Market Basket Survey of Arsenic Species in the Top Ten Most

Article Cite This: J. Agric. Food Chem. 2019, 67, 8253−8267

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Market Basket Survey of Arsenic Species in the Top Ten Most Consumed Seafoods in the United States Mesay Mulugeta Wolle,* Sarah Stadig, and Sean D. Conklin

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Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 5001 Campus Drive, College Park, Maryland 20740, United States ABSTRACT: The study focused on the determination of arsenic species in the top ten most consumed seafoods in the United States. Fifty-four samples were collected from local supermarkets, and their species identities were confirmed by DNA barcoding. The total arsenic in the samples varied greatly in the range of 8−22200 ng/g (wet mass). Speciation analysis based on extraction of water-soluble and nonpolar arsenic showed that inorganic arsenic (iAs) was found only in clams and crabs, while arsenobetaine (AsB) predominates in most samples. Among the other arsenicals, trimethylarsoniopropionate (TMAP) was found in most matrices with higher concentrations in crabs, and arsenosugars existed in most clams and crabs. Nonpolar arsenic accounted for 1−46% of the total arsenic in the samples. The accuracy of the analytical results was evaluated using standard reference materials and spike recovery tests. The survey showed that the iAs concentrations in America’s most consumed seafood products are much lower than the tolerable intake set by the Joint FAO/WHO Expert Committee, even at the highest levels found in this study. KEYWORDS: arsenic, DNA barcoding, seafood, speciation, survey juice,20 and wine21). However, the complex distribution of arsenic in seafoods suggests that risk assessment based only on iAs monitoring may not provide sufficient information14 as it leaves arsenicals of unknown or potential toxicity unidentified. It may also be difficult to certainly report the level of iAs in the product without ensuring that the unidentified fraction is free from iAs. Hence, meaningful risk assessment should aim at capturing a full picture of the distribution of arsenic species.14 Such comprehensive speciation analysis also helps us to understand the metabolic process of the analyte in the matrix and to initiate further investigations on the characterization of the chemical identities and/or toxic effects of unknown species. The U.S. is one of the largest seafood markets in the world where consumers have a wide choice of locally sourced as well as imported products.22 The National Fisheries Institute (NFI) reported that, among the wide variety of seafoods available in the marketplace, the top ten most consumed products represent more than 90% of the seafood consumption in the country.23 NFI identifies the top ten products based on per-capita consumption. Over the last nine years, where the list of the top ten seafood products has been consistent, the total annual per-capita consumption for the products ranged between 14.4 and 16.0 pounds. Throughout these years, shrimp is the leading product followed by salmon and canned tuna; the three represented more than 50% of total consumption. The other products in the list are tilapia, pollock, pangasius (swai), cod, crab, catfish, and clams. Considering their consumption, these products should be monitored for the levels of arsenic and its species.

1. INTRODUCTION Arsenic (As) is introduced to the environment from natural sources, such as volcanic activity and the weathering of minerals, and through a range of anthropogenic causes including smelting, fossil fuel combustion, pesticide use, and wood preservation.1,2 Humans are exposed to arsenic primarily through drinking water and dietary ingestion.2 Seafoods, defined here as marine and freshwater fish and shellfish, have long been known as significant sources of dietary exposure to arsenic.3 The Total Diet Study (TDS) from the United States (U.S.) Food and Drug Administration (FDA)4 and other reports5 show that about 90% of the arsenic in U.S. diets comes from seafoods. To the general public, the words “arsenic” and “toxic” are synonymous; however, the toxicity of arsenic is related to its chemical forms. Seafoods accumulate arsenic in a wide range of forms,6 among which arsenobetaine (AsB), a nontoxic and chemically stable species,7 is predominant in most cases.3,6 Inorganic arsenic (iAs; the sum of arsenite, As3+, and arsenate, As5+), classified as a group 1 carcinogen,8 is usually present at low levels.9,10 High levels of iAs, however, have been found in bivalves11−13 and crustaceans.13 Arsenoribosides (also known as arsenosugars) and arsenolipids, whose potential toxicological effects have yet to be fully elucidated,6,14 exist as major arsenicals in molluscs6,14,15 and oily products,6 respectively. Seafoods also contain other low molecular weight organo-arsenicals such as arsenocholine (AsC), dimethylarsinic acid (DMA), dimethylarsinoylacetic acid (DMAA), dimethylarsinoyl ethanol (DMAE), methylarsonic acid (MMA), tetramethylarsonium ion (TMA), trimethylarsine oxide (TMAO), and trimethylarsoniopropionate (TMAP), often as minor constituents.6 Speciation-based arsenic monitoring in seafoods often focuses only on iAs due to its established toxicity.9−11,16−18 Such an approach may be appropriate for products that accumulate arsenic in a few forms of known properties (e.g., rice,19 This article not subject to U.S. Copyright. Published 2019 by the American Chemical Society

Received: Revised: Accepted: Published: 8253

April 15, 2019 June 19, 2019 June 26, 2019 July 11, 2019 DOI: 10.1021/acs.jafc.9b02314 J. Agric. Food Chem. 2019, 67, 8253−8267

Journal of Agricultural and Food Chemistry

Article

dimethylarsinoyl acetate (DMAA), dimethylarsinoyl ethanol (DMAE), dimethylarsinoyl propionate (DMAP), and trimethylarsoniopropionate (TMAP) were purchased from the Institute of Chemistry, University of Graz, Austria. Solutions of arsenocholine (AsC), tetramethylarsonium ion (TMA), trimethylarsine oxide (TMAO), and glycerol-, sulfonate-, sulfate- and phosphate-arsinoylribosides (As-328, As-392, As-408 and As-482, respectively) were kindly obtained from FDA’s Forensic Chemistry Center in Cincinnati, Ohio. Spiking solutions were prepared with the following compositions: (I) 1000 ng/g AsB, (II) 200 ng/g As3+, (III) 200 ng/g As5+, (IV) 2.0 ng/g As-328, As-392, As-408, and As-482, and (V) 200 ng/g of each of the remaining species, i.e., AsC, DMA, DMAA, DMAE, DMAP, MMA, TMA, TMAO, and TMAP. All solutions were prepared in deionized water and kept at 4 °C. 2.3. Instrumentation. DNA was extracted from tissue samples with commercial extraction kits (QIAGEN), and Sanger sequencing was performed with a 3500 xL genetic analyzer (Applied Biosystems). Robot Coupe Blixer 3 or Retch GM200 food processor-type blenders were used to homogenize the samples. Aqueous extractions were performed in a DigiPREP MS 48-position hot block from SCP Science. A Multi-Purpose Rotator (Thermo Scientific) was used to shake samples for nonpolar extraction. Organic solvent was evaporated from nonpolar extracts using a benchtop vacuum concentrator (CentriVap, Labconco). Samples and extracts were acid-digested in UltraClave or UltraWave microwave system (Milestone). An Agilent 7900 ICP-MS equipped with a concentric nebulizer, Scott-type double pass spray chamber, Ni cones, octopole reaction system, quadrupole mass analyzer, and an orthogonal detector was used. The ICP-MS was tuned on the day of every analysis to ensure sufficiently low levels of oxide and doubly charged ions. Analyses were conducted in helium collision mode (4.5 mL/min) to avoid polyatomic interference from 40 35 + Ar Cl on 75As+. The operating conditions of the instrument were RF power (1550 W), RF matching (1.8 V), sampling depth (8 mm), plasma and carrier flow (15 and 1.0 L/min, respectively), and spray chamber temperature (2 °C). Ultrahigh purity (99.999%) argon (Roberts Oxygen Company) and helium (Airgas) were used. The HPLC instrument was an Agilent 1260 equipped with a temperaturecontrolled autosampler, a binary pump, and a vacuum degasser. For total arsenic determination by ICP-MS, analytical solutions were introduced using an ASX-500 Series autosampler (Agilent Technologies) and mixed with a germanium internal standard solution (20 ng/g) in a Teflon tee-fitting. Data were acquired using Agilent MassHunter software in spectrum mode at a 0.5 s integration time. The HPLC-ICP-MS system was set up by connecting the LC column outlet with the ICP-MS nebulizer inlet. The system was operated using Agilent MassHunter software, and data were acquired in timeresolved analysis (TRA) mode at 1.0 s integration time. A 10 ng/g arsenic solution injected post-column was used as internal standard to compensate for instrument drift over the course of the batch. The chromatographic conditions used with ICP-MS are described in Table 1. 2.4. Samples and Reference Materials. To get some diversity in geographical supply, samples of each of the top ten seafoods were purchased from local supermarkets in three different areas, namely, Lanham (Maryland), Lenexa (Kansas), and Alameda (California), see Table 2. The samples were collected in 2017. The list includes 18 domestic and 24 imported products and 12 with no specified origin (i.e., country of origin could not be determined from the label or sample information). Attempts were made to represent the available species of shrimp, salmon, tuna, tilapia, cod, clams, and crab in the sampling. Reference materials of tuna fish tissue (BCR 627) from the Institute for Reference Materials and Measurements in Belgium, fish protein (DORM-4) from the National Research Council of Canada, and mussel tissue (SRM 2976) from the U.S. National Institute of Standards and Technology (NIST) were used to evaluate the accuracy of the method. All the reference materials were certified for total arsenic, and additionally, BCR 627 was certified for AsB and DMA and DORM-4 for AsB. 2.5. Sample Homogenization. Samples (except canned) were shipped frozen overnight to the analysis laboratory in College Park, Maryland. After thawing, inedible parts (shells, skin, bones, etc.)

Market basket surveys help to evaluate the level of target substances and their estimated intake through the food supply. The FDA, in its TDS program, regularly monitors contaminants and nutrients, in foods and beverages collected from different parts of the country.24 The study, however, targets total arsenic and includes only a few of the top ten seafoods in its list. Survey studies on arsenic species in seafood products from the U.S. are also rare in the literature. In 1999, Schoof et al.16 reported a market basket survey of iAs in 40 food commodities including fish (i.e., canned tuna, catfish, and trout) and shrimp. Similarly, total and inorganic arsenic were monitored in fish and shellfish commonly harvested in the U.S. Territory of American Samoa17 and in estuarine waterbodies of the U.S. Mid-Atlantic region that support recreational and commercial fishing.25 Surveys of arsenic species were conducted in seafoods from other parts of the world, including Norway,9,11,26 France,13 Spain,18 Belgium,27,28 China,29 Italy,30 Japan,31 the western Arabian Gulf,32 and European marine ecosystems.18 In some of these studies, samples were collected based on their consumption level13,29−31 or commercial significance.26,28,30,32 Most of the studies monitored total and inorganic arsenic along with organo-arsenicals, such as DMA, MMA, and AsB,13,27−29,32 and arsenosugars,28,32 while the others focused only on total arsenic,26 total and inorganic arsenic,9,11,18 and arsenolipids.31 In the present study, a market basket survey of arsenic species was conducted in America’s top ten most consumed seafoods. A total of 54 samples were purchased from local supermarkets in three different parts of the country. The species identities of the samples were confirmed based on DNA barcoding. The samples were analyzed for total arsenic, water-soluble arsenic compounds, and total nonpolar arsenic using a method recently developed33 and single-lab validated34 by the FDA. The method involves stepwise extraction of water-soluble and nonpolar arsenic followed by speciating the arsenic in the aqueous extracts by anion and cation exchange high pressure liquid chromatography−inductively coupled plasma mass spectrometry (HPLC-ICP-MS). To the authors’ knowledge, this study is unique in that it represents the most comprehensive survey both in terms of the most relevant seafoods for U.S. consumers and the number of arsenic species evaluated.

2. MATERIALS AND METHODS 2.1. Safety. iAs is a carcinogen, and care must be taken to avoid exposure. Lab coat, safety glasses, gloves and fume hood should be used for adequate protection. Waste should be considered hazardous and disposed of accordingly. 2.2. Reagents and Standards. Optima grade nitric acid (HNO3, 67−70%), hydrogen peroxide solution (H2O2, 30−32%), acetone (certified ACS), ethyl acetate (certified ACS), and petroleum ether (pesticide grade) were purchased from Fisher. Methanol (HPLC grade) and 2-propanol (electronic grade) from J. T. Baker Chemicals, dichloromethane from Honeywell Burdick & Jackson, ammonium carbonate ((NH4)2CO3, 99.999% purity) from Alfa Aesar, ammonium bicarbonate (NH4HCO3, ≥ 99.0% purity) from Sigma-Aldrich, and pyridine (HPLC grade), diethyl ether (ACS reagent, anhydrous), and N-ethylmaleimide (99+%) from Acros Organics were used. Water deionized to >18 MΩ cm (Milli-Q Element, Millipore) was used throughout the experiment. A 10 μg/mL stock solution of arsenic from High Purity Standard and 1000 μg/mL separate stock solutions of As3+ and As5+ from Inorganic Ventures were used. AsB standard was obtained from the Institute for Reference Materials and Measurements of the European Commission’s Joint Research Centre. Salts of monomethylarsonic acid (MMA, 98.5% purity) and dimethylarsinic acid (DMA, 98.9% purity) were purchased from Chem Service. Standards of 8254

DOI: 10.1021/acs.jafc.9b02314 J. Agric. Food Chem. 2019, 67, 8253−8267


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Table 1. Chromatographic Methods Used in the Present Study LC guard column analytical column mobile phases gradient profile

column temperature autosampler temperature sample diluent injection volume

anion exchange

cation exchange

PRP-X100, Hamilton PRP-X100 (10 μm, 4.1 × 250 mm), Hamilton (A) 5 mM NH4HCO3; (B) 50 mM (NH4)2CO3 0−15 min (3.5% B, 1.0 mL/min), 15.5−24 min (80% B, 1.0 mL/min), 24.5−36 min (99% B, 1.0 mL/min), 36.5−40 min (3.5% B, 1.5 mL/min) ambient 4 °C deionized water 50 μL

Metrosep C4 Guard/4.0, Metrohm Metrosep C6 (5 μm, 4.0 × 250 mm), Metrohm (A) deionized water; (B) 50 mM pyridine (both at pH 2 with HNO3) 0−22 min (0% B, 0.7 mL/min), 22.5−34 min (10% B, 1.0 mL/min) and 34.5−44 min (0% B, 1.2 mL/min) ambient 4 °C mobile phase A 50 μL

Table 2. List of the Seafood Samplesa seafood shrimp

salmon

canned tuna

sample ID

common nameb

shrimp 1 shrimp 2

Whiteleg shrimp mix (brown, pink, white) shrimp brown shrimp* unknown rainbow shrimp brown shrimp whiteleg shrimp pink shrimp

Litopenaeus vannamei Farfantepenaeus aztecus, Farfantepenaeus duorarum, Litopenaeus setiferous Farfantepenaeus aztecus no PCR amplification Parapenaeopsis sculptilis Farfantepenaeus aztecus Litopenaeus vannamei Farfantepenaeus duorarum

Maryland Maryland

Belize USA

Kansas Kansas Kansas California California California

USA unspecified Indonesia USA India USA

Atlantic salmon* sockeye salmon sockeye salmon* Atlantic salmon* pink salmon* sockeye salmon* Atlantic salmon* pink salmon* Chunk Light tuna**

Salmo salar Onchorhynchus nerka Onchorhynchus nerka Salmo salar Oncorhynchus gorbuscha Oncorhynchus nerka Salmo salar Oncorhynchus gorbuscha no PCR amplification

Maryland Maryland Kansas Kansas Kansas California California California Maryland

Canada USA USA Chile unspecified USA Chile USA unspecified

farmed, peeled wild, labeled American white shrimp wild, gulf shrimp wild, gulf shrimp wild, tiny shrimp, canned wild, large shrimp farmed wild, large shrimp, peeled, deveined farmed, whole fish wild, canned wild, fillets farmed, fillets, color added wild, canned wild farmed, color added wild, canned canned in water

no PCR amplification

Maryland

unspecified

canned in water

no PCR amplification no PCR amplification no PCR amplification no PCR amplification no PCR amplification Oreochromis niloticus, Oreochromis mossambicus, Oreochromis aureus Oreochromis niloticus Oreochromis aureus Oreochromis niloticus, Oreochromis mossambicus

Maryland Kansas Kansas California California Maryland

Indonesia Thailand unspecified Thailand unspecified China

canned in water wild, canned in water canned in water canned in water packed in pouch farmed, fillets, frozen

Kansas Kansas California

China China Indonesia

farmed, fillets farmed, fillets farmed

pollock 1 pollock 2 pollock 3 swai 1 swai 2 swai 3 cod 1 cod 2 cod 3

Chunk Light Yellowfin tuna** white albacore tuna** Chunk Light tuna** white albacore tuna** Chunk Light tuna** white albacore tuna** mix (Nile, Mozambique, Blue) tilapia Nile tilapia blue tilapia mix (Nile, Mozambique) tilapia walleye pollock* walleye pollock* walleye pollock* swai* swai* swai* Atlantic cod Pacific cod unknown

Maryland Kansas California Maryland Kansas California Maryland Kansas California

USA USA unspecified Vietnam Vietnam Vietnam Iceland USA China

fillets, breaded, frozen fillets wild, breaded, beer battered fillets, frozen farmed, fillets fillets wild, fillets fillets, frozen wild, fillets

crab 1

snow crab*

Gadus chalcogrammus Gadus chalcogrammus Gadus chalcogrammus Pangasianodon hypophthalmus Pangasianodon hypophthalmus Pangasianodon hypophthalmus Gadus morhua Gadus macrocephalus Gadus morhua, Gadus microcephalus, Boreogadus saida Chionoecetes opilio

Maryland

USA

crab crab crab crab

blue crab* golden king crab* swimming crab snow crab*

Callinectes sapidus Lithodes aequispinus Portunus pelagicus Chionoecetes opilio

Maryland Kansas Kansas Kansas

USA unspecified Philippines Canada

legs and body meat, cooked, frozen jumbo lump legs wild, lump, pasteurized crab clusters, frozen

shrimp shrimp shrimp shrimp shrimp shrimp

3 4 5 6 7 8

salmon salmon salmon salmon salmon salmon salmon salmon tuna 1

1 2 3 4 5 6 7 8

tuna 2

tilapia

tuna 3 tuna 4 tuna 5 tuna 6 tuna 7 tilapia 1 tilapia 2 tilapia 3 tilapia 4

pollock

swai

cod

crab

2 3 4 5

scientific namec

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origin

remarks



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Table 2. continued seafood

catfish

clams

sample ID

common nameb

scientific namec

sampling area

origin

crab 6 crab 7

swimming crab swimming crab

Portunus pelagicus Portunus pelagicus

Kansas California

Indonesia Philippines

crab 8 crab 9 crab 10 catfish 1 catfish 2 catfish 3 clam 1 clam 2 clam 3 clam 4

dungeness crab* imitation crab** swimming crab channel catfish channel catfish channel catfish Manila clam softshell clam Ocean Quahog clam New Zealand cockle clam

Metacarcinus magister Gadus chalcogrammus Monomia haanii Ictalurus punctatus Ictalurus punctatus Ictalurus punctatus Ruditapes philippinarum Mya arenaria Arctica islandica Austrovenus stutchburyi

California California California Maryland Kansas California Maryland Maryland Kansas California

clam 5

unknown

no PCR amplification

California

unspecified USA Vietnam USA unspecified USA unspecified unspecified USA New Zealand USA

remarks lump, canned, pasteurized wild, jumbo lump, pasteurized whole cooked made from pollock lump, canned farmed, nuggets farmed, fillets farmed, fillets wild, whole baby, canned fresh steamer wild, canned, chopped wild, littlenecks wild, canned in clam juice, chopped

a

Samples are listed in order of their placement in the latest top ten most consumed seafood list from NFI.23 bFDA common names with one asterisk are from sample labels and verified by DNA barcoding, and those with two asterisks are from sample labels only. All the other common names are from DNA barcoding only. cScientific names were identified based on DNA barcoding. Shrimp 4 could not be barcoded due to bacterial contamination which resulted from a storage error prior to analysis. Most canned samples could not be barcoded due the DNA being too sheared.

and breading were removed, and canned samples were drained. The samples were then homogenized to smooth paste in a food processor and stored at −30 °C without freeze-drying. 2.6. DNA Barcoding. Portions of individual samples were taken for DNA barcoding prior to homogenization. DNA was extracted from tissue samples and amplified according to previously validated polymerase chain reaction (PCR) conditions.35−37 PCR products were verified on precast, 2% agarose gels. PCR product cleanup, cycle sequencing, and cycle sequencing product cleanup were performed under conditions listed in the validated methods.35,36 Sequences were analyzed in Geneious software with quality control steps described previously.35,36 Unknown sequences were identified using the FDA Reference Standard Sequence Library (RSSL) for seafood38 where possible. Species not contained in the RSSL were identified using public sequence libraries such as BOLD39 or GenBank.40 2.7. Total Arsenic Determination. The total arsenic concentration in the seafood samples was determined according to the method in the FDA’s Elemental Analysis Manual (EAM) Section 4.7.41 The test material was weighed out (0.25 g dry or 0.5 g wet) into Teflon microwave vessels in triplicate. A fourth portion was weighed out from selected samples and spiked with 0.1 g of a 10 μg/mL arsenic solution. Concentrated HNO3 (5 mL) and 30% hydrogen peroxide (1 mL) were added into the vessels, and the mixtures were heated in a microwave at 250 °C for 15 min with a 30 min linear ramp. Digests were cooled to room temperature, quantitatively transferred to polypropylene tubes (VWR), gravimetrically diluted to 50 g, and analyzed by ICP-MS. 2.8. Stepwise Extraction of Arsenic. 2.8.1. Extraction of Water-Soluble Arsenic. The test material (0.25 g dry or 1.0 g wet) was weighed out into 50 mL polypropylene centrifuge tubes in triplicate. A fourth portion was weighed out from selected samples and spiked with solutions of arsenic species (due to limited supply, the arsenosugars were spiked into small volumes of the fortified extracts). After adding deionized water (15 g) into each tube, mixtures were vortexed and then heated in a hot block at 90 °C for 30 min with a 45 min linear ramp. Extracts were cooled to room temperature and centrifuged at 3000 rpm for 10 min. Supernatants were carefully decanted, filtered through a 0.45 μm pore size polyvinylidene difluoride syringe filter (Whatman, GE Healthcare Life Sciences), and kept at 4 °C. The extracts were analyzed by anion and cation exchange HPLC-ICP-MS after 3-fold dilution as described in Table 1. Portions of the extracts were also acid-digested and analyzed by ICP-MS (EAM Section 4.7)41 to determine the total water-soluble arsenic. 2.8.2. Extraction of Nonpolar Arsenic. The solids left from aqueous extraction of the nonspiked samples were rinsed with three

portions of deionized water (to remove residual extract) and dried in an oven at 60−80 °C. The dry solids were mixed with 5 mL of a 2:1 (v/v) dichloromethane−methanol mixture and gently shaken for 60 min at room temperature. Supernatants were filtered into disposable borosilicate glass microwave vessels (VWR) and heated to dryness in a centrifugal evaporator at 45 °C. The solids left in the glass vessels were mixed with concentrated HNO3 (5 mL) and 30% hydrogen peroxide (1 mL) and digested in an UltraWave to determine the total nonpolar arsenic by ICP-MS (EAM Section 4.7).41 2.9. Analyte Quantification. Analytes were quantified based on external calibrations constructed using standards prepared on the day of the analysis. For total arsenic determination in the acid-digested samples and extracts, calibration standards (1.0−200 ng/g) were prepared by serially diluting the 10 μg/mL arsenic stock. For chromatographic analyses of aqueous extracts, calibration standards (0.5− 25 ng/g) were prepared in the dilution solvents (see Table 1) from the spike solutions of arsenicals (arsenosugars not included due to limited supply). All dilutions were made gravimetrically. Data acquisition, chromatographic peak integration, and analyte quantification were performed using Agilent MassHunter software. Data were normalized to the germanium internal standard (ICP-MS) or the post-column injected standard (HPLC-ICP-MS). Further calculations were performed using Microsoft Excel. Arsenicals with no available standards were quantified using the calibration curves of the nearest eluting species. Analyte concentrations were reported as mass fractions of arsenic. 2.10. Quality Control. Each sample digestion and extraction batch consisted of three portions of a sample, fortified portions of samples selected based on matrix type, three procedural blanks, a fortified blank, and one or more reference materials (in triplicates).

3. RESULTS AND DISCUSSION 3.1. DNA Barcoding. Methods have been validated for barcoding the “standard” cytochrome c oxidase 1 (COI) segment for fish35 and crustaceans.36 Fortunately, the crustacean method employs universal invertebrate primers, which will amplify most invertebrate species (clams in the present case). A method for barcoding the canned specimens was adapted from Leray et al.37 The results showed that, of the products that were identifiable using DNA barcoding, there were no instances of mislabeling. Furthermore, barcoding confirmed farmed versus wild samples of shrimp and salmon, as the products listed as farmed were the same species that are usually raised on farms. Likewise, nothing labeled as wild was identified as a primarily farmed species. There 8256



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are some wild harvests of L. vannamei and S. salar, but they are very rare and almost never seen in the U.S.. Certain canned samples (all tuna and clam-5) could not be barcoded as the DNA was likely fragmented into pieces during the canning process. The modified barcoding procedure utilized for these samples37 did successfully identify canned salmon and most canned shellfish. One shrimp sample (shrimp 4) was unidentifiable due to bacterial contamination which resulted from a storage error prior to analysis. 3.2. Total Arsenic. The concentrations of total arsenic in the seafood samples are summarized in Table 3. The concentrations varied greatly among individual samples, and the different seafood groups had values (ng/g) in the ranges of 236−7560 (shrimp), 90−348 (salmon), 756−2460 (tuna), 39−377 (tilapia), 830−1720 (pollock), 8−10 (swai), 581−4420 (cod), 94−22,200 (crab), 18−65 (catfish), and 748−2660 (clams). It can be seen that the matrices with the lowest arsenic levels were swai and catfish. Compared to the rest of the samples, the salmon and tilapia samples also had low total arsenic. On the other hand, most of the shrimp, tuna, pollock, cod, crab, and clam had more than 1000 ng/g arsenic, among which two crabs (5 and 7) had more than 10000 ng/g arsenic. On average, crabs contained arsenic at approximately 3 orders of magnitude higher than that of swai, 2 orders of magnitude higher than that of catfish, and an order of magnitude higher than those of salmon and tilapia. The wide variation of total arsenic level among seafoods may be due to several factors that include geographical and seasonal diversity,9,11−13,27 as well as habitat, diet, age, and size of organism.9,11,27,28 Seafoods feeding on organisms in a food chain based on macro-algae and those living on or close to the seabed are usually rich in arsenic.30 In the present case, the shrimp and crab species with high arsenic concentrations, such as brown shrimp (F. aztecus),42 snow crab (C. opilio)42,43 and swimming crab (P. pelagicus),43 belong to the benthic fauna living close to sandy and muddy bottoms. Previous studies also found high concentrations of arsenic in crabs13,27,29,44 and shrimps27,29,30 (their species identities were not specified). As expected, freshwater samples such as swai and channel catfish were found to have very low levels of arsenic. 3.3. Water-Soluble Arsenic. Optimization of the aqueous extraction step, which effectively maintains the integrity of arsenic species, is thoroughly discussed in a previous paper.33 The concentrations of total water-soluble arsenic in the samples were determined by analyzing the acid-digested extracts by ICP-MS. The swai samples were not included in this analysis due to their low total arsenic level (85%) extracted with water from all the tilapia, pollock, and cod samples. Similar extraction yields were obtained for most of the shrimp, tuna, and crabs; exceptions were shrimps 1 and 5, tuna 4, and crabs 2 and 10, which exhibited 19−65% extraction efficiency. Overall, the results of this study agreed with literature data for the fraction of water-soluble12,28,45−48 or polar and inorganic arsenic49 in shrimp,12,28,45−49 salmon,12,28 canned tuna,12 pollock,28 cod,28 and crab28 (the species identities of the seafoods were not specified in the studies). 3.4. Speciation Analysis of Water-Soluble Arsenic. The arsenic in the aqueous extracts was speciated by anion and

cation exchange HPLC-ICP-MS, see Table 1. The chromatographic methods were developed through in-depth evaluation of existing methods.33,34 As3+, As5+, DMA, DMAA/DMAP (coeluted), MMA, As-392, As-408, and As-482 were separated on the anion exchange column, and the cation exchange method separates AsB, AsC, DMA, DMAA, DMAP/DMAE (coeluted), TMA, TMAO, TMAP, and As-328. Analytes were identified by matching their retention times with those of standards. To address matrix effects, retention times were also verified by analyzing fortified analytical portions. Table 4 summarizes the concentrations of arsenic species found in the aqueous extracts. A total of 16 known and 12 unknown arsenicals were detected in the 51 samples (the swai samples were excluded due to their low total arsenic levels). While differences were observed in the distribution of arsenic species between the three groups of seafood, i.e., finfish, crustaceans, and mollusc, it can be seen from Table 4 that the crab and clam samples had the greatest diversity of arsenicals. Figure 1 shows chromatograms of a blue crab (crab 7) extract, which contained the largest number of arsenicals (including iAs, arsenosugars, and unknowns) among all the samples. 3.4.1. Inorganic Arsenic. A closer look at Table 4 shows that, regardless of the total arsenic level, iAs is either undetectable or exists at very low concentrations in the samples. Almost all the finfish and shrimps were free from detectable iAs. The arsenical was found in all the clams and crabs (except crab 9), but it was above the limit of quantification (LOQ) only in some of them. The highest level of iAs was found in golden king crab (crab 3; 145 ng/g). The present results are consistent with previous findings where iAs was either undetected or found at very low levels in several types of fish9,10,12,16,32,44,50,51 and shrimp10,32,51,52 but existed at relatively higher levels (mostly above 0.2 mg/kg) in crabs13 and clams.17,18,32,44,50 Relatively high levels of iAs have also been found in other seafoods such as mussel,11,12 oyster,12,13 and octopus.13 As can be seen from Table 4, in most of the samples where it was detected, iAs predominantly existed as arsenite (As3+). Such distinctive identification of As3+ and As5+ is not common in seafood analyses as studies often use extraction conditions that easily convert As3+ to As5+.9−11,18,51,52 For all the three seafood groups (finfish, crustaceans, and clams), no correlation was observed between the iAs and total arsenic concentrations. Since there is no specific guideline for seafood arsenic, potential exposure to iAs from samples with relatively high level of the arsenical was evaluated based on the provisional tolerable weekly intake (PTWI) set by the Joint FAO/WHO Expert Committee (15 μg iAs per kg of body weight).53 Conservative exposure estimate for the highest iAs level found here (145 ng/g) shows that even extreme seafood eaters (365 g seafood per day)10 would not be in danger of exceeding the PTWI; the weekly iAs intake for a 70 kg person will be 5.3 μg. To reach at the PTWI level, the same person of 70 kg would have to eat 1.03 kg of the crab (150 μg iAs) daily. 3.4.2. Arsenobetaine. Consistent with the literature,12,13,28,29 AsB, a chemically stable and nontoxic species, represented the highest fraction of the total arsenic in most of the samples, i.e., 44−94% (shrimp), 30−58% (salmon), 27−100% (tuna), 74− 92% (tilapia), 79−94% (pollock), 67−98 (cod), 42−94% (crab), and 24−69% (clam). Exceptions were rainbow shrimp (shrimp 5), sockeye salmon (salmon 2), swimming crab (crab 10), Manila clam (clam 1), and all catfish where AsB accounted for only 1−22% of the total arsenic. Several factors 8257



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Table 3. Concentrations of Total, Water-Soluble, and Non-Polar Arsenic in the Seafood Samples (n = 3; 95% CI)a total arsenic seafood

sample ID

shrimp

shrimp 1 shrimp 2 shrimp 3 shrimp 4 shrimp 5 shrimp 6 shrimp 7 shrimp 8 salmon 1 salmon 2 salmon 3 salmon 4 salmon 5 salmon 6 salmon 7 salmon 8 tuna 1 tuna 2 tuna 3 tuna 4 tuna 5 tuna 6 tuna 7 tilapia 1 tilapia 2 tilapia 3 tilapia 4 pollock 1 pollock 2 pollock 3 swai 1 swai 2 swai 3 cod 1 cod 2 cod 3 crab 1 crab 2 crab 3 crab 4 crab 5 crab 6 crab 7 crab 8 crab 9 crab 10 catfish 1 catfish 2 catfish 3 clam 1 clam 2 clam 3 clam 4 clam 5 BCR 627

salmon

tuna

tilapia

pollock

swai

cod

crab

catfish

clams

SRM’s

DORM-4

(ng/g) 333 1660 6320 2440 444 7560 236 2650 313 348 310 94 261 337 90 286 1130 756 2460 878 1510 1170 1060 377 39 146 173 1530 1720 830 8 10 8 581 2940 4420 6560 557 8360 7880 10100 4030 22200 8730 94 670 65 28 18 1480 1050 748 2660 988 4540 4800

± 21 ± 41 ± 211 ± 69 ± 40 ± 481 ± 28 ± 68 ±6 ± 17 ± 21 ±7 ± 31 ±5 ±8 ± 10 ± 24 ± 90 ± 138 ± 163 ± 65 ± 74 ± 39 ± 31 ±6 ±5 ±6 ± 74 ± 26 ± 72 ±3 ±1 ±1 ± 12 ± 117 ± 185 ± 613 ± 42 ± 607 ± 426 ± 164 ± 61 ± 760 ± 116 ±2 ± 24 ±2 ±6 ±1 ± 131 ± 41 ± 82 ± 109 ± 33 ± 44 ± 300

6270 ± 185 6870 ± 440

water-soluble arsenic (ng/g) 217 1700 5840 2120 156 7540 220 2430 211 124 229 67 160 247 93 210 1120 625 2790 288 1460 1040 989 402 37 128 178 1660 1790 760 − − − 494 3020 4090 6840 356 8076 7558 9605 3600 22256 8963 104 126 5 6 11 498 917 520 1585 855 4370 520012 410049 5508

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

22 49 134 157 9 242 31 63 14 22 24 2 10 39 8 6 172 55 61 23 56 177 51 5 3 4 5 79 65 59

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 108 347 36 19 297 286 256 185 6379 1231 32 13 1 1 2 83 17 17 14 44 147

nonpolar arsenic

(%) 65 102 92 87 35 100 94 92 68 36 74 71 61 73 103 74 98 83 113 33 97 89 93 107 96 88 103 108 104 92 − − − 85 103 93 104 64 97 96 95 89 100 103 111 19 7 23 64 34 87 70 60 87 91

± 83

(ng/g)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7 3 2 6 2 3 9 2 5 6 8 2 4 11 9 2 15 7 2 3 4 15 5 1 7 3 3 5 4 7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1 4 8 1 3 4 4 3 5 29 14 35 2 1 2 10 6 2 2 1 4 3

81 ± 1

8258

123 125 165 76 36 40 11 107 131 137 38 31 61 55 18 50 91 108 61 229 45 53 50 3 2 2 2 22 20 5 − − − 85 18 7 388 106 120 171 358 263 232 69 1 254 30 7 4 311 79 62 223 15 163

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

74 52 61 89 21 30 3 11 36 25 27 5 4 8 9 4 37 8 6 103 6 1 4 0 0 1 0 7 7 1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

73 13 6 72 51 128 57 190 44 182 53 0 19 1 1 2 323 23 8 23 19 18

378 ± 115

extraction efficiency

(%) 37 7 3 3 8 1 5 4 42 40 12 33 24 16 20 17 8 14 2 26 3 5 5 1 4 1 1 2 1 1 − − − 15 1 1 6 19 1 2 3 7 1 1 1 38 46 27 21 21 8 8 8 2 3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

22 3 1 4 5 0 2 0 12 7 9 6 2 2 11 2 3 1 0 12 0 0 0 0 0 1 0 0 0 0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

13 0 0 1 9 2 1 2 1 1 1 0 3 1 5 10 22 2 1 1 2 0

6±2

(%) 102 110 95 90 43 101 99 96 109 75 86 104 85 89 123 91 106 97 116 59 100 94 98 108 100 89 104 110 105 93 − − − 100 104 94 110 83 98 98 98 96 101 104 112 57 54 50 85 55 95 78 68 89 95

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

18 6 2 9 7 4 9 2 8 12 4 6 2 9 9 2 12 8 2 12 4 15 5 1 7 3 3 5 4 7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

13 3 8 1 7 2 3 3 4 30 14 34 4 2 5 14 24 2 3 1 5 3

87 ± 2



Article

Table 3. continued total arsenic seafood

water-soluble arsenic

nonpolar arsenic

extraction efficiency

sample ID

(ng/g)

(ng/g)

(%)

(ng/g)

(%)

(%)

NIST 2976

13423 ± 142 13300 ± 1800

12847 ± 830

97 ± 6

281 ± 53

2±0

99 ± 7

a Larger concentration values were rounded off. Percentages of water-soluble and non-polar arsenic were calculated relative to total arsenic (certified total arsenic for reference materials) based on non-rounded values. Bolded and underlined numbers represent certified and literature values, respectively.

Figure 1. (a) Anion and (b) cation exchange HPLC-ICP-MS chromatograms of an aqueous extract generated from blue crab (crab 7). IS represents the post-column injected standard peak.

predominance in these matrices as well as the metabolism and toxicity of the species as it appears that there is a significant exposure especially through the consumption of crabs. TMA was detected at relatively higher concentrations (25−90 ng/g) in a few samples (shrimp, cod, and crab), and MMA and TMAO were found almost exclusively in crabs. AsC, DMA, DMAA, and DMAE were present at trace levels throughout. Due to the lack of standards, many peaks could not be identified with certainty and remained “unknown”. These species were quantified using the calibration curves of the nearest eluting standards. The sum of the concentrations of the unknown arsenicals detected by anion and cation exchange chromatography are presented in Table 4. Swimming crab (crab 4) had the highest number of the unknowns, i.e., four anions and three cations (data not shown), and the total concentration of the unknowns was highest in Manila clam (clam 1) and brown shrimp (shrimp 6), i.e., 83 and 60 ng/g, respectively. The species eluted from the cation exchange column just before DMA (see Figure 1 (b)) primarily accounted for the high concentration of the unknowns in these samples. Our previous findings suggest that this species is likely a thiolated arsenical.56 Further investigations are required to characterize the unknown arsenicals as they represent non-negligible fractions of arsenic in some matrices. 3.5. Chromatographic Recovery. To evaluate mass balance in the separation and quantitation of water-soluble arsenicals, chromatographic (or column) recovery was calculated as the ratio of the sum of the concentrations of the chromatographed species (including unknowns) and the total arsenic concentration in the corresponding acid-digested aqueous extract. Table 4 shows that the chromatographic recovery values were in the ranges of 4−96% (shrimp), 58−89% (salmon), 86− 97% (tuna), 87−98% (tilapia), 83−92% (pollock), 85−98% (cod), 13−96% (crab), 20−108% (catfish), and 34−86%

may have contributed to the lower fraction of AsB in these matrices. In most cases, the fractions of water-soluble arsenic in these samples were too low such that either the individual water-soluble arsenicals including AsB were trace or the fraction of nonpolar arsenic exceeded AsB. As shown in Figure 2, in the finfish and crustaceans, the AsB concentration was directly correlated with the total arsenic in the corresponding matrix. No such correlation was observed for the clams which may be because the matrices contained other arsenicals (mainly arsenosugars) at relatively high fractions, see Table 4. Although it was predominant, the percentage of AsB in clams (median = 29%) was far below those in finfish (median = 77%), shrimp (median = 77%), and crab (median = 82%). 3.4.3. Arsenosugars. The other groups of arsenicals found in some of the seafoods were arsenosugars. The species were major components in most of the crabs and clams. Table 4 shows that, in particular, As-328 and As-482 were dominant in softshell and New Zealand cockle clams (clams 2 and 4, respectively). These two arsenosugars were also found at relatively higher concentrations in swimming crab (crabs 4 and 7). As-392 and As-408 were detected in a few of the crabs and clams; all were below the LOQ. None of the finfish and shrimp samples were found to contain any of the arsenosugars. Several studies identified arsenosugars as major arsenicals in clams29,32,54 and crab.55 In precise agreement with the present results, Li et al.29 found As-328 and As-482 as major arsenicals in clams. It has been highlighted that the potential toxicological effects of arsenosugars need full elucidation.6,14 3.4.4. Other Water-Soluble Arsenicals. Among the other water-soluble arsenic species, TMAP was found in most matrices with elevated concentrations in crabs (130−800 ng/g). To the authors’ knowledge, this is the first study to identify TMAP as a major arsenical in a range of seafoods. It is suggested that future experiments should evaluate the reason for its 8259


2 ± 0.3

3±1 23 ± 1

8260

1 ± 0.3

1 ± 0.2

1 ± 0.2

9±1

cod 1

391 ± 23

3±1

13 ± 4

19 ± 5

660 ± 29

cod

pollock 3

16 ± 1 14 ± 3

1610 ± 18

pollock 1

pollock 2

1330 ± 106

1 ± 0.3 8 ± 0.4

4±1

6±2

4±2

5±1

9 ± 0.4

1 ± 0.3 3 ± 0.3

153 ± 4

tilapia 4

7±2

3±2

1 ± 0.3

9±3

7±2

4 ± 0.5 16 ± 2

85 ± 5

92 ± 2

92 ± 4

83 ± 2

90 ± 1

98 ± 3

92 ± 8

94 ± 2

97 ± 3

95 ± 5

91 ± 3

89 ± 2

87 ± 8

86 ± 3

74 ± 28

58 ± 10

73 ± 11

79 ± 1

76 ± 4

89 ± 3

81 ± 2

68 ± 4

94 ± 1

71 ± 11

96 ± 3

4±0

92 ± 2

94 ± 2

87 ± 3

1 ± 0.5

pollock

71 ± 1 79 ± 7

2±5

5±1

4 ± 0.2

4 ± 0.5

2 ± 0.2

30 ± 1

1±1

1 ± 0.1

5 ± 0.2

6±1

7 ± 0.3

12 ± 2

12 ± 6

1 ± 0.1 11 ± 2

1 ± 0.1

3 ± 0.2

1 ± 0.2 16 ± 1

108 ± 2

7±3

7±1

4±1

4 ± 0.2

4±1

4±1

30 ± 64

2 ± 0.2 36 ± 2

2±1

8±1 15 ± 1

tilapia 3

7 ± 0.1

1 ± 0.1

2 ± 0.3

3 ± 0.3

2 ± 0.3

30 ± 3

tilapia 2

347 ± 12

tilapia 1

1 ± 0.4 2 ± 5

2 ± 0.1

918 ± 44

tuna 7

tilapia

1 ± 0.2 2 ± 0.3 2 ± 0.4

1380 ± 57 1000 ± 146

tuna 5

2 ± 0.2 10 ± 1

tuna 6

4±2

2 ± 0.2

1 ± 0.2 4 ± 0.4 11 ± 1

2 ± 0.4

2450 ± 99

tuna 3

2±1

2 ± 0.6 2 ± 0.2

238 ± 29

2 ± 0.4

526 ± 5

tuna 2

tuna 4

1 ± 0.2

945 ± 123

tuna 1

tuna

1 ± 0.1 8 ± 1

108 ± 6

salmon 8

salmon 6 3 ± 0.4

1 ± 0.2

77 ± 6

salmon 5 2 ± 0.3 6 ± 1 11 ± 1

1 ± 0.1

48 ± 1

salmon 4

41 ± 6

1 ± 0.3

180 ± 18

salmon 3

156 ± 10

10 ± 1

73 ± 17

salmon 7

5 ± 0.7 5 ± 1

132 ± 12

salmon 1

salmon 2

28 ± 1

2 ± 0.2 20 ± 3

2 ± 0.4

2210 ± 73

56 ± 5

8±1

shrimp 8

salmon

3±1

2 ± 0.4

3 ± 0.3 10 ± 0.4

3±1

unknown unknown chromatographic recovery (%)e anionsc cationsd

5±3

As-482

150 ± 13

11 ± 1

As392 As-408

shrimp 7

43 ± 2

As-328

2 ± 0.3

4±1

37 ± 1

12 ± 2

3±1

TMAP

7140 ± 357

3 ± 0.2 19 ± 2

TMAO

shrimp 6

shrimp 5

1 ± 0.4

25 ± 2

TMA

1 ± 0.5 61 ± 3

MMA

3 ± 0.3

DMAP

2±0

2 ± 0.1

2 ± 0.4 2 ± 0.3 7 ± 2

1910 ± 100

4 ± 0.1

5390 ± 125

DMAE

shrimp 4

DMAA

shrimp 3

DMA 2 ± 0.7

3 ± 0.1

AsC

2 ± 0.5

AsB 145 ± 16

1 ± 0.1

iAsb

1270 ± 111

1 ± 0.3

As5+

shrimp 1

1 ± 0.1

As3+

shrimp 2

shrimp

seafood

Table 4. Concentrations (ng/g) of Water-Soluble Arsenic Species in the Seafood Samples and Chromatographic Recovery Values (n = 3; 95% CI)a

Journal of Agricultural and Food Chemistry Article


5±1

4±0

5 ± 1.2

4 ± 0.1

crab 5

8261

65 ± 14

17 ± 3

8±2

28 ± 1

48 ± 11

45 ± 10

clam 3

clam 4

clam 5

171 ± 9

66 ± 7

DORM-4

NIST 2976 16 ± 6

11051

81 ± 2

171 ± 9

1064

663

5412

4±5

53 ± 12

9 ± 10

862

23 ± 2

41051 127065

887065

677 ± 12

617 ± 9

150

180 ± 4

1 ± 0.2

20 ± 1

1 ± 0.1

5 ± 0.4

13 ± 11

1 ± 0.1

2±1

2±1

1030051

8680 ± 233 20 ± 11

3950

3890 ± 39

3900

3470 ± 60

678 ± 32

640 ± 18

412 ± 46

311 ± 4

12 ± 2

6±1

12 ± 1

3±1 4 ± 0.2

11 ± 3

6±1

2±1

11 ± 1

7±1

4 ± 0.2

2 ± 0.2

47 ± 7

3 ± 0.1

1 ± 0.3

8180 ± 887

20100 ± 5988

3050 ± 159

8260 ± 187

6470 ± 295

7050 ± 89

235 ± 5

5720 ± 76

8±2

2±1

DMA

DMAE

7±1

12 ± 1

7±1

28 ± 3

15 ± 2

2 ± 0.5

3 ± 1 17 ± 1

2 ± 0.3 24 ± 8

2 ± 0.3

2 ± 0.4 6 ± 2

DMAA

80 ± 6

50 ± 1

17 ± 1

1 ± 0.3

DMAP

57 ± 3

11 ± 1

2 ± 0.3

65 ± 2

88 ± 4

70 ± 5

TMA

9±1

12051

66 ± 4

47 ± 1

863

6312

8±2

TMAP

150 ± 7

67 ± 4 691 ± 26

470 ± 22

2 ± 1 385 ± 7

93 ± 3 132 ± 1

TMAO

12 ± 1

15 ± 3

1±1

1±2

As-328

2±1

2 ± 0.2

As-482

9±2

6±1

8 ± 2 12 ± 1

As392 As-408

60 ± 1

63 ± 5

33 ± 2

80 ± 8

16 ± 1

2962

99 ± 3

31 ± 2

3 ± 0.3

44 ± 2 103 ± 2

96 ± 3

5112

9±5

4±4

21 ± 1

7 ± 1 259 ± 28

349 ± 19

16 ± 2

8±5

92 ± 15

85 ± 17

12 ± 12

4±1

2 ± 0.4

15 ± 2

7±1

13 ± 1

26 ± 3

13 ± 2

2±1

9±1

6±1

9±1

1 ± 0.4

6±1

9±3

11 ± 12

14 ± 12

15 ± 2

3±1

1 ± 0.3 83 ± 27

2 ± 0.5

2 ± 0.3 36 ± 4

5±2

7±1

9±1

29 ± 4

12 ± 1

2 ± 0.1

2 ± 0.2

2±1

88 ± 2

91 ± 2

9862

10012

88 ± 4

86 ± 4

64 ± 3

86 ± 7

70 ± 6

34 ± 9

20 ± 3

108 ± 12

38 ± 25

13 ± 2

50 ± 22

96 ± 6

95 ± 1

91 ± 3

95 ± 2

94 ± 2

95 ± 2

72 ± 3

88 ± 1

97 ± 2

98 ± 4

unknown unknown chromatographic recovery (%)e anionsc cationsd

17 ± 2 735 ± 18 117 ± 58 269 ± 193

1862

32 ± 3

169 ± 18

126 ± 28

10 ± 4 11 ± 11

5±1

46 ± 13 11 ± 3 799 ± 240 47 ± 15 3 ± 1 9 ± 3 54 ± 19

1 ± 0.1 10 ± 21

1 ± 0.2

9±1

27 ± 6

2 ± 0.3 10 ± 1

2±1

3±1

7±2

MMA

a For certified reference materials, n = 6−9. Larger concentration values were rounded off. Bolded and underlined numbers represent certified and literature values, respectively. Numbers in italics (x): LOD < x < LOQ. biAs = As3+ + As5+ cSum of the concentrations of unknown arsenicals detected by anion exchange chromatography. dSum of the concentrations of unknown arsenicals detected by cation exchange chromatography. eChromatographic recovery (%) = (sum of water-soluble arsenic species/total water-soluble arsenic) × 100. All recoveries were calculated based on original nonrounded concentrations of species.

4±5

BCR 627

SRM’s

28 ± 1

8±2

17 ± 2

7±3

9±1

21 ± 2

clam 1

28 ± 5

2±1

clam 2

clams

catfish 3

catfish 2

catfish 1

catfish

crab 10

2±1

crab 8

3±0

3 ± 0.2

crab 7

crab 9

3±1

10 ± 4

3±1

9 ± 3.1

crab 6

1±2

18 ± 12 145 ± 40

127 ± 30

crab 4

7±1

1 ± 0.5

crab 3

1±1

6±1

crab 2

crab 1

1 ± 0.5

3830 ± 261

crab

9±5

2870 ± 43

cod 3

iAsb

cod 2

As5+ AsC

As3+ AsB

seafood

Table 4. continued




Article

salmon, crab, and clams. Poor recoveries, however, were obtained for rainbow shrimp (shrimp 5), Atlantic salmon (salmon 7), imitation and swimming crab (crabs 9 and 10), Manila and New Zealand cockle clams (clams 1 and 4), and two catfish samples (1 and 3). The poor recoveries may be due to irreversible binding and/or nonretention of analytes on the column(s). To verify this, the aqueous extracts with poor chromatographic recoveries were analyzed by HPLC-ICP-MS with no column. For quality control purposes, three extracts with quantitative chromatographic recoveries (shrimp 8, salmon 3, and crab 3) were also analyzed. Figure 3 compares the percentage of arsenic detected in the chromatographed and direct-injected (with no column) extracts; recoveries were calculated relative to the total arsenic in the acid-digested extracts. For all the extracts with poor chromatographic recoveries (except salmon 7 and crab 9), the arsenic in the direct-injected extract was significantly higher than those quantified by chromatography. This confirmed that substantial amount of the watersoluble arsenic in these matrices was either irreversibly retained on the column or eluted without retention. Unretained components appear in the dead volume, whereas strongly bound analytes show no peaks. For the control samples, the fractions of arsenic in the direct-injected and chromatographed extracts were comparable, Figure 3. 3.6. Nonpolar Arsenic. The determination of nonpolar arsenic, which comprises arsenolipids and other water-insoluble species, serves two purposes. First, these groups of arsenicals (specifically arsenolipids) are reported to have potential toxicity and their determination is recommended.6,14 Second, it helps to confirm mass balance in the overall extraction of arsenic as leaving the water-insoluble arsenic unidentified creates information gap. The extraction condition for nonpolar arsenic was identified through evaluation of several organic solvents, see Figure 4. The solvents were mixed with methanol to increase the solubility of phospholipids as reported elsewhere.57 This nonpolar extraction was carried out after the aqueous extraction step (see Section 2.8) to avoid the dissolution of less polar water-soluble species into methanol. As can be seen from Figure 4, a 2:1 (v/v) mixture of dichloromethane and methanol provided better extraction from most of the matrices. In the samples analyzed in this study, nonpolar arsenic represented 1−40% (shrimp), 13−42% (salmon), 2−22% (tuna), 1−5% (tilapia), 1% (pollock), 1−16% (cod), 1−37%

Figure 2. Plots of AsB versus total arsenic concentration in the finfish, crustacean, and clam samples (one dot per sample).

(clams). Recoveries were sufficiently quantitative (>70%) for all the tuna, tilapia, pollock, and cod and for most shrimp,

Figure 3. Arsenic recovery in chromatographed and direct-injected (no column) aqueous extracts relative to the total arsenic concentration in acid-digested extracts. 8262



Article

Figure 4. Evaluation of solvents for nonpolar arsenic extraction from seafood.

and spiked with the analytes. It can be seen form Table 5 that the use of NEM significantly improved the recoveries of all the analytes susceptible to matrix-induced thiolation. In general, most of the recovery values in Table 5 were in the acceptance range set by CODEX59 and in the control limit (100 ± 20%) provided in FDA’s methods for arsenic speciation analyses in juice60 and rice.61 Here, it can be pointed out that the poor spike recovery of As3+ from some matrices (with no NEM) suggests that seafoods may contain thiol-bound arsenite which cannot be extracted by water. Determination of such tightly bound arsenic is challenging as it requires aggressive extraction conditions that potentially affect the chemical forms of other arsenicals. However, even if present, the thiolbound arsenite is unlikely to represent a significant exposure because the unextracted fractions of arsenic are relatively low in the tested samples. 3.8. Analysis of Reference Materials. The accuracy of the analytical data for total arsenic and its speciation was evaluated by analyzing certified reference materials of finfish (BCR 627 and DORM-4) and molluscs (NIST 2976). For total arsenic, the experimental results were 95%, 92%, and 101% of the certified values for the three materials, respectively (see Table 3). All the values were well within the CODEX acceptance criteria, i.e., 60−115% recovery for 10 μg/kg and 80−110% for 0.1−10 mg/kg.59 The water-soluble arsenic accounted for 92% (BCR 627), 80% (DORM-4), and 96% (NIST 2976) of the total arsenic. Only a small fraction of the total arsenic was extracted as nonpolar in the materials, i.e., BCR 627 (3%), DORM-4 (6%), and NIST 2976 (2%). Several studies found comparable values for water-soluble arsenic in BCR 627;12,62,63 however, data were not found for DORM-4 and NIST 2976. For arsenic species, the insufficiency in terms of having certified values only for two arsenicals has to be noted; BCR 627 was certified for AsB and DMA, and DORM-4 was certified for AsB. No seafood reference material is available with certified concentrations of iAs and other arsenicals. The production of reference materials for more arsenic species, especially iAs, is called upon as it provides a useful and mandatory tool in such analysis. The experimental results of AsB in BCR 627 and DORM-4 and of DMA in BCR 627 were 89%, 98%, and 122% of the certified values, respectively. The concentrations of the other arsenicals found in BCR 627 and NIST 2976 were compared with literature values; such data could not be found for DORM-4. Due to the abundance of literature results for arsenicals in BCR 627, those based on

(crab), 22−46% (catfish), and 2−20% (clams) of the total arsenic in the respective samples. On average, the highest nonpolar fractions were found in catfish (31%) and salmon (26%). Previous studies reported up to 4% and 9% nonpolar arsenic in shrimp and clam, respectively, extracted with acetone,49 and 1−4% arsenolipids in Gulf clam.32 Overall, the results of this study show that some seafoods contain nonpolar arsenic species at significant amounts. Future studies should focus on the characterization of the individual species in this fraction and investigation of their toxic effects. The mass balance (extraction efficiency) data in Table 3 show that stepwise extraction of water-soluble and nonpolar arsenic accounted for 44−110% (shrimp), 74−123% (salmon), 54−118% (tuna), 89−107% (tilapia), 93−109% (pollock), 93−104% (cod), 56−110% (crab), 46−83% (catfish), and 53−96% (clams) of the total arsenic. The nonquantitative extraction from some matrixes may be due to the presence of arsenicals strongly bound to the matrix (e.g., thiol-bound arsenic). Studies showed that extraction of such species require aggressive conditions,50,58 but this may affect the native chemical forms of analytes.33 For the purpose of this study, maintaining the chemical integrity of species took precedence over quantitative extraction with aggressive conditions. However, even using these relatively mild conditions, 75% of the samples had over 90% recoveries of water-soluble and nonpolar arsenic. 3.7. Spike Recovery. The spike recovery values for total arsenic were in the range 90−119% (data not shown). Spike recoveries for the 16 arsenic species are listed in Table 5. Except the four arsenosugars, all the arsenicals were spiked into the samples prior to extraction. Due to limited supply, the arsenosugars were spiked into small portions of extracts generated from the fortified samples. The values in Table 5 show that recoveries were mostly in the range of 70−125% for AsB, AsC, DMA, MMA, TMA, TMAP, As-328, As-392, As-408, and As-482. For As5+, 76−132% recoveries were obtained from the finfish and shrimp samples. The values were higher for clams and crabs may be due to As3+ oxidation. As3+, DMAA, DMAE, DMAP, and TMAO were poorly recovered from all the finfish and shrimps. As discovered previously,34,56 the poor recoveries were due to matrix-induced thiolation of the arsenicals. The poor recovery of the arsenicals from snow crab (crab 1) suggests that crabs are not entirely excluded from such effects. The effect of matrix thiols was prevented using a thiol-selective blocking agent, N-ethylmaleimide (NEM);34,56 samples were treated with 5 mL of a 1% (w/v) aqueous solution of NEM 8263


8264

As3+

87 (85) 9 (93) 8 (97) 69 (95) 16 (99) 47 (91) 48 (97) 44 (95) 67 (97) 68 (93) 24 (94) 10 (98) 28 (97) 44 (97) 10 (94) 20 (95) 15 (100) 34 (102) 15 (95) 4 (95) 17 (101) 14 (98) 25 (94) 83 69 (104) 24 (97) 32 (99) 21 (101) 72 80 92

sample

fortified blank shrimp 1 shrimp 3 shrimp 6 salmon 1 salmon 3 salmon 6 tuna 1 tuna 4 tuna 6 tilapia 1 tilapia 2 tilapia 4 pollock 1 pollock 2 pollock 3 swai 1 swai 2 swai 3 cod 1 cod 2 cod 3 crab 1 crab 3 crab 7 catfish 1 catfish 2 catfish 3 clam 1 clam 3 clam 4

114 (103) 108 (97) 121 (99) 103 (92) 87 (106) 106 (108) 87 (104) 76 (99) 111 (87) 77 (88) 86 (106) 107 (91) 105 (104) 132 (93) 123 (95) 97 (103) 119 (92) 108 (88) 126 (134) 109 (91) 112 (108) 129 (99) 148 (88) 189 124 (102) 110 (96) 85 (103) 122 (117) 239 149 208

As5+ 91 (88) 25 (93) 27 (98) 74 (94) 27 (100) 57 (94) 54 (98) 49 (96) 74 (95) 70 (92) 34 (97) 26 (96) 41 (98) 58 (96) 29 (94) 33 (97) 32 (99) 46 (99) 34 (102) 21 (94) 33 (103) 33 (98) 45 (93) 101 78 (103) 38 (97) 41 (100) 38 (104) 99 92 111

iAsb 100 102 115 97 113 100 102 98 114 112 113 100 95 105 99 105 100 103 100 97 110 113 74 71 107 102 104 100 99 96 84

AsB 100 100 103 106 105 102 102 95 100 105 101 85 99 96 88 99 96 98 98 93 91 112 89 85 100 99 90 96 77 92 95

AsC 99 101 100 101 101 100 100 96 101 97 97 101 96 113 111 100 111 103 101 104 101 99 105 103 98 108 107 106 102 106 110

DMA 91 (95) NC (100) NC (103) NC (107) NC (96) NC (106) NC (99) NC (97) NC (103) NC (97) NC (98) NC (105) NC (102) NC (97) NC (102) NC (102) NC (98) NC (99) NC (102) NC (99) NC (102) NC (101) NC (101) 104 71 (97) NC (103) NC (104) NC (100) 108 95 75

DMAA 98 (94) 7 (102) 28 (107) 42 (108) ND (98) 71 (115) 50 (98) 81 (108) 52 (103) 84 (108) 39 (96) 17 (121) 70 (104) 46 (93) 13 (97) ND (104) ND (109) 69 (105) ND (97) ND (94) 43 (115) 28 (110) 30 (105) 80 98 (94) 38 (104) 57 (113) ND (100) 85 77 91

DMAE 100 (105) ND (99) ND (97) 21 (97) ND (103) 15 (97) ND (98) ND (98) 47 (96) ND (99) ND (101) 25 (100) ND (98) ND (103) ND (97) 61 (98) ND (96) ND (102) 73 (103) ND (103) ND (101) 43 (98) ND (98) 95 122 (102) ND (103) 61 (105) 71 (104) 90 102 127

DMAP 101 116 104 102 107 103 104 90 99 95 109 99 97 114 102 102 114 102 102 100 103 103 107 108 99 105 102 105 105 105 106

MMA 97 101 102 98 109 100 99 96 104 101 102 98 94 95 98 94 96 101 92 94 97 89 97 94 98 98 88 95 90 92 103

TMA 99 (100) 6 (96) 16 (111) 27 (120) 9 (103) 35 (114) 15 (112) 29 (101) 45 (110) 26 (105) 19 (105) 22 (111) 36 (111) 30 (104) 10 (104) 29 (103) 3 (100) 39 (107) 21 (99) ND (103) 30 (106) 21 (104) 20 (91) 97 95 (112) 24 (104) 60 (111) 39 (102) 97 93 98

TMAO 100 99 105 104 107 102 97 101 107 104 102 104 100 95 101 103 97 105 100 99 104 110 93 95 92 104 104 101 101 99 102

TMAP 90 93 94 89 96 91 80 94 90 90 93 99 117 92 106 120 95 45 117 93 181 111 99 90 90 86 92 98 119 85 95

As-328 88 103 87 89 100 83 88 86 81 88 93 80 85 98 82 87 100 82 87 82 87 87 89 87 90 93 87 87 89 89 97

As-392 94 103 108 103 99 101 100 84 122 97 81 87 93 114 103 102 111 93 90 94 93 97 106 89 91 98 90 85 125 87 93

As-408

91 105 89 99 92 91 105 99 94 98 87 91 90 102 98 96 104 95 92 103 101 91 100 110 83 90 105 103 110 104 129

As-482

Numbers in parentheses are values for samples treated with N-ethylmaleimide (NEM). Spiking amount per a gram of sample: As3+ (200 ng/g, 0.5 g), As5+ (200 ng/g, 0.1 g), AsB (1000 ng/g, 0.2 g), and all other arsenicals except arsenosugars (200 ng/g, 0.5 g). NC: spike recovery of DMAA was not calculated due to its coelution with the transformation product of TMAO. ND: spiked analyte was not detected. biAs = As3+ + As5+

a

clams

catfish

crab

cod

swai

pollock

tilapia

tuna

salmon

shrimp

seafood

Table 5. Spike Recoveries (%) of Arsenic Species from Seafoodsa




Article

aqueous extractions were considered.12,62−64 Table 4 shows that good agreement was found between the present and the reported values for iAs58,63 and TMA.62 Two studies were found with values for iAs, AsB, DMA, and MMA in SRM 2976 based on dilute HNO351 and methanol−water65 extractions. The reported concentrations of iAs51 and AsB51,65 were in good agreement with the present values, see Table 4. As demonstrated previously,33 the high concentration of DMA in the acidic extract51 may be due to degradation of other arsenicals under such extraction conditions. To conclude, the seafood samples analyzed in this study exhibited large variation in their total arsenic levels and variable and complex distribution of arsenic species. Freshwater seafoods had the lowest total arsenic concentrations, while crabs and clams showed the most diversity in total arsenic and individual arsenic species. The survey looked at more arsenicals than those commonly targeted by most studies. Among the known and unknown arsenicals identified, AsB was predominant in most samples. iAs was detected only in clams and crabs, mostly as As3+. While clams and crabs were also found to be rich in arsenosugars, TMAP was found in most of the samples with relatively higher concentrations in crabs. Nonpolar arsenic existed at high fractions primarily in salmon and catfish. While most of the samples were successfully analyzed, low extraction efficiency and poor chromatographic recovery presented major obstacles to a more complete understanding of arsenic species in certain matrices. Future studies are needed to investigate the unextracted arsenic and to improve the column recoveries of arsenicals extracted from these samples. Moreover, since nonpolar arsenic was found to be a major fraction in some of the seafoods (e.g., catfish and salmon), further studies are needed to characterize the chemical forms and toxicities of the arsenic species in this fraction. Overall, the survey confirms that the levels of iAs in samples of the most commonly consumed seafoods in the U.S. are much lower than the tolerable intake set by the Joint FAO/WHO Expert Committee, even at the highest levels found in this study. The major arsenic species in the seafood samples in this study are organic forms with little or unknown toxicity. The presented data would be useful for several applications, including long-term arsenic exposure estimation studies.

■

inductively coupled plasma mass spectrometry; iAs, inorganic arsenic; IS, internal standard; LOD, limit of detection; LOQ, limit of quantification; MMA, methylarsonic acid; NEM, N-ethylmaleimide; NFI, National Fisheries Institute; NIST, National Institute of Standards and Technology; As-482, phosphate-arsinoylriboside; PCR, polymerase chain reaction; PTWI, provisional tolerable weekly intake; RSSL, Reference Standard Sequence Library; As-392, sulfonate-arsinoylriboside; As-408, sulfate-arsinoylriboside; TMA, tetramethylarsonium ion; TRA, time-resolved analysis; TMAP, trimethylarsoniopropionate; TMAO, trimethylarsine oxide; TDS, Total Diet Study; U.S., United States; WHO, World Health Organization

■

REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +1-240-402-5393. ORCID

Mesay Mulugeta Wolle: 0000-0001-5586-6643 Funding

The authors thank the Oak Ridge Institute for Science and Education (ORISE) for financial support. Notes

The authors declare no competing financial interest.

■

ABBREVIATIONS USED As5+, arsenate; As, arsenic; As3+, arsenite; AsB, arsenobetaine; AsC, arsenocholine; CI, confidence interval; COI, cytochrome c oxidase 1; DMA, dimethylarsinic acid; DMAA, dimethylarsinoylacetic acid; DMAE, dimethylarsinoyl ethanol; DMAP, dimethylarsinoyl propionate; EAM, Elemental Analysis Manual; FAO, Food and Agriculture Organization; FDA, Food and Drug Administration; As-328, glycerol-arsinoylriboside; HPLC, high pressure liquid chromatography; ICP-MS, 8265



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