Environ. Sci. Technol.
2010, 44, 4735–4740
Arsenic Speciation in Tissues of the
Hyperaccumulator P. calomelanos
var. austroamericana using X-ray
Absorption Spectroscopy
A N T H O N Y G . K A C H E N K O , * ,†
M A R K U S G R Ä F E , † B A L W A N T S I N G H , † A N D
STEVE M. HEALD‡
Faculty of Agriculture, Food and Natural Resources, The
University of Sydney, New South Wales 2006, Australia and
X-ray Science Division, Advanced Photon Source, Argonne
National Laboratory, Argonne, Illinois 60439
Received February 16, 2010. Revised manuscript received
April 28, 2010. Accepted April 29, 2010.
The fate and chemical speciation of arsenic (As) during
uptake, translocation, and storage by the As hyperaccumulating
fern Pityrogramma calomelanos var. austroamericana
(Pteridaceae) were examined using inductively coupled
plasma-atomic emission spectrometry (ICP-AES) and
synchrotron-based µ-X-ray absorption near edge structure (µ-
XANES) and µ-X-ray fluorescence (µ-XRF) spectroscopies.
Chemical analysis revealed total As concentration was ca. 6.5
times greater in young fronds (5845 mg kg-1 dry weight
(DW)) than in old fronds (903 mg kg-1 DW). In pinnae, As
concentration decreased from the base (6822 mg kg-1 DW) to
the apex (4301 mg kg-1 DW) of the fronds. The results from
µ-XANES and µ-XRF of living tissues suggested that more than
60% of arsenate (AsV) absorbed was reduced to arsenite
(AsIII) in roots, prior to transport through vascular tissues as
AsV and AsIII. In pinnules, AsIII was the predominant redox species
(72-90%), presumably as solvated, oxygen coordinated
compounds. The presence of putative AsIII-sulphide (S2-)
coordination throughout the fern tissues (4-25%) suggests
that S2- functional groups may contribute in the biochemical
reduction of AsV to AsIII during uptake and transport at a whole-
plant level. Organic arsenicals and thiol-rich compounds
were not detected in the species and are unlikely to play a
role in As hyperaccumulation in this fern. The study provides
important insights into homeostatic regulation of As following As
uptake in P. calomelanos var. austroamericana.
Introduction
Arsenic (As) hyperaccumulation has been reported in few
Pteridale (fern) species, with the Chinese brake fern (Pteris
vittata) being the most noteworthy example (1–3). The ferns
that exhibit As hyperaccumulation can efficiently accumulate
>1000 mg kg-1 As on a dry weight (DW) basis in the above-
ground biomass without induced metabolic stress (1).
Recently, As hyperaccumulation was reported in the gold
dust fern (Pityrogramma calomelanos var. austroamericana;
* Present address: Nursery & Garden Industry Australia, Epping,
New South Wales 2121, Australia; phone: +61 2 8922 7006; fax: +61
2 9876 6360; e-mail: anthony.kachenko@ngia.com.au
†
The University of Sydney.
‡
Argonne National Laboratory.
10.1021/es1005237  2010 American Chemical Society
Published on Web 05/11/2010
Pteridaceae), with As concentration of 3008 mg kg-1 DW
reported in fronds when exposed to 50 mg kg-1 As in a potting
medium (4). This species was considered a suitable candidate
for phytoremediation of soils with low levels (e50 mg kg-1)
of contamination.
Despite the widespread appeal of this potentially useful
biological resource, the physiological mechanisms which
enable As accumulation, translocation, and detoxification
in this species remain unclear. One important aspect to
consider is the biochemical transformation of As during
translocation from root to frond. Several studies have
employed wet chemical techniques to examine As speciation
in P. vittata and observed that 75-95% of total As in fronds
was present as arsenite (H3AsO3) (1, 5–7). Similarly, in the
silver back fern Pityrogramma calomelanos, 60-72% of
accumulated As was found as AsIII (8). Furthermore, these
studies did not observe a significant amount of organoarsenic
species such as dimethylarsenic acid (DMA) or monom-
ethylarsonic acid (MMA) in fern tissues.
The destructive nature of wet chemical techniques may
result in artifactural changes in As speciation. To overcome
limitations of chemical or physical treatments, micro-X-ray
absorption near edge structure (µ-XANES) and micro-X-ray
fluorescence (µ-XRF) spectroscopies have proven to provide
pertinent information on the in situ speciation of As. To
date, synchrotron-based studies of Pteridophytes have
examined As speciation in P. vittata and have indicated that
the accumulated As is mainly coordinated by oxygen (O) in
the reduced state, i.e., H3-nAsO30-n (9–13). Despite this
consensus, the pathway of transformation is not directly
apparent. Pickering et al. (10) and Hokura et al. (12) suggested
that arsenate (AsV) was translocated through vascular tissues
and stored predominantly as AsIII in fronds. Conversely, Webb
et al. (11) and Huang et al. (13) found that reduction of AsV
occurred immediately after uptake before transport into
aboveground tissues. The role of sulfur groups (e.g., thiols)
such as those found in phytochelatins is also unclear in As
hyperaccumulators. Webb et al. (11) utilized extended X-ray
absorption fine structure (EXAFS) spectroscopy and reported
that As was present in frond tissues as a mixture of AsIII-O
and AsIII-S coordinated compounds. In contrast, Pickering
et al. (10) using the EXAFS spectroscopy reported that the
majority of As in fronds was coordinated by oxygen rather
than thiolates.
The objectives of this study were to determine the in-
planta speciation dynamics of arsenate in P. calomelanos
var. austroamericana following its uptake to understand the
mechanism of As hyperaccumulation observed in this species.
We employed (1) inductively coupled plasma-atomic emis-
sion spectrometry to quantify total As in various fern tissues,
(2) µ-XANES spectroscopy to quantify As chemical species
in various fern tissues, and (3) µ-XRF spectroscopy to create
qualitative images of total As, AsIII, and AsV localization. To
our knowledge, this is the first report on the translocation
and speciation of As in tissues of this hyperaccumulating
fern species.
Materials and Methods
Selection of Fern Material. Spores of P. calomelanos var.
austroamericana were germinated on Debco general con-
tainer mix. Sporelings were divided and individually trans-
planted into plastic pots (Ø 14 cm; 2 L capacity) containing
Debco general container mix with 10% coarse perlite. After
4 months of growth, ferns at the 3-4 frond stage were selected
and subsequently transferred into pots (Ø 14 cm; 2 L capacity)
containing As at either 0 or 50 mg kg-1 dry weight (DW) of
VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4735
potting mix without added fertilizer. The potting mix was
spiked with an AsV solution supplied as Na2HAsO4 · 7H2O two
weeks prior to transplanting. The pots were placed in
containers to collect any possible leachate from irrigation
with deionized water (to 60-70% water holding capacity).
The treatments were replicated 3 times and ferns were grown
in greenhouse controlled conditions for 10 weeks with an
11-h daily photoperiod, a photon flux of >370 µmol m-2 s-1,
a day-night temperature range of 17-32 °C, and relative
humidity of ca. 65%.
Total Arsenic Determination by ICP-AES. After 10 weeks,
ferns were removed from pots and carefully washed to remove
all traces of potting mix. Portions of young (newly formed;
<5 weeks of age) and old (>5 weeks of age) frond biomass
from As treated ferns were digested in concentrated acids
according to Miller (14). Three new fronds (one per replicate)
from As treated ferns were also harvested and digested in
concentrated acids to determine As concentrations from
different regions along the length of the frond. Owing to the
small quantity of sample per replicate, samples were
combined. The digests were analyzed for As using a Varian
Vista CCD inductively coupled plasma-atomic emission
spectrometer (ICP-AES) as described elsewhere (4). In
addition, a selection of fronds from As treated ferns were
freeze-dried for synchrotron-based µ-X-ray absorption spec-
troscopy (µ-XAS) as described in Lombi et al. (9). The
remaining live ferns from both treatments were transported
to the Advanced Photon Sources (APS), Argonne, IL, and FIGURE 1. Concentration of As in (a) the pinnae and (b) stipe
examined with µ-XAS. (O) and rachis (•) tissues along the axis of P. calomelanos var.
Synchrotron-Based µ-X-ray Absorption Spectroscopy. austroamericana fronds exposed to 50 mg kg-1 AsV for 10
Synchrotron-based µ-XAS was conducted at beamline 20-ID weeks.
(PNC/XOR) of the APS. Upon arrival, As treated ferns were
placed in beakers containing ultrapure water, whereas control set of model compounds used for the AFA and LCF of
ferns were placed in beakers containing 5 mM AsV (supplied the experimental spectra included aqueous arsenate
as Na2HAsO4 · 7H2O) at ambient laboratory conditions. The (Na2HAsO4 · 7H2O), dimethylarsenic acid (DMA; C2H7AsO2),
ferns treated with 5 mM AsV were sampled after 40 h of monomethylarsonic acid (MMA; CH3AsO(OH)2), arsenite
exposure to examine the biochemical transformation of AsV (NaHAsO2), and AsIII-glutathione [As(Glu)3]. The AsIII-
after a short uptake period and translocation in various fern glutathione complex was prepared as described by Pickering
tissues. The remaining ferns that had been previously exposed et al. (10). Spectra were also collected on solid orpiment
to AsV for 10 weeks were examined to ascertain the trans- (As2S3). Micro-XRF data were normalized using incident
formation of AsV after long-term uptake. Excised fern samples X-rays (I0) and plotted using beamline 20 ID’s 2D scan plot
from both treatments were plunge-frozen in liquid nitrogen program (http://www.pnc.aps.anl.gov/downloads.htm).
and kept frozen during µ-XAS analysis to maintain cellular
integrity and minimize redistribution of elements, using a Results and Discussion
cryostat sample holder/chamber (-20 °C) that was fitted to The ability of P. calomelanos var. austroamericana to
an X-Y-Z axes step motor driven stage. Further details of hyperaccumulate As was demonstrated by As concentrations
the beamline specifications, data acquisition, and analysis in above-ground tissues exceeding 1000 mg kg-1 DW. Ferns
are given in the Supporting Information (SI). exposed to 50 mg kg-1 AsV for 10 weeks showed no visual
Data Analysis. Arsenic concentrations in fronds were symptoms of phytotoxicity. The concentration of As in these
statistically analyzed by t-tests using GenStat version 8.1.0.152 ferns was 2644 ( 417 mg kg-1 dry weight (DW) as compared
(15). To obtain qualitative µ-XRF speciation maps, windowed to 2 ( 0.9 mg kg-1 DW in the control ferns (t-test; P < 0.05).
µ-XRF AsIII and AsV data signals were corrected for background Arsenic concentrations observed in fronds are similar to those
noise as described in the SI. reported earlier for this species confirming that As was readily
Micro-XANES data were energy and background corrected translocated into above-ground tissues (4). In As treated ferns,
using WinXAS (version 2.3) (16). The resulting (normalized) concentrations were significantly higher in younger fronds
spectra were then analyzed by abstract factor analysis (AFA) than in older fronds with concentrations of 5854 ( 566 and
comprising principal component analysis (PCA) and target 251 ( 41 mg kg-1 DW, respectively. Along the length of each
transformation (TT) analysis using the software available from frond, As concentration decreased from basal pairs of pinnae
beamline 10.3.2 (Advanced Light Source, Lawrence Berkeley (6800 mg kg-1 DW) to the apical pinnae (4300 mg kg-1 DW;
National Laboratories, Berkley, CA) (17, 18). Target trans- Figure 1a). Conversely, the concentration of As increased
formation characterized the principal components (PCs) from 79 mg kg-1 DW in stipe tissues to 1512 mg kg-1 DW in
using SPOIL values as described by Manceau et al. (17). Linear the rachis tissues (Figure 1b). Lombi et al. (9) reported a
least-squares combination fit (LCF) analyses were then similar pattern in P. vittata and suggested that it was related
performed to reveal the composition of individual spectra to the maturation of pinnae with older pinnae positioned at
using beamline 10.3.2 linear combination fitting program the base of fronds. It may also be related to the relative
(http://xraysweb.lbl.gov/uxas/Beamline/Software/Software. distance of As translocation from roots to sites of accumula-
htm). This procedure computed the fractions of end-member tion and storage in above-ground tissues. Spores (reproduc-
(reference) spectra which, when summed, yielded the lowest tive tissues) contained 490 mg As kg-1 DW whereas the As
sum-square (SS) value. Inclusion of a reference spectrum concentration in croziers (uncoiling young fronds) was 4393
was only considered if the SS improved by g20% (17). The mg As kg-1 DW. Exclusion of accumulated metal(loid)s from

4736 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 12, 2010
FIGURE 2. Arsenic K-edge µ-XANES spectra of model
compounds and bulk P. calomelanos var. austroamericana
freeze-dried pinnule and stipe/rachis tissues. Dashed and solid
lines drawn at 11872.2 and 11875.5 eV indicate the positions of
the whitelines for arsenite (AsIII) and arsenate (AsV),
respectively.
reproductive tissues has been reported in several hyperac-
cumulating species (9, 19, 20), and might contribute to their
reproductive success on metal(loid)-enriched substrates.
Micro-XANES spectroscopy combined with µ-XRF analy-
ses were utilized as complementary techniques to examine
the in-planta localization and speciation of As in P. calom-
elanos var. austroamericana. Owing to the higher concentra-
tion of As in new fronds of this species, young tissues were
chosen for µ-XANES and µ-XRF analyses. Principal compo-
nent analyses conducted on the whole experimental µ-XANES
spectra suggested the data set was considered a 3-component
system explaining 93% of the variance (SI Table S4-S1).
Target transformation using three principal components
showed that arsenite (NaH2AsO3) and orpiment (As2S3) were
the only model compounds with a SPOIL value <3 while the
SPOIL value for arsenate (Na2HAsO4) was <4.5. Therefore,
inorganic arsenicals (arsenate [AsV], arsenite [AsIII], and
orpiment [As2S3]) were selected for analysis of individual
spectra. The As K-edge µ-XANES spectra of the model
compounds showed whiteline energies in the order As(Glu)3
< As2S3 < AsIII < DMA < MMA < AsV (Figure 2). The absence
of organic arsenicals (MMA and DMA) concurred with several
earlier studies of other As hyperaccumulating ferns that
indicated organic arsenicals were not present in significant
quantities (6–8). Arsenic methylation was, therefore, not
involved during As uptake, translocation, and storage in this
fern species. Similarly, AFA revealed that the As(Glu)3 complex
was not a significant component in this species and the role
of glutathione-conjugated AsIII in this fern species appears
limited. In P. vittata, Pickering et al. (10) observed As(Glu)3
in µ- XRF images as cylindrical sheaths around AsV loaded
veins however their contribution to overall As in leaf spectra
was 5 ( 1%. The putative role of thiols such As(Glu)3 or
phytochelatins in mediating vacuole sequestration of AsIII
has not been confirmed in As hyperaccumulation (21, 22).
FIGURE 3. Arsenic K-edge µ-XANES spectra for various regions
of P. calomelanos var. austroamericana frond tissues. Spectra
1-4 and 5-9 correspond to ferns exposed to 50 mg kg-1 AsV
for 10 weeks and 5 mM AsV for 40 h, respectively. Dashed and
dotted lines drawn at 11872.2 and 11875.5 eV indicate the
position of the whitelines for arsenite (AsIII) and arsenate (AsV),
respectively.
Micro-XANES spectra of bulk pinnule material was
characterized by a whiteline position consistent with that of
AsIII, whereas the µ-XANES spectrum of bulk stipe/rachis
tissue inferred the presence of both AsIII and AsV (Figure 2).
Arsenic in frond tissues sampled from ferns exposed to AsV
for 10 weeks was largely present as AsIII, as observed by the
coincidence of the whiteline at the energy of AsIII (Figure 3).
Similarly, in ferns exposed to 5 mM AsV, all spectra were
characterized by a dominant peak at the AsIII whiteline, however,
in root and stipe tissues, a peak at 11875.5 eV indicated the
additional presence of AsV (Figure 3). Linear least-squares
combination fits (LCF) confirmed that AsIII dominated sampled
tissues (59-90%; SI Table S5-S2). Micro-XRF images of ferns
exposed to 50 mg kg-1 AsV for 10 weeks and freshly exposed
ferns (5 mM AsV) also revealed that AsIII was the predominant
species in both cases (Figures 4 and 5).
Linear least-squares combination fits showed that As2S3
was present in all fresh tissues investigated (4-25%), however,
only as a secondary component to arsenite (NaAsO2, SI Table
S5-S2). Trivalent arsenic (AsIII) is well-known for its high
affinity for sulphide functional groups (S2-) in As tolerant
species (23), and possibly contributes to As homeostasis in
the studied fern. Webb et al. (11) indicated the presence of
As2S3 in P. vittata leaf tissues (6 ( 2%) based on linear fits
of As K-edge XANES spectra, but ruled out the presence of
actual orpiment due to the absence of secondary As-As or
As-S shell structures in the EXAFS region. Additional As-S
model compounds such as As(Glu)3 were not considered in
this analysis and Webb et al. (11) speculated that thiol-rich
compounds might be present. Inclusion of biologically
important As model compounds (24) might resolve the true
identity of As-S coordination and clarify the spectral
signature observed in P. calomelanos var. austroamericana
tissues. Biologically important model compounds should
include organic molecules (e.g., arsenocholine, arsenobe-
VOL. 44, NO. 12, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 4737

FIGURE 4. Qualitative As K-edge µ-XRF images of P.
calomelanos var. austroamericana tissues exposed to 50 mg
kg-1 AsV for 10 weeks. Tip of pinnule (A-C), two views of
costa (D-I), rachis (J-L), and stipe (M-O) each showing
optical micrograph and the relative concentration of arsenite
and arsenate. The µ-XRF signals were collected at 25 µm step
size. The normalized intensity scale is arbitrary with the black
and white pixel color corresponding to the lowest and highest
relative concentrations, respectively.
taine, or arsenosugars), which have not been included in
any previous µ-XAS studies of As hyperaccumulating species.
To elucidate the biochemical transformation of AsV during
uptake in the roots, ferns exposed to 5 mM AsV for 40 h were
analyzed. Presumably AsV entered P. calomelanos var. aus-
troamericana roots via the phosphate transport system as
reported for P. vittata (5, 25, 26). Meharg and Hartley-
Whitaker (27) hypothesized that the rhizosphere might play
a role in the transformation of As prior to uptake, however,
a role of the rhizosphere environment on As redox chemistry
has yet to be confirmed in ferns.
Following uptake, the band of AsIII observed in Figure 5K
(and less pronounced in Figure 5L), is consistent with the
position of the stele (vascular tissues) in ferns (28) and
suggests that both forms of As were loaded into vascular
tissues for transport into aboveground biomass. The spe-
ciation of As in the root revealed a mixture of AsIII (59%) and
AsV (32%), indicating that most of the As was reduced upon
uptake (SI Table S5-S2). Similar observations were made by
4738 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 12, 2010
FIGURE 5. Qualitative As K-edge µ-XRF images of P.
calomelanos var. austroamericana tissues exposed to 5 mM AsV
for 40 h. Uncoiling crozier (A-C), costa (D-F), stipe (G-I), and
root (J-L) each showing optical micrograph and the relative
concentration of arsenite and arsenate. The µ-XRF signals were
collected at 25 µm step size. The normalized intensity scale is
arbitrary with the black and white pixel color corresponding to
the lowest and highest relative concentrations, respectively.
Huang et al. (13) and Hokura et al. (29) investigating P. vittata
roots using µ-XANES and EXAFS spectroscopy. Hokura et al.
(29) reported that AsV was more dominant in the root cortex
tissues (outer root tissues) whereas spectral contributions
from AsIII increased from outer cortex tissues toward the
center of the root. These results suggested that the reduction
of AsV occurred between the cortex and central cylinder in
root tissues. The same process may also operate in AsV
reduction in P. calomelanos var. austroamericana root tissues.
Our results contrast those of Pickering et al. (10) who
indicated that AsV dominated the root tissues (90 ( 4%) of
P. vittata with only minor contributions from AsIII (10 ( 5%).
Similarly, earlier wet chemistry studies of P. vittata and P.
calomelanos roots have indicated the preponderance of AsV,
however, in both species <64% of the total As was extracted
from root tissues and these data should be interpreted with
caution (6, 8). The reduction of AsV in P. calomelanos root
tissues may be catalyzed by the enzyme arsenate reductase,
previously reported to reduce AsV to AsIII in P. vittata root
tissues (30). These authors reported that enzymatic reduction
occurred predominantly in roots with no measurable arsenate
reductase observed in frond tissue. Further investigation is
warranted to measure arsenate reductase activity in P.
calomelanos var. austroamericana root tissues extracts.
Following uptake into the roots, As was translocated into
aboveground tissues in the xylem stream. Xylem sap of P.
vittata extracted using HPLC indicated that AsV was the
dominant form transported in the sap following exposure to
inorganic arsenicals (31). In the present study, distinct strands
of AsIII and AsV in stipe (e.g., Figure 5H, I) tissues were
observed, which correspond with the position of vascular
bundles in this species (4). Similarly, distinct bands of both
As species were also observed in costa (e.g., Figure 4E, F) and
pinnule tissues (Figure 4B, C) which appeared to overlap
with the position of veins depicted in their corresponding
optical micrographs. Furthermore, the AsV µ-XRF image of
a crozier (Figure 5C) indicated that AsV was present through-
out the tissue, particularly in the tip region where active cell
division is occurring. Our findings are similar to those of
Pickering et al. (10) who observed that AsV was confined to
bundles of parallel strands in P. vittata rachis and leaf tissues.
Interestingly, these authors also observed interleaved strands
of AsIII and hypothesized that the AsV and AsIII strands
corresponded to xylem and phloem tissues, respectively. It
is unclear from the present study whether the same reasoning
can be applied due to the displacement of the X-ray beam
by the sample thickness and its 45° orientation.
In the present study, LCF did not identify AsV in rachis
tissues of ferns that had been exposed to AsV for 10 weeks.
These ferns were devoid of As for 4 days during transportation
to the beamline and were instead supplied with ultra pure
water. Consequently, this may have led to the diminished
AsV in the rachis tissues of ferns exposed to AsV for 10 weeks
(Figure 4L). To confirm this, root tissues of these ferns were
investigated using µ-XANES and the generated maps could
not clearly identify the presence of AsV and AsIII. Similar
observations were reported in P. vittata after ferns had been
given water for a period of 2 months (10). It may also offer
an explanation for the low concentration of As in roots of P.
vittata reported by Webb et al. (11).
In live P. calomelanos var. austroamericana pinnule
tissues, As accumulated predominantly as AsIII (72-75%),
presumably as the aqueous H3AsO3 species as observed by
Webb et al. (11) and consistent with previous studies that
examined As speciation in P. vittata and P. calomelanos fronds
(7, 8, 10). In the present study, As localization in the pinnule
tip increased from the central midrib to the pinnule margin,
which was consistent with quantitative µ-PIXE results
presented previously (4, 32). A similar accumulation trend
has been reported for P. vittata pinnules using µ-XRF (12).
It is possible that accumulation of excess As in the pinnule
margin may result in the onset of phytotoxicity symptoms
and cell death. For example, Tu and Ma (33) observed brown
coloration and necrosis at the tips and margins of P. vittata
fronds exposed to 1000 mg kg As1-. Similar symptoms have
been observed for P. calomelanos var. austroamericana when
exposed to >100 mg kg As1- (4). Micro-XANES spectra of
older browning P. vittata pinnae revealed that accumulated
AsIII can reoxidize into AsV in these regions (29). Similarly, in
senescent P. calomelanos fronds, a similar transformation of
AsIII to AsV has been reported (8). Tu et al. (7) suggested that
the reoxidation of AsIII might be related to a decline in the
levels of ascorbate (a reductant) in the old fronds. The nature
of this distribution and reoxidation, however, is not im-
mediately apparent.
The reduction of AsV to AsIII in P. calomelanos var.
austroamericana is intriguing considering AsIII is considered
more toxic to plants than AsV (34). Arsenite interferes with
sulfhydryl groups of enzymes and proteins, and may inhibit
cellular function leading to cell death (35). Conversely, AsV
can interfere with phosphate mediated processes such as
competing for phosphate sites in ATP and various phos-
phorolysis reactions, and can lead to the disruption of energy
flow in cells (34, 36). It is possible that the reduction of AsV
to AsIII in P. calomelanos var. austroamericana following
uptake may represent an efficient tolerance strategy to
minimize AsV cytoplasmic concentrations, thus ensuring
plant metabolic function is preserved. Presumably, the
transformation of AsV occurs in the cytoplasm (27). It is
possible that at least part of the AsIII present in P. calomelanos
var. austroamericana is complexed with S into a less toxic
form, and stored in vacuoles away from metabolic activity.
Lombi et al. (9) observed As compartmentation in P. vittata
vacuoles, and suggested that it may result in lower As
concentration in the cytoplasm. Vacuole compartmentation
in leaves has been reported as a possible mechanism of plant
metal tolerance for several hyperaccumulating species
(37, 38), and this may, in part, provide tolerance to ac-
cumulated As in this species.
An understanding of the nature and proportion of As
species present in above-ground harvestable biomass may
assist with the correct handling, storage, and disposal of fern
biomass harvested during phytoremediation of As contami-
nated soils. The results of the present study confirm the
preponderance of inorganic arsenicals throughout P. calom-
elanos var. austroamericana tissues that may, if combusted,
lead to the release of toxic As emissions and would inadvert-
ently translate into secondary contamination of As in the
environment (39). Alternative, environmentally sound strat-
egies are required to ensure the safe disposal of As con-
taminated (frond) biomass. Consideration of treatments to
minimize arsenic leakage from As contaminated (frond)
biomass during handling and storage is also warranted.
Acknowledgments
This work was supported by the Australian Synchrotron
Research Program, which is funded by the Commonwealth
of Australia under the Major National Research Facilities
Program. Use of PNC/XOR (Sector 20), Advanced Photon
Source (APS), Argonne National Laboratory was supported
by the U.S. Department of Energy, Office of Science, Office
of Basic Energy Sciences, under Contract DE-AC02-
06CH11357. A.G.K. acknowledges financial assistance from
the University of Sydney and the Australian Commonwealth
Government through an Australian Postgraduate Award
scholarship. Thanks are extended to Dr. Euan Smith (Uni-
versity of South Australia, Adelaide, Australia) for providing
the spectrum of aqueous monomethlyarsonic acid.
Supporting Information Available
Experimental details and two tables. This information is
available free of charge via the Internet at http://pubs.acs.org.
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