Arsenic Speciation in Rice and Soil Containing Related Compounds

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Anal. Chem. 2008, 80, 5768–5775

Arsenic Speciation in Rice and Soil Containing Related Compounds of Chemical Warfare Agents Koji Baba,*,† Tomohito Arao,† Yuji Maejima,† Eiki Watanabe,‡ Heesoo Eun,† and Masumi Ishizaka† National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8604, Japan Food Safety Commission Secretariat, Cabinet Office, Government of Japan, Tokyo 100-8989, Japan Diphenylarsinic acid, phenylarsonic acid, methylphenylarsinic acid (MPAA), dimethylphenylarsine oxide (DMPAO), and methyldiphenylarsine oxide (MDPAO) in soil and rice were extracted, separated by reversed-phase chromatography, and quantified by ICPMS with a membrane desolvating system. For the extraction of arsenicals from rice grain and straw, 68% HNO3 provided better extraction efficiency than water, 50% methanol, or 2.0 mol L-1 trifluoroacetic acid. For the extraction from soil, 68% HNO3 provided better extraction efficiency than H2O, 1 mol L-1 H3PO4, or 1 mol L-1 NaOH. The contaminated soil contained all five aromatic arsenicals along with inorganic arsenicals as main species (5.86 ( 0.19 µg of As kg-1: 60.8 ( 2.0% of total extracted As). After pot experiments, rice straw contained mainly DMPAO (7.71 ( 0.48 µg of As kg-1: 60.5 ( 3.7%), MDPAO (0.91 ( 0.07 µg of As kg-1: 7.2 ( 0.5%), and inorganic As (2.85 ( 0.20 µg of As kg-1: 22.3 ( 1.6%). On the other hand, rice grain contained mainly MPAA (1.17 ( 0.04 µg of As kg-1: 86.7 ( 2.7%). The root uptake of each species from the soil and transport from straw to grains were significantly related to the calculated log Kow values. Chemical warfare agents containing aromatic arsenicals (AAs), such as ADAMSITE (10-chloro-5,10-dihydrophenarsine), CLARK I (diphenylchloroarsine), CLARK II (diphenylcyanoarsine), and PFIFFIKUS (phenyldichloroarsine), are well-known. They were mainly produced as vomiting or vesicant agents during World Wars I and II, and after WW II, these agents as well as other chemical weapons were abandoned in Europe, China, Japan, and other countries by sea-dumping or earth-burying.1–3 The risk of leakage of these agents from the munitions to the environment has been pointed out. In Lo¨cknitz, Germany, where the Army Ammunition Plant of the former Third Reich was located, * To whom correspondence should be addressed. Phone: +81 29 838 7351. Fax: +81 29 838 7352. E-mail: [email protected]. † National Institute for Agro-Environmental Sciences. ‡ Food Safety Commission Secretariat, Cabinet Office, Government of Japan. (1) Stock, T.; Lohs, K. The Challenge of Old Chemical Munitions and Toxic Armament Wastes, SIPRI Chemical and Biological Warfare Studies, No. 16; Oxford University: Oxford, 1997. (2) Outline of the Project for the Destruction of Abandoned Chemical Weapons (ACW) in China (ACW Destruction Project), Abandoned Chemical Weapons (ACW) Office, Cabinet Office, Government of Japan, October 2002. (3) Bunnett, J. F.; Mikolajczyk, M. Arsenic and Old Mustard: Chemical Problems in the Destruction of Old Arsenical and ′Mustard′ Munitions, NATO ASI Series 1. Disarmament Technology; Springer: Berlin, 1998; Vol. 19.

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extremely high concentrations of arsenic (average: 923 mg of As kg-1) were detected in the soil. In addition, triphenylarsine and methyldiphenylarsine were detected in plants.4 In the Baltic Sea, ∼40000 tons of chemical munitions (13000 tons of arsenic chemical weapons) have been dumped, where fishermen have occasionally caught the munitions.5 Recently, at Kizaki area in Kamisu-town, Japan, prominent cerebellar symptoms were observed in the residents, presumably due to the presence of diphenylarsinic acid (DPAA).6 The maximum level of DPAA in the contaminated drinking well water in the area was 15 mg of As kg-1.7 Phenylarsonic acid (PAA) was also detected in a much lower range. There were facilities of the former Japanese Army at Kizaki area, but the relation between DPAA and the military facilities is yet uncertain. Initially, DPAA was assumed to result from the decomposition of CLARK; however, in the extensive digging investigation, the dumping of not CLARK but DPAA itself was confirmed. According to records of those days, DPAA might have been used as a material for synthesizing CLARK. In a further study by Morita et al., other AAs, such as methylphenylarsinic acid (MPAA), dimethylphenylarsine oxide (DMPAO), and methyldiphenylarsine oxide (MDPAO), were detected in some underground water in this area.8 These methylated species will be produced by microorganisms.9,10 In addition, the Japanese Ministry of the Environment reported that MPAA was detected in brown rice cultivated in paddy fields irrigated with the contaminated underground water.11 Analyses of DPAA and PAA in soil and well water were recently reported.12–14 However, quantitative methods of AAs, especially MPAA, DMPAO, and MDPAO, in high matrix samples such as soil and plants must be established. (4) Pitten, F. A.; Mu ¨ ller, G.; Ko ¨nig, P.; Schmidt, D.; Thurow, K.; Kramer, A. Sci. Total Environ. 1999, 226, 237–245. (5) HELCOM CHEMU. Report on chemical munitions dumped in the Baltic Sea. Report to the 16th meeting of Helsinki Commission. Danish Environmental Protection Agency. January 1994. (6) Ishii, K.; Tamaoka, A.; Otsuka, F.; Iwasaki, N.; Shin, K.; Matsui, A.; Endo, G.; Kumagai, Y.; Ishii, T.; Shoji, S.; Ogata, T.; Ishizaki, M.; Doi, M.; Shimojo, N. Ann. Neurol. 2004, 56, 741–745. (7) Ishizaki, M.; Yanaoka, T.; Nakamura, M.; Hakuta, T.; Ueno, S.; Komura, M.; Shibata, M.; Kitamura, T.; Honda, A.; Doy, M.; Ishii, K.; Tamaoka, A.; Shimojo, N.; Ogata, T.; Nagasawa, E.; Hanaoka, S. J. Health Sci. 2005, 51, 130–137. (8) Ministry of the Environment, Government of Japan, http://www.env. go.jp/chemi/report/h17-07/index.html (in Japanese), June 2005. (9) Nakamiya, K.; Nakayama, T.; Ito, H.; Edmonds, J. S.; Shibata, Y.; Morita, M. FEMS Microbiol. Lett. 2007, 274, 184–188. (10) Kohler, M.; Hofmann, K.; Volsgen, F.; Thurow, K.; Koch, A. Chemosphere 2001, 42, 425–429. (11) 11. Ministry of the Environment, Government of Japan, http://www.env.go.jp/press/press.php?serial)5965 (in Japanese), May 2005. 10.1021/ac8002984 CCC: $40.75  2008 American Chemical Society Published on Web 06/25/2008

The aim of our study is to develop a determination method for AAs in rice (grain and straw) and soil. Evaluation of AAs in rice grain and straw is important, because rice, a major foodstuff in Japan, is widely cultivated in the Kizaki area and the straw is generally plowed back into the soil or fed to livestock. To simultaneously quantify AAs and inorganic As with wide hydrophobicity, reversed-phase HPLC/ICPMS was used because of its high sensitivity and selectivity. EXPERIMENTAL SECTION Reagents and Standards. MPAA, DMPAO, and MDPAO were kindly donated by Dr. Morita of the National Institute for Environmental Studies (Tsukuba, Japan). Water was purified using a Milli-Q system (Nihon Millipore, Tokyo). The following reagents were purchased: 68% nitric acid, 35% hydrogen peroxide, and 38% hydrogen fluoride of TAMAPURE-AA-100 series ultrapure analytical reagents (TAMA Chemicals, Tokyo, Japan); electronics industry grade CH3OH (Kanto Chemicals, Tokyo, Japan); guaranteed reagent grade diarsenic trioxide, disodium arsenate (Na2HAsO3 · 7H2O), phenylarsonic acid (PAA), sodium hydroxide, and column chromatography grade formic acid (Nacalai Tesque, Kyoto, Japan); methylarsonic acid (MAA) and dimethylarsinic acid (DMAA) (Tri Chemical Laboratories, Yamanashi); DPAA analytical standard (Wako Pure Chemical Industries, Osaka); bis(diphenylarsine) oxide (BDPAO) analytical standard (Hayashi Pure Chemical Industries, Osaka); spectrophotometric grade trifluoroacetic acid (TFA) and Trace SELECT Ultra phosphoric acid (Sigma Aldrich, Japan, Tokyo). An arsenic solution standard of 1000 mg of As L-1 for ICPMS was purchased from SPEX CertiPrep. The arsenic solution standards of 0, 2, 5, 10, 20, 50, 100, 200, and 1000 µg of As L-1 in 0.1% formic acid or 1% HNO3 were prepared by stepwise dilution. The individual arsenicals were initially dissolved in 50% CH3OH to prepare proper stock solutions of 100 mg of As L-1. These solutions were stepwise diluted with 0.1% HCOOH to 50, 200, and 10 000 µg of As L-1. The concentrations were determined by flow injection (FI)-ICPMS with the arsenic solution standards in 0.1% HCOOH. Mixed standard solutions containing nine arsenicals, arsenite (As(III)), arsenate (As(V)), MAA, DMAA, PAA, DPAA, MPAA, DMPAO, and MDPAO, were prepared from individual 100 mg of As L-1 arsenical solutions and 0.1% HCOOH. Mixed standard solutions of 0, 1, 2, 5, 10, 20, 50, 100, 200, and 10 000 µg of As L-1 for each species were prepared by stepwise dilution. The mixed standards were prepared as solutions containing 20 mM H3PO4. For the analyses of extracts with HNO3, eight arsenicals, arsenate (As(V)), MAA, DMAA, PAA, DPAA, MPAA, DMPAO, and MDPAO, were prepared from individual 100 mg of As L-1 arsenical solutions and 2% HNO3. Standard reference materials of NIST SRM 1568a (rice flower) and NIST SRM 2709 (San Joaquin soil) were used for method validation of total arsenic analysis. Sample Preparations. The soil contaminated with AAs was obtained from a paddy field at Kizaki area, Kamisu town, Japan. (12) Kinoshita, K.; Shida, Y.; Sakuma, C.; Ishizaki, M.; Kiso, K.; Shikino, O.; Ito, H.; Morita, M.; Ochi, T.; Kaise, T. Appl. Organomet. Chem. 2005, 19, 287–293. (13) Hanaoka, S.; Nagasawa, E.; Nomura, K.; Yamazawa, M.; Ishizaki, M. Appl. Organomet. Chem. 2005, 19, 265–275. (14) Haas, R.; Schmidt, T. C.; Steinbach, K.; von Lo ¨w, E. Fresenius J. Anal. Chem. 1998, 361, 313–318.

The noncontaminated soil was obtained from a paddy field in the same area, which was not irrigated with contaminated water. Indeed, AAs were not detected in the soil. The soil was air-dried and sieved to 95%). Inorganic As in straw could not be fully extracted even with TFA; HNO3 gave a good overall recovery

(>80%). In 68% HNO3 extraction, the spike recoveries for each As species (4 mg of As kg-1) were 86-108 and 81-106% (n ) 3) for grain and straw, respectively, and all spiked As(III) were oxidized to As(V). Although a small unknown peak was observed at 19 min in the chromatogram of the straw extract by 68% HNO3, this is not derived from known arsenic species, because recovery tests did not reveal the peak. Column recovery was determined by comparison of sum of As species present in chromatographed extracts versus that present in total As analysis of the same extracts. For the grain and straw extracts with 68% HNO3, the column recoveries were 99.5 ± 3.6 and 100.2 ± 2.9%, which show no notable specific adsorption on the column. The concentrations obtained by external calibration were 95-110 (grain) and 94-111% (straw) of those obtained by standard addition; matrix effects were not considered. Dilution of the extracted solution might have reduced matrix effects. The results by standard additional and external calibration methods matched well for arsenic speciation in rice in some reports.22,37,38 Extraction of Arsenic Compounds in Soil. For the extraction of As in soil, various extraction solvents have been used;36,37,39–41 however, a method that is valid for both hydrophilic inorganic As and hydrophobic organic As has not been established. Figure 7 shows HPLC/ICPMS chromatograms of soil extracts. The results of extractions with different extraction solvents are summarized in Table 1. H2O extracted only a small part of each arsenic species. The 68% HNO3 provided the best result of 95.5% overall recovery. Due to the strong acidic conditions, it was observed that As(V) is eluted faster than MAA or DMAA. Extractions with 68% HNO3 at 30, 70, and 100 °C were preliminary examined, resulting in the highest extraction efficiency at 100 °C for inorganic As (Figure 8). Organic arsenicals were more efficiently extracted at 70 and 100 °C than at 30 °C. Extraction with 1.0 mol L-1 NaOH in 50% CH3OH provided a better result with low extraction efficiency for inorganic As. No difference was observed in the extract efficiencies between 1.0 and 1.0 mol L-1 NaOH in 50% CH3OH. Added CH3OH was helpful for smooth filtration. Kahakachchi et al. reported that 0.1 mol L-1 NaOH or a citrate buffer provided the best result in the case of soil.39 Oxidation of As(III) to As(V) was more outstanding in 0.1 mol L-1 NaOH extraction than in H2O or H3PO4, which could be explained from the Eh-pH diagram, which shows that As(V) is dominant in alkaline solution.42 In 68% HNO3 extraction, all of As(III) were oxidized to As(V) and the spike recoveries for each organic As species (4 mg of As kg-1) were 88-110% (n ) 3). No chemical transformation of AAs was found under harsh hot 68% HNO3 extraction as well as rice. For example, the chromatogram of extract with 68% HNO3 from noncontaminated soil (0.5 g) spiked with MDPAO (100 µL, 10 mg of As L-1) was shown in Figure 6c. The concentrations obtained by external calibration were 93-109% of those obtained by standard addition, suggesting that matrix (37) Pizarro, I.; Go´mez, M.; Ca´mara, C.; Palacios, M. A. Anal. Chim. Acta 2003, 495, 85–98. (38) Kohlmeyer, U.; Jantzen, E.; Kuballa, J.; Jakubik, S. Anal. Bioanal. Chem. 2003, 377, 6–13. (39) Kahakachchi, C.; Uden, P. C.; Tyson, J. F. Analyst 2004, 129, 714–718. (40) Bissen, M.; Frimmel, F. H. Fresenius J. Anal. Chem. 2000, 367, 51–55. (41) Garcia-Manyes, S.; Jime´nez, G.; Padro´, A.; Rubio, R.; Rauret, G. Talanta 2002, 58, 97–109. (42) Ferguson, J. F.; Gavis, J. Water Res. 1972, 6, 1259–1274.

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Table 1. Extraction of Arsenic Species with Various Media (n ) 3) extraction media (mg of As kg-1) grain

H2O

50% CH3OH

TFA

HNO3

inorganic As DPAA MPAA total overall recovery (%)a straw inorganic As MAA+DMAA MPAA DMPAO MDPAO total overall recovery (%)a

0.167 ± 0.005 0.048 ± 0.006 0.262 ± 0.013 0.475 ± 0.011 63.2 ± 1.5

0.164 ± 0.019 0.072 ± 0.005 0.323 ± 0.008 0.555 ± 0.021 73.8 ± 2.8

0.226 ± 0.034 0.076 ± 0.007 0.426 ± 0.024 0.732 ± 0.009 97.3 ± 1.2

0.255 ± 0.039 0.071 ± 0.005 0.408 ± 0.010 0.729 ± 0.027 96.9 ± 3.6

1.094 ± 0.100 0.064 ± 0.004 0.107 ± 0.007 0.834 ± 0.064 0.072 ± 0.005 2.189 ± 0.138 43.6 ± 2.8

1.068 ± 0.012 0.067 ± 0.004 0.125 ± 0.007 1.162 ± 0.089 0.115 ± 0.011 2.537 ± 0.100 50.6 ± 2.0

1.062 ± 0.105 0.091 ± 0.003 0.209 ± 0.003 1.112 ± 0.022 0.125 ± 0.005 2.599 ± 0.094 51.8 ± 1.9

1.661 ± 0.038 0.272 ± 0.028 0.216 ± 0.010 1.742 ± 0.032 0.124 ± 0.004 4.020 ± 0.096 80.1 ± 1.9

digest (mg of As kg)-1

0.752 ± 0.032

5.018 ± 0.134

extraction media (mg of As kg-1) soil inorganic As MAA+DMAA PAA DPAA MPAA DMPAO MDPAO total overall recovery (%)a a

H2O

H3PO4

NaOH

HNO3

ndb ndb 0.051 ± 0.005 0.107 ± 0.007 0.152 ± 0.007 0.071 ± 0.002 0.117 ± 0.007 0.498 ± 0.004 5.1 ± 0.0

0.863 ± 0.069 0.173 ± 0.018 1.130 ± 0.027 0.217 ± 0.008 0.626 ± 0.032 0.142 ± 0.014 0.232 ± 0.003 3.383 ± 0.137 34.5 ± 1.4

1.041 ± 0.048 0.208 ± 0.012 1.247 ± 0.066 0.267 ± 0.025 0.750 ± 0.031 0.188 ± 0.007 0.373 ± 0.027 4.074 ± 0.193 41.6 ± 2.0

5.856 ± 0.191 0.524 ± 0.015 1.355 ± 0.070 0.338 ± 0.022 0.762 ± 0.058 0.347 ± 0.019 0.449 ± 0.025 9.632 ± 0.364 98.3 ± 3.7

digest (mg of As kg-1)

9.803 ± 0.396

100 × [chromatographic total As]/[digested total As]. b Below instrumental detection limit.

Figure 6. Chromatograms of extracts with 68% HNO3 from noncontaminated grain (a), straw (b), and soil (c) spiked with MDPAO.

effects were negligible. For the soil extract with 68% HNO3, the column recovery was 98.9 ± 4.0%, which suggests no notable specific adsorption on the column. Plant Uptake of AAs. Total As concentrations of rice straw and soil in a pot experiment were over 7-fold larger than that of rice grain. This result agrees with the reports that arsenic in rice grain is much lower than that in rice straw.16,21 The concentrations of each As species in rice grain, straw, and soil are shown in Figure 9. MPAA was the only detected AAs in grain and 87% of total extracted As. MPAA in grain was 2-fold larger than that in straw. MPAA absorbed from soil was transported to grains without specific adsorption on shoots and roots. PAA and DPAA were detected only in soil. DMPAO and MPDAO were detected not in 5774

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Figure 7. Arsenic speciation analysis of the soil solutions extracted with (a) 68% HNO3, (b) 1 mol L-1 NaOH in 50% CH3OH, (c) 1 mol L-1 H3PO4 in 50% CH3OH,and (d) H2O (5-µL injection).

grain but in straw at 22- and 2-fold higher concentrations, respectively, than those in soil. Briggs et al. reported that the uptake by roots was larger for more hydrophobic chemicals and the translocation is maximized at log Kow ) 1.8 according to a Gaussian curve.43 The high uptake of DMPAO and MDPAO was due to the high log Kow values of 3.54 and 4.75, respectively. However, DMPAO and MDPAO were not translocated into grains because of adsorption on lipid due to high log Kow. On the other hand, MPAA in straw was translocated into grains because of the (43) Briggs, G. G.; Bromilow, R. H.; Evans, A. A.; Williams, M. Pestic. Sci. 1983, 14, 492–500.

Figure 8. Extraction of arsenicals in soil with 68% HNO3 at 30, 70, and 100 °C (n ) 3) (MAA (or DMAA) is under the quantification limit).

intermediate log Kow values (1.58). PAA (log Kow 0.03) has two dissociative protons (pKa 3.47 and 8.48), which must be in monoanion form (log Kow -2.92) in soil. Thus, it would be difficult for PAA to pass through cell membranes, and it would not be absorbed by roots. For ionizable organic acids, the cell permeabilities of the negatively charged dissociated forms are much lower than those of the undissociated forms.44–48 The calculated log Kow values for other arsenicals are 2.80 (DPAA), 0.37 (DMAA), -1.18 (MAA), -4.14 (monoanion form of MAA), -3.14 (As(III)(OH)3), and -5.69 (monoanion form of H3AsVO4). DPAA was not found in pot experiments, but the existence in field samples was reported.11 Plant uptake of inorganic As cannot be explained by only log Kow factor; some mechanisms are proposed.49,50 A detailed study to clarify the uptake and transport of AAs is in progress. CONCLUSIONS An analytical method to simultaneously determine inorganic and organic As containing AAs related to chemical warfare agents in rice grain, straw, and soil was established. The hydrophilic and hydrophobic arsenicals were separated with a reversed-phase column using a gradient program (1-70% (44) Briggs, G. G.; Rigitano, R. L. O.; Bromilow, R. H. Pestic. Sci. 1987, 19, 101–112. (45) Raven, J. A. New Phytol. 1975, 74, 163–172. (46) Bromilow, R. H.; Chamberlain, K.; Tench, A. J.; Williams, R. H. Pestic. Sci. 1993, 37, 39–47. (47) Tench, A. J.; Williams, R. H.; Bromilow, R. H.; Chamberlain, K. Pestic. Sci. 1993, 37, 31–37. (48) Trapp, S. Environ. Sci. Pollut. Res. 2004, 11, 33–39. (49) Meharg, A. A.; Jardine, L. New Phytol. 2003, 157, 39–44. (50) Zhu, Y. G.; Geng, C. N.; Tong, Y. P.; Smith, S. E.; Smith, F. A. Ann. Bot. 2006, 98, 631–636.

Figure 9. Uptake of arsenicals by rice cultivated with a contaminated soil. Arsenic concentrations in rice grain, straw, and soil were expressed by the white, gray, and black bars, respectively (n ) 3).

CH3OH). The mobile phase with high organic solvent was introduced into ICPMS through a membrane desolvating system. The extraction of inorganic and organic As was the most efficiently carried out with 68% HNO3. In the process of the harsh extraction, As(III) was fully converted to As(V), but no obvious decomposition of AAs was found. The selectivity of AAs in plant uptake showed that MPAA is much more important for human intake than other AAs, which suggests that the relevant authorities must execute countermeasures including the estimation of toxicity. Using this method, we are now studying the uptake by plant and the degradation of AAs in soil. ACKNOWLEDGMENT This research was supported by the Grant-in-Aid from the Ministry of the Environment, Japan. SUPPORTING INFORMATION AVAILABLE Additional experimental information containing two figures. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review February 12, 2008. Accepted May 21, 2008. AC8002984

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