International Journal of Phytoremediation

ISSN: 1522-6514 (Print) 1549-7879 (Online) Journal homepage: https://www.tandfonline.com/loi/bijp20

Citric acid as soil amendment in cadmium removal

by Salix viminalis L., alterations on biometric
attributes and photosynthesis

Danijela Arsenov, Milan Župunski, Milan Borišev, Nataša Nikolić, Andrej

Pilipovic, Saša Orlovic, Marko Kebert & Slobodanka Pajevic

To cite this article: Danijela Arsenov, Milan Župunski, Milan Borišev, Nataša Nikolić, Andrej
Pilipovic, Saša Orlovic, Marko Kebert & Slobodanka Pajevic (2019): Citric acid as soil amendment
in cadmium removal by Salix�viminalis L., alterations on biometric attributes and photosynthesis,
International Journal of Phytoremediation, DOI: 10.1080/15226514.2019.1633999

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INTERNATIONAL JOURNAL OF PHYTOREMEDIATION
https://doi.org/10.1080/15226514.2019.1633999

Citric acid as soil amendment in cadmium removal by Salix viminalis L.,

alterations on biometric attributes and photosynthesis


Danijela Arsenova , Milan Zupunski a
, Milan Bori
seva , Nata
sa Nikolica , Andrej Pilipovicb ,
Sa
sa Orlovic b
, Marko Kebert , and Slobodanka Pajevica
b

a
Faculty of Sciences, Department of Biology and Ecology, University of Novi Sad, Novi Sad, Serbia; bInstitute of Lowland Forestry and
Environment, University of Novi Sad, Novi Sad, Serbia

ABSTRACT KEYWORDS
During the past decade, the target in cleaning polluted sites is an application of chelating agents, Cd accumulation; chelating
such as citric acid (CA), which is proposed as a good candidate in the promotion of phytoremedia- agent; photosynthetic
tion. Among heavy metals, cadmium (Cd) is one of the most common and dangerous elements, performances; phytoreme-
diation; willow
which strongly disturbs morphophysiological properties in plants. A pot experiment was assessed
to evaluate the influence of CA in Cd phytoremediation in alkaline soil by Salix viminalis (clone
SV068). The effects of CA on Cd bioavailability, mobility, and distribution in plants, various mor-
phometric measurements, and physiological performances as photosynthesis, transpiration, water
use efficiency, and pigment content were tested. The highest Cd accumulation was evident after
60 days of growing, in plants subjected to combined treatment of CA with a higher dose of Cd.
Application of CA showed a beneficial effect in maintaining the photosynthetic rate as well as gas
exchange capacity in willows grown in Cd-contaminated soil. Furthermore, CA slightly increased
plant growth and biomass production, depending on applied Cd dose and harvest period. A che-
lating agent like CA showed strong influence in plant response to combat Cd toxicity.

Introduction capacity, which is predominantly influenced by soil pH val-

Environmental pollution by heavy metals is one of the lead-
ing problems at the global scale and requires the need for
successful and economically feasible decontamination. These
pollutants are very persistent in the biosphere and retain in
the soil for a long period. As a consequence, excess heavy
metal provokes detrimental effects on all life processes in
plants, whereas entering into trophic chains poses a risk to
animals and human health (Muhammad et al. 2009).
Cadmium (Cd) is considered as a widespread environ-
mental contaminant having highly poisonous and severe
negative effects on living organisms (Zacchini et al. 2009).
Increased concern about Cd toxicity on plants is due to its
role in triggering a series of changes in plant metabolism.
Cd can cause a decrease in biomass production, stunted
growth, imbalance in mineral nutrition, inhibition of photo-
synthesis and respiratory processes, disturbance in protein
synthesis, and changes in activation of antioxidative enzymes
(Borowiak et al. 2015; Yang et al. 2015; Mleczek et al. 2018).
Cd bioavailability and its uptake by plant roots is directly
correlated with the number of bioavailable metal forms in
the soil and depends on several soil properties such as pH,
aeration, humidity, and soil temperature (Zhang et al. 2018).
The main limiting factor in metal removal is sorption
ues. According to Trakal et al. (2012), in alkaline soil, heavy
metal ions are mostly immobilized, thus Cd mobility is sig-
nificantly reduced at a pH higher than 6–7. Furthermore,
Tang et al. (2006) reported that Cd extractability and avail-
ability decreased over time. During time, adsorbed Cd in
soil made rearrangement of soluble metal fractions, from
soil solution to solid surface, by forming complex with solu-
tion phases, causing diffusion of Cd through soil and mak-
ing strong chemical bonds and stable complex to clay or
organic matter and initialize its retention and lowering Cd
availability (Zhang et al. 2018). Therefore, successful phytor-
emediation requires high metal solubility and bioavailability.
Aiming to achieve this goal application of different chelates
can be performed.
Chelate-assisted phytoremediation has been proposed as a
successful tool in improving heavy metal removal from dif-
ferent media (Chen et al. 2003). Chelating agents stimulate
the release of heavy metals from the soil matrix, enhance
the metal solubility in the soil and uptake into the roots
(Lesage et al. 2005; Zhao et al. 2011). The main drawback of
assisted phytoremediation is the often use of synthetic acids
such as EDTA, which are proven to hasten metal solubility
in soil, which can, therefore, lead to leaching of these pollu-
tants to underground water (Meers et al. 2005). Also, this

CONTACT Danijela Arsenov danijela.arsenov@dbe.uns.ac.rs Faculty of Sciences, Department of Biology and Ecology, University of Novi Sad, Novi
Sad, Serbia.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/bijp
Supplemental data for this article can be accessed here
ß 2019 Taylor & Francis Group, LLC
2 D. ARSENOV ET AL.
approach is strongly criticized, because excessive and uncon-
trolled use of synthetic chelates may have a toxic effect on
plants and can adversely affect the number and variety of
rhizosphere microorganisms, possessing a threat to the over-
all environment (Zhao et al. 2011). With respect to this, low
molecular weight organic acids (LMWOAs), such as citric
acid (CA), have been proposed as an alternative to synthetic
chelators (Nascimento and Xing 2006; Muhammad et al.
2009). Several studies support the fact that organic acids
have a role as a chelating agent and can modify heavy metals
mobilization without a negative impact on the environment,
thus LMWOAs have a greater social acceptance (Freitas et al.
2013; Song et al. 2018). Enhanced metal uptake, accumula-
tion, and translocation in various plants due to the applica-
tion of CA are well documented (Gao et al. 2010; Ehsan
et al. 2014; Shakoor et al. 2014). On contrary to above men-
tioned, several authors emphasized the weak effectiveness of
LMWOAs in the phytoextraction process, which is provoked
by their rapid biodegradation and mineralization by soil
microorganisms (Lesage et al. 2005; Meers et al. 2008). This
inconstancy might be due to different influences of CA
depending on plant species, type, and concentration of heavy
metals, soil properties, and therefore, further studies are
mandatory aiming to define the efficiency of CA as a soil
amendment in assisted phytoremediation.
Numerous studies highlight that fast-growing trees pos-
sess desirable characteristics for phytoremediation based on
several points, that is, high biomass production, rapid
growth, deep root system, energy production, and so on
(Lux et al. 2004; Zacchini et al. 2009; Nikolic et al. 2017). A
number of studies confirmed the high efficacy of willows
(Salix spp.) in phytoremediation, especially toward Cd-con-
taminated soil and water (Yang et al. 2015; Mleczek et al.
2018). Furthermore, Cd effect on the growth rate, biomass
production, and photosynthetic attributes in Willows has
been a subject of numerous studies (Pietrini et al. 2009;
Zacchini et al. 2009). Willows are widely distributed, can be
easily propagated and hybridized, which allows clones selec-
tion and targeted breeding for various environmental condi-
tions that attend to enhance phytoextraction capacity
(Zalesny and Bauer 2007). Besides previously mentioned,
willows are often used in short rotation coppices, as a good
source of renewable bioenergy due to the large biomass yield
and high planting density, thus it can be established on mar-
ginal land (Casta~no-Dıaz et al. 2018). Furthermore, willows
are favorable candidates for decontamination of polluted
sites due to their facilities to grow fast, produce high bio-
mass yield, and possess the ability to adapt in a new envir-
onment as a pioneer species (Mleczek et al. 2018). However,
Table 1. Physicochemical properties of soil.
pH
Soil type
Sandy clay KCl dH2O CaCO3 (%)
Soil 7.62 8.16 10.89
Soil þ peat 7.18 7.88 8.01
Cd concentration [mg g–1] Control Cd3 Cd6
0 days 0.79 3.8 6.67
90 days 0.72 3.6 6.5
the data analyzing efficiency of CA application in phytore-
mediation trials using woody species, including willows, are
still lacking.
Taking into account everything aforementioned, the main
goal of this research was to test CA efficiency as a soil
amendment in Cd removal from contaminated soil by Salix
viminalis. In context to that, CA was added in single, dou-
ble, and multiple doses and removal of Cd from mild to
moderate contaminated soil was analyzed depending on
experimental duration. Photosynthetic activity, pigment con-
tent, and proline (Pro) accumulation were also addressed.
Material and methods
Experimental set-up
The 1-year-old cuttings of S. viminalis L. (clone SV068) were
selected from the Institute of Lowland forestry and
Environment, Serbia. The pot culture experiment was set-up
in the glasshouse. Willow cuttings and soil were obtained
from a woody plantation near Novi Sad (45.3025
N,
19.9385
E). Plants were grown in natural photoperiod, the
temperature was in range 20–30
C night/day, while plants
were watered periodically (drinking water, pH 7.82, CaCO3
75–150 mg L1, EC 425 mS cm1) to maintain optimal soil
moisture content of 60% (400 mL of water per pot per week).
Stem cuttings were rooted in Mitscherlich pots
(“Mitscherlich containers” for greenhouse experiments, size
15 cm diameter by 19 cm tall and having a double bottom,
into which excess fertilizer flow) and soil used for experi-
ment was mixed with peat, thus each pot contains 3 kg of
total substrate, 2.5 kg of soil, and 0.5 kg of peat, which was
homogenized before treatment exposure. The physicochemi-
cal properties of soil are given in Table 1. Cd was applied as
Cd(NO3)2  4H2O and sprayed onto the soil, the substrate
was mixed thoroughly, and left 10 days for stabilization
before plantation. Cd was applied in two concentrations:
3 mg kg1 (Cd3) and 6 mg kg1 dry soil (Cd6). The Cd che-
lation was provided by the addition of CA in water solution
and it was applied on soil surface a week before plants har-
vest. CA was added in concentrations of 20 mmol kg1 of
soil and its application was performed in three repetitions
after 30 days, following the same after 60 and 90 days of Cd
exposure. In total, six treatments included as follows: control
(soil without Cd, neither CA) and two doses of Cd (Cd3
and Cd6), followed by same with CA addition (CA without
Cd) and combined treatments with Cd (CA þ Cd3 and
CA þ Cd6). Each treatment was set-up in three replicates,
with six plants per pot. Plants were harvested three times,
Humus (%) Total N (%) AL-P2O5 (mg per 100 g) AL-K2O (mg per 100 g)
2.29 0.15 5.69 12.56
4.59 0.23 6.51 13.08
CA CA þ Cd3 CA þ Cd6
0.76 3.7 7.6
0.7 3.3 6.78

1 week after the single, double, and multiple additions of
CA; in total, 54 pots were analyzed.
Morphometric measurements and evaluations of
biometric parameters
Before analyses, plant material was washed in deionized
water, dried, and biomass of different organs (root, stem,
YL, and old leaves (OL)) was measured to calculate different
parameters. Separations of leaves to young and old were
obtained according to our previous research Arsenov et al.
(2017). Young leaves (YL) contained a group of four fully
developed youngest leaves and the other leaves are defined
as OL. The organ mass ratio (MR) was calculated according
to Iori et al. (2017), as a ratio of leaves (LMR), stem (SMR),
and roots (RMR) to total plant biomass, while roots to
shoots ratio (RSR) was calculated by dividing roots to
aboveground biomass (leaves þ stem).
Leaf area of both YL and OL was measured by leaf area
meter “ADC Bioscientific Ltd. AM350”. The specific leaf
area (SLA) was calculated according to Dijkstra and
Lambers (1989) as a ratio of leaf area to its biomass.
The tolerance index (TI) was calculated according to
Yang et al. (2015) as a ratio of plant biomass of Cd-treated
plants to biomass of controlled plants.
Photosynthetic pigment content and gas
exchange parameters
The chlorophyll and carotenoids concentrations were
assayed according to modified method by Von Wettstein
(1957) following extraction in absolute acetone and meas-
ured by UV-VIS spectrophotometer (Beckman DU-65, CA).
Total chlorophylls were calculated as a sum of chlorophyll
a þ b. Concentrations of photosynthetic pigments are
expressed in mg g1 fresh weight.
Gas exchange parameters such as net assimilation rate of
CO2 (PN), transpiration (E), water use efficiency (WUE),
and stomatal conductance (gs) were carried out on YL, using
infrared gas analyzer (LCProþ, ADC Bioscientific, UK). The
analyses were performed between 10 and 12 a.m., at a tem-
perature of 25 to 30
C. The photosynthetic active radiation
(PAR) was set to 1000 mmol m2 s1, while the flow of ambi-
ent air in the chamber was 100 mmol s1. The temperature,
air humidity, and the concentration of CO2 were depending
on their ambient values. WUE was calculated as the ratio of
net CO2 exchange and transpiration rate (PN/E)
Measurements were performed on six plants per treatment.
Cd content and phytoremediation factors
Separated plant organs (roots, stem, YL, and OL) and soil
samples were dried in an oven at 70
C for 48 h, followed by
complete mineralization to dry-ash at 450
C with the add-
ition of 30% of hydrogen peroxide and extraction with 25%
HCl. Cd content was determined using an atomic absorption
spectrophotometer with a flame atomizer (FSAAS240,
Varian, Canada).
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 3
On the basis of Cd content in plant tissue and soil, the
bioaccumulation factor (BAF) of the root (BAF root) and
the aerial plant parts (BAF aerial parts) was calculated as
mean of Cd concentration of both YL and OL, and stem
and translocation factor (TF) was calculated according to
Zacchini et al. (2009).
Proline content
The content of Pro amino acid was measured in YL and OL
by the modified method of Bates et al. (1973). Fresh leaves
were homogenized in 3% (w/v) sulfosalicylic acid and centri-
fuged at 3500 rpm for 10 min. The supernatant was mixed
with acid ninhydrin reagent and glacial acetic and incubated
for 15 min at 100
C. The reaction was stopped by cooling,
followed with the addition of toluene, and absorbance was
measured at k ¼ 520 nm. Pro concentration was expressed in
milligram per gram of fresh weight.
Statistical analysis
The obtained data were expressed as a mean of three inde-
pendent measurements (n ¼ 3) ± standard deviation (SD).
Mean values between treatments and experimental duration
were analyzed using two-way analysis of variance at a sig-
nificance level of p  .05. Multiple comparisons between
observed parameters were performed using the Fisher LSD
post hoc test. Different letters indicate significant differences
at p  .05.
A comparison between SLA, biomass production of YL,
OL, total biomass, and photosynthetic (PN), and transpir-
ation (E) rate was conducted by the Pearson correlation
coefficient for p  .05. Principal component analysis (PCA)
was performed to analyze the influence of Cd concentration
in various plants organs to different characteristics (biomass
production, chlorophylls, carotenoids, photosynthetic rate,
transpiration rate, WUE, intercellular CO2 concentration,
stomatal conductance, and Pro content). Analyses were per-
formed by STATISTICA 13.5 (StatSoft).
Results and discussion
Soil properties
The soil was mixed with peat, aiming to reduce the pH
value of the soil, and we observed a reduction in pH value
from 8.16 to 7.88 (in dH2O) and from 7.62 to 7.18 (in KCl).
At the same time, peat addition resulted in the reduction of
CaCO3 content and elevation of humus content and total
nitrogen (Table 1). These changes in soil properties could
have an impact on the production of biomass without affect-
ing Cd uptake and accumulation. According to Trevisan
et al. (2010), the addition of peat which is characterized by
a low pH value (pH value is about 3.5) can affect the bio-
availability of metal cations and contribute to their removal
from the soil. Furthermore, Stanislawska-Glubiak et al.
(2012) recorded increased adsorption of Cd and Pb due to
the addition of peat in sandy soils, compared with the same
4 D. ARSENOV ET AL.
treatment without peat. Cd content in the soil at the begin-
ning and at the end of experiment (90 days) was presented
in Table 1. The highest reduction in Cd content was
observed under CA þ Cd6 treatment and was 10.2% com-
pared with the initial Cd concentration.
CA and Cd influence on morphological traits
The biomass production and its partitioning as a plant
adjustment to heavy metal stress are vital in the selection of
favorable candidates for phytoremediation (Audet and
Charest 2008). Biomass production (YL, OL, stem, and
roots) showed variation depending on Cd dosage (3 and
6 mg kg1), and CA applications (CA or CA addition) and
experimental duration (Table S1). Similarly, the results of
this study revealed variation in plant biomass partitioning
depending on experimental duration and applied treatment
(Figure 1). The LMR was the highest after 30 days of Cd
exposure, RMR, and RSR after 60 days, while SMR showed
no significant differences regardless of treatments or time
exposure to Cd (Figure 1). Furthermore, after 30 days, RMR
was considerably decreased by Cd3 and Cd6, that is, 43 and
32% as compared to control. The adverse effect of Cd was
alleviated with CA addition, thus combined treatments did
not cause significant changes in RMR in comparison with
CA treatment. The exception of this trend was recorded
after 60 days when combined treatments caused a significant
decrease in RMR (25% and 20% by CA þ Cd3 and
CA þ Cd6, respectively) compared with CA treatment.
Nevertheless, CA addition elevated RSR in respect to Cd
applied alone, with exception after 60 days (Figure 1D).
Variations in plant biomass partitioning during time could
be the result of intrinsic ontogeny, as well as Cd content in
plant tissue. According to Audet and Charest (2008), fast-
growing species tend to shift biomass allocation and metal
distribution depending on heavy metals enrichment in soil,
Figure 1. Leaves biomass ratio (A), shoot biomass ratio (B), roots biomass ratio (C), and roots to shoots ratio (D) of S. viminalis depending on Cd dosage, CA applica-
tions, and experimental duration. Different letters indicate significant differences at p  .05.
as a consequence of stress-avoidance strategy. In line with
our findings, Iori et al. (2017) reported a reduction in RMR
by 65% in Eucalyptus plants exposed to 50 mmol CdSO4
grown hydroponically for 3 weeks. Morphological alterations
induced by CA application under heavy metal stress is well
established in many studies (Li et al. 2014; Shakoor et al.
2014). According to Melo et al. (2008), both single or mul-
tiple application of 5 mmol kg1 soil of CA in contaminated
soil has no negative effect to shoot biomass. A similar obser-
vation was reported by Freitas et al. (2013) who noted no
changes in dry biomass production of maize and vetiver
plants grown under Pb stress with the addition of
40 mmol kg1 soil CA. On contrary to this, the beneficial
role of CA in the alleviation of plant growth inhibition
induced by heavy metal stress was evident in various plants
(Ehsan et al. 2014; Farid et al. 2017; Gao et al. 2010).
Within biomass production and growth performances,
SLA is an important factor in defining plant tolerance to
heavy metal stress, since it can be correlated to photosyn-
thetic rate, relative growth rate, and nitrogen content in
leaves (Sharma et al. 2007; Iori et al. 2017). Based on the
Pearson correlation coefficient, a significant positive correl-
ation was observed between SLA and photosynthetic rate as
well as with biomass production (Table S2). In this study,
SLA tended to maintain on control level under both Cd
doses applied, thus no significant changes were observed
regardless leaves maturity or experimental duration
(Figure 2). The exception of this pattern was observed in
mature leaves after 90 days, when both Cd doses induced a
reduction in SLA by 34 and 36% compared with control
(Figure 2B). Similar findings were observed by Iori et al.
(2017), who reported no significant changes in SLA between
control and Cd-treated plants of Eucalyptus (velino clone)
grown hydroponically. Meanwhile, the CA application
caused changes in SLA depending on Cd doses applied,
leaves maturity, and experimental duration. After 30 days,

no significant changes in SLA was found neither to Cd nor
combined treatment applied. Furthermore, after double CA
application (60 days), CA þ Cd6 significantly reduced SLA in
YL by 19% in comparison with Cd6 treatment (Figure 2A).
The same trend was observed in mature leaves, and
CA þ Cd6 reduced SLA by 28% as compared to Cd6.
Meanwhile, multiple additions of CA (90 days) increased
SLA in YL by 19% under CA þ Cd3 and 31% under
CA þ Cd6 treatment, in comparison with respective treat-
ment without CA, while this trend is not observed in mature
leaves. Correlation coefficient showed a negative correlation
between SLA and applied treatment (Table S2). Reduction
in SLA might be the result of a higher accumulation of
carbohydrate compounds (Dijkstra and Lambers 1989).
According to Borowiak et al. (2015), enchanted accumula-
tion of carbohydrates under heavy metal stress can disturb
photosynthesis and lead to changes in water balance within
a plant, and consequently inhibit normal growth and devel-
opment. Therefore, the lowest SLA after 60 days of treatment
Figure 2. SLA of YL (A) versus OL (B) of S. viminalis depending on Cd dosage, CA applications, and experimental duration. Different letters indicate significant differ-
ences at p  .05.
Figure 3. Cd content in YL (A), OL (B), stem (C), and roots (D) of S. viminalis depending on Cd dosage, CA applications, and experimental duration. Different letters
indicate significant differences at p  .05.
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 5
under CA þ Cd6 treatment is probably attributed to high Cd
accumulation and reduced net photosynthetic rate.
Cd accumulation and phytoextraction potential
The metal allocation among plant organs is a useful tool in
plant species selection for phytoremediation and can play a
role as an indicator of detoxification mechanism under
heavy metal stress (Shamshad et al. 2018). Significant differ-
ences in Cd concentrations were evident depending on Cd
dose applied, the experimental duration and the presence of
CA (Figure 3). Cd accumulation showed a dose-dependent
pattern with decreasing order, respectfully, YL, followed by
roots, OL, and stem. The highest Cd content (25.23 mg g1)
was noticed in YL under CA þ Cd6 treatment after 60 days
and was increased by 67% in comparison with Cd6 applied
alone. Besides that, after 60 days, the almost linear elevation
of Cd amount in all tested plant organs with increasing Cd
in the soil was observed, whereas Cd content tends to
6 D. ARSENOV ET AL.
Figure 4. BAF of the root (A), aerial plant parts (B), TF (C), and TI (D) in S. viminalis depending on Cd concentration, CA applications, and experimental duration.
Different letters indicate significant differences at p  .05.
diminish after 90 days of growing (Figure 3). However, Cd
content in roots reached a maximum after 90 days under
Cd6 and CA þ Cd6 treatments and was almost twofold
higher with respect to Cd3 and CA þ Cd3. Combined treat-
ments elevated Cd concentration in roots, regardless of the
time of exposure, or Cd content in the soil (Figure 3D). In
contrast to these results, in our previous research Arsenov
et al. (2017), we observed no significant changes in Cd con-
tent in roots of S. viminalis exposed to combined treatment
of 6 mg kg1 Cd with a single dose of 20 mmol kg1 CA
compared with the same treatment without CA. In the line
with these results, Song et al. (2018) reported that the appli-
cation of organic acids in the soil can promote Pb uptake by
Larix olgensis, but their effectiveness depends on the acid
type, concentration, and duration of metal exposure.
Similarly, Melo et al. (2008) observed that multiple applica-
tion of CA showed more prominent effects in phytoextrac-
tion because it can alleviate the negative effect of its
biodegradation. Differences in Cd accumulation depending
on the time of experimental duration can be explained by
changes in Cd availability which occurs over time. Zhang
et al. (2018) noted rapid decreasing in EDTA-extractable Cd
in the soil after 30–60 days, while after 90 days, its content
tends to be stable. This strongly implies the need for taking
into consideration the aging time of Cd in soil when apply-
ing phytoremediation. Additionally, Wang et al. (2017)
observed that water-soluble Cd forms and metal-organic
acid complex are easily mobile and have a high adverse
impact on plants.
To evaluate the plant ability to accumulate Cd in a par-
ticular tissue, the most common factors such as BAF of the
root (BAF root) and the above-ground plant parts (BAF
aerial parts) were calculated. In general, lower BAF was
recorded in aerial plant parts (1.03–2.43) relative to roots
(1.42–3.79) (Figure 4A and B). Besides that, Cd chelation by
CA showed promoting role to BAF in both roots and aerial
parts depending on treatment and experiment exposure
(Figure 4). Chelating effect of CA and its beneficial role
on heavy metal uptake and accumulation was reported by
numerous research (Muhammad et al. 2009; Farid et al.
2017; Song et al. 2018). Melo et al. (2008) recorded a
lower metal accumulation by a single CA application
compared with its successive application. The authors
found that Cd accumulation in velvet bean increased by
32% in the case of threefold CA addition in relation to
individual treatment.
Various literature data revealed that Cd distribution
within a plant relies on selected clone properties, thus con-
trasting findings among willow clones ability to accumulate
and translocate Cd from roots to shoots and leaves occurred
(Lux et al. 2004; Yang et al. 2015). TF showed value higher
than 1, indicating that the selected clone represents a good
candidate for phytoextraction. Meanwhile, TF in Cd-treated
plants decreased in the dose-dependent pattern (Figure 4C).
CA addition significantly elevated Tf under CA þ Cd6 after
30 and 60 days, while under CA þ Cd3 after 90 days, in com-
parison with respective non-chelated Cd treatment (Figure
4C). The effectiveness of organic acids in the phytoextrac-
tion is reflected by their ability to form complexes with
heavy metals that are easily transported to the above-ground
plant parts (Song et al. 2018). The beneficial role of CA to
translocate Cd from roots to shoots was observed in differ-
ent plants, radish (Chen et al. 2003), Typha angustifolia
(Muhammad et al. 2009), and Brassica juncea (Mahmud

Figure 5. Total chlorophylls in young (A) versus old (B) leaves and carotenoids in YL (C) versus OL (D) in S. viminalis depending on Cd dosage, CA applications, and
experimental duration. Different letters indicate significant differences at p  .05.
et al. 2018). Organic acids can bind heavy metal ions and
thus promote their mobilization through xylem sap for
long-distance transport (Wang et al. 2017). Mahmud et al.
(2018) observed that metal soluble fractions of Cd and
metal-organic complex migrate rather than protein-inte-
grated Cd complex, which mainly remains in cell walls.
Consequently, it can be concluded that plants differently dis-
tribute Cd, depending on the chemical form of the analyzed
metal, its concentration in the plant tissue, as well as on the
time of Cd exposure.
Metal tolerance is an essential feature in plant evaluation
with high phytoremediation capacity, since the accumulation
and tolerance are independent traits (Yang et al. 2015).
According to Lux et al. (2004), TI > 0.60 is defined as a
threshold value for defining willow species as a highly toler-
ant to heavy metal stress. The analyzed S. viminalis clone
showed TI > 0.6 under both Cd doses applied (Figure 2D),
which strongly support its usage in decontamination of mild
to moderate Cd-polluted soil. Relatively high TI may be
related to willows ability to adapt to different environmental
conditions such as heavy metal stress as well as changed soil
properties due to the addition of chelating agents. However,
the lowest TI (0.63) was detected after 60 days under
CA þ Cd6 treatment and was 17% reduced in comparison
with the same treatment without CA (TI ¼ 0.76). Lowered
tolerance is directly linked to high Cd concentrations
observed in plants grown 60 days under combined treatment
CA þ Cd6, indicating that high Cd content in plant tissue
consequently can reduce plant tolerance to Cd stress. On
the other hand, no visual symptoms, such as chlorosis and
necrosis, were found due to the application of CA, even if
the Cd content in plant tissue was higher. These results
could be attributed to CA involvement in Cd detoxification
mechanism and metal compartmentalization in subcellular
structures. Several studies have shown that organic acids
INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 7
have an influence on plant tolerance to heavy metal stress
by chelating metal ions, forming organometallic complexes
and sequestrated them in the vacuole, thereby reducing the
negative impact of these pollutants on fundamental physio-
logical processes in plants (Shakoor et al. 2014; Guo
et al. 2017).
CA and Cd influence on pigment content and leaf gas
exchange parameters
Cd hampers plant productivity causing a disturbance in fun-
damental plant processes such as photosynthesis causing
reduction in CO2 fixation and gaseous exchange, transpir-
ation, as well as inhibition of chlorophyll biosynthesis
(Pietrini et al. 2009; Parmar et al. 2013). Chlorophyll pro-
duction can indicate the physiological and nutritional status
of plants as well as the occurrence of heavy metal toxicity,
therefore, it is a significant trait in estimations of plant abil-
ity to cope with heavy metal stress (Borowiak et al. 2015).
In this research, lower Cd dose applied (Cd3) mainly did
not cause significant changes in photosynthetic pigments
regardless to control, neither in YL nor OL, whereas Cd6
significantly decreased pigments content (Figure 5).
Furthermore, Cd6 treatment-induced reduction in chloro-
phylls in YL by 17%, 14%, and 48% after 30, 60, and
90 days, while in OL considerable reduction in total chloro-
phyll content was evident only after 90 days, 21% in com-
parison with control (Figure 5A and B). Reduced
chlorophyll production and its enhanced breakdown are one
of the prime consequences of Cd stress (Kaur et al. 2017).
In line with our results, Bori
sev et al. (2016) reported a sig-
nificant reduction in photosynthetic pigments in YL of S.
viminalis exposed to Cd, while their content in mature
leaves tends to maintain on control level. It was previously
8 D. ARSENOV ET AL.
Figure 6. Photosynthetic rate (A), transpiration rate (B), WUE (C), and stomatal conductance (D) in S. viminalis depending on Cd dosage, CA applications, and
experimental duration. Different letters indicate significant differences at p  .05.
reported that Cd2þ can substitute Mg2þ ions, disturbing
chlorophyll structure and its functionality, resulting in
graduate inhibition of photosynthesis (Parmar et al. 2013).
The time-responses of Cd and CA influence on pigment
content showed that total chlorophylls and carotenoids
reached a maximum after 90 days of growing under CA
treatment (Figure 5). Moreover, after 90 days combined
treatments induced significant elevation of total chlorophylls,
regardless leaves maturity, compared with Cd applied alone.
Exogenous application of CA under heavy metal stress
increased chlorophyll content in diverse plant species (Ehsan
et al. 2014; Kaur et al. 2017; Song et al. 2018). Possibly, CA
can preserve the chloroplast ultra-structure and thylakoid
membrane integrity in stress conditions by limiting Cd
transport in the cytosol (Li et al. 2014; Mahmud et al.
2018). CA can act as a Cd chelating agent leading to metal
accumulation in the vacuole, thereby preserving primary
metabolism and accelerating plant ability to overcome stress.
In contrary to our study, Shamshad et al. (2018) reported
lower pigment contents and less toxicity in younger leaves
in comparison with OL in pea plants subjected to Cd stress
with CA addition.
Excess heavy metals content can affect photosynthesis
causing damages in photosynthetic apparatus, inhibition of
electron transport, carbon fixation capacity, reduction in sto-
matal conductance, and transpiration rate (Borowiak et al.
2015; Nikolic et al. 2017). Results of this study noted
decreasing order in gas exchange attributes such as net
assimilation rate, transpiration rate, WUE, as well as stoma-
tal conductance with an elevation of Cd in plant tissue
(Figure 6). Net assimilation rate of CO2 showed Cd dose-
dependent decreasing, whereas CA addition diminishes
adverse effect of Cd, thus PN was higher under combined
treatments in comparison with Cd applied alone (Figure
6A). Combined treatment CA þ Cd6 elevated PN by 20%
after single CA application (30 days), 23% after multiple CA
application (90 days), while after 60 days of growing it was
recorded reduction in PN values by 14%, in comparison with
Cd6. Similarly, CA increased transpiration rate (E) after 30
and 90 days of growing on both Cd doses applied, while
after 60 days, combined treatment provoked decreases in
transpiration rate by 27 and 42% under CA þ Cd3 and
CA þ Cd6 treatment compared with respective treatment
without CA addition (Figure 6B). Furthermore, WUE
showed variation depending on the time of experiment
exposure, Cd doses applied, as well as on the presence of
CA (Figure 6C). The highest elevation of WUE (80%) was
recorded after 60 days under CA þ Cd3 compared with Cd3
treatment. Both Cd doses did not affect stomatal conduct-
ance (gs) after 30 and 60 days, whereas after 90 days, Cd
induced a significant reduction in gs by 32 and 42% under
Cd3 and C6 treatment relative to control (Figure 6D).
Stomatal conductance reduction due to Cd exposure is prob-
ably induced by stomatal closure or decrease in stomatal
aperture size which is related to restriction water vapor dif-
fusion and gases exchange (Nikolic et al. 2017).
Furthermore, Cd chelation with CA stress did not affect the
stomatal conductance after single nor double application in
comparison with the same treatment with no CA addition,
while after multiple application (90 days) gs values were
reduced by 33 and 47% in comparison with Cd3 and Cd6.
Gas exchange attributes can be modified by different
heavy metals and can lead to a reduction in plant growth
and biomass production. The inhibition of net photosyn-
thetic rate induced by excessive heavy metals is provoked by
disturbed CO2 assimilation through reduced stomatal con-
ductance of CO2 (Pietrini et al. 2009). Meanwhile, the appli-
cation of natural chelating agents in contaminated soils can
accelerate the efficiency of water use, plant growth, thus sta-
bilizing photosynthesis and transpiration processes at a high
level (Ehsan et al. 2014; Li et al. 2014). In context of that,
changes in photosynthetic performances revealed in this
 INTERNATIONAL JOURNAL OF PHYTOREMEDIATION 9

Table 2. Pro content in YL versus OL of S. viminalis depending on Cd dosage, CA applications, and experimental duration.
Young leaves Old leaves
30 days 60 days 90 days 30 days 60 days 90 days
Control 6.7 ± 1.6c 9.7 ± 2.9ghi 6.7 ± 1.2c 10.7 ± 4.4fgh 8.8 ± 2.6c 6.3 ± 0.9hi
Cd3 20.6 ± 6.1b 11.9 ± 2.1efg 9.3 ± 1.8c 22.1 ± 1.0b 10.1 ± 0.4c 7.3 ± 0.4ghi
Cd6 38.3 ± 6.0a 16.3 ± 1.9cde 8.4 ± 1.9c 32.2 ± 4.1a 10.2 ± 0.7c 5.9 ± 0.3hi
CA 9.1 ± 3.4c 15.1 ± 3.0def 9.4 ± 0.2c 5.4 ± 2.1i 8.3 ± 0.1c 7.7 ± 1.1ghi
CA þ Cd3 10.4 ± 1.1c 16.5 ± 2.2cd 11.2 ± 3.5c 10.1 ± 0.1ghi 8.9 ± 0.7c 7.1 ± 0.4ghi
CA þ Cd6 18.6 ± 5.9b 21.3 ± 7.3b 9.8 ± 0.7c 17.5 ± 2.5bcd 8.7 ± 0.8c 8.5 ± 1.ghi
Data were expressed as mean ± SD (n ¼ 3). Different letters indicate significant differences at p  .05 applying Fisher LSD post hoc test.

Figure 7. PCA of morphophysiological traits in Salix viminalis grown in soil culture under different Cd dosage (3 and 6 mg kg1), CA applications (CA addition or no
CA) and experimental duration. CA, citric acid addition of 20 mmol kg1 of soil; Acc: accumulation of Cd; YL: young leaves; OL: old leaves; Chl: chlorophyll; Car: caro-
tenoids; PN: photosynthetic rate; E: transpiration rate; WUE: water use efficiency; ci: intercellular CO2 concentration; gs: stomatal conductance.

study under combined treatment with CA may be a result
of plant adjustment to overcome Cd stress. This acclimatiza-
tion enables the plant to cope with heavy metal stress that
consequently leads to enhanced plant tolerance to these pol-
lutants. In the line to our findings, Farid et al. (2017)
reported the positive role of CA in mitigation of heavy met-
als to photosynthetic performances. Similarly, Guo et al.
(2017) reported that malic acid can mediate photosynthetic
rate and stomatal conductance in Miscanthus sacchariflorus
plants exposed to Cd stress.
CA and Cd influence on Pro content
Pro accumulation has been reported to occur in different
species mainly as a response to osmotic stress influenced by
drought, high temperature, salt, or heavy metals (Xu et al.
2009; Mahmud et al. 2018). High Pro content is proposed to
have an important role in plant stress tolerance, due to its
role as a compatible osmolyte, ROS scavenger, and in buf-
fering cytosolic pH (Verbruggen and Hermans 2008).
Therefore, the accumulation of this amino acid has been
considered a useful marker for assessing plant tolerance to
heavy metal stress. In this study, the highest Pro accumula-
tion was recorded after 30 days, in plants exposed to Cd6, in
both YL and OL, and was almost six- and threefold higher
in comparison with control (Table 2). The increased synthe-
sis of this amino acid under heavy metal stress can be con-
sidered as a non-enzymatic defense strategy due to its role
in scavenging of OH radicals and 1O2, indicating the anti-
oxidant role of this metabolite (Xu et al. 2009). However,
chelation of Cd by CA-induced lower production of Pro in
plants subjected to the combined treatment with respect to
Cd applied alone. At the same time, after 60 days, the add-
ition of CA caused elevation of Pro accumulation in YL, by
38 and 31% under CA þ Cd3 and CA þ Cd6, respectively,
while in OL combined treatments only slightly reduced Pro
accumulation, with no significant differences in relation to
10 D. ARSENOV ET AL.
the same treatments without CA (Table 2). The possible rea-
son is a significantly higher amount of Cd in YL exposed to
combined treatment with respect to Cd applied alone.
Contrary to this, after 90 days, no significant differences
were found irrespective of applied treatment or leaves
maturity (Table 2). Obtained results indicate that Pro accu-
mulation strongly depends on Cd accumulation, presence of
soil chelating agents, plant organs and maturity, as well as
on the experimental duration. According to Verbruggen and
Hermans (2008), Pro accumulation varied among plant spe-
cies, maturation stage, and leaf age. Authors declared that
genes involved in Pro biosynthesis are predominantly
expressed in apical meristem and YL, while in OL their acti-
vation is limited to certain parts of the leaf (veins, guard
cells, trichomes, etc.). In addition, Shamshad et al. (2018)
confirmed the hypothesis that Cd alter the plant metabolism
depending on its chemical forms present in plant tissue,
rather than total metal content. Furthermore, Song et al.
(2018) observed similar finding to our results, authors con-
cluded that CA can mitigate toxic effect induced by Pb, by
decreasing Pro content after 10 > 20 > 30 days of
100 mg kg1 Pb exposure in Larix olgensis. In addition,
Mahmud et al. (2018) reported the influence of CA in
osmotic adjustment and in mitigation of stress conditions
observing decreases in Pro content in Brassica juncea plants
exposed to Cd.
PCA analyses (Figure 7) showed that first variable (axis)
reflects 28.89% (eigenvalue 9.53), while the second variable
contributes by 16.65% (eigenvalue 5.49) with cumulative
45.54% of the total variability in the data set. The first axis
separates treatments with Cd addition regardless of CA add-
ition (Cd3, CA þ Cd3, Cd6, and CA þ Cd6) and no Cd
treatments (control and CA). Cd treatments are correlated
to Cd accumulation in plant organs and grouped in fourth
quarters. CA and control treatments are correlated to bio-
mass production and grouped in first and second quarters.
PCA analyses showed a negative correlation between Cd
accumulation and photosynthetic performances (chloro-
phylls, carotenoids, WUE, and PN) and a positive correlation
between Cd accumulation and Pro content.
Conclusions
In this study, it was observed that soil amendment with CA
in Cd-contaminated soil has an influence on morphological
traits and photosynthetic performances of S. viminalis
plants. As a consequence, chelation of Cd with CA reduced
the negative effect of this pollutant to plant metabolism,
leading to enhanced Cd accumulation and translocation
resulting in enhanced phytoextraction. Meanwhile, the effi-
ciency of CA was dose depended and showed time-response
manner; thus, further studies are necessarily aiming to
define plant defense machinery in Cd detoxification.
Acknowledgments
This research was conducted as a part of the project “Investigating the
climate changes and their impact to environment: tracking impact,
adaptation and reduction” III43007, financed by the Ministry of
Education and Science of the Republic of Serbia.
ORCID
Danijela Arsenov http://orcid.org/0000-0002-1200-5023


Milan Zupunski http://orcid.org/0000-0002-8576-1292
Milan Bori
sev http://orcid.org/0000-0002-1833-002X
Nata
sa Nikolic http://orcid.org/0000-0002-7326-589X
Andrej Pilipovic http://orcid.org/0000-0002-9458-0581
Sa
sa Orlovic http://orcid.org/0000-0002-2724-1862
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