Article pubs.acs.org/est
Field Testing of Arsenic in Groundwater Samples of Bangladesh Using a Test Kit Based on Lyophilized Bioreporter Bacteria Konrad Siegfried,† Carola Endes,† Abul Fateh Md. Khaled Bhuiyan,‡ Anke Kuppardt,† Jürgen Mattusch,§ Jan Roelof van der Meer,∥ Antonis Chatzinotas,† and Hauke Harms†,* †
Helmholtz Centre for Environmental ResearchUFZ, Department of Environmental Microbiology, Leipzig, Germany The ACME Specialized Pharmaceuticals Ltd., Dhulivita, Dhamrai, Dhaka, Bangladesh § Helmholtz Centre for Environmental ResearchUFZ, Department of Analytics, Leipzig, Germany ∥ Département de Microbiologie Fondamentale, University of Lausanne, Lausanne, Switzerland ‡
ABSTRACT: A test kit based on living, lyophilized bacterial bioreporters emitting bioluminescence as a response to arsenite and arsenate was applied during a field campaign in six villages across Bangladesh. Bioreporter field measurements of arsenic in groundwater from tube wells were in satisfying agreement with the results of spectroscopic analyses of the same samples conducted in the lab. The practicability of the bioreporter test in terms of logistics and material requirements, suitability for high sample throughput, and waste disposal was much better than that of two commercial chemical test kits that were included as references. The campaigns furthermore demonstrated large local heterogeneity of arsenic in groundwater, underscoring the use of well switching as an effective remedy to avoid high arsenic exposure.
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INTRODUCTION The provision with drinking and irrigation water of over 90% of the 160 million inhabitants of Bangladesh relies on groundwater that is extracted via an estimated 10 millions of tube wells.1 These wells are typically owned by the consumers of the water (households, farms), whereas centralized water supply is presently limited to parts of the major cities and unlikely to be expanded to larger parts of the country in the foreseeable future.2 Serious problems with infectious diseases in the densely populated country had motivated the installation of most of the tube wells during the last four decades.3 As a result, the prevalence of water-borne infections has decreased.2 However, a steadily increasing number of consumers of the groundwater developed a chronic intoxication syndrome characterized by discoloration and necrotic changes of the skin,4 which is frequently followed by different forms of cancer of the skin, lung, bladder, kidney, and other internal organs.5 These health problems are referred to as arsenicosis and can be attributed to the highly variable (from moderate to very severe) groundwater contamination with arsenic.6 Numerous test campaigns revealed the wide geographic distribution and local heterogenities of the arsenic contamination.7 While the enormous dimension of the arsenic problem has been well recognized, the arsenic content in the water of most tube wells in Bangladesh is still unknown. Results of past test campaigns indicate a high landscape and depth variability of the arsenic contamination,8 which makes testing of every individual tube well strongly advisible. Bangladesh is one of the most © 2012 American Chemical Society
water-rich countries in the world; in fact, large parts of the country are constantly threatened by flooding. Consumption of arsenic-contaminated groundwater can thus be avoided in most places by selecting alternative cleaner nearby wells,3 whereas arsenic removal by, e.g., filtration would also be technically feasible.9 Such mitigation measures, however, require knowledge of arsenic concentrations in the water that is presently consumed. Major obstacles for extensive arsenic monitoring are the limited reliability or practicability, and/or the relatively high costs of existing analytical methods. Certain commercially available chemical field test have been criticized for insufficient sensitivity and high rates of false-positives and -negatives,10 whereas lab-based spectroscopic methods are costly and require substantial logistics for sample labeling, transport, and results communication to tube well owners. A test that would substantially facilitate extensive and continuous arsenic monitoring in Bangladesh should be affordable, robust, mobile, safe for the user, environmentally friendly, easy to operate, and intuitive regarding the interpretation of its result. Besides this, it needs to be reliable at the Bangladesh and WHO (World Health Organization) guideline concentrations of 50 and 10 μg l−1 total arsenic, respectively.11 This combination of requireReceived: Revised: Accepted: Published: 3281
October 4, 2011 February 10, 2012 February 16, 2012 February 16, 2012 dx.doi.org/10.1021/es203511k | Environ. Sci. Technol. 2012, 46, 3281−3287
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Figure 1. Maps showing the geographic location of sampling sites in Bangladesh (inset) and the microscale distribution of tube wells in the village of Noapara. Values next to the sampling sites represent ICP-MS derived total arsenic concentrations (μg L−1) with standard deviations. A trend toward lower arsenic in deep tube wells and largely differing arsenic values in groundwater from neighboring tube wells are visible. Bold numbers and black squares indicate water contaminated above the local recommended value of 50 μg L−1 arsenic.
rural regions in Bangladesh, and (iii) testing its suitability for the analysis of large series of water samples, as they may arise during blanket test campaigns.
ments is not entirely fulfilled by the existing lab- or field-based methods and products. In our present study, we used a luminescent whole cell living bacterial biosensor, in the following referred to as bioreporter, for the sum of arsenite and arsenate.12 A related construct had been tested with groundwater in Vietnam in a laboratory-based study using fresh bioreporter.13,14 Our objective was to optimize the bioreporter-based test kit to form the central component of logistically simple test campaigns. We had a test in mind that would enable a single person to collect 150 water samples, corresponding to the number of tube wells in a typical village, measure arsenic, and communicate the results, all on the same day, while leaving time for advice to people owning arsenic-affected tube wells. Our strategy consisted in (i) designing a robust analytical kit based on existing arsenic bioreporters,12 (ii) testing its reliability under the infrastructural and climatic conditions of
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MATERIALS AND METHODS Design and Production of the Bioreporter Test Kit. The reporter strain E. coli DH5α-2697 was used throughout this study. It carries plasmid pSB403-arsR, in which the arsR gene and its cognate promoter are located upstream of the luxCDABE genes.12 The bioreporter thus synthesizes bacterial luciferase and, unlike an earlier construct used by Trang et al.14 regenerates the luciferase substrate in the presence of arsenite. Arsenate is also recognized after reduction to arsenite by the intracellular arsenate reductase ArsC. The reporter strain thus accounts for arsenite and arsenate in water samples without reporting the contributions of these two species. The plasmid confers tetracycline resistance to the strain and tetracycline was 3282
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added to all media at 10 μg mL−1 in reporter cell cultures in order to maintain the reporter plasmid. The bioreporter kit was produced as follows: Fifty mL LB medium in a 100-mL Erlenmeyer flask was inoculated with a colony of strain DH5α2697 from overnight cultivation on LB agar and incubated at 30 °C at 230 rpm on a rotary shaker overnight. When the culture turbidity (measured at 600 nm) reached around 1.5, 6 mL culture were transferred into 300 mL LB medium in a 500-mL Erlenmeyer flask and the incubation continued for another 4−5 h under the same conditions. The culture was harvested by centrifugation at 8000 × g for 6 min at room temperature and resuspended in 120 mL of a cryoprotectant solution prepared by mixing 40 mL LB medium, 8 mL distilled water, 60 mL of a 20% trehalose solution, and 12 mL of a 15% polyvinylpyrrolidone solution. A 1-mL portion of bioreporter suspension was filled into 4-mL 2R injection vials (Christ, Osterode, Germany), and a rubber stopper was placed carefully on top of each vial without closing the vial. Batches of 100 vials were cooled to −80 °C for 1 h and then lyophilized first for 16 h at 12 °C and 0.52 mbar and then for 1 h at 8 °C and 0.10 mbar in an Alpha 1−4 LSC (Christ, Osterode, Germany) freeze-dryer. Vials were closed manually at the end of the drying process but under continued vacuum. Then the vacuum was relieved and each vial was further sealed with an aluminum crimp cap. The vials were stored in polystyrene containers at 4 °C, shipped at ambient temperature and, once in Bangladesh, stored at 4 °C until use. On the day of the measurement the vials were carried to the field sites in an insulated bag at approximately 20 °C. The bioreporter assay vials were approximately six weeks old at the time of use. Description of Sampling Sites and Climatic Conditions. The sampling sites were located in six villages in different regions of Bangladesh and are shown in the inset of Figure 1. The geographic coordinates of all groundwater wells were recorded with an e-Trex H GPS device (Garmin, Southampton, U.K.). Groundwater in Kalaroa (7 m above sea level (asl), 22° 51.45′ N; 89°02.86′ E), Noapara (24 m asl, 23°46.95′ N 90°39.12′ E), Kamarpara (9 m asl, 24°08.01′ N 89°04.63′ E), Choropur (18 m asl, 24°03.075′ N 89°03.316′ E), and Kathali (16 m asl, 23°16.59′ N 90°52.46′ E), was expected to be affected by arsenic, whereas deep tube wells in Goshinga (4 m asl, 22°24.769′ N 90°30.902′ E) served as a validated arsenic-free control site.15 To test the robustness of the arsenic bioreporter assay, samples from arsenic-affected villages were either measured instantly without further treatment, instantly after addition of 100 μM Na2EDTA, or without treatment but 2 h after sampling. The distribution of sampled tube wells in Noapara and some of their features are illustrated in Figure 1. Arsenic content in the Noapara wells was further measured by ICP-MS or ICPOES (see below). Information on the age and depth of the individual groundwater wells was obtained by interrogation of the owners. The reliability of the depth information was judged satisfying, since most owners also recollected the costs of the drilling, which are correlated with the drilling depth. Groundwater Sampling. A plastic tube was connected to the mouth of a hand tube well. Before collecting a water sample for arsenic analysis, water was pumped for 5 min and discarded. The water flow was then guided through a flow cell equipped with an inserted battery-charged U10 Multi-Parameter Probe (Horiba Group, Kyoto, Japan), in order to simultaneously measure water temperature, pH, electric conductivity (EC), turbidity, dissolved oxygen, salinity, and turbidity. The ambient
air temperature at the time of sampling was further recorded. When pH and DO attained a stable value (typically after 5 min), 500-mL plastic sampling bottles were rinsed with well water and then filled and transported to a central place in the village, where the measurements took place. Samples for laboratory analyses in Germany were collected in triplicate (20 mL each) in PE scintillation vials. A 200-μL portion of 1 M phosphoric acid was added to each vial for stabilization, which was then stored in a refrigerator for up to one month at 2−8 °C before shipment to Germany. The sampling campaign in fall 2010 was documented by a team of the German television and radio station Deutsche Welle. Detailed impressions of a sample site, tube well drilling, groundwater sampling, biosensor measurements as well as various interviews with concerned locals and research team members can be watched on http:// futurenow.dw-world.de/english/category/health/arsenic-inthe-water/. Groundwater Testing with the Arsenic Bioreporter Kit. Field measurements were conducted with lyophilized bioreporter bacteria. Each production lot was calibrated individually with standards of known concentrations of arsenite prepared by dilution of a 0.05 M NaAsO2 stock solution (Titripur, Merck) in demineralized water. Calibration series included 8 concentrations ranging from 0 to 117 μg L−1 arsenite as NaAsO2. A 1-mL portion of arsenite standard or groundwater was filled into a plastic syringe and injected into a bioreporter vial by penetrating the stopper. Three replicate vials were filled this way. The vials were shaken 10 times by hand and kept at ambient temperatures ranging from 27 to 34 °C. Groundwater samples were occasionally 10-fold diluted prior to incubation to identify arsenite toxicity on the bioreporter cells, which would result in false-negative low bioluminescence. Na2EDTA corresponding to a final concentration of 100 μM was added to some water samples to release inorganic arsenic from iron precipitates. After exactly two hours incubation, the vials were carefully wiped with a paper tissue and inserted into a battery-driven Junior luminometer 9509 (Berthold Technologies, Bad Wildbad, Germany) to measure integrated bioluminescence over a 10 s interval. Bioluminescence data were first stored in the memory of the luminometer and later downloaded to a laptop computer. Arsenic concentrations in groundwater were inferred by comparison of luminescence values with those in the calibration series by using an automated linear regression in a customized Excel spreadsheet, and are thus expressed as arsenite equivalent concentration. Used vials and syringes were collected and autoclaved in a laboratory at the University of Dhaka. Groundwater Testing with Commercial, Chemical Field Tests. To evaluate the relative practicability of the arsenic bioreporter kit, measurement campaigns also included two commercial arsenic field tests for comparison. The Arsenator test kit (Wagtech, Palintest, London, U.K.) and the Merckoquant test kit (Merck, Darmstadt, Germany) quantify total arsenic in water samples using different variants of the Gutzeit method, where As (III) and As (V) are chemically transformed into arsine gas. Upon contact with a reagent, colored mixed arsenic/mercury halide compounds are formed, the intensity of which are compared to a color scale. To simplify interpretation of signals arising from arsenic below 100 μg L−1, the Arsenator test kit also includes a small portable photometer. Both tests were conducted according to the manufacturer’s protocols. The Merckoquant test was performed in duplicate. Results obtained with both chemical tests were in 3283
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established for the range between 0 and 117 μg L−1. Better quantitative results were obtained by dilution of groundwater samples to below 150 μg L−1 arsenic (not shown here14) or can be achieved by inference of bioreporter results from nonlinear calibration curves fitted to calibrations including arsenite concentrations above 150 μg L−1.14 However, the focus of the present study was on rigorous simplification of the test procedure, so we chose to go without dilution and to content ourselves with the good qualitative agreement between results from both types of measurements. In the meantime, we have included nonlinearity of calibration curves into the luminometer software so that arsenite equivalent concentration readings account for inhibition. There was only one slightly false negative and one slightly false positive bioreporter measurement at a threshold of 50 μg L−1 total arsenic standard of the Bangladesh authorities. With respect to the 10 μg L−1 total arsenic WHO standard, two false positives and no false negatives were obtained (as can be seen in the inset of Figure 2B). Tests performed 2 h after sampling but without addition of EDTA gave similar results, with one slightly false negative and five false positive measurements out of 41 analyses of water samples (not shown). Results from measurements performed immediately after sampling without addition of EDTA resulted in six false positives and no false negative measurements out of 33 analyses of water samples (not shown). As the number of false results and the overall scattering of bioreporter data were least in EDTA-treated samples, and water quality standards refer to total arsenic, we recommend this treatment for bioreporter field measurements. Earlier work had shown that EDTA releases inorganic arsenic from ferric iron precipitates thereby transferring it into a bioreporter-detectable form.17 As the bioavailability of iron-bound arsenate to the human body, e.g., during exposure to acidic gastric juice is difficult to judge, EDTA treatment of water samples is advised to mimic arsenic remobilization. Local Distribution of Arsenic in Groundwater. The spatial distribution of arsenic in the groundwater of a single village is shown in Figure 1. Arsenite equivalent concentrations above 50 μg L−1, with a maximum of 246.5 μg L−1 is present in the water of 17 out of 28 tube wells. Nine of the remaining 11 wells, among them all three tube wells of more than 200 m depth, contain arsenic even below the WHO guidance value of 10 μg L−1,11 whereas the remaining two values are between 10 and 50 μg L−1. Although one might recognize a cluster of alarmingly arsenic-contaminated tube wells surrounding the center, the erratic overall distribution strongly implies to conduct arsenic analyses in all shallow tube wells in this village (there are several hundred wells in Noapara besides those included in this study). Figure 1 also illustrates that arseniccontaining water can be avoided, since safer (arsenic levels below 50 μg L−1) groundwater from shallow tube wells is available for every household within less than hundred meter distance. Similar microscale arsenic distributions were also observed in the villages of Kamarpara and Choropur, whereas arsenic more homogenously contaminated the groundwater in Kalaroa and Kathali. In contrast, all wells of Goshinga contained arsenic below 50 μg L−1. Practical Aspects of Arsenic Field Testing. Bangladesh suffers from a poorly developed transportation infrastructure making the logistics of blanket test campaigns difficult, since these require substantial transport of people and materials, and correct sample labeling. However, due to the dense population, high numbers of tube wells are typically located closely
good quantitative agreement with ICPqMS analyses (not shown). Chemical Analysis in the Lab. To check the reliability of bioreporter analyses, total arsenic was also measured by two spectroscopic methods. Inductively coupled plasma atomic emission spectrometry ICPqMS (ELAN 6000 DRC-e, PerkinElmer), in some instance operated as flow injection analysis (FIA), and inductively coupled plasma quadrupole mass spectrometry ICPOES (CIROS, Spectro A.I.) were applied for arsenic concentrations below and above 100 μg L−1, respectively. The differentiation of arsenic species was performed by coupling HPLC online with ICPqMS.16
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RESULTS AND DISCUSSION Reliability of the Arsenic Bioreporter Kit. A typical field calibration of bioreporter bioluminescence as a function of known arsenite concentrations is shown in Figure 2A. A
Figure 2. Calibration and cross-analysis of arsenic groundwater from Bangladesh using a test kit based on lyophilized arsenic bioreporter bacteria and ICP-MS analysis. (A) Calibration curve of the light emission from arsenic bioreporter Escherichia coli DH5α-2697 as a function of arsenite concentration, established in the village of Noapara in October 2010. The line represents a linear fit of measured data. (B) Cross-analysis of 24 groundwater samples by ICP-MS and the bioreporter kit. Light emission was converted into arsenic concentrations by interpolation and extrapolation using the linear calibration line. The inset shows the results obtained around the WHO standard at higher resolution.
comparison of arsenite equivalent concentrations determined with the bioreporter assays (test variant with addition of EDTA) and total arsenic concentration by ICP-MS across 24 different water samples is shown in Figure 2B. Bioreporter results obtained at higher arsenic concentrations are systematically slightly lower than ICP-MS readings. This is due to some degree of toxic inhibition of the bioreporter bacteria by arsenic concentrations above approximately 150 μg L−1 which is not accounted for when extrapolating from linear calibration curves 3284
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Figure 3. Materials for one day field testing with the ARSOlux biosensor test kit (160 tests; left) and the Arsenator test kit (60 tests, right). The left panel shows 160 bioreporter test kits, the luminometer, syringes, a rack with test tubes for the calibration curve and vials containing the arsenic standard and EDTA solution. No waste container is needed in the field since the used vials are collected for later heat-inactivation of the bioreporter bacteria.
experienced person was capable of running six tests in parallel, so that eight consecutive series of 33 min each were possible in 4.5 h, which totals 48 tests in a working day. Considering that less test results require less time for their communication, one could conduct up to 60 Arsenator or Merckoquant measurements per person per day. Material Requirements and Waste Disposal. For 160 bioreporter tests including the calibration series, 160 4-mL bioreporter vials, five 2-mL disposable syringes, empty test tubes in a rack, two screw-cap tubes with EDTA solution and arsenic standard solution, and a luminometer are required. All this fits into a medium size trekking backpack. Figure 3 compares the different material requirements for a one day field testing with the bioreporter test kit (160 tests) and the arsenator test (60 tests). After use, the bioreporter vials and syringes are collected and autoclaved in the lab before disposal or recycling. It should be noted that the genetically modified bioreporter bacteria remain in the sealed vials throughout shipping, storage, application, and autoclaving. Referring to this product life cycle, the German Federal Office of Consumer Protection and Food Safety (BVL) has certified that the application of the present bioreporter kit for field testing does not represent a field release according to the German laws on genetically modified organisms. Analytical results of Arsenator or Merckoquant analyses were in good agreement with those of laboratory based spectroscopic measurements (data not shown). The volume of all materials required for 60 measurements of either commercial test kits was considerably bigger, particularly due to the much higher water volumes required per test (50 mL vs 1 mL). It also consisted of a much larger number of components, including flasks, test stripes, filters, holders of filters, and test stripes, portioned reagents, color scales, tools, gloves, waste bags, and a container for the collection of the processed water. In contrast to the bioreporter test, considerable amounts of packaging
together, which favors the application of high-throughput onsite methods. We therefore tested the bioreporter kit for its suitability for high-throughput analyses in the field. We discuss our experiences while considering critical issues identified during a field performance study conducted by Kabir.18 Particular emphasis was laid on the number of tests that can be conducted and evaluated by one trained person in a working day (8 h), the weight and volume of the employed materials and the ease and environmental friendliness of waste disposal. In these respects, the bioreporter test was compared with two chemical test kits. The subsequent description reflects a realistic scenario which is based on experience with approximately 2000 bioreporter analyses accompanied by 400 Arsenator and 400 Merckoquant tests that were conducted in Bangladesh in 2010. Operators of field test kits were briefly trained before start of the campaign with particular emphasis put on the preparation of calibration curves and the handling of the luminometer. Time Requirements. The time needed for a measurement series results partly from test-independent operations, i.e., the collection of water samples (1.5 h for sending out locals/ children to fetch water in PET-bottles) and the final communication of results to the tube well owners (2 h), thus leaving 4.5 h for the test-specific core operations. Within 4.5 h, bioreporter testing allows the preparation of a calibration series and the injection of the standard into approximately 10 bioreporter vials (20 min), the injection of 150 groundwater samples and EDTA solution into 150 bioreporter vials (2 h), the sequential 10 s measurement of each of the 150 vials exactly 2 h after injection (2 h), and automated data processing and collection of used vials and syringes (10 min). The luminometer delivers a numerical reading for arsenic. The core operations of the Arsenator or Merckoquant tests consisted in the filling of test flasks, addition of reagents and mounting of filters (10 min), incubation (20 min), reading of test results and rinsing of flasks for the next series (3 min). One 3285
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waste and used filters, and, most problematically, several liters of hazardous liquid waste (containing, e.g., mercury and zinc) representing the groundwater incubations (50 mL per measurement) and rinsing water were produced and had to be carried away for proper treatment. Support Infrastructure Requirements. Minimal infrastructural requirements for field campaigns consist in 4 °C cooling capacity for storage of bioreporter kits in the area of the campaign (roughly 1 m3 per 10 000 tests), a means to deactivate the kits after usage (boiling water) and occasional access to electricity for recharging the luminometer. Further requirements depend on biosafety conditions imposed by the approving authorities. Our permission for field testing in Germany requires that all handling of the bioreporter kits takes place in a mobile lab (a vehicle) equipped with waste containers to collect the used biosensor vials and syringes, safety and operating instructions, safety clothing, and disinfectant solution. Required Skills of Operators and Risk of Operation Errors. The requirements in terms of skills of the personnel operating the bioreporter test are significantly lower than those required for the commercial test kits applied for comparison. The test basically requires correctly measuring and injecting a predefined sample volume into a bioreporter vial by means of a syringe, marking biosensor vials for proper sample assignment, conducting the luminometric measurement after a precise incubation time, and recollecting the bioreporter kits for subsequent inactivation by boiling of the entire vials. The luminometer measurement in its easiest variant consists in inserting a bioreporter vial, closing the lid of the instrument, pushing a button and noting down a reading after 10 s. Optional automated data storage and download require basic computer skills and basic knowledge of a spreadsheet program. There is no particular dexterity or faculty of judgment needed along the entire procedure. Operation errors at any stage of this procedure might lead to false results, but hardly to environmental or health risks, since no toxic reagents are used. As the biosensor vials are extremely robust due to their small volume to surface ratio and since they remain firmly closed during the entire test procedure, there is very limited risk of an accidental spill of the bioreporter bacteria. This risk can be reduced further by using plastic vials. There is, however, the possibility that the bacteria are deliberately released by destroying or opening the vials or removing the bioreporter suspension using the syringe. Operating the test thus requires a certain degree of maturity and responsibility of the operator. Estimated Costs of the Bioreporter Test Kit. The bioreporter test kits are presently manufactured in our research lab at Leipzig (Germany) by a technician in daily batches of 100. The costs for labor and materials are presently around 1 € per test. We estimate that upscaled and partly automated production, local production in Bangladesh and shifting from laboratory grade materials (e.g., vials and stoppers) to lower grade materials would reduce the production costs by a factor of 5 to 10. The luminometer price amounts to 4000 €. Hence, assuming 400 days of operation at 100 measurements per day, the acquisition costs would add 0.10 € per test. It should also be noticed that the small volume and weight of the bioreporter kit promises low costs for storage and shipment. Regarding its satisfying analytical reliability at reasonable costs and its superior practicability in view of field testing in poorly developed regions, the bioreporter test thus represents a promising alternative to existing chemical test kits.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Mrs. Sonja Hahn-Tomer, Mr. Khorshed Alam, and Dr. Abdul Kader for their help with the organization of test campaigns in Bangladesh and Verena Jaschik and Nicole Schäfer for technical assistance. The Helmholtz Association is acknowledged for financial support.
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REFERENCES
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