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Arsenic release from foodstuffs upon food preparation Karlien Cheyns, Nadia Waegeneers, Tom Van de Wiele, and Ann Ruttens J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05721 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 9, 2017

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Journal of Agricultural and Food Chemistry

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Arsenic release from foodstuffs upon food preparation

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Karlien Cheyns1, Nadia Waegeneers1, Tom Van de Wiele2, Ann Ruttens*1

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Tervuren, Belgium

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9000 Ghent, Belgium

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*Corresponding author: [email protected] +32 27692234

Veterinary and Agrochemical Research Centre (CODA-CERVA), Leuvensesteenweg 17, 3080

Center for Microbial Ecology and Technology (CMET), Ghent University, Coupure Links 653,

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ACS Paragon Plus Environment


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Abstract

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In this study the concentration of total arsenic (As) and arsenic species (inorganic As,

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arsenobetaine, dimethylarsinate and methylarsonate) was monitored in different foodstuff

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(rice, vegetables, algae, fish, crustacean, molluscs) before and after preparation using

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common kitchen practices. By measuring the water content of the foodstuff and by

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reporting arsenic concentrations on a dry weight base we were able to distinguish between

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As release effects due to food preparation and As decrease due to changes in moisture

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content upon food preparation. Arsenic (species) were released towards the broth during

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boiling, steaming, frying or soaking of the food. Concentrations declined with a maximum of

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57 % for total arsenic, 65 % for inorganic As and 32 % for arsenobetaine. Based on a

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combination of our own results and literature data we conclude that the extent of this

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release of arsenic species is species specific with inorganic arsenic species being released

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most easily, followed by the small organic As species and the large organic As species.

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Keywords

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Arsenic; food; inorganic arsenic; speciation; preparation

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Introduction

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Arsenic (As) concentration in food was not regulated for a long time due to its high

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complexity and species dependent toxicity 1. Unlike other elements (cadmium, lead,

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mercury), it is not the total concentration of As (Astot) that determines the toxicity in food

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but the species distribution and concentration of the element 2. This awareness and the

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growing analytical possibilities during the last ten years resulted in the collection of As

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speciation data in food and the appearance of the first European legislation for inorganic As

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(Asi) in rice and some rice products 3.

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Both the total As concentration and the As speciation depend on the food matrix and

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cultivation conditions (soil type, water, pesticide use, cultivation practices, …). Relatively low

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concentrations of As are measured in plant-based food of terrestrial origin. Plants can take

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up Asi or methylated species (methylarsonate (MA), dimethylarsinate (DMA)) and

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accumulate this As in the leaves, roots, shoots or grains. Arsenic in plants is predominantly

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Asi, DMA and to a lesser amount MA or the tetramethylarsonium ion (TETRA)

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percentage of methylated species depends on the ability of the soil microbial community to

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methylate the Asi present in soil 7. Arsenic concentrations in rice are often a factor 10 higher

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compared to other grains (e.g. wheat and barley) because of a higher accumulation

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efficiency from the soil and the anaerobic cultivation conditions

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rice typically ranging between 0.08 mg kg-1 and 0.47 mg kg-1 WW (as sold; i.e. air dry)

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Less data are available for vegetables but the reported concentrations are generally low (
95.0%) (Fluka, Buchs, Switzerland), dimethylarsinic acid (99.5%) and

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monosodium acid methane arsonate (99.0%) (Chemservice, West Chester, USA),

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trimethylarsine oxide and tetramethyl arsonium iodide (Tri Chemical laboratories,

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Yamanashi, Japan). Multispecies stock solutions of 250 µg L-1 were prepared from these

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stocks by appropriate dilutions. Final calibration standards of 0, 2, 5, 10 and 20 µg L-1 were

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prepared daily from the multispecies stock solution.

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A multi-elemental Varian tuning solution (10 µg L-1) (Spectropure, Arlington, USA) was used

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to prepare tuning solutions for ICP-MS in 4% (v/v) nitric acid.

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Food sample collection and food preparation

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Different food samples, belonging to various food groups, were bought in Belgian

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supermarkets. The food matrices were: two types of rice (white and brown), three different

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vegetables (carrots, leeks, and onions), one type of mushroom (Agaricus bisporus), one type

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of potato, one alcoholic drink (red wine), two types of fish (cod and trout), one crustacean

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(scampi), two types of molluscs (mussels and scallops) and two types of edible seaweeds

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(nori-Porphyra sp. and hijiki-Hizikia fusiformis). Three independent batches were sampled for



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each food matrix, with the exception of hijiki were only one brand was available and hence

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only one batch was analysed.

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All samples were analysed as sold and after preparation, using some commonly applied

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kitchen practices. Each preparation was done in triplicate for all batches. The preparation

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methods included boiling, steaming, microwave preparation and pan frying. An overview of

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the treatments per matrix is presented in Table 1. Tap water was used for washing and

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boiling (As=0.15 ±0.03 µg L-1).

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The rice batches (1 kg in total for each rice type) were homogenized manually before further

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treatment. Prior to cooking, sub batches were made of 125 g each. The following rice/water

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ratios (by weight) were used: 10/32 w/w (excess water was evaporated; rice boiled to

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dryness) and 5/32 w/w (excess water was collected). Cooking time was as indicated on the

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packaging (10-20 minutes).

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Vegetables (1 kg in total for each vegetable) were pealed if relevant, rinsed twice, and

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chopped in pieces of about 1 cm³. Sub batches of ±300 g each were subjected to the various

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treatments. Carrots and leeks were boiled in stainless steel cooking wear for 10 minutes with

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a vegetable/water ratio of 12/23 w/w. Additional sub batches were steamed for 15 minutes

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using a kitchen steamer (Multi Gourmet, Braun).

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Onions and mushrooms were fried in vegetable butter for 8 minutes in aluminum-teflon

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frying pans.

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Fish, scallops and scampi were cleaned, with removal of non-edible parts, and sub batches

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(±300 g) were fried for 5 minutes in aluminum-teflon frying pans without the addition of any

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fat. In addition sub batches (±300 g) of the fishes were prepared in the microwave (5


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minutes 750 W) and sub batches of mussels were steamed in their own broth for 5 minutes

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in a stainless steel cooking pot.

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Nori seaweeds (±20 g/sub batch) were boiled for 3 minutes with a seaweed/water ratio of

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0.5/33 w/w. The soup was mixed prior to analysis.

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For Hijiki, only one batch was sampled which was divided in sub batches for different

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treatments. The first preparation technique was soaking, i.e. the sub batches were immersed

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in water for 15 minutes where after the water was removed. Other sub batches of Hijiki

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were boiled in water for 10 minutes with a seaweed/water ratio of 0.5/33 w/w. For one

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treatment the water was removed from the matrix and for another treatment the seaweed

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was mixed with the water as a soup.

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All preparations (except steaming) were done on individual electric cookers (Domo

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DO309KP). Water was always brought to boiling temperature before the foodstuff was

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added. The differences in water/solid ratios among foodstuffs are due to the difference in

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water absorbing capacities of the matrices, which asks for differences in optimal water

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quantities. During preparation the temperature of all foodstuffs was logged with a

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temperature probe connected with a Picolog® data registration system, temperature in the

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food matrices never exceeded 100°C.

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Methods

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Moisture content

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Moisture content of all samples was determined by oven drying sub samples of ± 20 g each

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(48h at 50°C; Memmert, Germany) and weighing them before and after drying. Arsenic



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concentrations determined on ‘whole weight’ base (mg kg-1 WW) could therefore be

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recalculated to ‘dry weight’ base (mg kg-1 DW).

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Sample preparation

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MINERALISATION OF TOTAL AS

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Homogenized samples were acid digested in teflon vessels (PTFE, polytetrafluoroethene) in a

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microwave oven (CEM MARS XPress, Matthews, USA), according to a validated and

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accredited ‘in house’ method. Samples were weighed (typical weights are presented in Table

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2) in duplicate in the microwave vessels. After addition of 4 ml nitric acid and 4 ml bidistilled

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water, the vessels were closed and placed into the microwave system. The samples were

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heated to 180°C in 15 min and maintained at that temperature during 30 minutes. After

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cooling, samples were diluted on a weight basis (1 g sample solution + 9 g bidistilled water)

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in analytical 15 ml tubes.

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EXTRACTION OF AS SPECIES

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Homogenized samples (± 0.25 g to 1 g, depending on the matrix, see Table 2) were weighted

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in PFA microwave tubes with magnetic stirrers. After addition of 9 mL 0.07 M HNO3 and 1 mL

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H2O2 (30%) for vegetable matrices and algae, or 10 mL H2O for wine, fish, crustacean and

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molluscs, the samples were extracted for 30 minutes in the microwave with activated

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stirring function (CEM, Mars XPRess). Extracts were centrifuged (10 minutes, 12500 g) and

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filtrated (0.45 µm, Millipore) before analysis. Extraction of Asi from marine animals is, up to

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now, associated with the presence of analytical difficulties. Recent proficiency tests for Asi in

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dogfish liver were found to be spread over a wide range and no certified reference material

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for Asi in marine animals exists yet

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. However, Pétursdóttir et al, 201449 showed that


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extraction of Asi from seafood is fairly robust when H2O or diluted acid is used as extractant.

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Wine cannot be measured as such by HPLC-ICP-MS, dilution with H2O is recommended50. In

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plants, As is apparent less soluble, which can be explained by the formation of As-

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phytochelatins complexes sequestered in the vacuoles51. We preferred to use diluted acid in

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case of vegetal matrices to assure sufficient extraction efficiency in these more resistant

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matrices52. In combination with the H2O2 added, AsIII oxidizes to AsV and only the total

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inorganic As can be quantified. This method is very similar to CEN method EN16802:2016 53.

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Consequently, acid extraction was used for vegetables and algae, and water extraction was

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used for seafood and wine, the latter allowing a separate quantification of AsIII and AsV 54.

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After analysis, both inorganic species can be summed to calculate the total Asi

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concentration.

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Sample analysis

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TOTAL AS ANALYSIS

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Determination of total As was performed by ICP-MS (VARIAN 820; Varian, Melbourne,

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Australia), with H2 as reaction gas. Quantification of As in the digests was performed by

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external calibration (calibration range: 1 µg L-1 -10 µg L-1) using acidified dilutions (4% nitric

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acid) of a multi-element stock solution from Analytika (Prague, Czech Republic). The matrix

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relevant certified reference material (CRM) IRMM 804 (rice flour; Astot = 0.049 ± 0.004 mg

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kg-1, IRMM, Belgium), NMIJ 7405 (Hijiki seaweed Astot= 35.8 ± 0.9 mg kg-1, National

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Metrology Institute of Japan) or BCR-627 (tuna fish; Astot = 4.8 ± 0.3 mg kg-1; IRMM, Belgium)

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was added to each sample batch. Although a good agreement was observed between the

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certified and the measured values (80-120%), sample results were corrected for the



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deviation of the measured values from the certified value, to allow an optimal comparison of

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results across different sample batches. The limit of quantification (LOQ) of the method for

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total As determination, calculated as 10 times the standard deviation of 20 blank samples,

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corresponds to 0.010 µg L-1 in solution. The LOQ in the matrix depends on the dilution factor

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applied (Table 2).

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AS SPECIES ANALYSIS

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The analysis of As species was performed using HPLC-ICP-MS (high performance liquid

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chromatography – inductively coupled plasma – mass spectrometry; Varian, Mulgrave,

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Austalia). The considered species in this study are: Asi (=AsIII+ AsV), MA, DMA and AB.

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The chromatographic separation of the As species was performed on anion exchange

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columns. Two different chromatographic methods were used, depending on the expected

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species in the matrix. Combined with the different extraction solutions, this resulted in three

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different analytical methods, elucidated in Table 3. The separation of AB on an anion

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exchange column is generally poor because AB does not interact with the column and tends

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to co-elute with other uncharged or cationic species. Their separation can be improved by

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using an ion pair reagent (benzenedisulfonic acid) in the mobile phase which creates

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differences in elution behavior

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method was preferred, because of the potential presence of this type of As species. In the

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other matrices a sufficient separation of the As species present can be obtained by using an

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ammoniumcarbonate solution 53 (Table 3). However, algae and mushrooms might contain a

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complex mixture of As species, and it was beyond the scope of this study to analyse all of

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them, only Asi, MA and DMA were quantified in these matrices. The calibration curve of AB

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was further used to quantify all cationic and uncharged species in these matrices. The

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. In fish, molluscs and crustacean the latter separation


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chromatographic software (GALAXY) of the ICP-MS instrument was used for quantification of

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the peak area. A five point external calibration (0-5 µg L-1) with the respective standard

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compounds was carried.

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The matrix relevant CRM NMIJ 7503a (rice flour; Asi = 0.0841 ± 0.0030 mg kg-1, DMA= 0.0133

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± 0.0009 mg kg-1; National Metrology Institute of Japan), NMIJ 7405 (hijiki seaweed; Asi =

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10.1 ± 0.5 mg kg-1; National Metrology Institute of Japan) or BCR-627 (tuna fish; AB = 3.897 ±

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0.225 mg kg-1; DMA = 0.145 ± 0.022 mg kg-1; IRMM, Belgium) was added to each sample

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batch. Although a good agreement was observed between the certified and the measured

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values (80-120%), sample results were corrected for the deviation of the CRM values, in

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order to reduce potential differences between the various analytical batches. The limit of

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quantification (LOQ) of the method for total As determination, calculated as 10 times the

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standard deviation of 20 blank samples, corresponds to 0.010 µg L-1 in solution. The LOQ in

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the matrix depends on the dilution factor applied to the sample (Table 2).

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Statistics

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A 2-factor ANOVA, followed by a multiple comparison test using a Bonferroni correction

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(Software: Graphpad prism 6.0) was used for statistical analysis of the results. The nominal

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factors were the processing treatments on the one hand and the 3 lots on the other hand for

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all matrices except for hijiki (in the latter case only 1 lot was studied and the treatment

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effect was the only factor considered). When the effect of the processing treatment was not

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the same for all lots (i.e. a significant interaction effect was observed between the 2 factors)

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then the effects of the processing treatments were analysed separately for each lot. When

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no significant interaction effect was observed main effects were interpreted.



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Results

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As (species) concentrations in the raw matrices

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Concentrations of total As and As species for all raw matrices are summarized in Table 4.

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Although rice has the reputation to show higher Asi concentrations compared to other

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terrestrial plant species, the results in Table 4 indicate that when concentrations are

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recalculated to dry weight base, total As and Asi overlap in vegetables and rice (0.02-0.18 mg

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Astot kg-1 DW in vegetables and 0.16-0.39 mg Astot kg-1 DW in rice; 0.01- 0.15 mg Asi kg-1 DW

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in vegetables and 0.12-0.17 mg Asi kg-1 DW in rice). This illustrates that the high moisture

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content of fresh vegetables tends to mask their sometimes relatively high Asi concentrations.

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Inorganic arsenic concentrations are low in mushrooms (0.03-0.04 mg Asi kg-1 DW), but can

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rise up to 90.8 mg Asi kg-1 DW in hijiki algae. In the marine organisms, the main As species

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was AB, with exception of mussels, in which large amounts of other species could be

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detected, including Asi (up to 0.13 mg Asi kg-1 DW).

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Effect of food preparation on moisture content

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Food preparation influences the moisture content of the food matrices. This can hence

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impact data interpretation when results are expressed on fresh weight basis. We therefore

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recalculated As release values to a DW basis. This allows distinguishing between As release

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from the foodstuff attributed to changes in water content and As release caused by the food

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preparation. The % DW before and after treatment are presented in Table 4 and Table 5.

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Boiling rice evidently led to an increase of the moisture content of the grains (% DW

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decreases from 89-90% to 37-52 %). The % DW dropped upon boiling and increased upon

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steaming in leeks and carrots. The % DW in onion increased after frying, indicating that

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water was lost out of the onions during the treatment. The values of the % DW after frying


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of the mushrooms, show that more water was lost in the second batch (% DW increase from

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6.2 to 19%) compared to the other two batches (increase from 6.7 to 11% and from 6.3 to

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11%). Preparing seaweed samples, which were bought as dried leaves, included hydratation

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during soaking and/or boiling; consequently, the % DW drastically decreased (from 83-90 %

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to minimum 6.2% in hydrated seaweed, or to 2 % in soup). The % DW consistently increased

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for all treatments of fish and crustacean (with maximum 6 %), which indicated a loss of

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water. Mussels, steamed in their own broth, showed an increase in % DW (from 8.5%-12% to

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28%-31%).

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Effect of food preparation on As (species) concentrations

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The % change in concentration after different treatments are given in Table 5, overall, no

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quantitative data on MA were obtained as MA concentrations were lower than LOQ before

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or after preparation.

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A significant decrease in concentration of Astot, Asi and DMA was seen in all three of the lots

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after boiling white rice in an excess of water. However, the extent of the effect was not the

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same for all lots (significant interaction effect). No effects on As species concentrations were

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observed when the same rice was cooked until dryness, except for one lot, where the DMA

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concentration was reduced. An unidentified peak with a short retention time (3.2 minutes)

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was observed in the latter case. Significant decreases of Astot, Asi and DMA were observed in

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all lots of brown rice when boiling in an excess of water.

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Boiling and steaming carrots also reduced the average concentrations of As and Asi in all lots,

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but was only significant in lot 3. Arsenic was detected in the broth of all treatments (results

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not shown). When boiling or steaming leek, a main treatment effect indicated also a

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decrease in As (species) concentrations. Arsenic was detected in the broth of all treatments



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(results not shown). Frying onion did not significantly affect As or Asi concentrations. Heating

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wine did not affect the Astot or Asi concentration.

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Frying mushrooms induced different effects on the As (species) concentration in the distinct

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lots (significant interaction effect). A clear and significant decrease of all As species was

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observed in the lot 2 (the lot where more moisture was lost), while this was not observed in

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the other batches.

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Making soup of nori or hijiki did not induce effects on Astot concentrations, because the

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boiling liquid was included in the analysis. Soaking or boiling hijiki (with removal of the

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excess water) led to significant decreases of Astot, Asi and DMA. The concentration of Asi was

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significantly more reduced when hijiki was boiled (-65%) compared with the soaking process

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only (-40%). No significant difference in DMA concentration was observed between soaking

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and boiling. Remarkably, the concentration of cationic and uncharged species was not

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significantly reduced by the different treatments.

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Preparation of fish and crustacean did not induce effects on Astot and AB concentrations.

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Frying scallops, caused a slight (

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