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Chapter 5

Development of a Test-Kit Method for the Determination of Inorganic Arsenic in Rice Downloaded by UNIV OF FLORIDA on November 17, 2017 | http://pubs.acs.org Publication Date (Web): October 23, 2017 | doi: 10.1021/bk-2017-1267.ch005

Julian Tyson,* Ishtiaque Rafiyu, and Nicholas Fragola Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, United States *E-mail: [email protected].

There is compelling evidence that in many countries, certain sectors of the populations are at risk of long-term adverse health effects from the ingestion of arsenic compounds in their diets. Most at risk are those whose diet consists predominantly of rice and rice products, a group that includes young children and infants, and there are calls for the introduction of arsenic-in-food regulations as well as the provision of advice on limiting consumption. For this advice to be meaningful, information about the chemical composition of rice is needed, with particular reference to the concentration of inorganic arsenic, a class 1 carcinogen, which is thus the species for which, at present, the availability of reliable methodology is crucial. In addition, to meet the needs of inhabitants of countries, such as Bangladesh, with limited (or no) capability to conduct high performance liquid chromatography with inductively coupled plasma mass spectrometry detection, simple inexpensive methodology is needed. As the problem is similar to that of measuring the arsenic concentration in ground water, one possible way forward is to adapt the field test kits that are available for water testing. A progress report on the development of a method for the determination of inorganic arsenic in rice in which the measurement is made with the Hach EZ kit is presented. The co-extracted starch and protein material causes a severe depression of the response when arsine is generated by the reaction of zinc with acid and so an alternative chemistry based on the reaction (well known to analytical atomic spectroscopists) with borohydride in acid is being investigated, © 2017 American Chemical Society Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

Downloaded by UNIV OF FLORIDA on November 17, 2017 | http://pubs.acs.org Publication Date (Web): October 23, 2017 | doi: 10.1021/bk-2017-1267.ch005

for which there is evidence from other researchers that the reaction is interference-free. A major problem is the control of the kinetics in a batch reaction vessel and procedures in which the borohydride is encapsulated prior to addition. A number of such procedures have been investigated and the most promising appears to be dissolution of borohydride in alkaline solution in an agar hydrogel. A number of parameters of these BAG (borohydride agar gels) are being optimized by three groups of students: those pursuing independent studies in the Tyson research group, those in a course-based research experience, and those in two chemistry classes in a local high school. The most promising composition appears to be 2% agar, 0.2% xanthan gum, to the cooling mixture of which is added (a) sodium hydroxide solution such that the final concentration in a 5 mL volume is 0.1 M and (b) 350 mg of solid sodium borohydride. The BAGS react with almost any acid extract of powdered rice grains to release arsine that forms a color on the mercuric bromide test strips that is close to that of standards of the same concentration.

Introduction The growing interest by all sectors of the scientific community in all aspects of the presence of arsenic compounds in rice is readily shown by interrogating the Web of Science database. When the search terms “arsen*” and “rice” are entered into the title field, some 672 items are retrieved from the core collection. Well over 90% of these items are the results of original research in peer-reviewed publications of one sort or another. The number of items published as a function of time is shown in Figure 1, from which it is readily seen that since around the year 2002 there has been a steady increase in output, such that in 2016, the year with the highest number of articles, just over 80 reports were published. The activity is worldwide: 60 countries are home to the 500 institutions at which the 1,830 researchers, whose names are associated with these reports, are (or were) working. The status of our knowledge of the causes, extent, and implications for this contamination of the world’s rice supply was summarized by Meharg and Zhao in a 170-page book published in 2012 (1). There is no doubt that the potential health problems are enormous, as all rice contains inorganic arsenic, a known human carcinogen, and, possibly even higher concentrations of dimethylarsinic acid, which even if not carcinogenic to humans is not harmless. Every recent article of the type mentioned above opens with some summary statement of the extent of rice consumption. For example Bralatei et al. (2) write, “rice is a staple food eaten by more than 50% of the world’s population,” and inform readers that China, Bangladesh, India, and Indonesia, whose populations get 70% of their caloric intake from rice consumption, produce nearly 70% of the world’s rice. Furthermore, we learn that some of these countries use groundwater contaminated with naturally occurring inorganic arsenic to irrigate the rice fields, 64 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


which compounds the problem as “rice, unlike most grains and cereals, has the ability to take-up and store arsenic [compounds] in the grain.” Those of us living in the USA have no cause for complacency, as rice grown in many parts of the USA has the highest arsenic contamination of any rice on the supermarket shelves. The most likely causes are (a) the past of use of arsenical pesticides, herbicides and desiccants that were widely and liberally applied to growing cotton, so that rice grown in the same parts of the country now takes up the legacy of these compounds (3), and (b) the high concentrations of naturally occurring arsenic in the soil (4). The extent of exposure in the US was summarized by Consumer Reports in a November 2012 article (4) to which a number of experts had contributed, entitled, “Arsenic in your food: our findings show a real need for federal standards for this toxin.” A more fine-grained picture was present two years later (5) when results of the US Food and Drug Administrations (FDA) analysis of 656 rice-containing products (6) were available. Both of these Consumer Reports articles include recommendations for limiting one’s intake, which are based on current information about the inorganic arsenic content of relevant foodstuffs and the resulting excess lifetime risk of getting lung cancer.

Figure 1. Number of papers published each year with the terms “arsen*” and “rice” in the title.

In early 2016, the US FDA issued guidance for industry on an action level of 100 µg kg-1 for inorganic arsenic in infant rice cereal (7), pointing out that this was the same as that introduced by the European Union (EU) in 2015 for rice to be used in the production of infant foods (8). The EU also has a limit of 200 µg kg-1 for inorganic arsenic in polished white rice (9), which is the same as that proposed by the Codex Alimentarius Commission [a joint initiative of the Food and Agriculture Organization (FAO) of the United Nations (UN) and 65 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


the World Health Organization (WHO)] (10). The Codex adopted a limit of 350 µg kg-1 for inorganic arsrenic in husked (bown) rice (11). The development of arsenic-in-food regulations around the world up to August 2105 was summarized by Petursdottir et. al. (12), who also discussed the implications for chemical measurement, pointing out the development of screening methods would be an important step because when large numbers of samples must be monitored, it is more efficient to determine inorganic arsenic by a rapid, but possibly imprecise, method and then use more precise (and accurate) methods for those samples who arsenic concentrations are possibly above the regulatory limit. For countries, such as Bangladesh, with limited access to facilities with the relevant analytical chemistry instrumentation, but who have a severe arsenic-contamination problem, a simple, low-cost measurement method will be the only way that large numbers of measurements of the arsenic content of rice can be made. On this basis, Chemists Without Borders (CWB) initiated a project in early 2016, whose goal was to devise a method for the determination of inorganic arsenic in rice based on a procedure that has been available for some time (and is widely used) for the determination of inorganic arsenic in ground water. This topic is the basis of an earlier CWB project that is described in Chapter 4 of this book (“Penny per Test” - Low Cost Arsenic Test Kits by Christopher Lizardi). Although a number of variations of the procedure have been described in the literature and are commercially available, CWB has selected the version in which the arsine, generated by the reaction with zinc powder in acid solution, reacts with mercuric bromide impregnated in a test strip exposed to the head-space gases of the reaction vessel to form a yellow/brown product. Although almost any acid (except nitric) is suitable, the kit comes with reagent packages of sulfamic acid. Two versions of a test based on this chemistry are available from the Hach Company of Loveland CO (13). They differ in the way they remove the potential interference from sulfide, which under the conditions of the test produces hydrogen sulfide, which also gives a colored product on the test strip. The more expensive, “5-reagent” version of the kit (product number 2800000) includes reagents to (a) oxidize sulfide to sulfate and then (b) remove the excess oxidant, whereas the lessexpensive “EZ” version (product number 2822800) removes the hydrogen sulfide from the gas phase by scrubbing with lead acetate solution supported on a cotton wool plug held in place immediately below the hole in the vessel lid over which the test strip is mounted. The company refers to the former product as the “low range test kit,” and the latter as the “high range test kit,” but the reality is that the EZ version will detect down to 10 µg L-1 of inorganic arsenic in a groundwater sample. Both test kits come with a chart on which the colors expected to develop on the strip for a limited number of concentrations are printed. The EZ test kit chart contains colors for 10, 25, 50, 100, 250 and 500 µg L-1, which are calibrated for a 50-mL sample volume and a 20-min reaction time. Researchers in our group have extensive experience of the performance of this test going back more than 10 years (14, 15), and we have used the EZ version of the test kit to support many of the research experiences for students (also described in Chapter 6 of this book: Arsenic in Food and Water: Promoting Awareness through Formal and Informal Learning by Julian Tyson). We have also examined the performance of the test itself and made some suggestions for improvement (16), 66 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


which can be summarized as run the test for longer and calibrate the response with the help of digital image analysis software. The Hach test kit, its basic operation and the basis of digital image analysis are shown in Figure 2.

Figure 2. A. Hach EZ test kit (reproduced with permission from the Hach Company). The carrying box contains two 50-mL reaction vessels with lids, 100 sachets of reagents 1 and 2, and 100 test strips, tipped with mercuric bromide, in a light-tight container. B. Exposed test strip and calibration chart for arsenic concentration in rice extract solution. Visual interpolation is needed. C. Plot of R, G, B values as a function of arsenic concentration in the rice extract solution. Graphical interpolation gives a more precise result. (see color insert)

Adapting the Hach EZ Test Kit for the Determination of Arsenic in Rice When rice is cooked (or extracted with dilute acid), four arsenic compounds appear in the solution: the two so-called inorganic arsenic compounds arsenate and arsenite, and two organic arsenic compounds monomethylarsonate and dimethylarsinate. As all of these compounds are weak acids, the exact forms in solution will depend on the pH. At the time of writing, all interest appears to be on the concentrations of the inorganic species, which range from low double-digit µg kg-1 values to high triple-digit µg kg-1 values. The good news is that (a) the chemistry of the Hach test kit ignores the methylated species: even if the hydrides are formed by reaction at the zinc surface, they do not react with mercuric bromide to form a yellow/brown product, and (b) the test does not distinguish between the two inorganic forms: arsenate and arsenite both react under the conditions of the test to give arsine. The bad news is that it is difficult to get less than a ten-fold dilution during the extraction process and so as the minimum concentration that can be detected by the test is about 10 µg L-1 in the extract, the minimum concentration that can be detected in the rice would be about 100 µg kg-1. If the goal is to screen rice against a statutory limit of 100 µg kg-1, then clearly some significant method development is needed to achieve the desired improvements in detection capability. Either the dilution during the extraction has to be decreased or the detection capability of the test has to be improved, or both. If the decision level is 200 µg kg-1, a method based on the procedure outlined above is clearly feasible and more detailed questions can be asked about, for example, the precision of the measurements as this is the parameter that limits the methods performance when the target analyte concentration is close to the decision-level 67 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


value in terms of the risks of false positives or false negatives. In addition to uncertainties in the various chemical stages of the method, there is another possible source of imprecision to consider, namely sampling. This is an important topic, which does not get the recognition it deserves in regards to the determination of arsenic compounds in rice, to be discussed later. We have preliminary results showing that sufficient inorganic arsenic can be extracted from rice grains by hot water to give a response with the Hach EZ test kit. Participants (about 30) in a citizen science activity in conjunction with a public lecture-demonstration (How Much Arsenic Do We Eat?) in December 2011 on the UMass campus (sponsored by the American Chemical Society as part of the celebrations of National Year of Chemistry) were given a Hach test kit and set of instructions as to how to make the measurement in their kitchens. About 10 of the participants emailed digital images of test strips clearly showing yellow colorations. Since that time, the development of this “citizen-science” method has been an on-going, but challenging, project because only readily available household chemicals and apparatus can be used. However, for the CWB project, the method developed is to be implemented by students working in a laboratory in the Asian University for Women in Chittagong in Bangladesh, and some of the “kitchen-method” restrictions can be relaxed. Method Development Of course, questions about precision are not the only ones to be asked. Accuracy is also a major concern. There are two stages that might be problematic: (a) the extraction of arsenic species from the rice into solution and (b) the generation of arsine in the presence of the co-extracted matrix components. Fortunately, the first of these is a feature common to all procedures in which the goal is to determine the individual arsenic species in rice, and as several research reports of this determination are published every year, the extraction of arsenic species from rice has been extensively studied by a large number of research groups. Not surprisingly, there is not universal agreement among the findings. Procedures for the determination of total arsenic may or may not be suitable. Some researchers claim that plasma-source instrumentation gives a response that is independent of the chemical form of the analyte, and so (a) it doesn’t matter what forms are produced by the extraction as long as all the arsenic is extracted, and (b) any convenient arsenic compound can be used for calibration. Some methods are clearly designed to convert all the arsenic species to an inorganic species and as much of the matrix as possible to products, such as carbon dioxide and water, that will not interfere in the subsequent measurement. Clearly such a sample preparation method is unsuitable for the determination of just the inorganic forms, but, looking ahead, if a future goal is to determine both inorganic and organic arsenic in rice by a Hach-test-type method, then a second stage in the overall procedure is needed in which all arsenic is converted to forms measurable by the test. The organic arsenic can now be determined from the difference between the total arsenic and the inorganic arsenic. On the other hand, there is almost no information in the peer-reviewed literature about the second stage, the generation of arsine in the presence of the 68 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


co-extracted matrix components. All of our preliminary work with the Hach test kit indicates that these components interfere. For the kitchen method, we have focused on water as the extractant and have been investigating some possible means of separating the arsenic species from the macromolecular species that we think are present by dialysis (the dialysis bag is placed in the extract with the goal of collecting small ionic arsenic species inside the bag and excluding the rice matrix). Although this is not the only possible procedure that we have in mind, the appearance of a paper in Analytical Chemistry in the fall of 2015 (2) has moved our research in a slightly different direction. It was also the impetus for CWB to raise the whole question of a simple, low-cost method for the determination of inorganic arsenic in Bangladeshi rice that could be implemented in a lab at the Asian University for Women.

The Bralatei et al. Paper The title of the report is “Determination of Inorganic Arsenic in Rice Using a Field Test Kit: A Screening Method,” which at first sight, might suggest that we had been “scooped.” The field test kit in question turns out to be the Arsenator, a system made by Wagtech Ltd in the UK, available from Palintest (17). The test is based on the same color-forming reaction as that of the Hach EZ kit, namely the reaction between arsine gas and mercuric bromide solid immobilized on a paper support. However, the arsine generation reaction is the hydride generation reaction widely used in analytical atomic spectrometry, in which arsenic species in solution react with tetrahydroborate (also known as borohydride). In the Arsenator version of the reaction, the borohydride is added as the sodium salt, which constitutes about 10% of the mass of a tablet that is dropped into the reaction vessel containing the acidified sample before securing the cap in which the test strip is mounted. As with the Hach EZ kit, the acid provided is sulfamic acid, and the color chart shows colors for 20 min-reaction time and 50-mL sample volume. An important difference between the two kits is that the Arsenator comes with a battery-operated, combined timer/reflectance spectrometer (called a DigiPAsS). When the holder containing the mercuric bromide strip is inserted into the spectrometer, not only is the blank reading established but also the timer is initiated. After the development of the color, the strip is reinserted in the device and the reading (µg L-1 of arsenic in solution) noted. The Arsenator also features a second strip to capture any excess arsine (users of the Hach kit are told to open in a well-ventilated space). Like the Hach kit, the Arsenator features an optional sulfide removal procedure: a filter that is placed in the cap of the reaction vessel immediately below the mercuric bromide test strip. The Arsenator may thus be considered the “Cadillac” version of arsenic field test kits, and, of course, is priced accordingly. It therefore, does not meet an important criterion of the CWB project: the costs must be as low as possible.

69 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


What the Results in the Bralatei et al. Paper Tell Us about the Prospects for a Hach Kit Method To determine inorganic arsenic in rice, the researchers ground 5 g (measured volumetrically) of rice grains in a coffee grinder or with a mortar and pestle, and boiled the powder in 50 mL of 1% nitric acid for 15 min. The solution was cooled (in a water bath) and the entire mixture (of solution and suspended rice matrix) was transferred to an Arsenator reaction vessel, 2 – 3 drops of antifoaming agent were added followed by one sachet of sulfamic acid and finally a borohydride tablet. The vessel was capped with a bung containing all three adsorbers (sulfide removal, mercuric bromide color development, and arsine collector). After 20-min reaction, the strip was read by the DigiPAsS device. The procedure is illustrated in Figure 3. A second sample for analysis by HPLC-ICP-MS was treated identically, followed by centrifugation at 3000 rpm and 100 µL was injected with no further treatment.

Figure 3. Outline of the Arsenator-based method. Adapted with permission from Bralatei, E; Lacan, S; Krupp, E; Feldmann, J. Determination of Inorganic Arsenic in Rice Using a Field Test Kit: A Screening Method. Anal. Chem. 2015, 87 11271–11276. Copyright 2015. American Chemical Society.

The researchers validated the method by the analysis of a very well characterized pseudo-reference material, the rice flour used in the IMEP-107 proficiency test (18), whose inorganic arsenic concentration is 107 ± 14 µg kg-1, where the ± term is the expanded uncertainty with a coverage factor of 2, corresponding to a confidence interval of approximately 95%. They analyzed 30 rice and rice products, purchased in local shops, by the methods described above for inorganic arsenic (Arsenator), for inorganic arsenic and DMA (HPLC-ICP-MS), and also for total arsenic (ICP-MS), for which a 200-mg sample was digested with a mixture of concentrated nitric acid (70%) and concentrated hydrogen peroxide (30%) in a microwave oven. 70 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


The results presented showed that the total arsenic ranged from 6 µg kg-1 to 477 µg kg-1 and that the inorganic arsenic in the nitric acid extract ranged between 5 and 301 µg kg-1. All results were presented as the mean of three replicates together with the standard deviation as the ± term. A comparison of the results for the inorganic arsenic determined by the Arsenator method and the HPLC method is described, based on a plot of Arsenator results (y-axis) vs HPLC-ICP-MS results (x-axis), as “accurate.” The slope of the plot presented is 0.928, but no ± term is given, nor is a value for the intercept. Comparing the results by a paired t-test (two-tailed) of the differences shows that the Arsenator values are significantly lower than those of the HPLC method, even at 99% confidence. The average difference is about 10%. In terms of the performance one would expect from a screening method, this is perfectly acceptable, given the spread of values likely to be encountered. In the 30-sample date set under discussion only 2 had mean values above 200 µg kg-1, with a further 2 whose 95% confidence intervals about the means included 200. Applying a “correction” of 10% does not change these numbers. For a decision level of 100 µg kg-1, 14 samples had concentrations above this value, with a further 6 whose 95% confidence intervals about the means includes 100. Again, these numbers do not change if a 10% correction is applied. The good news for our proposed Hach test kit method is that the Arsenator results are only in error by about -10% when compared with the results obtained for the same nitric acid extract by HPLC-ICP-MS, meaning that the presence of the rice matrix does not exert a severe depression on the field test kit method, which we interpret as a result the generation of arsine by reaction with borohydride rather than by reaction with zinc. In addition, we conclude that under the conditions of the test, there is no difference between the response for arsenite and arsenate, although such a difference is well-known in continuous-flow and flow-injection hydride generation (HG) with borohydride for detection by atomic spectrometry (19). It is also well known that arsine can be selectively generated from arsenite (by reaction with borohydride) in the presence of arsenate by controlling the acidity. As the acidity is decreased, the efficiency of HG from arsenate decreases, eventually falling to zero. We also deduce that methylated arsines do not cause any intereferene. It is well known that both DMA and MMA react with borohydride in acid solution to form dimethylarsine and monomethylarsine, both of which will be transferred to the headspace of the reaction vessel by the co-evolution of hydrogen from the decomposition of the excess borohydride. However, there is no evidence that samples that had a high concentration of DMA (as measured by HPLC-ICP-MS) gave a higher concentration by the Arsenator method than by the HPLC-ICP-MS method. In fact, for the three samples with the highest concentrations of DMA, the mean of the Arsenator results were lower than those obtained by HPLC-ICP-MS. The same evidence leads us also to conclude that boiling with 1% nitric acid for 15 min does not convert any of the DMA to inorganic arsenic. The evidence for the completeness of the nitric acid extraction is less compelling, as only one sample (the IMEP-107 material) whose inorganic arsenic content is known has been analyzed. Validation of a method that consumes 5 g of sample for each measurement by the analysis of certified reference materials represents a very considerable cost, as certified reference materials are expensive 71 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


(many hundreds of dollars for a few tens of grams). Furthermore, a fairly crucial aspect of the analysis is not discussed at all, namely accounting for the moisture content of the samples. As foodstuffs contain considerable percentages of water, it is important to know whether the results provided are on a “wet” (as received) or dry basis. Almost universally for rice, researchers report the results on a dry weight basis. In the case of the IMEP-107 material, the 107 ± 14 µg kg-1 mentioned earlier is the concentration of inorganic arsenic on a dry weight basis; on a “wet” weight basis, the inorganic arsenic concentration is 88 ± 15 µg kg-1, where the ± term is the 95% confidence interval (calculated from data in Table 1 is reference (16)). Bralatei et al. reported obtaining 119 ± 14 µg kg-1, where the ± term is not defined. Assuming this is one standard deviation for three replicates, the 95% confidence interval is ± 35 µg kg-1 and so, based on the overlap of the confidence intervals and the likely outcome of t-testing, there is no significant difference between the value measured by the Arsenator method and the “reference” values from the IMEP-107 material, regardless of whether the wet or dry value is considered. As most rice contains relatively low concentrations of MMA, to a first approximation the total arsenic concentration can be accounted for by the sum of the concentrations of DMA and inorganic arsenic, which in turn is the sum of arsenate and arsenite. Most rice contains more arsenite than arenate, but the arsenate concentration is not negligible. When the data presented by Bralatei et al. is examined there are 10 samples for which the sum of the inorganic and DMA arsenic is 85% or less than the total, determined by the more aggressive “nitric acid plus hydrogen peroxide” digestion followed by ICP-MS. This suggests that for some samples, boiling with 1% nitric acid for 15 min is not sufficient to extract all the species. It has been pointed out by Huang et al. that “substantial time length seems to be necessary to break AsIII-thiolate complexes with dilute HNO3” (20). Possible inaccuracies due to incomplete extraction are of less concern, as this step of the method is common to all methods that have been developed for the determination of arsenic species in rice. In addition to the many tens of articles describing such methods, there are several reports of studies in which the focus was on the accuracy of the extraction method.

Development of a Hach Kit Method for Inorganic Arsenic in Rice Our reading of the Bralatei et al. paper and other relevant reports has led to the following strategy for the development of a method in which the quantification is performed by the Hach EZ test kit (or the CWB version of this): step 1, replace the zinc with borohydride; step 2, investigate possible interference by the antifoam agent, step 3, ensure the extraction procedure solubilizes all the arsenic species; step 4, decrease the quantification limit of the Hach EZ test (from 10 µg L-1 to 5 µg L-1); and step 5, validate the method by the analysis of the same rice material by a different method. We also expect that creation of a new calibration relationship will be needed because the colors on the printed chart provided by Hach will no longer apply. 72 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


We also envisage a step 6: improve the precision demonstrated by the Arsenator method. Clearly, it is not possible to ignore precision as a figure of merit, but our expectation is that once steps 1 and 3 have been devised, the precision will also, to a large extent, have been optimized. We already know from our previous work with the Hach test how we can improve the test performance: longer reaction times and digital image analysis (16). We decided not to try to implement step 4 by increasing the ratio of mass of rice to volume of extractant. Many researchers have indicated that 1 : 10 (mass in g to volume in mL) is the limit, nor did we decide to try (at this stage) to increase the volume of solution. Though this is clearly a possible route, as the volume of the reaction vessel is such that, say, 100 mL could easily be accommodated. It would also seem relatively straightforward to decrease the volume of the solution by evaporation. Our initial experiments have been conducted for the most part on aqueous standards containing 50 µg L-1, made by serial dilution from a stock 1000 mg L-1 arsenite standard from Hach. We have an Arsenator device, purchased about 12 years ago, that was used in some comparative studies of field test kits (14). There were still some reagents available, including the borohydride tablets. As a first step in our investigations, we repeated the procedure described by Bralatei et al. except that the extracted rice suspension was transferred to a Hach reaction vessel, followed by a few drops of the same antifoam agent, one sachet of sulfamic acid from the Hach test kit (about 0.7 g) and one 12-year-old borohydride tablet from our Arsenator. A measurable color was obtained on the test strip. Iodometric titration (21) of the borohydride in a tablet showed that it contained 320 mg of sodium borohydride Step 1. Addition of Sodium Borohydride to the Reaction Vessel Almost all of our experiments to find a suitable way to carry out the hydride generation reaction have been motivated by the need to control the reaction kinetics. The Arsenator borohydride tablets contain about 300 - 400 mg of borohydride, but if a standard arsenic solution is placed in the reaction vessel and acidified with a sachet of sulfamic acid, it is impossible to get the lid of the reaction vessel secured sufficiently quickly after the addition of a few hundred mg of solid sodium borohydride to ensure that the appropriate color develops on the mercuric bromide strip. We think there are three reasons for this: 1) the arsine generated escapes from the reaction vessel before the lid can be screwed on to make a reasonably gas-tight seal, 2) borohydride also reacts sufficiently rapidly with the acid (to form hydrogen) near the surface of the liquid so that there is not enough reagent left to generate arsine from the arsenic in solution near the bottom of the reaction vessel, and 3) the rapid decomposition of borohydride releases too much hydrogen early in the process so that not all of the arsine generated is purged from solution (arsine is soluble in water to the extent of about 700 mg L-1). Quite possibly all three of these mechanisms are in operation simultaneously. That the successful generation and release into the gas phase of arsine produced by the reaction of inorganic arsenic species in acid solution with borohydride depends critically on the reaction kinetics and the use of a purge gas is well-known to the HG atomic spectrometry community (19). Most modern 73 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


instrumentation is based on a two-line continuous flow (or flow injection) system in which the acidified sample solution is merged with a borohydride solution (stabilized with sodium hydroxide). The hydride is generated downstream of the merging point, together with hydrogen gas, and the vapors are separated in a gas-liquid separator device aided by the merging of a purge gas (usually argon). Typically the sample and reagent are flowing at single-digit mL min-1 values, whereas the purge gas is flowing at many tens of mL min-1. The purge gas delivers the volatile hydride to the atomizer of the spectrometer. In the regular version of the Hach kit, the sulfamic acid is the limiting reagent and the reaction with the powdered zinc is relatively slow so that even well after the 20-min recommended reaction time has elapsed, bubbles of hydrogen are still forming and detaching. As the zinc sits on the bottom of the reaction vessel, the release of bubbles of hydrogen helps both to purge the arsine from solution into the headspace and to stir the solution so as to aid in the mass transfer of arsenic species from the bulk solution to the surface of the zinc. Despite a diligent internet search, we have been unable to find a supplier of borohydride tablets similar to those supplied with the Arsenator, which contain about 450 mg sodium borohydride and about 4 g of an “inert” filler material that disintegrates (and or dissolves) slowly when the tablet is in contact with the solution in the reaction vessel. It is possible to purchase tablets or caplets that are essentially 100% sodium borohydride, but we found that these reacted in much the same way as the powdered material does and low results were obtained. They are also more expensive than the powder. We were able to delay the reaction long enough to get the lid on the vessel by loading powdered sodium borohydride into gelatin capsules of various sizes, which were then dropped into the acidified sample. As might be imagined, there was little activity until the gelatin had dissolved, whereupon a vigorous reaction occured with considerable frothing and splashing. As a general precaution, we insert a small amount of the cotton wool (approximately 0.02 g) provided into the holder on the underside of the vessel lid to prevent liquid droplets from reaching the mercuric bromide strip. If the drops contain borohydride, the strip turns a dark brown/black color, under which circumstances the test has clearly failed. To some extent the transition from inactivity to rapid reaction could be smoothed out by perforating the capsule once or twice (at one or both ends) with a “push pin.” Results with these “holey capsules” were much better, but were still not as precise as nor developed the same extent of color as the regular Hach reaction with zinc. Despite ingenious modifications to the number and positions of the holes, the performance was always poorer than that of the regular Hach reaction. Two other methods of adding borohydride have been investigated. Attempts were made to prepare sodium borohydride in kaolin tablets according to the procedure described by Yamamoto and Kumamaru (22); however, this proved to be a challenge and after several attempts failed to produce usable tablets, this line of investigation was abandoned. It was considered too complicated for use in the Asian University for Women in Bangladesh. As our research group has developed a number of chemical vapor generation procedures in which the borohydride is immobilized on an anion-exchange resin (23), we have also investigated adding the borohydride to the reaction vessel in this form. Amberlite IRA-400 resin was 74 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


converted to the borohydride form by stirring in a saturated solution of sodium borohydride, washing and drying. Only 1.5 g of this material was needed to produce the same color with the Hach test kit for a 500 µg L-1 solution as was obtained with the regular operation of the test. In addition, slighter darker colors were produced by replacing the sulfamic acid with 0.1 M hydrochloric acid. It was also found that stirring was not necessary and that darker colors could be obtained by running the reaction for 40 minutes (instead of the recommended 20). However, problems were encountered with aerosol deposition on the strips that could not immediately be alleviated by the use of the cotton wool barrier and this line of investigation has been temporarily put aside in favor of the investigation of another promising procedure.

Gum-Based Gels The procedure currently being investigated by us and our student collaborators at Four Rivers Charter School, Greenfield MA, is the encapsulation of the required mass of sodium borohydride in a polysaccharide gel to which sodium hydroxide has been added as a stabilizer. Initial experiments were performed with agar, a hydrophilic colloid extracted from certain red-purple seaweeds of the class Rhodophyceae, and readily available from a number of suppliers. An agar gel may be easily prepared by transferring an appropriate mass of agar and 50 mL of water to a 50-mL centrifuge tube, which is then capped and immersed in boiling water (supported vertically, so that the cap is above the surface of the water) for about 20 minutes with occasional removal and shaking. Masses of agar corresponding to a final concentration of a few percent (m/V) have been used so far. Agar will also gel in sodium hydroxide solutions and up to 0.2 M NaOH solutions have been used to prepare gels. To procedure a borohydride agar gel (BAG), an appropriate mass of powdered sodium borohydride was transferred to an empty well in a 12-well polystyrene tray (Falcon brand, non-tissue culture treated plate, 12 well, flat bottom, Ref 351143). Each well is a cylinder of about 5-mL volume and diameter 23 mm. To this was added an appropriate volume of sodium hydroxide solution and the contents stirred with a glass rod. Finally 5-mL of the cooling molten agar gel was added and the mixture again stirred. The lid was secured with a rubber band and the tray placed in a refrigerator (approx 3 °C) to accelerate the setting process. Typical values for the mass of sodium borohydride and the final sodium hydroxide concentration are about 300 mg and 0.1 M, respectively. When dropped into a Hach reaction vessel containing an acidified inorganic arsenic solution a mild reaction occurs almost immediately with large bubbles of gas (presumably hydrogen) forming rapidly at the surface of the BAG, which floats. The reaction persists for at least 60 minutes, by which time the mercuric bromide strip will typically have been removed and “read.” Swirling the reaction vessel produces the evolution of small bubbles uniformly throughout the solution. This is interpreted as being due to the mixing of borohydride diffusing from the BAG with the acid in the remainder of the solution by the convection currents produced by the swirling action. At this stage, it is not known whether such swirling is necessary or not. 75 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


In addition to gels made just from agar, we have investigated some other gel compositions involving mixtures of materials. Both locust bean gum and xanthan gum have been added in smaller amounts to agar gels as a possible strategy for improving the mechanical stability and decreasing syneresis (seepage or weeping). Currently the best performance is obtained from a gel consisting of 2% (m/V) agar and 0.2% (m/V) xanthan gum, containing 400 mg of sodium borohydride in about 0.1 M sodium hydroxide, known as an XBAG. Chemical stability is related to the amount of sodium hydroxide, without which the borohydride reacts to liberate hydrogen, presumably because of the acidic nature of agar. Figure 4 shows a Hach test kit with an aqueous sample and an XBAG reagent.

Figure 4. A Hach test vessel containing a borohydride agar gel reagent. The color developed on the strip is shown in Figure 5 together with the color for the same concentration (200 µg L-1) measured by the standard Hach test reaction (zinc and acid). Both tests were run for the same time (40 minutes). Compared with the color on the printed chart, that produced by the XBAG is lighter and that produced by the zinc is darker. In Figure 6, the test in action with some rice extracts is shown, from which the potential problem due to foaming can be clearly seen.

Figure 5. Color developed with XBAG reagent (top) and with zinc (bottom). (see color insert) 76 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 6. Hach test in progress for several rice extracts with XBAG reagents. In addition to these gums we are also investigating the possibilities for gels made from a variety of other gums, including various carrageenans. There are a large number of possibilities (24). Other Preliminary Experiments: Acid, Rice Matrix, and Antifoam Agent As both the generation arsine and the extraction of arsenic species from rice depend on the concentration and maybe the type of acid, we have also been investigating the role of the acid. In the simplest version of the overall procedure, we envisage that the same acid concentration that would be used to extract the arsenic species from the rice would also be suitable for the arsine generation reaction. Our initial thoughts were that this might not be a parameter with much effect, in the sense that there would be a wide range of acid types and concentrations that would be suitable for both extraction and the hydride generation. We also considered that it might be possible to control the rate of the hydride generation reaction by adjusting the acidity, and we have results that show that for a perforated gelatin capsule, the reaction is less vigorous with 0.01% (1.2 x 10-3 M) hydrochloric acid than with higher concentrations. Reversing the order of addition of reagents does not offer any benefits. When an alkaline solution containing borohydride and arsenic was acidified by pouring in the contents of a sachet of sulfamic acid, a rapid vigorous reaction ensued and a (very) low result was obtained. We also have results that show there is a possible difference, in terms of the color developed for a given concentration of arsenic, between hydrochloric, nitric, and sulfuric acids, as sulfuric acid (0.1%, 1.8 x 10-2 M) gives results that are darker for a given concentration and reaction time than either nitric or hydrochloric acids. We are currently using 0.1% sulfuric acid to extract the arsenic compounds from rice. We confirmed that the reaction with borohydride tablets works in the presence of the rice matrix by preparing 400 mL of extract from 40 g of rice with 0.1% sulfuric acid (heat for 40 min). The remaining material was then poured into 6 Hach reaction vessels, 3 - 4 drops of antifoam agent were added and spikes of 5, 10, 15, 20, 25, and 30 µg L-1 inorganic arsenic were added and the test run with an Arsenator borohydride tablet with 40-minute reaction time. The results are shown in Figure 7, from which it may be deduced that not only was a measurable 77 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


concentration of inorganic arsenic extracted from in the rice, also that it might be possible (a) to apply the method of standard additions, and (b) to distinguish between 5 and 10 µg L-1 of arsenic in solution. Experiments in which the antifoam agent was added to standards indicate that there is a slight, but measureable, depression in the extent of color formation when antifoam is present. However, this feature of the method is essential, as without the antifoam agent the foaming overwhelms the reaction vessel and contacts the mercuric bromide strip even when a protective cotton wool plug is present. In these preliminary studies, we did not measure the moisture content, though in a parallel project (is arsenic lost on drying?) we have measured moisture contents of up to 12%. Typically, we grind the rice grains as received (i.e. without drying) in a domestic coffee grinder or food blender and then use the powdered material. We do not have any details of the particle-size distribution, but all the particles pass through a 1-mm sieve.

Figure 7. Colors developed on strips for additions of 5 (bottom), 10, 15, 20, 25 and 30 (top) µg L-1 inorganic arsenic spikes to 6 portions of the same rice extract. (see color insert)

Conclusions and Future Work From our results so far, we are convinced that the reaction between borohydride and inorganic arsenic to generate arsine does not suffer any major interferences from the components of rice that are co-extracted by dilute acids, and that provided the arsine can be purged from solution into the head-space of the reaction vessel, it may be detected by reaction with mercuric bromide. We are confident that by the judicious use of longer reaction times with, possibly, elevated temperatures and addition purge gas generation (for example by reaction between acid and a carbonate), the quantification capability of the Hach EZ kit can be decreased to 5 µg L-1 in the presence of both rice matrix and antifoam agent. We are also confident that a method that ensures complete extraction of 78 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


all inorganic arsenic species can be developed, though we think it will involve (much) longer heating than the 15 min of the Bralatei et al. method. Times of 90 minutes (20) and 2 hours (25) are reported by other researchers. We think it would be relatively easy to incorporate a pH/acidity adjustment step, if needed, between the extraction and the hydride generation stages. We have some concerns about the sampling of rice grains from the bulk material. A grain of rice weighs about 20 mg and so a 5-g sample contains 250 grains. If all rice grains contained the same concentration of inorganic arsenic there would be no sampling error, but we have evidence from experiments by a doctoral student (26) and more recently by undergraduates in our lab that there are very considerable differences between grains from the same bag, some of which have concentrations of thousands of µg kg-1. Suppose 10% of the grains contain 1000 µg kg-1 inorganic arsenic and the remaining 90% contain 0 µg kg-1. For a sample of 250 grains, the sampling standard deviation for the 25 grains (on average) that contain arsenic is the square root of (250 x 0.1 x 0.9), which is 4.74. So the relative uncertainty is about 19%. And this is the best-case scenario in which the two types of grain are thoroughly mixed. This is a really a problem for all rice analyses that start by taking a relatively small mass of grains. As almost no researchers report how the sample was taken, we have almost no idea of what the current practice is. Most descriptions of procedures simply indicate that the rice was ground and a few hundred mg were taken for the digestion/extraction procedure. Often no information about the particle size of the ground sample is provided. Drying is another important aspect of the method that is often omitted from reports in the peer-reviewed literature, but as has been discussed above is perhaps not of major concern for a screening method. Not correcting for moisture will cause results to be biased low by not more that about 10% or so. Although making tablets is a little more complicated than making agar gels, it is not out of the question even for a simple laboratory. So our future studies will certainly include making borohydride tablets with a variety of filler and excipient materials and evaluating their performance in our Hach EZ kit method.

Acknowledgments We gratefully acknowledge the contributions by UMass Amherst undergraduate students Da (Harry) Lu, Cassandra Martin, Thanh Mai, Jem Sibbick, Paul Sinno, Patrick Tonne, Alex White, and Chloe Zhang to various aspects of this method development. We thank Andrew Patari and his chemistry students at Four Rivers Charter School in Greenfield MA for their work on the polysaccharide gels. We also acknowledge the work of, and helpful discussions with, Richmond Ampiah-Bonney of Amherst College. Financial support, in the form of a Bradspies summer research fellowship for Ishtiaque Rafiyu, is gratefully acknowledged.


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