âPenny per Testâ - Low Cost Arsenic Test Kits - ACS Symposium

Chapter 4

“Penny per Test” - Low Cost Arsenic Test Kits

Downloaded by UNIV OF FLORIDA on November 1, 2017 | http://pubs.acs.org Publication Date (Web): October 23, 2017 | doi: 10.1021/bk-2017-1267.ch004

Christopher Lee Lizardi*,1,2 1Clear

Waters Testing, 3708 W. Bearss Ave., Suite B3422, Tampa, Florida 33618, United States 2Chemists Without Borders http://www.chemistswithoutborders.org/ *E-mails: [email protected], [email protected].

In late 2011, Chemists Without Borders had become concerned with the prevalence of high arsenic concentrations in the groundwaters of Bangladesh. A low cost arsenic abatement technology was implemented, however it required sustained testing to monitor arsenic levels. Also, widespread testing of domestic ring wells is still needed to determine at risk populations. In response to these issues, Chemists Without Borders sought to develop a low cost arsenic test kit, named the “Penny per Test”, describing the idealistic goal of a test that can be manufactured and purchased by a consumer for less than 1 US penny. Several methods were explored by both the Chemists Without Borders team and in collaboration with the company IdeaConnection®, with the best option proving to be a modified version of the Gutzeit test for arsenic. To this end, Chemists Without Borders set out to develop a semi-quantitative test for the Drinking Water Quality standard of 50 ppb that could be manufactured by local Bangladeshis. So far, Chemists Without Borders has begun working with the Asian University for Women, formulated the reagents for the modified Gutzeit test, which foregoes the use of the lead acetate oxidant, sourced reagents for purchase within the country, and have reached a current manufacturing cost of $0.21 US dollars per test.

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


Chemists Without Borders’ Ingress in Developing a Low Cost Arsenic Test Kit The development of the low cost arsenic test kit project began with investigations into a completely different public health issue in the country of Bangladesh. Chemists Without Borders (CWB) had been concerned about reports of Typhoid fever in the country in the past two decades, and began discussing humanitarian efforts to mitigate the spread of the disease. Typhoid fever is caused by the bacterium Salmonella enterica typhi, where infection commonly occurs by drinking contaminated water (1). Waters become contaminated with S. Typhi when human waste is discharged into the water body. Unfortunately this is a common practice in the slums and rural areas of the country. Highlighting the incidence of Typhoid fever in Bangladesh, a 5 year study from 2005-2009, measured rates of infection in the Dhaka Metropolitan Area (DMA). Dhaka is the capital and largest city in Bangladesh, and the population measured in the paper included some 8 million residents. In this work, Ongee and coworkers had shown incidence rates in an urban slum of Bangladesh, at 3.9/1000 persons/year (Figure 1 is a graphical representation of infection over the years) (2).

Figure 1. Incidence of Typhoid Fever in the Dhaka Metropolitan Area. Since Typhoid infection generally occurs from drinking contaminated surface waters, the initial solution was for CWB to look into the installation of wells to pull groundwater for domestic supply (see Figure 2 for an image of a ring tubewell in Bangladesh). Although groundwaters have provided people with potable water for millennia, the geology of Bangladesh introduces its own challenges for providing clean drinking water. Groundwater in the aquifers of the Bengal Basin contain high levels of arsenic adsorbed onto iron oxide minerals and clay. Changes in the oxidation-reduction potential (ORP) and pH are the major causes for speciation and release of arsenic from aquifer materials. Seasonal variations, and pumping of the aquifer can also result in the relase of arsenic from the aquifer and dissolution into the groundwater (3). An exemplary mechanism for arsenic release into the groundwater based on changes in oxidation state comes from microbial action. Sorped As on iron oxyhydroxide clays can release As when undergoing microbial reduction in the presence of organic carbon, producing As(III), As(V), and iron hydroxide (4). Equation 1 shows microbial 52 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


reduction promoting the release of As into the groundwater. As will be seen later, the reverse of this chemistry can be used to adsorb As back onto an iron matrix. Some aquitards are hydrogeologically leaky, and can sustain As transport to the aquifer in concentrations up to 500 ppb (5).

Figure 2. Ring Tubewell in Bangladesh.

A 2009 study by the Bangladesh Bureau of Statics (BBS) and the United Nations International Children’s Fund (UNICEF) illustrates the extent of arsenic contamination in the groundwaters of Bangladesh. In this study, wells were measured to determine if they contained arsenic in concentrations higher than the Bangladesh Department of Environment’s Drinking Water Quality standard (DWQ) of 50 ppb (6). The intent was to map out the degree of contamination in both rural and urban communities. The study chose 15,000 clusters of random geographic locations to measure, at 20 households per cluster, with a total sample size of 300,000 (7). Based on the results of the survey, an estimated 22 million people may have been drinking arsenic contaminated water beyond the 50 ppb DWQ in 2009. Even more astonishing was the estimate that another 5.6 million people were most likely drinking water with >200 ppb As. In response to this surmounting humanitarian issue, the search for an arsenic abatement technique was begun. CWB deployed the “SONO Filter” for arsenic removal of groundwater, developed by Prof. Abul Hussam at George Mason University. The SONO filter was employed in over 30,000 households in Bangladesh. Filter efficacy verification for the SONO filter was performed by both the Bangladesh government (Bangladesh Arsenic Mitigation Water Supply Project, BAMWSP) and third parties such as Grainger. Ten different filters were studied that treated 590,000 L of groundwater from wells in villagers’ homes. The studies found that the SONO filters arsenic removal brought concentrations down to below the Bangladesh DWQ standard of 50 ppb, and even the World Health Organization (WHO) and United States Environmental Protection Agency (USEPA) limit of 10 ppb (8). The primary active material in the SONO filter is a composite iron matrix (CIM) made of cast iron turnings (9). It is believed that the filter works by iron binding arsenates (HAsO42-, H2AsO4-) and arsenite (As2O3) to form bidentate binuclear complexes with solid phase FeOH and Fe(O)OH. The adsorbed arsenic 53 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


is stable and does not readily desorb, as illustrated by the large equilibrium constant, K = 1029. IR and X-Ray absorption structural studies led Hussam and coworkers to publish a hypothetical mechanism for the adsorbtion of arsenic species by the CIM. First, oxygen is converted to the superoxide radical by iron(II) catalysis in water (Equation 2). Two moles of the superoxide can then react with As(III) species such as arsenite, and convert them to As(V) arsenates. It is believed that the As(III) oxidation to As(V) is catalyzed by ~1-2% w/w Mn impurities in the CIM (Equation 3). Iron hydroxides and iron oxyhydroxides then adsorb this resulting arsenate (Equations 4 and 5).

Although the SONO filters have shown some initial succes in chemical removal of environmental arsenic species, it has still not been possible to install them in all contaminated homes’ and communities’ wells due to the cost of manufacturing. As of 2007, the SONO filter costs ~ $50 (USD) to manufacture. This led CWB to reconsider the strategy of combating arsenic poisoning in the country. Instead of treating all the wells with contaminated groundwater, it was decided that a simpler, more cost effective solution to helping people drink As free waters is to develop a low cost test kit. This low cost test kit could also sustain monitoring of wells undergoing treatment programs, such as those with installed SONO filters. Furthermore, a low cost readily available kit will allow users to switch from highly contaminated wells to wells that are safe according to the DWQ. This is when the genesis of the “Penny per Test” project began. The desired goal of the “Penny per Test” kit is as follows; develop a test kit that is operationally simple (i.e. operable by users with no formal education), the test should be safe (no personal protective equipment required), the test should have a clear endpoint (i.e. red is contaminated, green is safe), and finally, the test should be cheap (costs one US penny to manufacture each test). 54 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Finding the Best Method for the “Penny per Test” Arsenic Test Kit Current methods to determine arsenic concentrations may be conducted in the laboratory or in the field. Laboratory based methods include hydride generation-atomic absorption (10) and the silver diethyldithiocarbamate method (11). In the hydride generation-atomic absorption method, a suitable hydride reducing agent such as sodium borohydride (NaBH4) is used to convert arsenic species to their hydrides (i.e. arsine gas, AsH3) and are subsequently decomposed in an argon-hydrogen flame, allowing for atomic absorption measurement. The silver diethyldithiocarbamate method involves generating arsine gas (AsH3) as above with NaBH4, then by use of oxygen as a carrier gas, passing the arsine into first a glass wool or cotton scrubber with adsorbed lead acetate to remove interfering hydrogen sulfide. The arsine gas then proceeds through the apparatus to react in an absorber tube containing silver diethyldithiocarbamate and morpholine dissolved in chloroform. A red colored solution is formed which may be sampled and measured spectrophotometrically at 520 nm to determine the total inorganic arsenic concentration (12, 13). The most common field method is based on the traditional Gutzeit method for arsenic determination (14). The Gutzeit method was first established in 1879 as a means to detect the presence of antimony and arsenic, and is based on the even older Marsh-Berzelius test. Major suppliers of water quality test kits, such as Hach, LaMotte, and Taylor Technologies, manufacture field kits based on the Gutzeit method. CWB began investigation into finding the best method for the “Penny per Test” arsenic test kit with the help of IdeaConnection®. IdeaConnection® is a company that specializes in providing technical solutions and outsourcing research for other companies which lack the adequate technical resources. With consultation from IdeaConnection®, four methods were provided to CWB that best fulfilled the requirements of “Penny per Test”. These four solutions were an iodometric titration (15–17), an acoustic biosensor (18–20), an E. coli assay (21), and the aforementioned Gutzeit method. The iodometric titration uses iodine formed in situ from potassium iodide to quantitatively react with arsenite (Equation 6). The addition of bromine water (Br2 in H2O) then oxidizes the resulting NaI from the previous reaction to form sodium iodate (Equation 7), which can then react quantitatively with arsenates (Equation 8). With a modified starch indicator, the first excess of iodine can be measured as the color changes from green to a clear blue endpoint. The test is sensitive down to a concentration of 2 x 10-5 M I2.

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


Another method was proposed which used a biosensor and acoustic transducer. A bacterium that consumes As(III) and As(V) can be grown on a membrane attached to an input and output transducer, and adhered to a piezoelectric substrate. Changes in current or magnetic field by the consumption of arsenic would produce ultrasound waves that can be measured and directly related to arsenic concentration. A schematic of the device is shown in Figure 3 for representation.

Figure 3. A Biosensor and Acoustic Transducer for Arsenic Detection. The third solution proposed was an E. coli assay which involved growing two strains of the bacteria, one which is sensitive to arsenic, and the other which uses arsenic as a food source. The sensitive strain would die off in the ppb range of arsenic, implicating a concentration of concern. The other strain would thrive in the arsenic environment, informing the user that the pollutant in question is indeed arsenic and not a false positive for another contaminant. Lastly, a modification of the Gutzeit method was proposed as an ideal solution for the “Penny per Test”. As described previously, the Gutzeit method is a widely used field test, and is available in commercial arsenic test kits. The chemistry of this test will be examined in more detail in the next section of this chapter. The four solutions put forth by IdeaConnection® then underwent an analysis by CWB to weigh the pros and cons of each method, and how well they matched with the “Penny per Test” criteria. A metric system was devised with the more ideal solutions receiving a higher numerical value. The iodometric titration received a 50/100, although it was operationally simple and inexpensive, it lacked the sensitivity, and measurements began in the ppm range. The acoustic biosensor received a 61/100. It was a novel idea and was made of inexpensive materials (quartz, cellulose, etc.). However, the materials needed for the device required advanced manufacturing (360 μm silicon wafer). Also, the sensor had an expiration date, and was most accurate when used within 24 hours after manufacturing. 56 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

The E. coli assay received a 78/100, this test met the requirement of 1 US penny per test, and was operationally simple, but provided no real measurement of concentration. Although the sensitive strain died off in the ppb range, the exact concentration was not specified, and the ppb range is where arsenic is measured and given regulatory limits (i.e. 50 ppb for DPHE, 10 ppb for the WHO and USEPA).


Current Results and Moving Forward with the “Penny per Test” When it was determined that the Gutzeit method would prove to be the best for “Penny per Test”, CWB set out to optimize and modify the method for its eventual application in rural Bangladesh. The first task was to determine the reagents and formulations used in the Gutzeit method, or any modern variants thereof. Through a search of the literature (22, 23), and manufacturers of Gutzeit-based test kits, a test method was developed by CWB. Before listing the reagents in detail, it will be advantageous to describe the chemistry of the test first, so as to provide insight into why CWB chose these formulations. The Gutzeit method uses highly acidic and reducing conditions to both reduce As(V) down to As(III), and to generate arsine gas (AsH3). The arsine gas then travels its way up the reaction vessel to a mercuric bromide impregnated strip held at the top of the container. The strip will then change color as arsenic-mercury halogenides are formed from the reaction of arsine gas with mercuric bromide. The major interferences are antimony and hydrogen sulfide. Antimony reads the same as arsenic in the test, while H2S also reacts with the mercuric bromide producing a very dark color and false positive. Hydrogen sulfide is removed by the use of cotton, glass wool, or other porous support that is moistened with an oxidizing solution, such as lead acetate. Antimony can not be removed and is a known intereference that must be accounted for through other means. CWB’s choice of reagents to carry out this test were as follows: for a 50 mL volume in a 125 mL reaction flask, 1.5 g of sulfamic acid, ACS reagent grade for the acid, 1.0 g of Zn granules, 10-100 mesh, As free, to produce a reducing environment, and 5% w/v HgBr2 in EtOH impregnated on Whatman filter paper for the detection of arsine gas. The proposed reactions are illustrated in Equations 9 and 10 using the traditional sulfuric acid as a model acid.



One important feature to note of CWB’s test for arsenic is the lack of an oxidation step to remove interfering hydrogen sulfide. The rationale for this comes from van Geen and workers (2005) who discovered that when testing the waters of Bangladesh, it was possible to remove the oxidation step with little to no change in the outcome of the measurement. They tested 799 wells over five years and showed that using the Hach field kit for arsenic without the oxidation step produced the same results as found from ICP-MS for 88% of the tests (24). CWB decided then to omit the oxidation step to save on the costs of producing the “Penny per Test”. Figure 4 shows a schematic of how the kit is to be made, and a blank test being performed. First, in Figure 4 a) mercuric bromide is dissolved in EtOH while being shielded from light. The strip support portion of the test strip can be made from 110 lbs. paper or an equivalent material that is of low cost and easy to cut. Figure 4 b) show a cut strips of 110 lbs. paper at 3.8 cm x 8.9 cm, Whatman filter paper is then cut at 3.0 cm x 4.5 cm and adhered to the strip. At this time, the HgBr2 solution can now be added via pipet. Both the sulfamic acid and zinc can be measured out and added to heat-sealable Mylar® bags, 2 Mil thickness. Figure 4 c) shows sulfamic acid in a Mylar® bag as it is then impulse sealed in Figure 4 d). In Figure 4 e-g) the general procedure is shown, which operates the same way as the current commercial Gutzeit-based test kits. In Figure 4 e) the strip is fitted into a rubber cap with a bored hole to allow AsH3 to pass to the reagent containing strip. Zinc and sulfamic acid are added to a 50 mL sample taken, and the user then allows the reaction to proceed for 15 minutes with intermittent swirling (Figure 4 f)). Figure 4 g) shows the evolution of hydrogen gas, and in the case of an arsenic containing sample, arsine gas.

Figure 4. Making the “Penny per Test”. Once the formulations were had, CWB needed to build connections in the country of Bangladesh to facilitate the development and deployment of the kit. CWB had worked with the Asian University for Women on projects in the past, and decided that hiring on interns who could work in the lab would be ideal for beginning prototypes of the kits. In late 2015 two interns were hired from the university who spearheaded the Bangladesh portion of the project. The first goal was to find sources for raw 58 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


materials which could be easily purchased in the country for similar or lower costs than what would be found in the United States. The interns working under CWB sourced all the requisite reagents for the formulations, materials for manufacturing the packaging and cases, and equipment for furnishing the lab. All of this was done to a budget of $2000 US dollars. However, this dollar value equated only to the initial production of ~8-10 test kits which house 50-100 tests each. Further budgeting is required to produce a value that will describe how much it will costs to equip a lab, and purchase the materials required to make enough reagents until a return on investment (ROI) can be seen from a potential start-up company. This is one of CWB’s current milestones for this project, and proper budgeting with a small company in mind are at the fore of the work on “Penny per Test”. Another major goal of the project is the continued research into a test that costs 1 US penny to manufacture. As of September 2016 the current costs per test was ~ $0.89 US dollars to manufacture in the US and $0.48 US dollars to manufacture in Bangladesh. However, this was due to the misconception that 2.0 g of sulfamic acid were needed per test, and that the concentration of the mercuric bromide was 5% w/w. New insight showed that the kits could be manufactured in the US at $0.44 US dollars and at $0.21 US dollars in Bangladesh. Still, these values are far off from the idealized penny per test goal that CWB has envisioned. Continued work goes into optimizing the test kit, and more effectively, finding newer and cheaper suppliers for raw materials. Some reagents and items are actually more expensive purchasing in Bangladesh than the US, such as sulfamic acid which the interns found listed at 2500 BDT (Bangladeshi Taka) for 500 g vs $12.00 USD for 11.33 kg in the US. Ensuring quality control and product uniformity is another challenge for the project. As daily manufacturing of the kits will eventually occur without supervision from CWB team members, proper training of the students and future employees of AUW is imperative. One strategy for maintaining quality assurance is through CWB sponsored training at the university. By sending CWB volunteers to train students and staff at AUW, good manufacturing practices and proper quality control procedures can be passed down to those who will be involved with the daily production of the kits. Chemists Without Borders has already held online training seminars with the students via Skype and distributed electronic files of the manufacturing and quality control of the kits. Laboratory staff at AUW have also volunteered to assist CWB with training students. Chemists Without Borders has tasked themselves with aiding the rural and poor people of Bangladesh who are affected by the surmounting humanitarian issue of arsenic in their drinking water. Many strategies have been employed to mitigate arsenic in drinking water supplies, but as of now Chemists Without Borders has decided to develop low cost test kits for determining which drinking supplies are safe, instead of the gargantuan task of treating the vast number of wells in the country. To this end, the idea of a “Penny per Test” kit was envisioned for determining arsenic in drinking waters in Bangladesh. A kit that was operationally simple, safe to use, easy to understand the results, and above all, affordable to poor villagers (the cost to manufacture and purchase at one US penny per test). So far, Chemists Without Borders has teamed up with industry and academia to tackle this problem, and has come up with a test kit which works, can be used by trained 59 Grosse; Mobilizing Chemistry Expertise To Solve Humanitarian Problems Volume 1 ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

individuals in the country, and is rather inexpensive at $0.25 US dollars per test in Bangladesh. Continued efforts will reveal whether or not a “Penny per Test” kit is feasible, and how best it can be distributed to needing communities in the country of Bangladesh.

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âPenny per Testâ - Low Cost Arsenic Test Kits - ACS Symposium

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