Arsenic speciation in Chinese brake fern by ion-pair high-performance liquid chromatography inductively coupled plasma mass spectroscopy
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1 Analytica Chimica Acta 504 (2004) Arsenic speciation in Chinese brake fern by ion-pair high-performance liquid chromatography inductively coupled plasma mass spectroscopy Ruixue Chen a, Benjamin W. Smith a, James D. Winefordner a,, MikeS.Tu b, Gina Kertulis b, Lena Q. Ma b a Department of Chemistry, University of Florida, P.O. Box , Gainesville, FL , USA b Soil and Water Science Department, University of Florida, Gainesville, FL , USA Received 10 June 2003; received in revised form 30 September 2003; accepted 14 October 2003 Abstract Ion-pair reverse-phase HPLC inductively coupled plasma (ICP) MS was employed to determine arsenite [As(III)], dimethyl arsenic acid (DMA), monomethyl arsenic (MMA) and arsenate [As(V)] in Chinese brake fern (Pteris vittata L.). The separation was performed on a reverse-phase C18 column (Haisil 100) by using a mobile phase containing 10 mm hexadecyltrimethyl ammonium bromide (CTAB) as ion-pairing reagent, 20 mm ammonium phosphate buffer and 2% methanol at ph 6.0. The detection limits of arsenic species with HPLC ICP-MS were 0.5, 0.4, 0.3 and 1.8 ppb of arsenic for As(III), DMA, MMA, and As(V), respectively. MMA has been shown for the first time to experimentally convert to DMA in the Chinese brake fern, indicating that Chinese brake fern can convert MMA to DMA by methylation Elsevier B.V. All rights reserved. Keywords: Arsenic speciation; Ion-pair; High-performance liquid chromatography; Inductively coupled plasma mass spectrometry; Chinese brake fern 1. Introduction Arsenic contamination has been a global problem. Thousands of people suffer from chronic toxicity effects from the surrounding arsenic contaminated soil, ground water, and various foods. For example, approximately million people out of a population of 125 million in Bangladesh are at the risk of being exposed to arsenic in their drinking water [1]. The toxicity and bioavailability of arsenic compounds strongly depend on their chemical forms. For example, both inorganic and organic arsenic compounds are toxic to humans, but inorganic arsenic compounds tend to be more toxic than organic arsenic, and As(III) is more toxic than As(V). [2] Therefore, identification and quantification of individual arsenic forms are important to appropriately measure the arsenic toxicity, environmental impact, and health risk related to arsenic exposure. Coupling high-performance liquid chromatography (HPLC) to inductively coupled plasma mass spectrometry Corresponding author. Tel.: ; fax: address: jdwin@chem.ufl.edu (J.D. Winefordner). (ICP-MS) is a powerful technique for trace elemental speciation analysis in various sample matrices. HPLC ICP-MS combines the powers of high separation efficiency of HPLC with the superior selectivity and sensitivity of ICP-MS. HPLC ICP-MS has the ability to perform real-time analysis following the separation of species of interest. It also has multi-element capability and high detection power. In addition, compared to other chromatographic methods, HPLC is more suitable to couple with ICP-MS due to their compatible liquid flow rates. This is because the liquid flow rate of HPLC, which is typically in the range of ml/min, is consistent with the requirement of the ICP-MS nebulizer sample uptake rate ( ml/min). The coupling technique of HPLC ICP-MS is simple since only a short Teflon tube of small diameter is needed to connect the HPLC column to the ICP-MS nebulizer. Chinese brake fern (Pteris vittata L.) has recently been discovered as an arsenic hyperaccumulating plant [3]. Itcan effectively extract large amounts of arsenic from soils into its fronds in a short time. Since this plant is also hardy, versatile, and fast-growing, it holds great potential to commercially and cost-effectively clean up thousands of arsenic contaminated sites as a result of both natural and human activities /$ see front matter 2003 Elsevier B.V. All rights reserved. doi: /j.aca
2 200 R. Chen et al. / Analytica Chimica Acta 504 (2004) worldwide. Knowing only the total arsenic concentration in the plant is insufficient to understand the mechanisms of arsenic hyperaccumulation by this plant and effectively use the plant to clean up arsenic contaminated sites. The goal of this research was to develop a reliable and robust analytical method for routine arsenic speciation in a single run. Reverse-phase liquid chromatography (LC) is the most popular LC separation mode due to its high separation efficiency, good sample loading tolerance, and ability to separate a broad range of different polarity samples. Reverse-phase ion-pair chromatography has been developed to routinely separate both non-ionic and ionic compounds in a signal run using the same column [4]. Ion pairing reagents, including tetrabutylammonium hydroxide (TBAH) [5 7], tetrabutylammonium phosphate (TBAP) [8], sodium pentanesulfonate [9], methanesulfonic acid, and propanesulfonate acid [10], have been used by other research groups. In the present research, a novel ion-pair reverse-phase HPLC ICP-MS was employed to perform arsenic speciation, including arsenite [As(III)], dimethyl arsenic acid (DMA), monomethyl arsenic (MMA), and arsenate [As(V)], in environmental samples. The choices of HPLC separation conditions were based on the selection and optimization of ion-pairing reagent concentration, buffer concentration, methanol concentration, ph, and mobile phase flow rate. The organic solvent and flow rate effects as well as spectroscopic interferences were also considered for ICP-MS detection. This optimized ion-pair HPLC ICP-MS method was applied to determine arsenic species in Chinese brake fern. The objectives of this research are to: (1) develop a reliable analytical method for arsenic speciation in environmental samples; and (2) apply this method to determine arsenic speciation in the recently discovered arsenic hyperaccumulating plant, Chinese brake fern. The arsenic speciation information helps to better understand the mechanisms of arsenic accumulation, transformation, and detoxification in Chinese brake fern. The HPLC ICP-MS method can also be widely applied to determine various elemental species in environmental, biological, geological, and medical field. 2. Experimental 2.1. Instrumentation A VG plasma quadrupole II (VG Elemental, Winsford, Cheshire, UK) ICP-MS was used. The ICP-MS was computer controlled (Dell Dimension XPS 4100, Dell, TX, USA) and VG instrument control software (Plasma Quad, Version 4.30, VG elemental 1996) was operated under the OS/2 (IBM, USA) system. Tuning of the ICP-MS was performed daily using a 100 ppb arsenic solution with a peristaltic pump (Rainin, Woburn, MA, USA) and a Meinhard TR-30-A concentric nebulizer (Precision glassblowing, Englewood, CO, USA) to maximize the signal response. After nebulization, sample was transported to the ICP torch through a spray chamber held at 5 C. The quadrupole mass analyzer was constantly scanned at m/z 75 for arsenic analysis. Data were acquired in a time resolved acquisition (TRA) mode. There was no ArCl interference on arsenic speciation in these studies. The chromatography system consisted of a Spectra SYS- TEM P2000 binary gradient pump (Thermo Separation Production, Fremont, CA, USA), an Auzx 210 injector valve with a 20 l loop and a Haisil 100 (Higgins Analytical, Mountain View, CA, USA) C18 column with 150 mm 4.6mmi.d. 5 m particles. The optimized mobile phase contained 10 mm hexadecyltrimethylammonium bromide (CTAB) as the ion-pairing reagent, 20 mm ammonium phosphate buffer, and 2% methanol at ph Reagents The stock solutions (1000 ppm of As) of arsenite [As(III)], arsenate [As(V)], and DMA were prepared separately by dissolving g NaAsO 2 (MCIB, East Rutherford, NJ, USA), g Na 2 HAsO 4 7H 2 O (Sigma Chemical Co., St. Louis, MO, USA), and g C 2 H 6 AsO 2 Na (Supelco, Bellefonte, PA, USA) into 100 ml Milli-Q water. The stock solution (100 ppm of As) of MMA was prepared by dissolving g CH 3 AsNa 2 O 3 6H 2 O (Supelco) into 100 ml Milli-Q water. All stock solutions were stored in refrigerator at 4 C. The buffer solutions of HPLC ICP-MS were prepared by dissolving g NH 4 H 2 PO 4 and g (NH 4 ) 2 HPO 4 into 1 l Milli-Q water for 20 mm ammonium phosphate buffer. The ph of the buffer solutions was adjusted to 6.0 by drop wise addition of diluted phosphoric acid or ammonium hydroxide. The ion-pair reagent, CTAB, was prepared by directly adding g of C 19 H 42 BrN to 1 l buffer solutions to produce 10 mm CTAB solution. The mobile phase was mixed using 98% (v/v) 20 mm ammonium phosphate buffer and 10 mm CTAB solution with 2% (v/v) methanol by HPLC pump. Working solutions of arsenic were prepared daily by appropriate dilution from the stock solutions with Milli-Q water. All solutions and mobile phases were filtered through 0.45 m Teflon filter (Gelman Instrument Company, Ann Arbor, MI, USA). The mobile phases were degassed using an ultrasonic bath for 20 min and also a helium sparge for 10 min before starting the chromatography Sample preparation and collection Chinese brake fern cultivation and root exudate collection The spores of Chinese brake fern were germinated in an arsenic free soil mixture in a greenhouse for 3 months. The fern plants with five to six fronds were transported to a controlled hydroponic system with a relatively constant temperature of C, humidity of 70%, and equal amounts of artificial light. It took approximately 2 weeks
3 R. Chen et al. / Analytica Chimica Acta 504 (2004) for the ferns to grow new roots. The fern roots were washed free of soil by tap and deionized waters, respectively. Then the ferns were transferred to a 20% strength Hoagland nutrition solution and spiked with different levels of arsenic species. The solution was buffered by 5 mm 2-2(N-morpholino)ethanesulfonic acid (MES) at ph 6.0. After 2 days, the fern roots were washed again with tap water, deionized water and phosphate buffer to assure arsenic desorbed from the root free spaces. After washing with tap and deionized waters again, each fern plant was placed in 150 ml deionized water to collect root exudate for 6 h. The short collection time was used to minimize the impacts of microbial activities on arsenic speciation. The root exudate solution was filtered immediately by a 0.45 M filter and then analyzed immediately by the HPLC ICP-MS method, or stored below 80 C for future analysis Chinese brake fern xylem sap collection Chinese brake fern cultivation was the same as the procedure described in Section For this study, Chinese brake ferns with similar size and age were selected. After the fern roots were washed free of soil by tap and deionized waters, respectively, the ferns were transported to a 20% strength Hoagland nutrition solution for 1 week. Prior to harvesting, the ferns were transferred to a new 20% strength Hoagland nutrition solution, which was spiked with different levels arsenic species, and grown for 3 days. One or two fronds of similar size were cut from the fern and placed immediately into a pressure chamber for xylem sap collection. Xylem sap was excreted from the cut of the frond by nitrogen gas in the pressure chamber. Approximately ml of xylem sap was collected from each frond with a micropipette and was stored at 80 C for future analysis Method comparison Arsenic speciation in the root exudates were also analyzed by using an arsenic separation cartridge (Metal Soft Center, Highland Park, NJ, USA) as a method comparison. This disposable cartridge retained As(V) and allowed As(III) to pass through to the filtrate [11]. Then As(III) concentration was determined by a graphite furnace atomic absorption spectrometer (GFAAS) (SIMMA 6000, Perkin-Elmer, Norwalk, CT, USA) to analyze the total arsenic in the filtrate. Thus, this method is only capable of separating As(V) from As(III) in the sample. 3. Result and discussion 3.1. Ion-pair reverse-phase HPLC ICP-MS arsenic speciation The chromatographic behaviors of arsenic species are based on their acidic or basic properties (pk a value) as shown in Table 1 [12]. The elution order was predicted from Table 1 The formula and pk a value of arsenic species [12] Compound Formula pk a As(III) O=As OH 9.3 Dimethylarsinic acid (DMA) 6.2 Monomethylarsinic acid (MMA) 3.6; 8.2 As(V) 2.3; 6.9; 11.4 their pk a values and verified experimentally by injecting the four arsenic species individually. When the ph of the phosphate buffer solution was 6.0, arsenite was present as neutral HAsO 2 (pk a = 9.3), which is fully protonated and not retained by the column. Hence, As(III) eluted first with the void volume. DMA (pk a = 6.2) was partially ionized at ph 6.0 and retained on the column a little longer than As(III), therefore it eluted a little later than As(III); MMA (pk a1 = 3.6) and As(V) (pk a1 = 2.3 and present as H 3 AsO 4 ) were completely ionized and became anionic species, which reacted with hexadecyltrimethyl ammonium pairing cation and were retained longer on the column. However, As(V) eluted last due to its strong interaction with hexadecyltrimethyl ammonium pairing cation. Under the chromatographic conditions, the elution sequence was As(III), DMA, MMA, and As(V), respectively, as shown in Fig. 1. Hexadecyltrimethyl ammonium bromide was used as the ion-pairing reagent, which is a compound with a polar head (ammonium) and a non polar tail (hexadecyltrimethyl). The reverse-phase mobile phase (98% ammonium phosphate buffer and 2% methanol) was polar and the stationary phase (silica-based bonded phase with C18 as ligand) was non-polar. All of the four arsenic compounds formed anions. Hence, they could be separated by the anion ion-pairing reagent (CTAB), which could form hexadecyltrimethyl ammonium cations. The ion-pairing reagent (CTAB) was dissolved in the mobile phase, interacted with the stationary phase, and was strongly retained by the column after a period of time. Before CTAB dynamically coated the column, the four arsenic species were not retained on the column completely and rinsed out quickly. With longer equilibration times, the retention times of MMA and As(V) were longer. Once the interaction between the ion-pairing reagent and the column approached equilibrium, the retention times for four arsenic species did not change during the whole experiment. The concentration of the ion-pairing reagent, which is CTAB in the present research, affected the time for the column to achieve equilibrium. The higher the CTAB concentration, the sooner the column equilibrated. The concentration of the ion-pairing reagent was typically in the range of 1 5 mm [4]. As shown in Fig. 2, it took 240 min
4 202 R. Chen et al. / Analytica Chimica Acta 504 (2004) Fig. 1. Determination of arsenic species standard by HPLC ICP-MS (all compounds present at 10 ppb). to equilibrate the column with 1 mm CTAB; 130 min with 5 mm CTAB; but only 75 min with 10 mm CTAB holding the other entire HPLC ICP-MS conditions constant. After the column equilibrated, As(V) retention times were constant even at the different CTAB concentration conditions. Therefore, 10 mm CTAB concentration was chosen as the upper limit to equilibrate the column, and prevent clogging the sampler cone at the same time. The influence of other parameters such as buffer concentration, methanol concentration, ph, mobile phase flow rate, and column degradation were also studied. The organic solvent and flow rate effects on ICP-MS were considered in addition to achieve an effective separation and detection of As(III), DMA, MMA, and As(V) Arsenic speciation in the root exudates of Chinese brake fern after treatment with different levels of As(V) In these experiments, Chinese brake fern roots were treated with 1.5, 15, or 150 ppm As(V) solution for 2 days before root exudate collection (Fig. 3). When the fern roots were treated with As(V), the predominant arsenic species in the fern root exudate remained as As(V), ranging from 83 to 100% of the total arsenic concentration (Table 2). As(III) Fig. 2. Effect of CTAB concentration on column equilibrium time.
5 R. Chen et al. / Analytica Chimica Acta 504 (2004) Fig. 3. Concentrations of As(III) and As(V) in Chinese brake fern root exudates. Roots were treated with 1.5, 15, and 150 ppm As(V) solutions for 2 days. was presented in some cases ranging from 0 to 17% of the total arsenic concentration. This indicated that As(V) was the main species in the fern root though some of the As(V) was probably reduced to As(III) by the roots. This plant survived in the solution containing up to 150 ppm As(V). The total arsenic concentration in root exudates ranged from 1405 to 2955 ppb for the 150 ppm arsenic treatment, corresponding to 1 2% of the original treatment solution, which indicated that the fern roots released some of the arsenic taken up by the roots back into solution. The same fern root exudate samples were also analyzed using the arsenic cartridge-gfaas method (Table 2). The sum of As(III) and As(V) concentrations determined by HPLC ICP-MS and the total arsenic concentration deter- mined by arsenic cartridge-gfaas were in good agreement for the majority of the samples. However, the As(III) concentrations were slightly different. In some cases, the arsenic cartridge-gfaas method gave As(III) concentrations ranging from 1 to 6% of the total arsenic concentration. However, the HPLC ICP-MS results showed that there was no As(III) present. This was possible since the cartridge retained only As(V), which meant that all species other than As(V) passed through the column and were counted as As(III). Therefore, it is expected that the cartridge-gfaas method may overestimate As(III) concentration if As(V) in the fern sample is present in a complex form and hence passes through the column. The average recovery of As(III) was 98% using the cartridge, with arsenic concentrations less than 500 gl 1 [11]. The sensitivity of this GFAAS method was adequate to analyze total arsenic in most fern samples. However, DMA and MMA could not be determined by this arsenic cartridge-gfaas method due to the limitation of the cartridge s selectivity. Therefore, HPLC ICP-MS is an important analytical technique for arsenic speciation in Chinese brake fern Arsenic speciation in the root exudates of Chinese brake ferns after treatment with 15 ppm As(III), As(V), DMA, or MMA In this experiment, Chinese brake fern roots were treated with 15 ppm As(III), As(V), DMA, or MMA for 2 days before root exudate collection (Fig. 4). For the control, when no arsenic was applied, the only species present in the root exudate was As(V) at a small level of 2 7 ppb (Table 3). When the fern roots were treated with either As(V) or As(III), the main arsenic species in the root exudates was As(V) comprising of % of the total arsenic concentration. This Table 2 Concentrations of arsenic species in Chinese brake fern root exudates after being exposed to arsenic for 2 days HPLC ICP-MS Arsenic cartridge GFAAS Control As(III) As(V) Total As (As(III)/total As) 100 As(III) As(V) Total As (As(III)/total As) 100 R1-1 ND ND ND 1 2 R1-2 ND ND ND ND 1 R1-3 ND ND ND ND ppm R2-1 ND ND R2-2 ND ND T ppm R3-1 ND ND RT R ppm R4-1 ND ND R4-2 ND ND R R1-1: root exudate blank sample no. 1; (As(III)/total As) 100: (As(III) concentration/total arsenic concentration) 100; ND: not detected; detection limit for As(III), DMA, MMA, and As(V) is 0.5, 0.4, 0.3 and 1.8 ppb of arsenic.
6 204 R. Chen et al. / Analytica Chimica Acta 504 (2004) Table 3 Arsenic speciation in the root exudates of Chinese brake ferns after treatment with 15 ppm As(III), As(V), DMA, or MMA solution for 2 days Fig. 4. Concentrations of four arsenic species in Chinese brake fern root exudates. Roots were treated separately with 15 ppm As(III), DMA, MMA, or As(V) solutions for 2 days. was consistent with our hypothesis that arsenic reduction occurred mostly in the fronds [13]. In a study to determine the location of arsenic reduction in the Chinese brake fern, Tu and Ma [13] reported that As(III) accounted for 24 34% of the total As in the excised roots (detopped) that were treated with 50 ppm As(V) for 1 day, whereas 30 39% of As(V) was present when the roots were treated with 50 ppm As(III). Their data strongly suggest that both As(III) oxidation [when treated with As(III)] and As(V) reduction [when treated with As(V)] occurred in the roots. Oxidation of As(III) has rarely been reported in the plants, but it has been reported for soil bacteria [14] and mineral leaching bacteria [15]. In the study of Tu and Ma [13], As(III) concentrations in the solution spiked with As(V) in the presence of the roots increase by 3 17% in comparison to the control without plant [ % As(III)], suggesting an occurrence of As(III) oxidation/reduction possibly by microbial activity in the solution or direct root exudation of As(V)/As(III) into the solution. The fact that more As(V) was present in the roots (24 34%) than the solution (3.1 17%), and more As(III) was present in the solution (71 80%) than the roots (61 70%) suggested that As oxidation occurred inside the roots [13]. However, As(III) was also detected accounting for 0 3% of the total arsenic concentration when treated with either As(V) or As(III), which was consistent with the data discussed in Section 3.2. Such results indicated that significant oxidation of As(III) to As(V) occurred either in or outside the roots, which could not be verified in this experiment. Although the root exudates were collected with minimum contact hours (i.e. 6 h) to minimize microbial-mediated arsenic oxidation in the solution, such a process is still possible. Approximately 76 87% of DMA was detected in the root exudate when the plants were treated with DMA (Table 3). Additionally, there was no MMA present with such treat- Control As(III) DMA MMA As(V) Total C % C % C % C % R1-1 ND ND ND ND ND ND R1-2 ND ND ND ND ND ND R1-3 ND ND ND ND ND ND R1-3 ND ND ND ND ND ND As(III) R ND ND ND ND R ND ND ND ND R2-3 ND ND ND ND ND ND R2-4 ND ND ND ND ND ND As(V) R3-1 ND ND ND ND ND ND R3-2 ND ND ND ND ND ND R ND ND ND ND MMA R R R R DMA R5-1 ND ND ND ND R5-2 ND ND ND ND R5-3 ND ND ND ND R1-1: root exudate blank sample no. 1. ments. A typical chromatogram is shown in Fig. 5C. This suggests DMA was a stable arsenic form and the plant failed to convert it to other less toxic forms. This was consistent with our previous data [16]. In a study to determine the effects of arsenic species on plant growth and arsenic uptake, Chinese brake ferns were exposed to 50 ppm As(III), As(V), DMA or MMA for 12 weeks in a greenhouse study [16]. All the ferns survived such arsenic exposure except those treated with 50 ppm DMA, where all died after 12 weeks of growth. When the fern was treated with MMA, MMA was the main species comprising of 18 56% of the total arsenic concentration. In addition, DMA was presented when the fern was treated with 15 ppm MMA, as shown in Fig. 5B. The concentration of DMA was far above the detection limit of the HPLC ICP-MS method accounting for 8 22% of the total arsenic concentration. The original 15 ppm MMA treatment solution was also analyzed to verify there was no DMA contamination as shown in Fig. 5A. This experiment demonstrated that MMA converted to DMA in the presence of Chinese brake fern roots. However, it was unclear whether such the methylation occurred inside the roots or in the exudate solution. Odanaka et al. [17] also found same phenomenon in rice plants with GC MID-MS HG-HCT (gas chromatography with a multiple ion detection mass spectrometry and hydride generation-heptane cold trap).
7 R. Chen et al. / Analytica Chimica Acta 504 (2004) Fig. 5. Typical chromatograms of arsenic species (A) original 15 ppm MMA treatment solution and Chinese brake fern root exudate with (B) 15 ppm MMA treatment; and (C) 15 ppm DMA treatment Arsenic speciation in the xylem saps of Chinese brake ferns after treatment with 10 ppm or 50 ppm DMA or MMA solutions The role of xylem sap in the fern is to transport arsenic from the roots to the fronds. In this experiment, the fern roots were treated with 10 ppm or 50 ppm DMA or MMA solutions for 3 days before xylem sap collection (Fig. 6). For the control when no arsenic was applied, approximately 70 89% of the total arsenic concentration in the xylem sap was present as As(III) and 10 25% was As(V) (Table 4). It is important to note that As(V) was the only species in the root exudates for the Chinese brake fern without arsenic treatment (Table 3). This was in good agreement with previous research concerning the conversion of As(V) to As(III) in the plant [18]. Inorganic arsenic is the predominant form of arsenic in the Chinese brake fern xylem sap in the control. This also suggested that some arsenic reduction occurred either in the roots after uptake or in the xylem during translocation. However, a small amount of MMA, ranging from 1 to 7% of the total arsenic, was also detected, which may indicate that methylation occurred inside the plant. Furthermore, no DMA was detected. As shown in Table 4, the total arsenic concentration in xylem sap can increase up to 344 ppm with 50 ppm DMA treatment, which was approximately six times greater than the solution concentration. This further verified the hyperaccumulating property of Chinese brake fern.
8 206 R. Chen et al. / Analytica Chimica Acta 504 (2004) Fig. 6. Concentrations of four arsenic species in Chinese brake fern xylem sap. Roots were treated separately with 10 ppm or 50 ppm DMA or MMA solutions for 3 days. When the Chinese brake fern roots were treated with either 10 ppm or 50 ppm DMA, as expected, the predominant arsenic species in the fern xylem sap was DMA. DMA ranged from 78 to 100% of the total arsenic concentration, and there was no MMA present. This indicated that demethy- lation did not occur inside the plants. MMA remained the primary species ranging from 79 to 95% of the total arsenic concentration when treated with either 10 ppm or 50 ppm MMA. Furthermore, DMA was also discovered in the xylem sap treated with MMA. The concentration of DMA was far Table 4 Concentrations of arsenic species in Chinese brake fern xylem sap after the plants were treated with 10 ppm or 50 ppm DMA or DMA solutions for 3 days Control As(III) DMA MMA As(V) Total (ppm) C (ppm) % C (ppm) % C (ppm) % C (ppm) % S ND ND S ND ND S ND ND S ND ND ppm DMA S ND ND S ND ND S ND ND S ND ND ppm DMA S ND ND S ND ND S ND ND S ND ND ppm MMA S S S ppm MMA S S S S S1-1 Xylem sap sample blank no. 1.
9 R. Chen et al. / Analytica Chimica Acta 504 (2004) above the detection limit of this HPLC ICP-MS method, which was 0.4 ppb, and accounted for 2 5% of the total arsenic concentration. This is in agreement with the conversion of MMA to DMA in Chinese brake fern root exudate discussed in Section 3.3. Our results suggest that methylation is possible in the Chinese brake fern. The relative standard deviation of the total arsenic concentration ranged from 5 to 126% in either fern root exudate or xylem sap samples. This is due to the variation among different fern plants. Although demethylation from DMA to MMA was not observed in the Chinese brake fern, conversion of DMA or MAA to As(III) or As(V) was observed. However, it is unclear how the conversion occurred. microbial-mediated arsenic reduction and oxidation may play a role. The results with root exudates and xylem sap data suggested that DMA was more toxic to Chinese brake since it failed to convert to other less toxic forms, and the plant was capable of methylation (conversion from MMA to DMA) in addition to converting MMA to As(III) and As(V) in the roots or in the xylem. Acknowledgements This research was supported in part by the National Science Foundation (Grant BES ). 4. Conclusion HPLC ICP-MS has been successfully used to perform arsenic speciation in the root exudates and xylem saps of Chinese brake fern after they were exposed to arsenic for 2 3 days in a hydroponic system. Our results confirmed that HPLC ICP-MS was a reliable method that resulted in a low detection limit and rapid analysis for arsenic speciation in plant and aqueous samples. When Chinese brake ferns were treated with either As(V) or As(III), the primary arsenic species in the root exudates was As(V), i.e. little arsenic reduction occurred in or outside the roots, whereas significant arsenic oxidation occurred either in or outside the roots (Tables 2 and 3). This is consistent with our hypothesis that arsenic reduction mostly occurred in the fronds. When treated with DMA, DMA remained as the dominant arsenic species with no MMA being detected in the root exudates or in the xylem sap, suggesting DMA is a stable arsenic form that the plant was unable to convert to inorganic forms. On the other hand, when treated with MMA, DMA was detected in both the root exudates or the xylem sap (Tables 3 and 4), suggesting methylation was possible in this plant. The results from the root exudates suggested that both arsenic reduction and oxidation occurred either inside the roots or in the solution. If the latter is the case, then References [1] A.H. Smith, E.O. Lings, M. Rahman, Bull. World Health Organ. 78 (2000) [2] M. Bissen, F.H. Frimmel, Fresenius J. Anal. Chem. 367 (2000) 51. [3] L.Q. Ma, K.M. Komart, C. Tu, W. Zhang, Y. Cai, E. Kennelley, Nature 409 (2001) 579. [4] K.L. Sutton, J.A. Caruso, J. Chromatogr. A 856 (1999) 243. [5] S. Wangkarn, S.A. Pergantis, J. Anal. At. Spectrom. 15 (2000) 627. [6] S.A. Pergantis, E.H. Heithmar, T.A. Hinners, Analyst 122 (1997) [7] D. Beauchemin, K.W.M. Siu, J.W. Mclaren, S.S. Berman, J. Anal. At. Spectrom. 4 (1989) 285. [8] P. Thomas, K. Sniatecki, J. Anal. At. Spectrom. 10 (1995) 615. [9] C. B Hymer, K.L. Sutton, J.A. Caruso, J. Anal. At. Spectrom. 13 (1998) 855. [10] X.C. Le, M. Ma, J. Chromatogr. A 764 (1997) 55. [11] X. Meng, W. Wang, Presented at the 3rd International Conference on Arsenic Exposure and Health Effects, San Diego, CA, [12] R.L. David (Ed.), CRC Handbook of Chemistry and Physics, CRC Press, Boca Raton, FL, [13] M.S. Tu, L.Q. Ma, Environ. Exp. Bot., 2003, in press. [14] S.E. Phillips, M.L. Taylor, Appl. Environ. Microbiol. 32 (1976) 392. [15] H.M. Sehlin, E.B. Lindström, FEMS Microbiol. Lett. 93 (1992) 87. [16] C. Tu, L.Q. Ma, J. Environ. Qual. 31 (2002) 641. [17] Y. Odanaka, N. Tsuchiya, O. Matano, S. Goto, J. Agric. Food Chem. 33 (1985) 757. [18] W. Zhang, Y. Cai, C. Yu, L.Q. Ma, Sci. Total Environ. 300 (2002) 167.
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