extraction and speciation of arsenic in plants grown on arsenic contaminated soils
TRANSCRIPT
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Talanta 72 (2007) 1507–1518
Extraction and speciation of arsenic in plants grownon arsenic contaminated soils
Kalam A. Mir a, Allison Rutter b,∗, Iris Koch c, Paula Smith c,Ken J. Reimer c, John S. Poland b
a Department of Chemistry, Queen’s University, Kingston, ON, Canadab School of Environmental Studies, Queen’s University, Kingston, ON K7L 3N6, Canadac Environmental Sciences Group (ESG), Royal Military College, Kingston, ON, Canada
Received 18 October 2006; received in revised form 26 January 2007; accepted 28 January 2007Available online 13 February 2007
bstract
A sequential arsenic extraction method was developed that yielded extraction efficiencies (EE) that were approximately double those using currentethods for terrestrial plants. The method was applied to plants from two arsenic contaminated sites and showed potential for risk assessment
tudies. In the method, plants were extracted first by 1:1 water–methanol followed by 0.1 M hydrochloric (HCl) acid. Total arsenic in plant andoil samples collected from contaminated sites was mineralized by acid digestion and detected by inductively coupled plasma-atomic emissionpectrometry (ICP-AES) and hydride generation-atomic absorption spectrometry (HG-AAS). Arsenic speciation was done by high performanceiquid chromatography coupled with HG-AAS (HPLC–HGAAS) and by HPLC coupled with ICP-mass spectrometry (HPLC–ICP-MS). Spikeecovery experiments with arsenite (As(III)), arsenate (As(V)), methylarsonic acid (MA) and dimethylarsinic acid (DMA) showed stability of thepecies in the extraction processes. Speciation analysis by X-ray absorption near edge spectroscopy (XANES) demonstrated that no transformationf As(III) and As(V) occurred due to sample handling. Dilute HCl was efficient in extracting arsenic from plants; however, extraction andetermination of organic species were difficult in this medium. Sequential extraction with 1:1 water–methanol followed by 0.1 M-HCl was most
seful in extracting and speciating both organic and inorganic arsenic from plants. Trace amounts of MA and DMA in plants could be detected byPLC–HGAAS aided by the process of separation and preconcentration of the sequential extraction method. Both organic and inorganic arsenicompounds could be detected simultaneously in synthetic gastric fluid extracts (GFE) but EEs by this method were lower than those of the sequentialethod. The developed sequential method was shown to be reliable and applicable to various terrestrial plants for arsenic extraction and speciation.2007 Elsevier B.V. All rights reserved.
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eywords: Arsenic; Plants; Extraction; Speciation; Sequential extraction
. Introduction
Arsenic has been known to be toxic since ancient times.his toxicity can make the high levels of arsenic found inlants growing on many contaminated sites [1,2] problematicor plant consumers. Many arsenic compounds present in theerrestrial and marine environments have been detected [3,4].s(III), As(V), methylarsonic acid (MA) and dimethylarsinic
cid (DMA) are the predominant arsenic species found in terres-rial biota, including plants [2,3]. Chronic exposure to arsenic,articularly inorganic arsenite (As(III)) and arsenate (As(V)),
∗ Corresponding author. Tel.: +1 613 533 2642; fax: +1 613 533 2897.E-mail address: [email protected] (A. Rutter).
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as been implicated in many physiological disorders and variousypes of cancers [5–7]. Since the toxicity of arsenic is dependentn its chemical form, both speciation and quantitative determina-ion of arsenic are essential [8–10]. Furthermore, arsenic muste extracted from solid samples and presented in an aqueousorm for speciation analysis by the more common methods suchs high performance liquid chromatography (HPLC) with ele-ent specific detection [3,11]. The extraction of arsenic from
olid matrices is a critical step since high recoveries as wells species preservation are required [3,11–14]. The diversity ofrsenic and plant species in the environment as well as locations
f sampling only further complicate the situation [15–18].Solvent extractions aided by physical shaking or sonicationave been the traditional methods for extracting arsenic fromhe solid matrices. A number of extraction methods are based
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n mixtures of methanol and water, a relatively mild extrac-ant, aimed at maintaining the integrity of arsenic species inhe samples [3]. Despite the popularity of methanol–water asn extractant for most terrestrial plants, it has a poor extrac-ion efficiency [12,16–18]. Dilute (0.3 M) phosphoric acid wasound to be a convenient extractant of arsenic species from ter-estrial plants [15]. Combinations of acetonitrile–water [13,14]nd methanol–water–chloroform [10] have been used as well.xtraction efficiency varies widely depending on matrix andxtractant.
Pressurized liquid extraction (PLE), also known as acceler-ted solvent extraction (ASE), is a rapid technique of solventxtraction at elevated pressure and temperature. The extrac-ion efficiencies of PLE varied widely depending on sample
atrix; extraction efficiencies of 80–102% for freeze-dried car-ots [13], 33% for rice [19] and 30% for fresh plant materials [20]ere obtained. Modification of sample matrix with chemical
eagents such as acid [14,19] and base [14] for the improvementf extraction efficiency is another approach that may, however,ompromise species integrity.
Enzymes such as alpha-amylase for cellulose have also beensed for the modification of the plant matrices [13,19,21]. Treat-ent by the cellulase yielded an extraction efficiency of 104%
or freeze-dried apples [13] and 59% for rice samples [19]. Thenzymatic treatment did not improve the extraction efficiency ineaweed (kelp) [21] and enzymes are generally expensive.
Even if all the arsenic in a sample can be extracted and char-cterized, the toxicological risk of arsenic to either humansr ecological receptors is dependent on its bioavailability.ioaccessibility, the fraction of a dose that is soluble in theastrointestinal environment [22] is considered to be a good esti-ate of arsenic bioavailability, since dissolved arsenic is almost
ompletely absorbed into the bloodstream from the gastroin-estinal tract [23]. Most bioaccessibility methods involve thextraction of a matrix (predominantly soils) with synthetic gas-ric fluid (e.g., diluted HCl at pH 1.5–4.0) at typical solid/liquid
ixtures (e.g., 1:100) and at body temperature (37 ◦C forumans) for appropriate lengths of time (1 h for gastric phase,h for small intestinal phase) [24–27]. Bioaccessibility rangesidely for soils (<1–100%) and may be linked to pH and ironxide content; ranges are smaller for algae (30–80%) and areependent on algal species [27].
Although many individual plant species and foods have beenxtracted using the various methods, there remains a need forore effective extraction methods that can be applied to terres-
rial plants [3,28]. The goal of the present work was to developore effective but simple methods for arsenic extraction from
errestrial plants, particularly for plants from sites with high lev-ls of arsenic contamination. In order to be applicable to riskssessment approaches to arsenic impacted sites, these methodseed to be highly reproducible, rugged, and use instrumenta-ion that is readily available, and cost effective, in commercialaboratories. In the present study, various extractants were
ested for arsenic extraction, and a sequential extraction methodith 1:1 water–methanol and dilute HCl was developed andpplied to extract and speciate arsenic from terrestrial plantamples.
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. Experimental section
.1. Reagents and apparatus
All solutions were prepared with distilled deionized waterDDW) (Barnstead, E-pure, 18 M� or Millipore 18 M�).
ethanol was Fisher Scientific pesticide or HPLC grade. ICP-ES standards were procured from commercial sources. HNO3
nd HCl were ACS reagents from Fisher Scientific. H3PO4,H3 and HCl used for HPLC–HGAAS experiments were Fluka
nalytical reagents. NaBH4 (BDH analytical reagent) solutionsere prepared fresh before each analysis. Arsenic standardsere procured from commercial vendors. Arsenic species stan-ards were from different sources; As(III) was from arsenicxide AAS standard (SCP Science and Fluka Chemica), As(V)as from sodium arsenate (Na2HAsO4·7H2O, Sigma–Aldrich),MA was monomethylarsonic acid (CH5AsO3, Pfaltz andauer), DMA was dimethylarsinic acid (cacodylic acid,2H7AsO2, Fluka Chemica).
The plant samples were weighed in 15 mL extraction tubesCorning plastic), mixed with solvent by vortexing (Thermolyne
axi Mix II) and sonicated in a Fisher Scientific FS 2850/60 Hz) sonicator. Solid and liquid phases were separatedy centrifuging at 3000 rpm (IEC/Centra®/MP4 Internationalquipment Company, USA). The HG-AAS analysis for totalrsenic was carried out with an AA/AE spectrophotometerInstrumentation Laboratory, Allied Analytical Systems, USA)quipped with a vapor generation accessory (VGA-76, Var-an) for hydride generation and separation; atomization waserformed in an air–acetylene flame. The ICP analyses wereonducted by a simultaneous ICP-AES instrument (VISTAX CCD, Varian). HPLC–HGAAS experiments were per-
ormed using an anion exchange column (Hamilton PRP-x10050 mm × 4.6 mm). Mobile phase was 20 mM ammonium phos-hate buffer at pH 6.0 and flow rate 1.5 mL min−1. Arsines wereetected by a SOLAAR 969 AAS instrument equipped withP90 vapor generator accessory, an EC90 furnace and quartz-tube and run using SOLAAR software (Thermo Instruments,anada). HPLC–ICPMS analyses were performed by connect-
ng the HPLC to a PQ Excell ICPMS. All instrument operatingonditions were given elsewhere [29].
.2. Samples
Plant samples used for the method development were col-ected from an abandoned gold mining area at Deloro, ON,anada. The plant samples used to test the developed sequentialxtraction method were from Deloro and from gold mining areasn Yellowknife, NWT, Canada. Plant samples were hand pickednd stored for 1–3 days in plastic bags at 4 ◦C until processing.urface (0–10 cm) soil samples from Deloro were collected inhirlpaks® and stored at 4 ◦C. Plants were cut to remove roots
rom stems and washed with copious amounts of distilled water
o remove the adhered soil and dust particles. Washed plant andoil samples were air dried in the laboratory. Dried plants wereround with a laboratory grinder and were stored in plastic bagsn the freezer for up to one year at −20 ◦C to ensure stability
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f the arsenic species. Dried soil samples were homogenized byortar and pestle before sampling for analysis.
. Determination of total arsenic
Total arsenic in all plant and soil samples was determined bycid digestion. Portions of ground plant samples were weighedccurately (0.5 ± 0.0001 g) in 30 mL Vycor® glass cruciblesnd ashed in the muffle furnace (Isotemp® Programmable Muf-e Furnace, Fisher Scientific) using a temperature ramp to00 ◦C. The ashed plant samples were digested with 4 mL 1:3NO3/HCl (aqua regia) solution for 4 h on a hot plate underatch glass covers. After removing the watch glass covers,–3 drops of 50% H2O2 were added and the volumes reducedo approximately 2 mL. All samples were then quantitativelyransferred to graduated glass tubes and volumes were madep to 12.5 mL with DDW, filtered (Fisherbrand® Quantita-ive, medium porosity) and analyzed by ICP-AES or HG-AASepending on arsenic concentration. All dissolved arsenic in thealibration standards and sample solutions were reduced to AsIII) in the prereduction step using hydroxylamine hydrochlo-ide (masking agent), potassium iodide and concentrated HClo ensure maximum HG-AAS sensitivity. HG-AAS was usedor the low arsenic samples because of its higher sensitivity.ertified reference plant materials (CRM GBW 07603: Bushranches and Leaves and SRM NIST 1575: Pine Needles) werenalyzed following the above procedure. Dried soil samples0.5 ± 0.0001 g) were digested with 8 mL aqua regia solutionvernight in graduated digestion tubes heated on an aluminumlock. Digested residues were made up to 25 mL with DDW andfter filtration analyzed by ICPAES. The 1:1 water–methanolxtracts in batches were completely dried at ∼60 ◦C in an oveno remove solvent and regenerated in distilled-deionized waterDDW) in preparation for total arsenic analysis and speciation.ll other extracts were analyzed directly by HGAAS, ICPAESr HPLC–HGAAS.
. Extraction of arsenic from plants
.1. Solvent-sonication extraction
The solvents used to extract arsenic from plant matrices wereater (DDW), methanol, 1:1 water/methanol, 0.1 M-, 0.05 M-0.02 M- and 0.01 M-HCl solutions, and 0.1 molar sodium
ydroxide (M-NaOH). Initially, 0.5 g, and after method opti-ization 0.25 g, of powdered samples were accurately weighed
nto 15 mL extraction tubes and 10 mL of extractant was intro-uced to the samples. After closing the lids securely, samplesnd extractants were mixed thoroughly by a vortex apparatusor a minute. The samples were then sonicated for 20 min in therst step of extraction. The resulting mixtures were centrifugedt 3000 rpm for 10 min for all extraction steps. In the subsequentteps of extraction, undissolved sample matrices that settled at
he bottom of the tube due to centrifugation were redispersed intohe extractant solvent by vortexing and tapping/shaking if nec-ssary, and were sonicated for 10 min. The supernatant solutionsrom centrifugation from each step were collected in respectiveuawi
(2007) 1507–1518 1509
ample vials. Extraction of samples was carried out five times (5teps) initially, and then three times (3 steps) after method opti-ization. The extraction solutions were filtered before analysis.fter the initial trial of the time-consuming roto-evaporation
echnique, the 1:1 water–methanol extracts were dried in batchesn the oven (∼65 ◦C) to remove methanol and regenerated inmaller volume (3–5 mL) of DDW than the extractant volume30 mL). Spike recoveries were not affected by this process; thendividual standard spike results were within ±10% of the intro-uced concentrations. The drying and regeneration processesmproved the detection limits of the trace organic species by aactor of ten.
.2. Solvent-Soxhlet extraction
The powdered samples, 0.5 or 0.25 g portions, were accu-ately weighed into cellulose thimbles (Fisherbrand® 25 mL)or Soxhlet extraction. About 70 mL solvent was employedn 125 mL distillation flasks and heated on electric coils tochieve four cycles per hour. Water (DDW), methanol and 1:1ater–methanol were used as solvents for Soxhlet extractions
hat lasted for 6 h for each batch. Exhaustive Soxhlet extractionas demonstrated by extracting for 2, 4, 6 and 12 h with water
s solvent.
.3. Gastric fluid extraction (GFE)
GFE was carried out using synthetic gastric fluid solutiononsisting 1.25 g L−1 pepsin (Sigma P7000: activity 1:10000)nd 8.77 g L−1 NaCl. The pH of solution was adjusted to 1.8ith concentrated HCl [30]. Powdered plant samples wereeighed (0.5 ± 0.0001 g) into 50 mL centrifuge tubes and 20 mLortions of gastric fluid were added. The samples were vor-exed for one minute and then shaken at 250 rpm for 60 mint 37 ◦C in an incubator shaker (Innova Refrigerated Incubatorhaker, Brunswick). The resulting solutions were centrifugedt 3000 rpm for 10 min and the supernatants were decanted in00 mL plastic bottles. The extracts were filtered and analyzedor total arsenic and arsenic species.
. Arsenic speciation analysis (HPLC–HGAAS)
Standards and plant extracts were introduced in a 100 �Lample loop using 1 mL syringes fitted with the syringe filtersMillipore Milex-HV Hydrophilic PVDF 0.45 �m) and injectednto the HPLC column. The elution order in retention time wass(III) < DMA < MA < As(V), as reported in Fig. 3(A). Hydrideeneration was achieved by mixing the eluent from HPLC col-mn with 1 M-HCl and 1% NaBH4 stabilized in 0.1% NaOH.PLC chromatograms were generated from AAS data by usingmacro in MS Excel. Mixed standards of As(III), As(V), MA
nd DMA were prepared from concentrated stocks in 50, 100,00 and 500 �g L−1 concentrations by mixing appropriate vol-
mes in DDW. Identification of arsenic species in extracts wasccomplished by matching the peak retention times of standardsith those in the sample extracts. The reliable detection lim-ts (RDL = 2(t-stat)(S.D.)) were determined to be 7, 31, 15 and
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Fig. 2 illustrates extraction efficiencies for solvent–sonicationextractions using methanol, 1:1 water/methanol, water (DDW),GFE extractant, and 0.1 M-NaOH and HCl solutions of 0.1 M-,0.05 M-, 0.02 M- and 0.01 M concentrations. The extraction
510 K.A. Mir et al. / Tala
4 �g L−1 for As(III), As(V), MA and DMA, respectively. The t-tat is the value (at n − 1 degree of freedom and single-sided 95%onfidence limit) taken from Student’s t-table and S.D. is stan-ard deviation of n (8) replicate runs of the lowest concentration50 �g L−1) of calibration standards.
. Quality assurance and quality control (QA/QC)
The sample preparations and analytical procedures wereccompanied by blanks and spiked standards. Samples werenalyzed in two or three independent replicates to ascertaineproducibility of the analytical results. The number of dupli-ates and blanks in each analysis ranged from 15% to 25%f the samples analyzed. Sample and spike concentrationsere calculated using linear equations (r2 ≥ 0.999) obtained
rom the external calibration curves. The total amounts ofrsenic in a number of reference materials [CRM GBW 07603ush Branches and Leaves, 1.25 �g g−1 total As; SRM NIST575 Pine Needles, 0.21 �g g−1 total As; and IAEA-140/TMeaweed (Fucus sp.), 42.2–46.4 �g g−1 total As] were deter-ined. The four-point linear calibration range of ICP-AES was
.10–5.0 �g g−1. Except for the concentrations close to theetection limits, results were within 10% of the certified values.
The results of total arsenic in a number of plants determinedy the ashing and aqua regia digestion method and a concen-rated HNO3 acid digestion method [31] were compared. Theoncentrations of arsenic of the CRM by the wet method rangedrom 90% to 100% of the certified results and no significant dif-erence was found between the results from the two methods; andherefore the ashing method that removed plant organic materi-ls by dry ashing was used as it was more convenient to follown this lab than the wet HNO3 method.
Mass balance experiments comprising determinations ofrsenic in the extracts and plant residues accounted for the totalrsenic (99–111% recovery) and showed insignificant loss orain of arsenic during analyses involving several steps.
. X-Ray absorption near-edge spectroscopy (XANES)
XANES spectra of plant samples containing arsenic levelsbove the XANES detection limit of approximately 10 �g g−1
ere collected at the bending magnet beamline of Pacificorthwest Consortium Collaborative Access Team (PNC-CAT),ector 20, at the Advanced Photon Source, Argonne Nationalaboratory in Illinois [32].
Subsamples were loaded into wells of an aluminum sam-le holder, and sealed in place using KaptonTM tape. The plateontaining the samples was positioned in the path of the X-rayeam, at a 45◦ angle to both the beam and the detector (the latteras positioned 90◦ to the beam). A silicon (1 1 1) double-crystalonochromator (resolution ∼5000 E/dE at 12 keV, detuned to
5% of maximum at 12,100 eV) and a rhodium-coated har-
onic rejection mirror provided X-rays for measurement. Theonochromator was calibrated using the gold L3 absorptiondge (11919.7 eV)) [33] for measurements at the arsenic K-dge (11,868 eV). Incident X-ray beam size was defined by a
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mm vertical and 4 mm horizontal slit after the mirror. Nitrogen-lled transmission ionization chambers were present before andfter the samples for normalization to the incident intensitynd transmission measurements, respectively. Fluorescence dataere collected using a solid-state Ge(Li) detector (Canberraodel GL0055PS) or an argon-filled fluorescence ionization
hamber. Typically, five scans were collected and averagedefore background-removal and normalization-to-edge-jump.he WinXAS program [34] was used for processing the spectra.
. Results and discussion
.1. Extraction efficiencies of solvents by Soxhlet andonication processes
Arsenic concentrations in soil samples collected from theeloro site ranged from 335 to 100,000 �g g−1. The very highs concentration was found in the soil of an arsenic tailingond near the gold mining and processing area. Plant samplesad concentrations ranging from 2.3 to 241 �g g−1 (dry weight)rsenic. Three plants were selected for further extraction exper-ments because the species were representative of plants grownn the arsenic contaminated soils at this site and because theirigh concentration would simplify the analysis of arsenic speciesithin the plant. The three Deloro plants were P-1, field horse-
ail (Equisetum arvense, 241 �g g−1 total As), P-3, an alkalirass (Puccinellia sp., 146 �g g−1 total As) and P-6, variegatedorsetail (Equisetum variegatum, 24.4 �g g−1 total As).
Soxhlet and sonication methods are used in the extraction ofetals from plant matrices [35,36]. Of the two, currently the son-
cation technique has been more commonly employed in toxicetal extractions [3]. The extraction efficiency (EE) of a partic-
lar solvent is defined as the percentage of total arsenic extractedy the solvent from a plant sample. Results of extraction experi-ents for a number of solvents are shown in Figs. 1 and 2 for theoxhlet and sonication experiments, respectively. Of the threeolvents tested in the 6-h Soxhlet extractions, water extractedhe most arsenic from the plant samples. In contrast, methanolhowed considerably lower extraction efficiencies (Fig. 1).
ig. 1. Extraction efficiencies (EE) of different solvents in solvent-Soxhletethod for three plants are presented. Error bars are ±1 S.D. from three inde-
endent determinations.
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ig. 2. Extraction efficiencies (EE) of different solvents in solvent-sonicationethod for three plants are shown. Error bars represent ±1 S.D. from three
ndependent determinations.
esults showed that the water-Soxhlet method was moreffective than water-sonication method. The higher efficiency ofhe water-Soxhlet method was likely because the former was anxhaustive extraction technique carried out at a higher tempera-ure. The water–methanol solvent has been traditionally used asn extractant in terrestrial plant studies, particularly for the envi-onmentally focused studies [20,37]. While water–methanol haseen shown to be effective for biological samples such as lobsterissues [36], the data presented here indicated that other solventsere more effective for terrestrial plants.In another study [14] NaOH solution has been shown to pro-
ide higher EE compared to the other extractants (e.g., aceticcid, EDTA, tetrabutylammonium hydroxide, acetonitrile/H2O,nd MeOH/H2O) in the extraction of arsenic from plant materi-ls. EE’s of 22%, 32% and 36% were obtained from freeze-driedoplar leaves, pine shoots, and spruce shoots, respectively. Inhe present study, 0.1 M-NaOH performed similarly to waterith EE’s 12%, 56% and 79% for plants P-1, P-3 and P-6,
espectively. In contrast, introduction of 0.1 M-HCl induced aarked improvement (92%, 104% and 115%) in the EE’s for
he plants (Fig. 2). Concentrated HCl has been used to extractnorganic arsenic from biological samples [38,39] where HClelped to solubilize the sample and aided in breaking up theonds between As(III) and the thiol groups of proteins [13].ilute HCl solutions have not been used as an extractant for
rsenic from terrestrial plants. However, our data indicated thatt was an effective extractant for arsenic in terrestrial plants.
In a previous study [11], it was observed that the percentf arsenic extracted was inversely related to the total arsenicontent of the plant. A reason for this trend was thought to beue to the induced chemical and/or physical bonding of arsenico the plant matrix and/or sequestration in vascular plant tis-ues due to the increased exposure of the plant to arsenic. Inther studies, it has been demonstrated that both As(III) ands(V) efficiently induced the biosynthesis of phytochelatins
[�-glutamate-cysteine]n-glycine) synthesized from reducedlutathione (GSH) in plants and that arsenic–phytochelatinAs–PC) were present in weak acid extracts [40,27,41]. As(III) isound to coordinate with the sulphur (S) of thiol groups (SH) of
he PC in various proportions. The cause of higher arsenic extrac-ion efficiency of dilute HCl may be due to its ability to break uphe As–S bond of the various As–S bond-containing complexesn plant tissues. Interestingly, much less arsenic was complexedp[ti
(2007) 1507–1518 1511
o phytochelatins in an arsenic hyperaccumulator plant, whichs presumably more tolerant to arsenic [41].
.2. Study of arsenic species spiked on a plant matrix
Given the relationship between arsenic species and toxicity,t is critical to assess the effect any extraction method has on thendividual arsenic species. The arsenic concentrations in theselants are high and there are no similar terrestrial plant refer-nce materials with comparable arsenic values. Because theres no agreement on extraction methods, there are no referencealues for individual species in any of the available terrestriallant reference materials. Marine algae, which have proportionsf carbohydrates, lipids and proteins similar to terrestrial plants,re available but their speciation is vastly different. The pre-ominant arsenic species found in terrestrial plants are As(III),s(V), MA and DMA; therefore, these four compounds were
piked onto a plant matrix in a series of experiments designedo assess the effect of the solvents and extraction processes onpeciation. Spiking was done with prepared solutions of theseour individual arsenic species and results are shown in Table 1.
Recovery experiments were conducted using a plant matrixaving comparatively low arsenic concentration (1.8 �g g−1)nd spiking with the standard arsenic species. The spikedoncentrations, which ranged from 6.0 to 120 �g g−1, wereetermined on the basis of 0.5 g dry plant sample. Spikes werextracted following the same procedures that were used forhe extraction of plant matrices. Soxhlet extractions with waternd sonication extractions with water, 1:1 water–methanol and.05 M-HCl as extractants were carried out.
Total concentrations of the inorganic arsenic species, As(III)nd As(V), were determined by HG-AAS and those of therganoarsenic species, DMA and MA, were done by ICP-AESor the spike-experiments. The spike-speciation experimentsere performed by HPLC–HGAAS. Results of spike recovery
total arsenic), reported in Table 1, show quantitative recovery ofrsenic species ranging from 90 to 110% for the extraction mediand methods employed. Overall, the HPLC–HGAAS speciationata also showed good total arsenic recovery (sum of speciesoncentrations), suggesting that the extraction procedures usedere mild and did not significantly alter the arsenic speciation.xidized or reduced forms of the introduced inorganic speciesere not detected in the 1:1 water–methanol sonication and00% water-sonication extracts.
However, in the 0.05 M-HCl sonication and 100% water-oxhlet extracts a small amount of interconversion (2.5–5%) of
he inorganic arsenic species was observed. The reduced formf As(V), that is As(III), was detected in the extracts of 0.05 MCl-sonication (Table 1). The source of the arsenite could be thelant matrix spiked, but since As(III) was not observed in thether extracts analyzed, it is likely that in this case the extrac-ion process caused the reduction of arsenate to arsenite. It haseen shown that under slightly reducing conditions and/or lower
H, As(III) can become more stable mainly as neutral H3AsO342]. It appeared that species interconversion occurred also inhe 100% water-Soxhlet extraction where As(V) was detectedn the As(III) spike. Interconversion between As(III) and As(V)
1512 K.A. Mir et al. / Talanta 72 (2007) 1507–1518
Table 1Results of spike recovery experiments
Extraction medium-method Spikes Spiked conc. Recovered spike conc.(HG-AAS/ICP-AES)
Recovered spike conc. (HPLC–HGAAS)
As(III) As(V) DMA MA
1:1 Water–methanol-sonication As(III) 120 132 ± 6 144 ± 6 nd nd ndAs(V) 120 120 ± 12 nd 132 ± 6 nd ndDMA 60.0 58.8 ± 3.6 nd nd 56.4 ± 6.6 ndMA 12.0 11.4 ± 1.2 nd nd nd 14.4 ± 0.6
0.05 M-HCl-sonication As(III) 120 132 ± 6 144 ± 6 nd nd ndAs(V) 120 126 ± 6 7.8 ± 1.8 144 ± 6 nd ndDMA 12.0 11.7 ± 0.2 nd nd 13.2 ± 0.6 ndMA 12.0 11.9 ± 0.1 nd nd nd 16.2 ± 0.6
100% Water-sonication As(III) 120 126 ± 6 144 ± 6 nd nd ndAs(V) 120 129 ± 2 nd 132 ± 6 nd ndDMA 12.0 12.0 ± 0.6 nd nd 12.6 ± 0.6 ndMA 12.0 12.2 ± 0.2 nd nd nd 16.3 ± 0.2
100% Water-Soxhlet As(III) 120 120 ± 1 137 ± 1 3.6 ± 1.8 nd ndAs(V) 120 120 ± 6 nd 108 ± 6 nd ndDMA 60.0 52.8 ± 2.4 nd nd 60.0 ± 6.0 nd
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pAtwith the As(III) spike data for water-Soxhlet where some con-version of As(III) to As(V) was observed. The reducing and/orstabilizing nature of chloride ion for As(III) was shown by thepresence of As(III) in all HCl media. Both As(III) and As(V)
MA 6.0 6.5 ±
piked and recovered spike concentrations (average ± 1 S.D. from three indepetected.
n water [43] and the influence of pH and matrix on the stabilityf inorganic arsenic species has been demonstrated by spik-ng the species in wastewater samples [44].The transformationnd underlying uncertainties in predicting such transformationsf the inorganic arsenic species in aqueous samples have beeniscussed [3].
.3. Speciation of arsenic species in plant extracts
The results of arsenic in the extracts of P-1, P-3 and P-6 byhe various extraction methods have already been discussed (seeigs. 1 and 2). The extracts were analyzed for arsenic species byPLC–HGAAS, and the concentrations of individual species asell as total arsenic concentrations in the extracts are reported
n Table 2. The sums of arsenic concentrations of the speciesgree well with the totals determined by ICP-AES. The majorityf arsenic extracted was inorganic arsenite and arsenate. Therganoarsenic species, MA, was detected in 1:1 water–methanolxtracts of P-1 (Table 2).
Although the EE of 1:1 water–methanol was low, only therganoarsenic species (MA) that was detected in the targetlant P-1 was extracted in this medium. This observation wasmportant since it indicated that small concentrations or tracerganoarsenic species could be detected in the water–alcoholedium which suppressed the extraction of inorganic species.epresentative chromatograms of standard arsenic species alongith the chromatogram of arsenic in two plant extracts of 1:1ater–methanol are presented in Fig. 3. In terrestrial plants, therganoarsenic species that are usually encountered are MA andMA [45]. For example, up to 2% MA and DMA were mea-
ured in shoots and roots of Holcus lanatus and Arabidopsishaliana [46].
In the acid media, concentrations of As(V) predominated overhose of As(III) in all cases except for plant P-6. For P-6, As(III)
FsTcp
nd nd nd 7.8 ± 0.6
t determinations) are reported in �g g−1 dry sample. nd indicates species not
redominated over As(V) in all but the 0.1 M-HCl medium. Nos(III) was detected in water-Soxhlet extracts. This may be due
o the oxidizing capability of Soxhlet extraction and is consistent
ig. 3. Representative HPLC–HGAAS chromatograms. (A) Standard arsenicpecies, (B) 1:1 water–methanol extract of plant YK-11, and (C) DL-129 (seeables 4 and 5 for plant information). HPLC was performed with anion exchangeolumn (Hamilton PRP-X100 250 mm × 4.6 mm column), 20 mM ammoniumhosphate, pH 6.0 at 1.5 mL min−1.
K.A. Mir et al. / Talanta 72 (2007) 1507–1518 1513
Table 2Speciation of arsenic extracted from plant samples
Extraction method Plant ID Amount of As extracted As by HPLC–HGAASa Sum of species
As(III) As(V)
100% water-sonication P-1 24 ± 2 nd 21 ± 1 21 ± 1P-3 34 ± 3 nd 29 ± 3 29 ± 3P-6 18 ± 4 13 ± 1 6.3 ± 0.2 19 ± 1
0.01 M-HCl-sonication P-1 108 ± 2 5.9 ± 1.1 96.1 ± 0.2 102 ± 1P-3 117 ± 4 11.9 ± 0.9 97 ± 5 109 ± 9P-6 28 ± 2 16.7 ± 0.1 10 ± 1 27 ± 1
0.02 M-HCl-sonication P-1 184 ± 3 24 ± 14 169 ± 24 193 ± 38P-3 122 ± 8 36 ± 3 100 ± 21 136 ± 24P-6 24 ± 1 15 ± 1 12.3 ± 0.2 27 ± 1
0.05 M-HCl-sonication P-1 202 ± 6 15 ± 1 184 ± 6 199 ± 7P-3 153 ± 6 29 ± 1 114 ± 6 143 ± 7P-6 29.3 ± 1.3 21.6 ± 0.3 12.5 ± 1.4 34 ± 2
0.1 M-HCl-sonication P-1 223 ± 11 12 ± 1 208 ± 8 220 ± 9P-3 153 ± 7 31 ± 8 143 ± 8 174 ± 16P-6 28 ± 1 8 ± 2 27 ± 1 35 ± 3
100% water-Soxhlet P-1 160 ± 7 nd 150 ± 16 150 ± 16P-3 118 ± 4 nd 100 ± 14 100 ± 14P-6 26 ± 1 nd 22 ± 4 22 ± 4
1:1 water–methanol-sonication P-1 7.7 ± 1.0 1.7 ± 0.8 5.6 ± 0.4 8.8 ± 1.2b
P-3 10.0 ± 0.5 5.6 ± 1.5 6.5 ± 0.8 12 ± 2P-6 9.7 ± 0.5 10.8 ± 0.6 2.1 ± 0.3 13 ± 1
Results are average ±1 S.D. from three independent determinations. Concentrations are in �g g−1. nd = not detected.g g−1
wioawHgE
8
tahecseTTa
1e1p
altowdetci
8
iaocpit
m
a MA was detected only in the water–methanol extracts of P-1 (1.51 ± 0.04 �b Sum includes MA concentration. DMA was not detected in any extract.
ere present in 1:1 water–methanol extracts of all plants indicat-ng the milder nature of the solvent. HPLC–ICPMS speciationf arsenic in Deloro plants was performed to look for additionalrsenic species, however, only As(III), As(V), MA and DMAere detected, confirming the HPLC–HGAAS results. In Fig. 4,PLC–ICPMS chromatograms of two plants DL-119 (Canadaoldenrod, Solidago Canadensis) and DL-207 (field horsetail,quisetum arvense) are shown.
.4. Gastric fluid extraction
Extraction efficiencies of synthetic gastric fluid are usedo estimate the bioavailability of arsenic in the human stom-ch and intestines [47,48]. Gastric fluid extractions, modelinguman gastrointestinal conditions, have been carried out toxtract arsenic from plant matrices [11]. GFE extractions areurrently being used in risk assessments of arsenic contaminatedites [49]; therefore, it is important to compare the extractionfficiency of the various extracts used in this study to GFE.he speciation results of the GFE experiments are presented inable 3. The EE of GFE was comparable to that of 100% waters was shown earlier in Fig. 2.
The average (n = 3) amount of MA extracted from P-1 was
.4 ± 0.1 �g g−1 and compared favourably with the amountxtracted by the 1:1 water–methanol sonication method of.51 ± 0.04 �g g−1. Thus, the synthetic gastric fluid showedotential for simultaneous analysis of the organic and inorganiceaam
).
rsenic in plant samples. Although GFE extracted significantlyess arsenic from the terrestrial plants than 0.05 M-HCl did, it hadhe advantage of extracting both organic and inorganic arsenic inne extraction. Unlike water–methanol extracts where methanolas removed prior to analysis, GFE extracts were introducedirectly to the ICP and HPLC column for analysis. Furtherxperiments with GFE, however, showed generally low extrac-ion efficiencies from the various terrestrial plants, suggesting itould not be used to characterize the speciation of all the arsenicn a plant sample.
.5. Sequential extraction of arsenic from plants
Although As(III) and As(V) are known to be the predom-nant species in terrestrial plants [3,10,20], MA and DMAre also detected in lower concentrations [3,13,45], and otherrganoarsenic compounds such as arsenobetaine (AB), arseno-holine (AC), and different arsenosugars have been detected inlant tissues [3,50]. Therefore, it is important to extract bothnorganic and organic arsenicals to obtain a broader picture ofhe species in plants.
The present study has revealed that while the water–methanolixture can extract organoarsenic species that dilute HCl appar-
ntly cannot, HCl is more efficient in extracting inorganicrsenic, and therefore, an extraction that includes both is desir-ble. In addition, the organoarsenic species in general exist inuch smaller amounts than the inorganic species in the ter-
1514 K.A. Mir et al. / Talanta 72 (2007) 1507–1518
Fig. 4. HPLC–ICPMS anion exchange chromatograms of: (A) standard solu-tion (20 �g L−1), (B) Deloro plant DL-207 (field horsetail, Equisetum arvense)and (C) DL-119 (Canada goldenrod, Solidago Canadensis). HPLC–ICPMScH
rpiwm(t
etesvAHH
Fig. 5. HPLC–HGAAS chromatograms of field horsetail (P-1): 1:1water–methanol extract (A), water extract (B), and 0.1 M-HCl extract (C). Anal-yses of water extract (B) and acid extract (C) were performed in June (same day);ayt
iM
1eTusf
8plants
TA
S
PPP
A
onfirmed MA in DL-207 and DMA in DL-119 previously determined byPLC–HGAAS.
estrial plants which makes their determination difficult in theresence of concentrated inorganic species. This was evidentn the case of organoarsenic species MA in field horsetail asas demonstrated in the chromatograms in Fig. 5. The chro-atogram of the field horsetail 1:1 water–methanol extract
Fig. 5A) has a prominent peak of organic MA as compared tohe water and 0.1 M-HCl extracts in Fig. 5B and C, respectively.
In the procedures for optimization of the sequential method,xperiments were conducted to find out if a 1:1 v/v combina-ion of 0.1 M-HCl and methanol could be used as a potentialxtractant of both inorganic and organic arsenicals. The resultshowed that the total arsenic extracted by the experimental sol-ent was less than 50% of the amount extracted by 0.05 M-HCl.
rsenic speciation by HPLC–HGAAS revealed no MA in theCl–methanol extracts of P-1 indicating a suppressing effect ofCl on the extraction of the organoarsenic compound. Interest- aable 3rsenic extracted by GFE
ample ID Total As As extracted by GFE Species in GFE e
As(III)
-1 241 ± 9 29.4 ± 2.4 nd-3 146 ± 5 31.3 ± 5.1 4.8 ± 0.1-6 24 ± 1 20.0 ± 0.8 16.5 ± 0.4
rsenic speciation in GFE extracts was done by HPLC–HGAAS. Results are mean ±
nalysis of 1:1 water–methanol extract (A) was performed in August of the sameear. Difference in the retention time of As(V) in the chromatograms was dueo difference in the HPLC conditions (given in Fig. 3).
ngly, HCl was a component of GFE solvent, which releasedA; this contradictory behaviour is unexplained at present.Consequently, a sequential method in which extraction with
:1 water–methanol followed by 0.1 M-HCl was developed toxtract both organic and inorganic arsenic from plant matrices.he extraction by acid prior to water–alcohol was found to benfavourable to the extraction or determination of organoarsenicpecies; therefore, the extraction sequence of water–methanolollowed by acid was adopted.
.6. Application of the sequential method to terrestrial
The sequential extraction results of fifteen plant samples fromrsenic contaminated areas in Yellowknife, NWT and nine plant
xtracts Sum of species in GFE
As(V) MA DMA
28.4 ± 0.1 1.4 ± 0.1 nd 29.8 ± 0.226.8 ± 0.2 nd nd 31.6 ± 0.36.1 ± 1.3 nd nd 22.6 ± 1.7
1 S.D. (�g g−1) from three independent determinations and nd = not detected.
K.A
.Mir
etal./Talanta72
(2007)1507–1518
1515
Table 4Extraction of arsenic from the Yellowknife (YK) and Deloro (DL) plants by sequential extraction method
Plant ID Latin name Common name Total As in plant(�g g−1)
As in 1:1 water–MeOH,(�g g−1)
As in 0.1 M-HCl,(�g g−1)
EE of 1:1water–MeOH
EE of sequentialmethod
YK-1 Agropyron trachycaulum Slender wheat grass 95.2 8.2 20.2 9 30YK-2 Epilobium angustifolium Fireweed 4.6 1.5 nd 33 33YK-3 Archtostaphytos uva-ursi Common bearberry 18.5 1.8 4.8 10 36YK-4 Agropyron trachycaulum Slender wheat grass 8.0 1.5 1.5 19 38YK-5 Senecio vulgaris Common groundsel shoots 38.5 5.8 20.6 15 69YK-6 Agrostis scabra Rough hair grass shoots 47.5 24.9 ± 0.8 25.0 ± 0.8 52 ± 2 105 ± 3YK-7 Picea mariana Black spruce bough/cones 12.6 0.78 ± 0.20 2.8 ± 0.1 6.2 ± 1.5 29 ± 2YK-8 Hordeum jubatum Foxtail barley 4.4 1.2 nd 27 27YK-9 Calamagrostis canadensis Blue joint-1 4.0 2.5 nd 63 63YK-10 Calamagrostis canadensis Bluejoint-2 8.1 3.7 ± 0.2 nd 46 ± 2 46 ± 2YK-11 Calamagrostis canadensis Bluejoint-3 77.7 3.4 ± 0.4 20.2 ± 1.1 4.4 ± 0.5 30 ± 2YK-12 Calamagrostis canadensis Bluejoint-4 3.8 1.1 1.3 29 64YK-13 Calamagrostis canadensis Bluejoint-5 3.0 1.4 1.7 48 105YK-14 Hordeum jubatum Foxtail barley 0.90 0.80 nd 89 89YK-15 Epilobium angustifolium Fireweed 2.1 1.1 1.5 52 128DL-60 Equisetum arvense Field horsetail 24.9 18.5 3.3 74 88DL-101 Equisetum arvense Field horsetail 410 126 ± 12 222 ± 6 30 85DL-105 Verbena hastata Blue vervain 8.4 2.5 0.82 30 40DL-129 Solidago canadensis Canada goldenrod 8.2 1.6 nd 19 19DL-201 Onoclea sensibilis Sensitive fern 48.4 7.8 11.6 16 40DL-204 Equisetum arvense Field horsetail 19.4 6.4 13.4 33 102DL-205 Aster lanceolatus Panicle aster 2.9 0.37 13 nd 13DL-220 Lythrum salicaria Purple loosestrife 18.7 0.67 3.6 2.9 19Dl-221 Equisetum arvense Field horsetail 26.3 9.7 10.8 37 78
Results of mean ± average deviation from two independent analyses for a number of plants show reproducibility of analyses and nd = not detected.
1 nta 72 (2007) 1507–1518
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516 K.A. Mir et al. / Tala
amples from abandoned gold mining areas at Deloro, ON,anada are reported in Table 4. The total arsenic concentrationsetermined by ICP-AES ranged from 1 to 95 �g g−1 and from 1o 530 �g g−1 in the Yellowknife and Deloro plants, respectively.
ost of the Yellowknife plants were grass species; the grassessually consist of silica-rich hard-tissues and are difficult to pro-ess [51]. The 1:1 water–methanol extraction of the Yellowknifelants yielded EEs ranging from 4 to 89% with a median at 29%.he sequential extraction followed by 0.1 M-HCl extracted morersenic from the plants. Had the extractions been carried out onlyith 1:1 water–methanol, the traditional solvent used for plant
rsenic extractions, low extraction efficiency would have beenchieved. From these plants which were mostly grasses, the over-ll EE from sequential extractions ranged from 27 to 128% withmedian at 46% that was almost double of the median EE of 1:1ater–methanol extractions. The arsenic extraction efficienciesf Deloro plants obtained using the sequential method rangedrom 13 to 106% with a median at 70%.
Arsenic in selected plant extracts was speciated byPLC–HGAAS and the results are reported in Table 5. The
esults showed that most of the arsenic in plants was inorganic.he overall predominance of As(V) was observed in the extractsf the plants from both locations. The organoarsenic speciesere detected in the 1:1 water–methanol extracts of the plants;A in YK-1, YK-4, YK-11, DL-204 and DL-221 and DMA inL-105, DL-129, DL-205 and DL-220.The knowledge of chemical and physical states of the
nextracted arsenic in these plants may provide insight intonderstanding the factors that influence extraction of arsenicrom the plant matrix. It is generally believed that the low EE isue to a number of factors such as the insoluble forms of arsenic,he chemical and/or physical bonding of As to the plant matrix,nd the trapping of arsenic compounds inside the plant vascularissues [20,28]. A number of plant samples were examined byANES to better understand the arsenic speciation in the plant
amples.
.7. XANES experiments
The XANES method has been described in Section 7.ANES spectra were collected for a variety of plant samples
t different stages of preparation including: frozen fresh plantsplants frozen after sampling without drying), dry-ground sam-les, and dried residues from the sequential extraction of plants.ANES spectra of three plants are compared to standard arsenic
pecies in Fig. 6. The arsenic species in XANES spectra are gen-rally identified using the position of the main peak feature inhe spectra (Fig. 6).
A comparison of the X-ray absorption peaks of arsenicpecies in the arsenic standards and the fresh frozen and dryround samples of three plants in Fig. 6 revealed no significantransformation in the oxidation states of arsenic species fromhe fresh frozen to the dry ground plant samples. This demon-
trated, within the sensitivity of the XANES method, the stabilityf As(III)/As(V) species in plant samples during handling andample preparations. However, within the As(III) oxidation stateAs(Glu)3 and As2O3) some shifts, indicative of transformation, Table
5Sp
ecia
tion
ofa
Plan
tID
YK
-1Y
K-3
YK
-4Y
K-5
YK
-6Y
K-1
1D
L-6
0D
L-1
01D
L-1
05D
L-1
29D
L-2
01D
L-2
04D
L-2
05D
L-2
20D
l-22
1
Mea
n±
aver
ag
K.A. Mir et al. / Talanta 72
Fig. 6. XANES spectra of fresh frozen, dry ground and dry residue of sequen-tial extraction of sensitive fern (SF), field horsetail (FH) and purple loosestrife(PL). Aqueous standards of arsenic(III)-gluatathione (As(Glu)3 synthesized,s(i
wieprtrpsm
9
mtdmee(rspsecos
A
a
PCfAs9aAiS3w
R
[[
[
[[[
[
[
[
[
[
[
[
[
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[
ee acknowledgement), arsenite (As2O3–Fluka reagent grade) and arsenateKH2AsO4–Fluka reagent grade) are shown for reference. Extraction detailsn Tables 4 and 5.
ere evident particularly in purple loosestrife (PL). A compar-son of the XANES spectra of dry ground and dry residues ofxtraction of the same plants showed significant shifts in theeak intensities as well as absorption positions between the cor-esponding spectra of PL and sensitive fern (SF). It is clear thathe profile of the arsenic species remaining in the PL and SFesidues of extract are not consistent with that of the originallants. Further study will be required to come to any conclu-ions regarding the transformation of arsenic species in plantatrices due to extraction.
. Conclusions
The traditional method of arsenic extraction by water–ethanol mixtures from terrestrial plants was augmented by
he sequential extraction with dilute hydrochloric acid. Almostouble the amount of arsenic was extracted by the sequentialethod compared to the traditional method. Arsenic spike recov-
ry experiments demonstrated stability of the species during thextraction processes and XANES results showed that no speciesAs(III)/As(V)) transformation occurred due to sample prepa-ations (prior to extraction). Trace amounts of organoarsenicpecies could be detected by HPLC–HGAAS aided by therocess of separation and preconcentration: the organoarsenicpecies were separated from the large inorganic fractions byxtracting them first in the water–methanol mixture, which wasoncentrated. We demonstrated the applicability of the devel-ped sequential method to various terrestrial plants for arsenicpeciation.
cknowledgements
We would like to thank the Ministry of Environment, Ontariond in particular Robert A. Helliar, Project Engineer, Regional
[
[
(2007) 1507–1518 1517
rogram Unit, for allowing access to the Deloro site and Dr. W.ullen and Dr. H. Sun of the University of British Columbia
or providing the As(Glu)3 standard. PNC-CAT facilities at thedvanced Photon Source, and research at these facilities, are
upported by the US DOE Office of Science Grant No. DEFG03-7ER45628, the University of Washington, a major facilitiesccess grant from NSERC, Simon Fraser University and thedvanced Photon Source. Use of the Advanced Photon Source
s also supported by the U. S. Department of Energy, Office ofcience, Office of Basic Energy Sciences, under Contract No. W-1-109-Eng-38. We thank Dr. Robert Gordon for his assistanceith XANES analyses.
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[
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