.g. within or directly under an oil slick). Samples collected May 12 to June 16, 2010 for screening toxicity tests contained visible floating oil while those collected later in the year were identified by the fluorescence anomaly or the DO sag rather than by visual evidence of oil, sheen or oil droplets. Results also indicate that samples collected at or below 1000 m contained lower maximum total PAH concentrations at 0.170 lg L 1 and 0.076 lg L 1 for
Fig. 2. Total PAH concentrations (lg L 1) measured in OWDs and WAFs prepared from field-collected samples in relationship to the field collection date. Grey symbols represent samples where no PAHs were detected.
the OWDs and WAFs, respectively. The levels of total PAHs in the sample seems related to the amount of visible oil rather than the collection location. OWD preparations were slightly more acutely toxic than WAFs for both A. bahia and M. beryllina. Mysids were generally more sensitive than M. beryllina. Ten of the 14 water samples resulted in >20% mortality for A. bahia, following the 96 h static-renewal exposures to both undiluted WAF and OWD preparations. In tests with M. beryllina, eight of the 14 water samples caused >20% mortality following the 96 h static-renewal exposures to both undiluted WAF and OWD preparations. WAFs were slightly less toxic than OWDs for both species and A. bahia was more sensitive than M. beryllina. The maximum total PAH concentration for mysid OWD tests associated with 620% mortality was 59 lg L 1, while the maximum concentration in tests with >20% mortality was 2962 lg L 1. For A. bahia WAF tests, total PAH concentrations ranged from 0.02 to 15 lg L 1 in exposures with 620% mortality. In exposures with >20% mortality, up to 895 lg L 1 total PAH was observed. In silverside OWD tests, the maximum total PAH concentration associated with 620% mortality was 0.11 lg L 1, while the highest concentration observed in samples with >20% mortality was 11 175 lg L 1. For WAF tests using M. beryllina, 620% mortality was observed at total PAH concentrations up to 64 lg L 1. In samples with >20% mortality, the maximum total PAH concentration observed was 259 lg L 1. 3.2. Single-species definitive toxicity tests A variable dilution method was used to prepare both OWD and WAF exposure test media of 6.25–100% of the original sample. Use of variable dilution is the preferred method to prepare treatment exposure media for oil since it creates reproducible conditions and concentrations of PAHs in exposure test media compared to
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Table 2 Total PAH concentrations (lg L
1
) using a variable dilution series of a 100% sample.
Dilution (%)
Measured WAF concentration (lg L 1 TPAH)
Measured OWD concentration (lg L 1 TPAH)
100 50 25 12.5 6.25
45.61 19.34 10.68 4.64 2.60
1124.02 437.91 212.69 64.33 26.93
serial dilution (Barron and Ka’aihue, 2003; Neff, 1999). In support of this method dilutions of a single field-collected sample (GOM Field Sample JF2-2 km-surf-TOX-20100524-E125) were prepared in Table 2. Results indicate the use of this method as concentrations of WAF TPAH (lg L 1) were within 25% of expected nominal concentrations (Table 2). Thirty (collected July 10 through December 11, 2010) of the 44 GOM collected water samples were subjected to definitive acute toxicity testing with both species. Samples collected after June 16, 2010 contained total PAHs below 23.1 lg L 1, while samples collected after the well was capped (July 15, 2010) contained total PAHs below 0.22 lg L 1. For each species, a total of 60 definitive tests were conducted; 30 were with OWD and 30 with WAF preparation using variable dilutions. Total PAH concentrations for mysid OWD tests ranged from non-detect to 23.03 lg L 1 while WAF tests ranged from non-detect to 1.88 lg L 1. Total PAH concentrations for M. beryllina OWD tests ranged from non-detect to 0.64 lg L 1 while WAF tests ranged from non-detect to 0.17 lg L 1. Of the 30 OWD definitive tests with A. bahia, only five OWD preparations exhibited >20% mortality. Only three of the WAF definitive tests out of 30 exhibited >20% mortality. One OWD and two WAF exposure media showed a concentration-response in mysids. For M. beryllina, only tests from two water samples in both the WAFs and OWDs exhibited >20% mortality. However, since mortality in controls in all four tests exceeded the quality criterion of 10% and the total PAH concentrations were low or undetected in exposure media mortality in treatments was not attributed to oil. 3.3. MicrotoxÒ assays MicrotoxÒ assays with Vibrio fischeri showed both stimulatory (negative) and inhibitory effects on light production for both the Table 3 Responses of Vibrio fischeri (Microtox) to OWDs and WAFs of Field Collected Samples collected from May 12 to June 16, 2010 for Screening Toxicity Studies. Samples selected for Screening Studies Contained Visible Floating Oil. Sample No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14
OWDs % Inhibition of lighta,b 29.2 22.0 27.0 29.6 42.3 27.2 32.4 25.9 17.8 36.3 49.2 40.4 19.8 70.4
WAFs Total PAHs lg L 1 (T0)
% Inhibition of lighta,b
Total PAHs lg L 1 (T0)
14.7 0.92 0.26 0.69 65.4 0.11 2245 353 2824 1402 0.10 616 4152 18174
24.5 27.6 24.7 18.7 11.7 100 23.5 0.33 33.6 23.6 34.3 62.5 37.1 15.5
2.21 1.20 0.28 0.003 62.9 0.65 46.3 48.1 49.0 48.9 2.75 21.2 51.6 259.3
a Percent inhibition of light production at 15 min exposure. Based on Microtox protocol, these numbers correspond to an 18.1% dilution of original sample (81.9% of original sample). b Negative effect is a stimulatory effect.
Fig. 3. Total PAH concentrations in field samples and toxicity responses of mysid shrimp and silverside minnows in test samples collected over time (May 2010 to December 2010). Gray circles in the background indicate actual measurements in samples collected from the water column during the DWH incident. Green and blue circles correspond to OWD and WAF sample concentrations while the color of the edge shows toxicity in the reference species above the 20% threshold. Black circles are samples tested for toxicity that had TPAHs below the detection limits. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
5- and 15-minute exposures. Results of Screening Sample studies are provided in Table 3. Inhibition of normal metabolism, as indicated by decreased rate (%) luminescence did not appear to have a direct relationship with either PAH exposure concentrations, location of sample, or depth. Overall, the results were not consistent with the results from single species acute toxicity tests. This assay was not useful for evaluating the toxicity of field collected water samples from the GOM, especially when visible oil was present. 4. Conclusions Total PAH exposure media concentrations from WAFs and OWDs used for toxicity tests were compared to the 10,828 total PAH measurements from water samples collected in the Gulf of Mexico during the Deepwater Horizon incident in 2010 based on the empirical data presented in the Summary Report for Sub-Sea and Sub-Surface Oil and Dispersant Detection: Sampling and Monitoring (OSAT, 2010), as well as the field data collected as part of NRDA activities (Fig. 3). WAF and OWD exposure test media PAH concentrations used for toxicity testing were generally consistent with the total PAH concentrations measured in the field from May to July 2010 as well as following successful capping of the MC252 wellhead. The highest WAF and OWD exposure media total PAH concentrations used in single species toxicity tests mirror the highest (top 5%) total PAH concentrations out of the 10,828 water samples analyzed from the GOM (Fig. 3) from May to mid-July, 2010. Mysids were more sensitive to the field-collected samples than M. beryllina based on total PAH concentrations. Gala et al. (2001) also found that in the laboratory with spiked preparation exposures of light and heavy crude oils, mysids were also more sensitive than M. beryllina, when expressed as total PAHs and
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LC50s expressed as total PAHs (sum of 41 parent and alkylated PAHs) showed the least variability. Note since the aquatic organisms were exposed in this study to a mixture of hydrocarbons in WAFs and OWDs, acute toxicity expressed as the concentration of one subset of components (e.g., total PAHs) is not independent of the presence of other constituent component types in the mixture (Gala et al., 2001). For silversides, OWD studies were generally more toxic than WAFs in the screening tests (field samples collected May to June 2010) when there was observable oil in the sample, but were consistent for the remaining samples evaluated in the definitive tests. Mysid results were typically similar for the WAF and OWD studies. For OWD and WAF exposures with mysids, most of the mortality occurred in the first 48 h of the 96 h test. In nearly all samples analyzed, OWDs contained more PAHs than WAFs. Most mortality to mysids and silversides occurred initially (surface samples collected May to July 2010), with a clear decline in mortality associated with a decline in total PAH water concentrations following capping of the well in July 2010. It should be noted that most samples with measurable toxicity in the laboratory studies had visible oil, droplets or a surface sheen when collected in the field. It can be concluded from this study that there was minimal acute toxicity (mortality) to two standard toxicity test species during oil release and prior to closure of the well from the DWH incident. Following well closure, PAH concentrations decreased with little to no mortality. This is supported by results from the Operational Science Advisory Team (OSAT) assessment which is an interagency team selected to assess real-time data collected by the response of the DWH incident relative to specific indicators (OSAT, 2010). In the OSAT report from water samples collected (May and June 2010) during response, it was indicated that only six near-surface water samples (1 m) in the offshore sampling zone exceeded the EPA chronic aquatic life benchmark and only one exceeded the acute aquatic life benchmark for PAHs (OSAT, 2011). Furthermore, no significant mortality was observed in silversides and limited effects were observed in mysid tests (3 out of 99). Although sampling and acute toxicity testing are extensive but yet limited compared to the size of the GOM, if the USEPA standard toxicity test species are representative and accepted as a sound biological indicator, it can be concluded that the potential for acute toxicity from water exposures to MC252 were also limited. Acknowledgments This work was supported by BP Exploration and Production Inc. and the BP Gulf Coast Restoration Organization. References Anderson, J.W., Neff, J.M., Cox, B.A., Tatem, H.E., Hightower, G.M., 1974. Characteristics of dispersions and water-soluble extracts of crude and refined oils and their toxicity to estuarine crustaceans and fish. Mar. Biol. 27, 75–88. Barron, M.G., Káaihue, L., 2003. Critical evaluation of CROSERF test methods for oil dispersant toxicity testing under subarctic conditions. Mar. Poll. Bull. 46, 1191– 1199. Clark, J.R., Bragin, G.E., Febbo, E.J, Letinski, D.J., 2001. Toxicity of Physically and Chemically Dispersed Oils Under Continuous and Environmentally Realistic Exposure Conditions: Applicability to Dispersant Use Decisions in Spill Response Planning. In: Proceedings, 2001 International Oil Spill Conference. American Petroleum Institute, Washington, DC. pp. 1249–1255.
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