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Science of the Total Environment

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How do low doses of desferrioxamine B and EDTA affect the


phytoextraction of metals in sunower?
J.Y. Cornu a,, C. Dpernet a, C. Garnier b, V. Lenoble b, A. Braud c, T. Lebeau c
a
ISPA, Bordeaux Sciences Agro, INRA, 33140 Villenave d'Ornon, France
b
PROTEE, Universit de Toulon, CS 60584, 83041 Toulon cedex 9, France
c
LPG-NANTES, UMR 6112 CNRS, Universit de Nantes, 44322 Nantes, France

H I G H L I G H T S G R A P H I C A L A B S T R A C T

DFOB and EDTA efciency in increasing


metal phytoextraction was compared.
Adding DFOB did not increase mobility
of divalent metals in soil in contrast to
EDTA.
Adding EDTA considerably reduced the
labile Cd, Cu and Zn fraction in pore
water.
Low doses of EDTA selectively en-
hanced the phytoextraction of Cu and
Ni.
Metal-EDTA complexes were likely tak-
en up undissociated.

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this study was to compare the efciency of siderophore desferrioxamine B (DFOB) and EDTA in in-
Received 5 January 2017 creasing the phytoextraction of metals in sunower. A 28-day pot experiment was conducted in a metal-
Received in revised form 2 March 2017 contaminated soil supplied with 200 mol kg1 of DFOB or EDTA. Pore water was collected and pseudo-
Accepted 9 March 2017
polarographic analyses were conducted to assess the impact of the two chelators on the mobility and speciation
Available online xxxx
of metals in the liquid phase. Our results showed that DFOB is not an efcient mobilizing agent of divalent metals
Editor: D. Barcelo in soil. Adding DFOB selectively increased the mobility of trivalent metals while the supply of EDTA simulta-
neously increased the mobility of both trivalent and divalent metals. EDTA signicantly reduced the labile frac-
Keywords: tions of Cd, Cu, (Pb) and Zn measured in the porewater. The labile concentration of Cd and Zn measured in
Soil presence of EDTA was even less than that measured in the control. As expected from the pore water analysis,
Bacterial siderophore the addition of DFOB did not affect the phytoextraction of any divalent metals. In contrast, the addition of
Complexation EDTA enhanced Cu and Ni phytoextraction in sunower 2.0 to 2.8 fold for Cu and 1.3 to 2.3 fold for Ni, depending
Phytoavailability on the cultivar. This result supports different hypotheses regarding the forms and the related pathways in which
Pseudo-polarography
metals are taken up in presence of EDTA. Based on the results obtained for Ni, whose uptake is rate limited by its
Between-cultivar variability
internalization across the cell membrane, the direct uptake of metal-EDTA complexes via the non-selective
apoplastic pathway is hypothesized to contribute the most to the overall uptake of metals in presence of EDTA,
even added at low concentrations.
2017 Elsevier B.V. All rights reserved.

Corresponding author.
E-mail address: jean-yves.cornu@inra.fr (J.Y. Cornu).

http://dx.doi.org/10.1016/j.scitotenv.2017.03.092
0048-9697/ 2017 Elsevier B.V. All rights reserved.

Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.03.092
2 J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx

1. Introduction phytoavailability of divalent metals in soil and they led to contrasted


conclusions. Dimkpa et al. (2009) concluded that DFOB could increase
Phytoextraction, i.e. the use of plants to selectively remove metals the soil to plant transfer of Cd while Neubauer et al. (2000b) demon-
from contaminated matrices, has emerged as a cost effective and envi- strated little impact of DFOB on the mobility of divalent metals in soil.
ronmentally friendly alternative to destructive techniques (e.g. soil Further information is thus required concerning the inuence of DFOB
washing, soil capping and vitrication) for the in situ clean-up of mod- on metals' dynamics at the soil-root interface to enable recommenda-
erately contaminated soils. However, this technique is not really ef- tions on the use of DFOB-producing bacteria in phytoremediation.
cient because it is too slow (Glass, 2000) due to the low This study focuses on the way the addition of low doses (i.e. doses
phytoavailability of metals in soil (McGrath et al., 2002). lower than those usually used) of DFOB and EDTA can affect the soil to
Metal phytoextraction could be improved by adding either synthetic plant transfer of metals. The main objectives were (i) to compare the ef-
or natural chelators, providing that (i) the chelator (or ligand, usually ciency of two chelators in enhancing the mobility and phytoextraction
noted L) has the ability to complex the metal (M) in soil, (ii) the of metals in soil, (ii) to clarify the mechanisms underlying these differ-
resulting complex (ML) is less sorbed to soil particles than the free ion ences in efciency, and (iii), to test whether their phytoextraction ef-
(Mn+), and (iii) ML contributes to root uptake of M (Lin et al., 2014). ciency differs between two cultivars of the same plant species.
The synthetic chelator ethylenediaminetetraacetic acid (EDTA) usually Experiments were performed in pots using an agricultural topsoil con-
fullls the two rst conditions thanks to its high afnity for divalent taminated with Cd, Cu, Ni, Pb and Zn. Sunower was chosen since it is
metals (log K = 18.9, 18.32 and 16.68 for Cu, Pb and Zn, respectively) a good candidate for metal phytoextraction (January et al., 2008) and
and because the sorption of M-EDTA complexes to the soil solid phase two cultivars with apparently contrasted ability to translocate metals
is limited due to their negative charge. Conversely, it is suspected that aboveground (Laporte et al., 2015) were compared.
the last condition is only satised when EDTA is added at a concentra-
tion that also damages the endodermis of the plant roots (Nowack 2. Materials and methods
et al., 2006). Hence, when EDTA is added at several mmol kg1 soil
(e.g. Huang et al., 1997; Luo et al., 2005; Wu et al., 2004) the rate of 2.1. Experimental design
metal phytoextraction often increases 2 to 100 fold depending on the
soil to be remediated, the metal to be extracted, the plant used, and Two linoleic sunower cultivars cultivated in France: ES RICA
the dose of EDTA applied (Evangelou et al., 2007). Unfortunately, at (Euralis Semences) and Kapllan (RAGT Semences) were grown on a
this dose EDTA is toxic to numerous plants and to soil macro and micro- metal-contaminated soil supplied with either DFOB, EDTA or with no
fauna (Grman et al., 2001) and the risk that mobilized metals leach into chelator (as control) and sampled after 28 days to determine the
groundwater is drastically increased (Nowack et al., 2006). One way to amount of Cd, Cu, Ni, Pb and Zn phytoextracted, i.e. accumulated in
reduce the deleterious effects associated with EDTA, would be to reduce aboveground tissues. One plant was grown per pot and ve biological
its concentration, even if this requires more frequent additions during replicates were performed per cultivar and per treatment. DFOB and
the clean-up process. Strangely enough, this approach has rarely been EDTA were supplied at a concentration of 200 mol kg1 soil DW,
tested so far. Another option would be to replace EDTA by inoculating which is one order of magnitude lower than the concentration usually
the rhizosphere of accumulator plants with siderophore producing bac- used in EDTA-assisted phytoextraction (Evangelou et al., 2007).
teria (SPB). This approach is based on the ability of siderophores to che-
late other metals than iron, including divalent metals (Schalk et al.,
2011), and on the ability of SBP to up-regulate their siderophore synthe- 2.2. Soil sample
sis in presence of metals (Braud et al., 2010). The localized production of
siderophores in the near vicinity of the plant roots, where most SPB es- The soil was collected in the Pierrelaye-Bessancourt plain (24 km
tablish themselves (Deweger et al., 1995), and their rather rapid de- northwest of Paris, France) from agricultural topsoil contaminated
gradability could facilitate phytoextraction of metals with limited risk with trace elements. According to Bourennane et al. (2002), soils in
of leaching of the metal (Ferret et al., 2015). Several studies reported en- the study area were amended with urban sludge and smut compost in
couraging results regarding the ability of the siderophore pyoverdine the mid 1960s and irrigated with wastewater between 1899 and
(Pvd) to promote the mobility and the phytoavailability of metals in 2000. The characteristics of the soil sample were 70% sand, 20% silt
contaminated soils, either by direct addition of Pvd to the soil (Cornu and 10% clay, 5.7% organic matter, pH (H2O) 6.7, pH (CaCl2) 6.3, cation
et al., 2014) or by inoculation with Pvd-producing bacteria (Braud exchange capacity (CEC) of 8.8 cmol+ kg1, 4.5% carbonate content,
et al., 2009). and a water holding capacity (WHC) of 250 g kg1. Total metal concen-
Desferrioxamine B (DFOB), also known as desferal, is a bacterial trations (HF digestion) were (in mg kg1) 4.9 for Cd, 339 for Cu, 28 for
siderophore present in soil that is produced by the Actinobacteria Strep- Ni, 602 for Pb and 1120 for Zn. Available metal concentrations (0.01 M
tomyces pilosus. It belongs to the hydroxamate siderophores and has CaCl2 extraction) were (in g kg1) 87 for Cd, 288 for Cu, 524 for Ni,
medical applications as a chelating agent in the treatment of acute b7 for Pb and 14393 for Zn. All the analyses (except WHC) were per-
iron intoxication. The ability of DFOB to mobilize a broad spectrum of formed by the INRA soil testing laboratory (INRA Arras, France) using
metals (Cd, Co, Cr, Cu, Pb, Zn) from different puried mineral phases standardized procedures. The soil sample was sieved to b 2 mm and
(ferrihydrite, goethite, kaolinite, zeolite) has already been demonstrat- stored sheltered from light in knotted plastic bags at a moisture content
ed under certain conditions (notably pH) (Bi et al., 2010; Hazotte of 200 g kg1 (80% WHC) for about three months before the beginning
et al., 2016; Hepinstall et al., 2005; Karimzadeh et al., 2013; Maurice of the experiment.
et al., 2009; Neubauer et al., 2000a; Stewart et al., 2016). DFOB produc-
ing bacteria might therefore be used in phytoextraction, especially 2.3. Chelators
when plants are able to acquire metals directly from metal-DFOB com-
plexes, as shown for Fe in sunower (Cline et al., 1984). However, the DFOB was obtained as mesylate salt (C25H48N608CH4O3S, CAS Num-
impact of DFOB on the mobility of metals in the soil is difcult to estab- ber: 138-14-7, M = 656.79 g mol1) from Merck, and EDTA as disodium
lish in experiments conducted with puried minerals since in the soil, dihydrate salt (C10H14N2Na2O8.2H2O, CAS Number: 6381-92-6, M =
minerals are mixed with other minerals as well as with solid organic 372.24 g mol1) from Sigma-Aldrich. Stock solutions of DFOB and
matter, metals are numerous, and ligands other than DFOB are present EDTA (16 mM) were stored at 20 C for a few days prior to use. All
in pore water (notably humic substances). To our knowledge, only the glassware was soaked in 5% HNO3 and thoroughly rinsed with
two studies have tested how DFOB addition altered the mobility and ultra-pure water before being used for the study.

Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.03.092
J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx 3

2.4. Plant cultivation noted that these extra pots did not contain plants and that the analyzed
pore water was extracted on day 9. It was hypothesized that root exuda-
Seeds were washed with H2O2 (6% v/v), rinsed with deionized water tion had no effect on the complexation of metals in pore water and
and germinated on wet paper in the dark at room temperature for 96 h. hence, that the labile fraction of metals determined in unplanted pots
The seedlings were then transferred into a transient hydroponic device was representative of that in the pots containing plants. All
and left for about 72 h until the cotyledons were fully expanded (for voltammetric measurements were performed using a PGSTAT12
more details see Cornu et al., 2016). Thereafter, the seedlings were potentiostat (EcoChemie, The Netherlands) equipped with a Metrohm
transplanted into 0.2 L plastic pots lled with 240 g DW of soil (day 663 VA stand (Metrohm, Switzerland) on an Hg drop electrode. Analysis
0). On day 4, DFOB and EDTA were each supplied to one third of the of the voltammetric peaks' and construction of the pseudo-polarograms
pots (n = 10; 2 cultivars 5 replicates) at a concentration of were performed automatically using ECDSoft software. The experimen-
200 mol kg1 soil DW by adding 3 mL of the corresponding stock solu- tal procedure adapted from Nicolau et al. (2008) and Bravin et al. (2012)
tion to the soil surface. For controls, 3 mL of deionized water were added is explained briey below. Pseudo-polarographic measurements were
the same way. The pots were placed in a glass crystalliser (diameter initially performed on one control and one EDTA sample, diluted 10
7 cm) to avoid any loss of chelators from the bottom. The pots were and 100 times, respectively, with 0.01 M KClO4 (suprapur grade,
weighed and watered (with deionized water) three times a week to Merck). First, pseudo-polarograms were measured (details in
maintain the soil moisture at 80% WHC. The plants were left for Table T1) at natural pH in four separate runs (Zn, Cu, Cd and Pb). A
28 days in a growth chamber in the following conditions: 16 h photope- short conditioning time (1 s) at a deposition potential (Edep) of
riod at 300 mol photons m2 s1, 20 C and 75% relative humidity. 1.6 V was used between the deposition time and the potential scanning
to avoid any interference from organic surfactants (Louis et al., 2008).
The samples were then acidied with HNO3 suprapur grade, Merck to
2.5. Pore water extraction and analyses
pH b 2 to fully dissociate organic metal complexes, and a new set of
pseudo polarograms was measured at the original metal concentration
Pore water was extracted using Rhizon MOM soil moisture sam-
followed by repeated measurements with standard additions of metals.
plers (b0.2 m pore size) obtained from Rhizosphere Research Products
This enhances the sensitivity of the method and gives the total concen-
(Wageningen, the Netherlands) and previously cleaned with 5% HNO3.
tration of each metal in each sample. The acidication step also makes it
As the soil was being packed, the samplers were inserted obliquely
possible to determine the Edep corresponding to the electrochemically
into each pot and left in place throughout the 28-day culture period.
labile fraction (sum of free ions plus inorganically bound metal) in
Soil solution was collected on days 9, 16 and 23 that is 5, 12 and
non-acidied samples (Zn 1.2 V, Cd 0.7 V, Pb 0.7 V, Cu 0.3 V)
19 days after the chelator was added. Five hours before collection, the
for each metal. As previously dened (Feldmann et al., 2009; Nicolau
soil was watered to 80% WHC and 10 mL of extra deionized water
et al., 2008), the additional fraction of metals possibly measured at the
(10 mL) was supplied to the soil surface to facilitate sampling. On
most negative Edep (leading to an additional wave on the pseudo-
each occasion, collection started 8 h after the beginning of the photope-
polarograms) thus corresponds to the direct reduction of organic metal
riod and lasted for 6090 min in order to extract about 10 mL. Pore
complexes at the Hg drop electrode, electrochemically dissociable, here-
water pH and conductivity were measured directly after sampling
after referred to as the dissociable metal fraction. The difference be-
using the appropriate electrode (WTW Sentix 940 and WTW
tween the total metal concentration (determined at pH b 2) and the
TetraCon925, respectively). Total concentrations of Al, Cd, Cu, Fe, Ni,
sum of inorganic and dissociable metal concentrations correspond to
Pb and Zn were determined by ICP-MS (Varian 820-MS) and total con-
the presence of non-dissociable organic metal complexes, hereafter re-
centrations of Ca and Mg by ame-AAS (Solaar M6, Thermo Electron),
ferred to as the inert metal fraction. Cu pseudo-polarograms were mea-
on a 2% HNO3 acidied subsample. Dissolved organic carbon (DOC) con-
sured on EDTA samples at natural pH after addition of Triton X-100
centration was determined from non-purgeable organic C (NPOC) mea-
(200 L of a 0.2% (v/v) solution to 20 mL sample). This non-ionic surfac-
sured by combustion catalytic oxidation on a Shimadzu TOC-VCSH
tant is used to minimize the unwanted adsorption of free ligands during
analyzer. The specic UV-absorbance (SUVA) of dissolved organic mat-
the analytical process. With Triton X-100, the pseudo-polarographic
ter, a proxy of DOM aromaticity, was calculated from pore water absor-
wave corresponding to the dissociation of Cu-EDTA2 complex shifts
bance at 254 nm measured by UVVIS spectrometry (Cary 1 Bio, Varian,
to more negative potential and thereby the labile Cu fraction is better
quartz cells) and from the concentration of DOC in the pore water using
separated from that bound to EDTA (Croot et al., 1999).
the equation presented in Amery et al. (2008). Based on the assumption
that the supply of DFOB and EDTA did not affect organic matter dynam-
2.6.2. Ion-exchange measurements
ics in the soil, their concentrations in pore water were assessed from the
The free ionic fraction of Cd (Cd2+) was measured in each sample of
concentration of DOC. The micromolar concentration of DFOB (or EDTA)
pore water collected on day 9 according to the method of Schneider
in pore water i at a given time t was determined as follows:
(2006). This method is based on the Ca/Cd exchange properties of a cat-
DOCDFOBi;t or DOCEDTAi;t DOCCONTROLt
DFOBi;t or EDTAi;t nCDFOB or nCEDTA MC 0:001 (1)where ion exchange resin (Amberlite IR-120) converted to the Ca form. Briey,
2 mg of Ca resin were put in contact with 1 mL of pore water in a 5 mL
DOCCONTROLt is the mean concentration of DOC measured in the control
polyethylene tube. A known amount of radioactive 109Cd2+ was imme-
at a given time t, nc is the number of carbon atoms in the chelator
diately added to the suspension and the tube was shaken for 24 h at
(nc = 25 for DFOB and 10 for EDTA) and Mc is the molar mass of carbon
20 1 C on a roller. The free ionic fraction of Cd was determined
(Mc = 12).
from the radioactivity remaining in solution after contact with the
resin, from the total Ca concentration in solution before and after con-
2.6. Speciation assays tact and from the resin Vanselow selectivity coefcient for Ca/Cd ex-
change (vKCa/Cd = 0.661) (Schneider, 2006).
2.6.1. DPASV analyses
Differential pulse anodic striping voltammetry (DPASV) measure- 2.6.3. Geochemical calculation
ments were performed to assess to what extent the supply of EDTA al- Geochemical calculation was performed to assess the speciation of
tered the labile fraction of Cd, Cu, Pb and Zn in pore water. These metals in the pore water of the control soil. Modeling was performed
measurements required 10 mL of sample and were thus made in dedi- with Visual MINTEQ version 3.0 (Gustafsson, 2012) using the pH, DOC,
cated pore water extracted from 10 extra pots, with or without EDTA, total concentrations of Ca, Mg, Fe, Al, Cd, Cu, Ni, Pb and Zn of the pore
according to the procedure described above (Section 2.5). It should be water as input data. We assumed that NO 3 was the main anion present

Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.03.092
4 J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx

in pore water and its concentration was adjusted so that the difference Table 1
in charge between the sum of anions and cations was always b 5%. The Number of developed leaves, shoot biomass, root biomass, root to shoot biomass ratio
(RS), stem height and total leaf area, measured in two sunower cultivars (cv. ES RICA
NICA-Donnan model was used to evaluate metal binding to dissolved and Kapllan) grown for 28 days (392 growing degree days) on a metal-contaminated soil
organic matter, with generic NICA-Donnan parameters optimized for supplied either with desferrioxamine B (DFOB) or ethylenediaminetetraacetic acid
fulvic acids (FA). DOM was assumed to contain 50% carbon and was (EDTA). Each treatment (cultivar, chelator) was replicated ve times. For each cultivar,
simulated by a mixture of FA and hydrophilic compounds. The quality ve additional pots not supplied with chelators were used as controls.
of the DOM was introduced in the prediction of metal complexation Shoot Root Stem Total leaf
Number of
by linking the percentage of FA (%FA) to the SUVA of DOM (Amery biomass biomass RS height area
leaves
et al., 2008): g DW g DW cm cm2

%FA SUVA
SUVAFA (2) where SUVAFA is the specic UV-absorbance of the
DOM Control 8 1.02a 0.23a 0.23a 17.8a 193a
ES RICA DFOB 8 0.99a 0.20ab 0.20a 19.1a 200a
Suwanee River standard FA substance (55 L g1 cm1; Benedetti et al.,
EDTA 8 1.05a 0.16b 0.15b 16.0b 196a
2002). Cd binding to hydrophilic compounds was assumed to be negli- Control 8 1.02a 0.30a 0.29a 18.6a 205a
gible, as suggested by Groenenberg et al. (2010). Kapllan DFOB 8 0.98a 0.29a 0.29a 16.5ab 213a
EDTA 8 1.06a 0.17b 0.16b 16.3b 209a

2.7. Plant sampling and analyses For a given cultivar, different letters in the same column indicate signicant differences
(p b 0.05).
For a given chelator, values are underlined when they are signicantly higher (p b 0.05)
At harvest, the number of expanded leaves was counted and stem than that measured in the other cultivar.
length was measured with a tape measure. The xylem sap was collected
by root pressure (Nguyen et al., 2016): briey, shoots were cut just
above the cotyledons and the rst drop of the stem exudate was
on the control soil. In addition, the shoot dry matter was not affected
discarded. After that, all drops were collected with a micropipette
by EDTA but the stem height was, and sunower grown on the soil sup-
until ~ 200 L was collected, then stored in pre-weighed microtubes
plied with EDTA had a thicker stem. It is worth noting that the root to
bathed in ice. At the end of the collection, the microtubes were weighed
shoot biomass ratio of the control (0.23 for ES RICA and 0.29 for Kapllan)
again to determine the exact volume of sap. After centrifugation at
was in good agreement with that reported by Laporte et al. (2015) for
16,000g for 10 min, the supernatants were collected and stored at
the same two cultivars grown in hydroponics, suggesting that the root
20 C until analysis. After the xylem sap was collected, the plants
system was efciently retrieved from the soil.
were divided into roots and shoots. To isolate the roots from the soil
particles, the soil-root samples were placed in 100-m mesh sieves
3.2. Plant Cd, Cu, Ni, Pb, Zn
and washed by hand under a water jet. The roots that remained in the
sieve were further cleaned with deionized water and blotted dry. The
The amounts of Cd, Cu, Ni, Pb and Zn recovered in sunower shoots
shoots were washed in two successive baths of deionized water. The
are listed in Table 2. The supply of DFOB did not alter the
surface area of the leaf blades was measured with an area meter (LI-
phytoextraction rate of metals. There was no signicant variation
3100C) to determine the plant total leaf area. All plant tissues were
(p N 0.05) in the amounts of Cd, Cu, Ni, Pb and Zn recovered in sunower
then dried at 50 C until constant weight, weighed and milled before
shoots between plants grown on the soil supplied with DFOB and plants
wet digestion in a HNO3/H2O2 mix, according to the procedure de-
grown on the control soil. This was the case in both sunower cultivars
scribed in Laporte et al. (2015). The total concentrations of Al, Cd, Cu,
tested in this study. Conversely, the supply of EDTA selectively increased
Fe, Ni, Pb and Zn in the xylem sap and in the plant digests were deter-
the phytoextraction of Cu and Ni. The amount of Cu and Ni recovered in
mined by ICP-MS (Varian 820-MS). The validity and accuracy of the
shoots was higher (p b 0.05) in plants grown on the soil supplied with
digestion + elemental analysis procedures were checked using a certi-
EDTA than in plants grown on the control soil, whereas the amounts
ed sample of tomato leaves (NIST-SRM 1573a).
of Cd, Pb and Zn remained the same (p N 0.05) in the two treatments.
In presence of EDTA, the phytoextraction rate increased 2.0 to 2.8 fold
2.8. Statistical analyses for Cu and 1.3 to 2.3 fold for Ni depending on the cultivar. Interestingly,
the impact of EDTA on the phytoextraction of Cu and Ni was more pro-
Analyses of variance (ANOVA) with Tukey's HSD test at 5% were per- nounced in the cultivar ES RICA than in the cultivar Kapllan.
formed to identify signicant differences between the chelators and be- In the cultivar ES RICA, the concentration of metals in the xylem sap
tween the cultivars. Principal component analyses (PCA) were used to (Table 2) agreed fairly well with the results of metal phytoextraction. In
study groups of correlations between the pore water variables. this cultivar, EDTA increased (p b 0.05) the xylem sap concentrations of
SYSTAT 11 Edition 2004 (SPSS Inc., 233S. Wacker Drive, Chicago, USA) Cu and Ni (2.3 fold and 1.8 fold, respectively) while DFOB had no effect
was used for statistical analyses. (p N 0.05) regardless of the metal concerned. Surprisingly, in both culti-
vars, the concentration of Cd in the xylem sap was about 2 fold higher
3. Results when EDTA was supplied (p b 0.05).
The concentrations of Cd, Cu, Ni, Pb and Zn in sunower roots and
3.1. Plant growth shoots are listed in Supplementary Table T2. As expected, the concentra-
tion of metals was higher in roots than in shoots. In both tissues, except
No visible symptom of metal toxicity was observed in the above- for Cd and Ni, the concentration of metals did not vary signicantly
ground tissues, irrespective of the treatment (cultivar, chelator). The (p N 0.05) between the two cultivars. The concentration of Cd was
two cultivars grew at the same rate and were affected in the same higher (p b 0.05) in the roots of Kapllan but higher (p b 0.05) in the
way by the supply of chelators (Table 1). The supply of DFOB did not shoots of ES RICA. In roots, the concentration of metals did not signi-
alter either the growth of shoots or the growth of roots. There was no cantly vary (p N 0.05) between plants grown on the soil supplied with
signicantly difference (p N 0.05) in root dry matter, shoot dry matter, chelators (either DFOB or EDTA) and plants grown on the control soil.
stem height and total leaf area between plants grown on the soil sup-
plied with DFOB and plants grown on the control soil. Conversely, the 3.3. DFOB and EDTA concentrations in pore water
supply of EDTA compromised the growth of roots. Depending on the
cultivar, there was on average 3043% (p b 0.05) less root dry matter Changes in the concentrations of DFOB and EDTA in the pore water
in plants grown on the soil supplied with EDTA than in plants grown over time are listed in Table 3. The concentration of EDTA in the pore

Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.03.092
J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx 5

Table 2
Amount of Cd, Cu, Ni, Pb and Zn in shoots and total concentration of Cd, Cu, Ni, Pb and Zn in xylem sap, measured in two sunower cultivars (cv. ES RICA and Kapllan) grown for 28 days
(392 growing degree days) on a metal-contaminated soil supplied either with desferrioxamine B (DFOB) or ethylenediaminetetraacetic acid (EDTA). Each treatment (cultivar, chelator)
was replicated ve times. For each cultivar, ve additional pots not supplied with chelators were used as controls.

Cd Cu Ni Pb Zn
ab a a ab
Control 1.33 14.1 3.59 0.76 328a
ES RICA DFOB 1.14a 15.0a 3.81a 0.46a 349a
EDTA 1.55b 38.8b 8.37b 1.10b 381a
Metals accumulated in shoots (g)
Control 0.99a 14.8a 5.28a 0.67a 369a
Kapllan DFOB 0.98a 14.1a 4.87a 0.88a 315a
EDTA 1.11a 29.1b 6.94b 0.73a 321a
Control 0.34A 6.91A 3.43A 0.07A 251A
ES RICA DFOB 0.33A 9.19A 4.02A 0.05A 245A
EDTA 0.63B 16.1B 6.26B 0.08A 277A
Concentration of metals in xylem sap (M)
Control 0.21A 10.7AB 7.03A 0.09A 282A
Kapllan DFOB 0.21A 8.94A 5.46A 0.08A 234A
EDTA 0.42B 13.9B 5.07A 0.06A 257A

For a given cultivar, different letters in the same column indicate signicantly differences (p b 0.05).
For a given chelator, values are underlined when they are signicantly higher (p b 0.05) than that measured in the other cultivar.

water measured on day 9 was signicantly higher (p b 0.05) than that of water were signicantly higher (p b 0.05) in the soil supplied with
DFOB. Five days after the chelators were supplied, there was an average EDTA than in the control soil. This was particularly marked for Cu and
of three times more EDTA than DFOB in the soil solution. The concentra- Pb whose concentrations in the pore water collected on day 9 increased
tion of DFOB in the pore water decreased markedly during plant growth 90 fold and 74 fold, respectively, when EDTA was added. In contrast to
(p b 0.05) while that of EDTA remained almost constant (p N 0.05). The the concentrations of Fe and Al in the pore water measured in the
decrease in DFOB content averaged 75% between day 9 and day 23. As a DFOB treatment, in the presence of EDTA, the concentrations of Fe, Cd,
consequence, the concentration of EDTA in the pore water was on aver- Cu, Pb and Zn did not decrease (p N 0.05) over the culture period, as ob-
age 12 times higher than that of DFOB at the end of the culture period. It served for the concentration of EDTA itself. The concentration of Ni in
is worth noting that the concentration of chelators in the pore water did the pore water increased over time (p b 0.05), from 19 M in the pore
not differ signicantly (p N 0.05) between the two cultivars. water collected on day 9 to 52 M in that collected on day 23. The pH
of the pore water also increased over time (p b 0.05), from 6.7 on day
3.4. Al, Fe, Cd, Cu, Ni, Pb and Zn concentrations in the pore water 9 to 7.2 on day 23, but did not differ signicantly (p N 0.05) among the
three treatments. Similar results were obtained in the pore water col-
Changes in the concentrations of Al, Fe, Cd, Cu, Ni, Pb and Zn in the lected from the soil planted with the cultivar Kapllan (suppl. Table T3).
pore water over time are listed in Table 4. All these data concern the
soil planted with the cultivar ES RICA. The addition of DFOB selectively
promoted Fe and Al mobilization from the solid phase. The concentra- 3.5. Relationships between pore water characteristics
tions of Fe and Al in the pore water were signicantly higher
(p b 0.05) in the soil supplied with DFOB than in the control soil, where- The variables related to the pore water characteristics were analyzed
as no differences in Cd, Cu, Ni, Pb and Zn were observed (p N 0.05) be- by principal component analysis (PCA). Fig. 1 shows the soil planted
tween the two treatments. This increase was particularly marked for with the cultivar ES RICA. Fig. 1a presents the plot of factor loadings of
Fe whose concentration in pore water collected on day 9 increased the variables related to the DFOB treatment and the control. The rst
236 fold in presence of DFOB. Mobilization of Fe and Al was promoted two components accounted for 79% of total variance. The rst dimen-
throughout the culture period (i.e. up to day 23) even though concen- sion was associated with conductivity (cos2 = 0.97) and pH (cos2 =
trations of Fe and Al in the pore water decreased over time (p b 0.05) 0.81) and the second dimension with the concentration of DFOB
in parallel with the concentration of DFOB (see above). The addition of (cos2 = 0.70). Cd, Ni and Zn contents were strongly correlated with
EDTA favored the mobilization of all metals (except Al) from the solid conductivity (r = 0.99, 0.93, 0.97, respectively) and pH (r = 0.80,
phase. The concentrations of Fe, Cd, Cu, Ni, Pb and Zn in the pore 0.71, 0.82, respectively) and poorly correlated with the concentra-
tion of DFOB (r = 0.50, 0.40, 0.47, respectively). Conversely, Fe and Al
concentrations were strongly correlated with DFOB concentration
(r = 0.93 and 0.90, respectively) and poorly correlated with conductiv-
Table 3 ity (r = 0.47 and 0.51, respectively) and pH (r = 0.35 and 0.44, re-
Changes in the concentrations of dissolved organic carbon (DOC) and chelators (DFOB, spectively). It is worth noting that Cu and Pb concentrations were not
EDTA) over time in the pore water of a metal-contaminated soil supplied either with signicantly correlated (p N 0.05) with conductivity, pH or with the con-
desferrioxamine B (DFOB) or ethylenediaminetetraacetic acid (EDTA). Pore water was col- centration of DFOB. Fig. 1b shows the plot of factor loadings of the var-
lected 9, 16 and 23 days after sunower transplanting, i.e. 5, 12 and 19 days after the che-
lators were supplied. Each treatment (cultivar, chelator) was replicated ve times. For
iables related to the EDTA treatment and the control. The rst two
each cultivar, ve additional pots not supplied with chelators were used as controls. components accounted for 86% of total variance. The rst dimension
was associated with EDTA concentration (cos2 = 0.84) and the second
ES RICA Kapllan
with pH (cos2 = 0.90) and conductivity (cos2 = 0.78). Cd, Cu, Ni, Fe,
Control DFOB EDTA Control DFOB EDTA
Pb and Zn concentrations were strongly correlated with the concentra-
Day 9 46a 115a 108a 44a 83a 104a
DOC tion of EDTA (r = 0.87, 0.85, 0.81, 0.80, 0.85, 0.84, respectively). They
Day 16 56a 94a 105a 40a 62ab 110a
(mg C L1) were poorly correlated with conductivity (r = 0.34, 0.37, 0.09, 0.45,
Day 23 44a 63b 117a 52a 60b 109a
Day 9 230a 522a 128a 501a 0.44, 0.42, respectively) and were not signicantly correlated
Chelators (M) Day 16 126a 406a 75ab 585a (p N 0.05) with pH. Similar relationships between pore water character-
Day 23 65b 611a 26b 474a istics can be inferred from the plots of factor loading in Supplementary
Different letters in the same column indicate signicant differences (p b 0.05). Fig. F1 for the soil planted with the cultivar Kapllan.

Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
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6 J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx

Table 4
Changes in pH and in total concentrations of Al, Fe, Cd, Cu, Ni, Pb and Zn over time measured in the pore water of a metal-contaminated soil supplied either with desferrioxamine B (DFOB)
or ethylenediaminetetraacetic acid (EDTA). Pore water was collected 9, 16 and 23 days after sunower transplanting, i.e. 5, 12 and 19 days after chelators supply. Each treatment (chelator)
was replicated ve times. For each cultivar, ve additional pots not supplied with chelators were used as controls. The data shown are for the sunower cultivar ES RICA (data for the cul-
tivar Kapllan are shown in supplementary data).

pH Al Fe Cd Cu Ni Pb Zn
ES RICA
M

Day 9 6.64a 0.70a 0.72a 0.05a 1.97a 1.41a 0.03a 21.3a


Control Day 16 6.88b 1.94a 0.43a 0.04ab 2.70b 1.30a 0.01a 15.3ab
Day 23 7.19c 0.43a 3.31b 0.02b 2.90b 0.81a 0.01a 7.0b
Day 9 6.67a 23.2a 170a 0.07a 2.20a 1.56a 0.02a 25.5a
DFOB Day 16 6.96b 7.43b 133a 0.05ab 2.84b 1.37ab 0.01a 17.0ab
Day 23 7.26c 1.68c 25.6b 0.02b 3.12b 0.85b 0.02a 7.4b
Day 9 6.68A 0.73A 18.8A 0.98A 178A 19.0A 2.22A 378A
EDTA Day 16 6.86B 1.14A 14.1A 1.13A 192A 43.2B 1.97A 403A
Day 23 7.25C 0.51A 10.9A 1.08A 157A 51.9B 1.59A 315A

For a given chelator treatment, different letters in the same column indicate signicant differences (p b 0.05).

3.6. Metals pore water speciation assessed by DPASV 4. Discussion

Fig. 2 shows Cd pseudo-polarograms obtained for pore water col- 4.1. Speciation of metals in the control soil
lected from the control soil (Fig. 2a) and from the soil supplied with
EDTA (Fig. 2b). In the rst case, the Cd pseudo-polarogram recorded at Despite its sandy texture, a high fraction of metals was xed onto the
natural pH showed a unique wave with a half-wave position and an solid phase in the soil used for the present study. The total metal distri-
overall height similar to that obtained at pH b 2. This means that Cd bution coefcient (Ktotd ), calculated as the ratio of their total concentra-
chemical speciation in the pore water of the control soil was dominated tions in the solid phase (HF extraction) to that in the pore water, (in
by inorganic complexes. The DPASV-labile fraction of Cd was maximum L kg1) averaged 934 for Cd, 1981 for Cu, 359 for Ni, 145,270 for Pb
(i.e. 100%) in every replicate (Table 5). Conversely, in pore water collect- and 937 for Zn. These are high Ktotd values, comparable to those reported
ed from the soil supplied with EDTA, the Cd pseudo-polarogram record- by de Groot et al. (1998) for humic sandy soils. This high retention of
ed at natural pH differed markedly from that obtained at pH b 2. Two metals by the solid phase is probably due to the high soil organic matter
waves were observed at Edep below 0.7 V at natural pH, which content (N5%). According to CaCl2 extractions (see Section 2.2), only a
means that a substantial fraction of Cd in pore water was bound to or- very small part of the solid-phase metal (b 2% for Cd, Cu, Ni, Pb and
ganic ligands. Cd chemical speciation was even dominated by organic Zn) was available for plant uptake, which makes this soil a good candi-
complexes, either partially dissociable or inert, which were assumed date for chelator-assisted phytoextraction.
to be Cd-EDTA2 complexes. The DPASV-labile fraction of Cd was on According to DPASV measurements, all the Cd and Zn recovered in
average b 2% in the pore water of EDTA-treated soils (Table 5). the pore water was present as labile species in the control soil, mainly
Similarly to Cd, the Zn DPASV-labile fraction in the pore water was as free ion or inorganic complexes, even though the concentration of
maximum (i.e. 100%) in the control soil and b 2% in EDTA-treated soil DOC was fairly high (4056 mg L1). This apparent discrepancy is
(Table 5). Conversely, Cu speciation in the pore water of the control most probably due to low DOM reactivity. Indeed, the low specic UV-
soil was characterized by a dominant fraction (64%) of Cu bound to or- absorbance of DOM measured in pore water (Supplementary
ganic ligands (Table 5). Like for Cd and Zn, the DPASV-labile fraction of Table T5) suggests that dissolved organic matter mainly consisted in al-
Cu much decreased (p b 0.05) when EDTA was supplied. The largest iphatic compounds (sugars, amino acids and/or low molecular weight
fraction of the Cu-organic complexes belonged to the inert fraction in organic acids) whose reactivity towards divalent metals is much lower
the control soil while they were fully dissociable in the soil supplied than that of aromatic compounds like fulvic acids (Groenenberg et al.,
with EDTA (Table 5, suppl. Fig. F2). This may reect the presence of 2010). The hypothesis of low DOM reactivity is supported by geochem-
Cu-EDTA2 complexes, which are known to be dissociable at sufcient- ical calculations (Visual MINTEQ ver 3.0) performed using the pore
ly negative Edep (Croot et al., 1999), in the pore water of EDTA treated water characteristics measured on day 9. In fact, with a DOM reactivity
soils. Moreover the DPASV-labile concentrations of Cd and Zn in the adjusted from its aromaticity (for more details see Section 2.6.3), the
pore water were decreased (p b 0.05) while that of Cu was increased calculated Cd and Zn free ionic fractions were N80% and 90%, respective-
(p b 0.05) by the supply of EDTA (Table 5). ly, whereas that of Cu averaged 2530%. The prediction for Cu is in
agreement with its labile fraction measured by DPASV (Table 5) and
agrees with its known afnity for the organic matter in the soil solution
3.7. Pore water speciation of Cd assessed by ion exchange (Bravin et al., 2012). It is worth noting that the pH of the pore water in-
creased slightly over time. This slight alkalization, often attributed to
As a complement to DPASV measurements, Cd speciation in pore microbial activity (e.g. Cornu et al., 2007), led to an decrease in the con-
waters collected on day 9 was assessed by ion exchange (Supplementa- centrations of Cd and Zn in the pore water over time due to their pro-
ry Table T4). The Cd free ionic fraction was on average N 90% in pore moted sorption onto soil constituents.
water collected from the control soil and b1% in pore water collected
from the EDTA treated soil. These results are in agreement with the Cd 4.2. Impact of DFOB on the soil-to-plant transfer of metals
labile fraction measured by DPASV (see Table 5) thereby conrming
that Cd pore water speciation changed dramatically when EDTA was The concentration of DFOB in the pore water measured on day 9 av-
supplied. The Cd free ionic fraction was on average N 85% in the pore eraged 179 117 M. This means that ve days after having been sup-
water of DFOB treated soil (Supplementary Table T4) and did not signif- plied, up to 78 14% of DFOB was sorbed onto the solid phase. This
icantly vary (p N 0.05) from that measured in the pore water of the con- afnity probably derives from the electrostatic attraction that occurs
trol soil. All these results were observed in both the cultivar ES RICA and at pH 6.7 between the positively-charged DFOB (LH+ 4 ) and/or metal-
with the cultivar Kapllan. DFOB complexes (MLH+ or MLH+ 2 ) and the negatively charged sites of

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J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx 7

Cd, this was highlighted by speciation assays performed in pore water


demonstrating that the Cd free ionic fraction in the DFOB-treated soil
was as high as in the control. Furthermore, PCA analysis of the pore
water characteristics of the DFOB-treated vs. control soil underlined
the fact that the concentrations of Cd, (Cu), Ni, and Zn were not correlat-
ed with the concentration of DFOB but with pH, conductivity and Ca
concentration. This strongly suggests that in our conditions the mobility
of divalent metals (except Pb) in the soil was controlled by ion-
exchange rather than by complexation, even in presence of DFOB.
Conversely, the supply of DFOB strongly promoted the mobility of
trivalent metals, notably Fe(III). DFOB may have altered Fe/Al oxides
or more likely, the coating of Fe/Al on other minerals (e.g. quartz parti-
cles). The high afnity of DFOB for Fe(III) (log K = 30.99) and to a lesser
extent for Al(III) (log K = 24.5) may explain why DFOB did not complex
divalent metals and thereby failed to affect their mobility in soil. This
hypothesis agrees with that of Neubauer et al. (2002) who demonstrat-
ed in a batch experiment that the ability of DFOB to mobilize Cu from
montmorillonite and kaolinite was suppressed when Fe(III) was intro-
duced in excess (to DFOB). Hence, the non-complexation of divalent
metals by DFOB suggests that all the DFOB present in the pore water
was complexed to trivalent metals. The relationship between DFOB
and Fe + Al (Supplementary Fig. F3) shows that the two concentrations
were linearly related (r2 = 0.81) and that DFOB was present at nearly
the same concentration as Fe + Al (slope = 0.90) in the pore water.
The targeted effect of DFOB towards trivalent metals is in agreement
with the results of Neubauer et al. (2000b) and Zhong et al. (2013),
which, to our knowledge, are the only two studies that investigated
the effect of DFOB on the mobility of metals in soil. It implies that the
DFOB-induced alteration of Fe/Al oxides did not promote the mobility
of divalent metals, even though Fe/Al oxides are metal-bearing phases.
The absence of an effect of DFOB on the mobility of Cu contrasts with
that of the siderophore pyoverdine (Pvd) (Cornu et al., 2014; Tansupo
et al., 2008). This difference can be explained on one hand by the pres-
ence of a catecholate function within the chelating group of Pvd
(Albrecht-Gary et al., 1994), which confers a higher afnity for Cu (log
K = 20.1) than for DFOB (log K = 14.12) to this siderophore, and on
the other hand, by the fact that Cu-Pvd complexes are mostly
negatively-charged at pH N 6.
The supply of DFOB did not enhance the phytoextraction of any diva-
lent metals. This result was expected since DFOB had no effect on the
mobility of divalent metals in the soil or on plant growth. This result
contrasts with the results obtained in a study by Dimkpa et al. (2009)
Fig. 1. Principal component analyses of variables related to pore water characteristics of a conducted on a Cd-amended soil, in which the authors concluded that
metal-contaminated soil supplied either with desferrioxamine B (DFOB) or the supply of a bacterial culture ltrate containing hydroxamate
ethylenediaminetetraacetic acid (EDTA). The variables are pH, conductivity, siderophores (DFOE, DFOB and coelichelin), although 10 times less con-
concentrations of DFOB or EDTA and total concentrations of Al, Fe, Ca, Cd, Cu, Ni, Pb and
centrated than in the present experiment, enhanced Cd uptake by sun-
Zn. The plot of factor loadings in (a) concerns the variables measured in the DFOB
treatment vs. the control and the one in (b) concerns the variables measured in the ower. This discrepancy suggests that hydroxamate siderophores
EDTA treatment vs. the control. DFOB and EDTA concentrations in the control soil pore probably did not play a prominent role in enhancing plant uptake of
water were assumed to be null. The two PCA shown are of the cultivar ES RICA. Those of Cd in that study (Dimkpa et al., 2009). It is more probable that other bac-
the cultivar Kapllan are shown in Fig. F1 (Supplementary data). terial by-products than siderophores, for example, low-molecular
weight organic acids, or nutrients remaining in the culture ltrate, in-
creased Cd mobility in the soil by decreasing the pH and/or increasing
the soil constituents (either secondary clay minerals or solid organic the ionic strength of the pore water. Despite its strong impact on Fe mo-
matter) (Neubauer et al., 2000a). The concentration of DFOB in the bility in soil, DFOB supply did not increase the level of Fe in sunower
pore water decreased notably over time to concentrations below tissues (Supplementary Table T2). Hence, the ability of the plant to ac-
45 M at the end of the culture period (day 23). Accordingly to Zhong quire Fe directly from Fe-DFOB complexes (Cline et al., 1984) appears
et al. (2013), DFOB sorption on soil is rapid and was probably achieved to be restricted to Fe-decient conditions where sunower, like other
within the rst 24 h. Hence, the decrease in the concentration of DFOB Fe-efcient plant species, has shown increased capacity to reduce
over time most likely results from its degradation. Indeed, DFOB is Fe(III) in the rhizodermis (Rmheld et al., 1982).
suspected to rapidly degrade (Parra et al., 2008) in non-sterile environ-
ments like soils, due to indigenous microorganisms. 4.3. Impact of low doses of EDTA on the soil-to-plant transfer of metals
The supply of DFOB did not alter the mobility of divalent metals in
soil. This result contrasts with the results of batch experiments (e.g. In the present study, EDTA was supplied at a rate of 200 mol kg1
Neubauer et al., 2000a) in which DFOB strongly affected the mobiliza- soil DW, which is about 10 times lower than the dose usually used in
tion of divalent metals from puried minerals. In our study, it appears EDTA-assisted phytoextraction assays (Evangelou et al., 2007). The con-
that no divalent metals were complexed by DFOB in soil. In the case of centration of EDTA measured in the pore water on day 9 averaged

Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
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8 J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx

Fig. 2. Cd pseudo-polarograms obtained in pore water collected from (a) a control soil and (b) a soil supplied with EDTA. The conditions used to get these pseudopolarograms are
summarized in Table T1 (supplementary data). Vertical dashed lines at Edep = 0.7 V stand for the threshold between Cd occurring as organic complexes (left side) and labile Cd
(free ion + inorganic complexes, right side) (see Section 2.6.1 for further details).

Table 5
Total concentration, labile concentration and chemical speciation of Cd, Cu, Pb and Zn measured by DPASV in the pore water of a metal-contaminated soil with or without ethylenedi-
aminetetraacetic acid (EDTA). The data presented concern the pore water collected on day 9. Each treatment (chelator) was replicated ve times.

Chemical speciation (%)


Metal Total conc. (M) Labile conc. (M)
Labile Dissociable Inert

Cd 0.12 0.02 0.12 0.02 100 0 0


Cu 2.2 0.3 0.81 0.17 37 4 18 5 46 8
Control
Pb nd nd nd nd nd
Zn 46 4 46 4 100 0 0
Cd 1.08 0.26 0.012 0.005*** 1.3 0.6*** 42 6 56 6
Cu 149 15 2.8 0.7*** 1.9 0.6*** 98 1 0
EDTA
Pb 1.25 0.25 0.022 0.008 1.8 0.6 51 11 48 10
Zn 353 62 13 8*** 4.7 0.6*** 10 3 86 4

For a given metal, *** indicates that the labile fraction or the labile concentration of metals measured in EDTA-treated soil was signicantly different (p b 0.001) from that measured in the
controls.
nd: non detectable.

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J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx 9

511 172 M. This means that ve days after having been supplied, (e.g. Barrutia et al., 2010; Evangelou et al., 2007; Hernandez-Allica
only 38 21% of EDTA was sorbed onto the solid phase. This low et al., 2007; Nowack et al., 2006), it is surprising that Pb was not
EDTA afnity for the solid phase probably results from electrostatic re- among the metals whose phytoextraction was increased by EDTA.
pulsion at pH 6.7 between the negatively-charged EDTA (mainly Taken together with the data related to the speciation of Cd, Cu, Zn
HEDTA3 ) and/or metal-EDTA complexes (MEDTA2 or MEDTA) and Pb in pore water, these results support different hypotheses regard-
and the soil constituents. It is worth noting that the concentration of ing the forms (and the related pathways) in which metals are taken up
EDTA in the pore water was constant over the culture period in agree- (and translocated aboveground) in presence of low doses of EDTA.
ment with the well-known persistence of metal-EDTA complexes in First, the data suggest that the metal-EDTA complexes contributed to
the environment (Nowack et al., 2006). As expected from the literature the uptake of metals by sunower. Indeed, the addition of EDTA in-
(Evangelou et al., 2007), the supply of EDTA increased the mobility of di- creased Cu and Ni uptake whereas it probably decreased their free ion
valent metals in soil. A PCA analysis of the pore water characteristics of concentration in pore water. This suggests that Cu-EDTA and Ni-EDTA
the EDTA-treated vs. control soil, revealed that the concentrations of Cd, complexes contributed, respectively, to the additional uptake of Cu
Cu, Ni, Pb and Zn were highly correlated with the concentration of EDTA and Ni observed in presence of EDTA. Likewise, the fact that EDTA addi-
and not with pH and conductivity. This strongly suggests that the tion did not reduce Cd, (Pb) and Zn uptake, even though their free ion
mobility of divalent metals in EDTA treated soil was controlled by concentration in pore water was reduced in presence of EDTA
their complexation with EDTA. The lower afnity of metal-EDTA com- (Table 5), suggests that Cd-, (Pb-) and Zn-EDTA complexes managed
plexes (M-EDTA2 ) than that of their free ionic form (M2+) for the to compensate for this reduction through their respective contribution
negatively-charged soil constituents explains why supplying EDTA in- to the uptake of Cd, (Pb) and Zn. Second, they suggest that undissociated
creased the mobilization of divalent metals. Their mobilization factor, metal-EDTA complexes were taken up by sunower. This hypothesis is
calculated as the ratio of their pore water concentrations in the presence mainly based on the fact that Ni uptake was promoted in presence of
to that in the absence of EDTA, ranged from 10 to 100 and followed the EDTA. Indeed, contrary to most metals (e.g. Zn and Cd), Ni uptake is
sequence: Cu ~ Pb N Ni ~ Zn ~ Cd, which agrees fairly well with the sta- not rate-limited by the free ion diffusion to the root surface but mostly
bility constants reported for M-EDTA complexes (Supplementary by its internalization across the cell membrane (Degryse et al., 2012).
Table T6). For the sake of comparison, the mobilization factor of metals This means that the contribution of Ni-EDTA complexes to the Ni2+
like Pb can exceed 1000 when EDTA is supplied at several mmol kg1 symplastic uptake was probably small or even negligible. As a conse-
soil DW (e.g. Grman et al., 2001). This difference illustrates how the quence, the additional uptake of Ni observed when EDTA was added is
ratio of chelators to metals in soil inuences the rate of metal assumed to mainly result from the passive uptake of non-dissociated
mobilization. Ni-EDTA complexes by sunower roots. Considering the low EDTA
Our measurements of DPASV underlined the fact that adding EDTA dose added to the soil, and the resulting low concentration of EDTA
to the soil strongly increased the pore water fraction of Cd, Cu, (Pb) measured in pore water (b1 mM), it is unlikely that EDTA disrupted
and Zn complexed to organic ligands. It can reasonably be assumed root cell membranes (Schaider et al., 2006). Thereby, Ni-EDTA com-
that these organic complexes mainly consist in metal-EDTA complexes. plexes were probably taken up via a pure apoplasmic pathway through
For Cd and Zn, they probably almost entirely consist in metal-EDTA passage cells or at undifferentiated root tips where the Casparian strip is
complexes since no (non-labile) organic complexes were detected in not fully developed (Enstone et al., 2002). Given that apoplastic route
the control. The strong presence of metal-organic complexes in pore selectivity is minimal, other metal-EDTA complexes than Ni-EDTA
water therefore suggests that Cd, Cu, (Pb) and Zn were mainly mobi- were probably directly taken up by sunower roots. This uptake of
lized through complexation in EDTA treated soils. An important ques- metal-EDTA complexes would at least partly explain the deviations
tion to be addressed in the context of phytoextraction is whether the from the free-ion based equilibrium models observed for Cd, (Cu),
addition of EDTA increased the concentration of labile metals in the (Pb) and Zn when EDTA was added. In the case of Cd and Zn, whose
pore water. Indeed, the labile forms of metals are usually considered as symplastic uptake is often rate limited by the diffusion of free ions to
being available for biological uptake (Wilkinson and Bufe, 2004) and the root surface (Degryse et al., 2012), deviations from the free ion ac-
their concentration in pore water is a major determinant of the efciency tivity model (FIAM) could also result from the dissociation of metal-
of metal phytoextraction. Interestingly in our study, the labile concentra- EDTA complexes within the diffusion layer. However, considering the
tions of Cd and Zn were lower in the pore water of EDTA-treated soils. To high ratios of EDTA-complexed to free ion suspected in the pore water
our knowledge, this is the rst time the addition of EDTA has been exper- and the fairly slow dissociation of metal-EDTA complexes, the nonselec-
imentally shown to decrease the labile concentration of metals in soil tive uptake of metal-EDTA complexes is assumed to contribute the most
pore water. This decrease conrms the predictions of a mechanistic to the overall metal uptake in presence of EDTA. This has been reported
model developed for Cd by Lin et al. (2014) in which the sorption, trans- several times in hydroponics, notably for Pb (Barrutia et al., 2010;
port and uptake of Cd2+ and its complexed forms (CdL) are described. Hernandez-Allica et al., 2007), but more rarely in soil.
Conversely, the labile concentration of Cu in the pore water increased One pending question is why EDTA addition selectively promoted
when EDTA was added. This increase was the result of the strong Cu mo- Cu and Ni uptake by sunower. Assuming that in presence of EDTA,
bilization induced by EDTA but also of the fact that 2% of pore water Cu metals were only taken up via the non-saturating apoplastic pathway
remained DPASV-labile in presence of EDTA. Surprisingly, this fraction (see above), a linear relationship would be expected between the total
was higher than that of Cd measured in the pore water of EDTA- concentration in the pore water and the amount taken up by plant
treated soils, which does not agree with the higher afnity displayed roots. Given the low selectivity of the apoplastic route, it is sometimes
by EDTA for Cu than for Cd (Supplementary Table T6). This discrepancy proposed that this relationship is the same for all metals (Nowack
suggests that DPASV measurements slightly overestimated the labile et al., 2006), in other words that the apoplastic uptake of all metal-
fraction of Cu in pore water. This potential overestimation might be relat- EDTA complexes occurs at the same rate. According to this hypothesis,
ed to the analytical need for this element to forcibly separate the pseudo- the uptake of metals in presence of EDTA relies only on their total con-
polarographic wave corresponding to the labile fraction from that corre- centration in pore water, and the greater increase observed in Cu and Ni
sponding to the EDTA-bound one (see Section 2.6.1). uptake results entirely from their higher EDTA-induced mobilization
Even if added at a low dose, EDTA was expected to increase the from the solid phase. Simplied diagrams of metal uptake in absence
phytoextraction rate of all metals. Yet, EDTA addition selectively pro- and presence of EDTA (Supplementary Fig. F4) illustrate to what extent
moted Cu and Ni phytoextraction while that of Cd, Pb and Zn remained our results are consistent with this hypothesis. For instance, the fact that
unchanged. Given the large number of studies showing that the addi- Cu but not Zn uptake increased in presence of EDTA can be explained by
tion of synthetic chelators increased Pb accumulation in plant shoots the greater mobilization of Cu from the solid phase caused by EDTA.

Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.03.092
10 J.Y. Cornu et al. / Science of the Total Environment xxx (2017) xxxxxx

Conversely, to explain why EDTA addition did not increase Pb uptake, it Fe deciency. However, a more in depth investigation of how to pro-
has to be assumed that the apoplastic uptake capacity of sunower mote colonization of the rhizosphere by inoculated bacteria remains a
roots was lower for Pb-EDTA than for Cu-EDTA and Ni-EDTA complexes. key issue to guarantee the success of any process combining bioaug-
This type of selectivity along the apoplastic pathway to the stele has al- mentation with phytoremediation.
ready been described for metal-EDTA complexes of lower charge (Bell Conversely, this study conrmed that EDTA is an efcient mobilizing
et al., 2003) and, according to the Tanton and Crowdy (1971) route, agent of divalent metals in soil. EDTA increased the solubility of divalent
could be mediated by aquaporins in the plasmalemma of passage cells. metals in the soil 10 to 100 fold when added at the same concentration
as DFOB. This is presumably due to its lower specicity than DFOB for
4.4. Between cultivar differences Fe(III) and to the negative charge of metal-EDTA complexes at neutral
pH. One originality of this work is that EDTA was added at a concentra-
The two sunower cultivars grew at the same rate and were similar- tion one order of magnitude lower than the concentrations usually test-
ly affected by the addition of chelators. They were not sensitive to DFOB ed in EDTA-assisted phytoextraction. At this low dose, EDTA did not
but they were sensitive to EDTA, whose addition at a dose of compromise the shoot growth of sunower but enhanced only and
200 mol kg1 soil DW was detrimental to root growth. Given that moderately Cu and Ni phytoextraction. Consequently, it is probably
the concentrations of the metals in the plant tissues did not exceed not possible to reduce the dose of EDTA without altering the
the limits proposed by toxicologists for sunower, we suspect that phytoextraction yield of metals. Probably the best way to reduce the
EDTA was detrimental to root growth regardless of its impact on the harmful effects of EDTA (observed at higher dose levels) is to simply
phytoavailability of metals in soil. The higher ability of ES RICA to accu- avoid using it, and instead to use SPB producing siderophores that
mulate Cd aboveground was conrmed in soil by this study in which the form negatively charged metal complexes at neutral pH, like
concentration of Cd in the shoots was always higher in the ES RICA cul- pyoverdine. Based on the results we obtained on metal solubility,
tivar than in Kapllan. This trait appears to be specic to Cd, at least in ab- metal speciation in pore water, and the concentrations of metals in
sence of chelators, since the concentrations of Cu, Pb and Zn in the plant tissues, we conclude that metal-EDTA complexes contributed to
shoots of plants grown on the control were the same in both cultivars, the plant uptake of metals and that metal-EDTA complexes were likely
while that of Ni was higher in Kapllan. Given that the concentration of taken up undissociated via the non-selective apoplastic pathway. This
Cd in the roots was always lower in ES RICA than in Kapllan, this trait last point would need to be evidenced by analytical measurements of
is certainly related to the lower ability of ES RICA to sequestrate Cd in the concentration of EDTA in plant tissues (roots, xylem sap). We sus-
root tissues, which agrees with one conclusion of the sunower varietal pect that the uptake of metal-EDTA complexes even contributed most
screening conducted by Laporte et al. (2015) in hydroponics. to the overall uptake of metals in presence of EDTA, even at low con-
The two cultivars differed in how their phytoextraction ability was centrations. To explain how EDTA selectively promoted Cu and Ni
affected by the addition of EDTA. ES RICA was more sensitive to the ad- phytoextraction, we propose that the root apoplastic uptake capacity
dition of EDTA in the sense that the increase in Cu and Ni of sunower was higher for Cu- and Ni-EDTA complexes. The physiolog-
phytoextraction caused by adding EDTA was larger in this cultivar ical processes underlying the segregation between metal-EDTA com-
than in Kapllan. As a consequence, ES RICA was the most efcient culti- plexes along the apoplastic pathway to the stele and whether their
var for Cu and Ni phytoextraction in presence of EDTA. It remains to efciency may vary from cultivar to cultivar and with EDTA concentra-
be elucidated whether the difference in the sensitivity to the addition tion remain to be elucidated.
of EDTA arose from differential uptake and/or a differential root-to-
shoot translocation of Cu- and Ni-EDTA complexes between the two
Acknowledgments
lines. The rst hypothesis implies that the two cultivars differed in the
permeability of their roots to metal-EDTA complexes, whereas the sec-
This work was supported by funding from ANR 2011 CESA 008 01.
ond hypothesis suggests that the two cultivars did not transport metal-
The authors thank PhD Juan Carlos Raposo from SGIker of UPV/EHU,
EDTA complexes in the xylem with the same efciency. Given that
Sylvie Bussire and Ccile Coriou for technical assistance and Andr
metal-EDTA complexes are supposed to be transported with the water
Schneider for help in improving the quality of the manuscript.
stream, the greater sensitivity of ES RICA to the addition of EDTA may
also result from higher cumulative transpiration of this cultivar in pres-
Appendix A. Supplementary data
ence of EDTA.
Supplementary data to this article can be found online at http://dx.
5. Conclusions
doi.org/10.1016/j.scitotenv.2017.03.092.
This study compared the efciency of desferrioxamine B and EDTA in
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Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.03.092
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Please cite this article as: Cornu, J.Y., et al., How do low doses of desferrioxamine B and EDTA affect the phytoextraction of metals in sunower?,
Sci Total Environ (2017), http://dx.doi.org/10.1016/j.scitotenv.2017.03.092