Research Article Received: 26 January 2014
Revised: 14 May 2014
Accepted article published: 24 July 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6839
Arsenic toxicity in rice with special reference to speciation in Indian grain and its implication on human health Bishwajit Sinhaa* and Kallol Bhattacharyyab Abstract BACKGROUND: Rice is a potentially important route of human exposure to arsenic, especially in populations with rice-based diets. However, arsenic toxicity varies greatly with species. The initial purpose of the present study was to evaluate arsenic speciation in rice. RESULTS: It appeared very clear from the present study that inorganic arsenic shared maximum arsenic load in rice straw while in grains it is considerably low. As species recovered from rice grain and straw are principally As(III) and As(V) with a small amount of dimethylarsenic acid (DMA) and almost non-detectable monomethylarsonic acid (MMA) and arsenobetain (AsB). Discussion of the health risk of As in rice has largely been based on its inorganic arsenic content because these species have generally been considered to be more toxic than MMA and DMA and can be directly compared to As in drinking water, assuming equal bioavailability of inorganic As in the rice matrix and in water. The maximum dietary risk of exposure to inorganic arsenic through transplanted boro paddy in the present experiment was calculated to be almost 1706% of the provisional tolerable weekly intake for an adult of 60 kg body weight. CONCLUSION: As species recovered from boro rice grain and straw are principally As(III) and As(V) with a small amount of DMA and almost non-detectable MMA and AsB. Reductions in total As load through organic amendments in boro rice grain and straw samples were manifested predominately through reduced accumulations of inorganic As species [As(III) and As(V)], between which As(V) accounted for the larger share. © 2014 Society of Chemical Industry Keywords: arsenic; rice; speciation; methylation; risk assessment
INTRODUCTION The countries with the highest daily intake of total arsenic (t-As) are Spain and Japan followed by India and France, and the world’s two most significant cases of As contamination where the population suffers the most are located in Asia, particularly in Bangladesh and West Bengal in India.1,2 The cases in Bangladesh and India are different and more special than those in Spain or Japan, for instance. The speciation of As, which is related to the different sources of As, is responsible for such uniqueness. In general, As from seafood is organic (o-As) while As from drinking water and vegetables is inorganic (i-As). Therefore, dietary As can be categorised by As species (e.g. organic vs. inorganic) and by source (seafood vs. vegetables, mainly rice).3 Arsenic contamination in groundwater in the state of West Bengal has assumed the proportion of 12 endemic districts, 111 endemic blocks and more than 50 million people exposed to threats of arsenic-related health hazards.4 It is only the agricultural sector which enjoys the major share (>90%) of such contaminated groundwater as a source of irrigation and has received attention for quantifying the influence of arsenic in soil–plant systems.5,6 Arsenic contamination of water and soil can also adversely affect food safety. A global normal range of 0.08–0.2 mg As kg−1 has been suggested for rice,7 but values as high as 0.25 mg As kg−1 have been found in rice.8 The average daily intake of As from J Sci Food Agric (2014)
rice for an Indian adult is approximately 100 mg As (400 g dry weight × 0.25 mg As kg−1 ), which is five times higher than 20 mg As intake from consumption of 2 L of water as the WHO limit of 10 μg L−1 .9 Rice (Oryza sativa L.) is the most important crop of India and the second principal food crop of the world. In India, rice is predominantly grown in the Indo-Gangetic plains, over an area of 13.5 million ha which is 85% of the cultivated land area where groundwater is the principal source of irrigation. Most of the shallow aquifers of groundwater in southern Bangladesh and the eastern part of West Bengal, India, are geogenically contaminated with arsenic (As), exposing more than 40 million people at risk of As in drinking water.10 The WHO standard for As in drinking water of 10 μg L−1 has been adopted by many countries. Arsenic in water is generally inorganic and can be a mixture of arsenite [As(III)] and arsenate
∗
Correspondence to: Bishwajit Sinha, College of Agriculture, Orissa University of Agriculture and Technology, Bhawanipatna-766001, Odisha, India. E-mail: [email protected]
a College of Agriculture (OUAT), Bhawanipatna, Odisha, India b Department of Agricultural Chemistry and Soil Science, BCKV, Mohanpur, Nadia, West Bengal
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[As(V)]. The risk assessment for exposure to As-contaminated drinking water, as postulated by the U.S. Environmental Protection Agency, is based on carcinogenicity risk from inorganic As. No intake of inorganic As from food was considered while setting the drinking water standard, while it is now evident that significant amounts can be ingested this way. Arsenic in rice is of special concern because of the much higher levels of As in rice grain compared to other staple cereal crops, coupled with high levels of rice consumption by Asian populations. Moreover, knowledge of the speciation of As in rice is critical to understanding the potential toxicity of rice to humans. Adult daily rice intakes as high as 750 g uncooked weight have been reported in West Bengal, India,11 and between 400 and 650 g uncooked weight in Bangladesh.12 Rice grain As concentrations in Bangladesh vary widely, but 50% (between the 25th and 75th percentiles of the overall distribution from 871 samples) fall within the range of 0.15–0.36 mg kg−1 ,7 and values as high as 1.8 mg kg−1 have been reported.13 Daily consumption of 400 g dry weight of rice containing 0.25 mg As kg−1 would provide 100 μg As or five times the 20 μg As from consumption of 2 L of water at the acceptable WHO limit of 10 μg L−1 . Moreover, knowledge of speciation of As in rice is critical to understanding the potential toxicity of rice to humans as the toxicity of different arsenic containing compounds varies widely. For example, As concentrations in fish and shellfish can reach 10 mg kg−1 (Schoof et al.14 ) and are approximately 10–100 times the levels found in rice. However, most As in seafood is present in organic compounds such as arsenobetaine and arsenocholine, which are considered to be non-toxic to humans (acute toxicity 103 times less than inorganic arsenic). Four species of As are commonly reported in rice: arsenite, arsenate, monomethylarsonic acid (MMA), and dimethylarsenic acid (DMA). The dominant species are usually arsenite and DMA, although the sum of arsenite + arsenate is often reported.15 The inorganic As content in rice can vary from 10% to 90% of total As,16 but the reason for this variability has not been established. Discussion of the health risk of As in rice has largely been based on its inorganic arsenic content because these species have generally been considered to be more toxic than MMA and DMA17 and can be directly compared to As in drinking water, assuming equal bioavailability of inorganic As in the rice matrix and in water. Rice produced in Bangladesh/India contained a mean of 80% inorganic As.15 Risks of dietary exposure to arsenic through contaminated food are alarming in the Indian subcontinent, although arsenic (i-As, precisely) ingestion through processed foods is a global issue. Rice milk samples from national supermarket chains in the city of Aberdeen, UK, displayed a high i-As content, with values being above 60% of the t-As content (exceeding EU and US drinking water standards) and the remainder being dimethylarsinic acid (DMA). The i-As contents were significantly higher in gluten-free rice than in cereals mixtures with gluten, placing infants with coeliac disease at high risk.18 High contents of i-As were observed in Spanish gluten-free products based on rice with mean contents being 69 mg kg−1 . The finding of elevated contents of i-As in infant products and consequently elevated intakes of i-As in infants older than 4 months in China, USA and UK is of concern and deserves further attention.19 The purpose of the present study was to assess As speciation in rice from West Bengal, India, in order to improve understanding of the health risk posed by As in Indian rice. An effort has been made, through the present study, to take an account of species level accumulation of arsenic in rice in the arsenic affected villages
of Chakdaha block, Nadia district, West Bengal, India, having a total arsenic concentration of irrigation water, drifted by shallow tube wells, of 0.32 mg L−1 .
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MATERIALS AND METHODS Site description The experiment was conducted at a farmer’s field (2009–2010) in the village of Ghentugachhi, geographical location: 23∘ 2′ 7.1′′ N, 88∘ 35′ 4.8′′ E, district of Nadia, West Bengal, India. Details of crop management The crop summer rice, variety IET 4786, which is widely grown in the arsenic affected area of West Bengal, was selected for the study. The crop was sown during the first week of February 2010. The seed rate was 100 kg ha−1 and the spacing was maintained at 30 × 10 cm. Weeding was done twice [at 20 and 40 days after transplanting (DAT)]. Rice fields were irrigated both from shallow tube well water (As concentration, ∼0.32 mg L−1 ) and pond water (As concentration, ∼0.03 mg L−1 ). Experimentation The experiment was laid out in a two-factor randomised block design with three replications. Factorial experimental treatments were two levels of irrigation (irrigation through shallow tube well water and irrigation through surface water) and four levels of organic manures, namely farmyard manure at 10 t ha−1 , vermicompost at 3 t ha−1 , municipal sludge at 10 t ha−1 and mustard cake at 1.0 t ha−1 . The soils were amended with well-decomposed farmyard manure, vermicompost, municipal sludge and mustard cake in respective treated plots followed by two ploughing operations 25 days before sowing. The recommended doses of N, P, K fertilisers (N:P2 O5 :K2 O, 100:50:50) kg ha−1 were applied to the soils irrespective of treatments. The entire P and K fertilisers were applied basally while N fertiliser was applied in three splits (50% as basal and the remainder 50% top dressed at 30 DAT and 45 DAT). Weeding was done twice (at 20 and 40 DAT). Arsenic speciation in plant samples Sample digestion: total As, HNO3 digest About 0.2 g of rice grain or straw sample were weighed into a microwave Teflon vessel and 7 mL of concentrated nitric acid was added to it and left to stand overnight at room temperature. Samples were then digested in a microwave vessel maintained at 200 ∘ C for 20 min. Samples were then cooled and transferred to a 50 mL volumetric flask for total arsenic analysis by using a Perkin Elmer ELAN DRCe 6000 ICP-MS (Perkin Elmer, California, USA). Details of the operating parameters of the ICP-MS are shown in Table 1. Sample extraction for arsenic species For speciation analysis about 0.2 g of rice grain or straw sample were weighed into a microwave Teflon vessel and 2 mL of 2.0 mol L−1 trifluoroacetic acid (TFA) was added to it. Samples were then digested in a microwave maintained at 90 ∘ C for 20 min. Samples were then cooled and transferred to a 50 mL volumetric flask for analysis of different forms of arsenic in a HPLC-ICP-MS.5 Details of the operating parameters of HPLC are listed in Table 2. Total As recoveries from rice were validated by using the Perkin Elmer ELAN DRCe 6000 ICP-MS and compared with the NIST standard SRM 1568a (rice flour); the certified value was 290 ± 30 μg kg−1 and the observed value was 283 ± 8 μg kg−1 .
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Arsenic toxicity in rice
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Table 1. ICP-MS operating conditions and parameters
Table 2. HPLC isocratic method and operating parameters
Parameter
Operating condition
Parameter
Nebuliser
Meinhard Type A quartz , Part No.: WE02-4371 ELAN DRC II: Quartz Cyclonic, Part No.: WE02-5221 1500 W 15 L min−1 0.82 L min−1 1.2 L min−1 ELAN DRC II: 2.0 mm i.d. Quartz, Part No.: WE02-3915 m/z 91 (75 As, 16 O) for DRC 30 s 15 s 30 s Dual Scanning Nickel 50 ms 600 s straw > grain (Table 3). A similar trend was also reported by Sanyal.21 Such findings of relative proportions of arsenic accumulations can be substantiated by findings of Rahman et al.,22 who reported 28- to 75-fold higher As accumulation in roots than shoot and raw rice grain. The findings of Liu et al.23 also corroborate such findings where proportional accumulations remained at 1.17 to 4.15 mg kg−1 (straw), 0.56–1.35 mg kg−1 (husk) and 0.25 to 0.73 mg kg−1 (grain) of rice. The relative accumulation of arsenic in the underground and aboveground portions of plant systems has been observed to vary substantially across growth stages. Figure 1 shows that root arsenic accumulation gradually increased up to 60 days after transplanting (DAT) after which it underwent a steep increase. The shoot arsenic J Sci Food Agric (2014)
Mobile phase for soil sample Mobile phase for plant sample
Flow rate Run time Column Column temperature Auto-sampler flush solvent Sample injection volume Sample preparation Detection
30 mmol L−1 ammonium phosphate, pH 5.6 6.66 mmol L−1 ammonium nitrate and 6.66 mmol L−1 ammonium hydrogen phosphate, pH 6.2 1.0 mL min−1 9 min Anion exchange, Hamilton PRP-X100 26 ∘ C 5% Methanol/95% deionised water 100 μL 1:1 with mobile phase Perkin-Elmer Elan DRCe ICP_MS
280
t-As accumulation (mgkg–1)
Spray chamber
Setting
240 200
ROOT
SHOOT
160 120 80 40 0 –40
30 DAT
60 DAT
90 DAT
Days after transplanting Figure 1. Progressive changes in t-As accumulation in different plant parts of boro rice (cv. IET-4786) with advancement of growth.
content has been observed to decrease in the first phase (up to 60 DAT) and increased thereafter. The relative affinity of the plant to accumulate arsenic is closely related to phosphate. The P/As molar ratio (MR) (P molar concentration in plant) can be a good index for the relative abundance and roles in plant. Another important index for the relative preference of plant systems towards As/P accumulation is the bioaccumulation preference (BP): BP =
Asplant ∕Assoil Pplant ∕Psoil
where Asplant is the As concentration in plant tissue, Assoil is the concentration of As in soil, Pplant is the P concentration in plant tissue, and Psoil is the concentration of P in soil. The observed values of MRs and BP in different plant parts of boro rice are listed in Table 4 and Table 5. The MR values (P/As) in root, straw and grain of boro rice varied from 3.29 to 10.14, 43.95 to 107.49, and 1118.87 to 2349.45, respectively. Such higher MR values have previously been reported in corn seedlings (102–4400), tomato (500–4200), cord grass (800–8000).24 Table 4 shows that the MR values (P/As) in rice decreased significantly when irrigated through underground shallow tube well
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Table 3. Effect of selected organic interventions and elevated phosphate administration on arsenic accumulation (mg kg−1 ) in soil–plant system under boro rice (cv. IET-4786) subjected to varying irrigation sources in farmers’ fields Arsenic accumulation (mg kg−1 ) 30 DAT Treatment
Root
I1 I2 CD𝛼=0.05 P1 P2 CD𝛼=0.05 O1 O2 O3 O4 O5 CD𝛼=0.05 I1 P1 I1 P2 I2 P1 I2 P2 CD𝛼=0.05 I1 O1 I1 O2 I1 O3 I1 O4 I1 O5 I2 O1 I2 O2 I2 O3 I2 O4 I2 O5 CD𝛼=0.05 P1 O1 P1 O2 P1 O3 P1 O4 P1 O5 P2 O1 P2 O2 P2 O3 P2 O4 P2 O5 CD𝛼=0.05 I1 P1 O1 I1 P1 O2 I1 P1 O3 I1 P1 O4 I1 P1 O5 I1 P2 O1 I1 P2 O2 I1 P2 O3 I1 P2 O4 I1 P2 O5 I2 P1 O1 I2 P1 O2 I2 P1 O3 I2 P1 O4
74.08 66.16 0.043 69.31 70.92 0.043 75.99 69.25 72.84 66.53 65.98 0.069 73.65 74.51 64.98 67.34 0.060 79.33 73.87 76.54 68.59 72.05 72.65 64.63 69.15 64.46 59.91 0.097 75.63 67.12 73.29 66.11 64.41 76.35 71.38 72.40 66.94 67.54 0.097 79.76 74.13 77.18 67.49 69.67 78.91 73.60 75.89 69.69 74.43 71.50 60.10 69.40 64.73
Shoot + leaf 11.53 9.02 0.034 10.57 9.98 0.034 14.64 8.36 9.90 9.12 9.34 0.057 12.25 10.81 8.88 9.15 0.052 16.80 8.86 11.06 11.31 9.61 12.48 7.87 8.74 6.93 9.06 0.080 14.74 9.17 9.98 10.30 8.65 14.55 7.56 9.82 7.94 10.03 0.080 17.28 9.78 11.18 13.92 9.09 16.33 7.93 10.93 8.71 10.13 12.19 8.56 8.78 6.68
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60 DAT Root 87.46 76.44 0.077 78.59 85.31 0.077 87.04 79.20 76.45 87.98 79.08 0.120 82.35 92.56 74.83 78.05 0.109 89.76 83.67 78.79 103.33 81.74 84.33 74.73 74.11 72.63 76.42 0.172 87.44 79.65 75.42 71.15 79.29 86.65 78.75 77.48 104.80 78.86 0.172 88.05 84.74 79.16 74.77 85.04 91.47 82.60 78.42 131.89 78.43 86.83 74.56 71.67 67.53
Shoot 4.95 3.62 0.149 4.64 3.93 0.149 5.33 4.33 3.17 4.07 4.52 0.235 5.57 4.33 3.71 3.53 0.212 5.94 5.04 3.53 4.87 5.37 4.72 3.62 2.81 3.27 3.67 0.332 5.57 4.72 3.77 4.25 4.90 5.10 3.94 2.58 3.89 4.13 0.332 6.56 5.90 4.59 5.03 5.79 5.33 4.18 2.47 4.71 4.95 4.58 3.54 2.94 3.47
90 DAT Leaf 15.08 11.95 0.072 13.37 13.66 0.072 20.01 14.07 10.00 11.87 11.63 0.115 15.10 15.06 11.64 12.26 0.103 23.77 18.71 10.03 11.43 11.46 16.26 9.42 9.97 12.31 11.79 0.163 19.61 14.20 12.95 10.14 9.96 20.41 13.93 7.06 13.60 13.29 0.163 24.54 19.91 13.33 11.10 6.63 22.99 17.51 6.74 11.76 16.30 14.67 8.48 12.56 9.19
Root 236.10 214.20 1.363 221.30 229.00 1.363 245.92 218.83 208.08 212.25 240.67 2.156 231.00 241.20 211.60 216.80 1.930 250.33 229.67 232.50 219.83 248.17 241.50 208.00 183.67 204.67 233.17 3.049 229.83 215.17 197.67 224.83 239.00 262.00 222.50 218.50 199.67 242.33 3.049 240.00 224.33 220.67 233.67 236.33 260.67 235.00 244.33 206.00 260.00 219.67 206.00 174.67 216.00
Grain 1.41 1.03 0.010 1.30 1.15 0.010 1.37 1.13 1.17 1.21 1.24 0.029 1.54 1.28 1.06 1.01 0.029 1.53 1.25 1.41 1.39 1.47 1.20 1.00 0.94 1.03 1.00 0.029 1.48 1.09 1.26 1.36 1.32 1.26 1.16 1.09 1.07 1.16 0.029 1.69 1.28 1.56 1.60 1.59 1.37 1.23 1.27 1.19 1.36 1.27 0.90 0.96 1.11
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Post-harvest soil Straw 13.06 11.25 0.040 11.46 12.85 0.040 15.09 11.71 11.86 11.51 10.60 0.063 12.50 13.62 10.43 12.07 0.057 16.66 12.46 12.78 13.00 10.41 13.51 10.97 10.95 10.01 10.79 0.089 14.45 10.57 12.12 11.21 8.96 15.72 12.86 11.61 11.80 12.24 0.089 17.18 11.29 13.43 12.90 7.68 16.14 13.63 12.13 13.09 13.13 11.72 9.85 10.81 9.51
Av. As (mg kg−1 ) 2.38 3.08 0.06 2.52 2.95 0.06 3.06 2.69 2.70 2.60 2.60 0.09 2.18 2.58 2.85 3.31 0.08 2.72 2.38 2.25 2.32 2.23 3.40 3.01 3.15 2.88 2.97 0.13 2.91 2.49 2.34 2.39 2.45 3.22 2.89 3.07 2.81 2.75 0.13 2.56 2.11 1.77 2.22 2.24 2.88 2.65 2.73 2.43 2.23 3.25 2.88 2.90 2.57
Av. P2 O5 (mg kg−1 ) 28.73 34.05 0.28 29.32 33.47 0.28 31.96 32.47 30.52 31.55 30.48 0.44 26.17 31.31 32.47 35.64 0.39 29.10 29.45 28.04 29.05 28.06 34.82 35.49 33.01 34.06 32.91 0.63 29.99 30.54 28.19 29.12 28.76 33.92 34.4 32.85 33.99 32.21 0.63 26.33 27.15 25.23 26.28 25.83 31.86 31.75 30.84 31.82 30.28 33.65 33.93 31.15 31.95
Yield (t ha−1 ) 8.32 9.17 0.023 9.01 8.48 0.023 7.65 8.96 9.34 9.01 8.76 0.037 8.00 8.64 10.02 8.31 0.031 7.27 8.40 8.28 9.43 8.22 8.02 9.53 10.41 8.58 9.29 0.052 8.07 8.99 9.80 9.80 8.40 7.22 8.93 8.89 8.22 9.11 0.052 7.38 8.13 8.40 9.24 6.86 7.17 8.66 8.17 9.61 9.59 8.76 9.86 11.20 10.35
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Arsenic toxicity in rice
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Table 3. Continued Arsenic accumulation (mg kg−1 ) 30 DAT
Treatment
Root
I2 P1 O5 I2 P2 O1 I2 P2 O2 I2 P2 O3 I2 P2 O4 I2 P2 O5 CD𝛼=0.05
59.15 73.79 69.16 68.90 64.18 60.66 0.135
Shoot + leaf 8.20 12.77 7.18 8.70 7.17 9.93 0.112
Output of Tukey Multiple range test∗ O1 75.99E 14.64E C O2 69.25 8.36A D O3 72.84 9.90D B O4 66.53 9.12B A O5 65.98 9.34C
60 DAT
90 DAT
Root
Shoot
Leaf
Root
Grain
73.54 81.82 74.89 76.54 77.72 79.29 0.243
4.02 4.87 3.70 2.68 3.07 3.31 0.472
13.30 17.84 10.36 7.38 15.44 10.28 0.229
241.67 263.33 210.00 192.67 193.33 224.67 4.314
1.04 1.14 1.10 0.91 0.95 0.96 0.029
87.04C 79.20B 76.45A 87.98D 79.08B
5.33D 4.33BC 3.17A 4.07B 4.52C
20.01E 14.07D 10.00A 11.87C 11.63B
245.92E 218.83C 208.08A 212.25B 240.67D
1.37D 1.13A 1.17B 1.21C 1.24C
∗ Output of Tukey Multiple range test for means of organic amendments. A – E Results with different superscript letters are significantly different at 5% level. I1 , irrigation from shallow tube well; I2 , irrigation from pond; P1 , 50 kg P2 O5 ha−1 ; P2 , 75 kg P2 O5 O3 , vermicompost at 3 t ha−1 ; O4 , municipal sludge at 10 t ha−1 ; O5 , mustard cake at 1 t ha−1 .
water (STW) as compared to surface pond water (PW) irrigation which can be attributed to higher As concentration of STW water. Elevated levels of P administration significantly increased the root and grain MR values in rice while the straw MR was observed to reduce under higher P situation. Similar observations were obtained by Tu and Ma 25 who reported that at high arsenic levels the P/As molar ratio of the aboveground portion of a plant is increased by applying more phosphate. Organic amendments increased the MR values in rice root, grain and straw significantly over the non-organic control, although different organic compounds behaved differentially in this regard. The highest root MR remained associated with the application of vermicompost, while the highest grain and straw MR were obtained from farmyard manure and mustard cake treated situations. The Tukey multiple range test showed that effects of different organic amendments are different on root MR values while in grain and straw MR effects of vermicompost, mustard cake and sludge amendments were similar (Table 4). The bioaccumulation preference for arsenic in root, grain and straw of boro rice have been found to be suppressed with elevated application of phosphorus. The BP values were greater than 1 in roots but less than 0.5 in grain and straw. Similar observations were recorded by Tu and Ma25 in the arsenic hyper-accumulator Chinese brake fern (Pteris vitata). These authors concluded that phosphate was preferentially accumulated over arsenic in the aboveground portion which was further encouraged through application of additional phosphate. Organic manure, phosphate intervention and arsenic in the soil–plant system Results of Table 3 showed that administration of elevated levels of phosphate fertilisers increased As accumulations in rice root in all the growth stages. Grain As, on the other hand, has been J Sci Food Agric (2014)
Post-harvest soil Av. As (mg kg−1 )
Av. P2 O5 (mg kg−1 )
Yield (t ha−1 )
10.23 15.31 12.08 11.08 10.52 11.34 0.126
2.67 3.56 3.14 3.41 3.19 3.27 0.18
31.69 35.99 37.04 34.86 36.16 34.14 0.89
9.94 7.28 9.20 9.61 6.82 8.64 0.072
15.09E 11.71C 11.86D 11.51B 10.60A
3.46D 2.72B 2.56A 2.81B 2.96C
31.96BC 32.47C 30.52A 31.55B 30.48A
7.65A 8.96C 9.34D 9.01C 8.76B
Straw
ha−1 ; O1 , control; O2 , farmyard manure at 10 t ha−1 ;
found to decrease at higher P administration. An increase in available As in soil and subsequent greater P availability may possibly be responsible for greater As accumulation in rice root. The grain As accumulation, on the other hand, was found to be suppressed, probably due to reduced translocation of As to grain under a P-rich enzymatic pathway. Arsenic and phosphorus exist in the same periodic group and have similar chemical and physical properties as well as similar electron configurations.25 Among the two important oxyanions, arsenite [As(III)] and arsenate [As(V)], the latter is an analogue of phosphate (PO4 3− ), making it an important factor in the behaviour of arsenic in well-aerated soils.26 Arsenite [As(III)] is not an analogue of PO4 3− , and is less relevant to arsenic behaviour under flooded soil conditions.27 However, Heikens28 reported that PO4 3− can play an important role in the rhizosphere, where an oxygenated micro-environment prevails under flooded rice conditions. Clement and Faust29 showed that during water–sediment interactions, phosphate concentration in the system plays an important role in the release of arsenite from the sediments. Inorganic arsenic species are generally highly toxic to plants. Arsenate acts as a phosphate analogue and is transported across the plasma membrane via phosphate co-transport systems.30 Arsenate sensitivity is intimately linked to phosphate nutrition, with increased phosphate status leading to reduced arsenate uptake, through suppression of the high-affinity phosphate/arsenate uptake system.31 Observations recorded in Table 3 showed that soil amendment through selected organic manures significantly reduced the arsenic accumulation in different parts of rice across growth stages which has been most efficiently manifested through vermicompost application regardless of the growth stage or the plant part(s) under consideration. The addition of organic amendments to soils reduce the bioavailability of heavy metals by changing them from bioavailable forms to fractions associated with organic matter or metal oxides or carbonates.32 Cao et al.33 reported that when
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Table 4. Molar ratios (MRs) of phosphorus to arsenic in different plant parts of boro rice
B Sinha and K Bhattacharyya
Table 4. Continued Treatment
Treatment I1 I2 CD (P = 0.05) P1 P2 CD (P = 0.05) O1 O2 O3 O4 O5 CD (P = 0.05) I1 P1 I1 P2 I2 P1 I2 P2 CD (P = 0.05) I1 O1 I1 O2 I1 O3 I1 O4 I1 O5 I2 O1 I2 O2 I2 O3 I2 O4 I2 O5 CD (P = 0.05) P1 O1 P1 O2 P1 O3 P1 O4 P1 O5 P2 O1 P2 O2 P2 O3 P2 O4 P2 O5 CD (P = 0.05) I1 P1 O1 I1 P1 O2 I1 P1 O3 I1 P1 O4 I1 P1 O5 I1 P2 O1 I1 P2 O2 I1 P2 O3 I1 P2 O4 I1 P2 O5 I2 P1 O1 I2 P1 O2 I2 P1 O3 I2 P1 O4 I2 P1 O5 I2 P2 O1 I2 P2 O2 I2 P2 O3 I2 P2 O4
Root MR 4.52 7.42 1.21 5.16 6.78 0.37 4.64 6.62 7.04 5.87 5.69 0.25 4.04 5.00 6.29 8.56 0.12 3.80 5.01 4.94 4.33 4.53 5.48 8.24 9.13 7.41 6.86 0.13 4.37 5.49 5.91 4.81 5.24 4.91 7.76 8.16 6.93 6.15 0.98 3.69 4.54 4.40 3.45 4.11 3.90 5.47 5.48 5.21 4.96 5.06 6.43 7.43 6.18 6.36 5.91 10.05 10.84 8.65
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Grain MR 1318.99 2058.43 78.27 1587.21 1790.20 58.98 1378.69 1848.39 1717.29 1728.67 1770.50 93.55 1161.71 1476.26 2012.72 2104.14 35.63 1127.38 1432.91 1325.67 1380.58 1328.40 1630.00 2263.87 2108.91 2076.76 2212.60 31.92 1258.00 1877.67 1608.51 1547.90 1643.99 1499.38 1819.10 1826.07 1909.44 1897.01 34.98 959.92 1335.34 1130.93 1169.47 1212.91 1294.83 1530.47 1520.41 1591.69 1443.90 1556.07 2420.01 2086.09 1926.33 2075.08 1703.93 2107.73 2131.72 2227.19
Root MR
Grain MR
Straw MR
Straw MR 62.38 72.07 1.34 68.86 65.59 1.53 48.13 66.99 68.36 75.98 76.66 3.80 64.56 60.20 73.16 70.98 3.39 43.03 63.49 69.90 57.60 77.87 53.24 70.49 66.81 94.36 75.45 3.76 47.56 68.27 65.53 62.99 99.93 48.71 65.71 71.19 88.96 53.39 10.11 35.70 60.87 65.92 47.74 112.55 50.37 66.12 73.89 67.45 43.19 59.42 75.66 65.14 78.25 87.32 47.06 65.31 68.48 110.46
I2 P2 O5 CD (P = 0.05)
7.35 0.92
Output of Tukey multiple range test∗ O1 4.64A O2 6.62D O3 7.04E O4 5.87C O5 5.69B
959.92 31.93
63.59 5.74
1378.70A 1848.40D 1717.30B 1728.70B 1770.50C
48.14A 66.99B 68.36C 75.98D 76.66D
∗ Output of Tukey Multiple range test for means of organic amendments. A – E Results with different superscript letters are significantly different at 5% level. I1 , irrigation from shallow tube well; I2 , irrigation from pond; P1 , 50 kg P2 O5 ha−1 ; P2 , 75 kg P2 O5 ha−1 ; O1 , control; O2 , farmyard manure at 10 t ha−1 ; O3 , vermicompost at 3 t ha−1 ; O4 , municipal sludge at 10 t ha−1 ; O5 , mustard cake at 1 t ha−1 .
Table 5. Bioaccumulation preference for arsenic observed in different plant parts of boro rice Phosphorus
Root
Grain
Straw
Standard P (at 50 kg ha−1 ) Elevated P (at 75 kg ha−1 )
2.2851 1.7193
0.0078 0.0065
0.1789 0.1772
bio-solid was added to either acidic or neutral soil the adsorption of arsenic was increased and water soluble arsenic was reduced. Shiralipour et al.34 reported that the application of organic matter to soil would increase soil cation and anion exchange capacity, which may increase arsenic adsorption by increasing the amount of positive charge on the oxide surface and/or forming a positively charged surface35 and enhanced sorption capacity of the soil matrix. Enriched soil humic fractions in manure amended soils may be active in retaining As(III) and As(V) through adsorption. So organo-arsenic complexations with humic/fluvic colloids of the native soil and the incorporated organic manures moderate the hazards of arsenic toxicity.36 In a very recent instance a similar observation has been cited by Rahaman et al.,37 who showed that combined applications of lathyrus + vermicompost + poultry manure reduced arsenic transport in plant parts (root, straw, husk, whole grains and milled grain). Such findings and the relative efficiencies of selected organic manures in off-loading arsenic from rice may be well corroborated with our findings38 relating to characterisation of humic/fulvic components extracted from the selected organic matters and their complexation with arsenic. Bioavailability of arsenic in boro rice irrigated through underground water and surface water (pond water) The inorganic [i.e. As(III) and As(V)] and organic (i.e. DMA and MMA) arsenic accumulation in rice grain and straw were determined from the TFA (at pH 6.2) extract by using HPLC-ICP-MS (Perkin Elmer ElanDRCe 6000) and the results are given in Table 6. Selected samples of the present experiment showing good response of off-loading As accumulation in rice grain and straw
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Table 6. Recoveries of arsenic species from boro rice under varying irrigation regimes and selected organic amendments Recovery of arsenic species in boro rice (μg kg−1 )
Source of irrigation
Selected interventions As B
Grain Pond (PW)
Shallow tube well (STW)
Straw Pond (PW)
Shallow tube well (STW)
Characterisation∗ STW PW SOIL
As(III)
DMA
MMA
As(V)
Sum of species
Total As (μg.kg−1 ) (HNO3 digest)
Species recovery (%)
1 461.60 ± 44.10 864.00 ± 25.04 918.00 ± 48.99 1 542.00 ± 35.86
93.97 ± 1.20 90.21 ± 2.44 86.14 ± 2.82 93.26 ± 1.90
C VC FYM C
ND 750.70 ± 6.76 140.50 ± 4.74 ND 516.00 ± 7.45 45.60 ± 6.50 ND 489.00 ± 9.06 34.80 ± 2.80 ND 1 223.50 ± 8.02 41.50 ± 2.27
ND ND ND ND
482.20 ± 7.13 217.80 ± 8.18 267.00 ± 4.60 173.00 ± 8.34
1 373.40 ± 11.42 779.40 ± 12.49 790.80 ± 12.50 1 438.00 ± 4.56
VC FYM
ND 589.50 ± 5.87 ND 1 040.90 ± 7.88
53.00 ± 3.65 57.20 ± 5.31
ND ND
136.90 ± 6.66 217.60 ± 3.89
779.40 ± 8.83 1 082.30 ± 54.43 72.01 ± 2.50 1 315.70 ± 10.67 1 180.00 ± 32.55 111.50 ± 2.48
C VC FYM C
ND 865.80 ± 12.4 ND 1 693.00 ± 7.8 ND 586.80 ± 4.9 ND 969.00 ± 7.2
443.00 ± 14.7 ND 7 600.30 ± 43.1 8 909.10 ± 54.6 367.30 ± 12.9 ND 2763.30 ± 23.3 4 823.60 ± 29.1 292.80 ± 4.8 ND 5 073.00 ± 31.5 5 952.60 ± 34.44 792.60 ± 14.5 194.0 8 146.80 ± 45.8 10 102.80 ± 39.6
VC FYM
ND ND
577.00 ± 12.9 294.70 ± 14.5 126.00 ± 3.9 133.20 ± 8.5
ND ND
– – –
ND ND ND
278.78 ± 6.4 ND 900.15 ± 9.4
ND 73.10 ± 3.5 351.88 ± 9.4 ND 23.65 ± 2.1 23.65 ± 2.1 ND 13 300.70 ± 125.8 14 200.85 ± 134
ND ND ND
5 080.30 ± 44.4 6 756.60 ± 23.7
5 952.00 ± 29.7 7 015.80 ± 18.9
8 823.00 ± 128.6 100.98 ± 2.3 4739.60 ± 145.2 101.77 ± 5.1 6 048.00 ± 157.9 98.42 ± 2.5 9 684.00 ± 120.0 104.32 ± 3.2 6 308.00 ± 138.8 7 326.00 ± 126.5
94.36 ± 2.2 95.77 ± 2.6
320.00 ± 4.7 109.96 ± 5.2 31.50 ± 2.4 75.08 ± 3.1 19 400.00 ± 168.9 73.20 ± 4.3
∗ Characterisation of arsenic sources (STW/PW) and sink (soil) of the experimental site. ND, not detectable.
through interventions of organic amendments (manures) have been extracted through TFA (at pH 6.2). A microwave digestion system (Multiwave 3000; Anton Par, Ashland, USA) with a rotor of 48 Teflon digestion vessels was used for sample digestion and extraction. Arsenic species were determined by using HPLC-ICP-MS (Perkin Elmer ElanDRCe 6000). All chemicals used were reagent grade. All the solutions were prepared with Milli-Q water (Millipore, Bedford, MA, USA). For the speciation studies, standard solutions (100 mg L−1 ) of As compounds were prepared from: (1) arsenite (NaAsO2 ; Perkin Elmer), (2) arsenate (Na2 HAsO4 · 7H2 O; Perkin Elmer), (3) monomethyl arsonate (CH3 AsNa2 O3 ; Sigma–Aldrich), (4) dimethyl arsenate [(CH3 )2 AsO(OH); Sigma] and arsenobetaine (AsB; Sigma). TFA was purchased from Aldrich (St. Louis, MO, USA) Results of Table 6 show that As compared to the species recoveries from TFA extract of rice grain ranged from 72.01 ± 2.50 to 111.50 ± 2.48 as a % of the total As recoveries from HNO3 extract, which are quite appreciable for a particular extractant in the back-drop of such recoveries reported by Abedin et al.5 They observed that the use of TFA resulted in >80% extraction efficiency. In TFA extracts, the proportions of arsenate, arsenite and DMA were 72–84%, 15–26%, and 1–4%, respectively. It appears very clear from Table 6 and Fig. 2 that i-As species recovered from rice grain and straw are principally As(III) and As(V) with a small share of DMA and almost non-detectable MMA and AsB. Rice grain As has been found to be principally As(III) while in straw As(V) predominated over As(III). Sanz et al.39 observed that for rice and paddy samples, inorganic arsenic accounted for up to 70–98% of the total arsenic content, with As (III) being the major species. The levels of arsenic obtained J Sci Food Agric (2014)
from straw and soil samples are significantly higher than the background levels, with As(V) being the major species, thus increasing human exposure to arsenic via the soil–plant–animal–human pathway. Table 7 shows significant lower recoveries of i-As, o-As and t-As in rice grain and straw when irrigated with pond water as compared to STW (underground) water irrigation. Interventions through organic manures significantly reduced t-As (HNO3 extract), i-As and o-As in rice grain and straw both under STW and pond water irrigated situations (Table 7), brought through application of farmyard manure and vermicompost at 39.59% and 28.04% compared to the control counterparts. Such reductions in t-As load in rice grain and straw samples were manifested predominately through reduced accumulations of i-As. The intervention through farmyard manure was observed to be more capable of reducing the As(III) and DMA loads in rice grain and straw samples, while vermicompost was more successful in reducing As(V) and total As (HNO3 digest) in rice samples (Table 7), although such changes brought through differential organic amendment could not be explained with the present set of data, and neither can they be substantiated through any citation in this regard. Arsenic methylation It is interesting to note that characterisation of arsenic source (STW/PW) and sink (soil) did not show any recoveries of organic As, although boro grain and straw accumulates organic As species (Table 6). The recoveries of such organic species in rice grain and straw may be due to transformation of inorganic As to organic forms in the plant body. The % recoveries of organic arsenic (out of
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Table 7. Recoveries of i-As and organic As in boro rice under varying irrigation regimes and selected organic amendments
Treatment Grain Main effect means PW STW CD (P = 0.05) C VC FYM CD (P = 0.05) Interactions PW × C PW × VC PW × FYM STW × C STW × VC STW × FYM CD (P = 0.05)
Figure 2. The % share of As species in boro rice (cv. IET-4786).
total arsenic from the HNO3 digest) ranged from as low as 1.75 to 15.53 depending upon sources of irrigation, organic interventions and their interactions (Table 7). It is further noteworthy that rice, irrigated through more arsenic-rich underground water (STW), accumulated more t-As and i-As but less o-As. On the other hand, rice irrigated through low-arsenic surface water (PW), accumulated less t-As and i-As but significantly higher o-As than STW-irrigated rice grains and straw (Table 7). The % recoveries of organic arsenic were observed to be higher in rice straw as compared to rice grain. Interventions of organic manures significantly reduced such recoveries as compared to the control which may be attributed to low total arsenic recoveries. Such reductions in t-As, i-As and o-As in rice grain and straw were a maximum through vermicompost amendment. The observations obtained from the present study are not sufficient to amply describe whether conversion of inorganic arsenic fractions into organic moieties were manifested through organic interventions employed (Table 7). Arsenic is metabolised from inorganic to organic forms by a wide range of organisms with limited evidence that this occurs in plants.40 Factors which can influence the As species present in a plant are arsenic species present in soil; the ability of the compound to enter the plant, actively or passively; the ability of plant to synthesise As species; and the presence of As species absorbed to the outside surface of plant roots.41 It should be noted here that no organic As species was recovered from the soil matrix in the present investigation. There is significant evidence that exposure to i-As results in the generation of reactive oxygen species, probably through the conversion of As(V) to As(III), a process which readily occurs in plants42 as has also been shown
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Straw Main effect means PW STW CD (P = 0.05) C VC FYM CD (P = 0.05) Interactions PW × C PW × VC PW × FYM STW × C STW × VC STW × FYM CD (P = 0.05) Output of Tukey test∗ Grain C VC FYM Straw C VC FYM
i-As
Organic As
Total As (μg kg−1 ) (HNO3 digest)
925.03 1135.71 14.24 1321.15 736.12 1033.85 17.44
114.43 56.98 5.67 97.05 52.85 107.22 6.94
1097.44 1267.67 44.34 1501.17 973.17 1073.33 54.31
1232.97 738.90 803.23 1409.33 733.33 1264.47 24.67
140.50 52.70 150.10 53.60 53.00 64.33 9.81
1461.67 864.00 966.67 1540.67 1082.33 1180.00 NS
5951.83 7211.18 39.63 8815.68 4689.17 6239.67 48.53
368.36 359.90 NS 616.03 329.00 147.35 19.96
6623.56 7853.44 258.22 9246.17 5499.67 6969.67 316.26
– – – – – – –
8466.17 3732.00 5657.33 9165.20 5646.33 6822.00 68.63
443.03 367.33 294.70 789.03 290.67 133.20 26.82
8823.00 4739.67 6308.00 9669.33 6259.67 7631.33 NS
5.02 7.76 4.68 8.16 4.65 1.75 –
1321.15C 736.12A 1033.85B
97.05B 52.85A 107.22C
1501.17C 973.17A 1073.33B
– – –
8815.68C 4689.17A 6239.67B
616.03C 329.00B 147.35A
9246.17C 5499.67A 6969.67B
– – –
Organic As (%)
– – – – – – – 9.61 6.09 15.53 3.48 4.91 5.47 –
∗ Output of Tukey multiple range test for means of organic amendments. PW, pond water; STW, shallow tube well water; C, no organic amendment; VC, vermicompost at 3 t ha−1 ; FYM, farmyard manure at 10 t ha−1 soil.
from the higher ratio of As(III) and As(V) in plant systems compared with As(III)/As(V) recovered from the sink (soil). Following the reduction of arsenate to arsenite in plants, arsenic may potentially be further metabolised to methylated species leading to further oxidative stress.43 In terrestrial plants it is not clear how organic As species appear in plant systems. Reduction of arsenate to arsenite has been definitely demonstrated in plants but methylation has not.44 Organic As species
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Arsenic toxicity in rice
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Table 8. Assessment of risk for dietary exposure to As-contaminated boro rice
i-As conc. (mg kg−1 ) Control 1.40 (max.) Vermicompost 0.73 (min.)
Daily rice consumption (g) 1564.80∗ 1564.80∗
Weekly i-As ingestion (mg) 15.35 8.01
% PTWI†
CONCLUSION
1706.07 890.16
The present study has made a modest effort to understand the arsenic toxicity profile in rice, grown in contaminated area of rural Bengal (India) and the possible risk of its dietary exposure. The unique character of the anaerobic rice ecosystem results in a significant build-up of i-As in soil and underground water used for irrigation and its concomitant accumulation in rice. The recoveries of i-As is dominated by As(III) in rice grain and As(V) in rice straw, leading to a toxicity profile that presents more risk as there are higher levels of the more toxic As(III) in the edible portion. Recoveries of organic As species in rice grain and straw without having any organic As in the source and sink in detectable range, emerged with possibilities of methylation of i-As in the plant system. The risk of dietary exposure to i-As through rice, the staple food in the experimental area, poses an almost equal threat to human health as that posed by contaminated drinking water. Organic amendments and augmented P fertilisation showed considerable promise in reducing t-As and i-As accumulation in rice and the dietary risk.
∗ National Nutrition Monitoring Bureau (NNMB) Diet Survey and Nutritional Status of rural populations, 2006 (www.nnmbindia.org). † % provisional tolerable weekly intake (PTWI) for a 60 kg adult.
present in field samples of plants may have been taken up from soil solution in that particular form as they can be present in soil through microbial activity;45 however, it is also possible that plants themselves could potentially transform arsenic species. In a study conducted by Koch et al.,46 no evidence of methylation was found in the surrounding soil and water, and yet a number of plant species contained MMA, DMA, tetramethyl arsonium ion and trimethylarsenium oxide (tetra). Dietary risk assessment The paradigm of the assessment of risk associated with human exposure to arsenic contamination through diets starts with weighing the arsenic load ingested through diet. Since rice is the main staple food in West Bengal, India, the possibility of arsenic ingestion through consumption of rice by people in the contaminated region cannot be ignored. The estimates of As exposure via dietary and drinking water routes show that even when people are drinking As-safe water (
Revised: 14 May 2014
Accepted article published: 24 July 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6839
Arsenic toxicity in rice with special reference to speciation in Indian grain and its implication on human health Bishwajit Sinhaa* and Kallol Bhattacharyyab Abstract BACKGROUND: Rice is a potentially important route of human exposure to arsenic, especially in populations with rice-based diets. However, arsenic toxicity varies greatly with species. The initial purpose of the present study was to evaluate arsenic speciation in rice. RESULTS: It appeared very clear from the present study that inorganic arsenic shared maximum arsenic load in rice straw while in grains it is considerably low. As species recovered from rice grain and straw are principally As(III) and As(V) with a small amount of dimethylarsenic acid (DMA) and almost non-detectable monomethylarsonic acid (MMA) and arsenobetain (AsB). Discussion of the health risk of As in rice has largely been based on its inorganic arsenic content because these species have generally been considered to be more toxic than MMA and DMA and can be directly compared to As in drinking water, assuming equal bioavailability of inorganic As in the rice matrix and in water. The maximum dietary risk of exposure to inorganic arsenic through transplanted boro paddy in the present experiment was calculated to be almost 1706% of the provisional tolerable weekly intake for an adult of 60 kg body weight. CONCLUSION: As species recovered from boro rice grain and straw are principally As(III) and As(V) with a small amount of DMA and almost non-detectable MMA and AsB. Reductions in total As load through organic amendments in boro rice grain and straw samples were manifested predominately through reduced accumulations of inorganic As species [As(III) and As(V)], between which As(V) accounted for the larger share. © 2014 Society of Chemical Industry Keywords: arsenic; rice; speciation; methylation; risk assessment
INTRODUCTION The countries with the highest daily intake of total arsenic (t-As) are Spain and Japan followed by India and France, and the world’s two most significant cases of As contamination where the population suffers the most are located in Asia, particularly in Bangladesh and West Bengal in India.1,2 The cases in Bangladesh and India are different and more special than those in Spain or Japan, for instance. The speciation of As, which is related to the different sources of As, is responsible for such uniqueness. In general, As from seafood is organic (o-As) while As from drinking water and vegetables is inorganic (i-As). Therefore, dietary As can be categorised by As species (e.g. organic vs. inorganic) and by source (seafood vs. vegetables, mainly rice).3 Arsenic contamination in groundwater in the state of West Bengal has assumed the proportion of 12 endemic districts, 111 endemic blocks and more than 50 million people exposed to threats of arsenic-related health hazards.4 It is only the agricultural sector which enjoys the major share (>90%) of such contaminated groundwater as a source of irrigation and has received attention for quantifying the influence of arsenic in soil–plant systems.5,6 Arsenic contamination of water and soil can also adversely affect food safety. A global normal range of 0.08–0.2 mg As kg−1 has been suggested for rice,7 but values as high as 0.25 mg As kg−1 have been found in rice.8 The average daily intake of As from J Sci Food Agric (2014)
rice for an Indian adult is approximately 100 mg As (400 g dry weight × 0.25 mg As kg−1 ), which is five times higher than 20 mg As intake from consumption of 2 L of water as the WHO limit of 10 μg L−1 .9 Rice (Oryza sativa L.) is the most important crop of India and the second principal food crop of the world. In India, rice is predominantly grown in the Indo-Gangetic plains, over an area of 13.5 million ha which is 85% of the cultivated land area where groundwater is the principal source of irrigation. Most of the shallow aquifers of groundwater in southern Bangladesh and the eastern part of West Bengal, India, are geogenically contaminated with arsenic (As), exposing more than 40 million people at risk of As in drinking water.10 The WHO standard for As in drinking water of 10 μg L−1 has been adopted by many countries. Arsenic in water is generally inorganic and can be a mixture of arsenite [As(III)] and arsenate
∗
Correspondence to: Bishwajit Sinha, College of Agriculture, Orissa University of Agriculture and Technology, Bhawanipatna-766001, Odisha, India. E-mail: [email protected]
a College of Agriculture (OUAT), Bhawanipatna, Odisha, India b Department of Agricultural Chemistry and Soil Science, BCKV, Mohanpur, Nadia, West Bengal
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[As(V)]. The risk assessment for exposure to As-contaminated drinking water, as postulated by the U.S. Environmental Protection Agency, is based on carcinogenicity risk from inorganic As. No intake of inorganic As from food was considered while setting the drinking water standard, while it is now evident that significant amounts can be ingested this way. Arsenic in rice is of special concern because of the much higher levels of As in rice grain compared to other staple cereal crops, coupled with high levels of rice consumption by Asian populations. Moreover, knowledge of the speciation of As in rice is critical to understanding the potential toxicity of rice to humans. Adult daily rice intakes as high as 750 g uncooked weight have been reported in West Bengal, India,11 and between 400 and 650 g uncooked weight in Bangladesh.12 Rice grain As concentrations in Bangladesh vary widely, but 50% (between the 25th and 75th percentiles of the overall distribution from 871 samples) fall within the range of 0.15–0.36 mg kg−1 ,7 and values as high as 1.8 mg kg−1 have been reported.13 Daily consumption of 400 g dry weight of rice containing 0.25 mg As kg−1 would provide 100 μg As or five times the 20 μg As from consumption of 2 L of water at the acceptable WHO limit of 10 μg L−1 . Moreover, knowledge of speciation of As in rice is critical to understanding the potential toxicity of rice to humans as the toxicity of different arsenic containing compounds varies widely. For example, As concentrations in fish and shellfish can reach 10 mg kg−1 (Schoof et al.14 ) and are approximately 10–100 times the levels found in rice. However, most As in seafood is present in organic compounds such as arsenobetaine and arsenocholine, which are considered to be non-toxic to humans (acute toxicity 103 times less than inorganic arsenic). Four species of As are commonly reported in rice: arsenite, arsenate, monomethylarsonic acid (MMA), and dimethylarsenic acid (DMA). The dominant species are usually arsenite and DMA, although the sum of arsenite + arsenate is often reported.15 The inorganic As content in rice can vary from 10% to 90% of total As,16 but the reason for this variability has not been established. Discussion of the health risk of As in rice has largely been based on its inorganic arsenic content because these species have generally been considered to be more toxic than MMA and DMA17 and can be directly compared to As in drinking water, assuming equal bioavailability of inorganic As in the rice matrix and in water. Rice produced in Bangladesh/India contained a mean of 80% inorganic As.15 Risks of dietary exposure to arsenic through contaminated food are alarming in the Indian subcontinent, although arsenic (i-As, precisely) ingestion through processed foods is a global issue. Rice milk samples from national supermarket chains in the city of Aberdeen, UK, displayed a high i-As content, with values being above 60% of the t-As content (exceeding EU and US drinking water standards) and the remainder being dimethylarsinic acid (DMA). The i-As contents were significantly higher in gluten-free rice than in cereals mixtures with gluten, placing infants with coeliac disease at high risk.18 High contents of i-As were observed in Spanish gluten-free products based on rice with mean contents being 69 mg kg−1 . The finding of elevated contents of i-As in infant products and consequently elevated intakes of i-As in infants older than 4 months in China, USA and UK is of concern and deserves further attention.19 The purpose of the present study was to assess As speciation in rice from West Bengal, India, in order to improve understanding of the health risk posed by As in Indian rice. An effort has been made, through the present study, to take an account of species level accumulation of arsenic in rice in the arsenic affected villages
of Chakdaha block, Nadia district, West Bengal, India, having a total arsenic concentration of irrigation water, drifted by shallow tube wells, of 0.32 mg L−1 .
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MATERIALS AND METHODS Site description The experiment was conducted at a farmer’s field (2009–2010) in the village of Ghentugachhi, geographical location: 23∘ 2′ 7.1′′ N, 88∘ 35′ 4.8′′ E, district of Nadia, West Bengal, India. Details of crop management The crop summer rice, variety IET 4786, which is widely grown in the arsenic affected area of West Bengal, was selected for the study. The crop was sown during the first week of February 2010. The seed rate was 100 kg ha−1 and the spacing was maintained at 30 × 10 cm. Weeding was done twice [at 20 and 40 days after transplanting (DAT)]. Rice fields were irrigated both from shallow tube well water (As concentration, ∼0.32 mg L−1 ) and pond water (As concentration, ∼0.03 mg L−1 ). Experimentation The experiment was laid out in a two-factor randomised block design with three replications. Factorial experimental treatments were two levels of irrigation (irrigation through shallow tube well water and irrigation through surface water) and four levels of organic manures, namely farmyard manure at 10 t ha−1 , vermicompost at 3 t ha−1 , municipal sludge at 10 t ha−1 and mustard cake at 1.0 t ha−1 . The soils were amended with well-decomposed farmyard manure, vermicompost, municipal sludge and mustard cake in respective treated plots followed by two ploughing operations 25 days before sowing. The recommended doses of N, P, K fertilisers (N:P2 O5 :K2 O, 100:50:50) kg ha−1 were applied to the soils irrespective of treatments. The entire P and K fertilisers were applied basally while N fertiliser was applied in three splits (50% as basal and the remainder 50% top dressed at 30 DAT and 45 DAT). Weeding was done twice (at 20 and 40 DAT). Arsenic speciation in plant samples Sample digestion: total As, HNO3 digest About 0.2 g of rice grain or straw sample were weighed into a microwave Teflon vessel and 7 mL of concentrated nitric acid was added to it and left to stand overnight at room temperature. Samples were then digested in a microwave vessel maintained at 200 ∘ C for 20 min. Samples were then cooled and transferred to a 50 mL volumetric flask for total arsenic analysis by using a Perkin Elmer ELAN DRCe 6000 ICP-MS (Perkin Elmer, California, USA). Details of the operating parameters of the ICP-MS are shown in Table 1. Sample extraction for arsenic species For speciation analysis about 0.2 g of rice grain or straw sample were weighed into a microwave Teflon vessel and 2 mL of 2.0 mol L−1 trifluoroacetic acid (TFA) was added to it. Samples were then digested in a microwave maintained at 90 ∘ C for 20 min. Samples were then cooled and transferred to a 50 mL volumetric flask for analysis of different forms of arsenic in a HPLC-ICP-MS.5 Details of the operating parameters of HPLC are listed in Table 2. Total As recoveries from rice were validated by using the Perkin Elmer ELAN DRCe 6000 ICP-MS and compared with the NIST standard SRM 1568a (rice flour); the certified value was 290 ± 30 μg kg−1 and the observed value was 283 ± 8 μg kg−1 .
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Arsenic toxicity in rice
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Table 1. ICP-MS operating conditions and parameters
Table 2. HPLC isocratic method and operating parameters
Parameter
Operating condition
Parameter
Nebuliser
Meinhard Type A quartz , Part No.: WE02-4371 ELAN DRC II: Quartz Cyclonic, Part No.: WE02-5221 1500 W 15 L min−1 0.82 L min−1 1.2 L min−1 ELAN DRC II: 2.0 mm i.d. Quartz, Part No.: WE02-3915 m/z 91 (75 As, 16 O) for DRC 30 s 15 s 30 s Dual Scanning Nickel 50 ms 600 s straw > grain (Table 3). A similar trend was also reported by Sanyal.21 Such findings of relative proportions of arsenic accumulations can be substantiated by findings of Rahman et al.,22 who reported 28- to 75-fold higher As accumulation in roots than shoot and raw rice grain. The findings of Liu et al.23 also corroborate such findings where proportional accumulations remained at 1.17 to 4.15 mg kg−1 (straw), 0.56–1.35 mg kg−1 (husk) and 0.25 to 0.73 mg kg−1 (grain) of rice. The relative accumulation of arsenic in the underground and aboveground portions of plant systems has been observed to vary substantially across growth stages. Figure 1 shows that root arsenic accumulation gradually increased up to 60 days after transplanting (DAT) after which it underwent a steep increase. The shoot arsenic J Sci Food Agric (2014)
Mobile phase for soil sample Mobile phase for plant sample
Flow rate Run time Column Column temperature Auto-sampler flush solvent Sample injection volume Sample preparation Detection
30 mmol L−1 ammonium phosphate, pH 5.6 6.66 mmol L−1 ammonium nitrate and 6.66 mmol L−1 ammonium hydrogen phosphate, pH 6.2 1.0 mL min−1 9 min Anion exchange, Hamilton PRP-X100 26 ∘ C 5% Methanol/95% deionised water 100 μL 1:1 with mobile phase Perkin-Elmer Elan DRCe ICP_MS
280
t-As accumulation (mgkg–1)
Spray chamber
Setting
240 200
ROOT
SHOOT
160 120 80 40 0 –40
30 DAT
60 DAT
90 DAT
Days after transplanting Figure 1. Progressive changes in t-As accumulation in different plant parts of boro rice (cv. IET-4786) with advancement of growth.
content has been observed to decrease in the first phase (up to 60 DAT) and increased thereafter. The relative affinity of the plant to accumulate arsenic is closely related to phosphate. The P/As molar ratio (MR) (P molar concentration in plant) can be a good index for the relative abundance and roles in plant. Another important index for the relative preference of plant systems towards As/P accumulation is the bioaccumulation preference (BP): BP =
Asplant ∕Assoil Pplant ∕Psoil
where Asplant is the As concentration in plant tissue, Assoil is the concentration of As in soil, Pplant is the P concentration in plant tissue, and Psoil is the concentration of P in soil. The observed values of MRs and BP in different plant parts of boro rice are listed in Table 4 and Table 5. The MR values (P/As) in root, straw and grain of boro rice varied from 3.29 to 10.14, 43.95 to 107.49, and 1118.87 to 2349.45, respectively. Such higher MR values have previously been reported in corn seedlings (102–4400), tomato (500–4200), cord grass (800–8000).24 Table 4 shows that the MR values (P/As) in rice decreased significantly when irrigated through underground shallow tube well
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Table 3. Effect of selected organic interventions and elevated phosphate administration on arsenic accumulation (mg kg−1 ) in soil–plant system under boro rice (cv. IET-4786) subjected to varying irrigation sources in farmers’ fields Arsenic accumulation (mg kg−1 ) 30 DAT Treatment
Root
I1 I2 CD𝛼=0.05 P1 P2 CD𝛼=0.05 O1 O2 O3 O4 O5 CD𝛼=0.05 I1 P1 I1 P2 I2 P1 I2 P2 CD𝛼=0.05 I1 O1 I1 O2 I1 O3 I1 O4 I1 O5 I2 O1 I2 O2 I2 O3 I2 O4 I2 O5 CD𝛼=0.05 P1 O1 P1 O2 P1 O3 P1 O4 P1 O5 P2 O1 P2 O2 P2 O3 P2 O4 P2 O5 CD𝛼=0.05 I1 P1 O1 I1 P1 O2 I1 P1 O3 I1 P1 O4 I1 P1 O5 I1 P2 O1 I1 P2 O2 I1 P2 O3 I1 P2 O4 I1 P2 O5 I2 P1 O1 I2 P1 O2 I2 P1 O3 I2 P1 O4
74.08 66.16 0.043 69.31 70.92 0.043 75.99 69.25 72.84 66.53 65.98 0.069 73.65 74.51 64.98 67.34 0.060 79.33 73.87 76.54 68.59 72.05 72.65 64.63 69.15 64.46 59.91 0.097 75.63 67.12 73.29 66.11 64.41 76.35 71.38 72.40 66.94 67.54 0.097 79.76 74.13 77.18 67.49 69.67 78.91 73.60 75.89 69.69 74.43 71.50 60.10 69.40 64.73
Shoot + leaf 11.53 9.02 0.034 10.57 9.98 0.034 14.64 8.36 9.90 9.12 9.34 0.057 12.25 10.81 8.88 9.15 0.052 16.80 8.86 11.06 11.31 9.61 12.48 7.87 8.74 6.93 9.06 0.080 14.74 9.17 9.98 10.30 8.65 14.55 7.56 9.82 7.94 10.03 0.080 17.28 9.78 11.18 13.92 9.09 16.33 7.93 10.93 8.71 10.13 12.19 8.56 8.78 6.68
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60 DAT Root 87.46 76.44 0.077 78.59 85.31 0.077 87.04 79.20 76.45 87.98 79.08 0.120 82.35 92.56 74.83 78.05 0.109 89.76 83.67 78.79 103.33 81.74 84.33 74.73 74.11 72.63 76.42 0.172 87.44 79.65 75.42 71.15 79.29 86.65 78.75 77.48 104.80 78.86 0.172 88.05 84.74 79.16 74.77 85.04 91.47 82.60 78.42 131.89 78.43 86.83 74.56 71.67 67.53
Shoot 4.95 3.62 0.149 4.64 3.93 0.149 5.33 4.33 3.17 4.07 4.52 0.235 5.57 4.33 3.71 3.53 0.212 5.94 5.04 3.53 4.87 5.37 4.72 3.62 2.81 3.27 3.67 0.332 5.57 4.72 3.77 4.25 4.90 5.10 3.94 2.58 3.89 4.13 0.332 6.56 5.90 4.59 5.03 5.79 5.33 4.18 2.47 4.71 4.95 4.58 3.54 2.94 3.47
90 DAT Leaf 15.08 11.95 0.072 13.37 13.66 0.072 20.01 14.07 10.00 11.87 11.63 0.115 15.10 15.06 11.64 12.26 0.103 23.77 18.71 10.03 11.43 11.46 16.26 9.42 9.97 12.31 11.79 0.163 19.61 14.20 12.95 10.14 9.96 20.41 13.93 7.06 13.60 13.29 0.163 24.54 19.91 13.33 11.10 6.63 22.99 17.51 6.74 11.76 16.30 14.67 8.48 12.56 9.19
Root 236.10 214.20 1.363 221.30 229.00 1.363 245.92 218.83 208.08 212.25 240.67 2.156 231.00 241.20 211.60 216.80 1.930 250.33 229.67 232.50 219.83 248.17 241.50 208.00 183.67 204.67 233.17 3.049 229.83 215.17 197.67 224.83 239.00 262.00 222.50 218.50 199.67 242.33 3.049 240.00 224.33 220.67 233.67 236.33 260.67 235.00 244.33 206.00 260.00 219.67 206.00 174.67 216.00
Grain 1.41 1.03 0.010 1.30 1.15 0.010 1.37 1.13 1.17 1.21 1.24 0.029 1.54 1.28 1.06 1.01 0.029 1.53 1.25 1.41 1.39 1.47 1.20 1.00 0.94 1.03 1.00 0.029 1.48 1.09 1.26 1.36 1.32 1.26 1.16 1.09 1.07 1.16 0.029 1.69 1.28 1.56 1.60 1.59 1.37 1.23 1.27 1.19 1.36 1.27 0.90 0.96 1.11
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Post-harvest soil Straw 13.06 11.25 0.040 11.46 12.85 0.040 15.09 11.71 11.86 11.51 10.60 0.063 12.50 13.62 10.43 12.07 0.057 16.66 12.46 12.78 13.00 10.41 13.51 10.97 10.95 10.01 10.79 0.089 14.45 10.57 12.12 11.21 8.96 15.72 12.86 11.61 11.80 12.24 0.089 17.18 11.29 13.43 12.90 7.68 16.14 13.63 12.13 13.09 13.13 11.72 9.85 10.81 9.51
Av. As (mg kg−1 ) 2.38 3.08 0.06 2.52 2.95 0.06 3.06 2.69 2.70 2.60 2.60 0.09 2.18 2.58 2.85 3.31 0.08 2.72 2.38 2.25 2.32 2.23 3.40 3.01 3.15 2.88 2.97 0.13 2.91 2.49 2.34 2.39 2.45 3.22 2.89 3.07 2.81 2.75 0.13 2.56 2.11 1.77 2.22 2.24 2.88 2.65 2.73 2.43 2.23 3.25 2.88 2.90 2.57
Av. P2 O5 (mg kg−1 ) 28.73 34.05 0.28 29.32 33.47 0.28 31.96 32.47 30.52 31.55 30.48 0.44 26.17 31.31 32.47 35.64 0.39 29.10 29.45 28.04 29.05 28.06 34.82 35.49 33.01 34.06 32.91 0.63 29.99 30.54 28.19 29.12 28.76 33.92 34.4 32.85 33.99 32.21 0.63 26.33 27.15 25.23 26.28 25.83 31.86 31.75 30.84 31.82 30.28 33.65 33.93 31.15 31.95
Yield (t ha−1 ) 8.32 9.17 0.023 9.01 8.48 0.023 7.65 8.96 9.34 9.01 8.76 0.037 8.00 8.64 10.02 8.31 0.031 7.27 8.40 8.28 9.43 8.22 8.02 9.53 10.41 8.58 9.29 0.052 8.07 8.99 9.80 9.80 8.40 7.22 8.93 8.89 8.22 9.11 0.052 7.38 8.13 8.40 9.24 6.86 7.17 8.66 8.17 9.61 9.59 8.76 9.86 11.20 10.35
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Arsenic toxicity in rice
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Table 3. Continued Arsenic accumulation (mg kg−1 ) 30 DAT
Treatment
Root
I2 P1 O5 I2 P2 O1 I2 P2 O2 I2 P2 O3 I2 P2 O4 I2 P2 O5 CD𝛼=0.05
59.15 73.79 69.16 68.90 64.18 60.66 0.135
Shoot + leaf 8.20 12.77 7.18 8.70 7.17 9.93 0.112
Output of Tukey Multiple range test∗ O1 75.99E 14.64E C O2 69.25 8.36A D O3 72.84 9.90D B O4 66.53 9.12B A O5 65.98 9.34C
60 DAT
90 DAT
Root
Shoot
Leaf
Root
Grain
73.54 81.82 74.89 76.54 77.72 79.29 0.243
4.02 4.87 3.70 2.68 3.07 3.31 0.472
13.30 17.84 10.36 7.38 15.44 10.28 0.229
241.67 263.33 210.00 192.67 193.33 224.67 4.314
1.04 1.14 1.10 0.91 0.95 0.96 0.029
87.04C 79.20B 76.45A 87.98D 79.08B
5.33D 4.33BC 3.17A 4.07B 4.52C
20.01E 14.07D 10.00A 11.87C 11.63B
245.92E 218.83C 208.08A 212.25B 240.67D
1.37D 1.13A 1.17B 1.21C 1.24C
∗ Output of Tukey Multiple range test for means of organic amendments. A – E Results with different superscript letters are significantly different at 5% level. I1 , irrigation from shallow tube well; I2 , irrigation from pond; P1 , 50 kg P2 O5 ha−1 ; P2 , 75 kg P2 O5 O3 , vermicompost at 3 t ha−1 ; O4 , municipal sludge at 10 t ha−1 ; O5 , mustard cake at 1 t ha−1 .
water (STW) as compared to surface pond water (PW) irrigation which can be attributed to higher As concentration of STW water. Elevated levels of P administration significantly increased the root and grain MR values in rice while the straw MR was observed to reduce under higher P situation. Similar observations were obtained by Tu and Ma 25 who reported that at high arsenic levels the P/As molar ratio of the aboveground portion of a plant is increased by applying more phosphate. Organic amendments increased the MR values in rice root, grain and straw significantly over the non-organic control, although different organic compounds behaved differentially in this regard. The highest root MR remained associated with the application of vermicompost, while the highest grain and straw MR were obtained from farmyard manure and mustard cake treated situations. The Tukey multiple range test showed that effects of different organic amendments are different on root MR values while in grain and straw MR effects of vermicompost, mustard cake and sludge amendments were similar (Table 4). The bioaccumulation preference for arsenic in root, grain and straw of boro rice have been found to be suppressed with elevated application of phosphorus. The BP values were greater than 1 in roots but less than 0.5 in grain and straw. Similar observations were recorded by Tu and Ma25 in the arsenic hyper-accumulator Chinese brake fern (Pteris vitata). These authors concluded that phosphate was preferentially accumulated over arsenic in the aboveground portion which was further encouraged through application of additional phosphate. Organic manure, phosphate intervention and arsenic in the soil–plant system Results of Table 3 showed that administration of elevated levels of phosphate fertilisers increased As accumulations in rice root in all the growth stages. Grain As, on the other hand, has been J Sci Food Agric (2014)
Post-harvest soil Av. As (mg kg−1 )
Av. P2 O5 (mg kg−1 )
Yield (t ha−1 )
10.23 15.31 12.08 11.08 10.52 11.34 0.126
2.67 3.56 3.14 3.41 3.19 3.27 0.18
31.69 35.99 37.04 34.86 36.16 34.14 0.89
9.94 7.28 9.20 9.61 6.82 8.64 0.072
15.09E 11.71C 11.86D 11.51B 10.60A
3.46D 2.72B 2.56A 2.81B 2.96C
31.96BC 32.47C 30.52A 31.55B 30.48A
7.65A 8.96C 9.34D 9.01C 8.76B
Straw
ha−1 ; O1 , control; O2 , farmyard manure at 10 t ha−1 ;
found to decrease at higher P administration. An increase in available As in soil and subsequent greater P availability may possibly be responsible for greater As accumulation in rice root. The grain As accumulation, on the other hand, was found to be suppressed, probably due to reduced translocation of As to grain under a P-rich enzymatic pathway. Arsenic and phosphorus exist in the same periodic group and have similar chemical and physical properties as well as similar electron configurations.25 Among the two important oxyanions, arsenite [As(III)] and arsenate [As(V)], the latter is an analogue of phosphate (PO4 3− ), making it an important factor in the behaviour of arsenic in well-aerated soils.26 Arsenite [As(III)] is not an analogue of PO4 3− , and is less relevant to arsenic behaviour under flooded soil conditions.27 However, Heikens28 reported that PO4 3− can play an important role in the rhizosphere, where an oxygenated micro-environment prevails under flooded rice conditions. Clement and Faust29 showed that during water–sediment interactions, phosphate concentration in the system plays an important role in the release of arsenite from the sediments. Inorganic arsenic species are generally highly toxic to plants. Arsenate acts as a phosphate analogue and is transported across the plasma membrane via phosphate co-transport systems.30 Arsenate sensitivity is intimately linked to phosphate nutrition, with increased phosphate status leading to reduced arsenate uptake, through suppression of the high-affinity phosphate/arsenate uptake system.31 Observations recorded in Table 3 showed that soil amendment through selected organic manures significantly reduced the arsenic accumulation in different parts of rice across growth stages which has been most efficiently manifested through vermicompost application regardless of the growth stage or the plant part(s) under consideration. The addition of organic amendments to soils reduce the bioavailability of heavy metals by changing them from bioavailable forms to fractions associated with organic matter or metal oxides or carbonates.32 Cao et al.33 reported that when
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Table 4. Molar ratios (MRs) of phosphorus to arsenic in different plant parts of boro rice
B Sinha and K Bhattacharyya
Table 4. Continued Treatment
Treatment I1 I2 CD (P = 0.05) P1 P2 CD (P = 0.05) O1 O2 O3 O4 O5 CD (P = 0.05) I1 P1 I1 P2 I2 P1 I2 P2 CD (P = 0.05) I1 O1 I1 O2 I1 O3 I1 O4 I1 O5 I2 O1 I2 O2 I2 O3 I2 O4 I2 O5 CD (P = 0.05) P1 O1 P1 O2 P1 O3 P1 O4 P1 O5 P2 O1 P2 O2 P2 O3 P2 O4 P2 O5 CD (P = 0.05) I1 P1 O1 I1 P1 O2 I1 P1 O3 I1 P1 O4 I1 P1 O5 I1 P2 O1 I1 P2 O2 I1 P2 O3 I1 P2 O4 I1 P2 O5 I2 P1 O1 I2 P1 O2 I2 P1 O3 I2 P1 O4 I2 P1 O5 I2 P2 O1 I2 P2 O2 I2 P2 O3 I2 P2 O4
Root MR 4.52 7.42 1.21 5.16 6.78 0.37 4.64 6.62 7.04 5.87 5.69 0.25 4.04 5.00 6.29 8.56 0.12 3.80 5.01 4.94 4.33 4.53 5.48 8.24 9.13 7.41 6.86 0.13 4.37 5.49 5.91 4.81 5.24 4.91 7.76 8.16 6.93 6.15 0.98 3.69 4.54 4.40 3.45 4.11 3.90 5.47 5.48 5.21 4.96 5.06 6.43 7.43 6.18 6.36 5.91 10.05 10.84 8.65
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Grain MR 1318.99 2058.43 78.27 1587.21 1790.20 58.98 1378.69 1848.39 1717.29 1728.67 1770.50 93.55 1161.71 1476.26 2012.72 2104.14 35.63 1127.38 1432.91 1325.67 1380.58 1328.40 1630.00 2263.87 2108.91 2076.76 2212.60 31.92 1258.00 1877.67 1608.51 1547.90 1643.99 1499.38 1819.10 1826.07 1909.44 1897.01 34.98 959.92 1335.34 1130.93 1169.47 1212.91 1294.83 1530.47 1520.41 1591.69 1443.90 1556.07 2420.01 2086.09 1926.33 2075.08 1703.93 2107.73 2131.72 2227.19
Root MR
Grain MR
Straw MR
Straw MR 62.38 72.07 1.34 68.86 65.59 1.53 48.13 66.99 68.36 75.98 76.66 3.80 64.56 60.20 73.16 70.98 3.39 43.03 63.49 69.90 57.60 77.87 53.24 70.49 66.81 94.36 75.45 3.76 47.56 68.27 65.53 62.99 99.93 48.71 65.71 71.19 88.96 53.39 10.11 35.70 60.87 65.92 47.74 112.55 50.37 66.12 73.89 67.45 43.19 59.42 75.66 65.14 78.25 87.32 47.06 65.31 68.48 110.46
I2 P2 O5 CD (P = 0.05)
7.35 0.92
Output of Tukey multiple range test∗ O1 4.64A O2 6.62D O3 7.04E O4 5.87C O5 5.69B
959.92 31.93
63.59 5.74
1378.70A 1848.40D 1717.30B 1728.70B 1770.50C
48.14A 66.99B 68.36C 75.98D 76.66D
∗ Output of Tukey Multiple range test for means of organic amendments. A – E Results with different superscript letters are significantly different at 5% level. I1 , irrigation from shallow tube well; I2 , irrigation from pond; P1 , 50 kg P2 O5 ha−1 ; P2 , 75 kg P2 O5 ha−1 ; O1 , control; O2 , farmyard manure at 10 t ha−1 ; O3 , vermicompost at 3 t ha−1 ; O4 , municipal sludge at 10 t ha−1 ; O5 , mustard cake at 1 t ha−1 .
Table 5. Bioaccumulation preference for arsenic observed in different plant parts of boro rice Phosphorus
Root
Grain
Straw
Standard P (at 50 kg ha−1 ) Elevated P (at 75 kg ha−1 )
2.2851 1.7193
0.0078 0.0065
0.1789 0.1772
bio-solid was added to either acidic or neutral soil the adsorption of arsenic was increased and water soluble arsenic was reduced. Shiralipour et al.34 reported that the application of organic matter to soil would increase soil cation and anion exchange capacity, which may increase arsenic adsorption by increasing the amount of positive charge on the oxide surface and/or forming a positively charged surface35 and enhanced sorption capacity of the soil matrix. Enriched soil humic fractions in manure amended soils may be active in retaining As(III) and As(V) through adsorption. So organo-arsenic complexations with humic/fluvic colloids of the native soil and the incorporated organic manures moderate the hazards of arsenic toxicity.36 In a very recent instance a similar observation has been cited by Rahaman et al.,37 who showed that combined applications of lathyrus + vermicompost + poultry manure reduced arsenic transport in plant parts (root, straw, husk, whole grains and milled grain). Such findings and the relative efficiencies of selected organic manures in off-loading arsenic from rice may be well corroborated with our findings38 relating to characterisation of humic/fulvic components extracted from the selected organic matters and their complexation with arsenic. Bioavailability of arsenic in boro rice irrigated through underground water and surface water (pond water) The inorganic [i.e. As(III) and As(V)] and organic (i.e. DMA and MMA) arsenic accumulation in rice grain and straw were determined from the TFA (at pH 6.2) extract by using HPLC-ICP-MS (Perkin Elmer ElanDRCe 6000) and the results are given in Table 6. Selected samples of the present experiment showing good response of off-loading As accumulation in rice grain and straw
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Arsenic toxicity in rice
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Table 6. Recoveries of arsenic species from boro rice under varying irrigation regimes and selected organic amendments Recovery of arsenic species in boro rice (μg kg−1 )
Source of irrigation
Selected interventions As B
Grain Pond (PW)
Shallow tube well (STW)
Straw Pond (PW)
Shallow tube well (STW)
Characterisation∗ STW PW SOIL
As(III)
DMA
MMA
As(V)
Sum of species
Total As (μg.kg−1 ) (HNO3 digest)
Species recovery (%)
1 461.60 ± 44.10 864.00 ± 25.04 918.00 ± 48.99 1 542.00 ± 35.86
93.97 ± 1.20 90.21 ± 2.44 86.14 ± 2.82 93.26 ± 1.90
C VC FYM C
ND 750.70 ± 6.76 140.50 ± 4.74 ND 516.00 ± 7.45 45.60 ± 6.50 ND 489.00 ± 9.06 34.80 ± 2.80 ND 1 223.50 ± 8.02 41.50 ± 2.27
ND ND ND ND
482.20 ± 7.13 217.80 ± 8.18 267.00 ± 4.60 173.00 ± 8.34
1 373.40 ± 11.42 779.40 ± 12.49 790.80 ± 12.50 1 438.00 ± 4.56
VC FYM
ND 589.50 ± 5.87 ND 1 040.90 ± 7.88
53.00 ± 3.65 57.20 ± 5.31
ND ND
136.90 ± 6.66 217.60 ± 3.89
779.40 ± 8.83 1 082.30 ± 54.43 72.01 ± 2.50 1 315.70 ± 10.67 1 180.00 ± 32.55 111.50 ± 2.48
C VC FYM C
ND 865.80 ± 12.4 ND 1 693.00 ± 7.8 ND 586.80 ± 4.9 ND 969.00 ± 7.2
443.00 ± 14.7 ND 7 600.30 ± 43.1 8 909.10 ± 54.6 367.30 ± 12.9 ND 2763.30 ± 23.3 4 823.60 ± 29.1 292.80 ± 4.8 ND 5 073.00 ± 31.5 5 952.60 ± 34.44 792.60 ± 14.5 194.0 8 146.80 ± 45.8 10 102.80 ± 39.6
VC FYM
ND ND
577.00 ± 12.9 294.70 ± 14.5 126.00 ± 3.9 133.20 ± 8.5
ND ND
– – –
ND ND ND
278.78 ± 6.4 ND 900.15 ± 9.4
ND 73.10 ± 3.5 351.88 ± 9.4 ND 23.65 ± 2.1 23.65 ± 2.1 ND 13 300.70 ± 125.8 14 200.85 ± 134
ND ND ND
5 080.30 ± 44.4 6 756.60 ± 23.7
5 952.00 ± 29.7 7 015.80 ± 18.9
8 823.00 ± 128.6 100.98 ± 2.3 4739.60 ± 145.2 101.77 ± 5.1 6 048.00 ± 157.9 98.42 ± 2.5 9 684.00 ± 120.0 104.32 ± 3.2 6 308.00 ± 138.8 7 326.00 ± 126.5
94.36 ± 2.2 95.77 ± 2.6
320.00 ± 4.7 109.96 ± 5.2 31.50 ± 2.4 75.08 ± 3.1 19 400.00 ± 168.9 73.20 ± 4.3
∗ Characterisation of arsenic sources (STW/PW) and sink (soil) of the experimental site. ND, not detectable.
through interventions of organic amendments (manures) have been extracted through TFA (at pH 6.2). A microwave digestion system (Multiwave 3000; Anton Par, Ashland, USA) with a rotor of 48 Teflon digestion vessels was used for sample digestion and extraction. Arsenic species were determined by using HPLC-ICP-MS (Perkin Elmer ElanDRCe 6000). All chemicals used were reagent grade. All the solutions were prepared with Milli-Q water (Millipore, Bedford, MA, USA). For the speciation studies, standard solutions (100 mg L−1 ) of As compounds were prepared from: (1) arsenite (NaAsO2 ; Perkin Elmer), (2) arsenate (Na2 HAsO4 · 7H2 O; Perkin Elmer), (3) monomethyl arsonate (CH3 AsNa2 O3 ; Sigma–Aldrich), (4) dimethyl arsenate [(CH3 )2 AsO(OH); Sigma] and arsenobetaine (AsB; Sigma). TFA was purchased from Aldrich (St. Louis, MO, USA) Results of Table 6 show that As compared to the species recoveries from TFA extract of rice grain ranged from 72.01 ± 2.50 to 111.50 ± 2.48 as a % of the total As recoveries from HNO3 extract, which are quite appreciable for a particular extractant in the back-drop of such recoveries reported by Abedin et al.5 They observed that the use of TFA resulted in >80% extraction efficiency. In TFA extracts, the proportions of arsenate, arsenite and DMA were 72–84%, 15–26%, and 1–4%, respectively. It appears very clear from Table 6 and Fig. 2 that i-As species recovered from rice grain and straw are principally As(III) and As(V) with a small share of DMA and almost non-detectable MMA and AsB. Rice grain As has been found to be principally As(III) while in straw As(V) predominated over As(III). Sanz et al.39 observed that for rice and paddy samples, inorganic arsenic accounted for up to 70–98% of the total arsenic content, with As (III) being the major species. The levels of arsenic obtained J Sci Food Agric (2014)
from straw and soil samples are significantly higher than the background levels, with As(V) being the major species, thus increasing human exposure to arsenic via the soil–plant–animal–human pathway. Table 7 shows significant lower recoveries of i-As, o-As and t-As in rice grain and straw when irrigated with pond water as compared to STW (underground) water irrigation. Interventions through organic manures significantly reduced t-As (HNO3 extract), i-As and o-As in rice grain and straw both under STW and pond water irrigated situations (Table 7), brought through application of farmyard manure and vermicompost at 39.59% and 28.04% compared to the control counterparts. Such reductions in t-As load in rice grain and straw samples were manifested predominately through reduced accumulations of i-As. The intervention through farmyard manure was observed to be more capable of reducing the As(III) and DMA loads in rice grain and straw samples, while vermicompost was more successful in reducing As(V) and total As (HNO3 digest) in rice samples (Table 7), although such changes brought through differential organic amendment could not be explained with the present set of data, and neither can they be substantiated through any citation in this regard. Arsenic methylation It is interesting to note that characterisation of arsenic source (STW/PW) and sink (soil) did not show any recoveries of organic As, although boro grain and straw accumulates organic As species (Table 6). The recoveries of such organic species in rice grain and straw may be due to transformation of inorganic As to organic forms in the plant body. The % recoveries of organic arsenic (out of
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B Sinha and K Bhattacharyya
Table 7. Recoveries of i-As and organic As in boro rice under varying irrigation regimes and selected organic amendments
Treatment Grain Main effect means PW STW CD (P = 0.05) C VC FYM CD (P = 0.05) Interactions PW × C PW × VC PW × FYM STW × C STW × VC STW × FYM CD (P = 0.05)
Figure 2. The % share of As species in boro rice (cv. IET-4786).
total arsenic from the HNO3 digest) ranged from as low as 1.75 to 15.53 depending upon sources of irrigation, organic interventions and their interactions (Table 7). It is further noteworthy that rice, irrigated through more arsenic-rich underground water (STW), accumulated more t-As and i-As but less o-As. On the other hand, rice irrigated through low-arsenic surface water (PW), accumulated less t-As and i-As but significantly higher o-As than STW-irrigated rice grains and straw (Table 7). The % recoveries of organic arsenic were observed to be higher in rice straw as compared to rice grain. Interventions of organic manures significantly reduced such recoveries as compared to the control which may be attributed to low total arsenic recoveries. Such reductions in t-As, i-As and o-As in rice grain and straw were a maximum through vermicompost amendment. The observations obtained from the present study are not sufficient to amply describe whether conversion of inorganic arsenic fractions into organic moieties were manifested through organic interventions employed (Table 7). Arsenic is metabolised from inorganic to organic forms by a wide range of organisms with limited evidence that this occurs in plants.40 Factors which can influence the As species present in a plant are arsenic species present in soil; the ability of the compound to enter the plant, actively or passively; the ability of plant to synthesise As species; and the presence of As species absorbed to the outside surface of plant roots.41 It should be noted here that no organic As species was recovered from the soil matrix in the present investigation. There is significant evidence that exposure to i-As results in the generation of reactive oxygen species, probably through the conversion of As(V) to As(III), a process which readily occurs in plants42 as has also been shown
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Straw Main effect means PW STW CD (P = 0.05) C VC FYM CD (P = 0.05) Interactions PW × C PW × VC PW × FYM STW × C STW × VC STW × FYM CD (P = 0.05) Output of Tukey test∗ Grain C VC FYM Straw C VC FYM
i-As
Organic As
Total As (μg kg−1 ) (HNO3 digest)
925.03 1135.71 14.24 1321.15 736.12 1033.85 17.44
114.43 56.98 5.67 97.05 52.85 107.22 6.94
1097.44 1267.67 44.34 1501.17 973.17 1073.33 54.31
1232.97 738.90 803.23 1409.33 733.33 1264.47 24.67
140.50 52.70 150.10 53.60 53.00 64.33 9.81
1461.67 864.00 966.67 1540.67 1082.33 1180.00 NS
5951.83 7211.18 39.63 8815.68 4689.17 6239.67 48.53
368.36 359.90 NS 616.03 329.00 147.35 19.96
6623.56 7853.44 258.22 9246.17 5499.67 6969.67 316.26
– – – – – – –
8466.17 3732.00 5657.33 9165.20 5646.33 6822.00 68.63
443.03 367.33 294.70 789.03 290.67 133.20 26.82
8823.00 4739.67 6308.00 9669.33 6259.67 7631.33 NS
5.02 7.76 4.68 8.16 4.65 1.75 –
1321.15C 736.12A 1033.85B
97.05B 52.85A 107.22C
1501.17C 973.17A 1073.33B
– – –
8815.68C 4689.17A 6239.67B
616.03C 329.00B 147.35A
9246.17C 5499.67A 6969.67B
– – –
Organic As (%)
– – – – – – – 9.61 6.09 15.53 3.48 4.91 5.47 –
∗ Output of Tukey multiple range test for means of organic amendments. PW, pond water; STW, shallow tube well water; C, no organic amendment; VC, vermicompost at 3 t ha−1 ; FYM, farmyard manure at 10 t ha−1 soil.
from the higher ratio of As(III) and As(V) in plant systems compared with As(III)/As(V) recovered from the sink (soil). Following the reduction of arsenate to arsenite in plants, arsenic may potentially be further metabolised to methylated species leading to further oxidative stress.43 In terrestrial plants it is not clear how organic As species appear in plant systems. Reduction of arsenate to arsenite has been definitely demonstrated in plants but methylation has not.44 Organic As species
© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
Arsenic toxicity in rice
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Table 8. Assessment of risk for dietary exposure to As-contaminated boro rice
i-As conc. (mg kg−1 ) Control 1.40 (max.) Vermicompost 0.73 (min.)
Daily rice consumption (g) 1564.80∗ 1564.80∗
Weekly i-As ingestion (mg) 15.35 8.01
% PTWI†
CONCLUSION
1706.07 890.16
The present study has made a modest effort to understand the arsenic toxicity profile in rice, grown in contaminated area of rural Bengal (India) and the possible risk of its dietary exposure. The unique character of the anaerobic rice ecosystem results in a significant build-up of i-As in soil and underground water used for irrigation and its concomitant accumulation in rice. The recoveries of i-As is dominated by As(III) in rice grain and As(V) in rice straw, leading to a toxicity profile that presents more risk as there are higher levels of the more toxic As(III) in the edible portion. Recoveries of organic As species in rice grain and straw without having any organic As in the source and sink in detectable range, emerged with possibilities of methylation of i-As in the plant system. The risk of dietary exposure to i-As through rice, the staple food in the experimental area, poses an almost equal threat to human health as that posed by contaminated drinking water. Organic amendments and augmented P fertilisation showed considerable promise in reducing t-As and i-As accumulation in rice and the dietary risk.
∗ National Nutrition Monitoring Bureau (NNMB) Diet Survey and Nutritional Status of rural populations, 2006 (www.nnmbindia.org). † % provisional tolerable weekly intake (PTWI) for a 60 kg adult.
present in field samples of plants may have been taken up from soil solution in that particular form as they can be present in soil through microbial activity;45 however, it is also possible that plants themselves could potentially transform arsenic species. In a study conducted by Koch et al.,46 no evidence of methylation was found in the surrounding soil and water, and yet a number of plant species contained MMA, DMA, tetramethyl arsonium ion and trimethylarsenium oxide (tetra). Dietary risk assessment The paradigm of the assessment of risk associated with human exposure to arsenic contamination through diets starts with weighing the arsenic load ingested through diet. Since rice is the main staple food in West Bengal, India, the possibility of arsenic ingestion through consumption of rice by people in the contaminated region cannot be ignored. The estimates of As exposure via dietary and drinking water routes show that even when people are drinking As-safe water (
