J Soils Sediments (2016) 16:581–591
DOI 10.1007/s11368-015-1248-6
SOILS, SEC 4 • ECOTOXICOLOGY • RESEARCH ARTICLE
Phytoavailability of lead altered by two Pelargonium cultivars
grown on contrasting lead-spiked soils
Muhammad Arshad 1,2,3 & Georges Merlina 2,3 & Gaëlle Uzu 5 & Sophie Sobanska 6 &
Géraldine Sarret 7 & Camille Dumat 4 & Jérome Silvestre 2,3 & Eric Pinelli 2,3 &
Jean Kallerhoff 2,3
Received: 16 April 2015 / Accepted: 20 August 2015 / Published online: 4 September 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract
Purpose This study assesses the potential of two contrasted
fragrant Pelargonium cultivars to induce pH and dissolved
organic carbon (DOC) changes in the soil solution, Pb speci-
ation, and their subsequent effects on rhizosphere
phytoavailable Pb.
Materials and methods Rooted plantlets were grown in spe-
cial devices, floating on aerated nutrient solution in PVC
tanks. This setup allows roots to be physically separated,
through a mesh, from a 3-mm soil matrix layer that can be
considered as rhizosphere soil. Two contrasted soils, each
spiked with Pb-rich particles, emitted from a battery recycling
Responsible editor: Dong-Mei Zhou
* Jean Kallerhoff
jean.kallerhoff@ensat.fr
1
Institute of Environmental Sciences and Engineering, School of Civil
and Environmental Engineering, National University of Sciences and
Technology, Sector H-12, Islamabad 44000, Pakistan
2 association of Pb with P in cylinder-like structures. Extended
UPS, INP; EcoLab (Laboratoire d’écologie fonctionnelle et
environnement); ENSAT, Université de Toulouse, Avenue de
l’Agrobiopôle, 31326 Castanet-Tolosan Cedex, France
3
CNRS; EcoLab (Laboratoire d’écologie fonctionnelle et
environnement), 31326 Castanet-Tolosan Cedex, France
4
Certop, CNRS UMR 5044, Université Toulouse J. Jaurès,
Toulouse, France
5
CNRS, IRD, LTHE, Université de Grenoble Alpes,
38000 Grenoble, France
6
LASIR UMR 8516, Université Lille 1, Bâtiment C5,
59655 Villeneuve d’Ascq Cedex, France
7
ISTerre, UMR-5275, Université J. Fourier & CNRS, BP. 53,
38041 Grenoble Cedex 9, France
industry, were used at total burdens of 500 and
1500 mg Pb kg−1 in addition to a control unspiked soil. Soil
solution pH, phytoavailable Pb, DOC, Pb adsorption, precip-
itation on roots, and Pb phases in soil and plant were
investigated.
Results and discussion Attar of Roses (Attar) cultivar acidi-
fied its rhizosphere by 0.4 pH units in both spiked soils.
Concolor Lace (Concolor) was unable to change soil solution
pH on soil-1 and increased it by 0.7 units on soil 2. Concen-
trations of Pb in soil solution from Attar plants were always
higher than those of Concolor ones. DOC contents of both
unspiked soil-1 and soil-2 without plants were not significant-
ly different. In the case of spiked samples, DOC contents in
the rhizosphere soil were increased by three and two times for
Attar and Concolor, respectively, compared to the unspiked
soil without plant. Both cultivars were able to increase DOC
contents, independent of soil type and level of contamination.
Accumulation of Pb in shoots and roots was higher in Attar as
compared to Concolor due to enhanced available Pb as a result
of pH and DOC modifications of the rhizosphere soil. Signif-
icant amounts of Pb were adsorbed on roots of both cultivars.
X-ray elemental analysis of precipitates on roots revealed the
X-ray absorption fine structure (EXAFS) spectroscopy re-
vealed that Pb was present, to a major extent in the inorganic
form, mainly as PbSO4 in the soil, whereas it was complexed
with organic species within plant tissues. The conversion of
Pb into organic species could decrease toxicity, may enhance
plant tolerance, and could increase translocation.
Conclusions Plant-induced changes were responsible for
the modification of lead phases within the soil. Immo-
bile forms present in the source leaded particles as well
as in the soils were converted into soluble species,
ultimately improving the phytoavailable or soil solubi-
lized Pb.
582
Keywords DOC . Pb . Phytoavailability . Rhizosphere pH .
Soil solution
1 Introduction
Anthropogenic activities including mining, smelting, agricul-
tural usages (application of insecticides), and urban wastes
(municipal sewage sludge) have been the main sources of Pb
contamination to soils (Fernández et al. 2012). Recently, bat-
tery manufacturing and recycling (Uzu et al. 2010) and shoot-
ing ranges (Strømseng et al. 2009) have emerged as major
contributors to soil contamination, as bullets contain about
90 % Pb (Sorvari 2007). Unlike organics, Pb is non-degrad-
able, and its persistence can result in its accumulation in the
terrestrial and aquatic biota, threatening human and environ-
mental health (Dixit et al. 2015).
The study of behavioral properties of Pb in soils could
hence be relevant to understanding processes involved in its
uptake by roots and translocation to aerial parts of plants.
Indeed, bioavailability is determined by soil physicochemical
characteristics, redox potential, genetic background of the
plant species, environmental factors, and their altogether in-
teractions (Kabata-Pendias 2004). Unravelling the underlying
mechanisms, such as pH dynamics (Cecchi et al. 2008) and
factors regulating bioavailable Pb (Khan et al. 2006), would
contribute to gaining insights into differential plant species
responses to heavy metal exposure.
Total Pb contents in soil cannot provide accurate infor-
mation on readily available or phytoavailable fractions con-
trarily to estimations of soil solution and rhizosphere status.
Moreover, its soluble forms could be vital for plant avail-
ability and uptake (Uzu et al. 2009). In our earlier work, we
showed that cultivars of Pelargonium species displayed dif-
ferent abilities for uptake of Pb from contaminated soils and
its transport to aerial parts. Attar cultivar appeared more
performant than Concolor, for lead translocation and accu-
mulation in leaves (Arshad et al. 2008). In this context, the
objective of the present study was to investigate the poten-
tial of these two contrasted Pelargonium cultivars to modify
soil solution pH, dissolved organic carbon (DOC), specia-
tion of Pb in soil and plant, and their effects on
phytoavailable Pb in the rhizosphere.
2 Materials and methods
2.1 Soils and spiking with leaded particles from the battery
recycling factory
Two uncontaminated top soils (0–30 cm) of contrasted calcic
cambisol profile (FAO 1998), produced by quaternary alluvial
deposits from the vicinity of Toulouse (southwest of France),
J Soils Sediments (2016) 16:581–591
were collected, air-dried, and sieved to 2 mm. Both soils
(named as soil-1 and soil-2) were chosen on the basis of their
contrasted physicochemical properties (Table 1). Soil-1 was
collected from a maize-cultivated organic farm and was a
calcareous clayey soil with 3.2 % of organic matter (OM)
and soil-2 was a sandy clay loam soil having 1 % of OM
cultivated with sunflower crops. Soil-1 had a higher cation
exchange capacity (30.0 cmol+ kg−1) compared to soil-2
(10.7 cmol+ kg−1).
Both soils were spiked with Pb-rich particles emitted from
a secondary Pb smelter which currently recycles batteries,
being run by the Chemical Metal Treatment Company
(STCM) located in the urban area of Toulouse, France. These
particles are the potential atmospheric fallouts responsible for
soil Pb contamination and contained 334 g Pb kg−1 in total and
had a particle size within the micrometer range (Uzu et al.
2009, 2011). Major phases consisted of approximately 55 %
PbS and around 25 % of PbSO4 +PbO·PbSO4. Calculated
amounts of particles were added to the soils to achieve final
concentrations of 500 and 1500 mg Pb kg−1, respectively.
These contamination levels were selected on the basis of pre-
vious field study of Arshad et al. (2008), where Pelargonium
cultivars showed potential for Pb uptake from moderately
contaminated soils, i.e., ~2000 mg Pb kg−1. Spiked soils were
placed at room temperature without addition of any moisture.
The soils were mixed every week to homogenize over a total
period of 1 month to reach equilibrium between particles and
exchanges sites. The uniformity of mixing was checked by
analyzing Pb concentration in five replicates. Variability did
not exceed 10 % of the average value. Uncontaminated soil-1
and soil-2 were treated similarly and contained naturally 19
and 24 mg Pb kg−1, respectively, and were used as controls.
2.2 Preparation of plant material for rhizosphere
experiments
Potted plants of both cultivars (Attar of Roses and Concolor
Lace referred hereinafter as Attar and Concolor, respectively)
were obtained from Heurtebise nursery, Clansayes, France.
Cuttings (12–15 cm in length) of both cultivars were grown
for 4 weeks on Fertis®, a low-nutrient substrate that favors
root development of the cuttings. After 4 weeks, rooted plants
were transferred to special cropping devices (Chaignon and
Hinsinger 2003). The device (Fig. 1) is of practical use for
rhizosphere studies, as roots are physically separated from the
2–3-mm soil layer through a mesh. Not only can the soil be
considered as rhizosphere soil, but complete separation of
roots from the soil is straightforward. The cropping devices,
containing one plant each, were placed in containers holding
20 L of nutrient solutions to allow development of the root
mat (Fig. 1). The composition of the nutrient solution was
5000 μM KNO3, 5000 μM Ca(NO3)2, 2000 μM KH2PO4,
1500 μM MgSO4, 46 μM H3BO3, 9 μM MnSO4·H2O,
J Soils Sediments (2016) 16:581–591 583

Table 1 Physicochemical characteristics of the two soils used for bottom parts. Root mat and soil were separated from each
rhizosphere experiments
other through a 30-μm mesh (Fig. 1). The experiment was
Characteristic Unit Soil-1 Soil-2 conducted in a growth chamber under the following condi-
tions: temperature 24±0.5 °C/18±0.5 °C day/night cycles;
pHH2O – 8.2 6.4 photoperiod 16 h under daylight fluorescent lamps providing
pHCaCl2 – 7.4 5.4 400 μmol m−2 s−1 (Philips 600 W, Eindhoven, Netherlands)
CEC cmol+ kg−1 30 10.7 and 8-h darkness; and relative humidity 70 %.
Clay g kg−1 420 230 For rhizosphere experiments, smaller containers having 4 L
Silt g kg−1 345 265 of nutrient solution were covered with aluminum paper. Five
−1
Sand g kg 235 510 devices, enclosing one plant each, were placed on the alumi-
C/N – 9.3 8.1 num foil. Humidification of soils was achieved through the
Organic matter g kg−1 32 10 filter paper, placed below the soil, and dipped into the nutrient
Total carbonates g kg−1 268 <1 solution (Fig. 1). Controls (soil in round plates without plants)
(P2O5) Joret Hebert g kg−1 0.07 0.035 were maintained under the same conditions in order to evalu-
Pb (HF extraction) mg kg−1 19 24 ate pH changes induced by the nutrient solution. For this pur-
pose, pH was measured after 0, 4, 8, 12, and 15 days by
CEC cation exchange capacity, HF hydrofluoric acid
collecting a representative sample from soil controls.
During soil-plant contact experiments, concentrations of
KNO3, Ca(NO3)2, KH2PO4, and MgSO4 were reduced to
0.1 μM MoNaO4·2H2O, 0.9 μM CuSO 4·5H2O, 15 μM
1/10th and, to half for iron of the original nutrient concentra-
ZnSO4·7H2O, and 180 μM Fe–EDTA. After 2 weeks of cul-
tions used for plant material preparation phase. These modifi-
ture in hydroponics, the root mats were well developed within
cations in the nutrient solution were done to favor Pb uptake
the cropping devices and plants were ready for rhizosphere
by reducing its competition with metal ions. The nutrient so-
studies. The level of nutrient solution was maintained with
lution was changed after 1 week over a culture period of
distilled water. These preparations were performed in a green-
2 weeks. The levels in the containers were maintained with
house with a photoperiod of 14 h, a temperature of 25±1 °C,
nutrient solution sufficiently to keep the filter paper dipped in
and with a relative humidity of 60–70 %.
the solution. The pH of nutrient solution ranged between 5.5
and 6.0. After 2 weeks, the rhizosphere soils, shoots, and roots
2.3 Rhizosphere experiments were collected for further analysis. There were five replicates
for each treatment.
Ten grams of soils was placed in the detachable round plate, at
the bottom of the 60-mm-internal-diameter devices, resulting 2.4 Soil and plant analysis
in a 3-mm-thick soil layer (Fig. 1). Each soil was sampled to
determine initial pH and to perform chemical analysis at the Eight grams of humid soil (58 % moisture) was taken in two
start of experiment. The upper parts of the devices, containing Eppendorf®tubesforeach sampleandcentrifugedat 15,000g
plants having developed root mats, were placed on round plate for 15 min at 20 °C for soil solution extraction (method

Fig. 1 Schematic diagram of the

cropping device used for
rhizosphere experiments, adapted
from Chaignon and Hinsinger
(2003)

aa PVC
Mat Cylinders

Soil
Polyamide net∅ 900μm
Filter Paper 30 μm mesh
Support

Nutrient Solution
584
modified from Angeles et al. 2006). Supernatants from both
tubes harboring the same sample were collected as Bsoil solu-
tion.^ The pH was determined using a digital pH meter. Soil
solution Pb concentrations were measured by IRIS Intrepid II
XDLICP-OESspectrophotometer.Dissolvedorganiccarbon
(DOC) was determined using a Shimadzu 5000A TOC
Analyzer.
After 15-day culture, shoots and roots were collected sep-
arately. Roots were washed with 0.01 M HCl to determine Pb
bound to outer root cell walls, called [Pb]adsorbed (Ferrand et al.
2006). Roots and shoots were then oven-dried at 80 °C during
48 h and weighed for dry weight (DW). Dried plant material
was ground to powder form, and 125 mg of each sample was
mineralized in a 5:1.5 mixture of HNO3 and H2O2 at 80 °C for
4 h. After filtration on a 0.22 μm pore size MinisartR syringe
filters, elemental concentrations were determined with an IRIS
Intrepid II XDL ICP-OES spectrophotometer. The accuracy of
the acidic digestion and analytical procedures was verified
using a known reference material (Virginia tobacco leaves,
CTA-VTL-2, ICHTJ).
2.5 Lead speciation through environmental scanning
electron microscopy and extended X-ray absorption fine
structure spectroscopy
Secondary and backscattering electronic images and X-
ray elemental maps of roots were obtained using an envi-
ronmental scanning electron microscopy–energy disper-
sive X-ray spectroscopy (ESEM-EDX) at BGeosysteme^
laboratory (UMR CNRS 8157), Lille University, France.
The roots of Attar plants, after 15 days of exposure, were
put on carbon substrates and analyzed. The Quanta 200
FEI instrument was equipped with an EDX Quantax
Brucker system working in low-vacuum mode at 25 kV.
Because of the ESEM configuration, only the qualitative
distribution of elements can be provided and the detection
of light element (C, N, and O) is difficult.
Spectra from extended X-ray absorption fine structure
(EXAFS) spectroscopy were recorded at the Pb LIII-edge
(13.055 keV) at the French National Synchrotron Facility
(SOLEIL, St. Aubin, France) on the SAMBA beam line
equipped with a Si(111) double crystal monochromator (Belin
et al. 2005). The bulk spiked soil was dried, ground in an agate
mortar, and pressed as 5-mm-diameter pellets using a hydrau-
lic press generating a pressure of 30 bars. Fresh roots of Attar,
cultivated on Pb-contaminated soil-1 with 1500 mg kg−1 were
frozen in liquid N2, ground using a mortar immersed in liquid
N2, pressed as 5-mm-diameter pellets, and transferred in a
cryostat cooled with liquid N2 by keeping the material in fro-
zen state at all times. This is crucial to avoid changes of Pb
speciation. Various Pb reference compounds were recorded,
including PbSO 4 , αPbO, Pb oxysulfate, aqueous Pb-
glutathion (15 mM Pb(NO 3 ) 2 + 15 mM GSH, i.e.,
J Soils Sediments (2016) 16:581–591
glutathione +30 mM ascorbic acid, pH =3), and aqueous
Pb2+ (10 mM Pb(NO3)2, pH=6.5). The solutions were mixed
with 30 % glycerol to avoid the formation of ice crystals.
Spectra were recorded in fluorescent mode using a silicon drift
detector (RONTEC) or in transmission depending on Pb con-
centration, at 77 K for the roots and aqueous reference and at
room temperature for soil, particles, and inorganic references.
The EXAFS spectroscopy interference function χ(k) was ex-
tracted using the ATHENA program, version 9, a part of the
IFEFFIT package (Ravel and Newville 2005). Sample spectra
were fitted by linear combinations using the standard spectra
mentioned above and other mineral and organic Pb references
recorded previously (Manceau et al. 1996; Sarret et al. 1998a,
b). For a given compound, the spectrum recorded at 77 K had
only slightly higher amplitude than the one recorded at 300 K,
so we combined both types of measurements in the linear
combination fits. Each spectrum was first fitted with one com-
ponent, and an additional component was allowed if the fit
quality was improved significantly, i.e., if the normalized sum
of squares residual parameter (NSS = ∑[k3x(k)exp – k3x(k)fit]2/
∑[k3x(k)exp]2 ×100) was decreased by at least 10 %. Using this
procedure, the spectra for the soil and roots were correctly
simulated by four components. Satisfactory fits were defined
by NSS increase within 5 % of that for the best fit. Using this
criterion, one and five good fits were obtained for the soil and
roots, respectively. For the roots, average and standard devia-
tion of the proportions of Pb species were calculated on the
basis of these good fits.
2.6 Statistical analysis
Statistical analysis was performed following the ANOVA pro-
cedures with the test of least significant difference (LSD)
using STATISTICA software (Stat Soft 2008).
3 Results
3.1 Soil solution pH and phytoavailable Pb
Changes in the soil solution pH are displayed in Fig. 2a. The
pH of unspiked soil, without plants, was unaffected by the
nutrient solution at the end of experiment (data not shown).
When plants were cultured on soil-1, a decrease of 0.35±0.11
pH units was observed for Attar cultivar as compared to the
unspiked soil without plants. In contrast, Concolor cultivar
could not modify pH on soil-1. On soil-2, Attar cultivar acid-
ified its rhizosphere down to 0.4 pH units (ΔpH=−0.4±0.05).
Contrastingly, a significant increase in pH (ΔpH=+0.7±0.13)
was observed when Concolor was cultivated on Pb-
contaminated soils both at 500 and 1500 mg Pb kg−1.
Lead concentrations measured in the soil solution are pre-
sented in Fig. 2b. For both soils, phytoavailable
J Soils Sediments (2016) 16:581–591 585

Fig. 2 Rhizosphere pH changes 8.0

and phytoavailable Pb after a
15 days culture of two scented
Pelargonium cultivars. Plants
were cultured on unspiked soils-1
and -2 (S0), and on spiked soils
with 500 (S500) and 1500
7.5

Soil Solution pH
(S1500) mg kg−1 of Pb,
respectively. Unspiked soils with
no plants (US) were used as
controls. a The soil solution pH in
the rhizosphere of Attar of Roses 7.0
(triangles) and Concolor Lace
(squares). b Soil solution or
phytoavailable Pb contents for
Attar of Roses (open bars) and
Concolor Lace (dark-spotted 6.5
bars). Different alphabetical
letters indicate significant
difference (p<0.05) among
values as measured by
LSD Fisher test 6.0
US S0 S500 S1500 US S0 S500 S1500
Soil 1 Soil 2
Pb concentration (mg kg -1)
200
b a
Soil Solution [Pb] mg kg-1

150

100
c c
cd c

50 d d

e e
0
S0 S500 S1500 S0 S500 S1500
Soil 1 Soil 2
Pb concentration (mg kg -1)

concentrations were higher for Attar cultivar compared to cultivar at a contamination level of 1500 mg Pb kg−1. The
those for Concolor at respective levels of contamination, i.e., minimum value of 33±9 mg Pb kg−1 was measured for soil-
500 and 1500 mg Pb kg−1. The maximum value of 160± 2 in contact with Concolor with a contamination level of
21 mg Pb kg−1 was recorded for soil-1 in contact with Attar 500 mg Pb kg−1.
586 J Soils Sediments (2016) 16:581–591

3.2 Dissolved organic carbon
Results of DOC measurements in soil solution are given in
Fig. 3. In unspiked soils without plant, DOC values after
15 days were 40±4 and 46±6 mg L−1 for soil-1 and soil-2,
respectively. DOC values were increased three times for Attar
(126±20 and 140±11 mg L−1 for soil-1 and soil-2, respective-
ly) and approximately two times for Concolor cultivar (75±8
and 78±6 mg L−1 for soil-1 and soil-2, respectively) as com-
pared to the unspiked soils without plant. There were no sig-
nificant differences between levels of DOC produced by Attar
on both soils and whatever the Pb load was. Concolor pro-
duced lesser DOC amounts than Attar, independently of the
soil properties and Pb contents.
3.3 Lead accumulation by plants
Results showing Pb concentrations in plant parts of Attar and
Concolor cultivars are presented in Table 2. For all conditions,
Attar cultivar accumulated more Pb in shoots than Concolor,
with a maximum of 284±39 mg Pb kg−1 DW for soil-1 spiked
with 1500 mg Pb kg−1 soil. Likewise, the roots of Attar
contained significantly more Pb than Concolor, with a maxi-
mum of 4042 ± 518 mg Pb kg − 1 DW for soil-2 at
1500 mg Pb kg−1 soil. Lead concentrations in roots increased
significantly with increasing Pb concentration for both
cultivars.
3.4 Lead adsorption on roots, precipitation, and speciation
Results on the amount of Pb bound to root cell walls
(Pbadsorbed), extracted by 0.01 M HCl, are shown in Fig. 4a.
Attar cultivar had significantly higher levels of adsorbed Pb
when cultured on soil-1 as compared to those on soil-2, with a
maximum value of 375±37 mg kg−1 DWroot for soil-1 spiked
with 1500 mg Pb kg−1. On the contrary, Concolor cultivar
adsorbed more Pb when cultured on soil-2 as compared to
soil-1. The maximum value determined was 443 ±
59 mg kg−1 DWroot on soil-2 containing 1500 mg Pb kg−1.
For all conditions, a higher Pb adsorption was measured at
1500 mg Pb kg−1 as compared to that at 500 mg Pb kg−1.
Secondary electrons (SEs) and backscattered electron
(BSE) images and EDX spectra were obtained through envi-
ronmental scanning electron microscopy (ESEM) from air-
dried roots (Fig. 4b–e) of Attar cultured in soil-1 at
1500 mg Pb kg−1 for 2 weeks. Some isolated fine particles
(<10 μm) containing Pb were observed on the root surface. In
addition, Pb was present on roots under various forms: aligned
cylinder-like particles of about 10-μm length and diameter,
aligned with the root elongation direction (Fig. 4b),

Fig. 3 Dissolved organic carbon 150

contents in the soil solution of two a ab
scented Pelargonium cultivars. ab ab
Attar of Roses (open bars) and ab
DOC in rhizosphere soil (mg L -1)

Concolor Lace (dark-spotted 125

bars) were cultured for 2 weeks b
on unspiked contrasted soils,
namely, soils-1 and 2 (S0), and on
bc
spiked soils containing 500 100
(S500) and 1500 (S1500) c
mg kg−1 of Pb, respectively. c c c
Controls were also maintained
c
with unspiked soils (US) with no 75
plants (gray filled bars) Different
alphabetical letters indicate
significant difference (p<0.05)
among values as measured by 50 d
LSD Fisher test d

25

0
US S0 S500 S1500 US S0 S500 S1500
Soil 1 Soil 2
Pb concentration (mg kg -1)
J Soils Sediments (2016) 16:581–591
Table 2 Lead concentrations in
different parts of Attar and Pb levels
Concolor cultivars after 2-week
culture on two soils spiked with (mg kg−1)
Pb particles
Soil-1 Control
500
1500
Soil-2 Control
500
1500
Different alphabetical letters indicate significant difference (p<0.05) among values as measured by LSD Fisher
test. Two-way ANOVA was used for the statistical analysis considering soil type, treatment levels, and cultivars.
ANOVA procedures were performed separately for shoots and roots
precipitates of fine particles (<1 μm) (Fig. 4c), and diffuse
ultrafine particles intimately associated with the root tissues.
The chemical composition was quite the same, i.e., Pb, P, Cl,
Ca, and Si (Fig. 4d) with a higher proportion of P in the
cylinder-like particles (Fig. 4e). The distribution of Pb and P,
observed on the same location through ESEM images, indi-
cates the association of Pb with P in the precipitates while
carbon compounds (probably carbonates or humic precipi-
tates) compete for the place on the same sites (Fig. 4e).
Figure 5 presents EXAFS spectra of some reference com-
pounds used in the linear combination fits and the spectra for
the amended soil and for the roots with their fits. The EXAFS
spectra for bulk soil and roots clearly differ by the shape of the
second oscillation and position of the third and higher oscil-
lations. Both spectra were correctly simulated by four compo-
nents. For the amended soil, the best fit was obtained with
30 % PbSO4 +33 % PbO.PbSO4 +47 % α PbO+12 % Pb
oxalate studied (NSS=0.038). Other four-component fits ob-
tained were not satisfactory because NSS was increased by
10 % and more and did not reproduce correctly the shoulder
on the second oscillation. Similarly, three-component fits did
not reproduce correctly the spectral features (NSS=0.052), so
the four Pb species including PbSO4, PbO·PbSO4, αPbO, and
organic Pb are effectively present in the soil.
For the roots, five linear combinations were retained, and
the following proportions were obtained: 52±5 % αPbO+33
±4 % Pb-sorbed ferrihydrite+50±3 % Pb-thiols. In the five
fits, Pb-thiols were a combination of two references including
Pb-gluthatione and Pb-cysteine. If Pb-thiols were excluded
from the fit, NSS was increased by 25 %. Therefore, the roots
likely contained Pb bound to thiol groups.
4 Discussion
Total metal concentrations in soils are not good indicators of
phytoavailable fraction of metals due to complex chemical
speciation of metals in soils (Chen et al. 1996). Soil solution
587
Shoot (mg Pb kg−1) Root (mg Pb kg−1)
Attar Concolor Attar Concolor
0.4±0.3f 0.2±0.1f 2.3±0.5f 8.5±2.9e
b e b
199.7±16.0 35.9±7.7 2095.8±323.1 958.0±82.8d
284.3±39.0a 53.1±3.9d 3707.3±695.8a 1492.8±122.7c
0.5±0.1f 0.2±0.1f 6.8±2.9e 8.3±3.0e
134.2±17.3c 32.4±8.7e 1499.1±206.0c 936.9±55.1d
201.3±15.2b 37.1±8.2e 4042.6±518.0a 1904.9±168.8b
Pb is widely accepted as phytoavailable. In the present study,
Attar cultivar probably released H + ions to acidify and
Concolor exudated OH− ions to alkalinize the rhizosphere
(Fig. 2a). The acidification potential of Attar cultivar led to
an increase in phytoavailable Pb in both soils as compared to
Concolor cultivar (Fig. 2b). The pH changes by Attar cultivar
are also responsible for oxidizing conditions, favoring the
conversion of PbS to PbSO4. Concolor was unable to change
the pH of soil-1. Contrastingly, it was capable to alkalinize the
rhizosphere in soil-2. These results would explain why
Concolor poorly mobilizes Pb in both soils. The higher Pb
uptake by Attar compared to Concolor could be, at least partly,
attributed to the difference in pH since acidification likely
favors metal phytoavailability, whereas alkaline pH reduces
it (Kidd and Monterroso 2005). As EDTA is present in the
nutrient solution, its presence in the medium and in the
rhizospheric compartment could also explain the difference
of levels in the observed phytoavailable Pb concentrations
due to the relation between chelation and pH. Genetic differ-
ence could be another reason for the different responses of
both cultivars. Moreover, Concolor cultivar promoted reduced
conditions through increasing pH that could also stop the con-
version of PbS into PbSO4, a relatively mobile entity. This
could also be the metal exclusion strategy used by this cultivar
to create conditions for Pb stabilization, rather than accumu-
lation (Rascio and Navari-Izzo 2011).
Root exudation, possibly by modifying DOC contents
(Fig. 3), can play a vital role by altering the redox conditions
and Pb speciation. Leaded particles, used for spiking of soils,
contained PbS as major component (55 %), but in the soil,
EXAFS spectroscopy results showed that PbSO4 dominated
(Fig. 5). Another important form in the soil was PbO, which
can easily be hydrolyzed and converted into PbOH+ or free
Pb2+ under neutral and mild acidic conditions. Soil character-
istics can also be an important factor of mobility for Pb and
other elements (Sungur et al. 2014). Soil-1 had a higher cation
exchange capacity and a higher carbonate and organic matter
content compared to soil-2 (Table 1). As pH influences metal
588 J Soils Sediments (2016) 16:581–591

500
a a
bb
a
400
Adsorbed Pb (mg kg-1)

300 cd
c cd
d
200
e e
100

f f
0
S0 S500 S1500 S0 S500 S1500
Soil 1 Soil 2
Pb concentration (mg kg -1)

c d
d

Fig. 4 Lead adsorption, precipitates on root surfaces, and their
characterization. Adsorbed Pb (a) on root surfaces of scented
Pelargonium cultivars, Attar of Roses (open bars) and Concolor Lace
(dark-spotted bars) after 2-week culture. Precipitates of adsorbed Pb on
the root surface of Attar cultivar were observed by ESEM (b).
Magnified view (c) indicates fragment cylindrical structures. ESEM-
XRD allowed chemical analysis of the precipitates (d). Backscattered
electron (BSE) images of precipitates (e) showing elemental
distribution. Different alphabetical letters indicate significant difference
(p<0.05) among values as measured by LSD Fisher test
J Soils Sediments (2016) 16:581–591
Fig. 5 Pb LIII-edge EXAFS spectra for the bulk soil amended with Pb-
containing particles, roots of the scented Pelargonium cultivar, Attar of
Roses, and representative Pb standard spectra used for the linear
combination fits (dashed lines)
solubility and transfer (Wang et al. 2006), rhizosphere acidifi-
cation could have displaced equilibrium toward bicarbonates
under oxidized conditions thereby increasing Pb mobility.
This could be due to the lower stability of bicarbonates with
respect to that of carbonates (Sauve et al. 1998).
Higher Pb concentrations in roots compared to shoots
(Table 2), resulting in low translocation, might be due to the
fact that soil-plant contact experiments only ran over a 2-week
period, which is the maximum possible duration for experi-
ments using the rhizosphere devices. Attar has shown the
ability to accumulate more Pb over the longer periods (Shahid
et al. 2012). Considerable amounts of Pb were adsorbed onto
root cell walls of both cultivars (Fig. 4a). Attar cultivar had
adsorbed more Pb on soil-1 whereas Concolor had higher
values on soil-2. The percentage of adsorbed Pb was calculat-
ed as follows:
% Pbadsorbed ¼ ½Pbadsorrbed =½Pbroot  100 ð1Þ
The values calculated were 6–12 % for Attar and 14–23 %
for Concolor. The Pb adsorbed to root cell walls may strongly
affect Pb2+ uptake (Pendergrass and Butcher 2006). Ultra-
scanned roots by ESEM revealed the presence of cylindric
precipitates. The chemical analysis of the precipitate
(Fig. 4d) highlights the presence of Pb and P close to each
other as well as two other different peaks for Pb. The possible
forms that could be here are Pb, αPbO, PbOH+, ferrite deriv-
atives, and carbonates. Interestingly, sulphur is not detected
confirming the changes in Pb speciation. Pb phosphate
589
precipitates have previously been reported on/in the roots of
various species (Cotter-Howells et al. 1999; Meyers et al.
2009). Pb sulfides, carbonates, and As-Pb sulfosalts were ob-
served in the rhizosphere of plants growing in mining waste
disposal sites by Cabala and Teper (2007).
Despite the major presence of PbS in the leaded particles,
four Pb species including PbSO4, PbO·PbSO4, αPbO, and
organic Pb were observed in the soil (Fig. 5). The first three
species have been identified in the original particles (Uzu et al.
2009). Organic Pb may result from the weathering of the par-
ticles and redistribution on the soil organic matter. In the roots
of Attar after exposure, Pb-sorbed ferrihydrite may corre-
spond to iron precipitates forming the root plaque as observed
in aquatic plants (Hansel et al. 2001). The αPbO form, which
was identified in the amended soil, likely corresponds to the
mineral particles attached to the roots. Finally, Pb-thiols could
correspond to complexes present in the root cells and could
have been the result of a detoxification process involving S-
containing ligands (cystein, GSH, phytochelatins,
metallothioneins). Wang et al. (2007) have shown, using
EXAFS spectroscopy in wheat roots of hydroponic cultures,
that Pb(II) could be bound to carboxylic groups. Zinc-organic
acid complexes have also been reported in tobacco roots by
Straczek et al. (2008).
The findings of this work could have important implica-
tions in Pb phytoextraction. Uptake of mineral elements is
critical, as the fate of a plant’s life is determined by the resul-
tant of all the interacting molecular cross talk between the
rhizosphere soil and the given trace elements that also govern
the overall responses, leading to tolerance and resistance
mechanisms of the plant species to the heavy metal elements
(Maestri et al. 2010). Due to low phytoavailability of Pb is
soil, remediation time increases alarmingly and ultimately dis-
courages the use of plant-based strategies in the field (Arshad
et al. 2008; Cheng et al. 2015). Tailoring strategies to manage
factors responsible for Pb availability, as highlighted in the
present study, could contribute to improve phytoextraction
efficiencies, in view of reducing the time required for heavy
metal soil decontamination.
5 Conclusions
The enhanced acidification and the presence of DOC in the
rhizosphere of the plant were responsible for mobilizing Pb
present in different forms and reload the phytoavailable pool.
Phase changes under oxidized and reduced conditions ap-
peared to be the key mechanism controlling phytoavailable
Pb. The ESEM-EDX and EXAFS spectroscopy analysis for
source particles, the secondary Pb minerals, and the binding to
organic molecules observed on/in the roots of Attar confirmed
the changes in Pb speciation and the binding to thiols inside
the root. From the fits obtained by EXAFS spectroscopy, it
590
can be concluded that Pb was bound to organic species in
roots. Its binding with cysteine and glutathione predicts the
potential presence of phytochelatins and metallothioneins
which can also play an important role in metal accumulation
and tolerance in plants. These findings could provide startup
information for investigations leading to the characterization
of various organic acids contributing to the total DOC in the
rhizosphere. Furthermore, the identification of the different
organic acids in the exudates, responsible for Pb mobilization,
would help to control Pb mobility through either external ap-
plication of the desired acids or identification and overexpres-
sion of relevant gene(s) to enhance its production through
genetic approaches. Improved understanding of the Pb mobil-
ity can ultimately help to optimize Pb phytoextraction from
contaminated soils.
Acknowledgments The authors would like to thank the Higher Educa-
tion Commission of Pakistan (www.hec.gov.pk) for sponsoring
Muhammad Arshad. The Society of Chemical Treatment of Metals
(STCM, http://www.stc-metaux.com/) is acknowledged for providing
Pb particles used in the experiments. We also thank Dr. Sohail Ejaz,
University of Cambridge, UK, for constructive proof reading and
English language corrections.
References
Angeles OR, Johnson SE, Buresh RJ (2006) Soil solution sampling for
organic acids in rice paddy soils. Soil Sci Soc Am J 70:48–56
Arshad M, Silvestre J, Pinelli E, Kallerhoff J, Kaemmerer M, Tarigo A,
Shahid A, Guiresse M, Pradere P, Dumat C (2008) A field study of
lead phytoextraction by various scented pelargonium cultivars.
Chemosphere 71:2187–2192
Belin S, Briois V, Traverse A, Idir M, Moreno T, Ribbens M (2005)
SAMBA a new beamline at SOLEIL for x-ray absorption spectros-
copy in the 4–40 keV energy range. Phys Scr T115:980–983
Cabala J, Teper L (2007) Metalliferous constituents of rhizosphere soils
contaminated by Zn-Pb mining in southern Poland. Water Air Soil
Pollut 178:351–362
Cecchi M, Dumat C, Alric A, Felix-Faure B, Pradere P, Guiresse M
(2008) Multi-metal contamination of a calcic cambisol by fallout
from a lead-recycling plant. Geoderma 144:287–298
Chaignon V, Hinsinger P (2003) A biotest for evaluating copper bioavail-
ability to plants in a contaminated soil. J Environ Qual 32:824–833
Chen B, Shan XQ, Qian J (1996) Bioavailability index for quantitative
evaluation of plant availability of extractable soil trace elements.
Plant Soil 186:275–283
Cheng S-F, Huang C-Y, Lin Y-C, Lin S-C, Chen K-L (2015)
Phytoremediation of lead using corn in contaminated agricultural
land–An in situ study and benefit assessment. Ecotox Environ Saf
111:72–77
Cotter-Howells JD, Champness PE, Charnock JM (1999) Mineralogy of
Pb-P grains in the roots of Agrostis capillaris L. by ATEM and
EXAFS. Mineral Mag 63:777–789
Dixit R, Wasiullah MD, Kuppusamy P, Singh UB, Sahu A, Shukla R,
Singh BP, Rai JP, Sharma PK, Lade H, Paul D (2015)
Bioremediation of heavy metals from soil and aquatic environment:
an overview of principles and criteria of fundamental processes.
Sustainability 7:2189–2212
J Soils Sediments (2016) 16:581–591
FAO (1998) World reference base for soil resources. Food and
Agriculture Organization of the United Nations, Rome
Fernández R, Bertrand A, García JI, Tamés RS, González A (2012) Lead
accumulation and synthesis of non-protein thiolic peptides in select-
ed clones of Melilotus alba and Melilotus officinalis. Environ Exp
Bot 78:18–24
Ferrand E, Dumat C, Leclerc-Cessac E, Benedetti MF (2006)
Phytoavailability of zirconium in relation to its initial added form
and soil characteristics. Plant Soil 287:313–325
Hansel CM, Fendorf S, Sutton S, Newville M (2001) Characterization of
Fe plaque and associated metals on the roots of mine-waste impact-
ed aquatic plants. Environ Sci Technol 35:3863–3868
Kabata-Pendias A (2004) Soil-plant transfer of trace elements-an envi-
ronmental issue. Geoderma 122:143–149
Khan S, Cao Q, Chen BD, Zhu YG (2006) Humic acids increase the
phytoavailability of Cd and Pb to wheat plants cultivated in freshly
spiked, contaminated soil. J Soils Sediments 6:236–242
Kidd PS, Monterroso C (2005) Metal extraction by Alyssum
serpyllifolium ssp lusitanicum on mine-spoil soils from Spain. Sci
Total Environ 336:1–11
Maestri E, Marmiroli M, Visioli G, Marmiroli N (2010) Metal tolerance
and hyperaccumulation: costs and trade-offs between traits and en-
vironment. Environ Exp Bot 68:1–13
Manceau A, Boisset MC, Sarret G, Hazemann JL, Mench M, Cambier P,
Prost R (1996) Direct determination of lead speciation in contami-
nated soils by EXAFS spectroscopy. Environ Sci Technol 30:1540–
1552
Meyers DER, Kopittke PM, Auchterlonie GJ, Webb RI (2009)
Characterization of lead precipitate following uptake by roots of
Brassica juncea. Environ Toxicol Chem 28:2250–2254
Pendergrass A, Butcher DJ (2006) Uptake of lead and arsenic in food
plants grown in contaminated soil from Barber Orchard, NC.
Microchem J 83:14–16
Rascio N, Navari-Izzo F (2011) Heavy metal hyperaccumulating plants:
how and why do they do it? And what makes them so interesting?
Plant Sci 180:169–181
Ravel B, Newville M (2005) ATHENA and ARTEMIS: interactive
graphical data analysis using IFEFFIT. J Synchrotron Radiat 12:
537–541
Sarret G, Manceau A, Cuny D, Haluwyn CV, Deruelle S, Hazemann JL,
Soldo Y, Eybert-Bérard L, Menthonnex JJ (1998a) Mechanism of
lichen resistance to metallic pollution. Environ Sci Technol 32:
3325–3330
Sarret G, Manceau A, Spadini L, Roux JC, Hazemann JL, Soldo Y,
Eybert-Bérard L, Menthonnex JJ (1998b) EXAFS determination
of Pb, Zn complexing sites of Penicillium chrysogenum cell walls.
Environ Sci Technol 32:1648–1655
Sauve S, Dumestre A, McBride M, Hendershot W (1998) Derivation of
soil quality criteria using predicted chemical speciation of Pb2+ and
Cu2+. Environ Toxicol Chem 17:1481–1489
Shahid M, Arshad M, Kaemmerer M, Pinelli E, Probst A, Baque D,
Pradere P, Dumat C (2012) Long-term field metal extraction by
Pelargonium: phytoextraction efficiency in relation to plant maturi-
ty. Int J Phytorem 14:493–505
Sorvari J (2007) Environmental risks at Finnish shooting ranges – a case
study. Hum Ecol Risk Assess 13:1111–1146
Straczek A, Sarret G, Manceau A, Hinsinger P, Geoffroy N, Jaillard B
(2008) Zinc distribution and speciation in roots of various genotypes
of tobacco exposed to Zn. Environ Exp Bot 63:80–90
Strømseng AE, Ljønes M, Bakka L, Mariussen E (2009) Episodic dis-
charge of lead, copper and antimony from a Norwegian small arm
shooting range. J Environ Monitor 11:1259–1267
Sungur A, Soylak M, Ozcan H (2014) Investigation of heavy metal mo-
bility and availability by the BCR sequential extraction procedure:
relationship between soil properties and heavy metals availability.
Chem Speciat Bioavailab 26:219–230
J Soils Sediments (2016) 16:581–591
Uzu G, Sobanska S, Aliouane Y, Pradere P, Dumat C (2009) Study of lead
phytoavailability for atmospheric industrial micronic and sub-
micronic particles in relation with lead speciation. Environ Pollut
157:1178–1185
Uzu G, Sobanska S, Sarret G, Munoz M, Dumat C (2010) Foliar lead
uptake by lettuce exposed to atmospheric fallouts. Environ Sci
Technol 44:1036–1042
Uzu G, Sobanska S, Sarret G, Sauvain JJ, Pradere P, Dumat C (2011)
Characterization of lead-recycling facility emissions at various
591
workplaces: major insights for sanitary risks assessment. J Hazard
Mater 186:1018–1027
Wang G, Su MY, Chen YH, Lin FF, Luo D, Gao SF (2006) Transfer
characteristics of cadmium and lead from soil to the edible parts of
six vegetable species in southeastern China. Environ Pollut 144:
127–135
Wang HH, Shan XQ, Liu T, Xie YN, Wen B, Zhang SZ, Han F, van
Genuchten MT (2007) Organic acids enhance the uptake of lead
by wheat roots. Planta 225:1483–1494