Arsenic Accumulation and Metabolism in Rice (Oryza sativa L

Jan 30, 2002 - Department of Plant and Soil Science and Department of Chemistry, University of Aberdeen, Aberdeen, AB24 3UU United Kingdom, Department...
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Environ. Sci. Technol. 2002, 36, 962-968

Arsenic Accumulation and Metabolism in Rice (Oryza sativa L.) M D . J O I N A L A B E D I N , * ,† MALCOLM S. CRESSER,‡ ANDY A. MEHARG,† JO ¨ RG FELDMANN,§ AND JANET COTTER-HOWELLS| Department of Plant and Soil Science and Department of Chemistry, University of Aberdeen, Aberdeen, AB24 3UU United Kingdom, Department of Environmental Science, University of York, York, United Kingdom, and Department of Biological Sciences, University of Exeter, Exeter, United Kingdom

The5 use of arsenic (As) contaminated groundwater for irrigation of crops has resulted in elevated concentrations of arsenic in agricultural soils in Bangladesh, West Bengal (India), and elsewhere. Paddy rice (Oryza sativa L.) is the main agricultural crop grown in the arsenic-affected areas of Bangladesh. There is, therefore, concern regarding accumulation of arsenic in rice grown those soils. A greenhouse study was conducted to examine the effects of arsenic-contaminated irrigation water on the growth of rice and uptake and speciation of arsenic. Treatments of the greenhouse experiment consisted of two phosphate doses and seven different arsenate concentrations ranging from 0 to 8 mg of As L-1 applied regularly throughout the 170)day post-transplantation growing period until plants were ready for harvesting. Increasing the concentration of arsenate in irrigation water significantly decreased plant height, grain yield, the number of filled grains, grain weight, and root biomass, while the arsenic concentrations in root, straw, and rice husk increased significantly. Concentrations of arsenic in rice grain did not exceed the food hygiene concentration limit (1.0 mg of As kg-1 dry weight). The concentrations of arsenic in rice straw (up to 91.8 mg kg-1 for the highest As treatment) were of the same order of magnitude as root arsenic concentrations (up to 107.5 mg kg-1), suggesting that arsenic can be readily translocated to the shoot. While not covered by food hygiene regulations, rice straw is used as cattle feed in many countries including Bangladesh. The high arsenic concentrations may have the potential for adverse health effects on the cattle and an increase of arsenic exposure in humans via the plant-animal-human pathway. Arsenic concentrations in rice plant parts except husk were not affected by application of phosphate. As the concentration of arsenic in the rice grain was low, arsenic speciation was performed only on rice straw to predict the risk associated with feeding contaminated * Corresponding author phone: 0044-1224-272264; fax: 00441224-272703; e-mail: [email protected]. † Department of Plant and Soil Sciences, University of Aberdeen. ‡ University of York. § Department of Chemistry, University of Aberdeen. | University of Exeter. 962

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straw to the cattle. Speciation of arsenic in tissues (using HPLC-ICP-MS) revealed that the predominant species present in straw was arsenate followed by arsenite and dimethylarsinic acid (DMAA). As DMAA is only present at low concentrations, it is unlikely this will greatly alter the toxicity of arsenic present in rice.

Introduction Naturally occurring arsenic-contaminated groundwater from water wells drilled into Ganges alluvial deposits in Bangladesh and West Bengal (India), the main source of drinking water locally, has received significant attention in recent years (1, 2) with reports of widespread arsenic-related health effects on millions of people (3-7). In the arsenic-affected areas of Bangladesh, groundwater contains up to 2 mg of As L-1 (8) as compared to the WHO recommended provisional limit of 0.01 mg of As L-1 (9). The focus of studies assessing the importance of arsenic in Bangladesh has been direct ingestion of tube-well (shallow water well) derived groundwater. However, as the groundwater is also used extensively for crop irrigation in these areas, there is considerable potential for elevated arsenic concentration in agronomic products. Generally, the normal irrigated soil of Bangladesh contains 4-8 mg of As kg-1 soil, while in areas where irrigation is performed with the arsenic-contaminated water, soil arsenic concentration can be up to 83 mg kg-1 (10). There is also report of elevated arsenic concentrations up to 57 mg of As kg-1 in soils collected from different locations of four districts of Bangladesh (11). High arsenic concentrations in soil and the use of irrigation water with high arsenic may lead to elevated concentrations of arsenic in cereals, vegetables, and other agricultural products of arsenic-affected areas. Arsenic in groundwater is mainly inorganic with arsenate comprising about 50% of the total (12). Phosphate and arsenate are analogues, have similar physicochemical behavior in soils, and compete directly for the same sorption sites on soil particle surfaces (13). Addition of phosphate to soil may enhance downward movement of arsenic, leading to increased leaching from the topsoil (14, 15), or increase availability of arsenic in the soil solution resulting in higher uptake by the plants (16). Arsenate also acts as a phosphate analogue with respect to transport across root plasma membrane, with phosphate competing much more effectively for transport sites (17). In Bangladesh, 75% of the total cropped area and 83% of the total irrigated area are used for rice cultivation (18); much groundwater is used to irrigate this crop, especially in the dry season. Rice is the staple food, and rice straw (leaves and stalk) is used as cattle feed. Therefore, it is necessary to evaluate the impact of using arsenate-containing irrigation water and application of phosphate on the arsenic uptake and accumulation in rice grain and straw. The toxic effect of arsenic in any foodstuff is highly dependent on its chemical speciation. Inorganic arsenic compounds are generally thought to be more toxic than organic forms (19). The toxicity of arsenic species follows the order AsH3 > As(III) > As(V) > MMAA (monomethylarsonic acid) > DMAA (dimethylarsinic acid). There are reports of transformation of arsenic species in the plant system (2024). However, information on the transformation of arsenic species within the rice plant is limited. A greenhouse study was conducted to evaluate the effects of different concentrations of arsenic in irrigation water on the growth of rice, including the partitioning of arsenic between different plant parts (grain, husk, root, and straw). 10.1021/es0101678 CCC: $22.00

 2002 American Chemical Society Published on Web 01/30/2002

TABLE 1. Analytical Results of Initial Soil for Different Properties soil properties

values

soil pH (in water) soil pH (in CaCl2) total N (%) organic carbon (%) CEC (mequiv/100 g) exchangeable Ca (mequiv/100 g) exchangeable Mg (mequiv/100 g) exchangeable Na (mequiv/100 g) exchangeable K (mequiv/100 g) available P (µg g-1) available Fe (µg g-1) available Mn (µg g-1) available Cu (µg g-1) available Zn (µg g-1) total As (µg g-1)

7.80 7.50 0.03 1.13 12.70 23.30 7.90 0.26 0.78 4.00 13.20 6.40 1.67 1.00 31.30

Arsenic species in rice straw were determined using liquid chromatography ICP-MS.

Materials and Methods Greenhouse Experimental Methodology. The Aman paddy rice (Oryza sativa L.), variety BR11, was selected for study as it is widely grown in Bangladesh. After germination on compost, three 30-day-old seedlings were transplanted to 14 cm deep 1-L plastic pots (with no perforations) uniformly packed with 1.1 kg of dry clay-rich soil (Cruden Bay, NE Scotland, GR NK 087 371). The principal physicochemical properties of this soil are presented in Table 1. The plant pots were placed in a greenhouse with overall temperature fluctuation between 22 and 35 °C. Daylight was supplemented with sodium lamps, which were on for 8 h during the day. After transplantation, the plants were grown under flooded conditions (i.e., saturation to permanent immersion of the soil under up to 3-4 cm of solution). There were altogether 14 treatments that were the combination of seven arsenate and two phosphate doses. Arsenate was supplied as a solution of Na2HAsO4‚7H2O in distilled water in concentrations of 0 (control treatment), 0.2, 0.5, 1.0, 2.0, 4.0, and 8.0 mg of As L-1 until the rice grain was ripe (170 days). A measured amount of arsenate solution was added each week. The amount added was between 400 and 600 mL pot-1 week-1 (total 12.6 L for each treatment) and was sufficient to maintain flooded conditions. Two phosphate doses used for this study were to simulate field application rates in Bangladesh. Phosphate was added as a solution of Ca(H2PO4)2‚H2O in distilled water and was applied once before transplantation of the rice seedlings to give 14.3 mg of P kg -1 (equivalent to 30 kg of P ha-1) and 28.6 mg of P kg -1 (equivalent to 60 kg of P ha-1). The design was completely randomized, and each treatment was replicated three times. The range of arsenate concentrations was chosen to encompass the concentrations occurring in underground waters of the arsenic-affected areas of Bangladesh. Reported concentrations of arsenic in well waters are up to 2 mg of As L-1 (8, 25), well below the highest arsenate treatment level (8 mg of As L-1) of this study. However, arsenic may build up as a consequence of continued use of arsenatecontaminated water for a prolonged period of time. Nitrogen and K were also applied because these fertilizers would normally be added to rice-growing soils in Bangladesh. Nitrogen was supplied as a solution of CO(NH2)2 (in distilled water) to give a total of 76.3 mg N kg-1 (equivalent to 160 kg ha-1). Nitrogen fertilizer was applied in four equal splits of 21 g of N pot-1, with the first application during transplantation of the rice seedlings, the second 30 days after transplanting, the third application 60 days after transplanting,

and the last 90 days after transplanting. KCl solution (in distilled water) was applied once during transplantation of the seedlings to give 28.6 mg of K kg-1 (equivalent to 60 kg ha-1). In the field, fertilizers are usually added in solid form to prevent excessive runoff. However, in this experiment, all pots were sealed units, and solutions were required for nutrients to be readily available. Harvesting Rice Plants, Recording Data for Different Agronomic Parameters, and Sample Preparation for Chemical Analysis. Arsenic applications ceased 10 days before harvest because irrigation would normally stop at this point in the field. After measuring plant height (top of the panicle to level of soil in the pot), rice plants were harvested by cutting at 4 cm above the soil (to avoid basal tissue contaminated by applied arsenate solution). Rice spikelets (which should each contain one grain of rice) were separated from the panicles by hand. Empty spikelets were separated from the filled spikelets, and the number of filled spikelets was recorded for each pot. The mass of filled spikelets (i.e., grain including husk) per pot (grain yield) was recorded after freezedrying the spikelets. Thousand-spikelet weight (i.e., the mass of 1000 rice grains including husks) was calculated from the weight and number of spikelets for each pot. Rice grains were separated from their husks using a pestle and mortar, and then both were ground using a Retsch grinder (type: MM2, made in Germany). The straw biomass (defined as the remaining aboveground portion of the rice plant after the spiklets have been removed) was recorded per pot after drying at 50 °C for 48 h. After being harvested, the soil in the plant pots was left to dry in the greenhouse and then further dried in an oven at 50 °C. Roots were separated from soil during disaggregation and sieving to 0.05) effect on the height of plant (Figure 1a), the number of spikelets pot-1 (Figure 1b), or the thousand-spikelet weight (Figure 1c). The grain yield of rice (mass of filled spikelets pot-1) was affected by the application of arsenate in irrigation water. Grain yield was found to vary significantly (p < 0.001) from 6.72 to 1.83 g pot-1 from the control to the highest arsenate concentration (Figure 1d), but the highest yield was comparable with that found in the 0.2 and 0.5 mg of As L-1 treatments. Application of arsenate did not significantly (p > 0.05) reduce the yield of rice straw, despite the general trend of a decrease in straw yield (Figure 1e). The treatment receiving no arsenate yielded the most straw (10.11 g pot-1), while the lowest straw production (8.81 g pot-1) was observed in the highest arsenate treatment (8 mg of As L-1). Root biomass varied significantly (p < 0.001) with concentration of arsenate in irrigation water. The lowest root biomass of 2.22 g pot-1 was found in the highest arsenate treatment (8 mg of As L-1) followed by the 4 mg of As L-1 treatment (2.24 g pot-1) and the highest biomass (5.05 g pot-1) in the treatment having no arsenate (Figure 1f). No significant differences (p > 0.05) were observed in grain yield (Figure 1d), straw biomass (Figure 1e) or root biomass (Figure 1f) as a result of P application. Analytical Quality Control Data. Quality control data for analysis of arsenic in soil and plant samples were quite good. The mean recovery for reference soil and plant materials was 87.8% (n ) 8) and 86.1% (n ) 2), respectively. The spikes recovery was 95.8% (n ) 5). The percent mean standard deviation for the duplicate samples was 8.2% (n ) 42) in roots, 6.7% (n ) 42) in straw, 4.8% (n ) 2) in husk, and 10.1% (n ) 3) in grain samples. Arsenic Concentration in Soils. Because of application of arsenate-containing irrigation water to soil, the arsenic concentration in soil increased significantly (p < 0.001) at harvest. The mean soil arsenic concentrations of the 0.2, 0.5, 1.0, 2.0, 4.0, and 8.0 mg of As L-1 treatments were 30.2, 34.5, 35.9, 45, 60, and 102 mg of As kg-1, respectively. Arsenic Concentration in Rice Plant Parts. Arsenic concentration in rice root, straw, and husk increased significantly (p < 0.001) with increasing arsenate concentration in irrigation water (Figure 2a-c). However, arsenic concentrations in rice grain remained statistically similar with increasing arsenate dose (Figure 2d). Regardless of arsenate dose, rice tissue arsenic concentration followed the trend: root > straw > husk > grain. No difference in arsenic concentrations in rice plant parts except in husk was observed due to phosphate application. In the lower arsenate treatments (0-0.5 mg of As L-1), the concentration of arsenic in roots ranged between 18.6 and 21.9 mg kg-1, but in the 1.0 mg of As L-1 treatment, it increased to 35.1 mg of As kg-1, which further increased with increasing

FIGURE 1. Effect of arsenate-contaminated irrigation water on rice biomass parameters (error bars represent ( SE) under two phosphate doses [(hatched) 14.3 and (solid) 28.6 mg kg-1]. arsenate dose (Figure 2a). The highest arsenic concentration (107.5 mg of As kg-1) in roots was observed in the highest arsenate treatment. Arsenic concentration in rice straw in the lowest arsenate treatment was only 3.9 mg kg-1, which increased progressively with increasing arsenate application and reached to 91.8 mg kg-1 in the highest arsenate treatment (8.0 mg of As L-1) (Figure 2b). The concentration of arsenic in husks increased significantly with increasing arsenate application (Figure 2c). The lowest arsenic concentration of 0.7 mg kg-1 was observed in the treatment where no arsenate was added (control), whereas the highest concentration of 6.1 mg of As kg-1 was found in the highest arsenate treatment. Arsenic concentration in rice husk was significantly (p < 0.05) increased in the higher phosphate dose. The mean arsenic concentration in rice husk at lower phosphate dose was 2.01 mg kg-1, which increased to 2.82 mg of As kg-1 in the higher phosphate application. The lowest arsenic concentration (0.15 mg kg-1) in rice grain was observed in the control treatment and slightly increased with increasing arsenate doses to approximately 0.24 mg kg-1 in the 4.0 mg of As L-1 treatment. The mean arsenic concentration reached 0.42 mg kg-1 in the highest treatment (Figure 2d). Arsenic Speciation in Rice Straw. Arsenic species extracted by both methanol/water and TFA (Table 2) are arsenate, arsenite, and DMAA. In the samples extracted by methanol/water, the predominant species was found to be arsenate (about 90% or more of the total), followed by arsenite (0-8%) and DMAA (0-4%). The extraction efficiency of methanol/water for rice straw was however very low (approximately 10-20%). Because of the lower extraction

efficiency of the methanol/water mixture, TFA was used as it extracts >80% of the arsenic. In TFA extracts, the proportions of arsenate, arsenite, and DMAA were 72-84%, 1526%, and 1-4%, respectively. Conversion Rate of Arsenic Species during TFA Extraction Method. Although the TFA digestion method has a higher extraction efficiency than methanol/water, there is a limitation of this technique as it reduces about 20% of the arsenate to arsenite, whereas arsenite, DMAA, and MMAA are almost stable (Table 3). However, there has been some conversion of arsenite and DMAA. About 4.8% of arsenite converted to arsenate, and an almost similar amount (approximately 2-4%) of arsenite and arsenate was observed from the conversion of DMAA.

Discussion Significant reduction in grain yield (defined as mass of filled spikelets per pot) in the highest arsenate treatment is caused by both lower numbers of filled spikelets per pot and lower thousand-spikelet weight (Figure. 1b,c). There are similar reports of yield reduction in rice (30, 31) and other crops (32) due to arsenic treatment. In this experiment, rice plants subjected to higher arsenate treatment produced lower shoot biomass and reduced plant height. Despite stunted plant height, rice straw biomass did not decrease significantly with increasing arsenate dose. Although there are number of reports of reduced shoot biomass/growth in rice (30, 33), apricot (34), and tomato plants (35) due to application of arsenate, the reason for VOL. 36, NO. 5, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Effect of arsenate-contaminated irrigation water on the concentration (error bar represents ( SE) of arsenic in rice plant parts under two phosphate doses [(hatched) 14.3 and (solid) 28.6 mg kg-1].

TABLE 2. Arsenic Speciation in Rice Strawa extractant methanol

TFA

a

As dose (mg L-1)

P dose (mg kg-1)

total As in straw (mg kg-1)

extraction efficiency (%)

arsenite

4 4 4 8 8 8 8 8 8 4 4 4 8 8 8 8 8 8

14.3 14.3 14.3 14.3 14.3 14.3 28.6 28.6 28.6 14.3 14.3 28.6 14.3 14.3 14.3 28.6 28.6 28.6

41.4 105.8 30.3 99.8 86.1 102.3 105.9 79.5 77.4 41.4 30.3 36.3 99.8 86.1 102.3 105.9 79.5 77.4

9.2 16.0 8.7 10.9 10.1 7.2 18.4 9.7 10.5 111.0 98.0 106.0 117.0 118.0 107.0 91.0 124.0 81.0

0.0 0.0 0.0 5.7 0.0 0.0 4.8 0.0 8.3 14.8 22.2 26.0 23.0 17.7 17.2 23.4 14.8 22.9

As species as % of total DMAA MMAA 0.0 0.0 0.0 2.9 0.0 0.0 3.5 0.0 4.3 0.8 2.9 2.3 4.3 0.9 0.8 4.9 0.8 4.4

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

arsenate 100.0 100.0 100.0 91.4 100.0 100.0 91.7 100.0 87.4 84.4 74.9 71.6 72.8 81.4 82.0 71.7 84.4 72.7

Detection limit for the arsenic species in methanol and TFA extraction methods were 0.125 and 0.10 mg kg-1 in the samples

TABLE 3. Conversion of As Species during Digestion of Rice Straw by TFA % species found after digestion As species

% recovery

As(III) As(V) DMAA MMAA

95.16 78.35 91.70 99.25

As(III)

As(V)

DMAA

MMAA

95.16 ( 0.05 21.65 ( 2.06 4.09 ( 0.79 0.75 ( 1.06

4.84 ( 0.05 78.35 ( 2.06 4.21 ( 2.40 0

0 0 91.70 ( 1.61 0

0 0 0 99.25 ( 1.06

comparable shoot biomass in higher arsenate treatments as compared to the control treatment in this experiment was obscure. However, Onken and Hossner (36) reported a contrasting result of increased dry matter production of rice plants grown in the Beaumont clay treated with 5 mg of As 966

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kg-1 as sodium arsenite or sodium arsenate over the plants that received no arsenic (control). Tsutsumi (37) in his pot experiment, with different concentrations of arsenate in soil, observed no reduction of rice plant height up to 125 mg of As kg-1 but did observe 63% reduction of height at 312.5 mg

of As kg-1. Marin et al. (38) in their hydroponic experiment found shorter rice plants when grown with 0.8 mg L-1 arsenite and MMAA. However, they found no significant reduction in plant height when plants were exposed to arsenate. The effect of arsenate-containing irrigation water on rice root development was so prominent that the treatment receiving only 0.2 mg of As L-1 produced significantly lower root biomass as compared to the control treatment. The reduction in root biomass was more pronounced in other high arsenate treatments. In our experiment, application of arsenate at a lower dose reduced root biomass significantly, but rice straw (shoot plus leaf biomass), rice yield, and yield contributing characters were not affected considerably at the same level of application. This result might not be true for other rice cultivars. Marin et al. (38) reported a differential response in root dry matter production between the two rice cultivars Lemont and Mercury when treated with different concentrations of arsenite and arsenate in hydroponic culture. At 0.2 and 0.8 mg of arsenate L-1 treatment, root dry matter weight in Mercury was significantly increased, while it was not the case in Lemont. They found a significant reduction in Lemont root dry weight at 0.8 mg of arsenite L-1, while no root weight reduction in Mercury at the same level of exposure. Significantly reduced root growth with increasing soil As was found by Tang and Miller (26) for rice. Sneller et al. (39), however, did not observe inhibited root growth with up to 0.58 mg of As L-1 in high-phosphate (3.1 mg L-1) treatment (on Silene vulgaris) but did observe 75% inhibited root growth in low-phosphate (0.31 mg L-1) treatment with 0.19 mg of As L-1. The data for root arsenic concentrations clearly show that, irrespective of arsenate dose (including the control), roots contain higher concentrations of arsenic than any other part of the rice plant. This effect is quite marked for all arsenate treatments except for the highest arsenate treatment where concentrations of arsenic in both root and straw are similar (Figure 2a,b). This suggests that the rice cultivar used in this study has an ability to store a certain amount of arsenic in its root system. However, at the highest arsenate treatment, this storage ability may have been exceeded, and arsenic is readily translocated to the shoot, resulting in similar arsenic concentrations for both straw and root. Marin et al. (33, 38) and Xie and Huang (40) in rice, Sachs and Michael (41) in beans, and Carbonell et al. (42) in Spartina alterniflora (a wetland grass) also observed higher arsenic accumulation in roots than any other parts. It is also possible that a similar kind of protection mechanism is employed by rice straw and husks: arsenic concentrations in grain (up to approximately 0.4 mg kg-1) are an order of magnitude lower than those found in husks (up to approximately 6.1 mg kg-1), which in turn are an order of magnitude below those found in rice straw (up to approximately 90 mg kg-1). The uptake of arsenic by rice plant found in this study might be different in the field as exact field environment cannot be maintained in the controlled greenhouse condition. The arsenic accumulation in the straw of up to more than 100 mg kg-1 in the highest arsenate treatment shows that rice straw has a potential to accumulate a very high level of arsenic. Accumulation of arsenic at such a high level corroborates to the result reported by Tsutsumi (37), who found 149 mg of As kg-1 in the rice straw when soil arsenic concentration was 312.5 mg kg-1. Straw arsenic concentration of 25 mg kg-1 in the 2 mg of As L-1 treatment (i.e., equivalent dose to reported highest contamination of Bangladesh groundwater) is 25-fold higher than the legal limit for foodstuffs (43). Straw given in the U.K. to cattle contained less than 0.20 mg of As kg-1 (44). How the arsenic is metabolized by the cattle is dependent however on the arsenic species in the straw and on the metabolism of the cattle. Only limited information is available on the arsenic me-

tabolism of the cattle. In another experiment, sheep that were fed organoarsenicals, monosodium methylarsonate (which has been used as a pesticide), showed a significant increase of the arsenic concentration in tissue and milk (45), whereas sheep exposed to arsenosugars from seaweed (arsenic concentration of up to 100 mg kg-1) did not show highly elevated level of arsenic in the meat; however, there was accumulation in the fat (27, 46). Although mammals can methylate inorganic arsenic to MMAA and DMAA, which can be subsequently excreted through urine or feces (47, 48), the above-mentioned studies (27, 45, 46) suggest not to ignore the probable risk associated with feeding contaminated food to cattle. To do a risk assessment for the potential of arsenic accumulation in the food chain, arsenic speciation in the foodstuff (e.g., straw) is necessary. The major arsenic species in irrigation water used in Bangladesh or India is likely to be in the form of inorganic (mixture of arsenate and arsenite) (12) as with our experiment. Although we applied arsenic as arsenate, soil solution will contain a significant proportion of arsenite due to submerged soil redox condition (49, 50). In our recent soil solution speciation study (unpublished data), we have also found percentage levels of DMAA in the paddy soils. Whether the presence of arsenite and DMAA in the rice straw is due to either direct uptake of these species from soil solution or conversion of arsenate in the rice plant system cannot be assessed in this present study. In this study, only a small amount of DMAA was found; the concentration and proportion of DMAA did not show any correlation to the applied arsenate concentration. In the speciation study, we used the straw from the two highest arsenate treatment, so the relative proportion of DMAA could have been different if straw samples from lower arsenate treatments were included. Other terrestrial plants have been shown to contain small quantities of DMAA when grown on contaminated sites (51) or nutrientdeficient conditions (21). Although the toxicity of DMAA is lower than the inorganic species, the proportion of DMAA is too small to have a significant influence on the toxicity of the straw. Our results on rice straw speciation suggest that TFA can be used successfully in extracting the arsenic species from the plant tissue. The speciation data on rice straw also revealed that the TFA extraction method is more acceptable than the methanol extraction for rice straw. However, the TFA extraction method has a drawback of reducing partial arsenate to arsenite. The reason for reduction of arsenate to arsenite by TFA is not very clear. TFA extraction while hydrolyzing carbohydrates might have released other organic compounds responsible for reducing arsenate to arsenite, or simply the low pH of the TFA solution can convert arsenate to arsenite due to a shift in redox potential of the arsenic species involved. This pH effect has been shown in spiked urine samples (52). Despite the limitation of reduction of some arsenate to arsenite during the extraction process, the measurement of total inorganic species may still be significant to do risk assessment on biological samples. The grain arsenic concentrations ranging from 0.15 to 0.42 mg kg-1 are comparable with the arsenic concentration (0.303 mg kg-1) in rice grain from Schoof et al.’s (53) market basket survey. In their speciation study, they measured a significant proportion of DMAA and MMAA in the rice grain. However, the total concentration of arsenic and the proportion of arsenic species in their study varied greatly depending on the source of grain. The grain arsenic concentration in this experiment did not exceed the maximum permissible limit of 1.0 mg of As kg-1 (43). As we have not done any speciation on rice grain, it is unwise to predict the relative proportion of organic and inorganic species from our study. Whatever the relative proportion of arsenic species, the rice grain may be considered as harmless for the people of riceVOL. 36, NO. 5, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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growing countries such as Bangladesh where rice is their staple food and most of the people take rice at least twice a day and even more. Although rice grain has a permissible concentration of arsenic, cooking rice with the arseniccontaminated water could pose the same danger as from the direct ingestion of contaminated water. Despite this, the people of Bangladesh are still at risk of being affected by arsenic through their food. The majority (about 87%) of the population of this country are Muslim, and they used to eat beef as a principal source of meat. Irrespective of religious identity, people are drinking cow’s milk as a source of their protein requirement. Although at this stage there are no data on the arsenic concentration of meat and milk of the cows of Bangladeshi cattle and those that imported from West Bengal, India (predominantly a non-beef-eating Hindu population), high tissue-arsenic concentration through high arsenic-containing straw feeding may be a risk. Growing rice with arsenic-contaminated water could pose a potential health hazard to the cattle population as rice straw is being used as cattle feed. The Bangladeshi government, WHO (World Health Organization), and other international organizations have not yet considered the possible health effects of arsenic-contaminated irrigation water on the livestock population. These organizations are dominantly concerned with the adverse health effects on humans caused by drinking arsenic-contaminated water. However, this study has shown that it is also necessary to consider animal health issues and the possibility of arsenic contamination of bovine meat and milk through feeding cattle with rice straw containing high levels of arsenic.

Acknowledgments This research is funded by Agricultural Research Management Project (ARMP), Bangladesh Rice Research Institute (BRRI) component (IDA Credit 2815-BD).

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Received for review June 13, 2001. Revised manuscript received October 24, 2001. Accepted November 26, 2001. ES0101678