Science of the Total Environment 505 (2015) 952961

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Science of the Total Environment

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Selenium distribution and speciation in plant parts of wheat

(Triticum aestivum) and Indian mustard (Brassica juncea)
from a seleniferous area of Punjab, India
E. Eiche a,, F. Bardelli b, A.K. Nothstein a, L. Charlet b, J. Gttlicher c, R. Steininger c, K.S. Dhillon d, U.S. Sadana d
a

Institute of Mineralogy & Geochemistry, Karlsruhe Institute of Technology, Adenauerring 20b, 76131 Karlsruhe, Germany
Institut des Sciences de la Terre, Universit Grenoble I, 1381 rue de la Piscine, 38400 Grenoble, France
c
Karlsruhe Institute of Technology, ANKA Synchrotron Radiation Facility, Hermann-von-Helmholtz-Platz 1, D-76344 Eggenstein-Leopoldshafen, Germany
d
Department of Soil Science, Punjab Agricultural University, Ludhiana 141 004, India
b

H I G H L I G H T S

Wheat plants can also accumulate very high amounts of Se (b 390 mg/kg)
Under Se stress wheat plants can synthesize similar species like accumulator plants
Excess selenate is not transformed into non-toxic forms in upper plant parts
Se is mainly present in a form that is highly absorbable by humans and animals

a r t i c l e

i n f o

Article history:
Received 28 July 2014
Received in revised form 22 October 2014
Accepted 22 October 2014
Available online 7 November 2014
Editor: Charlotte Poschenrieder
Keywords:
Selenium
Speciation
Wheat
Indian mustard
Synchrotron
XANES

a b s t r a c t
The concentration, distribution, and speciation of selenium in different parts of wheat and Indian mustard, grown
in a seleniferous area in Punjab, were investigated using synchrotron based (XAS) and classical acid digestion and
extraction methods.
The analyses revealed a high Se enrichment in all investigated plant parts, with Se levels in the range of 133
931 mg/kg (dry weight, dw). Such high Se enrichment is mainly due to the considerable amounts of easily available Se detected in the soil, which are renewed on a yearly basis to some extent via irrigation. Speciation analysis
in soil and plants indicated selenate and organic Se as major Se species taken up by plants, with a minor presence
of selenite. The analyses also revealed that the highest Se enrichment occurs in the upper plant parts, in agreement with the high uptake rate and mobility of selenate within plants. In both wheat and mustard, highest Se enrichments were found in leaves (387 mg/kgdw in wheat and 931 mg/kgdw in mustard). Organic species
(dimethylselenide and methylselenocysteine) were found in different parts of both plants, indicating that an active detoxication response to the high Se uptake is taking place through methylation and/or volatilization. The
high proportion of selenate in wheat and mustard leaves (47% and 70%, respectively) is the result of the inability
of the plant metabolism to completely transform selenate to non-toxic organic forms, if oversupplied.
Methylselenocysteine, a common Se species in accumulating plants, was detected in wheat, suggesting that, in
the presence of high Se concentration, this plant develops similar response mechanisms to accumulator plants.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Selenium is an essential element for humans and animals, provided
that the tolerable upper level of intake is not exceeded. The USA and the
World Health Organization (WHO) have set this upper safe intake level
to 400 g/d (WHO, 1996; FNB and IM, 2000). An optimal supply of Se is
important because it can support protection against several illnesses,
like different type of cancers (prostate, lung, gastrointestinal, Clark et al.,
Corresponding author. Tel.: +49 721 608 43327.
E-mail address: elisabeth.eiche@kit.edu (E. Eiche).

http://dx.doi.org/10.1016/j.scitotenv.2014.10.080
0048-9697/ 2014 Elsevier B.V. All rights reserved.

1998; Della Rovere et al., 2006; Grau et al., 2006) and cardiovascular diseases, assure a proper functioning of the immune system, inhibit HIV progression to AIDS (Rayman, 2000), or counteract heavy metal or semimetal poisoning (As, Hg, Cd, Merali and Singhal, 1975; Gailer et al.,
2000). The uptake of Se is mainly determined by the type of food consumed; consequently, individual Se levels depend on its concentration
in the food derived from plants (Rayman, 2000; Van Hoewyk, 2013).
Even though it is known that plants can readily take up Se in the
form of selenite, selenate, or as organic Se species (Sors et al., 2005;
Hawrylak-Nowak, 2013), it has never been proven so far that it plays
an essential role for their growth and health. Although several studies

E. Eiche et al. / Science of the Total Environment 505 (2015) 952961

have indicated that small amounts of Se could be benecial for plant

growth and stress tolerance (Xue et al., 2001; Pennanen et al., 2002;
Kong et al., 2005), it is widely accepted that excessive Se doses induce
pathological effects in plants, like stunted root growth, reduced biomass,
chlorosis, and reduced photosynthetic efciency ultimately leading to
plant death (Van Hoewyk, 2013). Younger plants are more sensitive to
high Se concentration compared to mature plants (Rosenfeld and Beath,
1964). The stress symptoms are thought to be caused by malfunction of
proteins and enzymes which incorporate Se amino acids (Se-Cysteine
(SeCys), Se-Methionine (SeMet)) (Brown and Shrift, 1981; Terry et al.,
2000).
The uptake of Se from soils into plants and, therefore, its concentration in food, depends on several parameters including soil characteristics, bioavailable Se concentration and Se speciation, concentration of
competing ions, and plant species (Terry et al., 2000; Zhao et al., 2005;
Renkema et al., 2012). Selenate is thought to be the most available
species for plants because of its low afnity towards adsorption onto
soil particles (Balistrieri and Chao, 1987; Hayes et al., 1987; Plant
et al., 2005). It is taken up from soil via sulfate transporters and quickly
transported from roots to shoots or leaves where it can be assimilated as
organic species (De Souza et al., 1998; Terry et al., 2000; Sors et al.,
2005). Conversely, selenite is thought to be less available because of
its stronger sorption onto soil particles (Balistrieri and Chao, 1987;
Hayes et al., 1987; Plant et al., 2005). For a long time, selenite uptake
from soil was considered to be a purely passive process (Shrift and
Ulrich, 1969; Arvy, 1993; Terry et al., 2000). More recently, it has been
shown that selenite can also actively be taken up by phosphate (Li
et al., 2008) or silicon transporters (Zhao et al., 2010). In contrast to
selenate, most of the selenite remains in the root because it is quickly
converted into organic species (De Souza et al., 1998; Li et al., 2008).
Organic species can also be taken up actively and further transported
within plants (Sors et al., 2005). Kikkert and Berkelaar (2013) demonstrated in their experiments using wheat that the rate of Se uptake is
up to 100 times higher for organic species compared to that of selenate.
Once taken up, however, selenate is more mobile within the plant
(Shrift, 1969).
Organic species are also readily formed within the plant by reduction
of inorganic Se species. If selenate is present its reduction to selenite is
both the rst as well as the rate limiting step to occur (De Souza et al.,
1998). Either in an enzymatic or non-enzymatic assisted process,
selenite is further reduced to SeCys (Ng and Anderson, 1978). SeCys can
either be incorporated into various selenoproteins or further transformed
into other organic species. These include SeMet, methylselenocysteine
(SeMeCys), methylselenomethionine (MeSeMet) and elemental Se
(Se0) (Shrift, 1969; Pilon et al., 2003). The conversion of SeCys into
other organic species is believed to be a detoxication mechanism,
because it prevents its incorporation into proteins (Neuhierl
&Bck, 1996; Van Hoewyk, 2013). SeMet and SeMeCys can be further
converted into volatile selenide species (dimethylselenide (DMeSe)
or dimethyldiselenide (DMeDSe)) (Pilon-Smits and Quinn, 2010). Volatilization is the only process which eventually removes Se from plants
and, therefore, from the whole soilplant system. Metabolic intermediates, like selenocystathione, glutamyl-methylselenocysteine, and various selenoproteins, can be found as further organic species in plants
(Shrift, 1969; Pickering et al., 2000; Whanger, 2004).
A detailed knowledge on the individual organic and inorganic Se
species occurring in food crops is important to understand the metabolic processes of plants in the presence of Se, but also for human health
risk assessment. In fact, Rayman (2008) highlighted that, since the actual absorption of Se from food by the mammalian metabolism depends
on its speciation, not only the amount of Se in food is of interest to estimate its benet, but also the exact knowledge of the individual Se
species. As indicated by a higher Se level in the blood, organic forms are
more bioavailable compared to inorganic Se species (Swanson et al.,
1991; Rayman, 2008). One reason might be that SeMet can be stored
within the body for longer periods of time, or reutilized, preventing Se

953

depletion during Se shortage periods (Swanson et al., 1991). In animal

tissue higher Se concentrations are reported for SeMet compared to
SeCys, selenite, or selenate (Whanger and Butler, 1988; Ip and Hayes,
1989). Apart from the actual absorption, Se efcacy for cancer prevention
and against toxic effects also depends on its speciation. SeMeCys is
believed to be the most effective anticarcinogenic Se compound, followed
by selenite and SeMet, which was shown to be only half as active at suppressing mammary tumors in rodents compared to SeMeCys (Thompson
et al., 1984; Whanger, 2002, 2004). With regard to toxicity to mammals,
inorganic forms are more toxic compared to organic forms, with selenite
being four times more toxic than SeMet (Ammar and Couri, 1981).
Among the inorganic species, selenite is indicated to be slightly more
toxic than selenate (Ammar and Couri, 1981; Barceloux, 1999). Consequently, also the actual upper safe intake limit of Se highly depends on
its speciation in the food.
From an animal and human health perspective it is, therefore, of the
utmost importance to reveal the uptake, translocation, transformation,
and accumulation of Se within crop plants. In this study we investigated
the total Se concentration, distribution, and speciation in different parts
of wheat and mustard, two important crops cultivated in the seleniferous area in Punjab, India. This knowledge is crucial to understand and
elucidate the mechanisms responsible for Se uptake, transformation,
and toxicity in plants, especially in seleniferous areas, which can have
both economical and toxicological implications: If too much Se is
taken up it can be disadvantageous for growers due to reduced harvest;
consumers, on the other hand, might suffer from illnesses related to Se
intoxication (e.g. selenosis). Furthermore, information on Se speciation
in the food can help to determine the upper safe limit of Se intake,
because different Se species are resorbed by the organism in a different
way and to a different degree.
2. Materials and Methods
2.1. Plant Sampling and Preparation
Wheat (Triticum aestivum) and Indian mustard (Brassica juncea)
plants were collected between the villages of Jainpur and Barwa in
March 2011. Both villages are located in a known seleniferous area
with high Se concentrations in soils, plants and irrigation water (Bajaj
et al., 2011), rst described by Dhillon and Dhillon (1991). The wheat
plants and grains were sampled at the rim of a eld next to an irrigation
channel and showed signs of white chlorosis. Soil samples were taken to
determine their Se concentration and speciation. Plant samples were air
dried in the laboratory of the Department of Soil Science (Punjab Agricultural University) before transportation to Germany to avoid mold
formation during transport and storage, while grain samples were
shipped as such. The plants were then separated into roots, stem, leaves
and owers and weighed before and after freeze-drying for 24 h to
determine the dry weight. Air dried samples were used for XAS measurements and freeze dried to determine the total Se concentration.
Plant material of several neighboring plants was ground to a ne powder and stored appropriately for further analyses. It has to be mentioned
that drying could affect the speciation in plant material leading to a
moderate over or underestimation of some species.
2.2. Selenium Speciation in Soil
It is important to unravel the selenium speciation in the soil because
it determines the extent of Se uptake by plants and its translocation
within the plant. A six step sequential extraction scheme of Zhang and
Moore (1996), modied according to Wright et al. (2003), was used to
reveal Se speciation in the soil. Easily soluble Se (using 0.25 M KCl),
adsorbed Se (0.1 M K2HPO4), elemental Se (0.25 M Na2SO3), organically
associated Se (5% NaOCl), Se associated with amorphous oxides and
carbonates (4 M HCl) and residual Se (HNO3, H2O2, HF microwave
digestion) were extracted respectively in steps one to six. Air dried

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E. Eiche et al. / Science of the Total Environment 505 (2015) 952961

composite samples (2 g) from the top 15 cm soil prole from wheat or

mustard elds were used for extraction. The concentration of Se in each
fraction was measured by ICP-MS (X-Series2, Thermo Scientic). The
accuracy of the ICP-MS analysis (Se mainly 5%) was tested using the
certied standard HPS CRM-TMDW (High Purity Standards, USA). The
quality of the extraction was checked including the GXR4 reference material (Park City, Utah, USA) into the extraction procedure, and 94 11%
of the nominal Se content was detected (this check was repeated three
times). Sequential extraction was performed in duplicate for both soils.
2.3. Total Selenium Concentration in Plant Tissue
The total Se concentration of dried and ground plant material was
determined using ICP-MS analysis (X-Serie2, Thermo Scientic) after
microwave digestion. One milliliter Milli-Q water, 5 mL HNO3 (65%,
subboiled) and 1 mL H2O2 (30%, suprapur) were added successively
to 0.2 mg of each sample and heated with a microwave system (Start
1500, MLS) for sample digestion. The samples were cooled down
completely over night before transferring into sample containers in
order to avoid any volatilization of Se. The accuracy of the ICP-MS analysis (Se 8%) was tested by including the certied standard HPS CRMTMDW (High Purity Standards, USA) into the measurements. Two
samples were digested in duplicate. The measured Se concentration of
the two replicates was within 1%, which is the error associated to the
concentrations.

glutathione following the procedure by Chakraborty et al. (2010).

Organic Se references, kindly furnished by G. Sarret, included: dimethyl
selenide (in solution), selenomethionine, S-methyl seleno L-cysteine,
seleno-DL-cystine, selenocystamine, selenourea, and selenoguanosine
(Fig. 1). Selenodi-glutathione was prepared according to Sarret et al.
(2005), while all other organic Se references were purchased from
Sigma-Aldrich.
Linear combination tting (LCF) was applied to the XANES spectra
(t range 20 to +40 eV, force weighs to sum up zero) and consisted
in a weighted linear combination of the references' spectra to reconstruct a given sample's spectrum. Provided that the set of reference
compounds is sufciently representative, LCF can reveal the relative
amounts of the main Se species present in the samples. Starting from
the best two components t, the best t with n + 1 components was
considered to be signicantly better than the best n-component one, if
the reduced -square (2) was at least 10% lower than that of the best
n-component t (Isaure et al., 2002; Bardelli et al., 2011). The precision
of the fractions obtained from LCF was previously estimated to be
between 10 and 20% (Isaure et al., 2002; Panli et al, 2005; Bardelli
et al., 2011), and strongly depends on the signal to noise ratio and on
possible errors in the calibration and normalization procedures. Fractions present in amounts b5% were neglected since they were much
lower than the error associated with this technique and were found to
improve the ts negligibly.

2.4. Selenium Speciation in Plants

Se speciation in plants was determined by X-ray absorption spectroscopy (XAS). Samples of different plant parts were prepared for
XAS measurements in the form of pressed pellets using ground material
from several neighboring plants. This was done to obtain samples as
representative and homogenous as possible. The bulk experiments
were carried out at the Angstrmquelle synchrotron (ANKA) at the
Karlsruhe Institute of Technology (KIT) and at the European Synchrotron Radiation Facility (ESRF) at the SUL-X (Wiggler as source) and
GILDA (Bending magnet) beamlines, respectively. Selenium K-edge spectra were recorded using a monochromator with Si(111) crystals at SUL-X
and Si(311) crystals at GILDA. The beam spot size was 1 1 mm2 at SULX and 250 100 m2 at GILDA. The photon ux density at SUL-X was
1.51011 phtonss1. XAS measurements were performed in uorescence mode (Se K X-ray uorescence) using solid state detectors (One
element Vortex silicon drift detector (SUL-X) and Ortec 13-element Ge
detector (GILDA)). Each spectrum is the average of two to four measurements at slightly different spots on the pellet. A spectrum of elemental Se
(8 wt-%) was collected simultaneously with each energy scan for reliable
energy calibration. The XAS scan energy ranged from ~200 eV below the
rst inection point of elemental Se (12,658 eV) to ~12,900 eV. Short
scan times of ~ 10 min were chosen in order to avoid beam-induced
redox reactions or volatilization of organic Se species. To prevent beam
damages spectra collected at GILDA were acquired in vacuum at low temperature (77 K). X-ray Absorption Near Edge Structure (XANES) spectra
of mustard and wheat roots, stems, leaves, and mustard owers were acquired at the SUL-X beamline (ANKA) using powdered plant material
pressed into pellets. Fresh grains were bisected and measured in vacuum
at low temperature (77 K) on the GILDA beamline at ESRF, without further manipulations.
The occurrence and contribution of the individual Se species to the
measured spectra were calculated by linear combination tting (LCF)
using several Se references and the IFEFFIT software package (Ravel
and Newville, 2005). Sodium selenate (SeVI, Na2SeO410H2O, Merck),
selenite (SeIV, Na2SeO3, Merck) and selenide (Se II, Na2Se, SigmaAldrich), crystalline grey Se0 (Alfa-Aesar), Se oxide (SeO2, SigmaAldrich), Se sulde (SeS2, Sigma-Aldrich), and amorphous Se0 were
used in a diluted form as inorganic references. Amorphous red Se
nanoparticles were synthesized by chemical reduction of Na2SeO3 by

Fig. 1. XANES spectra of the Se references: DMeSe: Dimethyl-selenide; SeCys: Se-cysteine;

SeMeCys: Se-methyl-seleno-L-cysteine; SeMet: Se-methionine. The vertical dashed lines
indicate the main absorption peaks corresponding to Se in the Se4+ (12.6642 keV) and
Se6+ (12.6675 keV) oxidation states.

E. Eiche et al. / Science of the Total Environment 505 (2015) 952961

3. Results
3.1. Selenium Speciation in Soil
In the wheat soil (Table 1), the fraction extracted from step 3, which
is associated with elemental Se, is dominating with 7.9 0.22 mg/kg
(60%). Also occurring in signicant amounts are fraction 1 (easily available Se) with 1.2 0.03 mg/kg (9%), fraction 4 (organically associated
Se) with 1.1 0.02 mg/kg (9%), and fraction 5 (oxidically bound Se)
with 1.8 0.33 mg/kg (14%). Fraction 2 (adsorbed Se) and fraction 6
(residual Se) have minor importance, with 0.7 0.01 mg/kg (5%) and
0.4 0.03 mg/kg (4%), respectively. Summing up, a total of 13.1
0.07 mg/kg Se was determined in the wheat soil.
Mustard's soil was also dominated by fraction 3 (Table 1), with
2.3 0.1 mg/kg (33%), followed by fraction 5, with 1.9 0.04 mg/kg
(28%), and fraction 4, with 1.1 0.02 mg/kg (16%). The other three
fractions are in a similar range, with 0.5 0.01 mg/kg (7%) in fraction
1, 0.6 0.01 mg/kg (9%) in fraction 2 and 0.4 0.04 mg/kg (6%) in
fraction 6. The single fraction results in a total Se content in the soil of
6.8 0.01 mg/kg.

3.2. Total Selenium Concentration in Different Plant Parts

The Se concentration in the wheat plant was similar in root and stem,
with 196 and 191 mg/kg (dry weight), respectively. Much higher concentrations were measured in wheat leaves, with 387 mg/kg (Table 2). Selenium concentrations in mustard roots were comparable to the wheat,
with 186 mg/kg, while lower concentrations were present in the stem,
with 133 mg/kg. Highest total enrichment was found in the leaves and
owers of mustard, with 931 and 541 mg/kg, respectively (Table 2).

3.3. Selenium Speciation in Wheat

As can be seen in Fig. 2, two main absorption peaks are clearly visible
in the XANES spectra of wheat roots: the rst, at 12,662.6 eV, can mainly
be assigned to organic species, and the second, at 12,667.5 eV, can be
attributed to selenate. The linear combination tting suggested the presence of approximately 75% organic Se (54% DMeSe and 21% SeMeCys)
and about 25% inorganic Se (13% selenate and 12% selenite) (Fig. 3,
Table 2).
In spectra from the stem, two peaks occur at comparable energies
as in the root spectra, but with a higher intensity of the selenate peak
(Fig. 2). Several linear combination ts were carried out, but none
was able to fully reproduce the rst peak, meaning that one or
more important organic references are probably missing. However,
in the best t it was possible to reproduce the spectra using the
organic reference spectra, which accounted for 71% of the tted
weight (namely 30% SeMeCys and 41% SeMet). The remaining 29%
was tted with inorganic Se (18% selenate and 11% to selenite,
Fig. 3, Table 2).

Table 1
Selenium concentration and its relative percentage in each fraction of the two soils
(Eiche, 2014).
Selenium fraction

Wheat soil
mg/kg

Easily available
Adsorbed
Elemental
Organically associated
Oxidically bound
Residual
Setot

1.2
0.7
7.9
1.1
1.8
0.4
13.1

0.03
0.01
0.22
0.02
0.33
0.03
0.07

Mustard soil
%

mg/kg

9
5
60
9
14
4
100

0.5
0.6
2.3
1.1
1.9
0.4
6.8

%
0.01
0.01
0.1
0.02
0.04
0.04
0.01

7
9
33
16
28
6
100

955

In the leaves the peak at 12,667.5 eV dominates, indicating that

selenate is predominant, while the peak at 12,662.6 eV is only barely
visible and appears as a shoulder of the main absorption peak
(Fig. 2). The ts conrmed selenate as major species (47%), while
the remaining fraction was attributed to selenite (10%) and SeMet
(43%) (Table 2).
XANES spectra of wheat grains acquired on their internal part and on
their surface were almost identical. A representative spectrum acquired
on the internal part is shown in Fig. 2. Best linear combination tting
indicated the presence of SeMeCys (~ 70%) and SeCys (~ 30%), while
no inorganic species where found (Table 2).
There is a clear trend in the proportion of organic and inorganic species from root to leaves with a dominance of organic species in roots and
stems and an increasing fraction of inorganic species towards the upper
parts of the wheat plant. The picture is different in the grains where only
organic Se species were detected (Fig. 3). The increase in inorganic
species from roots to leaves reects the increase of selenate (from 13%
in the roots to 47% in the leaves), while the proportion of selenite is
more or less equal in all plant parts. Among the organic species,
SeMeCys occurs in signicant amounts in roots and stems, where it
reaches 2030% of the total Se, and in the grains, where it is the dominant organic species (70%). In the roots DMeSe is the main Se species
(54%) along with SeMeCys (21%). In the stem DMeSe is absent, while
SeMet (41%) and SeMeCys (30%) are the main species. Finally, SeMet
becomes the only organic species in leaves and SeCys was only detected
in the grains (Table 2).
3.4. Selenium Speciation in Indian Mustard
In the brassica roots, the peak at 12,667.8 eV is clearly visible along
with a shoulder at 12,662.5 eV (Fig. 4). The best LCF t suggested the
presence of 26% SeCys, 30% SeMeCys, 28% selenate, and 16% selenite
(Table 2). Probably the organic species are still underestimated because
the ts cannot fully reproduce the organic part of the spectra; still, they
are dominant (56%) (Fig. 5).
In the stem the peak at 12,667.8 eV dominates with higher intensity
compared to the root's spectrum, indicating that more selenate is present
(Fig. 4). This is conrmed by the linear combination t, resulting in 52%
selenate. Further 10% can be attributed to selenite, and 38% of the Se in
the stem is present in an organic form, probably as SeMeCys (Table 2).
In the leaves, the peak at 12,667.8 eV, corresponding to selenate, is
even higher compared to roots and stem (Fig. 4). Accordingly, the linear
combination t resulted in 70% selenate and 24% SeMeCys. The remaining 6% can be attributed to selenite (Table 2).
In the ower the situation is different compared to the other
plant parts, with a higher peak at 12,662.3 eV and a smaller peak at
12,667.8 eV (Fig. 4), indicating that the organic species are dominant
(Fig. 4). The best t revealed 83% of organic species (75% DMeSe and 8%
SeMet), and 17% of selenite (Table 2). However, although too weak to
be tted, some selenate could be present because the t was not able
to reproduce the shoulder at 12,667.8 eV.
With the exception of the ower, the proportion of organic and inorganic species from root to leaves is conrmed, with a dominance of
organic species in roots and stems and an increasing fraction of inorganic species towards the upper parts of the plant (Fig. 5). Inorganic species
gradually increase from root and stem to leaves, reaching N70% in the
leaves. On the other hand, with more than 80% of the Se in an organic
form (DMeSe and SeMet), Se speciation in the ower is clearly different,
and does not follow the above trend.
The trend in inorganic species is controlled by the amount of selenate
which gradually increases from less than 30% in the roots to more than
70% in the leaves, while no selenate is present in the ower. Minor
amounts of selenite (615%) were found in roots and stems. SeCys and
SeMeCys dominate in roots, while in stems and leaves only SeMeCys
was found in a considerable amount. On the other hand, DMeSe and
SeMet were found in the owers only (Table 2).

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E. Eiche et al. / Science of the Total Environment 505 (2015) 952961

Table 2
Total Se concentrations and relative proportion of Se species in different plant parts (root, stem, leaves, ower, and grain) of wheat (Wh) and Indian mustard (Mu). Concentrations are
given on dry weight basis. The error on the relative fractions is estimated to be around 10%.
Setot
mg/kg

Roota
Stema
Leavesa
Flowera
Grainb
a
b

Selenate
%

Wh

Mu

196 2
191 2
387 3

186
133
931
541

2
3
4
3

Selenite
%

SeMeCys
%

SeMet
%

SeCys
%

DMeSe
%

Wh

Mu

Wh

Mu

Wh

Mu

Wh

Mu

Wh

Mu

Wh

Mu

13
18
47

28
52
70

12
11
10

16
10
6
17

21
30

68

30
38
34

41
43

32

26

54

75

Pellets measured at SUL-X beamline, ANKA.

Fresh bisected grains measured as such at GILDA, ESRF.

4. Discussion
4.1. Selenium Uptake and Translocation in Wheat and Mustard
The high total Se concentrations in all plant parts (133196 mg/kg
in the roots and stems and 387931 mg/kg in the leaves of wheat and
mustard, respectively) indicate that the concentration of bioavailable

Fig. 2. XANES spectra (solid lines) and linear combination ts (dashed lines) of different
plant parts of wheat. Root, stem and leaves were measured as pellets at SUL-X beamline
(ANKA). Fresh bisected grains were measured as such at GILDA (ESRF).

Se must be high in the seleniferous soil on which the plants were

grown. This is conrmed by sequential extraction experiments indicating that both Se associated with organic species and easily available Se
(selenate) are abundant in the soil (Table 1), and explaining the high
enrichment in the upper plant parts. Easily available Se is probably
mainly introduced through irrigation. The organic pool largely results
from the decay of organic matter that remains on the eld each year
after harvest.
The fact that Se was found in the plants at concentrations 1530
times higher than in the soil indicates active Se accumulation in plants.
In addition, Se concentration was found to increase moving from
the roots to the stem and leaves, indicating that Se is taken up and
transported from the roots to the leaves via the stem, where it nally
accumulates.
The high enrichment of Se in the wheat roots and its presence mainly as organic species (75%) (Fig. 3, Table 2), both suggest a substantial
proportion of Se taken up from the soil in the form of selenite. This is
in line with the results of other studies, which have shown that selenite
is mainly retained within the roots and quickly transformed to organic
forms like SeCys or SeMet (De Souza et al., 1998; Zayed et al., 1998; Li
et al., 2008; Kikkert and Berkelaar, 2013; Wang et al., 2013). According
to Li et al. (2008) and Kikkert and Berkelaar (2013) only a small fraction
(b12%) of selenite migrates to the shoots. The concentration of selenite
(adsorbed Se) within the soil is relatively low with 0.7 mg/kg (Table 1),
which could be due to previous substantial plant uptake of this species.
The fact that the highest Se concentration, by far, is found in the
leaves indicates that most of the Se must be present in a form that can
easily be taken up and transported within plants to sink tissues. Indeed,
selenate, which is considered to be the most mobile species within
wheat plants (Li et al., 2008; Kikkert and Berkelaar, 2013), was detected
as the major species in the leaves (47%). SeMet, also reported to be
mobile within plants (Sors et al., 2005), was also found in the leaves
in a high proportion (41%). According to Kikkert and Berkelaar (2013),
SeMet leads to the highest enrichment in wheat shoots when present

Fig. 3. Proportion of organic and inorganic Se species in different plant parts of wheat
based on the results of linear combination ts.

E. Eiche et al. / Science of the Total Environment 505 (2015) 952961

Fig. 4. XANES spectra (solid lines) and linear combination ts (dashed lines) of different
plant parts of Indian mustard. All plant parts were measured in the form of pellets at
SUL-X beamline (ANKA).

as the only Se species in a hydroponic system. However, SeMet could

also originate from transformation of selenate within the plants,
which is a well-known process (De Souza et al., 1998; Terry et al.,
2000; Sors et al., 2005). In the grains only organic Se species were

Fig. 5. Proportion of organic and inorganic Se species in different plant parts of Indian
mustard based on the results of linear combination ts.

957

detected, indicating that selenate is completely transformed into

organic species either within the grains or before being transported to
them. One reason for the dominance of organic Se in grains might be
the fact that Se, like sulfur, is typically mainly enriched in the endosperm of wheat grains and stored as part of the proteins (Moore et al.,
2010).
The presence of about 10% selenite in stem and leaves could indicate
its migration to the upper plant parts, despite the fact that it is assumed
to have a low mobility within plants. This, however, would imply that a
high amount of Se in the soil must be present as selenite. Assuming that
10% of Se in the leaves is present as selenite, corresponding to 39 mg/kg,
and that less than 12% of the selenite reaches the leaves (Li et al., 2008;
Kikkert and Berkelaar, 2013), this would result in a total selenite uptake
of 325 mg/kg. Such an amount, however, is extremely high and not in
line with the observed Se content in roots (190 mg/kg), which are
typically enriched in selenite and its transformation products, and
with the observation that selenate is one of the major species taken
up by mustard and wheat plants. Since the amount of selenite in root,
stem, and leaves is about the same (roughly 10%, i.e. close to the experimental error associated to linear combination tting) its presence
could be the result of an analytical artifact. Selenite could also originate
from beam induced photoreduction of selenate, as shown by Wang et al.
(2013).
Selenium concentration in root and stem of the mustard and wheat
plants is similar (133196 mg/kg), while higher concentrations were
found in the leaves of both plants, where it accumulates, with the
highest concentrations in mustard leaves (up to 931 mg/kg). In mustard
roots Se is nearly equally present as organic (56%) and inorganic (44%)
species (Fig. 5). The high proportion of organic Se in the form of
SeMeCys (30%) and SeCys (26%), as well as selenite (16%) (Table 2),
suggests that Se was taken up in the form of selenite or organic Se
(SeCys), because these Se species are known to be retained in the
roots once taken up (De Souza et al., 1998; Zayed et al., 1998). This
assumption is supported by the relatively high concentration of Se in
the organic pool (1.1 mg/kg, Table 1) of the soil, which typically largely
consists of organic Se species (Zhang and Moore, 1996). In mustard
roots, a large fraction of Se is also present in the form of selenate
(28%) indicating that it was also readily taken up from the soil. The
amount of easily available selenate was found to be relatively low in
the soil (0.5 mg/kg, Table 1). This could be due to the high Se removal
ability of these plants. Once taken up, selenate does not accumulate in
the roots, but is rather quickly transported through the vascular bundles
in the stem towards above-ground plant parts, becoming the dominant
species in the leaves. Substantial selenate uptake further explains the
ve times higher Se content in the leaves and the three times higher
Se concentration in the ower, compared to the roots. According to
Zayed et al. (1998) shoot/root Se concentration ratios N1.4, a value
which is exceeded in this study, are indicative of large uptake of
selenate. This is in agreement with Montes-Bayn et al. (2002), who
found that selenate has the highest uptake rate in mustard plants.
High shoot-to-root Se ratios are typical of hyperaccumulator plants.
The results of this work show that this might also be valid for semiaccumulator plants like mustard, in agreement with White et al.
(2007). The relatively low Se concentration in the stem compared to
the other plant parts suggests that the stem is mainly used as passage
for Se on its way towards the leaves and owers, where it eventually
accumulates, as occurs for other nutrients, like sulfur. Still, a considerable proportion of Se is retained in the stem through transformation
into organic Se in the form of SeMeCys (38%).
Although the total and easily available Se concentration in the
mustard soil (Setot 6.8 mg/kg, selenate 0.5 mg/kg) was found to be only
half of that in the wheat soil (Setot 13.3 mg/kg, selenate 1.2 mg/kg), the
total Se concentration in the upper plant parts of the mustard is up to
three times higher. Conversely to wheat leaves, mustard leaves did
not show any sign of chlorosis, indicating that mustard can take up and
tolerate higher Se concentrations compared to wheat.

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E. Eiche et al. / Science of the Total Environment 505 (2015) 952961

4.2. Adaption of Selenium Transformation mechanisms Towards High

Selenium Uptake
Selenium concentrations measured in wheat plants (up to
390 mg/kg) are higher than those reported in studies on wheat in
other seleniferous areas (b 63 mg/kg, Moxon et al., 1943; b263 mg/kg,
Dhillon and Dhillon, 1991; b185 mg/kg, Cubadda et al., 2010). Furthermore, the maximum concentration is above the threshold of toxicity of
Se in wheat tissue determined by Lyons et al. (2005) in pot experiments
(325 mg/kg), which explains the visible signs of white chlorosis.
Stunted growth, however, was not visible during sampling. Therefore,
our results strongly suggest that wheat can be considered as relatively
tolerable towards high Se uptake, especially in comparison with other
crop plants. One explanation for the high Se tolerance compared to
other studies could be a concurrent enrichment of sulfur. Kikkert et al.
(2013) concluded from their study on durum wheat that a high accumulation of sulfur enhances the resistance of plants towards Se toxicity,
allowing much higher Se uptake. This is because more sulfur can be
allocated to necessary plant functions leading to a protective effect.
Therefore, the role of sulfur should be included in further studies on
highly Se-contaminated wheat plants.
Typically, SeMet is reported to be the major Se species in different
parts of wheat plants or wheat based food with relatively low Se uptake
and no signs of chlorosis (Lintschinger et al., 2000; Whanger, 2002;
Warburton and Goenaga-Infante, 2007; Cubadda et al., 2010). This
is also true for the stem and leaves analyzed in this study, where
the SeMet fraction was determined to be 41 and 43%, respectively
(Table 2). However, the observed presence of methylated species
(DMeSe, SeMeCys) in all plant parts, except in the leaves, indicates an active response of the plants to minimize Se induced toxicity. Wang et al.
(1999), for example, have shown that methylation increases the Se tolerance of Astragalus bisulcatus. Methylated Se species are also reported to
be considerably less toxic than SeMet, SeCys, and inorganic Se species,
because they are not incorporated into proteins (Wilber, 1980; De
Souza et al., 1998; Neuhierl et al., 1999; Montes-Bayn et al., 2002).
Volatile DMeSe will eventually leave the plant, thereby leading to an
actual Se reduction (De Souza et al., 1998; Pilon-Smits and Quinn, 2010).
The fact that DMeSe is the dominant species in the wheat root (54%)
suggests that the plant tries to reduce toxicity by volatilization. For animals, DMeSe is reported to be 500 times less toxic compared to inorganic species (Wilber, 1980). Typically, SeCys or SeMet should be the major
organic species in roots, as primary conversion products of selenite or as
directly taken up Se species (De Souza et al., 1998; Zayed et al., 1998; Li
et al., 2008; Kikkert and Berkelaar, 2013; Wang et al., 2013). Zayed et al.
(1998), however, have shown that several plant species like rice, broccoli, and mustard volatilize considerable amounts of Se already in the
root, especially if selenite or SeMet is taken up. The rate of volatilization
depends not only on the Se speciation, but also on the Se and sulfate
concentration in the tissue and the soil solution (De Souza et al., 1998;
Terry et al., 2000; Cubadda et al., 2010). The presence of SeMeCys as
another important species in roots, stem, and especially in grains
(Table 2), is in agreement with the active detoxication interpretation
of the results. In fact, SeMeCys is a precursor of DMeDSe, another volatile species (Terry et al., 2000; Sors et al., 2005), and is typically formed
in semi-accumulator plants like mustard or garlic as detoxication
product in amounts up to 80% (Whanger, 2004). However, it is usually
not present in non-accumulator plants (Terry et al., 2000; Whanger,
2002). SeMeCys was detected for the rst time in wheat grains by
Cubadda et al. (2010), who found an increasing amount of SeMeCys
with increasing Se supply. However, the proportion of SeMeCys was
less than 1% in their study compared to 2168% in this study (Table 2).
The different results could partly be due to differences in the applied
speciation method (enzymatic extraction followed by HPLCICP-MS).
Furthermore, the wheat investigated by Cubadda et al. (2010) had
lower Se concentrations (b 185 mg/kg in the grains), and did not show
any signs of chlorosis. Consequently, the high proportion of SeMeCys

in our study is most likely the result of the metabolic response of the
wheat plant to Se levels well above the reported tolerance concentration
of 325 mg/kg.
Selenate is typically a minor species in wheat or wheat based food
(Lintschinger et al., 2000; Rayman et al., 2008). In accumulator plants,
like A. bisulcatus, selenate is reported to be a major constituent in older
leaves, whereas organic species largely dominate in younger ones (up
to 95%) (Pickering et al., 2000, 2003a; Ellis and Salt., 2003). In this
study a mixture of leaves of different ages was investigated. The high
selenate content in stem and leaves of the investigated wheat plants
(up to 47%) (Table 2) could, therefore, be the result of mixing plant
material of a different age. However, taking into consideration the proposed active detoxication process and the fact that selenate is typically
found in low amounts in wheat, it is more probable that the high selenate
content observed in this study results from the extremely high Se uptake.
It seems that, if the selenate concentration exceeds a certain threshold
value, the plant is unable to metabolize all incoming selenate into organic
Se species. A similar observation was made by Whanger (2002), who
showed that the rate of selenate conversion diminished with increasing
selenate supply. The high proportion of selenate could also be due to
the fact that plants store excess selenate in their vacuoles like they typically due with sulfate (Rennenberg, 1984). In the sulfur metabolism the
amount of sulfate that actually is reduced to organic sulfur compounds
depends on the need and physiological status of chloroplasts. As plants
cannot distinguish between sulfate and selenate a similar behavior can
be assumed for selenate. Based on our data it is, however, not possible
to determine the maximum tolerable selenate uptake, which is probably,
inuenced by several parameters other than the Se concentration. It is
also possible that the lack of selenate conversion is linked to the signs
of chlorosis.
In mustard's leaves and owers the total Se concentration (931 mg/kg
and 541 mg/kg, respectively) is much higher than those in wheat, whereas in root (189 mg/kg) and stem (133 mg/kg) Se concentrations are comparable. High Se enrichment in mustard, a secondary accumulator plant,
frequently occurs because mustard is able to take up and accumulate
the bioavailable Se very efciently. This is possible thanks to a detoxication process based on the conversion of more toxic inorganic Se or Se
amino acids into organic and non-protein-compatible Se compounds,
mainly by methylation (De Souza et al., 1998; Terry et al., 2000; Ellis
and Salt, 2003). Moreover, Brassicaceae are known to have high sulfur
levels and unique S compounds which could also contribute to their
high Se accumulation capacity and might explain the higher enrichment
compared to wheat (Cobbet and Goldsbrough, 2002; Cappa et al., 2014).
Indian mustard has repeatedly been reported to have a high ability
to methylate Se to SeMeCys or DMeSe, with the highest rate when
SeMet is the dominant species (Terry et al., 1992; Zayed et al., 1998;
De Souza et al., 1998). SeMeCys has also been described as a characteristic Se species produced by Se-accumulator plants (Terry et al., 2000)
with a proportion of up to 80% of total Se (Whanger, 2004). Consequently, the high proportion of SeMeCys (3038%) observed in mustard roots,
stem, and leaves, and of DMeSe (75%) in the ower (Table 2), is not
unusual, and originates from a detoxication process. The amount and
distribution of SeMeCys in different plant parts could also be determined by translocation within the plant. It has been reported from studies on A. bisulcatus that SeMeCys can be translocated within the plant
from older to younger tissues and to seeds during reproductive stage
(Pickering et al., 2003b). Volatilization is reported to be not only linearly
increasing with soil, but also root Se concentration (De Souza et al.,
1998). Zayed and Terry (1994) highlighted in their study that roots
are the main site of volatilization in mustard, and the Se speciation of
our study conrms this result. In fact, the proportion of SeMeCys, a precursor of volatile DMeDSe, in mustard roots is 30%, indicating that the
plant tries to transform Se into less toxic forms already in the root.
Rates of methylation are especially high in the presence of selenite or
organic Se compounds, which is the case of the mustard root samples
studied in this work (Table 2). Comparable methylation rates can also

E. Eiche et al. / Science of the Total Environment 505 (2015) 952961

be observed in stem and leaves (3834% SeMeCys), indicating that

detoxication, and probably also volatilization, occurs in the whole
plant. This could be due to the high uptake of easily transportable selenate, whose proportion increases from roots to stem, reaching 70% in
the leaves (Table 2). During methylation the reduction of selenate is
the rate limiting step. Consequently, the high proportion of selenate in
stem and leaves is not only a result of the large Se uptake, but also displays the inability of the plant to convert all selenate to non-toxic forms.
The highest methylation and volatilization take place in the
mustard's ower heads, as indicated by the large fraction of DMeSe
(75%) detected (Table 2). The high volatilization rate shows that mustard is highly effective in transferring Se into the atmosphere, therefore,
permanently removing it from the soilplant system. Since the rate of
volatilization depends on both Se speciation and concentration, the
much higher volatilization in the head compared to other plant parts
could be explained by the higher amount of total Se. It could also be
due to a preferential transport of organic species to the head, like in
the case of A. bisulcatus, where organic species are preferentially
translocated to seeds during reproductive stage (Pickering et al,
2003b). In fact, if only organic Se is present, selenate reduction, which
is the rate limiting step, does not occur. Consequently, the rate of volatilization is higher in plant parts where selenate is absent (De Souza
et al., 1998; Pilon-Smits et al., 1999).
4.3. Health and Economic Aspects
Typically, SeMet is the major species in many food crops like wheat
(Levander, 1987; Whanger, 2002). In our study, however, the major
species detected in grains, which are the main part of the plant consumed by humans, were SeMeCys (68%) and SeCys (26%). Because of
the high total Se concentration detected in the wheat, the presence of
Se in a less bioavailable form would be benecial from a health aspect.
SeMeCys is reported to be absorbed by humans as well as SeMet,
but not so well stored (Finley, 2003; Rayman et al., 2008). Moore et al.
(2010) have shown that Se is mainly enriched in the endosperm of
wheat grains which is the main part that is transferred into the food
chain during milling for our production. Consequently, the consumption of wheat grains from the studied area should result in a high transfer of Se into the human metabolism, but in no long-term accumulation.
In addition, from a toxicological point of view the high proportion
of SeMeCys, instead of SeMet or SeCys, is positive, because of its very
low toxicity and its high efcacy towards cancer prevention (Dong
et al., 2001; Medina et al., 2001; Whanger, 2004; Rayman et al., 2008;
Rayman, 2012). Ip et al. (1992) have reported that SeMeCys is twice
as active as SeMet at suppressing mammary tumors, and SeCys seems
to be more toxic, but also more effective with regard to cancer prevention compared to SeMet (Ip and Hayes, 1989; Hasegawa et al., 1996;
Barceloux, 1999). Due to the generally high Se concentration, these
positive aspects, however, do not imply that high consumption of
wheat based food from the studied seleniferous area is safe.
Animals will also consume wheat stem and leaves as part of their
fodder. In these plant parts SeMet and selenate were found to be
dominant. The high Se levels in the leaves (387 mg/kg), mostly in the
form of highly bioavailable SeMet (Ip and Hayes, 1989) in both stem
and leaves (4143%), imply a considerable Se transfer into the animal
metabolism. In addition, SeMet strongly accumulates in mammalian
bodies, worsening the problem (Combs, 1988). Selenate, which was
found to be the dominant species in the leaves, is generally excreted
more rapidly by the mammalian metabolism compared to SeMet, but
possesses a relatively high toxicity (Yang et al., 1983; Ip and Hayes,
1989). Consequently, a considerable threat for animal health has to be
expected from regular consumption of regional green fodder (Dhillon
and Dhillon, 1991; Bajaj et al., 2011).
The same problem applies to the studied mustard if used as animal
forage. Even though no highly transferable and accumulative species
like SeMet was found in mustard, its regular consumption would pose

959

a high health risk for cattle. This is due to the extremely high Se concentration (up to 931 mg/kg), to the high fraction of easy absorbable
SeMeCys, and to the high proportion of selenate (5270%), a relatively
toxic species. Humans mainly consume the seeds of mustard (Bansal
et al., 2010), which were not investigated in this study; nevertheless,
comparably high Se concentrations are expected also in the seeds and
its consumption without mixing with seeds from non-seleniferous
sources is not recommendable. In addition, in some countries the
consumption of green parts of mustard is common as well. Again, due
to the high total Se concentration and to the high proportion of relatively toxic inorganic Se species (5270%), their consumption has to be
considered a health hazard.
Other than being dangerous for human and animal health, high
transfer of Se into plants also has an economic relevance for the farmers.
Due to selenosis, which also makes animals more susceptible towards
other illnesses, the livestock might be reduced. Furthermore, relatively
high levels of Se in forage plants can cause stunted growth and lead to
fewer leaves, rosettes, and owers (Sharma et al., 2010). Rani et al.
(2005) reported a signicant yield reduction beginning with 18.9 mg
Se/kg in wheat shoots (Triticum aestivum L.) or 104.8 mg Se /kg in
raya (Brassica juncea Czern L.), which are much lower concentrations
than that reported in this study (Table 2). However, the studied Seenriched plants could be a good source of Se to be exported to Se-poor
areas after mixing them with plants from non-seleniferous areas to
reduce the Se content. In addition, the presence of high proportion of
SeMeCys, which is highly bioavailable and effective in cancer prevention,
would enhance even more the health benets.
It is not to be expected that the situation will change in the near
future since the pool of selenate, the major Se species taken up by the
plants in the studied area, and of organic Se, is renewed each year.
Selenate is introduced into the soil mainly via irrigation, whereas organic Se results from the decay of Se-rich plant material. High selenate and
SeMet levels in soil imply high Se concentration in the above-ground
plant parts (e.g. leaves), which are the most used by farmers, e.g. as
forage crops.
5. Conclusion
This study has shown that not only well-known Se accumulating
plant species like mustard are able to uptake and accumulate Se, but
also crop plants like wheat. The high Se enrichment is mainly due to
the fact that considerable amounts of easily available Se is present in
the soil and refreshed on a yearly basis. The high Se enrichment
observed in the upper plant parts suggests that selenate or organic species (mainly SeMet) are abundant in the soil and are, consequently, the
major Se species taken up. This is also conrmed by sequential extractions, which showed a signicant fraction of easily available Se. The
dominance of selenate in the leaves of wheat and mustard could result
from its preferential uptake and translocation within plants, but could
also be due to the inability of the plant metabolism to transform all
selenate into non-toxic organic Se species at high uptake rates or the
storage of Se in vacuoles. Methylated species like DMeSe and SeMeCys
were found in all wheat and mustard plant parts, indicating that active
detoxication is taking place. SeMeCys is a typical detoxication product in accumulator plants. However, the high proportion of SeMeCys
in wheat, especially in the wheat grains, shows that the wheat plant is
able to adapt its metabolism to the high Se concentration, similarly to
accumulator plants.
Selenium was found to be mainly present in a form that is highly
absorbable by human and animal metabolism. In addition to the generally high Se concentration observed in all investigated plant parts, this
leads to a considerable health risk for humans and livestock if only local
Se-contaminated food is consumed. One positive aspect for human
health is the high proportion of SeMeCys, which has a low toxicity, but
high efcacy towards cancer prevention. To prevent wasting and bring
benets for human health, the studied plants could be mixed with plants

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E. Eiche et al. / Science of the Total Environment 505 (2015) 952961

from non-seleniferous areas to reduce the Se content and obtain

SeMeCys-enriched food to be exported to Se-decient areas.
Acknowledgments
We want to thank the International Bureau of the BMBF for nancially supporting the sampling campaign in Punjab, enabling the cooperation with our Indian colleagues. Furthermore, we would like to thank
Dr. Mini Bajaj from the Institute of Biology for Engineers and Biotechnology of Wastewater (KIT) for her important support to start up the cooperation and during the research trip to Ludhiana. We are also grateful to
all students from the Department of Soil Science from the PAU for their
help during the eld work. The authors are also grateful to the Italian
beamline's staff (BM8 GILDA) at the ESRF, Dr. Francesco D'Acapito
and Dr. Angela Trapananti, and the German beamline staff (FLUO) at
ANKA (KIT), Dr. Rolf Simon and Dr. David Batchelor, for providing
beamtime and assistance during -XRF measurements. Finally, we are
grateful to Dr. Geraldine Sarret for the useful discussions and for sharing
the spectra of some Se standards.
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