Polycyclic Aromatic Hydrocarbons (PAHs) in Mississippi Seafood

On May 24, 2010, the federal government declared a fisheries disaster for the states of .... extraction and cleanup procedures developed by the MSCL (...
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Polycyclic Aromatic Hydrocarbons (PAHs) in Mississippi Seafood from Areas Affected by the Deepwater Horizon Oil Spill Kang Xia,†,* Gale Hagood,‡ Christina Childers,‡ Jack Atkins,‡ Beth Rogers,‡ Lee Ware,‡ Kevin Armbrust,‡ Joe Jewell,§ Dale Diaz,§ Nick Gatian,⊥ and Henry Folmer⊥ †

Department of Crop and Soil Environmental Sciences, Virginia Tech., Blacksburg, Virginia 24060, United States Mississippi State Chemical Laboratory, P.O. Box CR, Mississippi State, Mississippi 39762, United States § Mississippi Department of Marine Resources, 1141 Bayview Ave., Biloxi, MS, USA 39530 ⊥ Mississippi Department of Environmental Quality, P.O. Box 2261, Jackson, MS, USA 39225 ‡

S Supporting Information *

ABSTRACT: Seafood samples from the fishing ground closure areas of Mississippi Gulf Coast that were affected by the Deepwater Horizon Oil Spill Disaster were collected and analyzed for twenty-five 2- to 6-ring PAHs, about one month after the first day of incident. A total of 278 seafood samples consisting of 86 fishes, 65 shrimps, 59 crabs, and 68 oysters were collected and analyzed weekly from May 27, 2010 until October 2010 and monthly thereafter until August 2011. Statistically higher levels of total PAHs were detected in all four types of seafood samples during early part of the sampling period compared to the later months. There was no significant concentration difference between PAHs detected in the oyster samples for the current study and the 10-year historical data from the NOAA Mussel Watch program. The PAH levels in the tested seafood samples were similar to those detected in commonly consumed processed foods purchased from local grocery stores and restaurants. Overall, the levels of PAHs in all the tested seafood samples collected within one-year period after the Oil Spill incident were far below the public health Levels of Concern (LOC) established jointly by the NOAA/FDA/Gulf Coast states under the protocol to reopen state and federal waters.

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INTRODUCTION The Deepwater Horizon Oil Spill released an estimated 4.9 million barrels (780 000 m3) of crude oil into the Gulf of Mexico from April 22 to July 15, 2010, before the gushing wellhead was finally capped.1 This was the largest offshore oil spill in the U.S. history and at least the second or perhaps third largest oil spill worldwide.2,3 This event immediately triggered heightened © 2012 American Chemical Society

public concern for the safety of consuming seafood from areas in the Gulf of Mexico affected by the Deepwater Horizon Oil Received: Revised: Accepted: Published: 5310

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Figure 1. Seafood sampling locations in the Mississippi Gulf Coast areas affected by the Deepwater Horizon Oil Spill. Open square, fish sampling sites; open triangle, oysters sampling sites, open circle with black dot, shrimp sampling sites; gray triangle with black dot, crab sampling sites. The black triangle symbol indicates the NOAA Mussel Watch program Mississippi sampling locations.

the impact of the Deepwater Horizon Oil Spill on PAH levels in the seafood from the oil spill-affected areas due to its unprecedented size and widespread use of oil dispersants below water. In response to the seafood safety concern due to the Deepwater Horizon Oil Spill incident, a concerted effort was immediately established among the Mississippi Department of Marine Resources (MDMR), the Mississippi Department of Environmental Quality (MDEQ), and the Mississippi State Chemical Laboratory (MSCL) to monitor the levels of twenty-five 2- to 6ring PAHs (Table S1 of the Supporting Information) in the seafood samples from the Mississippi Gulf Coast areas affected by the incident. All twenty five target PAHs were included in the standard National Oceanic and Atmospheric Administration (NOAA) protocol for monitoring selected aromatic hydrocarbons in tissues and sediment21 and were the required target analytes in the Food and Drug Administration (FDA)’s protocol for reopening oil-impacted areas closed to seafood harvesting due to the Deepwater Horizon Oil Spill.22 Sixteen of the twenty-five target PAHs are listed in the US EPA priority pollutant list (Table S1 of the Supporting Information).9 The goal of this investigation was to provide long-term monitoring information on the PAH levels in Mississippi seafood after the Deepwater Horizon Oil Spill.

Spill and resulted in the closure of a vast area of commercial and recreational fishing grounds in affected Federal and state waters. At the height of the closure (June 2, 2010), fishing activity was banned in approximately 36.6% (229 270 km2) of Federal waters in the Gulf of Mexico, extending along the coast from Atchafalaya Bay, Louisiana to Panama City, Florida,4,5 resulting in billions of dollars of losses to the seafood industry in the affected states. On May 24, 2010, the federal government declared a fisheries disaster for the states of Alabama, Mississippi, Louisiana, and Florida.6 Polycyclic aromatic hydrocarbons (PAHs) consisting of over 100 different chemicals are components of crude oil (between 0.2 and 7%, depending on location).7 Sixteen PAHs are included in the European Union and US Environmental Protection Agency (US EPA) priority pollutant list because of their mutagenic and carcinogenic properties, environmental persistence, and bioaccumulation.8,9 Hence, they were among the chemicals of the most health concern after the Deepwater Horizon Oil Spill. Elevated concentrations of PAHs in marine organisms often occur in areas receiving chronic hydrocarbons discharges10 and in areas that are heavily contaminated due to anthropogenic activities.11 However, investigations after major oil spill events have demonstrated different impact of oil spill on the levels of PAH in marine organisms: from none detectable impact to initial elevated PAH levels followed by significant decrease to their baseline levels within a few months to a few years.12−19 Accumulation, biotransformation, and elimination of PAHs in marine organisms are largely affected by environmental factors such as route of exposure, water and sediment chemistry, and chemistry of PAHs, as well as by biological characteristics of the affected organisms (e.g., lipid contents, organism species, sex, and age).20 Therefore, it is difficult to predict and assess, based on historical data,

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EXPERIMENTAL SECTION Sampling sites. The first set of samples consisting of shrimp, crab, oysters, and finfish were collected by MDMR and MDEQ and sent to MSCL on May 27, 2010. Samples were collected and analyzed weekly until October 2010, and monthly thereafter. Over the reported period of this work (May 2010 to

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Table 1. Quantification Results for NIST Reference Material SRM 1974b (n = 11); Calculation of Upper and Lower Control Limits Is Based on the NOAA Technical Memorandum NMFS-NWFSC-77.26

August 2011), a total of 278 seafood samples consisting of 86 fishes, 65 shrimps, 59 crabs, and 68 oysters were collected offshore in the fishing ground closure areas of the Mississippi Gulf Coast due to the Deepwater Horizon Oil Spill (Figure 1). NOAA’s decision on fishing ground closure was based on the presence of observable oil or oil sheens in the affected areas. Seafood species were selected based primarily on species that are harvested recreationally and commercially and normally consumed by the public. The entire Mississippi sound and state waters south of the barrier islands were impacted by the oil spill in varying degrees and at one point were closed to seafood harvesting. Samples were collected from throughout the sound. Bays were included for those species that migrate throughout the area waters. Each sample has an associated latitude and longitude to identify the precise location of collection (Figure 1). The crustacea samples collected were brown shrimp (Farfantepenaeus aztecus), white shrimp (Farfantepenaeus setiferus), and blue crabs (Callinectes sapidus). The oysters collected were Crassostrea virginica, native to the Gulf of Mexico. The finfish samples collected consisted of Atlantic croaker (Micropogonias undulatus), Black drum (Pogonias cromis), Red drum (Sciaenops ocellatus), Cobia (Rachycentron canadum), Gulf Menhaden (Brevoortia patronus), Red Snapper (Lutjanus campechanus), Southern Flounder (Paralichthys lethostigma), Spotted seatrout (Cynoscion nebulosus), Striped Mullet (Mugil cephalus), Tripletail (Lobotes surinamensis), and White seatrout (Cynoscion Arenarius). Samples collected by the MDMR personnel were stored with ice in coolers and immediately transported to the MDMR processing lab located in Biloxi, MS. Each species was processed, composited, and homogenized separately. Sample sizes were chosen to obtain a sample size of at least 200 g for each composited sample, as required by the FDA/NOAA/ Trustee protocol for reopening oil impacted areas developed after the Deepwater Horizon spill (composited sample size of at least 200 g).22 Number of individuals making up a single composite sample varied by species depending on the size of the individuals collected but typically consists of two fishes, 0.5 pounds of shrimp, 15 oysters, and 10 crabs. The heads and shells of shrimps collected were discarded. Approximately 200 to 500 g of tail meat with vein was composited and homogenized using a food processor. Each finfish was scaled. The heads, tails, and viscera of each finfish were discarded. Approximately 200 to 500 g of finfish fillet was composited and homogenized using a food processor. The oyster shells were first rinsed in clean water, shucked, and all inner tissue retained and frozen. The frozen oyster inner tissue was then coarsely chopped. Approximately 200 to 500 g of chopped tissue was composited and homogenized with a mortar and pestle. Approximately 200 to 500 g of crab meat tissue from the body and claws as well as hepatopancreas were collected, composited, and homogenized using a food processor. All processed samples were frozen, packed on dry ice in coolers, and transferred to MDEQ for overnight shipment to the MSCL for analysis of PAHs. As a comparison study, single packages of processed food including smoked chicken, smoked pork, smoked catfish, smoked brisket, smoked shrimp, sandwich turkey, and sandwich ham were randomly selected and purchased from local grocery stores and restaurants in Starkville, Mississippi. Contents of each package were homogenized using a food processor similar to the ones used for the seafood samples. All the homogenized

Concentration (μg kg‑1, wet weight basis)

Napthalenea Fluorene Phenanthrene Anthracene 1-methylphenanthrene Fluoranthene Pyrene Benzo(a)anthracene Chrysene + triphenylene Benzo(b)fluoranthrene Benzo(k)fluoranthrene Benzo(e)pyrene Benzo(a)pyrene Perylene Dibenz(a,h)anthracene Benzo(ghi)perylene Indeno(1,2,3,-cd) pyrene

average detected expected

upper control limitc

lower control limitd

5.56 (±2.77)b 1.30 (±1.05) 7.81 (±7.42) 1.92 (±1.07) 0.901 (±0.413) 20.2 (±5.18) 17.9 (±5.03) 7.89 (±3.03) 9.67 (±1.54)

2.43 0.494 2.58 0.527 0.98 17.1 18.04 4.74 10.63

3.315 0.689 3.497 0.777 1.443 23.14 24.232 6.851 16.055

1.617 0.321 1.729 0.319 0.595 11.48 12.208 2.947 6.237

6.51 (±2.52) 2.85 (±0.431) 11.3 (±2.17) 2.71 (±0.784) 0.753 (±0.0981) 0.398 (±0.663) 2.81 (±0.742) 1.81 (±1.09)

6.46 3.16 10.3 2.8 0.99 0.327 3.12 2.14

9.165 4.342 14.82 4.134 1.469 0.465 4.485 2.925

4.109 2.086 6.44 1.694 0.595 0.207 1.953 1.421

a

Compound name in italic indicates average detected concentration was out of the control limits. bNumber in parentheses indicates standard deviation. cUpper control limit = [1.3 × (certified value + uncertainty value for 95% confidence)]. dLower control limit = [0.7 × (certified value - uncertainty value for 95% confidence)].

seafood samples and processed food samples were kept in a −80 °C freezer until ready (within a week) for sample extraction and cleanup for PAH analysis on gas chromatography-tandem mass spectrometer (GC/MS/MS). In order to maintain legal integrity of all samples and data, the U.S. EPA Chain of Custody procedures were followed.23 Sample Extraction and Cleanup. A flowchart of the sample extraction and cleanup procedures developed by the MSCL (SOP No. 1.681) is provided in Figure S1 of the Supporting Information. Each sample was extracted using an accelerated solvent extraction system (ASE 350, Dionex, Sunnyvale, CA). Approximately five grams of homogenized tissue sample were transferred into a beaker and the sample weight recorded to the fourth decimal point. An appropriate amount of stock surrogate mixture, described under preparation of standards of Section S1 of the Supporting Information, was added to the sample to bring the concentration to 50 ng mL−1. To absorb the moisture from the sample, 5−6 grams of hydromatrix were added into the beaker. The sample-hydromatrix mixture was stirred with a spatula until it became a flowable powder and the well mixed samplehydromatrix mixture was transferred into a 22 mL ASE cell with a glass fiber filter at the bottom. The beaker was rinsed with a small amount of methylene chloride and was add to the ASE cell. The ASE cell was loaded onto the instrument and PAHs were extracted from the sample using a 5 min heating cycle and two 2 min static cycles followed by a 100% solvent flush and 60 s purge cycle. The samples were extracted at 100 °C and 2000 psi using a methylene chloride/acetone (60:40, v/v) mixture as the extraction solvent. The extract was transferred to an evaporative flask attached with a concentration tube and the ASE collection vial was rinsed 5312

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Figure 2. Average percent recovery rates for shrimp (n = 18), oyster (n = 19), fish (n = 20), and crab (n = 17) samples spiked with PAHs at 50 μg kg−1.

three times with acetone into the evaporative flask. A Snyder column was then attached to the evaporative flask and the sample was concentrated by a Kuderna-Danish (KD) apparatus (Fisher Scientific) to ∼10 mL. Once completed, the KD-concentrated extract was poured from the concentration tube through a Drydisk (Horizon Technology, Salem, New Hampshire) to remove residual water. The concentration tube was rinsed three times with methylene chloride, and poured through the Drydisk. The Drydisk was rinsed with methylene chloride. The eluent from the DryDisk was transferred from the collection vial to another KD flask, followed by rinsing the collection vial with methylene chloride three times into the KD flask. The sample was then concentrated to ∼4 mL. At this point, 10 mL of hexane was added into the KD flask followed by concentrating the sample to ∼5 mL via KD to remove methylene chloride. If methylene chloride odor was detected, another 10 mL hexane was added into the KD flask followed by concentrating the sample to ∼5 mL via KD. This step was repeated until methylene chloride odor was not detected. The concentrated sample was further concentrated under a gentle stream of N2 using a N-EVAP (model 115, Organomation Associates, Inc., Berlin, MA) to ∼2 mL before final cleanup on a silica/alumina gel column. To prepare the silica/ alumina gel column, the 1% silica gel (VWR, Radnor, PA) was deactivated by heating at 700 °C for 18 h to a final water content of 1%. After deactivation, the silica gel was stored at 170 °C. It was cooled to room temperature in a desiccator just prior to use. To make a silica/alumina gel column, a slurry consisting of 20 g of 1% deactivated silica gel and 50 mL petroleum ether was transferred into a glass pipet (61 cm in length, 2.7 cm in diameter) followed by washing the residue silica gel into the column with an additional 50 mL of petroleum ether. Each column was topped with 5 mL of neutral alumina (VWR, Radnor, PA). The column was then conditioned with 50 mL of petroleum ether before transferring the concentrated extract to the column. A quantity of 100 mL petroleum ether was then added to the column and eluted at a rate of 5 mL min−1. The eluate was discarded and after exchanging the collection vial with a clean one, 100 mL

methylene chloride/petroleum ether (40/60, v:v) was eluted through the column at a rate of 5 mL min−1 into the collection vial. Another 50 mL methylene chloride was then added to the column, eluted at a rate of 5 mL min−1 into the collection vial and the final eluate was transferred into a KD flask followed by rinsing the collection vial with methylene chloride three times into the KD flask. The sample was concentrated to ∼5 mL via KD and transferred to a test tube. The KD-flask was rinsed three times with methylene chloride into the same test tube. The sample was concentrated under a gentle stream of N2 to exactly one mL. The final concentrate was transferred into a one mL GC vial for analysis of PAHs on GC/MS/MS. Twentyfive target 2- to 6-ring PAHs were analyzed (Table S1 of the Supporting Information) of which 16 are listed on the US EPA priority pollutant list.9 Instrumental Method. An Agilent 7000B Triple Quadrupole GC/MS/MS system (Agilent Technologies, Inc., Santa Clara, CA) was used for the analysis of PAHs. Individual PAHs were chromatographically separated using a DB-EUPAH CF column (20 m X 0.18 mm id X 0.14 um, Agilent Technologies, Inc., Santa Clara, CA) under the instrument parameters listed in Table S2 of the Supporting Information. Each sample was analyzed using the multiple reaction monitoring (MRM) mode. Multiple reaction monitoring in a triple quadrupole GC/MS/MS is inherently much more selective and sensitive than either scan or selected ion monitoring (SIM) with a single quadrupole GC/MS or ion trap GC/MS as many matrix interferences are minimized or even removed. Each analyte was quantified by means of calibration curves with a standard in solvent using the internal standard method.24 Table S3 of the Supporting Information lists the GC/MS/MS quantification ions, the confirmation ions, and the expected parent/daughter peak intensity ratio of confirmation for each analyte. Appropriate amounts of Naphthalene-d8, Acenaphthene-d10, Chrysene-d12, Perylene-d12, and Phenanthrene-d10 were added as internal standards to the final extract of each sample before GC/MS/MS analysis. Concentrations of analytes were 5313

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Figure 3. Box plot concentrations of PAHs in fish, shrimp, and crab samples from the Mississippi Gulf Coast areas affected by the Deepwater Horizon Oil Spill Disaster (May 27, 2010−August 02, 2011). The line within each box, the lower and upper boundaries of each box, the error bars below and above the box, and the black circles below and above the error bars mark the median (50th percentile), the 25th and 75th, 10th and 90th, 5th and 95th percentiles, respectively. Instrument detection limit was 0.5 ppb.

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calculated by using the response factors of multiple concentration levels of GC/MS/MS calibration standards. The response factor of an analyte is defined as the ratio of the instrument response of the analyte and the internal standard. The GC/MS/MS is considered stable if the response of an analyte relative to the response of its internal standard in a given repetition is within +15% of the respective average for the repetitions. The quality assurance/quality control (QA/QC) protocols used for this study followed the established EPA proceedures25 and the NOAA criteria for PAH analysis in seafood samples.24 Detailed information is described in Section S1 of the Supporting Information. One-way single factor ANOVA statistical analysis (α = 0.01) was conducted using the Microsoft Excel Analysis ToolPak.

RESULTS AND DISCUSSION

Method Performance. The certified values of PAHs in the NIST Mussel standard reference material (SRM) were compared with the values detected in our laboratory (Table 1). Most detected values fell within the upper and lower control limits established by the National Oceanic and Atmospheric Administration (NOAA) with the exception for napthalene, fluorene, phenanthrene, anthracene, and benzo(a)anthracene. The accuracy of the SRM result was considered acceptable if the concentrations of at least 70% of the analytes were within their NIST control limits.28 The duplicate samples relative percent difference (RPD) for all four matrixes were consistently crustaceans > molluscs.28−30 Statistically higher levels of total PAHs were detected in all four types of seafood samples during early part of the sampling period compared to the later months. This decrease might be possibly due to dissipation of the spilled oil with time. However, as the result of natural geological activity (oil seeps) and/ or inputs from typical anthropogenic activities (e.g boating traffic, urban runoff, etc) along the coast, similar levels of PAHs have also been historically detected in the seafood samples from the region before the Deepwater Horizon Oil Spill.26 Comparison with Results from the NOAA National Mussel Watch Program and Common Processed Food. The PAH levels measured in the oysters collected off shore in the Mississippi Gulf Coast areas affected by the Deepwater Horizon Oil Spill were compared with the published levels of PAHs detected in the oysters collected nationwide from 1998 to 2008 and in those sampled at three locations of Mississippi coast in 2008, all of which were part of the NOAA Mussel Watch program26 (Figure 4). Some of the oysters samples collected for the current investigation were from the vicinity of the three locations where samples were collected for the NOAA Mussel Watch program in 2008 (Figure 1). Compared with the average 10-year nationwide data, the PAH levels in the oyster samples from the Oil Spill affected areas were similar or even less (Figure 4). So was the case when compared with the average PAH levels in the oysters from the Mississippi Gulf Coast prior to the Deepwater Horizon Oil Spill.

(4%) at above the IDL for the oyster samples (Figure S3 of the Supporting Information). The PAHs with molecular weights higher than benzo(a)anthracene (MW = 228) were detected at above the IDL in less than 25% of the tested samples for all four sample types. As shown in Figures 3 and 4, for each matrix tested the concentrations of all 25 target PAHs were at far below the public health Levels of Concern (LOC) established jointly by NOAA/ FDA/Gulf Coast states under the protocol to reopen state and federal waters (Table 2). Of all 25 PAHs, benzo(a)pyrene has the lowest LOC values of 35 ug kg−1 for finfish, 132 ug kg−1 for shrimp and crab, and 143 ug kg−1 for oyster. However it is important to note that even this lowest threshold was never exceeded in any sample. In fact, the maximum benzo(a)pyrene concentration detected was below the IDL (0.5 ug kg−1), 6.35 ug kg−1, 1.38 ug kg−1, and 1.11 ug kg−1 for finfish, shrimp, crab, and oyster, respectively. This is also true for all other PAHs measured in this investigation; all were typically at least 2 orders of magnitude below their LOC values. The levels of total PAHs detected in the fish samples were statistically consistent temporally until January 2011 (average: 16 ug kg−1) followed by a significant decrease (P value