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Use of Zea mays L. in phytoremediation of trichloroethylene

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DOI: 10.1007/s11356-016-7570-8

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Environ Sci Pollut Res
DOI 10.1007/s11356-016-7570-8
RECENT ADVANCES IN CHEMISTRY AND THE ENVIRONMENT
Use of Zea mays L. in phytoremediation of trichloroethylene
Emanuele Moccia1 · Adriano Intiso1 · Angela Cicatelli1 · Antonio Proto1 ·
Francesco Guarino1 · Patrizia Iannece1 · Stefano Castiglione1 · Federico Rossi1
Received: 3 February 2016 / Accepted: 31 August 2016
© Springer-Verlag Berlin Heidelberg 2016
Abstract Trichloroethylene (TCE) is a chlorinated alipha-
tic organic compound often detected as pollutant in soils
and ground water. “Green technologies” based on phytore-
mediation were proven to be effective to reclaim organic
pollutants (e.g. TCE) and heavy metals from different envi-
ronmental matrices. In this work, we use Zea mays L. for the
removal of high TCE concentrations from medium cultures.
In particular, we investigated a sealed bioreactor where
the growth medium was contaminated with an increasing
amount of TCE, in the range 55–280 mg/L; the removal
capability of the maize plants was assessed by means of
GC-MS and LC-MS analyses. An accurate mass balance
of the system revealed that the plants were able to remove
and metabolise TCE with an efficiency up to 20 %, depend-
ing on the total amount of TCE delivered in the bioreactor.
Morphometric data showed that the growth of Z. mays is
not significantly affected by the presence of the pollutant
up to a concentration of 280 mg/L, while plants show sig-
nificant alterations at higher TCE concentrations until the
growth is completely inhibited for [TCE]  2000 mg/L.
Finally, the presence of several TCE metabolites, includ-
ing dichloroacetic and trichloroacetic acids, was detected
in the roots and in the aerial part of the plants, reveal-
ing that Z. mays follows the green liver metabolic model.
These results encourage further studies for the employment
Responsible Editor: Philippe Garrigues
Emanuele Moccia and Adriano Intiso contributed equally
 Federico Rossi
frossi@unisa.it
1 Department of Chemistry and Biology, University of Salerno,
Via Giovanni Paolo II, 132 - 84084 Fisciano (SA), Italy
of this plant species in phytoremediation processes of soils
and waters contaminated by TCE and, potentially, by many
other chlorinated solvents.
Keywords Trichloroethylene · DNAPL ·
Phytoremediation · Zea mays L. · Plants · Organic
pollutants · Remediation
Introduction
Trichloroethylene (TCE) is a chlorinated solvent that
belongs to the class of dense non-aqueous phase liquids
(DNAPLs) (Schwille 1988), and it is an ubiquitous envi-
ronmental pollutant (Russell et al. 1996). The physical
and chemical properties of DNAPLs, including their rela-
tively low solubility, high specific density and the attitude
to remain sorbed to organic materials, make their removal
from polluted sites extremely difficult (Russell et al. 1996);
despite the fact that TCE is banned from industrial and civil
application since the middle 1980s, its widespread pollution
in different environmental matrices is still a major problem
(Chiu et al. 2012).
Several techniques were developed for the remediation
of DNAPL polluted sites, including air stripping, adsorption
processes, pump and treat and surfactant-enhanced disso-
lution (Russell et al. 1996; Hunt et al. 1988; Khachikian
and Harmon 2000; Huang et al. 2011). However, in the
past 10 years, phytoremediation is rapidly growing as a
potential “green technology” for the cost-effective removal
of TCE from soils and waters (Chappell 1998; Newman
et al. 1998; Gerhardt et al. 2009; Cruz et al. 2014). For
example, Gordon and coworkers studied the remediation
capability of several plant species, such as poplars (Gordon
et al. 1998; Newman et al. 1997, 1999; Shang and Gordon
 Environ Sci Pollut Res

2002), tobacco (Shang et al. 2001) and leguminous trees set of experiments and finally the presence of TCE metabo-
(Doty et al. 2003); Doucette and coworkers studied the TCE lites in the the different plant organs was assayed by LC-MS
uptake of fruit trees (Chard et al. 2006; Doucette et al. 2007) analyses.
and devised a special reactor to assess the in situ TCE reme-
diation by poplars (Orchard et al. 2000a, b); Schnabel et al.
studied the use of horticulture plant species (Schnabel et al. Experimental
1997); Zhang et al. studied the use of a grass-like alfalfa
(Zhang et al. 2013), while recently, Schöftner et al. investi- To perform a balance of the TCE used as contaminant, we
gated the potentiality of willows (Schöftner et al. 2016); in devised a closed experimental system where we could moni-
all these cases, the used plants were found to be effective in tor [TCE] in three different compartments, namely the plant,
the removal process and tolerant to considerable amount of the growth medium (GM) and the atmosphere.
TCE. The use of phytoremediation still presents some limits Figure 1 shows a sketch of the experimental bioreactor
with respect to other physicochemical techniques, such as consisting of a sealed glass jar (Bormioli, V = 1057 mL)
the long time required for the remediation process, the appli- where germinated Z. mays seeds (Pioneer PT32B10, Mon-
cability limited to sites with low level of pollution and the santo) were exposed to different amounts of TCE (Sigma
problem related with the dumping of the contaminated plant analytical grade) delivered by sterile glass syringes directly
after the harvesting (Gerhardt et al. 2009). For this reason, in the growth medium. The atmospheric [TCE] was moni-
researchers aim at finding plants having a fast adsorption tored by means of a passive sampling apparatus. The passive
kinetics, a high tolerance towards the targeted pollutants and sampler, RING (Aquaria Research srl), consists of an adsor-
the ability to degrade or transform the pollutants in inert or, bent cartridge packed with 300 mg of activated coconut
at least, less harmful species. charcoal inserted in a microporous polyethylene membrane.
In this work, we evaluated the phytoremediation per- TCE diffuses through the membrane towards the cartridge
formances of a cultivar of maize (Pioneer PT32B10) for driven by the gradient of concentration between the ambient
its capability to remove TCE from a polluted medium. air and the inner cartridge. Air TCE concentration (mg/m3 )
Zea mays presents some distinctive features and advantages can be calculated by applying Eq. 1, derived from Fick’s
with respect to many other plant species previously used first law of diffusion (Cussler 2009; Rossi et al. 2015)
for phytoremediation studies. Mays is a monocot and a
C4 plant, it has a wide fasciculate root apparatus able to md − mb
[TCE] = (1)
deeply penetrate the soil and reach aquifers, and last but not P × t × 10−6
least, its genetics is continually improved by seed industries
(Vamerali et al. 2010; Wuana et al. 2010; Baldantoni et al.
2011). For our study, we devised a sealed bioreactor where
all the compartments (atmosphere, medium and organisms)
could be easily monitored to understand the fate of TCE
introduced in the system and its effects on the used organ-
isms. In particular, we decided to add to the medium
TCE concentrations in the range of about 50–300 mg/L
with respect to the total volume of the bioreactor (with
check experiments up to 2000 mg/L). This concentration
range roughly corresponds to the lethal dose for rats and
mice (Friberg et al. 1953; Siegel et al. 1971) and it is
far above the concentrations commonly found in waters
(0.002–20 mg/L) and of the same order of that found in soils
(up to 1500 mg/L) as reported, for example, in the US EPA
National Priorities List (EPA 2016).
The removal capability of the plants was assessed by
means of GC-MS analyses, and an accurate TCE balance in
the system was carried out by taking into account the dis-
tribution of TCE in all the compartments of the bioreactor,
either in the presence or in the absence of maize plants. Fur-
thermore, in order to evaluate a potential inhibitory effect of Fig. 1 Sketch of the experimental bioreactor used for the mass
the contaminant, morphometric data were recorded for each balance assessment
Environ Sci Pollut Res
where md is the adsorbed mass in milligrams of the analyte
sampled during the time t (min) while mb is the mass of
the analyte on a non-exposed cartridge, and P (mL/min) is
the diffusive uptake rate of the substances (69 mL/min for
TCE); P values were supplied by the RING manufacturer.
Samplers were exposed at 10 cm above the growth substrate.
The GM consisted in 4.4 g of a Murashige and Skoog
(MS) medium (Murashige and Skoog 1962) dissolved in
1 L of distilled water containing 8 g of phytoagar (Duchefa-
Biochemie, Haarlem, NL) and 30 g of sucrose (Sigma) as
carbon source.
The Z. mays seeds were surface sterilised in axenic con-
ditions by stirring in ethanol (Sigma analytical grade) 70 %
for 3 min; successively, they were washed twice with sterile
double-distilled water for 1 min, stirred in a 1.5 % sodium
hypochlorite (Sigma) solution for 10 min and finally rinsed
five times for 1 min with double-distilled sterile water.
The seeds were pre-germinated on water sterile imbibed
filter paper in Petri dishes; when the rootlet was evident
(roughly 3 days), they were sown on solid MS medium in
the bioreactor.
Three replicates for each experimental thesis with
increasing TCE amount (58.4, 100, 116.8, 200, 300 mg)
were prepared. Given the volume of the bioreactor, the
delivered TCE corresponded to a total concentration of
contaminant of 55.25, 94.6, 110.5, 189.2 and 283.8 mg/L
for each experimental thesis. In each bioreactor, five pre-
germinated maize seeds were sown at regular distance on
100 mL of solid GM; in parallel, a series of blank systems
was prepared, namely three replicates of bioreactors without
plants for each dose of TCE and three replicates of a non-
contaminated bioreactor. All the bioreactors were finally
placed in a plant growth chamber at 25 ± 1◦ C for 9 days
(the same temperature was kept both during the day and the
night), with a photoperiod of 8 h of dark and 16 h of light
ensured by a fluorescence lamp (Mazda TFP 36W JR/865
G13) placed 40 cm from the plants.
To have a better control on the morphometric parame-
ters of the plants, we also prepared a series of glass vials
(V = 30 mL) containing the contaminated GM (10 mL) and
only one seed. Even in this case, three replicates for each
TCE dose were set up.
Analyses
The three different matrices (air, medium and plants) of
each bioreactor were separately analysed for the detection
of TCE and of TCE metabolites by means of GC-MS and
LC-MS, respectively.
After the harvesting, each plant was separated in
stem/leaf (aerial part) and roots, weighted, immediately
frozen in liquid nitrogen and then stored at −80 ◦ C until use.
Samples were ground to powder in liquid nitrogen to pre-
vent analyte loss. One hundred milligrams of ground sample
was treated with 2 mL of 1 M H2 SO4 /NaCl (10/1 w/w)
(Sigma-Aldrich) solution and then extracted by shaking the
sample for 10 min at 6000 rpm with 10 mL of methyl tert-
butyl ether (MTBE, Sigma analytical grade) (Odom et al.
2013). In the case of metabolite analyses, the samples were
extracted with 200 μL of ethyl acetate (Sigma analytical
grade) and vortexed for 1 min (Odom et al. 2013). In both
cases, a small amount of MgSO4 (CARLO ERBA) was
added to the mixture to remove the residual water; samples
were vortexed for another minute, shaken for further 15 min
at 12,000 rpm and finally the supernatant was injected into a
GC-MS for TCE analysis or LC-MS for metabolite analysis.
In order to check whether any TCE residual was still
present in the growth medium at the end of the experiment,
the agar was homogenised by mechanical stirring in a sealed
vial and three samples of GM (2.8 g, corresponding to a vol-
ume of ∼3 cm3 ) were extracted by shaking for 10 min at
6000 rpm with 10 mL of MTBE; 1 μL of the supernatant
was injected into the GC-MS apparatus. The deviation of
the results for the three different samples was within 5 %.
Passive diffusive samplers were extracted according to
the NIOSH methodology (NIOSH 2003). The inner car-
tridge was desorbed in 2 mL of carbon sulphide (CS2 , Sigma
analytical grade) for 30 min, and 1 μL of the supernatant
was directly injected in the GC-MS apparatus.
All samples for TCE detection were analysed by means
of a GC-MS apparatus (Agilent Technologies 7890A) by
using a DB17MS 30-mt column with an internal diameter of
0.25 mm and with a stationary phase of 0.25 μm thickness.
One microlitre of each sample was injected in split-less
mode; runs lasted 10 min with a flow rate of 0.5 mL/min
and with a temperature ramp of 20.5 ◦ C/min up to 240 ◦ C.
Calibration curves were built by injecting TCE (Sigma ana-
lytical grade) standards at known concentrations prepared in
MTBE or CS2 . The limit of detection (LOD) was estimated
from the ratio signal to noise (S/N = 3) and corresponded to
0.15 mg/L in our experimental conditions.
Samples for metabolite analyses were run on a LC-MS
apparatus (Waters quattro micro) by direct injection of the
sample in the ESI-quadrupole mass detector, both in positive
and in negative modes.
Results and discussion
To evaluate the absorption capabilities of the Z. mays plants
towards the TCE added to the GM, we performed a balance
of the system, by taking into account that the bioreactor
was thermodynamically closed and could not exchange mat-
ter with the external environment. Therefore, at the end of
 Environ Sci Pollut Res

Table 1 Residual TCE found in the GM (2) and in atmosphere (3), at the end of the experiments for each TCE initial dose

TCE0 (mg) 58.4 100 116.8 200 300

TCE2 (μg) 30 ± 2 60 ± 3 <LOD <LOD 2200 ± 300

TCE3 (mg) 49 ± 2 82 ± 4 98 ± 3 161 ± 3 235 ± 1

Blanks
TCE2 (μg) 120 ± 5 160 ± 8 140 ± 3 160 ± 3 2700 ± 400
TCE3 (mg) 57 ± 1 98 ± 4 114 ± 4 198 ± 3 297 ± 5

the experiments, the initial TCE must be distributed among
three different compartments: the plants (1), the GM (2) and
the atmosphere (3), either in its initial form or in the form
of metabolites eventually produced by the plants. Table 1
shows the residual TCE found in the GM (2) and in atmo-
sphere (3), at the end of the experiments for each TCE initial
dose. Both in the blank systems and in the presence of the
plants, TCE was mainly detected in the atmospheric com-
partment, and it was found in traces (< 0.05 % of TCE0 ,
except for TCE0 = 300 mg where TCE2  0.7 %) in the GM,
while it was not detected into the plant tissues, as revealed
by GC-MS analyses of the extracted solutions.
We defined a removal efficiency (RE, %) of the Z. mays
as a direct measure of the phytoremediation abilities of the
plants. RE was defined in terms of absolute mass (mg) of the
removed TCE from the environment (compartments 2 and
3), i.e. the TCE accumulated or metabolised by the plants
   
TCE0 − i TCEi TCEi
RE = ×100 = 1 − i ×100 i = 2, 3
TCE0 TCE0
(2)
Z. mays mediated by the total duration of the experiment, t
(days), for each TCE dose

TCE0 − i TCEi
RR = i = 2, 3 (3)
t × mp
where mp is the total biomass in the bioreactor, as weighted
at the end of the experiments; mp was calculated as the aver-
age of the total biomass in each bioreactor at a given TCE
amount.
Figure 2b shows that the specific removal rate increased
with the amount of TCE added in the bioreactor and it
reached a value of ∼1 mgTCE g−1 p day
−1 when plants were
exposed to 300 mg of TCE; the same trend was observed
when RR was calculated separately with respect to the fresh
weight of the roots and of the aerial part (leaves + stems,
data not shown). A comparison with the blank systems
revealed that the morphometric data, both in terms of fresh
weight (see Table 2) and in terms of the length of the roots
and the aerial parts (see Fig. 3c, d), is not significantly
affected by the addition of TCE in the concentration range
0–300 mg/L.

TCE3 mass has been calculated by multiplying [TCE]3 from

Eq. 1 by the volume of air in the bioreactor (V bioreactor −
V GM ).
Figure 2a shows the removal efficiency of maize plants
for different expositions to the contaminant. In particular,
RE is reported as the average of three replicates for each
experimental thesis (three sets) containing five seeds for
each bioreactor. In all cases, RE was found to be in the range
of 15–20 % with the highest efficiency at the higher doses
of TCE. RE values take into account a loss of about the 1–
3 % of TCE, as revealed by the analyses of the blanks. This
was most probably due to the absorption of TCE by the rub-
ber ring placed underneath the jar caps and used for sealing
the bioreactors and/or by a photo-degradation process.
A further indicator of the performances of Z. mays is the
specific removal rate (RR, mgTCE g−1 −1
p day ) defined as the Fig. 2 a Removal efficiency of Z. mays for the different amounts of
ratio between the absolute mass (mg) of the removed TCE TCE. b Removal rate of Z. mays for the different amounts of TCE
from the environment and the averaged fresh biomass (g) of expressed per units of plants fresh weight
Environ Sci Pollut Res

Table 2 Average fresh biomass and residual TCE at the end of the the experiments, and it clearly shows how the stems and
experiments the roots tended to get thicker and shorter when plants are
TCE0 (mg) TCE removed (mg) Plant biomass (g) exposed to higher [TCE], as also highlighted in Fig. 3b.
Figure 3c, d reports the length of the aerial parts (la ) and
0 7±2 of the roots (lr ) of Z. mays at the end of experiment. Mor-
58.4 10 ± 2 6±2 phometric data suggested that RR could increase with the
100 18 ± 1 8±2 amount of the contaminant until TCE did not significantly
116.8 19 ± 1 7±2 inhibit the plant growth. Therefore, in the investigated con-
200 39 ± 3 9±3 centration range (0–283.8 mg/L), the more TCE was added
300 65 ± 3 7±2 in the bioreactor, the more Z. mays could efficiently remove
the contaminant.
To understand the fate of the TCE up taken by maize,
we analysed several samples extracted from the plants by
To study the effect of the TCE on maize plant mor- means of the GC-MS apparatus; no traces of the contami-
phology, we set up a single seed bioreactor with increasing nant were detected neither in the aerial part nor in the roots.
amount of TCE with respect to the total volume of the A possible explanation to this observation is that Z. mays
bioreactor, namely [TCE] = 667, 1330, 2000 mg/L; TCE plants were able to completely metabolise the adsorbed TCE
concentration was far above those used in the five seed during their growth process; therefore, we performed exper-
bioreactors, and these experiments only aimed to establish iments aimed to recognise and characterise metabolites
the maize lethal TCE dose, and therefore, RR and RE were derived from the metabolic modification of the contaminant.
not calculated. Figure 3a gives a clear glimpse of the growth Gordon and coworkers (Newman et al. 1997; Shang et al.
inhibition of the plants caused by increasing amounts of 2001; Doty et al. 2007) investigated the uptake of TCE by
TCE. The picture was taken 3 days after the beginning of different plant species, including poplars and tobacco, and

Fig. 3 a Comparison of the

effects of TCE on the growth of
Z. mays plants exposed to
increasing concentrations of
contaminant. The picture was
taken 3 days after the beginning
of the experiments. b
Comparison between the roots
of a blank system and the roots
of a system containing
TCE = 667 mg/L. The picture
was taken at the end of the
experiment. c, d Variation of the
aerial part (la ) and root (lr )
lengths of Z. mays plants
exposed to increasing doses of
TCE as measured after 9 days
from the beginning of the
experiment

they found a common metabolic pathway for the adsorbed
TCE, mainly operated through the cytochrome P450. The
pathway basically followed the 3-step green liver model
proposed by Sandermann (1994): (i) oxidation, (ii) conjuga-
tion and (iii) excretion. Trichloroethylene could be initially
oxidised to dichloroacetic acid (CHCl2 COOH, 128.9 g/mol)
or trichloroacetic acid (CCl3 COOH, 163 g/mol) to finally
yield oxalic acid ((COOH)2 , 90 g/mol), or be oxidised to
trichloroethanol (C2 Cl3 H2 OH) to finally be conjugated with
glucose to yield trichloroethanol glucoside (C8 H13 Cl3 O6 ,
311.5 g/mol); both the oxalic acid and the glucoside then
could enter in other biological processes or be excreted by
the plants.
To validate this hypothesis, we performed a prelimi-
nary screening of samples extracted with ethyl acetate by
means of a LC-MS apparatus and we found several TCE
metabolites both in root, leaf and stem samples. Thorough
and quantitative analyses are still in progress; however,
Fig. 4 shows, as an example, the comparison between the
mass spectra of the aerial parts of a blank system with a
system containing [TCE] = 110.5 mg/L. Four main metabo-
lites, namely oxalic acid, dichloroacetic acid, trichloroacetic
acid and the glucoside, were easily identified through their
molecular masses, and the overlapping of the two spec-
tra revealed a higher concentration of these metabolites
in the plants exposed to TCE, as shown in the insets
of Fig. 4. These analyses seem to confirm the metabolic
pathway followed by TCE as proposed by Sandermann,
even in the case of Z. mays, as well as for tobacco and
poplars. The absence of any TCE trace in all analysed plant
organs allows to speculate that Z. mays, in the TCE con-
centration range we investigated, is able to metabolise the
contaminant.
Fig. 4 Mass spectra of the solutions extracted from the aerial parts
of Z. mays exposed to 0 mg/L (black line) and 110.5 mg/L (red line).
Insets show the details of the spectra of the different metabolites
identified together with their chemical structure and molecular weight
Environ Sci Pollut Res
Conclusions
In our work, we proved that Z. mays is a good candidate
for the removal of high TCE amounts from an artificial
environment. In particular, we set up a bioreactor where all
the compartments (atmosphere, growth medium and plants)
could be easily monitored to understand the fate of known
amount of trichloroethylene (55–280 mg/L) added to the
growth medium, either in the presence or in the absence
of Z. mays plants. The bioreactor used in our experiments
represented an unnatural method of growth and did not
reflect the field conditions. As demonstrated by Doucette
and coworkers (Orchard et al. 2000a, b), many naturally
occurring types of root stress can drastically influence the
root permeability and hence the uptake performances of the
plants. However, starting from our results, several improve-
ments can be made to the experimental design to gradually
increase the complexity of the system and have a better
picture of the plant performances before the use of maize
for in situ remediation of TCE. The GC-MS analyses and
the accurate TCE balance of the systems revealed that the
plants were able to remove and metabolise TCE with an effi-
ciency (RE) ranging from 15 to 20 %, depending on the total
amount of the contaminant added to the bioreactor. More-
over, the specific RR increased with the increment of TCE
concentrations (up to 1 mg of TCE removed by 1.0 g of fresh
biomass per day) while the total biomass was unaffected by
the presence of the contaminant in the TCE concentration
range we employed. The RR trend suggests that Z. mays
could be employed for phytoremediation of TCE even for
high contaminations, until the pollutant becomes too toxic
for the plants.
A preliminary LC-MS study on the plant extracts re-
vealed the presence of several TCE metabolites, including
dichloroacetic and trichloroacetic acids, both in the roots
and in the aerial part of the plants; this suggested that Z.
mays followed the Sandermann green liver metabolic model
in accordance with previous study on poplar and tobacco
plants. Because of the different experimental conditions, a
direct comparison of the performances of the Z. mays with
previous studies is difficult. However, the specific removal
rate RR was found in line with other plants; for example,
in the case of transgenic alfalfa, RR was found to be about
10 μg of TCE for 1 g of fresh biomass per day of exposition
(Zhang et al. 2013); in the case of spinaches, RR was about
0.05 μgTCE g−1 p day
−1 (Schnabel et al. 1997) while in the
case of transgenic poplars, it was about 20 μgTCE g−1
p day
−1
(Doty et al. 2007). Because RR strongly depends on the
initial dose of TCE delivered in the bioreactor, for a bet-
ter assessment of the Z. mays performances with respect to
the other plants, we are planning a series of experiments
at lower TCE0 to match the experimental conditions of the
previous studies.
Environ Sci Pollut Res
The values of RR, together with the fast growth speed and
the large biomass typical of maize, make Z. mays a poten-
tial good candidate for an efficient and extensive removal of
TCE from polluted soils. Moreover, GC-MS and the LC-MS
analyses revealed that the maize plants do not accumulate
TCE in the tissues or on the leaves’ surface, thus promoting
an effective degradation of TCE following the removal step.
The bioreactor we employed was specifically designed
for soil remediation, but with few simple modifications, it
can be adapted to study hydroponic cultures for the cleaning
of surface and ground waters. Our results encourage further
studies for the employment of Z. mays in phytoremediation
processes of soils and waters contaminated by TCE and,
potentially, by other chlorinated DNAPLs.
Acknowledgments F. R. and A. P. gratefully acknowledge the
projects ORSA149477 and ORSA158121 funded by the University of
Salerno (FARB ex 60 %). S. C. gratefully acknowledges the project
ORSA148032 funded by the University of Salerno (FARB ex 60 %).
Thanks are due to Mr. Gianmatteo Fortunato, Ms. Filomena Giannelli
and Ms. Yleana Govetosa for their experimental help.
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