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Detection of inorganic arsenic in rice using a field test kit: a screening method Edi Bralatei, Severine Lacan, Eva M. Krupp, and Jörg Feldmann Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02386 • Publication Date (Web): 13 Oct 2015 Downloaded from http://pubs.acs.org on October 14, 2015
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Analytical Chemistry
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Detection of inorganic arsenic in rice using a field test kit: a screening method
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Edi Bralatei, Severine Lacan, Eva M Krupp and Jörg Feldmann*
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TESLA (Trace Element Speciation Laboratory), Department of Chemistry, University of
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Aberdeen, Meston Walk, Aberdeen, AB24 3UE, Scotland, UK
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Abstract
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Rice is a staple food eaten by more than 50% of the world’s population and a daily dietary
8
constituent in most South East Asian countries where 70% of the rice export comes from
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and where there is high level of arsenic contamination in ground water used for irrigation.
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Research shows that rice can take up and store inorganic arsenic during cultivation and is
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considered to be one of the major routes of exposure to inorganic arsenic, a class I
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carcinogen for humans. Here we report the use of a screening method based on the Gutzeit
13
methodology to detect inorganic arsenic (iAs) in rice within 1 hour. After optimisation, 30 rice
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commodities from the UK market were tested with the field method and compared to the
15
reference method (HPLC-ICPMS). In all but three rice samples, iAs compound can be
16
determined. The results show no bias for iAs using the field method. Results obtained show
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quantification limits of about 50 µg kg-1, a good reproducibility for a field method of ± 12 %
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and only a few false positive and negatives (< 10%) could only be recorded at the 2015 EC
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guideline for baby rice of 100 µg kg-1, while none were recorded at the maximum level
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suggested by the WHO and implemented by the EC for polished and white rice of 200 µg kg-
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1
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method in the field for pre-selection of rice which violates legislative guidelines.
. The method is reliable, fast and inexpensive hence it is suggested to use it as a screening
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Introduction
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Arsenic is always associated with the word poison1,2 and is confirmed to be toxic with
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carcinogenic capability in humans, causing cancer of the skin, lungs, liver and bladder.3 Its
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toxicity depends mainly on the chemical form in which it is present as, the solubility of the
5
species, the physical state, purity, and, the rate of absorption and elimination.4 It exists
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naturally in more than 100 different organo-arsenic species5 and inorganic species, arsenite
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and arsenate, mostly reported as inorganic arsenic (iAs). The inorganic species are more
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toxic compared to the organic species and are classified as non-threshold class 1
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carcinogen.5-8
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In the environment, it is mobilised through natural processes such as rock weathering,
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volcanic emission, biologically aided mineralisation and anthropogenic sources such as
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smelting, burning of coal and use of arsenic containing growth promoters and pesticides.9,10
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The predominant species in surface and ground water are the inorganic arsenic (iAs)
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species (sum of arsenite and arsenate) and natural As in ground water above the WHO limit
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of 10 µg L-1 is not uncommon.11 Hence, field kits for on-site measurements of traces of
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arsenic in drinking water have been developed over the years and successfully used in
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Bangladesh and other
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potable drinking water.12-14
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Rice, unlike most grain and cereal plants has the ability to take up and store As in its grains
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during cultivation.15 It is consumed by more than half of the world’s population with nearly
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70% of world rice production concentrated in China, Bangladesh, India and Indonesia where
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inorganic As contamination in ground water used for irrigation is a major issue and, rice
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provides about 70% of daily caloric intake from food.6 The dominant As species found in rice
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are the inorganic arsenic (iAs) and the organic species dimethylarsinic acid (DMA). Also
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present
are
geographical regions with problems of arsenic contamination in
methylarsonate 16,17
(MMA)
in
trace
amount
and
in
some
cultivars,
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tetramethylarsonium.
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regulatory limits for As in food and especially rice have only been established recently.18
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China has set a legal limit for iAs in rice and rice based products at 150 µg kg-1,19 and in July
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2014 the FAO/WHO (Food and Agriculture Organisation/World Health Organisation) joint
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committee set the maximum contaminant limit (MCL) for inorganic arsenic in polished rice at
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200 µg kg-1.20 In June 2015, the Commission Regulation for the European Union adopted the
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regulatory limit of 200 µg kg-1 for polished or white rice, and 100 µg kg-1 for rice destined for
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the production of food for infants and young children.21
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An analytical challenge for iAs determination in rice in comparison to water is that iAs must
35
be determined solely amongst variable amounts of organo-arsenic species such as DMA,
While arsenic in drinking water has been regulated worldwide,
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Analytical Chemistry
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MMA and even tetramethylarsonium species. Different analytical tools have been used for
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the detection of As in rice.17,22-24 These methods have been shown to be robust in proficiency
3
tests.25 Most commonly used for the determination of iAs is HPLC-ICP-MS which can used
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for separation (via a column) and detection of different As species. The ICP-MS has multi-
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element analysis capability, provides elemental isotopic ratio information, and has a good
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linear dynamic range with a limit of detection in the sub µg kg-1 range. Hydride generation is
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another popular method for the detection of arsenic in rice. This involves the reduction of
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arsenic species to their volatile hydrides in the presence of an acid (HCl) and a reductant
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(NaBH4). Often AFS or AAS is employed as the detection system,22 and to eliminate matrix
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interference ICP-MS can be used as the detection system.17 SPE HG-AAS and SPE HG-
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AFS which involves the separation of inorganic and organic species using a strong anion
12
exchange SPE (solid phase extraction) cartridge followed by HG-AAS or HG-AFS for
13
detection.23,26 These are all reliable and robust analytical tools but are all laboratory based.
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They require steady power supply and well trained personnel to run the instruments, they
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also require longer sample preparation time and in most cases are very expensive to run
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hence they are not suitable for use in remote parts of the world where steady power supply
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is still a major problem or for field based analysis. Furthermore, the instruments are too bulky
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to transport, they need a substantial amount of reagents for sample preparation, extra
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cooling units and high pressure gas cylinders for gas delivery hence their application in the
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field is not possible. Due to the regulatory guidelines for iAs in rice being passed into law by
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different regulatory bodies and extreme analysis cost for most established analytical
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methods, there is a need for a faster screening method, an
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alternative analytical tool that be used in the field and for remote regions.. With this method,
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direct answers can be given to rice producers regarding the amount of inorganic As in the
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rice produced and help in decisions making on consumption of produce and further analysis
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in cases where the iAs is above the regulatory limit.
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Aim of this study was to develop a reliable field based screening method for the
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determination of iAs in rice grains and also, obtain results under an hour to assist in
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regulatory decision making for iAs in rice with regards to recent legislative guidelines. The
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method will employ sample preparation and measuring tools which do not require electricity.
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It involves
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determination in drinking water,27 which is based on the Gutzeit reaction28 ( Equation I and II
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below).
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2H3AsO4 + 2H3O + 2NaBH4 → 2AsH3 + 2B (OH) 3 + 2H2O + 4Na+
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inexpensive but reliable
the use of a gas-fired stove and a commercial field-kit used for arsenic
AsH3 + HgBr2 → H2As–HgBr + HBr
(I) (II) 3
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As detection and quantification with the field kit depends mainly on the color stain on a
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mercury bromide impregnated filter paper which is as a result of bromine being replaced by -
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AsH2 group as outlined in equation (II). The colour varies from light yellow to dark brown
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depending on the concentration of iAs in samples analysed and can be compared to a pre-
5
existing colour chart or a battery powered digital spectrometer for quantification.
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Experimental Section
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Materials and Methods
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Chemicals and Reagents
9
Disodium methylarsenate (MMA) and sodium arsenate were from ChemService (West
10
Chester, USA), Cacodylic acid (DMA) was from Strem Chemicals (Newburyport, USA).
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Sodium Borohydride (99%) was from Acros Organics (Geel, Belgium), Rhodium used for
12
internal standard was from Specpure (Alfa Aesar, Germany), Antifoam B emulsion was from
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Sigma Aldrich (Missouri, USA) and Ammonium carbonate was from BDH UK. Sodium
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borohydride tablets and sulfamic acid were from Palintest UK.
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Double distilled water was used for preparation of standards and solutions. 1000 mg L-1 of
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AsIII, AsV, MMA and DMA standards were prepared from their sodium salts and further
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diluted to desired concentrations.
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Samples
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Rice samples and rice based products including rice based baby food were purchased from
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local shops and supermarkets around Aberdeen as a proof of concept (see table A1). For
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quality assurance a rice flour which was generated as a proficiency testing material in IMEP-
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107 trial (Institute for Reference Materials and Measurements, Geel, Belgium) was analysed
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along with the samples.
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Sample preparation for field method
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5 g of rice grains were milled with a coffee grinder or ground in a mortar with a pestle to
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obtain homogeneity before extraction. The mass of the rice was determined by the use of a
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graduated measuring spoon to make it very simple and adaptable for use in field like
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conditions or; by a balance. The milled rice sample was extracted with 50 mL 1% HNO3 by
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boiling for between 15-45 minutes. Samples were allowed to cool in a water-bath before 4 ACS Paragon Plus Environment
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Analytical Chemistry
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analysis. To further validate the use of the graduated spoon, 5 individuals were made to
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scoop same rice sample 10 times using a graduated measuring scoop and the analysis for
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iAs using the field kit performed.
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Sample Preparation for Speciation analysis using HPLC-ICP-MS
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The sample preparation for the rice samples were identical to that for the field method but
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additionally the extracts were centrifuged at 3000 rpm after cooling and the supernatants
7
were analysed directly without dilution using HPLC-ICP-MS.
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Sample preparation for total As determination on the ICP-MS
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To determine the total As in rice samples, 200 mg of sample was digested in 2 mL HNO3
11
(70%) and 1 mL H2O2 (30%). Microwave digestion was carried out on the CEM Mars 5
12
system (CEM microwave technology Ltd, UK). The digestion program was; 55°C for 5 min,
13
75°C for 5 min and 95°C for 30 min with a 5 min temperature ramp time.
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iAs determination with the field-method
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The Arsenator (Wagtech LTD UK) was used to determine the iAs in the rice extract
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selectively by using the Gutzeit method. The entire sample including the residue was
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emptied into the reaction flask, 2-3 drops of antifoam was added to sample in reaction flask
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followed by one sachet of sulfamic acid and one tablet of NaBH4. The flask was closed with a
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tri-filter bung device fitted with lead acetate filter for removal of H2S, HgBr2 loaded filter paper
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for trapping generated arsines (AsH3) and a scrubber for excess AsH3 removal. The sample
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was allowed to stand for about 20 minutes to let the reaction come to completion after which
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the holder for the HgBr2 is taken out, compared alongside the colour chart or inserted into the
24
digital detector and the concentration is read out in µg L-1. The Arsenator is zeroed with a
25
blank filter paper before the start of every analysis.
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Arsenic speciation using HPLC-ICP-MS
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The HPLC-ICP-MS system (Agilent 1100 and Agilent 7500c) was used for speciation
29
analysis. Species separation was via the Hamilton PRP-X100 anion exchange column
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(Dimensions: 10 µm, 4.1 x 250 mm) with 30 mM carbonate buffer (pH 9.2) as the mobile
31
phase (Flow rate: 1 mL min-1) and a sample injection volume of 100 µL. Rhodium was used
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as the internal standard which was added to the sample stream between the column and the
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nebuliser via a T-piece and mass to charge (m/z) ratios, As 75, Se 77, 82 and Rh 103 were
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selected for detection. Using DMA standards (10, 25, 50, 100 µg L-1) for calibration, peaks
35
were integrated with Origin 61 software and quantified accordingly. 5 ACS Paragon Plus Environment
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Total arsenic determination in rice
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Total As determined by the Agilent 7500c in reaction cell mode with hydrogen as the reaction
4
gas and argon as the carrier gas. Sample and internal standard were injected directly into the
5
plasma via the nebulizer. Mass to charge (m/z) ratios As 75, Se 77, 82 and Rh 103 were
6
selected for detection.
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Hazard identification: There is risk of burns during extraction of iAs by boiling and, the
8
determination of iAs using the Arsenator contain highly toxic compounds and should not be
9
handled by untrained personnel.
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Results and Discussions
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Firstly the field method needed to be optimised before it is tested on market rice samples.
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The parameters optimised are sensitivity, accuracy, precision, and analysis time.. The field
15
method should be able to detect 50 µg iAs kg-1 considering the regulatory maximum limits of
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iAs in rice of 100, and 200 µg kg-1 for the EC regulation for rice destined for the production of
17
food for infant and young children, and the WHO ML which is the same as the EC regulation
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for polished and white rice.21 Based on an estimated detection limit for the Arsenator of
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about 250 ng, which results when 50 mL of water is measured, 5 g of rice flour was
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analysed. This amount can easily be measured with a measuring spoon so that the method
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would not need the use of a balance. The data from the five individuals for the inter-person
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reproducibility for using the measuring spoon are less than around +/- 5% different from that
23
of the trained individual and is acceptable for a screening method (see supporting
24
information Table S2). The subsequent tests were studying the steeping time (the time the
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sample is in the extracting solution before boiling) and the boiling time for the extraction of
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iAs from the rice flour. Here rice flour S-21 was used and it can be summarised from figure
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1a and 1b that a longer steeping time is not changing the analysis significantly, while 15-20
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min is the time needed for the rice being boiled in 1 % HNO3 to extract the maximum amount
29
of iAs from the rice. To keep the analysis time to a minimum, no steeping time and 15 min
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boiling time was chosen for extraction. The reaction time for the Arsenator to transfer all iAs
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via AsH3 to the HgBr2 strip was previously optimised to be 20 min. Hence, the entire analysis
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can be performed in 60 minutes in the field considering additional time for sampling time with
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the scoop (1 min), grinding time (10 min), cooling time (10 min), flask set-up and conditioning
34
time (3 min), and read out time (1 min).
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Analytical Chemistry
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Table 1: Total As and iAs in rice. Error is given as SD from 3 replicates.
Rice samplesa
Total As µg kg-1
iAs from total Asc (%)
Recovery of iAs of field methodd (%)
(field kit) 126 ± 12 112 ± 12 140 ± 18
85 78 70
103 120 94
DMA µg kg-1
iAs µg kg-1
iAs2 µg kg-1
S-1 S-2 S-3
143 ± 11 120 ± 6 213 ± 3
19 ± 7 25 ± 14 44 ± 14
(Ref.) 122 ± 3 93 ± 5 149 ± 15
S-4
233 ± 30
53 ± 12
144 ± 1
113 ± 9
62
79
S-5 S-6 S-7
120 ± 11 129 ± 6 138 ± 14
41 ± 11 30 ± 3 52 ± 34
73 ± 1 91 ± 15 135 ± 1
57 ± 10 55 ± 8 143 ± 16
61 71 98
79 61 106
S-8
212 ± 4
47 ± 10
165 ± 10
136 ± 17
78
82
S-9 S-10
477 ± 21 117 ± 3
312 ± 1 19 ± 1
124 ± 18 85 ± 1
92 ± 15 83 ± 16
26 73
74 98
S-11
101 ± 6
14 ± 2
78 ± 6
95 ± 14
77
122
S-12 S-13 S-14
152 ± 31 419 ± 3 188 ± 6
10 ± 1 118 ± 6 57 ± 15
54 ± 6 217 ± 1 143 ± 2
50 ± 24 192 ± 17 100 ± 13
36 52 76
93 89 70
S-15
142 ± 2
45 ± 5
99 ± 0
59 ± 8
70
59
S-16 S-17
268 ± 2 160 ± 6
42 ± 22 34 ± 10
200 ± 15 129 ± 14
210 ± 26 121 ± 15
75 81
105 94
S-18
442 ± 5
61 ± 16
301 ± 1
272 ± 32
68
90
S-19 S-20 S-21 S-22 S-23 S-24
317 ± 7 115 ± 15 228 ± 23 265 ± 10 100 ± 1 142 ± 7
93 ± 38 17 ± 0 63 ± 7 74 ± 20 21 ± 2 53 ± 3
202 ± 5 74 ± 14 111 ± 0 106 ± 8 73 ± 7 80 ± 7
189 ± 15 50 ± 0 135 ± 18 116 ± 13 63 ± 2 59 ± 1
64 64 49 40 73 56
94 68 121 109 86 73
S-25
293 ± 2
188 ± 14
106 ± 3
98 ± 13
36
92
2
S-26 137 ± 5 20 ± 2 96 ± 5 84 ± 13 S-27 130 ± 3 47 ± 2 58 ± 3 52 ± 7 S-28 32 ± 1 7±3 24 ± 3 < l.o.q S-29 6±0