Total arsenic, arsenic species, and trace elements in crop and vegetable grown in areas irrigated with arsenic contaminated water in Bangladesh and West Bengal-India

Authors

• Uttam K Chowdhury School of Environmental Studies (SOES), Jadavpur University, Kolkata 700032, India.

Abstract

In this study, tube well water, soil, crop, and vegetable were collected from agricultural field where irrigated with arsenic contaminated water. Estimation of total arsenic and other metals and metalloids in soil, vegetable, and paddy (rice & husk) samples by using ICP-MS after microwave digestion. But arsenic species in paddy (rice + husk), rice, husk, and vegetable by IC-ICP-MS after TFA extraction.

Results show that the average arsenic concentration in contaminated soil, rice, and vegetable were 3.81, 3.62, and 5.66 times higher than the control samples, respectively. The overall observations indicated that arsenic concentration in vegetable and paddy were positively correlated with arsenic in soil. Also, for paddy arsenic concentration decreases shoot > seed (rice) > husk and in vegetables the distribution is leaf> stem > fruit.

The regression analysis was carried out between arsenic and other metals in soil samples.  However, no significant co-relation was observed between As & Mn, As & Cu, As & Ni, or As & Pb. But a significant (<0.05) positive correlation found between As & Zn (r =+0.763, p = 0.027) and also a strong negative correlation was observed between As & Hg (r = -0.802, p = 0.009).

Arsenic along with Se, Mn, Cu, Hg, Pb & Ni were analyzed in rice & husk of 3 paddy samples cultivated with arsenic contaminated water. The regression analysis was carried out between arsenic and other metals. Linear regression shown negative correlation between As & Se (r= -0.999, p = 0.018), As & Pb (r = -0.992, p = 0.078) and positive correlation between As & Cu (r =+0.998, p = 0.03). But no satisfactory correction observed between As & Mn, As & Hg, and As & Ni. It has been observed selenium concentration decreases with increase arsenic concentration in both rice and husk, collected from As contaminated field in Bangladesh. This has also been observed in two vegetable samples those we had studied. All analyzed elements concentration (µg/gm) were less in “Kachu (Taro)” comparing “Data (Stem amaranth)” except arsenic. Arsenic was very high in “Kachu” comparing “Data” even though they were irrigated with same water containing arsenic 205 µg/L.

The overall conclusion from arsenic species analysis in rice, paddy (rice + husk), and some vegetables are that inorganic arsenic is the dominating species of arsenic. It appears from all four-rice analysis that inorganic arsenic is the major portion of arsenic in rice. All four-husk analysis shows only presence of inorganic arsenic. No methylated form of arsenic was found in any husk samples, but arsenic species in a paddy (rice+ husk) sample shows high inorganic arsenic (76.46%) and 19.37% DMA & 4.23% MMA. It shows presence of inorganic arsenic & DMA, and possibility of an unknown arsenic species in Lady's Finger. It was very high arsenic concentration in a vegetable named “Kachu” which grows inside soil a popular food in West Bengal-India and Bangladesh. Most interesting, its inorganic arsenic concentration is quite high, but it has no detectable amount of methylated form of arsenic and possible of an unknown arsenic species.

Rice and vegetable are the staple food for poor villagers of Bangladesh and West Bengal-India. This is true for the villagers in Kolsur gram-panchayet (G.P.) in Deganga block of North 24-Parganas district, West Bengal-India, where we studied for arsenic in soil, rice, and vegetable from 10 plots cultivated with arsenic contaminated water. From the results of total arsenic (drinking water + rice + vegetable + Pantavat (rice mixed with water) + water added for food preparation) body burden to North Kolsur villagers is 1185.0 µg for per adult per day and 653.2 µg for per child per day. Amount of arsenic coming from rice, vegetable, and water added for Pantavat and food preparation is 485 µg i.e., 41% of total for adult and 253.2 µg i.e., 38.8% for child, and from rice and vegetable 285 µg i.e., 24% of total for adult and 153.2 µg i.e., 23.4% for child (around age 10 years). Our findings show most of the arsenic coming from food is inorganic in nature. As toxicity of most of the organic arsenic compounds in food is less compared to inorganic arsenic.

Therefore, compared to worldwide arsenic consumption from food, it appears Kolsur villagers are also consuming high amount of inorganic arsenic from food and vegetable, and people appears also at risk from arsenic in food. Kolsur village is an example of many such villages in West Bengal-India and Bangladesh.

Further, products from arsenic irrigated water- soil system rich in arsenic are also coming to common marketplace far away from contaminated areas and even people who are not drinking arsenic contaminated water may get arsenic from food products produced from contaminated fields. In West Bengal-India and Bangladesh rice, vegetable, and other products are coming to cities (including Kolkata in West Bengal-India and Dhaka in Bangladesh) from villages and possibility that city people consuming arsenic contaminated products from contaminated areas cannot be ruled out.

Abbreviation: IC-ICP-MS, Ion chromatography-inductively coupled plasma-Mass spectrophotometry; FI-HG-AAS, Flow injection-hydride generation -atomic absorption spectrometry

Introduction

In West Bengal-India out of its total 18 districts in 9 districts arsenic in groundwater has been found over 50 µg/L. Total area and population of these 9 districts are 38,865 km2 and 42.7 million (approx.), respectively while the area and population of West Bengal are 88,000 km2 and 68 million (approx.). This does not mean that 42.7 million people in these 9 districts are drinking arsenic contaminated water and will suffer from arsenic toxicity, but no doubt they are at risk. In 9 affected districts of West Bengal, approximately 6 million people are drinking arsenic contaminated water at levels >50 µg/L. For arsenical skin lesions, we examined approx. 86,000 people in 7 affected districts out of 9 and 8500 (9.8%) people were registered with arsenical skin lesions. But we expect, from extrapolation of our generated data that nearly 300,000 people may have arsenical skin lesions in West Bengal-India.

State, West Bengal is prosperous in agriculture. The state has surplus food production and main crops are paddy and vegetable. Land of these 9 contaminated districts of West Bengal is very fertile and all are in recent gangetic deltaic plain. Major quantum of food production of West Bengal is coming from these 9 districts and tens of thousands of small and big diameter tube wells are in use for irrigation purpose.  Plant needs a small fraction of the total water we pour to the field. Groundwater is considered to be the main source of water for agriculture and its use is increasing day by day. It has been noticed that even if surface water is available for irrigation from nearing source farmers are reluctant to use those sources if they must spend some extra money for this purpose. Except 5 months of rainy season (June-October) rest of the 7 months of the year farmers use groundwater for agriculture. Even during June to October if there are no rain farmers use groundwater.

During 1996 it has been reported¹ by School of Environmental Studies (SOES), Jadavpur University, and Kolkata that from a single Rural Water Supply Scheme (RWSS), Govt. of W. Bengal in Malda district, supplying water to a few villages, and 147.8 kg of arsenic came out during a year with groundwater. Therefore, it is expected that huge quantity of arsenic is falling on agricultural land from contaminated tube wells in use for irrigation. A follow-up study was made by SOES to know how much arsenic is falling on irrigated land in one year during cultivation from all 3200 tube wells that exist in the block Deganga of North 24-Parganas².  The basis of calculation was like this: 3200 shallow tube wells of 7 cm to 10 cm diameter were used in 1997 in Deganga block for agriculture and average discharge rate was 20 m3/hr. electric / diesel pumps (average 5HP) were used.  These shallow tube wells used to run in average 7 hours per day for 7 months in a year.  We had analyzed 597 irrigation tube wells out of total 3200. Out of those 597 tube wells, 574 tube wells contain arsenic ≥10 µg/L.  The average arsenic concentration of the 574 tube wells was 70 µg/L (range 10 -840 µg/L). A calculation was   made to know how much arsenic is falling on soil from 3200 tube wells based on measurement of 574 tube wells (total analyzed) and extrapolating to 3200. The overall result shows from the block Deganga alone 6.4 tons of arsenic is falling on agricultural land in one year from 3200 agriculture tube wells. Thus, it appears that in West Bengal-India and Bangladesh a few thousand tons of arsenic is falling on agricultural land in every year.

Total arsenic contribution through food for many developed countries have been reported³. Although sea food contains mainly nontoxic organic forms of arsenic and rapidly excreted

 

 

Figure 1. Location of Madaripur district of Bangladesh, and North 24-Parganas and Medinipur districts of India

 

through urine, but other than sea food inorganic arsenic may be the major contribution of arsenic in many foods. A study from Canada^4,5 indicates that arsenic content of many foods is mainly inorganic in nature and typically in the range 65-75%. US-EPA reported that percentage of inorganic arsenic in rice, vegetables and fruits are 35%, 5% and 10% respectively⁶. It is reported that absorption of arsenic by plant is influenced by the concentration of arsenic in soil⁷.

In arsenic affected areas of West Bengal-India and Bangladesh huge quantity of arsenic is falling on agricultural land and thus it will be interesting and very important to know whether there is an increase concentration of arsenic in vegetable and crops that grow in this region.

 

In this paper, I will report (a) the total arsenic concentration in soil, paddy (shoot, rice and husk), vegetable (including edible root, stem, leaf, and fruit) in 10 plots of land irrigated with arsenic contaminated water, (b) arsenic species in paddy (rice + husk), rice, husk, and in vegetable (also in edible root, stem, and fruit), (c) some metals and metalloids in soil, vegetable, rice. and husk, and (d) Arsenic body burden.

METHODS AND MATERIALS

Selection of study area

Demography of the State West Bengal is 18 administrative districts and North 24- Parganas is one of the districts. North 24-Parganas is one of the 9 arsenic affected districts of West Bengal-India also. In North 24-Parganas there are 22 blocks/ police stations. Each block has several Gram Panchayets (G.P.) and in each G.P., there are several villages. We have chosen Deganga block of North 24- Parganas district as our study area. The reasons are, (A) we have the detail 8785 hand tube-wells water analysis report of Deganga one of the 22 blocks of North 24-Parganas (Table 1) and also 597 irrigation tube wells report out of 3200 which were used for irrigation in Deganga block alone (Table 2), (B) Deganga block is close (about 60 km) from our institute (SOES) with good road connection, and (C) we are working in this block for a long time and have good report with the villagers. Figure 1 shows arsenic affected block of North 24-Parganas and Deganga block. Deganga block has 13-gram panchayets and groundwater of all these gram pahchayets are arsenic contaminated (>50 µg/L). Out of 13-gram panchayets (G. P.), in 11 G. Ps we have identified patients with arsenical skin lesions.   We had chosen 10 fields in Kolsur (North) village of Deganga block under Kolsur gram panchayet. Table 3 shows the crops from 10 fields we had analyzed and other related information. For control study, we had chosen the district Medinipur, an area was groundwater arsenic concentration <3 µg/L; soils of the control area show arsenic in the range 5.31 µg/gm to 6.60 µg/gm (n=6). Overall information of control area is given in Table 4.

 

 

 

 

 

Table 1: Distribution of arsenic in tube wells water in Deganga, North 24-Parganas, West Bengal, India

 

Table 2: Distribution of arsenic in irrigated tube wells water in Deganga block, North 24-Parganas, West Bengal-India

 

 

 

 

 

Table 3: Type of crops and other information of 10 selected agricultural fields in Kolsur village of Deganga block, West Bengal, India      where arsenic contaminated ground water was used for irrigation purpose.

Table 4:   Overall information of control area, where groundwater contains arsenic below 3 µg/L

 

21 months study report

Studies had been carried for 21 months (August 1998 to April 2000). Table 5 shows the number of samples and time schedule of the whole study.

Table 5: The number of samples and time schedule of the whole study (from August 1998 to April 2000) *

 

Instrumental Techniques

The FI-HG-AAS was used in School of Environmental Studies (SOES), Jadavpur University, Kolkata, India

A flow injection-hydride generation -      atomic absorption spectrometry (FI-HG-AAS) technique was used in our laboratory for analysis of total arsenic in water, soil, crop, and vegetable samples. The FI-HG­AAS system was assembled from commercially available instruments and accessories in our laboratory. A Perkin-Elmer Model 3100 spectrometer equipped with a Hewlett-Packard Vectra computer with GEM software, Perkin-Elmer EDL System-2, arsenic lamp (lamp current 400 mA), and Varian AAS Model Spectra AA-20 with hollow-cathode As lamp (lamp current 10 mA) were used. The flow injection assembly consists of an injector, Teflon T-piece, tigon tubing and other parts for the FI system from Omni-fit UK. The peristaltic pump (VGA-76) from Varian and Minipuls-3, Gilson, Model M 312 (France) were incorporated into the FI system. Details of the instrumentation have been discussed in our earlier publications^3,4. A Heraeus muffle furnace fitted with manual temperature control was used for dry ashing.

The ICP-MS was used in National Institute of Health Sciences (NIHS), Tokyo, Japan

A microwave digestion system (MDS-2100) from CEM Innovators in Microwave Technology, USA with a rotor for twelve Teflon digestion vessels HP-500, was used for sample digestion using HNO₃ and H₂O₂.

The ICP-MS was used from Department of Environmental Chemistry, National Institute of Health Sciences, Tokyo, Japan. Estimation of arsenic and other metals and metalloids in soil, vegetable, and paddy, (rice & husk) have been carried out by ICP-MS method. Analytical results of SRM digested similarly and analyzed by ICP-MS. The instrumental conditions of this system are shown in Table 6.  

 

Table 6: Instrumental conditions for ICP-MS

 

The IC-ICP-MS was used from Department of U.S. Food & Drug Administration, Forensic Chemistry Center, Cincinnati, USA.

The IC-ICP-MS was used from Department of U.S. Food and Drug Administration (FDA), Forensic Chemistry Center, Cincinnati, USA. The chromatographic system used consisted of a model GP 50 ion chromatography pump (Dionex, Sunnyvale, CA, USA), AS 3500 autosampler (Thermo Separations Products, San Jose, CA, USA) and a non-metallic automated switching valve (Waters, Milford, MA, USA) with 20 µL injection loop between the Column outlet and ICP-MS nebulizer. Operation of the valve was controlled using signal relays from the IC pump.

A model PQ3 ICP-MS (VG Elemental) operating under normal multi-element tuning condition was used for chromatographic detection. Column effluent was directed to the concentric nebulizer and cooled conical spray chamber of the ICP-MS using a length (~60 em) of 0.25 mm. i.d. PEEK® tubing. An anion exchange column, Hamilton PRP-X 100 (4.6 x 150 mm), was used for separation of arsenic species.

 

Chemicals and Reagents

School of Environmental Studies, Jadavpur University, West Bengal, India

All reagents were of analytical grade. Distilled demonized water was used throughout. Standard arsenic solutions were prepared by dissolving appropriate amounts of As₂O₃ (Merck, Germany) and standard arsenic (V) Titrisol (Merck, Germany).  Standard stock solutions were stored in glass bottles and kept refrigerated. Dilute arsenic solutions for analysis were prepared daily. The reducing solution was sodium tetrahydroborate (Merck, Germany) 1.5% (m/v) in 0.5% (m/v) sodium hydroxide (E. Merck, India Limited). The HCl (E. Merck, India Limited) concentration was 5M. Ashing acid suspension was prepared by stirring 10% (w/v) Mg(NO3)₂. 6H₂O and 1% (w/v) MgO in water until homogeneous⁴.

Standard reference materials were used to check the accuracy of the method. Pond Sediment NIES- 2, from the National Institute for Environmental Studies, Japan; Standard Chinese River Sediment 81-101 (of 1981); San Joaquin Soil SRM 2709, Citrus Leaves SRM 1572, Rice Flour SRM 1568a, Spinach Leaves SRM 1570a and Tomato Leaves SRM 1573a are from the National Institute of Standards and Technology (NIST), USA.

In National Institute of Health Sciences, Tokyo, Japan

All reagents were of analytical reagent grade. Milli-Q Water (Yamato Millipore filter, WT 100) was used throughout. Stock solutions (10 mg/L in water) of arsenic and other elements (Se, Cu, Zn, Pb, Mn, Ni, Hg) were prepared separately from 1000 mg/L stock standard (Cica-Merck, Kanta Chemical Co. Inc, Japan) of each element. All stock solutions were stored in polyethylene bottles and kept at 4°C. Dilute solutions (2.5, 5, 10, 20, 30, and 50 µg/L) for analysis were prepared daily.

The samples digestion was carried out with concentrated nitric acid (HNO₃) (Wako Pure Chemical Industries Ltd., Osaka, Japan) and high purity hydrogen peroxide (H₂O₂) (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Standard reference materials were used to check the accuracy of the method for multi element analysis. Rice flour SRM 1568a, Apple leaves SRM 1515, Tomato Leaves SRM 1573a, San Joaquin Soil SRM 2709, and Spinach leaves SRM 1570a are from the National Institute of Standards and Technology (NIST), USA.

 

In U.S. Food and Drug Administration, Cincinnati, U.S.A.

Ultrapure deionized water was used (DIW, Millipore, MA, USA). High-purity ammonium hydroxide was from Fisher Scientific (Fair Lawn, NJ, USA). Anhydrous trifluoroacetic acid (TFA) was from Sigma (St. Louis, MO, USA), and ammonium nitrate, and monobasic ammonium phosphate were from J.T. Baker (Phillipsburg, NJ, USA).

Commercial stock standards of As(III) and As(V) (1000 µg As/mL as As₂O₃ in 2% HCl and H₃AsO₄. ½ H₂O in 2% HNO₃, respectively) were obtained from Spex Industries (Metuchen, NJ, USA). Dimethylarsinic acid (98.0%) and disodium methyl arsenate (99%) were obtained from Chem Service (West Chester, PA, USA). The mobile phase consisted of 10 mM NH₄H₂PO₄ and 10 mM NH₄PO₃ adjusted to a pH of 6.3 with NH₄OH. NIST SRM 1568a rice flour was used for method validation

Sample collection and preservation

Water samples

Tube well water samples were collected in pre-washed (with 1:1 HNO₃) polyethylene bottles. After collection concentrated nitric acid (1.0 ml per liter) was added as preservative. Samples, which were not analyzed immediately, were kept in a refrigerator at 4°C. Details of the collection procedure have been described in our earlier publications^3,4.

Vegetable and crop samples

Vegetable and crop samples were collected from selected agricultural fields where arsenic contaminated water was used for agricultural purposes. Vegetable, crop, and plant samples were picked by hand, stored in polyethylene bags, and kept cool until processing in the lab. Samples were washed thoroughly with tap water to remove soil and other particles, and finally washed in sonicator with demonized water for several times. Prior to sample washing, plants consisting of both roots (below soil surface) and shoots (above soil surface) were separated into root, stem, leaf, and fruit as a sub samples and dry matter yields after drying at 60°C for about 72 hours. Dry samples made fine powder by Agate pestle and mortar, sieved, and stored in polyethylene bags with proper leveling at room temperature.

Soil samples

Surface soil samples and samples at different depth were collected from agricultural fields, where arsenic contaminated water was used for agricultural purposes and samples were also collected from non-arsenic contaminated agricultural fields (arsenic concentration in agricultural water was <3 µg/L). We collected four soil samples from different part of the same field and made a mixture. Samples were picked by non-metallic spoon, stored in polyethylene bags, and kept cool until processing in the lab. First soil samples were dried at room temperature and finally at 60°C for several hours. Dried samples were made fine powder by Agate pestle and mortar, sieved, and stored in polyethylene bags with proper leveling at room temperature.

 

Sample treatment for analysis

In SOES Laboratory

Sand bath digestion of soil samples

Approximately 0.10 to 0.50 gm of soil sample was placed in a 25cc conical flask (glass), 2 mL deionized water added, followed by 2 mL concentrated HNO₃ and 1 mL concentrated H2SO4 (Analar Grade, E. Merck, India). The mouth of the flask was covered with a small glass funnel. Then it was heated on a sand bath until the fumes of SO₃ evolved. When fumes of SO₃ evolved, heating was discontinued and after cooling, the solution was diluted and filtered through a millipore membrane (0.45µm pore size) filtering apparatus, then adjusted to fixed volume. Standard Reference Materials (SRM) was analyzed in the same way to test the accuracy.

Dry ashing digestion of vegetable and crop samples

Approximately 0.20 to 0.70 gm of dry vegetable/ crop sample or 1.5 gm to 3.0 gm of wet/weight vegetable sample was taken into a 100 ml Borosil Conical Flask and 10 ml ashing aid [10% (w/v) Mg(NO₃)₂. 6H₂O (Riedel-De Haenag, Seelze- Hannover, Germany) + 1% (w/v) MgO (E. Merck, Darmstadt, Germany) in deionized water] was added with continuous agitation. Then 5 mL HNO₃ (40%, v/v) was added and evaporated to almost dryness on a hot plate at about 90-100°C. When the sample dried, the beakers were covered with a pre-washed watch glass and transferred to the muffle furnace².

The following temperature program was adopted for proper ashing of the sample: 150°C for l hr, 200°C for 30 min, 250°C for 30 min, 300°C for 2 hrs, 400°C for l hr, and 450°C for 12-14 hrs. After cooling to room temperature 2 mL (10% v/v) HNO₃ was added to the carbonaceous residue. The mixture was dried again on the hot plate to dryness and transferred to the furnace. The ash was subjected again to the following temperature program: 150°C for 1 hr and 30 min, 300°C for 30 min, and 450°C for 12- 14hrs. After cooling the white ash was moistened with a few drops of water, dissolved in 1-2 mL 6M HCI and filtered through a Millipore filter (0.45 µm) and finally made the proper volume with 6M HCl. Triplicate blanks and Standard Reference Materials (SRM) were-prepared following the same digestion procedure.

Microwave digestion of soil, vegetable, and crop samples in NIHS, Tokyo, Japan

Samples for digestion were weighed (0.2 gm to 0.5 gm of dry sample) in the Teflon vessel and added 2:1 v/v of nitric acid and hydrogen peroxide. The vessels were closed using the lid provided. For safety of the vessel, rupture membrane was inserted in the lid. Vessels were set in the turn table of the micro-wave digestion machine and the below settings were programmed (Table 7).

 

 

Table 7: Optimum parameters for sample digestion by microwave system

 

 

 

After cooling for 30 minutes, the vessels were opened carefully. Each digested solution was transferred quantitatively to a 10 mL volumetric flask and adjust to fixed volume with Milli-Q water. Finally, it was filtered through a Millipore membrane (0.45 µm) (CASL 45 2.5 CMD, membrane: Acetyl Cellulose) and kept in plastic container for analysis. The Standard Reference Materials (SRM) were digested under the same digestion procedure.

Preparation and extraction of crop and vegetable samples for arsenic speciation (FDA lab, USA)

The rice and vegetable samples were grounded in an acid-washed glass mortar and pestle. A larger amount of each of the paddy sample was shipped; therefore, they were milled and sieved (0.5 mm) in a model ZM 100 ultra-centrifugal mill (Retsch, Haan Germany). Prepared samples were stored in HDPE bottles at ambient temperature prior to use.

Sample (0.1-0.5gm) were mixed with 0.5-2 mL of 2M trifluoroacetic acid (TFA) and allowed to stand for 6 hours at 100°C in either a 15 mL or a 60 mL capped HDPE centrifuge tube. The TFA extract was allowed to cool and diluted to volume (10-25 mL) with Ultrapure deionized water (DIW, Millipore, MA USA). Extracts were filtered through a 0.45 µm Nylon syringe filter prior to analysis by IC-ICP-MS.

 

Sample analysis

Flow injection-hydride generation-atomic absorption spectrometry (Fl-HG-AAS)  (SOES Lab.)

The sample was injected into a carrier stream of 5M HCl by means of a six-port sample injection valve fitted with a 50 µL sample loop. The injected sample, together with carrier solution met subsequently with a continuous stream of sodium tetrahydroborate. Mixing with sodium tetrahydroborate generated hydride, which subsequently entered the ice water bath and then the gas-liquid separator apparatus, which was cooled with ice-cold water. This cooling procedure and the design of the gas-liquid separator are more efficient than conventional FI-AAS and the possibility of water vapor entering the quartz cell is reduced to a great extent. Inside this apparatus a continuous flow of N₂ carrier gas assists mixing and the reaction and subsequently carries hydride to the quartz tube mounted in the air-acetylene flame for As measurement. Peak signals were recorded using a computer linked to the atomic absorption spectrophotometer (AAS) that is capable of both peak height and peak area measurement.  The peak height signals were measured and the concentrations of arsenic of the samples were measured/calculated against the standard curve. The experimental condition for FI-HG-AAS system is given in the Table 8.

 

Table 8: Optimum-Parameters for arsenic determination by flow infection (FI) system

 

 

Water Samples

Preserved water samples (in 1 mL HNO₃ per liter of water) were analyzed by FI-HG-AAS against arsenite and arsenate mixture (1:1) as the standard.

Soil, vegetable, and crop samples

Digested samples were analyzed by Fl-HG-AAS method against arsenate as the standard.

Inductively Coupled Plasma-Mass Spectrophotometry (ICP-MS) (NIHS Lab., Japan)

ICP-MS Analysis

ICP-MS is an element selective detector. Twenty microliters (20 µL) of the microwave acid digested sample were injected into a carrier stream of Milli-Q water with a sample loop. The Chromatographic areas were measured, and the concentrations of elements were calculated against the individual element standard curve. The experimental conditions of ICP-MS are given in Table 6.

 

Ion chromatography-inductively coupled plasma-Mass spectrophotometry (IC-ICP-MS) (Food & Drug Lab., USA)

Figure 7 shows the chromatogram obtained for a standard mixture of As(III), DMA, MMA and As(V) in our experimental condition (Table 9). Each of the arsenic species is present at an arsenic concentration of 2 ng/mL. Data was collected for 10 minutes. The four arsenic species are baseline resolved in under 8.5 minutes; however, arsenobetaine (AsB) is not resolved from As(III) under these separation conditions. AsB is generally found in fish and shellfish samples and is not expected to be present in vegetable or rice samples collected from arsenic contaminated agricultural fields. The precision of peak areas measured over a period of 4 hours (n=5) using replicate 25 µL injections of a standard containing 2 ng/mL each of As(III), DMA, MMA and As(V) was 4.8%, 4.4%, 4.1%, and 9.0%, respectively. As estimate of the instrumental detection limit (IDL) for each of the arsenic species was calculated based on 3 times, the standard deviation of peak area measurements for replicate 25 µl injections of a standard containing 0.2 ng As/mL each of As(III), DMA, MMA and As(V). The IDLs were 0.1, 0.1, 0.1 and 0.2 ng/mL for As(III), DMA, MMA and As(V), respectively.

 

Table 9. IC-ICP-MS operational condition

 

 

Samples were analyzed using calibration standards at 0.5, 1, 2, 5 and 10 ng/mL concentrations (in some cases a 20 ng/mL standard was also used). Peak areas were corrected for any drift associated with the flow injection signal at the beginning of each chromatogram. The concentrations of several samples were also checked using standard addition.

 

Spikes of each of the arsenic species were taken through the method and spike recoveries of 83%, 88%, 100%, and 93% were obtained for As(III), As(V), MMA and DMA, respectively. However. it should be noted that As(V) can be partially reduced during the extraction procedure. The As(V) recovery was calculated in spiked samples using the sum of the increase in the As(III) and As(V) concentrations. Because of this partial reduction, the method is only capable of providing total inorganic arsenic as the sum of the As(III) and As(V) concentrations. Standard NIST SRM 1568a Rice Flour also analyzed by the same procedure and the sum of the arsenic species concentrations for SRM Rice Flour (0.27 µg/gm) compared well with the certified total arsenic value (0.29 µg/gm).

 

Total arsenic in irrigation tube wells, soil, vegetable, paddy samples were analyzed by using FI-HG-AAS after dry ashing (vegetable, rice, and husk) and acid digestion (soil)

Water samples used for cultivation had been collected from 10 locations and analyzed by FI-HG­ AAS method and their results are given in Table 10. Similarly, soil samples had

 

Table 10: Analysis of arsenic and iron for 10 irrigation tube-well used in 10 fields for cultivation

 

 

 

 

Table 11: Analysis of arsenic in soil where arsenic contaminated groundwater was used for cultivation

 

Table 12: Distribution of arsenic concentration in different parts of the vegetable collected from 6 fields out of 10 irrigated with arsenic contaminated water

Table 13:  Distribution of arsenic concentration in different parts of paddy plant collected from 3 selected location of Kolsur (N) in Deganga block, North 24-Parganas, West Bengal, India during monsoon period and rainwater was used for cultivation

Table 14: Distribution of arsenic concentration in different parts of paddy plant collected from 5 selected fields of Kolsur (N) village in Deganga block, North 24-Parganas, West Bengal, India during pre-monsoon period and cultivated with arsenic contaminated groundwater

Table 15: Analytical results of Standard Reference Matarial (SRM) by FI-HG-AAS

 

Arsenic concentration in irrigation tube well and soil before and after a devastating flood

During September 2000 to October 2000 of our 10 experimental Fields were submerged with water due to a devastating flood. The analytical results are given in Table 16.

Table 16: Arsenic concentration in irrigation tube well and soil before and after a devastating flood

 

 

been collected from 10 fields where contaminated underground water was used for cultivation. The analytical results of those soil samples are given in Table 11. Vegetable and paddy samples (including various parts) cultivated with arsenic contaminated water had been collected time to time from selected fields. Their analytical results are given in Table 12, 13, & 14, respectively. Analytical results of Standard Reference Material (SRM) digested similarly & analyzed by FI-HG-AAS given in Table 15.

Total arsenic and other metals and metalloids in soil, vegetables, and paddy  (rice & husk) samples by using ICP-MS after microwave digestion.

Estimation of arsenic and other metals and metalloids in soil, vegetable, and paddy, (rice & husk) have been carried out by ICP-MS method. Total arsenic and other metals and metalloids concentrations in soil, rice (also in husk), and vegetable are given in Table 17, 18, and 19, respectively. Analytical results of SRM digested similarly and analyzed by ICP-MS given in Table 20.

 

Table 17: Concentration (µg/gm) of 7 elements in 9 contaminated soils collected from arsenic contaminated fields of West Bengal- India and Bangladesh

Field No.

As in water (µg rl)

Type of sample

As

Mn

Cu

Hg

Pb

Ni

Zn

Fl

805

Soil

34.15

732.9

58..44

1.13

67.36

72.29

80.38

F2

570

Soil

43.08

508.20

49.07

1.13

52.87

53.76

105.00

F3

156

Soil

17.46

388.33

38.25

1.36

39.67

40.48

58.53

F4

488

Soil

34.11

538.68

54.65

1.20

52.38

53.71

70.08

F6

215

Soil

25.63

514.93

49.16

1.26

57.12

61.44

68.54

F7

218

Soil

25.54

457.46

46.34

1.47

40.33

51.12

45.62

Fl0

195

Soil

23.41

415.47

42.60

1.61

43.87

44.59

52.12

Bangladesh

492

Soil     ':I

40.98

484.29

51.05

1.15

46.89

50.08

-

Bangladesh

525

Soil    '

42.13

473.62

49.20

0.96

55.92

61.67

71.47

Table 18:   Concentration (µg/gm) of 7 elements in rice and husk collected from arsenic contaminated agricultural fields of West Bengal-India and Bangladesh

 

BDL: Below Detection Limit

 

 

 

Table 19: Concentration (µg/gm) of 7 elements in vegetable samples collected from arsenic contaminated agricultural fields of Bangladesh

 

* A kind of vegetable, whole portion (stem + leaf) is used for food and local name 'Data'

** A kind of popular vegetable, whole portion (root +stem+ leaf) is used for food and local name 'Kachu'.

 

Table 20: Analytical results of NIST Standard Reference Material (SRM) by ICP-MS

 

Arsenic species in paddy (rice+ husk), rice, husk, and vegetable by IC-ICP-MS after TFA (trifluoroacetic acid) extraction

The analytical results are given in Tables 21 and 22.

Table 21: Concentration of arsenic species (ng/gm) and percentage of inorganic arsenic and methylated arsenic in rice, husk, paddy (rice+ husk) & vegetable (dry) collected from arsenic contaminated fields in Kolsur (N) village of Deganga block, North 24-Parganas, West Bengal, India

Table 22:  Concentration of arsenic species (ng/gm) and percentage of inorg-As and methylated arsenic in paddy (rice+ husk) and vegetable (dry) collected from arsenic contaminated fields in Datterhat village of Madaripur district, Bangladesh

 

       Results & Discussion

Table 10 shows analysis of arsenic from August 1998 to April 2000 of 10 tube wells used in 10 fields for irrigation purpose. Although from the results of arsenic in tube wells it appears that in some of the tube wells there is increase of arsenic with time but this type of variation, we had experienced in many tube wells during our work in West Bengal-India and Bangladesh.

It has also been observed that arsenic concentration in same field varies locations to location (Table 23) and concentration of arsenic is higher at the surface than at any depth (Table 24 and Figure 2). Table 24 and Figure 2 indicate that arsenic concentration is higher at surface and at depth 18-inch and 36-inch the variation is minimum.

 

Table 23. Distribution of arsenic concentration (µg/gm) of soil samples collected from different location of the same arsenic contaminated fields during August 1998.

 

Table 24. Distribution of arsenic concentration (µg/ gm) in contaminated soil with depth

 

 

 

Figure 2: Distribution of arsenic concentration (µg/ gm) in soil with increasing depth (in inch.).

 

       Table 16 shows arsenic concentration in irrigation tube wells and soil measured in April 2000 and again in April 2001 after flood. Our 21 months study on arsenic in soil, crop, and vegetable finished during April 2000. However, during September 2000 - October 2000 there was a devastating flood in West Bengal. Most of the parts of North 24-Parganas including our 10 experimental

fields were submerged in floodwater. During April 2001 we went to the fields and collected water and soil samples from the same location of our experimental fields. Table 16 indicates that there is almost no change of arsenic in irrigation tube well, but drastic change of arsenic in soil. Thus, it appears that arsenic from soil after flood washed away but six months was not sufficient for aquifer dilution.

Table 25 shows arsenic concentration in rice & husk (a) cultivated with arsenic contaminated underground water & elevated arsenic concentration in soil, (b) cultivated with rainwater & arsenic concentration in soil is low, and (c) controlled cultivation: cultivated by ground water having arsenic <3 µg/L and soil arsenic concentration 5.31-6.13 µg/gm.     From the Table 25 it appears that higher the arsenic concentration in irrigation water & soil, higher is the arsenic concentration in rice & husk. In pre-monsoon cultivation paddy was grown in arsenic rich irrigated water with elevated arsenic in soil. But in monsoon cultivation, contribution of arsenic from water for irrigation was not there. So, also arsenic in soil has gone down as rainwater washed away some arsenic from soil. It shows that the average arsenic concentration in contaminated soil, rice, and vegetable were 3.81, 3.62, and 5.66 times higher than the control samples, respectively.

Estimation of arsenic in different parts of the vegetable & paddy had also been carried out to know the distribution of arsenic in stem, leaf, and fruit for vegetable and shoot, rice, & husk for paddy. Analytical results of few are represented in Tables 12-14. Table 13 is the arsenic concentration in paddy irrigated with rainwater (monsoon) and Table 14 is for paddy irrigated with arsenic contaminated groundwater (pre-monsoon).

The overall observations from Tables 12-14 are arsenic concentration in vegetable and paddy increases when arsenic in soil is higher and when cultivated with arsenic contaminated groundwater. Also, for paddy arsenic concentration decreases shoot > seed (rice) > husk and in vegetables the distribution is leaf> stem > fruit. Although it is reported⁸ that root contains maximum amount of arsenic, but we could not analyze root for paddy plant as we were not sure about adhered soil remove from root. Arsenic in different parts of paddy with increasing concentration of arsenic in soil is given in Figures 3 (pre-monsoon and higher concentration of arsenic in soil) & Figure 4 (monsoon and arsenic concentration in soil is low) and that of vegetable given in Figure 5.

 

 

Figure 3: Distribution of arsenic in different parts of paddy (pre-monsoon) with increasing arsenic concentration in soil.

 

Figure 4: Distribution of arsenic concentration in different parts of paddy (monsoon) with increasing arsenic concentration in soil.

 

 

 

Figure 5: Distribution of arsenic concentration in different parts of vegetable with increasing arsenic concentration in soil.

 

Arsenic analysis of few rice and vegetable samples were done from different laboratories.  Results are given in Tables 26-28. FI-HG-AAS after Teflon bomb digestion was done from our laboratory; microwave digestion followed by ICP-MS was done from National Institute of Health Sciences (NIHS) laboratory, Tokyo, Japan & IC-ICP-MS for various species done from US Food and Drug Administration Laboratory, Forensic Chemistry Center, Cincinnati, USA. Results of IC-ICP-MS represents only sum of inorganic arsenic (In-As) + MMA + DMA. It is expected IC­ ICP-MS results will be low compared to FI-HG-AAS & ICP-MS results after digestion.

 

 

Table 26: Arsenic concentration in same rice and paddy (rice+ husk) samples measured by using FI-HG-AAS and IC-ICP-MS in different laboratories.

 

 

 

 

Table 27: Arsenic concentration in same rice and paddy (rice+ husk) samples (contaminated) measured by using FI-HG-AAS and IC-ICP-MS in different laboratories.

Table 28: Arsenic concentration in same vegetables samples measured by using FI-HG-AAS, ICP-MS, and IC-ICP-MS in different laboratories.

 

 

Total arsenic and other metal and metalloid in soil, vegetable, and paddy in some samples of West Bengal-India and Bangladesh

Along with arsenic in soil samples (n = 9), Mn, Cu, Hg, Pb, Ni & Zn were analyzed by ICP-MS after microwave digestion (Table 17). Our analysis of NIST soil sample (Table 20) by the same procedure is in well agreement. The regression analysis was carried out between arsenic and other metals.  However, no significant positive or negative co-relation was observed between As & Mn (r=0.382, p=0.309), As & Cu (r=0.632, p=0.067), As & Ni (r = 0.460, p= 0.212) and As & Pb (r = 0.493, p = 0.177). A significant (<0.05) positive correlation found between As & Zn (r = 0.763, p = 0.027) and also a strong negative correlation was observed between As & Hg (r = -0.802, p = 0.009).

Table 18 shows analysis of As along with Se, Mn, Cu, Hg, Pb & Ni in rice & husk of 3 paddy samples cultivated with arsenic contaminated water. The regression analysis was carried out between arsenic and other metals. Linear regression shown negative correlation between As & Se (r= -0.999, p = 0.018), As & Pb (r = -0.992, p = 0.078) and positive correlation between As & Cu (r = 0.998, p = 0.03). But no satisfactory correction observed between As & Mn (r = -0.980, p =0.126), As & Hg (r = -0.452, p 0.70) and As & Ni (r = -0.233, p = 0.849). It has been observed selenium concentration decreases with increase arsenic concentration in both rice and husk (Table 18 & Figure 6). This has also been observed in two vegetable samples those we had studied (Table 19). All analyzed elements concentration (µg/gm) were less in “Kachu” comparing “Data” except arsenic. Arsenic was very high in “Kachu” comparing “Data” even though they were irrigated with same water containing arsenic 205 µg/L.

 

Figure 6: Co-relation between arsenic and selenium in rice cultivated by using arsenic contaminated water.

 

 

Arsenic species in vegetable, rice, and paddy in some samples of West Bengal, India and Bangladesh

 

Table 21 & 22 show total arsenic in groundwater used for irrigation and arsenic in soil. The sum of arsenic species in vegetable, paddy (rice + husk), rice, husk, and arsenic species in those samples from West Bengal-India & Bangladesh also shown in these table respectively. In Table As(III) and As(V) presented together as inorganic arsenic. The reason show portion of As(V) is reduced to As(III) during extraction.

 

 

Figure 7 shows the chromatogram of As(III), MMA, DMA and As(V) measured by IC-ICP­ MS.

 

 

 

Figure 8 shows arsenic species in rice (Sample no. 1 in Table 21). It appears from all four-rice analysis that inorganic arsenic is the major portion of arsenic in rice.

 

 

 

 

Figure 9 shows arsenic species in husk (Sample no.6 in Table 21). All four-husk analysis shows only presence of inorganic arsenic. No methylated form of arsenic was found in any husk samples.

 

Figure 10 shows arsenic species present in lady's Finger (Sample no.18 in Table 21).  It shows presence of inorganic arsenic & DMA, and possibility of an unknown arsenic species.

 

Figure 11 shows arsenic species in a paddy (rice+ husk) sample (Sample no.11 in Table 21). It has high inorganic arsenic (76.46%) and 19.37% DMA & 4.23% MMA.

 

Figure 12: Sample No. 5B in Table 22; Peak identities: 1, As(III); 2, DMA; 4, As(V) and an unknown peak

 

Figure 12 shows very high arsenic concentration in a vegetable named “kachu” (Sample no.5B in Table 22) which grows inside soil a popular food in West Bengal-India & Bangladesh. Most interesting, its arsenic concentration in very quite high but it has no detectable amount of methylated form of arsenic and possible of an unknown arsenic species.

The overall conclusion from arsenic analysis in rice, paddy (rice + husk) and some vegetables are that inorganic arsenic is the dominating species of arsenic.

Conclusion

Food habit and arsenic intake from food and water to those living in arsenic contaminated villages.

Rice and vegetable are the staple food for poor villagers of West Bengal-India and Bangladesh. This is true for the villagers in Kolsur gram-panchayet (G.P.) in Deganga block of North 24-Parganas district, West Bengal-India, where we studied for 21 months arsenic in soil, rice, and vegetable from 10 plots cultivated with arsenic contaminated water. Normally, 3 times villagers eat rice with mainly vegetable per day. Adults (male or female) normally eat 250 gm of rice every time i.e., 750 gm of rice per day and child (around 10 years) eat 400 gm of rice during whole day. Adults eat in average 165 gm of cooked vegetable to each meal and child around 100 gm.    Average water intake per day for adult male, adult female, and child are 4 liters, 3 liters, and 2 liters, respectively. Those who work in field consume more water (average 6.0 liters) and during summer the average water intake to those work in field may go as high as 10 liters. Villagers are also consuming arsenic from Pantavat# and water added for food preparation like rice, soup, curry, and drinks like tea, lemon water, etc. After thorough discussion with the villagers, it appears that this is equivalent to consumption of 1 liter of water for adult and 500 ml for child. Normal daily average food intake for adult to each meal is as follows:

 

#PANTAVAT: This is common food for villagers of W. Bengal-India and Bangladesh at breakfast. Pantavat is rice mixed with water. Normally they pour water to the rice cooked on the previous night and have it as their breakfast. Normally, villagers eat Pantavat with vegetable/ smashed Potato/ Chili and Onion.

Total arsenic burden to the villagers from water and food

We have classified arsenic intake in three categories:

Category I Arsenic alone from contaminated drinking water.

Category II Arsenic from food (rice and vegetable).

Category III Arsenic from contaminated water added to 'Pantavat' and for food preparation (rice, soup, curry, drinks like tea, lemon water).

Altogether, we had analyzed 571 hand tube wells from North Kolsur village and out of these 80%

•

 

 tube wells contains arsenic above 10 µg/L and 70% tube wells contain above 50 µg/L. The average

•

 

 arsenic concentration in contaminated   hand tube wells (n=399) is 200 µg/L. When we started our work in this village, villagers were drinking arsenic contaminated water. We informed them about contamination of their hand tube-wells and identified safe tube wells for them in their village. We do not know what percentage of villagers consuming safe water are, but we know for irrigation purpose they are still using contaminated tube well water. Arsenic concentration in hand tube well they were drinking is given in Table 29.

We had collected and analyzed soil, rice, vegetable, and water used for irrigation for our 10 studied fields in North Kolsur village. Arsenic concentration in 10 fields shown in Tables 3 & 10. Villagers close to our studied areas are consuming rice and vegetable from these 10 fields. Average arsenic concentration in soil (n=68), rice (n=8) and vegetable (n=30) are 22.30 µg/gm (range 7.91µg/gm to 43.08 µg/gm, dry weight), 0.358 µg/gm (range 0.120 µg/gm to 0.663 µg/gm, dry weight) & 0.034 µg/gm (range 0.008 µg/gm to 0.120 µg/gm, wet/weight), respectively. Our preliminary study indicates that edible portion that vegetable grow inside soil contain high amount of arsenic. Villagers were also using the contaminated hand tube well's water for PANTAVAT (average 200

)

 

 µg As/L). Control soil (n=6), rice (n=3), and vegetable (n=3) samples contain arsenic (average) 5.84 µg/gm (range 5.31 to 6.60 µg/gm), 0.089 µg/gm (range 0.083 to 0.959 µg/gm), and 0.009 µg/gm (range 0.007 to 0.012 µg/gm), respectively.

Our preliminary study indicates, we have arsenic concentration in irrigated water of control area is <3 µg/L. Our control area was Medinipur district, which is not arsenic contaminated. It appears that the average arsenic concentration in contaminated soil, rice, and vegetable are 3.81, 3.62, and 5.66 times higher than the control samples, respectively.

Table 30 to Table 32 show arsenic burden to each villager from contaminated tube well water alone, from rice, vegetable, and from water added for Pantavat and food preparation, respectively.

 

 

Table 29: Distribution of arsenic in hand tube well water samples of North Kolsur village in Deganga block of North 24-Parganas district, West Bengal-India

 

North Kolsur villagers body burden of total arsenic, inorganic arsenic, and organic arsenic from water, food, and water added for food preparation

Our earlier study9 shows that arsenic compounds present in hand tube well from our study area are arsenite and arsenate. Our present rice and vegetable analysis show [preliminary study on total inorganic and organic arsenic compounds in some rice and vegetable samples measured by using IC-ICP-MS10 from US Food and Drug Administration, Forensic Chemistry Centre, Cincinnati, USA] 95% and 5% are inorganic arsenic and organic arsenic in rice, and 96% and 4% of inorganic arsenic and organic arsenic in vegetable.

 

 

Table 30: Arsenic burden to each adult male, adult female, and child from water alone at North Kolsur village of Deganga block of North 24-Parganas, West Bengal-India

Table 31: Arsenic burden to each adult male, adult female, and child from contaminated rice and vegetable per day

Table 32: Arsenic burden to each adult male, adult female and child from contaminated water added for pantavat and food preparation per day

Table 33 shows arsenic burden (total, inorganic arsenic, and organic arsenic) to each adult male, adult female, and child combining category I, II and III.

 

 

Table 34: Arsenic from food to North Kolsur villager and from other countries

Table 34 shows a comparative study of inorganic arsenic and total arsenic intake of North Kolsur villagers along with some other countries³. The high concentration of arsenic to population in Japan, Spain, Manaus region of Brazil is due to high intake of seafood. It is also obvious that organic arsenic contributions from other countries represented in Table 34 are also substantial from seafood. North Kolsur villagers rarely consume seafood.

From the results of total arsenic (drinking water + rice + vegetable + Pantavat + water added for food preparation) body burden to North Kolsur villagers (1185.0 µg for per adult per day, 653.2 µg for per child per day), amount of arsenic coming from rice, vegetable, and water added for Pantavat and food preparation  is 485 µg i.e., 41% of total for adult and 253.2 µg i.e., 38.8% for child and from rice and vegetable 285 µg i.e., 24% of total for adult and 153.2 µg i.e., 23.4% for child (around  age l0 years). Our findings show most of the arsenic coming from food is inorganic in nature. As toxicity of most of the organic arsenic compounds in food is less compared to inorganic arsenic, North Kolsur people appears also at risk from arsenic in food.

According to WHO¹¹, 1.0 mg of inorganic arsenic per day may give rise to skin effects within a few years. It has been estimated that based upon the current U.S. Environmental Protection Agency (EPA) standard of 50µg/L, the lifetime risk  of  dying  from  cancer  of  the  liver,  lung,  kidney,  or bladder, from drinking 1 liter per day of water could be as high as 13 per 1000 persons¹². Using the same methods, the risk estimate for 500 µg/1 of arsenic in drinking water would be 13 per 100 persons¹³. In its latest document on arsenic in drinking water, the U.S. National Research Council (NRC) concluded that exposure to 50 µg/1 could easily result in a combined cancer risk¹⁴ of 1 in 100. Comparing to the WHO, EPA, and NRC document with arsenic burden to Kolsur villagers from water and food it appears that Kolsur villagers’ risk of suffering from arsenical skin effect and cancer is there. Compared to worldwide arsenic consumption from food, it appears Kolsur villagers are also consuming high amount of inorganic arsenic from food and vegetable. Kolsur village is an example of many such villages in West Bengal-India and Bangladesh.

Further, products from arsenic irrigated water- soil system rich in arsenic are also coming to common marketplace far away from contaminated areas and even people who are not drinking arsenic contaminated water may get arsenic from food products produced from contaminated fields. In West Bengal-India and Bangladesh rice, vegetable, and other products are coming to cities (including Kolkata in West Bengal-India and Dhaka in Bangladesh) from villages and possibility that city people consuming arsenic contaminated products from contaminated areas cannot be ruled out.

In one of our studies² we mentioned that arsenic in urine (metabolites) to a group of controlled population using water for drinking and cooking having arsenic <3 µg/L, have arsenic in their urine higher than normal level. This explains that a high background level of arsenic is resulted to the surroundings of arsenic contaminated area due to arsenic coming from food chain.

Acknowledgement: The Author wants to dedicate this paper to the memory of Prof. Dipankar Chakarborti who passed away on February 28, 2018. He was the founder and Director of the School of Environmental Studies (SOES), and Professor in the Department of Chemistry at the Jadavpur University, Kolkata, India. Dr. Chakarborti was my Ph.D. supervisor, and this research work was done under his sole supervision with great contributions.

I would like to thank to the Chairman of the Jawaharlal Nehru Memorial Fund, Teen Murti House, New Delhi, India for the financial assistance throughout my research work. I gratefully acknowledge Dr. Masanori Ando, Dr. Hiroshi Tokunaga, and the authorities of the National Institute of Health Sciences, Tokyo, Japan for their laboratory support and cooperation. I express my deep gratitude to Dr. Douglas T. Heitkemper, U.S. Food and Drug Administration, Forensic Chemistry Centre, USA for his help to analyze vegetable and crop samples for arsenic species.

 

Reference

 

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