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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
1
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
5 6
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
12
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
25
present
are
geographical regions with problems of arsenic contamination in
methylarsonate 16,17
(MMA)
in
trace
amount
and
in
some
cultivars,
26
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 -
3
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
30
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
3
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
6
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
32
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.
14
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
22
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
