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Analytica Chimica Acta 504 (2004) 199–207

Arsenic speciation in Chinese brake fern by ion-pair high-performanceliquid chromatography–inductively coupled plasma mass spectroscopy

Ruixue Chena, Benjamin W. Smitha, James D. Winefordnera,∗,Mike S. Tub, Gina Kertulisb, Lena Q. Mab

a Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, FL 32611-7200, USAb Soil and Water Science Department, University of Florida, Gainesville, FL 32611-7200, 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 ona 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 withHPLC–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 forthe first time to experimentally convert to DMA in the Chinese brake fern, indicating that Chinese brake fern can convert MMA to DMA bymethylation.© 2003 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. Thou-sands of people suffer from chronic toxicity effects from thesurrounding arsenic contaminated soil, ground water, andvarious foods. For example, approximately 35–77 millionpeople out of a population of 125 million in Bangladesh areat the risk of being exposed to arsenic in their drinking water[1]. The toxicity and bioavailability of arsenic compoundsstrongly depend on their chemical forms. For example, bothinorganic and organic arsenic compounds are toxic to hu-mans, but inorganic arsenic compounds tend to be more toxicthan organic arsenic, and As(III) is more toxic than As(V).[2] Therefore, identification and quantification of individualarsenic forms are important to appropriately measure the ar-senic toxicity, environmental impact, and health risk relatedto arsenic exposure.

Coupling high-performance liquid chromatography(HPLC) to inductively coupled plasma mass spectrometry

∗ Corresponding author. Tel.:+1-352-3920556; fax:+1-352-3924651.E-mail address: [email protected] (J.D. Winefordner).

(ICP-MS) is a powerful technique for trace elemental speci-ation analysis in various sample matrices. HPLC–ICP-MScombines the powers of high separation efficiency of HPLCwith the superior selectivity and sensitivity of ICP-MS.HPLC–ICP-MS has the ability to perform real-time analysisfollowing the separation of species of interest. It also hasmulti-element capability and high detection power. In addi-tion, compared to other chromatographic methods, HPLCis more suitable to couple with ICP-MS due to their com-patible liquid flow rates. This is because the liquid flow rateof HPLC, which is typically in the range of 0.1–10 ml/min,is consistent with the requirement of the ICP-MS nebulizersample uptake rate (0.5–1.0 ml/min). The coupling tech-nique of HPLC–ICP-MS is simple since only a short Teflontube of small diameter is needed to connect the HPLCcolumn to the ICP-MS nebulizer.

Chinese brake fern (Pteris vittata L.) has recently beendiscovered as an arsenic hyperaccumulating plant[3]. It caneffectively extract large amounts of arsenic from soils into itsfronds in a short time. Since this plant is also hardy, versatile,and fast-growing, it holds great potential to commerciallyand cost-effectively clean up thousands of arsenic contami-nated sites as a result of both natural and human activities

0003-2670/$ – see front matter © 2003 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2003.10.042

200 R. Chen et al. / Analytica Chimica Acta 504 (2004) 199–207

worldwide. Knowing only the total arsenic concentration inthe plant is insufficient to understand the mechanisms of ar-senic hyperaccumulation by this plant and effectively usethe plant to clean up arsenic contaminated sites.

The goal of this research was to develop a reliable androbust analytical method for routine arsenic speciation ina single run. Reverse-phase liquid chromatography (LC) isthe most popular LC separation mode due to its high sep-aration efficiency, good sample loading tolerance, and abil-ity to separate a broad range of different polarity samples.Reverse-phase ion-pair chromatography has been developedto routinely separate both non-ionic and ionic compoundsin a signal run using the same column[4]. Ion pairingreagents, including tetrabutylammonium hydroxide (TBAH)[5–7], tetrabutylammonium phosphate (TBAP)[8], sodiumpentanesulfonate[9], methanesulfonic acid, and propanesul-fonate acid[10], have been used by other research groups.

In the present research, a novel ion-pair reverse-phaseHPLC–ICP-MS was employed to perform arsenic speci-ation, including arsenite [As(III)], dimethyl arsenic acid(DMA), monomethyl arsenic (MMA), and arsenate [As(V)],in environmental samples. The choices of HPLC separationconditions were based on the selection and optimizationof ion-pairing reagent concentration, buffer concentration,methanol concentration, pH, and mobile phase flow rate.The organic solvent and flow rate effects as well as spectro-scopic interferences were also considered for ICP-MS detec-tion. This optimized ion-pair HPLC–ICP-MS method wasapplied to determine arsenic species in Chinese brake fern.

The objectives of this research are to: (1) develop areliable analytical method for arsenic speciation in envi-ronmental samples; and (2) apply this method to determinearsenic speciation in the recently discovered arsenic hyper-accumulating plant, Chinese brake fern. The arsenic specia-tion information helps to better understand the mechanismsof arsenic accumulation, transformation, and detoxificationin Chinese brake fern. The HPLC–ICP-MS method can alsobe widely applied to determine various elemental speciesin 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 com-puter controlled (Dell Dimension XPS 4100, Dell, TX, USA)and VG instrument control software (Plasma Quad, Ver-sion 4.30, VG elemental 1996) was operated under the OS/2(IBM, USA) system. Tuning of the ICP-MS was performeddaily using a 100 ppb arsenic solution with a peristaltic pump(Rainin, Woburn, MA, USA) and a Meinhard TR-30-A con-centric 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 wasconstantly scanned atm/z 75 for arsenic analysis. Data wereacquired in a time resolved acquisition (TRA) mode. Therewas no ArCl interference on arsenic speciation in these stud-ies.

The chromatography system consisted of a Spectra SYS-TEM P2000 binary gradient pump (Thermo SeparationProduction, Fremont, CA, USA), an Auzx 210 injectorvalve with a 20�l loop and a Haisil 100 (Higgins An-alytical, Mountain View, CA, USA) C18 column with150 mm× 4.6 mm i.d. × 5�m particles. The optimizedmobile phase contained 10 mM hexadecyltrimethylammo-nium bromide (CTAB) as the ion-pairing reagent, 20 mMammonium phosphate buffer, and 2% methanol at pH 6.0.

2.2. Reagents

The stock solutions (1000 ppm of As) of arsenite [As(III)],arsenate [As(V)], and DMA were prepared separately bydissolving 0.1734 g NaAsO2 (MCIB, East Rutherford, NJ,USA), 0.4164 g Na2HAsO4·7H2O (Sigma Chemical Co., St.Louis, MO, USA), and 0.2133 g C2H6AsO2Na (Supelco,Bellefonte, PA, USA) into 100 ml Milli-Q water. The stocksolution (100 ppm of As) of MMA was prepared by dis-solving 0.0389 g CH3AsNa2O3·6H2O (Supelco) into 100 mlMilli-Q water. All stock solutions were stored in refrigeratorat 4◦C.

The buffer solutions of HPLC–ICP-MS were prepared bydissolving 2.1166 g NH4H2PO4 and 0.2133 g (NH4)2HPO4into 1 l Milli-Q water for 20 mM ammonium phosphatebuffer. The pH of the buffer solutions was adjusted to 6.0 bydrop wise addition of diluted phosphoric acid or ammoniumhydroxide. The ion-pair reagent, CTAB, was prepared bydirectly adding 3.6445 g of C19H42BrN to 1 l buffer solu-tions to produce 10 mM CTAB solution. The mobile phasewas mixed using 98% (v/v) 20 mM ammonium phosphatebuffer and 10 mM CTAB solution with 2% (v/v) methanolby HPLC pump.

Working solutions of arsenic were prepared daily by ap-propriate dilution from the stock solutions with Milli-Q wa-ter. All solutions and mobile phases were filtered through0.45�m Teflon filter (Gelman Instrument Company, AnnArbor, MI, USA). The mobile phases were degassed usingan ultrasonic bath for 20 min and also a helium sparge for10 min before starting the chromatography.

2.3. Sample preparation and collection

2.3.1. Chinese brake fern cultivation and root exudatecollection

The spores of Chinese brake fern were germinated inan arsenic free soil mixture in a greenhouse for 3 months.The fern plants with five to six fronds were transportedto a controlled hydroponic system with a relatively con-stant temperature of 23–28◦C, humidity of 70%, and equalamounts of artificial light. It took approximately 2 weeks

R. Chen et al. / Analytica Chimica Acta 504 (2004) 199–207 201

for the ferns to grow new roots. The fern roots werewashed free of soil by tap and deionized waters, respec-tively. Then the ferns were transferred to a 20% strengthHoagland nutrition solution and spiked with different lev-els of arsenic species. The solution was buffered by 5 mM2-2(N-morpholino)ethanesulfonic acid (MES) at pH 6.0.After 2 days, the fern roots were washed again with tap wa-ter, deionized water and phosphate buffer to assure arsenicdesorbed from the root free spaces. After washing with tapand deionized waters again, each fern plant was placed in150 ml deionized water to collect root exudate for 6 h. Theshort collection time was used to minimize the impacts ofmicrobial activities on arsenic speciation. The root exudatesolution was filtered immediately by a 0.45�M filter andthen analyzed immediately by the HPLC–ICP-MS method,or stored below−80◦C for future analysis.

2.3.2. Chinese brake fern xylem sap collectionChinese brake fern cultivation was the same as the proce-

dure described inSection 2.3.1. For this study, Chinese brakeferns with similar size and age were selected. After the fernroots were washed free of soil by tap and deionized waters,respectively, the ferns were transported to a 20% strengthHoagland nutrition solution for 1 week. Prior to harvesting,the ferns were transferred to a new 20% strength Hoaglandnutrition solution, which was spiked with different levels ar-senic species, and grown for 3 days. One or two fronds ofsimilar size were cut from the fern and placed immediatelyinto a pressure chamber for xylem sap collection. Xylem sapwas excreted from the cut of the frond by nitrogen gas inthe pressure chamber. Approximately 0.7–1.0 ml of xylemsap was collected from each frond with a micropipette andwas stored at−80◦C for future analysis.

2.4. Method comparison

Arsenic speciation in the root exudates were also analyzedby using an arsenic separation cartridge (Metal Soft Center,Highland Park, NJ, USA) as a method comparison. This dis-posable cartridge retained As(V) and allowed As(III) to passthrough to the filtrate[11]. Then As(III) concentration wasdetermined by a graphite furnace atomic absorption spec-trometer (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 arsenicspeciation

The chromatographic behaviors of arsenic species arebased on their acidic or basic properties (pKa value) asshown inTable 1 [12]. The elution order was predicted from

Table 1The formula and pKa value of arsenic species[12]

Compound Formula pKa

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 pKa values and verified experimentally by injecting thefour arsenic species individually. When the pH of the phos-phate buffer solution was 6.0, arsenite was present as neu-tral HAsO2 (pKa = 9.3), which is fully protonated and notretained by the column. Hence, As(III) eluted first with thevoid volume. DMA (pKa = 6.2) was partially ionized at pH6.0 and retained on the column a little longer than As(III),therefore it eluted a little later than As(III); MMA (pKa1 =3.6) and As(V) (pKa1 = 2.3 and present as H3AsO4) werecompletely ionized and became anionic species, which re-acted with hexadecyltrimethyl ammonium pairing cation andwere retained longer on the column. However, As(V) elutedlast due to its strong interaction with hexadecyltrimethyl am-monium pairing cation. Under the chromatographic condi-tions, the elution sequence was As(III), DMA, MMA, andAs(V), respectively, as shown inFig. 1.

Hexadecyltrimethyl ammonium bromide was used as theion-pairing reagent, which is a compound with a polar head(ammonium) and a non polar tail (hexadecyltrimethyl). Thereverse-phase mobile phase (98% ammonium phosphatebuffer and 2% methanol) was polar and the stationaryphase (silica-based bonded phase with C18 as ligand) wasnon-polar. All of the four arsenic compounds formed anions.Hence, they could be separated by the anion ion-pairingreagent (CTAB), which could form hexadecyltrimethylammonium cations. The ion-pairing reagent (CTAB) wasdissolved in the mobile phase, interacted with the stationaryphase, and was strongly retained by the column after a pe-riod of time. Before CTAB dynamically coated the column,the four arsenic species were not retained on the columncompletely and rinsed out quickly. With longer equilibra-tion times, the retention times of MMA and As(V) werelonger. Once the interaction between the ion-pairing reagentand the column approached equilibrium, the retention timesfor four arsenic species did not change during the wholeexperiment. The concentration of the ion-pairing reagent,which is CTAB in the present research, affected the time forthe column to achieve equilibrium. The higher the CTABconcentration, the sooner the column equilibrated. The con-centration of the ion-pairing reagent was typically in therange of 1–5 mM[4]. As shown inFig. 2, it took 240 min


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 with5 mM CTAB; but only 75 min with 10 mM CTAB holdingthe other entire HPLC–ICP-MS conditions constant. Afterthe column equilibrated, As(V) retention times were con-stant even at the different CTAB concentration conditions.Therefore, 10 mM CTAB concentration was chosen as theupper limit to equilibrate the column, and prevent cloggingthe sampler cone at the same time.

The influence of other parameters such as buffer concen-tration, methanol concentration, pH, mobile phase flow rate,and column degradation were also studied. The organic sol-vent and flow rate effects on ICP-MS were considered in

Fig. 2. Effect of CTAB concentration on column equilibrium time.

addition to achieve an effective separation and detection ofAs(III), DMA, MMA, and As(V).

3.2. Arsenic speciation in the root exudates of Chinesebrake fern after treatment with different levels of As(V)

In these experiments, Chinese brake fern roots weretreated with 1.5, 15, or 150 ppm As(V) solution for 2 daysbefore root exudate collection (Fig. 3). When the fern rootswere treated with As(V), the predominant arsenic species inthe fern root exudate remained as As(V), ranging from 83to 100% of the total arsenic concentration (Table 2). As(III)


Fig. 3. Concentrations of As(III) and As(V) in Chinese brake fern rootexudates. Roots were treated with 1.5, 15, and 150 ppm As(V) solutionsfor 2 days.

was presented in some cases ranging from 0 to 17% of thetotal arsenic concentration. This indicated that As(V) wasthe main species in the fern root though some of the As(V)was probably reduced to As(III) by the roots. This plantsurvived in the solution containing up to 150 ppm As(V).The total arsenic concentration in root exudates rangedfrom 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 thearsenic taken up by the roots back into solution.

The same fern root exudate samples were also analyzedusing the arsenic cartridge-GFAAS method (Table 2). Thesum of As(III) and As(V) concentrations determined byHPLC–ICP-MS and the total arsenic concentration deter-

Table 2Concentrations of arsenic species in Chinese brake fern root exudates after being exposed to arsenic for 2 days

HPLC–ICP-MS (ppb) Arsenic cartridge GFAAS (ppb)

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 2R1-2 ND ND ND ND 1R1-3 ND ND ND ND 2

1.5 ppmR2-1 ND 38 38 ND 1 50 3R2-2 ND 38 38 ND 3 48 6T2-3 6 29 35 17 6 44 14

15 ppmR3-1 ND 84 84 ND 2 97 2RT3-2 2 118 120 2 10 170 6R3-3 6 276 282 2 11 287 4

150 ppmR4-1 ND 1483 1483 ND 12 1430 1R4-2 ND 1405 1405 ND 30 1340 2R4-3 234 2721 2955 8 389 2810 14

R1-1: root exudate blank sample no. 1; (As(III)/total As)× 100: (As(III) concentration/total arsenic concentration)× 100; ND: not detected; detectionlimit for As(III), DMA, MMA, and As(V) is 0.5, 0.4, 0.3 and 1.8 ppb of arsenic.

mined by arsenic cartridge-GFAAS were in good agreementfor the majority of the samples. However, the As(III) con-centrations were slightly different. In some cases, the ar-senic cartridge-GFAAS method gave As(III) concentrationsranging from 1 to 6% of the total arsenic concentration.However, the HPLC–ICP-MS results showed that there wasno As(III) present. This was possible since the cartridgeretained only As(V), which meant that all species otherthan As(V) passed through the column and were counted asAs(III). Therefore, it is expected that the cartridge-GFAASmethod may overestimate As(III) concentration if As(V) inthe fern sample is present in a complex form and hencepasses through the column. The average recovery of As(III)was 98% using the cartridge, with arsenic concentrations lessthan 500�g l−1 [11]. The sensitivity of this GFAAS methodwas adequate to analyze total arsenic in most fern samples.However, DMA and MMA could not be determined by thisarsenic cartridge-GFAAS method due to the limitation ofthe cartridge’s selectivity. Therefore, HPLC–ICP-MS is animportant analytical technique for arsenic speciation in Chi-nese brake fern.

3.3. Arsenic speciation in the root exudates of Chinesebrake ferns after treatment with 15 ppm As(III), As(V),DMA, or MMA

In this experiment, Chinese brake fern roots were treatedwith 15 ppm As(III), As(V), DMA, or MMA for 2 days be-fore root exudate collection (Fig. 4). For the control, when noarsenic was applied, the only species present in the root ex-udate was As(V) at a small level of 2–7 ppb (Table 3). Whenthe fern roots were treated with either As(V) or As(III), themain arsenic species in the root exudates was As(V) com-prising of 97–100% of the total arsenic concentration. This


Fig. 4. Concentrations of four arsenic species in Chinese brake fernroot 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 reductionoccurred mostly in the fronds[13]. In a study to determinethe 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 weretreated with 50 ppm As(V) for 1 day, whereas 30–39% ofAs(V) was present when the roots were treated with 50 ppmAs(III). Their data strongly suggest that both As(III) oxida-tion [when treated with As(III)] and As(V) reduction [whentreated with As(V)] occurred in the roots. Oxidation ofAs(III) has rarely been reported in the plants, but it has beenreported for soil bacteria[14] and mineral leaching bacteria[15]. In the study of Tu and Ma[13], As(III) concentrationsin the solution spiked with As(V) in the presence of theroots increase by 3–17% in comparison to the control with-out plant [99.8–100% As(III)], suggesting an occurrenceof As(III) oxidation/reduction possibly by microbial activ-ity in the solution or direct root exudation of As(V)/As(III)into the solution. The fact that more As(V) was present inthe roots (24–34%) than the solution (3.1–17%), and moreAs(III) was present in the solution (71–80%) than the roots(61–70%) suggested that As oxidation occurred inside theroots[13].

However, As(III) was also detected accounting for 0–3%of the total arsenic concentration when treated with eitherAs(V) or As(III), which was consistent with the data dis-cussed inSection 3.2. Such results indicated that significantoxidation of As(III) to As(V) occurred either in or outsidethe roots, which could not be verified in this experiment. Al-though the root exudates were collected with minimum con-tact hours (i.e. 6 h) to minimize microbial-mediated arsenicoxidation in the solution, such a process is still possible.

Approximately 76–87% of DMA was detected in the rootexudate when the plants were treated with DMA (Table 3).Additionally, there was no MMA present with such treat-

Table 3Arsenic speciation in the root exudates of Chinese brake ferns aftertreatment with 15 ppm As(III), As(V), DMA, or MMA solution for 2 days

Control As(III) DMA MMA As(V) Total(ppb)

C(ppb)

% C(ppb)

% C(ppb)

% C(ppb)

%

R1-1 ND ND ND ND ND ND 7 100 7R1-2 ND ND ND ND ND ND 3 100 3R1-3 ND ND ND ND ND ND 3 100 3R1-3 ND ND ND ND ND ND 2 100 2

As(III)R2-1 5 2 ND ND ND ND 221 98 226R2-2 5 3 ND ND ND ND 143 97 147R2-3 ND ND ND ND ND ND 165 100 165R2-4 ND ND ND ND ND ND 205 100 205

As(V)R3-1 ND ND ND ND ND ND 173 100 173R3-2 ND ND ND ND ND ND 188 100 188R3-3 9 3 ND ND ND ND 271 97 280

MMAR4-1 4 6 14 22 32 50 15 23 64R4-2 5 1 42 8 95 18 386 73 528R4-3 4 5 13 14 42 46 32 36 91R4-4 4 3 29 21 77 56 27 20 136

DMAR5-1 ND ND 120 87 ND ND 18 13 138R5-2 ND ND 84 87 ND ND 12 13 96R5-3 ND ND 58 76 ND ND 19 24 77

R1-1: root exudate blank sample no. 1.

ments. A typical chromatogram is shown inFig. 5C. Thissuggests DMA was a stable arsenic form and the plant failedto convert it to other less toxic forms. This was consistentwith our previous data[16]. In a study to determine the ef-fects 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 thosetreated with 50 ppm DMA, where all died after 12 weeks ofgrowth.

When the fern was treated with MMA, MMA was themain species comprising of 18–56% of the total arsenic con-centration. In addition, DMA was presented when the fernwas treated with 15 ppm MMA, as shown inFig. 5B. Theconcentration of DMA was far above the detection limit ofthe HPLC–ICP-MS method accounting for 8–22% of the to-tal arsenic concentration. The original 15 ppm MMA treat-ment solution was also analyzed to verify there was no DMAcontamination as shown inFig. 5A.

This experiment demonstrated that MMA converted toDMA in the presence of Chinese brake fern roots. How-ever, it was unclear whether such the methylation oc-curred inside the roots or in the exudate solution. Odanakaet al. [17] also found same phenomenon in rice plantswith GC–MID-MS–HG-HCT (gas chromatography witha multiple ion detection mass spectrometry and hydridegeneration-heptane cold trap).


Fig. 5. Typical chromatograms of arsenic species (A) original 15 ppm MMA treatment solution and Chinese brake fern root exudate with (B) 15 ppmMMA treatment; and (C) 15 ppm DMA treatment.

3.4. Arsenic speciation in the xylem saps of Chinese brakeferns after treatment with 10 ppm or 50 ppm DMA orMMA solutions

The role of xylem sap in the fern is to transport arsenicfrom the roots to the fronds. In this experiment, the fernroots were treated with 10 ppm or 50 ppm DMA or MMAsolutions for 3 days before xylem sap collection (Fig. 6).For the control when no arsenic was applied, approximately70–89% of the total arsenic concentration in the xylem sapwas present as As(III) and 10–25% was As(V) (Table 4).It is important to note that As(V) was the only species inthe root exudates for the Chinese brake fern without arsenictreatment (Table 3). This was in good agreement with previ-

ous research concerning the conversion of As(V) to As(III)in the plant[18]. Inorganic arsenic is the predominant formof arsenic in the Chinese brake fern xylem sap in the control.This also suggested that some arsenic reduction occurred ei-ther in the roots after uptake or in the xylem during translo-cation. However, a small amount of MMA, ranging from 1to 7% of the total arsenic, was also detected, which mayindicate that methylation occurred inside the plant. Further-more, no DMA was detected.

As shown inTable 4, the total arsenic concentration inxylem sap can increase up to 344 ppm with 50 ppm DMAtreatment, which was approximately six times greater thanthe solution concentration. This further verified the hyper-accumulating property of Chinese brake fern.


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 MMAsolutions for 3 days.

When the Chinese brake fern roots were treated with ei-ther 10 ppm or 50 ppm DMA, as expected, the predomi-nant arsenic species in the fern xylem sap was DMA. DMAranged from 78 to 100% of the total arsenic concentration,and there was no MMA present. This indicated that demethy-

Table 4Concentrations 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) %

S1-1 1.0 89 ND ND 0.015 1 0.12 10 1.2S1-2 0.29 70 ND ND 0.031 7 0.093 23 0.41S1-3 0.055 84 ND ND 0.003 5 0.007 11 0.066S1-4 0.14 72 ND ND 0.007 4 0.047 25 0.19

10 ppm DMAS2-1 0.17 2 7.7 97 ND ND 0.093 1 8.0S2-2 0.36 20 1.4 78 ND ND 0.042 2 1.8S2-3 0.032 1 4.4 99 ND ND 0.024 1 4.4S2-4 0.068 2 4.3 98 ND ND 0.011 0 4.4

50 ppm DMAS3-1 0.091 0 65 100 ND ND 0.23 0 65S3-2 0.083 0 38 100 ND ND 0.055 0 38S3-3 0.11 0 30 100 ND ND 0.058 0 30S3-4 0.11 0 344 100 ND ND 0.043 0 344

10 ppm MMAS4-1 0.14 0 0.87 2 44 95 1.5 3 47S4-2 0.52 7 0.40 5 5.9 80 0.54 7 7.3S4-3 0.33 10 0.16 5 2.7 79 0.21 6 3.4

50 ppm MMAS5-1 1.3 4 1.4 4 29 87 1.8 5 33S5-2 0.43 5 0.15 2 8.3 87 0.71 7 9.6S5-3 0.29 1 0.35 2 18 90 1.3 6 21S5-4 1.2 13 0.31 3 7.2 80 0.32 4 9.0

S1-1–Xylem sap sample blank no. 1.

lation did not occur inside the plants. MMA remained theprimary species ranging from 79 to 95% of the total arsenicconcentration when treated with either 10 ppm or 50 ppmMMA. Furthermore, DMA was also discovered in the xylemsap treated with MMA. The concentration of DMA was far


above the detection limit of this HPLC–ICP-MS method,which was 0.4 ppb, and accounted for 2–5% of the total ar-senic concentration. This is in agreement with the conversionof MMA to DMA in Chinese brake fern root exudate dis-cussed inSection 3.3. Our results suggest that methylationis possible in the Chinese brake fern. The relative standarddeviation of the total arsenic concentration ranged from 5to 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 notobserved in the Chinese brake fern, conversion of DMAor MAA to As(III) or As(V) was observed. However, it isunclear how the conversion occurred.

4. Conclusion

HPLC–ICP-MS has been successfully used to performarsenic speciation in the root exudates and xylem saps ofChinese brake fern after they were exposed to arsenic for2–3 days in a hydroponic system. Our results confirmed thatHPLC–ICP-MS was a reliable method that resulted in a lowdetection limit and rapid analysis for arsenic speciation inplant and aqueous samples.

When Chinese brake ferns were treated with either As(V)or As(III), the primary arsenic species in the root exudateswas As(V), i.e. little arsenic reduction occurred in or outsidethe roots, whereas significant arsenic oxidation occurred ei-ther in or outside the roots (Tables 2 and 3). This is consistentwith our hypothesis that arsenic reduction mostly occurredin the fronds. When treated with DMA, DMA remained asthe dominant arsenic species with no MMA being detectedin the root exudates or in the xylem sap, suggesting DMAis a stable arsenic form that the plant was unable to convertto inorganic forms.

On the other hand, when treated with MMA, DMAwas detected in both the root exudates or the xylem sap(Tables 3 and 4), suggesting methylation was possible inthis plant. The results from the root exudates suggested thatboth arsenic reduction and oxidation occurred either insidethe roots or in the solution. If the latter is the case, then

microbial-mediated arsenic reduction and oxidation mayplay a role. The results with root exudates and xylem sapdata suggested that DMA was more toxic to Chinese brakesince it failed to convert to other less toxic forms, and theplant was capable of methylation (conversion from MMAto DMA) in addition to converting MMA to As(III) andAs(V) in the roots or in the xylem.

Acknowledgements

This research was supported in part by the National Sci-ence Foundation (Grant BES-0132114).

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