Assessment of the bioavailability of toxic and non-toxic arsenic species in seafood samples

Food Chemistry 130 (2012) 552–560

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Assessment of the bioavailability of toxic and non-toxic arsenic species in seafood samples Jorge Moreda–Piñeiro a,⇑, Elia Alonso-Rodríguez a, Vanessa Romarís-Hortas b, Antonio Moreda-Piñeiro b, Purificación López-Mahía a,c, Soledad Muniategui-Lorenzo a, Darío Prada-Rodríguez a,c, Pilar Bermejo-Barrera b a

Department of Analytical Chemistry, Faculty of Sciences, University of A Coruña, Campus da Zapateira, s/n, 15071 A Coruña, Spain Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias, s/n, 15782 Santiago de Compostela, Spain c University Institute of Environment, University of A Coruña, Pazo de Lóngora, Liáns, 15179 Oleiros, Spain b

a r t i c l e

i n f o

Article history: Received 24 September 2010 Received in revised form 30 May 2011 Accepted 19 July 2011 Available online 23 July 2011 Keywords: In vitro bioavailability Simulated gastric and intestinal digestion Dialyzability Arsenic speciation Seafood Nutrient content

a b s t r a c t Bioavailability of total arsenic, toxic (arsenite, As(III); and arsenate, As(V)), and non-toxic (monomethylarsonic acid, MA; dimethylarsonic acid, DMA; arsenobetaine, AB; and arsenocholine, AC) arsenic species has been assessed in different raw seafood samples (white fish, cold water fish and molluscs) by using an in vitro model that combines simulated gastric and intestinal digestion/dialysis methods. Correlations between arsenic species bioavailability and seafood nutrient contents (fat and protein) have also been established. Total arsenic content in seafood samples, and dialyzable and non-dialyzable fractions, were analyzed by inductively coupled plasma – mass spectrometry (ICP–MS) after a microwave-assisted acid digestion treatment. The determination of the different arsenic species concentrations in the samples (after an optimised matrix solid phase dispersion (MSPD) approach) and in the dialyzable fraction was done by high performance liquid chromatography (HPLC) coupled to ICP-MS as a selective detector. Accuracy of the procedure (total arsenic determination) was assessed by analyzing DORM-2 and BCR-627 certified reference materials. The accuracy of the in vitro procedure was established through a mass-balance study. After statistical evaluation (95% confidence interval), good accuracy of the whole in vitro process, for total arsenic and for arsenic speciation, was observed. High dialyzability percentages for total arsenic and for arsenic species were found (i.e. from 84.6 ± 1.7% to 106 ± 2.6%). Bioavailability of arsenic exhibits a negative correlation with the fat content of the seafood. However, no correlation was observed between the bioavailable fraction of total arsenic and arsenic species and the protein content of the seafood studied. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Human intake of arsenic occurs mainly via seafood ingestion (Ysart et al., 2000). Arsenic in marine organisms is found at concentrations ranging from 1 to 100 mg kg1, as a result of bioaccumulation and bio-transformation processes (Leonard, 1991). Due to the wide-ranging levels of toxicity exhibited by the different arsenic species, knowledge of the actual forms present, rather than total arsenic, is necessary for assessing dietary risks (Merian, 1999). Therefore, the development of accurate and precise arsenic speciation procedures, for biological and environmental materials, is a current trend in analytical chemistry (Cullen & Reimer, 1989; Leermakers et al., 2006; McSheehy, Szpunar, Morabito, & Quevauviller, 2003). ⇑ Corresponding author. E-mail address: [email protected] (J. Moreda–Piñeiro). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.07.071

Bioavailability is a term used to describe the proportion of a nutrient in food that can be utilized for normal body function (Promchan & Shiowatana, 2005). Specifically, the term bioaccessibility indicates the maximum fraction of a trace element, or other substances, in food that is theoretically released from the matrix in the gastrointestinal tract, and thus available for intestinal absorption (i.e. enters the blood stream) (Oomen et al., 2002). In general, bioavailability/bioaccessibility is affected by the type and/or composition of food, the different cooking procedures of the foodstuff and, also, the simulated gastrointestinal conditions (Intawongse & Dean, 2006). The determination of trace element bioavailability/bioaccessibility in a foodstuff is mainly assessed by in vitro methods. Most in vitro methods involve two different steps: a gastric digestion stage, for simulating the stomach, and a subsequent intestinal digestion stage, for simulating the digestion in the intestine and absorption of nutrients. In vitro methods use conditions similar to those found

J. Moreda–Piñeiro et al. / Food Chemistry 130 (2012) 552–560

in the human body during digestion. These procedures can measure the bioavailable fraction through two different approaches. First, the in vitro method can assess the maximum concentration of soluble substances in the simulated gastrointestinal medium (simulated extract isolated from the treated foodstuff after a filtration/centrifugation step). In this case, the in vitro procedures determine the bioaccessible fraction. Second, some in vitro methods use a semipermeable membrane with a specified pore size (a certain molecular weight cut-off, MWCO) during the intestinal digestion step to simulate the nutrient absorption mechanism. In this case, the nutrient is assessed in the liquid medium inside the membrane (dialyzate fraction), and the in vitro methods commonly determine the dialyzable fraction of the nutrient. This last approach can involve equilibrium (Miller, Schricker, Rasmussen, & Van Campen, 1981) or non-equilibrium (Shen, Luten, Robberecht, Daeel, & Deelstra, 1994; Shiowatana, Kitthikhun, Sottimai, Promchan, & Kunajiraporn, 2006; Wolters et al., 1993) conditions during the intestinal digestion/absorption stage. Both in vitro methods provide an effective approximation to in vivo situations and offer the advantages of simplicity, rapidity, ease of control, low cost, high precision, and good reproducibility. From the first method developed by Miller et al. (1981) to predict the bioavailability of iron in different foods, the number of papers on the subject has increased considerably, and other essential and toxic elements have also been studied (Intawongse & Dean, 2006). However, there are no data on As species’ bioavailability based on assessing the dialyzate fraction in seafood (As speciation after an in vitro dializability procedure). For this element, studies are mainly focused on assessing bioaccessible As species in raw and cooked vegetables (rice grain, chard, radish, lettuce, mung beans) (Juhasz et al., 2006, 2008; Laparra, Vélez, Barberá, Farré, & Montoro, 2005; Rees et al., 2009), edible seaweed (Almela et al., 2005; Koch et al., 2007; Laparra, Vélez, Montoro, Barberá, & Farré, 2003; Laparra, Vélez, Montoro, Barberá, & Farré, 2004), and also seafood (Koch et al., 2007; Laparra, Vélez, Barberá, Montoro, & Farré, 2007; Dufailly, Guérin, Noël, Frémy, & Beauchemin, 2008). Raw and cooked seafood (sole and Greenland halibut and DORM-2 fish protein) were subjected to an in vitro gastrointestinal digestion by Laparra et al. (2007). The proposed procedure used pepsin for gastric digestion and pancreatin and bile extracts for intestinal digestion, and the bioaccessible fraction was then obtained by centrifugation before determination of AB, MA, DMA, TMAO and TETRA (tetramethylarsonium ion). Koch et al. (2007) have also assessed the bioaccessibility of As(III) and As(V) in soft shell clams by using glycine, pancreatin and bile extracts. Other bioaccessability approaches for As(III), As(V), MA, DMA, AB, AC and TMAO in seafood reference materials, such as TORT-2 (lobster hepatopancreas), CRM 627 (tuna fish), DOLT-3 (dogfish liver), and DORM-3 (fish muscle), were developed by Dufailly et al. (2008). The assessment of the bioaccessible fraction of As(III), As(V), MA, DMA, AB and TETRA in the abdominal muscle of yabbies (freshwater crayfish) was established by Williams, West, Koch, Reimer, and Snow (2009). One of the objectives of the current work has been the development of an in vitro procedure based on dialyzability for assessing the bioavailability of arsenic species (As(III), As(V), DMA and AB) in seafood. The establishment of dialyzability percentages for arsenic species from seafood has not yet been reported. In addition, as previously mentioned, food constituent can exhibit a great influence on the bioaccessibility/bioavailability, due to the fat, protein, carbohydrate and dietary fibre contents of food. These major components affect the solubility of minerals released in the digestive/ intestinal fluids (Intawongse & Dean, 2006). Therefore, an additional objective of the current work has been the study of the effects of major nutrient constituents in seafood (protein and fat) on the bioavailability of arsenic and arsenic species. To the best of our knowledge, the systematic investigation of the influence of

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major food constituents on the bioavailability of arsenic species in seafood has not been previously undertaken. 2. Materials and methods 2.1. Instrumentation HPLC-ICP-MS analysis was performed with a Dionex HPLC UltiMateO 3000 LC (Dionex, Sunnyvale, CA, USA), equipped with a GP50 gradient pump (Dionex), an AS50 thermal compartment (Dionex) and an AS50 auto-sampler (Dionex). Arsenic species separation was carried out with an IonPac AS7 (250  4 mm i.d.) anion-exchange column (Dionex) and a guard column IonPac AG7 (Dionex). The chromatographic system was coupled to an ICP-MS Thermo Finnigan X Series (Thermo Fisher Scientific Inc., Waltham, MA, USA). Total arsenic content was measured using an 820-MS inductively coupled plasma quadrupole mass spectrometre (Varian, Mulgrave, Australia) equipped with an SPS3 autosampler (Varian) and a MIcroMist nebulizer type (Varian). A Boxcult incubator situated on a Rotabit orbital-rocking platform shaker (J.P. Selecta, Barcelona, Spain) was used to control the temperature of the enzymolysis procedure. Cellu SepÒ H1 high grade regenerated cellulose tubular membranes (molecular weight cutoff 10 kDa, 50 cm in length, dry diametre 25.5 mm, and a volume to length ratio of 5.10 ml cm1) were from Membrane Filtration Products Inc (Seguin, TX, USA). A vibrating ball mill, Retsch (Haan, Germany), equipped with zircon cups (15 ml in size) and zircon balls (7 mm diametre), was used for sample pulverization. Seafood samples were freeze-dried by using a LYPH–LOCK 6 l freeze dry system, model 77530 from Labconco Corporation (Kansas City, MO, USA). An ORION 720A plus pH-metre with a glass-calomel electrode (Cambridge, UK) was used for pH measurements. Methanol/ultrapure water extracts from seafood samples (As speciation) were pre-concentrated by using a RotavaporÒ, Büchi R-210, equipped with a heating bath Büchi B-491 and a vacuum pump Büchi V-740 (Büchi Laboryechnik AG, Flawil, Switzerland). Finally, an Ethos Plus microwave lab-station (Milestone, Sorisole, Italy), with 100 ml closed Teflon vessels and Teflon covers, HTC adapter plate and HTC safety springs (Milestone), was used for acid digestion of seafood samples. 2.2. Reagents Ultra-pure water of 18 MX cm resistance was obtained from a Milli-Q purification device (Millipore Co.). Methanol (gradient grade) was from Merck (Poole, U.K.). Arsenite and arsenate stock standard solutions, 1000 mg l1, were from Panreac (Barcelona, Spain). Standard solutions of MA, DMA, AB and AC (1000 g l1) were prepared by dissolving the appropriate amounts of MA (CH3AsO(ONa2)6H2O), purchased from Carlo Erba (Milan, Italy), DMA (C2H6AsNaO23H2O), purchased from Merck, and AB (AsC5H11O2) and AC (AsC5H14O), both purchased from Tri Chemical Laboratory Inc. (Yamanashi, Japan). The organic arsenic standard solutions were stored in amber glass bottles and were kept at 4 °C. Diluted standard solutions were prepared daily from stock solutions. Digestive enzymes (porcine pepsin, p-7000, porcine pancreatin, P-1750), bile salts (approx. 50% sodium cholate and 50% sodium deoxycholate) and piperazine-NN-bis(2-ethane-sulfonic acid) di-sodium salt (PIPES), were obtained from Sigma Chemicals (St Louis, MO, USA). Sodium hydrogen carbonate was from Merck. AnalaR nitric acid (69%), hydrochloric acid (37%) and hydrogen peroxide (33%, m/v) were from Panreac. DORM-2 (dog–fish muscle) CRM was from National Research Council of Canada (Ottawa, Canada). BCR 627 (form of As in tuna fish tissue) was from Community Bureau of Reference – Commission of the European Communities

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(Brussels, Belgium). To avoid metal contamination, all glassware and plastic ware were washed and kept in 10% (v/v) nitric acid for 48 h, and then rinsed several times with ultra-pure water before use. 2.3. Seafood samples Mollusc (edible cockle, Cerastoderma edule; razor shell, Ensis ensis; variegated scallop, Chlamys varia; carpet-shell clam, Tapes decussatus; scallop, Pecten maximus), white fish (hake, Merluccius merluccius; cod, Gadus gadidae; anglerfish, Lophius piscatorius; Atlantic pomfret, Brama brama; poor cod, Trisopterus minutus;), and cold water fish (tuna, Thunnus thynnus; sardine, Sardina pilchardus; Atlantic mackerel, Scomber scombrus and Atlantic horse mackerel, Trachurus trachurus), were obtained from a local supermarket. The byssus and/or shells were removed from molluscs (around 1 kg) and a composite sample of the soft tissues was prepared by pooling, together, all specimens. The soft tissues were then homogenized by mechanical blending and freeze-dried. Similarly, the bone and entrails were removed from fish (around 1 kg), and the flesh was homogenized and freeze-dried. Finally, dry seafood samples were pulverized by using a vibrating ball mill and were kept in polyethylene amber bottles with hermetic seals. 2.4. Microwave-assisted acid digestion of seafood samples and dialyzate extracts Microwave-assisted acid digestion was carried out by adding 2 ml of ultra-pure water, 4 ml of nitric acid 69% and 2 ml of hydrogen peroxide (33%, m/v) to 0.5 g of a pulverized seafood sample or to 1.0 ml of a dialyzated fraction. The reactors were then subjected to a conventional microwave programme (Moreda-Piñeiro et al., 2008). After acid digestion was completed, the acid digests were made up with ultra-pure water to 25 ml (seafood samples) or 10 ml (dialyzated fraction). Seafood samples were subjected to the microwave-assisted acid digestion procedure in triplicate and at least 2 different blanks were performed for each set of microwave conditions. Before ICP-MS measurements, acid seafood digests were filtered through 0.45 lm cellulose acetate syringe filters (Millipore). 2.5. Matrix solid phase dispersion (MSPD) procedure A previously developed MSPD procedure was applied (MoredaPiñeiro et al., 2008). Around 0.25 g of sample were weighed and then blended thoroughly with 1.75 g of diatomaceous earth (DE) in a glass mortar (50 ml capacity) for 5 min, using a glass pestle to obtain a homogeneous mixture. This mixture was quantitatively transferred by using a powder funnel to a 20 ml syringe containing 2.0 g of C18 between two polyethylene frits. Finally, a third polyethylene frit was placed at the top of the syringe and was slightly compressed with a syringe plunger to remove air and avoid preferential channels. Arsenic species were eluted from the syringes by gravity with 10 ml of 50/50 methanol/ultra-pure water (MeOH/ W). The eluted extracts were concentrated by rotary evaporation (water bath at 25 °C, vacuum pressure of 10 mm Hg) to around 2 ml (methanol removal) and were then made up to 5 ml with ultra-pure water (Moreda-Piñeiro et al., 2008). Three replicates were performed for each set of experiments. At least two different blanks were performed for each set of MSPD conditions. 2.6. In-vitro digestion procedure The in vitro digestion procedure was performed, in triplicate, by weighing 0.5 g lots of powdered seafood into 100 ml Erlenmeyer flasks. A volume of 20 ml of ultra-pure water was then added. After

15 min, the pH was adjusted at 2.0 with a 6.0 M hydrochloric acid solution. Afterwards, 0.15 g of a freshly prepared gastric solution (6.0% (m/v) pepsin dissolved in 6.0 M hydrochloric acid) was added, and the sample-enzyme mixture inside the covered flasks was heated at 37 °C in an incubator coupled to an orbital-rocking platform shaker (orbital – horizontal shaking at 150 rpm) for 120 min. The amount of pepsin used in the current study agrees with the enzyme mass to sample mass ratio proposed by Luten et al. (1996). Flasks were then placed in an ice-water bath to stop the enzymatic digestion. The supernatant obtained was the in vitro gastric digest. The following step is the addition to the gastric digest of 5 ml of an intestinal solution, consisting of 4.0% (m/v) pancreatin and 2.5% (m/v) bile salts dissolved in 0.1 M sodium hydrogen carbonate according to recommended enzyme mass to sample mass ratios (Luten et al., 1996). At this point, dialysis membranes (10 kDa MWCO), filled with 20 ml of a 0.15 N PIPES solution (pH 7.5 adjusted with hydrochloric acid) were placed inside the flasks (Domínguez-González et al., 2010; Romarís-Hortas et al., 2010). Intestinal digestion took place at 37 °C with orbital – horizontal shaking at 150 rpm for 120 min. After the specified time of 120 min, the intestinal enzymatic digestion was stopped by immersing the flasks in an ice-water bath. Membranes were then removed, their outer surfaces rinsed with ultra-pure water, and the membrane containing solution (dialyzate) and the residual or non-dialyzable fraction (remaining slurries in the flasks) transferred to polyethylene vials and weighed separately. Both dialyzate and the residual fractions were kept at 20 °C before measurements. Reagent blanks were also obtained to control possible contamination. 2.7. ICP-MS measurements Total arsenic in acid digests from seafood samples and total arsenic in dialyzate fractions were measured by ICP–MS under the operating conditions listed in Table 1. Determinations were performed by using aqueous standard solutions in 2.0 M nitric acid, covering arsenic concentrations from 0 to 1000 lg l1.

Table 1 Operating ICP–MS conditions. General

Radiofrequency power/W Peristaltic pump speed/rpm Stabilization delay/s Number of replicates Nebulizer type

1400 3.0 35 3 MIcroMist

Gas flows/l min1

Plasma Auxiliary Sheath Nebulizer

17.0 1.65 0.27 0.99

Torch alignment/mm

Sampling death

7.0

Ion optics/V

First extraction lens Second extraction lens Third extraction lens Corner lens Mirror lens right Mirror lens left Mirror lens bottom Entrance lens Fringe bias Entrance plate Pole bias

32 164 231 206 25 24 27 3 4.9 3.4 0

CRI/ml min1

Skimmer gas source Sampler gas source Skimmer flow Sampler flow

H2 OFF 80 0

Mass-to-ratio

As

75

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J. Moreda–Piñeiro et al. / Food Chemistry 130 (2012) 552–560 Table 2 Anion-exchange HPLC-ICP-MS conditions.

a b

ICP-MS

Radiofrequency power/W Peristaltic pump speed/rpm Nebulizer type

1400 2.5 Beat impact (cooled spray chamber)

Gas flows/l min1

Plasma Auxiliary Nebulizer

14.0 0.8 0.85

Torch alignment/mm

Horizontal Vertical Sampling death

117 317 210

Ion optics/V

Extraction Lens 1 Lens 2 Focus D1 D2 Pole bias Hexapole bias

102 1150 62 7.8 55.7 140 20 17

CCT/ml min1

H2/He, 5.85

Mass-to-ratio

As Ge (internal standard), post-column addition at 5 lg l1

75 72

HPLC

IonPac AS7 (250  4 mm i.d.) anion-exchange column Injection volume Column temperature/°C Mobile phases flow rate/ml min1

50a/10b ll 25 1.35

Mobile phase A

1.0 mM nitric acid (pH 2.9), 1%(v/v) methanol

Mobile phase B

80 mM nitric acid (pH 1.3), 1%(v/v) methanol Gradient programme

100% A, 3.5 min. 10% A, 5.0 min. 100% A, 1.0 min.

Injection volume for As speciation in seafood. Injection volume for As speciation in dialyzate fraction.

Germanium, at a concentration of 10 mg l1, was selected as an internal standard for As determination. The use of H2 in the collision cell, at a flow rate of 5.85 ml min1, gave the best sensitivity and linear ranges for arsenic determination. ICP-MS determinations gave a limit of detection (LOD) and a limit of quantification (LOQ) based on the 3 SD/10 SD criteria, respectively (S.D. standard deviation of eleven measurements of a reagent blank), of 24.6 and 82.0 ng g1, respectively. Accuracy of the method was assessed by analyzing DORM-2 (dog–fish muscle) and BCR 627 (forms of As in tuna fish tissue) in triplicate. Values of 17.5 ± 0.2 and 4.6 ± 0.1 lg g1 were found for DORM-2 and BCR 627, respectively; which agree with the certified values (17.2 ± 0.4 and 4.8 ± 0.4 lg g1 for DORM-2 and BCR 627, respectively).

Table 3 Mean slopes for calibration, and limits of detection and quantification.

As(III) MA DMA AB As(V) AC a b

LODb/ng g1

LOQb/ng g1

Mean calibration slope ± SDa

MSPD

In vitro digestion

MSPD

In vitro digestion

3778 ± 530 5062 ± 733 4827 ± 694 4656 ± 504 3780 ± 435 1685 ± 160

6.4 17.4 13.5 12.9 23.3 9.7

12.8 34.8 27.0 25.8 46.6 19.4

21.3 58.1 45.2 43.2 77.8 32.5

42.6 116 90.4 86.4 156 65.0

n = 7. n = 11.

2.8. HPLC–ICP–MS measurements Anion-exchange HPLC conditions were used to obtain the separation of six arsenic species (As(III), As(V), MA, DMA, AB and AC) in a single chromatographic run (Moreda-Piñeiro et al., 2008) with a gradient elution consisting of diluted nitric acid solutions as mobile phases. The anion-exchange HPLC conditions, as well as ICPMS settings, are summarized in Table 2. Different calibration curves, using germanium (5 lg l1) as an internal standard, were obtained by covering As(III), As(V), MA, DMA and AC concentrations of 0, 5, 10, 25, 50, 100 and 200 lg l1, expressed as As, and AB concentrations of 0, 125, 250, 500, 750, 1000 and 2000 lg l1, also expressed as As. Table 3 lists the means and standard deviations of the slopes of calibration graphs for each analyte. A good repeatability of the calibration curves can be seen over 6 different days, with RSD around 10% for all cases. Similarly, the LODs and LOQs, expressed as ng g1 in accordance with sample mass weight and final volumes, are also listed in Table 3 for both MSPD and HPLC-ICP-MS (As speciation in seafood), and in vitro digestion and HPLC-ICP-MS (As speciation in the dialyzable fraction from

seafood) methods. It can be seen that the values are low enough to perform As speciation in seafood and the dialyzate fraction from seafood. Accuracy was assessed by analyzing different CRMs offering certified concentrations for some arsenic species, such as DORM-2 (certified AB concentration) and BCR 627 (certified AB and DMA concentrations). Each CRM was prepared seven times, following the MSPD procedure, and each extract was analyzed twice by HPLC-ICP-MS. Good agreement was found between AB concentrations (16.2 ± 0.39 and 3.7 ± 0.28 lg g1, in DORM-2 and BCR-627, respectively) and certified AB contents in DORM-2 (16.4 ± 0.5 lg g1) and BCR 627 (3.9 ± 0.2 lg g1). In addition, the DMA concentration in BCR 267 (0.15 ± 0.0069 lg g1) agreed with the certified DMA content (0.15 ± 0.023 lg g1). 2.9. Determination of fat and protein Fat content in seafood samples was determined according to AOAC Official Method 991.36 (AOAC Official Method, 1992),

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Table 4 Total As concentrations in seafood after microwave – assisted acid digestion and ICPMS determination (MAD/ICP-MS), as a sum of the concentrations of different As species after MSPD and HPLC-ICP-MS determination (MSPD/HPLC-ICP-MS), and after in vitro digestion and dialysis step and ICP-MS determination (in vitro dialysis – ICPMS).

ranging from 0.57–10.4 mg kg1 (wet weight) in clams (Edmonds & Francesconi, 1993; Gagon, Tremblay, Rouette, & Cartier, 2004; Koch et al., 2007); 5.0–60 mg kg1 (dry weight) in sole (Laparra et al., 2007; De Gieter et al., 2002), and 4.4–4.8 mg kg1 (dry weight) in Greenland halibut (Laparra et al., 2007).

Total As/lg g1

Sample

Edible cockle Razor shell Variegated scallop Carpet-shell clam Scallop Hake Cod Anglerfish Atlantic pomfret Poor cod Tuna Sardine Atlantic mackerel Atlantic horse mackerel

MAD/ICPMSa

MSPD/HPLC-ICPMSb,c

In vitro dialysis – ICP-MSa

3.2. Arsenic species in seafood

2.94 ± 0.17 3.86 ± 0.37 3.36 ± 0.16 8.65 ± 0.26 3.26 ± 0.12 3.58 ± 0.35 34.9 ± 0.1 3.62 ± 0.28 0.373 ± 0.041 7.57 ± 0.15 1.49 ± 0.16 4.43 ± 0.38 0.92 ± 0.05 4.23 ± 0.26

3.12 ± 0.056 3.74 ± 0.067 3.42 ± 0.059 8.24 ± 0.10 3.45 ± 0.062 3.57 ± 0.031 33.5 ± 1.0 3.71 ± 0.090 0.352 ± 0.0060 7.50 ± 0.071 1.40 ± 0.010 4.36 ± 0.045 0.85 ± 0.023 4.38 ± 0.092

3.01 ± 0.08 3.76 ± 0.11 3.54 ± 0.09 8.51 ± 0.12 3.31 ± 0.08 3.50 ± 0.12 35.2 ± 1.4 3.61 ± 0.2 0.356 ± 0.01 7.33 ± 0.17 1.24 ± 0.012 4.26 ± 0.06 0.84 ± 0.014 4.19 ± 0.13

The MSPD-clean-up method was applied to molluscs, white fish, and cold water fish. Each sample was subjected to the MSPD in triplicate and each extract was measured twice by HPLC-ICPMS. Table 5 lists the concentrations of the different arsenic species in the studied samples, while Table 4 lists the total As concentration in each sample as a sum of the concentrations of the different As species present in each sample (Table 5). It can be seen that AB was present in all analyzed samples and was the major arsenic species (AB concentrations ranging from 0.352 lg g1 in Atlantic pomfret to 33.2 lg g1 in cod). AB concentrations found in the analyzed seafood samples are within the broad range for AB (3.0– 21.6 mg kg1) reported by Laparra et al. (2007) (for sole). DMA was found in molluscs, cold water fish and in cod, while As(III) was found in all molluscs and only in cod (white fish) and in Atlantic horse mackerel (cold water fish). Low AC contents were also observed in molluscs, except in razor shell, and also in hake and Anglerfish (white fish) and in Atlantic mackerel (cold water fish). Finally, As(V) was found in some of the analyzed molluscs and in cod, while MA was not detected in any analyzed sample. It must be said that, as shown in Table 4, the sum of concentrations of the different As species, after MSPD and HPLC-ICP-MS analysis, is quite similar to the total As concentration after microwave-assisted acid digestion and ICP-MS determination. Total As concentrations, after microwave-assisted acid digestion and ICP-MS, were higher than those obtained as the sum of As species after MSPD and HPLC-ICP-MS analysis for razor shell, carpet shell clam, cod, Atlantic pomfret and Atlantic mackerel. To test if there was some undetected/unresolved As species in these samples after HPLC-ICP-MS determination, total arsenic, as the sum of the resolved As species, and total arsenic, after microwave-assisted acid digestion, were statistically compared. At a 95% confidence interval, the Bartlett’s and Cochran’s C tests (variance comparison) showed that there were statistically significant differences

a

N = 3. N = 6. c Concentration obtained as a sum of concentrations of As(III), As(V), DMA, AB and AC. b

involving solvent extraction (submersion method) with petroleum ether, extract drying by boiling, final heating in an oven, and weighing, whereas, protein content was assessed by nitrogen determination by Kjeldahl (block digestion), AOAC Official Method 976.06 (AOAC Official Method, 1979).

3. Results and discussion 3.1. Total arsenic in seafood Total arsenic concentration in seafood samples was obtained by ICP-MS after microwave-assisted acid digestion. Total arsenic content was within the 0.37–34.9 mg kg1 (dry weight) range (Table 4). Previous studies have reported total arsenic concentrations

Table 5 As species concentrations in seafood after MSPD procedure and after in vitro digestion and dialysis step. Sample

As species concentration in seafood samples after MSPD procedure/lg g1

As species concentration in seafood dialyzate fraction/lg g1

As(III)

DMA

AB

As(V)

AC

As(III)

DMA

AB

As(V)

AC

Edible cockle Razor shell Variegated scallop Carpet-shell clam Scallop

0.132 ± 0.01 0.124 ± 0.02 0.102 ± 0.02

0.127 ± 0.02 0.186 ± 0.02 0.346 ± 0.01

2.54 ± 0.05 3.30 ± 0.06 2.73 ± 0.05

0.231 ± 0.01 0.132 ± 0.01 0.106 ± 0.02

0.085 ± 0.007 <0.032 0.138 ± 0.006

0.141 ± 0.01 0.125 ± 0.01 0.097 ± 0.01

0.145 ± 0.01 0.210 ± 0.02 0.338 ± 0.03

2.48 ± 0.07 3.34 ± 0.1 2.89 ± 0.08

0.210 ± 0.02 <0.156 <0.156

0.093 ± 0.01 <0.065 0.154 ± 0.02

0.153 ± 0.02

0.296 ± 0.02

7.41 ± 0.1

0.164 ± 0.01

0.221 ± 0.004

0.161 ± 0.02

0.290 ± 0.01

7.65 ± 0.2

0.186 ± 0.01

0.200 ± 0.01

0.097 ± 0.01

0.281 ± 0.01

2.98 ± 0.06

<0.078

0.091 ± 0.006

0.085 ± 0.01

0.269 ± 0.02

2.87 ± 0.1

<0.156

0.083 ± 0.01

Hake Cod Anglerfish Atlantic pomfret Poor cod

<0.021 0.052 ± 0.003 <0.021 <0.021

<0.045 0.136 ± 0.01 <0.045 <0.045

3.48 ± 0.03 33.2 ± 1.0 3.48 ± 0.09 0.352 ± 0.006

<0.078 0.094 ± 0.01 <0.078 <0.078

0.085 ± 0.007 <0.032 0.234 ± 0.007 <0.032

<0.043 <0.043 <0.043 <0.043

<0.090 <0.090 <0.090 <0.090

3.51 ± 0.08 34.3 ± 1.1 3.51 ± 0.9 0.371 ± 0.01

<0.156 <0.156 <0.156 <0.156

0.078 ± 0.01 <0.065 0.248 ± 0.01 <0.065

<0.021

0.120 ± 0.01

7.38 ± 0.07

<0.078

<0.032

<0.043

<0.090

7.46 ± 0.1

<0.156

<0.065

Tuna Sardine Atlantic mackerel Atlantic horse mackerel

<0.021 <0.021 <0.021

<0.045 0.252 ± 0.02 0.181 ± 0.01

1.40 ± 0.01 4.11 ± 0.04 0.56 ± 0.02

<0.078 <0.078 <0.078

<0.032 <0.032 0.106 ± 0.006

<0.043 <0.043 <0.043

<0.090 0.274 ± 0.01 0.176 ± 0.01

1.00 ± 0.01 4.23 ± 0.08 0.542 ± 0.01

<0.156 <0.156 <0.156

<0.065 <0.065 0.060 ± 0.01

0.040 ± 0.002

0.111 ± 0.02

4.23 ± 0.09

<0.078

<0.032

<0.043

0.124 ± 0.01

4.21 ± 0.07

<0.156

<0.065

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120

Bioavailability / %

100

%As total

80

%AB %DMA

60

%As(III) %As(V)

40

%AC

20

Atlantic horse mackerel

Atlantic mackerel

Sardine

Tuna

Poor cod

Atlantic pomfret

Anglerfish

Cod

Hake

Scallop

Carpet-shell clam

Variegated scallop

Razor shell

Edible cockle

0

Fig. 1. Bioavailability of total As, AB, DMA, As(III), As(V) and AC in 3 types of seafood determined by an in vitro method.

between the standard deviations (SD) of both groups (total As concentration by microwave-assisted acid digestion and ICP-MS, and the sum of As species by MSPD and HPLC-ICP-MS) for cod and Atlantic pomfret (p-values lower than 0.05), while there was no statistically significant differences between SD for razor shell, carpet shell clam and Atlantic mackerel (p-values higher than 0.05). After applying the multiple range test (statistically significant different SD) or ANOVA (statistically significant similar SD), to compare the means (total As concentration and the sum of concentrations of As species), no statistically significant differences were obtained at the 95% confidence level. This finding implies that, although MA and other As species not separated/identified, such as TMAO, were not determined, the levels of these As species in the studied samples must be low. 3.3. Bioavailable total arsenic in seafood The bioavailability, as a percentage, was calculated using the following equation:

Bavð%Þ ¼

½Asdialyzate extract  100 ½Asacid digest

where Bav (%) is the percentage of bioavailability, and [As]dialyzate extract and [As]acid digest are the As concentrations after the in vitro digestion and after the microwave-assisted acid digestion procedures, respectively. As shown in Table 4, the bioavailable arsenic concentration, referred to the mean of three assays for each seafood sample, was within the 0.35–35.2 lg g1 (dry weight) range. Fig. 1 shows the bioavailability percentages of total arsenic in the three types of seafood (white fish, cold water fish and molluscs). In general, the bioavailability of arsenic was high in all samples, ranging from 83% to 100% in white fish and molluscs, and from 91% to 99% in cold water fish. Since the in vitro method implies a dialyzability stage during the intestinal digestion, it can be said that most of the arsenic present in the samples is easily dialyzed. These results agree with most published data for bioaccessibility of total arsenic in seafood, such as percentages of 97.7%, 98.3% and 79.2% reported for DORM-2 CRM, sole and Greenland halibut, respectively (Lapa-

rra et al., 2007). However, it must be said that other studies have shown low bioaccessibility percentages in clams (from 36% to 44% for gastric and gastric plus intestinal digestion, respectively) (Koch et al., 2007). 3.4. Bioavailable arsenic species in seafood The bioavailable arsenic species concentrations, expressed as

lg g1 (dry weight), were within 0.37–34.3, <0.09–0.338, <0.043– 0.161, <0.156–0.210 and <0.065–0.200 for AB, DMA, As(III), As(V) and AC, respectively (Table 5). The bioavailability (dialyzability) percentages for the different As species are also plotted in Fig. 1, which shows high bioavailability percentages for AB, DMA, As(III), As(V) and AC in all the samples studied. These bioavailability percentages are from 71% to 106% for AB, from 95% to 114% for DMA, from 87% to 106% for As(III), from 90% to 113% for As(V), and from 56% to 111% for AC. The high bioavailability percentage values agree with reported data for As bioaccessibility in seafood. Therefore, reported bioaccessible percentages of inorganic As (As(III) + As(V)) in clams were 98% and 86% for gastric and gastric plus intestinal digestion, respectively (Koch et al., 2007), while AB bioaccessibility percentage was high in DORM-2, sole, and Greenland halibut (68%, 107% and 100%, respectively) (Laparra et al., 2007). In addition, the same authors have reported a bioaccessibility percentage of approximately 80% for TMAO in Greenland halibut (Laparra et al., 2007). 3.5. Mass balance To assess the accuracy in the current bioavailability study, a mass-balance approach was performed by using the Atlantic horse mackerel sample as a model. After the in vitro procedure (Section 2.6.), total arsenic and arsenic species (AB, DMA, As(III), As(V) and AC) concentrations were determined in the dialyzates (Sections 2.7 and 2.8, respectively), while the residual fraction was acid-digested in triplicate (Section 2.4), and then analyzed for total arsenic (Section 2.7). The determination of total As in the residual fraction from the in vitro method gave a concentration for total As of

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Table 6 ANOVA results for mass balance studies for total arsenic and arsenic speciation. Source Total arsenic Between groups Within groups Total

Sum of squares

Degrees of freedom

Mean squares

pvalue

0.0216

1

0.0216

0.5141

0.169 0.1906

4 5

0.04225

1

0.01815

4 5

0.0067525

Arsenic speciation Between 0.01815 groups Within groups 0.02701 Total 0.04516

0.1765

Table 7 The contents (%) of food constituents. Sample

Fat/%

Protein/%

Edible cockle Razor shell Variegated scallop Carpet-shell clam Scallop Hake Cod Anglerfish Atlantic pomfret Poor cod Tuna Sardine Atlantic mackerel Atlantic horse mackerel

1.36 2.31 3.77 1.79 3.27 0.98 0.22 1.95 2.20 0.28 21.93 4.73 13.11 5.59

47.2 67.1 65.4 64.5 63.7 84.9 83.3 65.4 79.1 84.3 45.9 72.9 76.3 69.6

0.16 ± 0.01 lg g1. Therefore, a mass balance study was performed by a statistical comparison between the As concentration as the sum of total As concentrations in the dialyzable and non-dialyzable fractions, and the total As in the seafood sample (4.35 ± 0.13 and 4.23 ± 0.26 lg g1, respectively). First, a statistical comparison of the standard deviation was established by means of the Cochran’s C and Bartlett’s tests. These two statistics test the null hypothesis that the S.D. values are the same within each of the two concentrations (total As and the sum of As concentrations in the dialyzate and the non-dialyzate fractions). The p-values, after the Cochran’s C and Bartlett’s tests at a 95% confidence level (a = 0.05) for variance check, were 0.4000 and 0.3981, respectively. As can be seen, the smallest of the p-values is higher than 0.05 (95.0% confidence level), which indicates that there is no statistically significant difference between the SD values, and an ANOVA test can be performed to compare means. ANOVA results are listed in Table 6, where it can be seen that the p-value 0.5141 is higher than 0.05 (95% confidence interval), which means that there is no statistically significant difference between the total As concentration, and the sum of As species concentrations in the dialyzate and the non-dialyzable fraction. Good accuracy is therefore assessed throughout the in vitro procedure. Similarly, a mass balance was also performed but, considering the total As concentration as the sum of the different arsenic species (As(III), DMA and AB in Atlantic horse mackerel according with Table 5) after HPLC-ICP-MS (4.38 ± 0.092 lg g1), instead of the total As concentration in the acid digest. In addition, the sum of DMA and AB concentrations in the dialyzate from Atlantic horse mackerel (Table 5) after HPLC-ICP-MS (4.33 ± 0.071 lg g1) was also used instead of the total As concentration in the dialyzate. Therefore, 4.33 ± 0.071 lg g1 (sum of the DMA and AB in the dialyzate), and 0.16 ± 0.01 lg g1, which is the total As concentration in the

Table 8 Matrix correlation of the variables, fat and protein contents (% (m/m)), and percentage of bioavailability of total As, DMA, AB and AC. Marked correlation (in bold) are statistically significant at 95% confidence interval (p < 0.05).

% Protein % Fat Bav% total As Bav% AB Bav% DMA Bav% AC

% Protein

% Fat

Bav% Total As

Bav% AB

Bav% DMA

Bav% AC

1.00 0.54 0.16

0.54 1.00 0.83

0.16 0.83 1.00

0.58 0.86 0.71

0.34 0.36 0.04

0.29 0.85 0.84

0.58 0.34 0.29

0.86 0.36 0.85

0.71 0.04 0.84

1.00 0.03 0.79

0.03 1.00 0.59

0.79 0.59 1.00

non-dialyzable fraction (concentration of 4.49 ± 0.071 lg g1) were statistically compared to the total As concentration 4.38 ± 0.092 lg g1 (sum of As(III), DMA and AB concentration after MSPD and HPLC-ICP-MS determination). Again, the application of Cochran’s C and Bartlett’s tests proved that there was no statistically significant difference between the S.D at the 95% confidence interval (p-values of 0.7465 and 0.7445, respectively). The ANOVA test (Table 6) showed, as a result, that the sum of As species in Atlantic horse mackerel (As(III) plus DMA plus AB) was statistically similar to the sum of total As in the non-dialyzable fraction and the contents of DMA and AB in the dialyzate. Therefore, adequate mass balance for As speciation throughout the in vitro process is also proved. 3.6. Influence of major food constituents on arsenic bioavailability It has been assumed that the composition of food strongly influences the bioavailability of nutrients and trace elements (HorneroMéndez & Mínguez-Mosquera, 2007; Vitali, Dragojevic´, & Šebecˇic´, 2008). To explore if there was a correlation between the bioavailability of arsenicals and the composition of food, the contents of total protein and total fat in the fourteen seafood samples were assessed (Section 2.9), and a statistic based on matrix correlation was performed between the bioavailability percentages and the contents of fat and protein (Table 7). The matrix correlation (Table 8) was obtained for the bioavailability of total As and AB (fourteen cases), and also for DMA (eight cases) and AC (seven cases). However, correlation between fat/protein and bioavailability of As(III) and As(V) could not be assessed because of the low number of samples with As(III) and As(V) concentrations (lower than 5). As shown in Table 8, statistical correlation between fat content and bioavailability percentages for total As, AB and AC (correlations r(X, Y) of 0.83, 0.86 and 0.85, respectively) at the 95% confidence interval was observed. It can be seen that correlation between fat content and bioavailability of total arsenic and arsenic species (AB and AC) is negative, which means that arsenic bioavailability decreases when increasing the fat content of seafood. Data adjusted to a linear model are given in Fig. 2a–c, where negative slopes can be seen (in accordance with negative correlation as shown in Table 8), and regression coefficients (r2) ranging from 0.6861 to 0.7417, which shows moderate linear relationships between fat content and arsenic bioavailability. The higher fat content in fish can lead to greater micellarization efficiency, and to a major surface interaction with the solubilized arsenic species in micelles. Micelle protection during intestinal digestion is therefore achieved. Moreover, it is interesting to note that the percentage of the arsenic bioavailable is low for high fat content. The major fat content in fish can mean higher micellarization efficiency, leading to a major surface interaction with the solubilized arsenic species in micelles, leading to protection during intestinal digestion. One argument that could explain this fact is the major lipophilicity of the organic arsenicals (Maier, Pepper, & Gerba, 2009) that led to

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(b)

120

120

100

100

80

80

Bav % (AB)

Bav % (Total As)

(a)

60 40 20

60 40 20

r2 = 0.6861; Bav % (Total As) = 100.8 - 0,743 x % Fat 0 0

5

10 15 Fat content (% m/m)

20

25

120

0

(d)

100

80

80

60 40 20

r2 = 0.7190; Bav % (AC) = 108.3 - 3,752 x % Fat

0

5

10 15 Fat content (% m/m)

20

25

120

100 Bav % (AB)

Bav % (AC)

(c)

r2 = 0.7417; Bav % (AB) = 104.5 - 1,231 x % Fat

0

60 40 20 r2 = 0.3375; Bav % (AB) = 69.55 + 0,424 x % Protein 0

0

5

10 15 Fat content (% m/m)

20

25

40

50

60 70 Protein content (% m/m)

80

90

Fig. 2. Linear correlation between fat content and bioavailability of total As (a), AB (b) and AC (c), and between protein content and bioavailability of total AB (d).

a greater retention in the non-soluble fat fraction and lower water phase solubilization, indicating a minor potential bioaccesibility (Ortega, Reguant, Paz-Romero, Macià, & Motilva, 2009). Weak negative correlation (r2 = 0.43 and 0.53 for total As and AB, respectively) was found when considering samples with fat contents up to 20% (n = 13, excluding the sole sample from the model). Therefore, a theoretical threshold of 20% of fat can be established to observe correlation between As bioavailability and fat content. Regarding protein content, low correlation r(X,Y) was observed between protein in seafood and arsenicals bioavailability (Table 8). Only low positive correlation (r(X,Y) of 0.58) was observed for the protein content and the bioavailability percentage of AB. However, as shown in Fig. 2e, a low regression coefficient (r2 = 0.3375) was attained, which implies poor correlation. As commented, major nutrients can play a predominant role in the bioabsorption of minerals. Results from the current work show that fat affects As bioavailability, and low lipophilic compounds, such as arsenicals, are less bioavailable when the fat content is higher. 4. Conclusions Bioavailability of total As and arsenic species (AB, DMA, As(III) and AC), based on an in vitro approach consisting of using a dialysis membrane during the simulated intestinal digestion (dializability), was evaluated in different seafood products (molluscs, white fish and cold water fish). High bioavailability percentages for total As (values higher than 83%) was obtained, while high bioavailability percentages for AB (between 71% and 106%), for DMA (between 95% and 114%), for As(III) (between 87% and 106%), and for As(V) (between 90% and 113%), were also obtained. The bioavailability percentage for AC was broad; and it ranged from 56% to 111%. Fat content in the samples mainly affects the bioavailability of total

As and also arsenic species (AB and AC), and higher bioavailability percentages were observed in seafood samples containing less fat. However, bioavailability of total arsenic and different arsenic species was not influenced by the protein content. Finally, accuracy of the in vitro procedure was assessed by means of a mass balance study. Good results have been obtained for total As bioavailability, and also for the bioavailability of the different As species present in the seafood samples studied. Acknowledgements The authors wish to thank the Xunta de Galicia (Programa de Consolidación y Estructuración de Unidades de Investigación Competitivas 2010-2012, 2010/52, and Programa Sectorial de investiagación aplicada, Peme I+D e I+D Suma do Plan Galego de Investigación, Desenvolvemente e Innovación Tecnolóxica (Incite) 09MDS038103PR), and Ministerio de Ciencia y Tecnología (Project number AGL-2006-11034) for financial support. We are also grateful to Alicia María Cantarero-Roldán (Servicios Xerais de Apoio a Investigación at the University of A Coruña) for HPLC-ICP-MS technical support. We also thank Dr. Carlos Franco-Abuín (Department of Analytical Chemistry, Nutrition and Bromatology, University of Santiago de Compostela) for technical assistance in the determination of major nutrient components in marine product samples. Vanessa Romarís-Hortas is grateful to the Ministerio de Ciencia e Innovación for a FPU pre-doctoral grant. References Almela, C., Laparra, J. L., Vélez, D., Barberá, R., Farré, R., & Montoro, R. (2005). Arsenosugars in raw and cooked edible seaweed: Characterization and bioaccessibility. Journal of Agricultural and Food Chemistry, 53, 7344–7351. AOAC Official Method 976.06 (1979). Protein (Crude) in animal feed: Semiautomated Method. Journal of AOAC International, 62, 290. AOAC Official Method 991.36 (1992). Fat (Crude) in meat and meat products. Journal of AOAC International, 75, 289.

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