The Possibility of Using Paulownia elongata S. Y. Hu × Paulownia fortunei Hybrid for Phytoextraction of Toxic Elements from Post-Industrial Wastes with Biochar

Abstract

The potential of the Paulownia hybrid for the uptake and transport of 67 elements along with the physiological response of plants cultivated in highly contaminated post-industrial wastes (flotation tailings—FT, and mining sludge—MS) was investigated. Biochar (BR) was added to substrates to limit metal mobility and facilitate plant survival. Paulownia could effectively uptake and translocate B, Ca, K, P, Rb, Re and Ta. Despite severe growth retardation, chlorophyll biosynthesis was not depleted, while an increased carotenoid content was noted for plants cultivated in waste materials. In Paulownia leaves and roots hydroxybenzoic acids (C6-C1) were dominant phenolics, and hydroxycinnamic acids/phenylpropanoids (C6-C3) and flavonoids (C6-C3-C6) were also detected. Plant cultivation in wastes resulted in quantitative changes in the phenolic fraction, and a significant drop or total inhibition of particular phenolics. Cultivation in waste materials resulted in increased biosynthesis of malic and succinic acids in the roots of FT-cultivated plants, and malic and acetic acids in the case of MS/BR substrate. The obtained results indicate that the addition of biochar can support the adaptation of Paulownia seedlings growing on MS, however, in order to limit unfavorable changes in the plant, an optimal addition of waste is necessary.

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plants
Article
The Possibility of Using Paulownia elongata S. Y. Hu ×
Paulownia fortunei Hybrid for Phytoextraction of Toxic
Elements from Post-Industrial Wastes with Biochar
Kinga Drzewiecka 1, *, Monika G ˛asecka 1, Zuzanna Magdziak 1, Sylwia Budzy ´nska 1, Małgorzata Szostek 2,
Przemysław Niedzielski 3, Anna Budka 4, Edward Roszyk 5, Beata Doczekalska 6, Marta Górska 5
and Mirosław Mleczek 1, *


Citation: Drzewiecka, K.; G ˛asecka,
M.; Magdziak, Z.; Budzy´nska, S.;
Szostek, M.; Niedzielski, P.; Budka,
A.; Roszyk, E.; Doczekalska, B.;
Górska, M.; et al. The Possibility of
Using Paulownia elongata S. Y. Hu ×
Paulownia fortunei Hybrid for
Phytoextraction of Toxic Elements
from Post-Industrial Wastes with
Biochar. Plants 2021,10, 2049.
https://doi.org/10.3390/
plants10102049
Academic Editors: Juan Barcelóand
Jaco Vangronsveld
Received: 18 August 2021
Accepted: 24 September 2021
Published: 29 September 2021
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Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Chemistry, Faculty of Forestry and Wood Technology, Pozna´n University of Life Sciences,
Wojska Polskiego 75, 60-625 Pozna´n, Poland; monika.gasecka@up.poznan.pl (M.G.);
zuzanna.magdziak@up.poznan.pl (Z.M.); sylwia.budzynska@up.poznan.pl (S.B.)
2
Department of Soil Science, Environmental Chemistry and Hydrology, University of Rzeszów, Zelwerowicza
8b, 35-601 Rzeszów, Poland; mszostek@ur.edu.pl
3Department of Analytical Chemistry, Faculty of Chemistry, Adam Mickiewicz University in Pozna´n,
Uniwersytetu Pozna´nskiego 8, 61-614 Pozna ´n, Poland; pnied@amu.edu.pl
4Department of Mathematical and Statistical Methods, Pozna´n University of Life Sciences, Wojska Polskiego
28, 60-637 Pozna´n, Poland; anna.budka@up.poznan.pl
5Department of Wood Science and Thermal Techniques, Faculty of Forestry and Wood Technology,
Pozna´n University of Life Sciences, Wojska Polskiego 38/42, 60-637 Pozna´n, Poland;
edward.roszyk@up.poznan.pl (E.R.); marta.gorska@up.poznan.pl (M.G.)
6
Department of Chemical Wood Technology, Faculty of Forestry and Wood Technology, Pozna ´n University of
Life Sciences, Wojska Polskiego 38/42, 60-637 Pozna´n, Poland; beata.doczekalska@up.poznan.pl
*Correspondence: kinga.drzewiecka@up.poznan.pl (K.D.); miroslaw.mleczek@up.poznan.pl (M.M.)
Abstract:
The potential of the Paulownia hybrid for the uptake and transport of 67 elements along
with the physiological response of plants cultivated in highly contaminated post-industrial wastes
(flotation tailings—FT, and mining sludge—MS) was investigated. Biochar (BR) was added to
substrates to limit metal mobility and facilitate plant survival. Paulownia could effectively uptake and
translocate B, Ca, K, P, Rb, Re and Ta. Despite severe growth retardation, chlorophyll biosynthesis
was not depleted, while an increased carotenoid content was noted for plants cultivated in waste
materials. In Paulownia leaves and roots hydroxybenzoic acids (C6-C1) were dominant phenolics, and
hydroxycinnamic acids/phenylpropanoids (C6-C3) and flavonoids (C6-C3-C6) were also detected.
Plant cultivation in wastes resulted in quantitative changes in the phenolic fraction, and a significant
drop or total inhibition of particular phenolics. Cultivation in waste materials resulted in increased
biosynthesis of malic and succinic acids in the roots of FT-cultivated plants, and malic and acetic
acids in the case of MS/BR substrate. The obtained results indicate that the addition of biochar can
support the adaptation of Paulownia seedlings growing on MS, however, in order to limit unfavorable
changes in the plant, an optimal addition of waste is necessary.
Keywords: hybrid; Oxytree; phytoextraction; trace elements; uptake
1. Introduction
The genus Paulownia includes mainly herbs with only a few woody plants. Fast-
growing, deciduous trees of this genus naturally occur in Southeast Asia, but some species
have been successfully introduced in Poland and can be found in the warmest areas in
the country, i.e., Paulownia tomentosa (Thunb.) Steud. (known as the princess tree) and
Paulownia fortunei (Seem.) Hemsl. (commonly called the dragon tree). Both of them
grow up to 20 m tall, and their crowns are typically spherical-shaped. The Paulownia
tree is difficult to propagate since its seeds are highly vulnerable to soil fungi. Therefore,
in vitro
reproduction techniques are implemented to produce seedlings. To maximize the
Plants 2021,10, 2049. https://doi.org/10.3390/plants10102049 https://www.mdpi.com/journal/plants

Plants 2021,10, 2049 2 of 25
advantages of various species, some new artificial species and their varieties have been
developed, e.g., Paulownia “Shan Tong” and Paulownia “Cotta Vista 2” (Paulownia tomentosa
and Paulownia fortunei hybrid) [
1
], Paulownia Clon in Vitro 112 (Paulownia elongata
×
fortunei
hybrid) developed by the Spanish In Vitro S.L. company. Trees of the latest clone assimilate
more CO
2
than other taxa of the Paulownia genus, and consequently produce more O
2
,
earning them the name of “Oxytree”, a commercial name given by Carbon Solutions Global
Ltd. in 2016 [
2
]. The extremely fast-growing Oxytree was introduced in Poland in 2015,
and is considered as an alternative in biomass, wood, pulp production, as well as the
recovery processes of soils exploited by mining. Thus, Paulownia plantations have become
increasingly popular [3].
Along with poplar and willow, plants of the Paulownia genus are usually described as
good candidates for phytoremediative purposes due to their tolerance to high concentra-
tions of metals, rapid growth, and above all, high biomass production [
4
]. This aspect has
been described for selected hybrids and most often concerns assisted phytoremediation
using particular agents added to substrate [
5
–
7
]. Trees of the genus Paulownia may be suit-
able for metal phytoextraction due to their belonging to the C4 plant group, whose highly
reductive internal environment of cells prevents the oxidative conversion of metals [
8
].
As part of numerous studies in this area, significant differences in the phytoextraction of
harmful elements have been identified. In soil heavily contaminated with copper com-
pounds, the Paulownia elongata
×
fortunei hybrid showed good resistance and reduced
the Cu content in the soil by 17% [
3
]. It was found that the addition of EDTA caused a
significantly higher uptake of metals compared to citrate [
9
]. Attention has also been paid
to the use of fungi or peat as growth-stimulating agents influencing the phytoextraction
potential of Paulownia [
10
,
11
]. Further, Paulownia has so far only been investigated to a
limited extent on mining waste materials and there is lack of extensive research on the
accumulation of noble or rare earth elements. However, bioconcentration and transloca-
tion factors exceeding one for common metallic pollutants as Cd, Cu, Pb and Zn, make
Paulownia an increasingly attractive research subject [
10
]. Wang et al. [
12
] showed that
during revegetation, the immobility and bioavailability of heavy metals were enhanced in
the rhizosphere soils of Paulownia fortunei.
An effective reduction of metal load in soil using Paulownia tomentosa, almost 2-fold
higher than for Cytisus scoparius or Populus alba was documented [
13
]. The high potential of
two lines of Paulownia was also described [
14
] with clear differentiation in phytoextraction
of Pb and Zn. Nevertheless, despite valuable morphological features and significantly
higher phytoremediative abilities than most woody non-hyperaccumulators, the practical
use of Paulownia is clearly limited due to non-native trees are now prominently on the lists
of invasive plants in many parts of the world [
15
]. Possibilities for the use of the Paulownia
genus can be seen in hybrids such as Paulownia elogata
×
fortunei that produce non-viable
seeds and limiting the invasion risks [16].
Plant growth on contaminated soil is already problematic at the adaptation stage when
the plant experiences contact with high concentrations of toxic elements. A promising
solution undertaken in several works is the use of biochar. Many studies have reported
that biochar has been effectively used to immobilize metal(loid)s in contaminated soils and
influences their bioavailability and bioaccessibility for plants [
17
,
18
]. Biochar amended
bioremediation is one of the critical remedial technologies to remediate soils contaminated
by metal(loid)s. Biochar-enhanced phytoremediation shows excellent potential to immo-
bilize cationic metal(loid)s in mine wastes and tailing soils, particularly those with high
acidity. The moderate specific surface area of biochar and a pH higher than 9.0 may help
plants to survive in new unfavorable growth conditions by reducing the concentration
of toxic elements in the rhizosphere and their gradual dosing. Biochar may also improve
soil fertility and revegetation and create a suitable environment for soil microbial diver-
sity. The potential benefits of biochar for phytoremediation are (1) physical adsorption of
cationic metal(loid)s from soil pore water; (2) (co)precipitation with phosphate, carbon-
ates, silicate, and chloride, e.g., the formation of pyromorphite; (3) complexation with

Plants 2021,10, 2049 3 of 25
functional groups on the surface of biochar; and (4) the release of nutrients such as N,
P, K, Ca.
Processes (1)–(3)
can reduce the bioavailable metal concentrations in soil pore
water and further minimize phytotoxicity. Process (4) can produce nutrients for plant roots
and microorganisms in the rhizosphere, which is a crucial point in the development of
cost-effective remediation strategies [19].
The search for plants capable of adaptation and growth on post-industrial waste is
necessary due to the need for the decontamination of polluted matrices and their use in
phytomining. The use of species recognized at present as invasive, although in the context
of progressive climate change potentially non-invasive species in the future, seems rational,
providing these plants are characterized by high resistance and accumulation of pollutants
while growing on waste with moderate concentrations of toxic elements.
The aim of this study was to characterize the potential of the Paulownia elongata
×
Paulownia fortunei hybrid to uptake 67 elements (including rare earth elements (REEs)
and noble elements (NEs) together with an estimation of the physiological response of
plants cultivated in highly contaminated post-industrial waste materials. Due to the high
concentrations of metals and metalloids in the applied waste materials, biochar was used to
assess its use as a factor limiting metal mobility and availability for plants, and ultimately
facilitating the survival of Paulownia seedlings.
2. Materials and Methods
2.1. Plant Material
Oxytree (Paulownia elongata S. Y. Hu
×
Paulownia fortunei (Seem.) Hemsl.) seedlings
with the trade name of in Vitro 112, were obtained from an authorized supplier, Oxytree
Solutions Poland Ltd. (formerly Carbon Solutions Poland Ltd.). Seedlings (6–8-month-old)
were used after their preliminary selection from a population of 10,000 specimens according
to similar biomass (14–16 g), height (15 cm) and leaf count (5 pcs).
2.2. Experiment Design
Five experimental systems were prepared in hydroponic pots (23
×
23 cm, diameter
×
height) as follows: (1) control—6.1 kg of reference soil, (2) FT—4.2 kg of flotation tailings,
(3) FT/BR—3.99 kg of FT and 0.21 kg of biochar (BR), (4) MS—4.6 kg of mining sludge
and (5) MS/BR—4.37 kg of MS and 0.23 kg of BR. Biochar addition was set at 5% (w/w)
based on preliminary trials where its application increased the porosity of waste materials
and eventually led to undisturbed water flow in the substrate. Biochar was obtained from
miscanthus (Miscanthus gigantheus). The lignocellulosic material was ground with a roller
mill and sieved. The crushed plants were subjected to pyrolysis and carbonization in a
chamber reactor in an oxygen free atmosphere by heating up to 600
◦
C at the temperature
rate of 3
◦
C min
−1
and then holding for 1 h. The pore structure of the biochar was
characterized by the nitrogen adsorption-desorption method at 77.4 K in a sorptometer
ASAP Micromeritics 2020. Prior to gas adsorption measurements, the biochar was degassed
at 300 ◦C in a vacuum condition for 24 h. The Brunauer-Emmett-Teller (BET) surface area
was calculated from the isotherms using the BET equation. The pH of the aqueous biochar
solution was also determined. The mixtures of waste materials and BR (characterized by
the specific surface area of 50.1 m
2
g
−1
) were carefully blended using a POLYMIX PX-SR
90 D stirrer (KINEMATICA AG, Littau-Luzern, Switzerland). Detailed characteristics of
the resulting experimental substrates are shown in Tables 1 and 2 in the Results section.
Each experimental system combined 6 plants growing one plant per pot. The ex-
periment was set up on 5th April 2018 and terminated on 27th September 2019. Plants
were cultivated in a ventilated greenhouse of the Botanical Garden administered by the
Adam Mickiewicz University in Pozna´n. The humidity of substrates was continuously
measured, and the plants were constantly watered with tap water using an automatic
irrigation system to maintain a constant humidity. The temperature was controlled by a
mechanical ventilation system and automatic data loggers recording ambient parameters

Plants 2021,10, 2049 4 of 25
on an hourly basis. Mean temperature, air relative humidity and concentration of CO
2
were 22.8 ◦C, 50.5% and 459 ppm, respectively.
After the growing season of 2018, 3 randomly selected plants from each experimental
system were harvested, gently washed with ultrapure water (Milli-Q, Millipore, Saint Luis,
MO, USA) and divided into roots, stems and leaves. The biomass of separated organs was
determined by weighing. For physiological and biochemical investigations, fresh organs
were in situ frozen in liquid nitrogen and stored at
−
80
◦
C till analyses. Before extraction,
each plant sample was homogenized in liquid nitrogen. For the elemental investigations,
collected tissue was dried at 55
±
2
◦
C to a constant weight using an electric oven (SLW 53
STD, Pol-Eko, Wodzisław ´
Sl ˛aski, Poland). The dry material was ground using an SM 200
Cutting Mill (Retsch GmbH, Haan, Germany) until a powder fraction was obtained. The
remaining 3 plants from each group were left for the next growing season (2019), and the
harvested plants were investigated for biomass parameters and metal accumulation.
2.3. Analysis of Experimental Substrates
Substrate samples were air dried and sieved through a 2 mm sieve. Particle size
distribution was performed with the laser diffraction method using the Laser Particle Sizer
ANALYSETTE 22 (Fritsch, Idar-Oberstein, Germany). pH was analyzed in a 1:2.5 substrate-
water suspension using a Hanna Instruments (Nusfalaucity, Romania) 4221 pH-meter.
Electrical conductivity (EC) was analyzed in a 1:5 substrate-water suspension with a HI 2316
EC-meter by Hanna Instruments (Nusfalau, Romania). Total carbon (TC), nitrogen (Nt) and
sulfur (St) were determined by the dry combustion method using the auto analyzer Vario
El CUBE (Elementar Analysensysteme GmbH, Langenselbold, German) [
20
]. Soil organic
carbon (SOC) was determined using the Walkley-Black procedure [
20
]. Inorganic carbon
(SIC) was calculated as a difference between total carbon (TC) and organic carbon (SOC).
Humic substances (HS)—humic acids (C
HA
), fulvic acids (C
FA
) and humins (C
HUMIN
) were
determined according to Kononowa [
21
]. The base cations (Ca
2+
, Mg
2+
, K
+
, Na
+
) were
determined by an inductively coupled plasma optical emission spectrometer (ICP-OES
5110, Agilent, Santa Clara, CA, USA) following the extraction with 1 M ammonium acetate.
Cation exchange capacity (CEC) and base cation saturation ratio were calculated according
to Culman et al. [22].
2.4. Analysis of Elements in Soil and Plant Samples
In the experimental substrates and plant organs, 67 elements were analyzed as follows:
Al, As, B, Ba, Be, Bi, Ca, Cd, Co, Cr, Cs, Cu, Fe, Ga, Ge, Hf, Hg, In, K, Li, Mg, Mn, Mo, Na,
Ni, P, Pb, Rb, Re, Sb, Se, Si, Sn, Sr, Ta, Te, Th, Ti, Tl, V, W, Zn, Zr, rare earth elements (REEs):
Ce, Dy, Er, Eu, Gd, Ho, La, Lu, Nd, Pr, Sc, Sm, Tb, Tm, Y and Yb and noble elements (NEs):
Ag, Au, Ir, Pd, Rh, Ru, Os, Pt.
For the fractionation of elements in all experimental substrates, the four steps se-
quential extraction proposed by the Commission of the European Communities Bureau of
Reference (BCR) procedure [23] was performed as follows:
Step 1—Exchangeable/extractable fraction (F1): 40 mL 0.11 M acetic acid was added to
a 100 mL centrifuge tube containing 1 g of dry soil sample and sieved through a 2-mm
grid. The samples were then shaken at room temperature for 16 h. The supernatant and
solid were decanted and kept for further analysis. The residual solid was rinsed twice with
distilled water (2
×
10 mL) by shaking for 15 min. After centrifugation, the liquid was
decanted and discarded.
Step 2— Reducible fraction bound Fe–Mn oxides (F2): 40 mL 0.5 M hydroxyloammonium
chloride solution was added to the centrifuge tube containing the residue from step 1. The
samples were shaken once more at room temperature for 16 h. The samples were then
centrifuged and treated as in step 1.
Step 3— Oxidizable fraction bound to organic matter (F3): 10 mL 8.8 M hydrogen perox-
ide solution was added to the Step 2 residue. The contents were digested first at room
temperature for 1 h, then at 85
◦
C in a water bath until approximately 1 mL of solution

Plants 2021,10, 2049 5 of 25
was obtained. Then 50 mL 1 M ammonium acetate was added and shaken for 16 h at
room temperature. The supernatant was collected after centrifugation and kept for further
analysis. The solid was rinsed as before.
Step 4— Residual fraction (F4): The residue remaining at the end of step 3 was digested in
aqua regia solution and the concentration of REEs was determined using an inductively
coupled plasma optical emission spectrometer (ICP-OES 5110, Agilent, USA).
2.4.1. Sample Processing
Samples of dry material (0.300
±
0.001 g) were digested with concentrated nitric acid
(65%; Sigma-Aldrich, Saint Louis, MO, USA) in Teflon containers using a closed microwave
sample preparation system (Mars 6 Xpress, CEM, Matthews, NC, USA). After digestion,
the samples were diluted with Milli-Q water (Merck Millipore, Darmstadt, Germany)
to a final volume of 15 mL and filtered (Qualitative Filter Papers Whatman, Grade 595).
Concentration of all determined elements is expressed as mg kg−1DW.
2.4.2. Instruments and Quality Control
The inductively coupled plasma optical emission spectrometry (Agilent 5110 ICP-OES,
Agilent, USA) was used for analysis. The following conditions of analytical procedure
were set: radio frequency (RF) power 1.2 kW; 0.7, 1.0 and 12.0 L min
−1
, respectively for
nebulizer gas, auxiliary gas and plasma gas flows; detector charge coupled device (CCD)
temperature
−
40
◦
C; the time of signal accusation 5 s for 3 replicates. The detection limit
for all elements determined was set (as 3-sigma criteria) at the level of 0.01–0.09 mg kg
−1
of dry weight (DW). The uncertainty for the total analytical procedure (including sample
preparation, for uncertainty budget calculation, the coverage factor k = 2 was used) was
at the level of 20%. The recovery for certified reference materials analysis (CRM NCSDC
73349—bush branches and leaves, CRM S-1 – loess soil, CRM 2709 – soil) was acceptable
(80–120%) for most of the determined elements.
2.5. Determination of Arsenic Species in Soil and Plant Samples
2.5.1. Preparation of Samples
Dry samples (0.300
±
0.001 g) were extracted in a glass flask with 10 mL of phosphoric
acid (1 M) in an ultrasonic bath for 30 min. Next, the solution was diluted with water to a
final volume of 10 mL and filtered using filter paper.
2.5.2. Instruments and Quality Control
The following arsenic species: inorganic As(III), As(V) and dimethylarsenic (DMA)
were determined using high performance liquid chromatography with hydride generation
optical emission spectrometry detection (HPLC-HG-ICP-OES). The HPLC instrument
was a liquid chromatograph (Shimadzu, Kyoto, Japan) with an anion-exchange column
(Supelco, Bellefonte, PA, USA) LC-SAX1 (250
×
4.6 mm). The chromatographic elution
was isocratic with a flow rate of 1 mL min
−1
of phosphate buffer (5 mM Na
2
HPO
4
and
50 mM KH2PO4×2H2O
) and an injection volume of 200
µ
L. PEEK (polyetheretherketone)
tubing was inserted into a Tygon sleeve for transfer of the eluent from the LC column to the
in-spray chamber of the hydride generation unit (MSIS, Agilent, USA). The concentration
of NaBH
4
was 1% in 1% NaOH, the concentration of HCl was 5 M (all reagents Merck,
Darmstadt, Germany). An inductively coupled plasma optical emission spectrometer
Agilent 5110 ICP-OES (Agilent, USA) was used for signal detection with the parameters
listed above. As an analytical signal, the peak height corrected by the noise level was used.
The limits of quantification were 6, 18, 21
µ
g L
–1
for As(III), DMA, and As(V), respectively.
Due to a lack of certified reference materials for arsenic speciation in the analyzed matrix,
the standard addition method was used for accuracy and traceability studies. Recovery at
the level of 80–120% was found as satisfactory.
A detailed description of the equipment, reagents, analytical procedure and obtained
metrological data has been presented in previous work [24].

Plants 2021,10, 2049 6 of 25
2.6. Biochemical and Physiological Parameters of Plant Response to Substrate
Pigment content was assayed in methanolic extracts with colorimetric method [
25
].
Approximately 0.5 g of fresh leaf tissue was homogenized in 5 mL of 96% methanol using
a mortar and a pestle and then centrifuged to obtain a clear supernatant (
3600 rpm/min
,
15 min
). 0.05 mL of the extract was diluted with 0.95 mL of 90% methanol and the ab-
sorbance at
λ
= 470, 653 and 666 nm was measured using a Cary 300 Bio UV-Vis spec-
trophotometer. Chlorophyll a (Chl-a), b (Chl-b) and total carotenoid (Caro) contents were
calculated using the following formulae:
Chl-a (mg L−1) = 15.65 A666 −7.34 A653;
Chl-b (mg L−1) = 27.05 A653 −11.21 A666;
Caro (mg L−1) = 1000 A470 −2.86 Chl a −129.2 Chl b/245.
Leaf samples of ~0.5 g were ground in liquid nitrogen and extracted with 6 mL of 80%
methanol. Extracts were centrifuged (10,000 g, 20 min), and the obtained supernatant was
used for the total phenolic content (TPC) and relative antioxidant activity assays.
Folin–Ciocalteu reagent was employed in order to determine the TPC in methanolic
extracts [
26
]. The extracts (0.2 mL) were mixed with diluted (1:1 with water, v/v) Folin-
Ciocalteu reagent (2 mL) and after 3 min 10% Na
2
CO
3
(2 mL) was added. Following
incubation in a dark (60 min, room temperature), the absorbance at
λ
= 765 nm was
measured using a Varian Cary 300 Bio UV-Visible scanning spectrophotometer. The results
were expressed as mg of gallic acid equivalents (GAE) per g of tissue fresh weight (mg
GAE g−1FW). The assay was performed in triplicate.
The relative antioxidant activity of methanolic extracts was measured based on their
scavenging activity towards a DPPH radical using a Cary 300 Bio UV-Vis spectrophotome-
ter [
27
]. The reaction mixture comprised 0.5 mL of the extract, 3 mL of absolute ethanol
and 0.3 mL of 0.5 mM DPPH ethanolic solution. After the incubation (100 min, in dark),
the absorbance at
λ
= 517 nm was measured. Ethanol and the extract mixture was used as
a blank. The reaction was performed in triplicates. The relative scavenging capacity (RSC)
was calculated using the following formula:
RSC [%]=Ac −Ae
Ac ×100,
where: Acis the absorbance of the control; Aeis the absorbance of the extract.
The contents of salicylic acid (SA) in leaves was determined with a HPLC-FLD
method [
28
,
29
]. Paulownia leaves were ground in liquid nitrogen using a mortar and
a pestle to obtain a fine powder and ~0.50 g was taken for analysis. SA was extracted twice
with methanol (90% followed by a straight solvent) and each time sonicated for 15 min.
After centrifugation (10,000 g, 20 min), the supernatants were combined, and the solvent
was evaporated to dryness under a stream of nitrogen (industrial grade,
≥
99.95% by Air
Products, Poland). Dry extracts were redissolved in 3 mL of TCA (5%, w/v) for pigment
precipitation and centrifuged (10,000 g, 10 min). SA was extracted from the aqueous phase
three times with a mixture of ethyl acetate, cyclopentane and isopropanol (100:99:1, v/v/v).
The organic phases were combined, and the solvent was evaporated to dryness under a
stream of nitrogen. Dry extracts were stored at
−
24
◦
C until analyses. Before analysis,
dry residues were dissolved in the mobile phase (0.2 M KAc buffer, pH 5.0) and analyzed
with a Waters Alliance 2695 Chromatograph coupled with a Waters 2475 Multi-
λ
Fluores-
cence Detector (Waters Corporation, Milford, MA, USA). The separations were performed
using a Waters Spherisorb ODS2 column (100
×
4.6 mm, 3
µ
m) at a 1.5 mL min
−1
flow
rate. The fluorometric detection was performed at
λEx
= 295 and
λEm
= 405 nm. SA was
identified according to the retention time and quantified by comparing the peak area using
an appropriate calibration curve.

Plants 2021,10, 2049 7 of 25
Rhizosphere, root and leaf sample preparation for phenolic acids and low-molecular-
weight organic acids (LMWAOs) analyses was carried out [
30
]. The samples were homoge-
nized in 80% methanol and HCl (99:1), sonicated and shaken for 5 h. Extracts were dried
and redissolved in water for organic acids and 80% methanol for phenolic acids. Aqueous
and methanolic solutions were centrifuged (at 3600 rpm/ min for 15 min at 25
◦
C), and
the resulting samples were filtered through 0.2
µ
m nylon filters prior to chromatographic
analysis. For determination of phenolic compounds and organic acid a Waters Acquity H
class UPLC system coupled with a Waters Photodiode Array Detector (Waters Corporation,
Milford, MA, USA) and a Waters Acquity UPLC BEH C18 column (150
×
2.1 mm, 1.7 mm)
were used. The gradient of water and acetonitrile (both containing 0.1% formic acid) was
applied at a flow rate of 0.4 mL min
−1
. The quantification was conducted at
λ
= 280 and
320 nm for LMWOAs and phenolic compounds, respectively (Magdziak et al. 2020).
2.7. Statistical Analysis and Calculations
All statistical analyses were performed using STATISTICA 13.3 software (StatSoft,
USA). To show the existence of uniform groups of objects (
α
= 0.05), the multiple com-
parison Tukey’s HSD test was performed following one-dimensional analysis of variance
(ANOVA).
To define the effectiveness of element uptake by Paulownia, the bioconcentration
factor (BCF) was calculated. It is the ratio of particular elements or their groups (NEs,
REEs) concentration in plant organs (leaves, stem and root) to their concentration in soil.
Additionally, to characterize the effectiveness of element transport from the root system
to aerial plant parts, the translocation factor (TF) was calculated as the ratio of studied
elements/groups of elements concentration in leaves and stem to their concentration
in roots [
31
,
32
]. To perform a quantitative analysis of the element extraction ability of
Paulownia, a Metal Extraction Ratio (MER) was calculated [
33
,
34
]. The MER value shows
the suitability of the plants for phytoremediation [
35
], and is calculated according to the
following formula:
MER (%) = {cplant ×mplant} / {csoil ×mrootzone}×100,
where: c
plant
is the element concentration in the harvested organs of the plant (stems and
leaves), m
plant
is the biomass of harvestable organs produced in one harvest, c
soil
is the
element concentration in the experimental system, and m
rootzone
is the soil mass rooted by
the plant during studies.
The plant effective number (PEN) was calculated to estimate, how many plants are
needed to extract 1 g of determined elements from the substrate, taking into account the
biomass of the above-ground organs [33,36].
3. Results
3.1. Substrates Properties
The control soil was characterized as sandy soil with a low content of total car-
bon and nitrogen (0.91 and 0.05%, respectively), slightly acidic (pH = 5.39), low salinity
(
EC = 0.27 mS
) and low cation exchange capacity (CEC = 22.6 meq 100 g
−1
) (Table 1). Com-
pared to the other variants, this soil was characterized by a content of the determined
elements similar to geochemical background of Polish soils (Table 2).
The remaining substrates (FT, MS and their mixtures with BR) were characterized by
an alkaline reaction with a pH ranging from 7.94 for MS/BR to 8.49 for FT/BR (Table 1),
which resulted from alkali waste addition and was characterized by a pH of the aqueous so-
lution of 10.1. Additionally, significantly higher salinity was noted for the MS and MS/BR
substrates compared to the control soil (EC = 6.75 and 6.53 mS, respectively). Substrate pH
and EC were directly related to a high sodium and calcium content. Sodium concentration
in FT and MS was over 3 and nearly 5-fold higher than in control soil. Significantly higher
calcium concentrations were also found in FT and MS samples compared to the control,
i.e., ~22 and 19 times higher, respectively. The high concentration of these elements also

Plants 2021,10, 2049 8 of 25
influenced the base cation saturation ratio. MS and MS/BR substrates showed signifi-
cantly higher concentrations of TC, SOC, SIC, N
t
, S
t
compared to other variants (
Table 1
).
The results obtained for MS most likely result from the presence of organic pollutants
characteristic of this kind of waste, that disturb the correct estimation of the SOC value.
With only a few exceptions, the detected elements were present in significantly higher
concentrations in FT, MS and their mixtures with BR compared to control soil (Table 2).
Further, their concentration in MS was higher compared to FT except for Cs, K, Li, Mg, Mn
and Sr. Extremely high concentrations of As, Cd, Pb, Zn were found in MS and MS/BR
substrates and in the case of As it was ~1780 and 370 fold higher compared to the control
for MS and FT, respectively. This metalloid was present mainly in inorganic forms in
MS and MS/BR substrates, and the concentration of the most toxic As(III) in MS reached
1020 mg kg−1and was ~165 fold higher than FT (Table 2).
Considering the particular fraction of elements determined by the BCR procedure, it
was observed that for substrates FT and MS, most of analyzed elements was associated with
the F1 fraction, which is the most soluble and most bioavailable for plants. For example,
the F1 fraction for the MS substrate was associated with ~90%, 74% and 49% of total Cd, Cu
and As, respectively. Biochar application for these substrates influenced metal speciation,
especially for the F1 fraction. Biochar application led to a decrease in the F1 fraction for
most analyzed elements (Table S1).
Table 1. Characteristics of experimental substrates.
Parameter Control FT FT/BR MS MS/BR
Granulometric
composition of soil
sand 2–0.05 % 84 34 39 1 7
silt 0.05–0.02 (%) 14 60 54 89 85
clay < 0.002 (%) 2 6 7 10 8
granulometric fraction
S SiL SiL Si Si
pH H2O 5.39 e±0.34 8.30 b±0.04 8.49 a±0.01 8.19 c±0.04 7.94 d±0.02
EC mS 0.27 d±0.01 0.44 c±0.01 0.51 e±0.04 6.75 a±0.16 6.53 b±0.06
CEC meq/100g 22.6 c±4.01 186 b±6.9 188 ab ±12.5 198 ab ±5.74 201 a±4.28
Base cations
(meq/100g)
Ca2+ 2.60 c±0.03 181 a±6.66 182 a±12.5 142 b±5.13 144 b±3.25
Mg2+ 0.29 c±0.02 4.15 b±0.16 4.26 b±0.05 43.2 a±0.53 43.9 a±1.04
K+0.04 d±0.00 0.82 c±0.01 0.84 c±0.02 3.63 a±0.04 3.42 b±0.04
Na+0.32 d±0.01 0.54 c±0.04 0.74 b±0.03 9.62 a±0.11 9.54 a±0.11
Base cations
saturation
(%)
Ca2+ 11.8 c±2.38 97.0 a±0.02 96.9 a±0.16 71.5 b±0.53 71.6 b±0.52
Mg2+ 1.34 c±0.36 2.23 b±0.01 2.27 b±0.14 21.8 a±0.42 21.9 a±0.42
K+0.20 d±0.04 0.44 c±0.02 0.45 c±0.04 1.83 a±0.03 1.70 b±0.04
Na+1.42 b±0.30 0.29 c±0.01 0.39 c±0.05 4.86 a±0.10 4.76 a±0.15
Total 14.8 b±3.05 100 a±0.00 100 a±0.00 100 a±0.00 100 a±0.00
Bio-P mg P2O5/100 g 37.9 a±1.61 4.26 d±2.11 2.73 d±1.04 19.7 b±1.55 14.4 c±1.78
Bio-K mg K2O/100 g 1.98 c±0.06 20.5 b±0.59 20.7 b±0.33 119 a±2.53 116 a±0.62
ST
%
0.01 c±0.00 0.09 b±0.01 0.10 b±0.00 1.86 a±0.26 1.92 a±0.04
TN 0.05 b±0.01 0.03 bc ±0.01 0.03 c±0.01 0.25 a±0.01 0.26 a±0.01
TC 0.91 c±0.08 5.97 b±1.13 5.82 b±0.22 11.4 a±0.16 11.9 a±0.34
TOC 0.80 d±0.04 1.01 c±0.14 1.41 b±0.53 4.27 a±0.06 4.28 a±0.05
SIC 0.10 d±0.04 4.96 b±1.00 4.41 d±0.37 7.13 a±0.18 7.62 a±0.28
TOC/TN 16.1 c±2.90 30.5 b±1.96 41.9 a±8.96 17.3 c±0.46 16.5 c±3.18
CHA 0.31 a±0.02 0.11 b±0.01 0.14 b±0.01 0.32 a±0.03 0.30 a±0.05
CFA 0.20 a±0.07 0.04 bc ±0.01 0.04 c±0.00 0.05 b±0.01 0.04 c±0.00
CHA/CFA 1.72 c±0.57 2.82 c±0.53 3.94 b±1.66 6.54 a±1.86 7.50 a±1.25
CHumins 0.29 d±0.09 0.79 c±0.04 1.75 b±0.05 3.90 a±0.18 4.14 a±1.06
Mean values (n= 3)
±
standard deviations. Identical superscripts denote no significant (p> 0.05) differences according to a post-hoc
Tukey’s HDS test; EC—electrical conductivity, S—sand, LS—loamy sand, SL—sandy loam, SiL—silt loam, Si—silt; Bio-P—bioavailable
phosphorus, Bio-K—bioavailable potassium, ST—total sulfur, TN—total nitrogen, CEC—cation-exchange capacity, TC—total carbon,
TOC—total organic carbon, SOC—soil organic carbon, SIC—soil inorganic carbon, C
HA
—carbon of humic acids, C
FA
—carbon of fulvic
acids, CHumins—carbon of humins.

Plants 2021,10, 2049 9 of 25
Table 2. Concentration of major and trace elements (mg kg −1DW) in substrates used in the experiment (n= 3).
Element(s) Control FT FT/BR MS MS/BR
Ca 1240 c26,900 a26,100 a23,300 b22,700 c
K 279 c7170 a6840 a3160 b2950 b
Mg 188 c4190 a4020 a3210 b3220 b
Na 118 c383 b365 b566 a476 a
P 438 a350 b346 b439 a405 a
Al 2217 c18,100 a17,700 a9220 b9130 b
As 6.46 c31.1 b25.5 b11,500 a11,100 a
As(III) bDL 6.18 b5.82 b1020 a987 a
As(V) bDL 4.98 b4.52 b10,290 a9970 a
DMA bDL bDL bDL 42.4 a8.06 b
Asorg bDL 19.9 b15.2 b150 a135 a
B 1.60 c4.48 b3.00 bc 117 a110 a
Ba 92.0 c564 b543 b4750 a4600 a
Be 1.29 c1.84 a1.65 b1.47 bc 1.31 c
Bi 1.67 b1.47 b1.41 b5.62 a5.38 a
Cd 1.31 b1.54 b1.35 b1730 a1670 a
Co 1.30 c8.65 b8.23 b102 a95.9 a
Cr 4.26 c21.5 b21.4 b619 a616 a
Cs 157 c933 a901 a586 b528 b
Cu 3.18 c5009 b4780 b7870 a7710 a
Fe 2520 c7550 b7290 b27,900 a27,300 a
Hf 1.35 a1.51 a1.49 a2.00 a1.92 a
Hg 1.54 b1.90 b1.64 b70.9 a67.3 a
In 2.98 a4.05 a3.16 a5.95 a5.26 a
Li 2.50 c24.6 a23.9 a19.6 b17.8 b
Mn 116 d970 a873 ab 731 bc 697 c
Mo 1.89 c6.61 b3.75 bc 19.4 a18.2 a
Ni 2.23 c14.1 b12.4 b572 a496 a
Pb 17.1 c56.8 b53.4 b1666 a1646 a
Rb 3.23 c42.4 a37.8 a18.2 b18.1 b
Re 1.48 a1.75 a1.56 a2.02 a1.64 a
Sb 4.37 b4.85 b3.88 b229 a207 a
Se 20.3 c25.6 b22.5 b179 a171 a
Si 348 c819 ab 743 b850 a723 b
Sn 2.08 c31.7 b22.5 b179 a171 a
Sr 4.74 c361 a340 a193 b152 b
Ta 1.31 b1.59 b1.49 b4.06 a3.83 a
Te 5.15 c9.15 b6.71 bc 21.4 a19.9 a
Th 3.48 c12.7 b12.2 b31.0 a32.6 a
Ti 239 c350 a316 b334 a322 ab
Tl 3.27 b4.16 b4.00 b191 a182 a
V 7.37 c25.5 b23.7 b123 a122 a
W3.00 b3.58 b3.15 b1230 a1220 a
Zn 14.2 c53.7 b52.9 b12,500 a12,300 a
Zr 4.08 c8.66 b7.09 b65.7 a63.3 a
NE 49.8 c114 b76.2 bc 808 a535 b
REEs 32.5 c82.9 a72.1 ab 76.4 a55.8 b
Identical superscripts denote no significant (p> 0.05) differences between elements content in particular substrates (within a rows) according
to a post-hoc Tukey’s HDS test; bDL—below detection limit.
3.2. Paulownia Hybrid Growth
The highest biomass in the first and the second year of the experiment was noted for
control seedlings (57.0 and 46.2 g, respectively) (Figure 1). For FT, FT/BR and MS/BR

Plants 2021,10, 2049 10 of 25
substrates, significant inhibition of overall plant growth was observed with a mean biomass
yield of 24.7, 25.2 and 26.5 g for FT, FT/BR and MS/BR in the first year and 24.1 and 22.9 g
for FT and FT/BR in the second year. Seedlings cultivated in MS were unable to survive
during the first year (Figure 1A) and also in MC/BR in the second year (Figure 1B) of the
experiment. The biomass of particular organs of seedlings cultivated in waste materials
(all variants) was similar and significantly lower compared to the control.
Figure 1.
Characteristics of fresh biomass (g FW) of Paulownia growing under particular experimental
systems in the first (
A
) and the second (
B
) year of investigation (identical lettering denote no
significant (p> 0.05) differences according to a post-hoc Tukey’s HDS test).
In the first year, plant height reached 34.4 cm for the control and 25.9, 25.4 and
23.6 cm
for the FT, FT/BR and MS/BR systems, respectively (Figure 2A). In the second year of
cultivation, a significant difference in the biomass of the control and FT/BR seedlings
was observed (41.2 and 26.6 cm, respectively) (Figure 2B). The surface of main and side
stem leaves also reflected the inhibitory effect of the applied substrates (Figure 2A,B).
Additionally, leaf/stem ratios both for main and side stems were significantly higher for
the control and FT/BR than for FT and MS/BR plants in the first year of the experiment.
Moreover, for these parameters, no significant differences were noted between the control,
FT and FT/BR systems in the second year.

Plants 2021,10, 2049 11 of 25
Figure 2.
Characteristics of main stem height [cm], main and side stem leaves surface (cm
2
) and
leaves/stem ratio of Paulownia cultivated in particular experimental systems in the first (
A
) and
the second (
B
) year of investigation (identical lettering denote no significant (p> 0.05) differences
according to a post-hoc Tukey’s HDS test).
In the first year of cultivation, plants suffered no visible leaf damage resulting from
applied waste materials. In contrast, in the second year, leaf necrosis and chlorosis, along
with root thinning, was observed for plants cultivated in FT/BR (Figure 3).

Plants 2021,10, 2049 12 of 25
Figure 3. Leaves and roots of Paulownia after second year of the experiment.
3.3. Element Uptake and Distribution in Paulownia Plants
Depending on the variant, a specific distribution of individual elements was found
(Tables S2 and S3). After the first year of the experiment, elements were accumulated to
a different extent in roots and shoots. At the same time, Ag, B, Ba, Bi, Ca, Co, Re and Ta
were found in the aboveground organs exclusively. Plants cultivated in FT accumulated
the majority of elements in roots or leaves, while their content in stems was significantly
lower with the exception of Hg, Sn, Ta and Zn. The addition of BR altered the distribution
of numerous elements in favor of their transport to the stem. The uptake of the majority
of elements in plants cultivated in MS/BR was generally limited to the root system with
effective transportation to the leaves observed only for particular elements (Ba, Bi, Hg, Mn,
Si, Sn, Th, Tl, W and Zn).
In the second year of studies, both control plants and plants cultivated in flotation
tailings (FT and FT/BR) were characterized by effective uptake to roots observed for the
majority of elements, and to a different extent, to leaves. Effective accumulation in the stem
was limited to selected elements, i.e., As, Bi, Cd, Co, Cs, Hg, Rb, Sn, Ta and W.
BCF and TF values higher than 1 were calculated for B, Ca, K, P, Rb, Re and Ta
(
Figure 4
). Effective uptake of NE, Hf, Mo, Na, Pb, Sr, Te, W and Zn was noted, and
translocation for Ba, Bi, Cs, Hf, Hg, In, Mn, Mo, P, Rb, Sn, Sr, Te, W and Zn. Uptake and
translocation of REEs (sum), Al, Be, and V were greatly limited compared to other elements.

Plants 2021,10, 2049 13 of 25
Figure 4.
Bioconcentration factor (BCF) and translocation factor (TF) values calculated for determined
elements in Paulownia cultivated in particular experimental systems.
Considering metal accumulation per plant, the highest content of the majority of
elements was determined for MS/BR (2018), and in the case of B, Ca, Cu, In and Si, for
the control (2018 and 2019). Further, despite significant differences in Be and Bi content
in substrates, their accumulation was strongly inhibited and found to be at a similar level
(Table 3).
In the second year of the study, the similarities and differences between the control
plants and FT and FT/BR were less visible. Content of REEs and also Al, As, B, Cu, K, Mn,
Na, Ni, Sb, Se, Tl, V, Zn and Zr were similar in the control and seedlings growing under
FT. It was also observed that BR addition to FT substrate modified metal concentration on
plant biomass. Compared to the FT substrate plants growing on the FT/BR substrate had a
significantly lower concentration per plant of most of the analyzed elements. This trend
was observed in both 2018 and 2019.

Plants 2021,10, 2049 14 of 25
Table 3.
Content of major and trace elements (mg per plant DW) in Paulownia plants growing in particular experimental
systems (n= 3).
Element(s) 2018 2019
Control FT FT/BR MS/BR Control FT FT/BR
Ca 652 a412 b320 b610 a585 a442 b270 c
K241 b245 b209 c283 a272 a264 a172 b
Mg 66.6 b43.3 c21.0 d92.2 a104 a49.6 b22.1 c
Na 38.6 b39.2 b40.9 b54.7 a55.5 a59.8 a43.3 a
P 131 a7.10 b7.04 b12.3 b97.6 a12.3 b7.06 b
Al 18.9 ab 17.3 ab 10.8 b58.8 a21.7 a21.3 a13.3 b
As 0.08 b0.10 b0.05 b1.42 a0.12 a0.13 a0.06 b
B 1.40 a0.47 b0.35 c1.85 a0.72 a0.46 b0.28 c
Ba 0.62 d0.96 b0.79 c1.30 a0.78 b1.21 a0.73 b
Be <0.0 a<0.0 a<0.0 a<0.0 a<0.0 a<0.0 a<0.01 a
Bi 0.01 a0.01 a0.01 a0.01 a0.01 a0.02 a0.01 a
Cd <0.0 c0.01 b<0.01 c1.03 a0.01 b0.01 a<0.01 b
Co 0.02 c0.05 b0.03 c0.12 a0.04 b0.08 a0.04 b
Cr 0.06 b0.04 c0.04 c0.11 a0.09 a0.06 b0.04 b
Cs 2.72 b1.60 c1.26 d3.18 a3.03 a2.01 b1.24 c
Cu 5.48 ab 4.14 bc 3.43 c7.04 a7.60 a6.62 a3.54 b
Fe 94.2 a23.0 c22.7 c71.0 b94.1 a27.8 b24.1 b
Hf 0.01 c0.02 b0.02 b0.03 a0.02 c0.03 a0.02 a
Hg 0.03 b0.01 c0.01 c0.03 a0.04 a0.02 b0.01 b
In 0.07 a<0.01 b<0.01 b0.06 a0.04 a<0.01 b<0.01 b
Li 0.03 b0.01 c0.01 c0.10 a0.06 a0.02 b0.01 b
Mn 1.42 b1.37 b1.04 b4.83 a1.86 a1.65 a1.12 b
Mo 0.06 c0.20 ab 0.12 b0.23 a0.11 c0.26 a0.16 b
Ni 0.11 b0.13 b0.13 b0.32 a0.17 ab 0.20 a0.14 b
Pb 0.46 d3.37 b2.02 c9.36 a0.79 c4.59 a2.17 b
Rb 0.31 c0.49 b0.28 c0.78 a0.50 b0.61 a0.30 c
Re 0.06 b0.07 b0.07 b0.22 a0.06 b0.11 a0.05 b
Sb 0.02 b<0.01 c0.01 c0.05 a0.04 a0.03 a0.01 b
Se <0.01 b<0.01 b<0.01 b0.02 a<0.01 a<0.01 a<0.01 a
Si 7.12 a1.95 b2.16 b7.86 a9.71 a5.32 b2.10 c
Sn <0.01 c0.06 ab 0.04 b0.09 a<0.01 c0.08 a0.04 b
Sr 2.05 c2.74 b1.90 c5.78 a2.83 a3.16 a1.72 b
Ta 0.16 b0.08 c0.02 c0.18 a0.15 a0.12 b0.05 c
Te 0.11 c0.18 b0.14 bc 0.26 a0.14 b0.28 a0.16 b
Th <0.01 c0.03 b0.03 b0.10 a0.05 b0.06 a0.03 c
Ti 0.11 b0.61 b0.55 b1.39 a0.15 c0.94 a0.57 b
Tl <0.01 b<0.01 b<0.01 b0.67 a<0.01 a<0.01 a<0.01 a
V0.03 bc 0.04 b0.01 c0.07 a0.06 a0.05 a0.02 b
W0.22 b0.13 c0.06 d0.31 a0.30 a0.21 b0.07 c
Zn 1.05 bc 1.28 b0.89 c5.04 a1.44 a1.47 a0.93 a
Zr 0.03 b0.02 c0.01 c0.05 a0.05 a0.04 a0.02 a
NE 1.53 c3.61 b3.48 b12.0 a1.85 b4.53 a3.72 a
REEs 0.14 b0.10 bc 0.05 c0.38 a0.15 a0.13 a0.06 b
Identical superscripts denote no significant (p> 0.05) differences between elements content in whole plant biomass (within a row, separately
for particular years of samples collection) according to a post-hoc Tukey’s HDS test.
3.4. Quantitative Analysis of the Extraction Ability of Paulownia
Estimation of the uptake of particular elements using BCF or their transport from
roots to shoots using TF does not explain the extraction ability of the studied Paulownia
hybrid with respect to polluted substrates enriched or not with biochar. For this reason,
MER values were calculated to show the % of elements removed from the control and

Plants 2021,10, 2049 15 of 25
especially polluted substrate during one harvest. Values higher than 0.001% and in at least
one experimental system are shown in Table 4.
Table 4.
Metal extraction ratio (MER) values (%) for major and trace elements determined in Paulownia plants growing
under particular experimental systems ("-“ means MER value below 0.001).
Element(s) 2018 2019
Control FT FT/BR MS Control FT FT/BR
Ca 0.332 0.021 0.164 0.038 0.355 0.017 0.122
K 0.423 0.044 0.041 0.074 0.479 0.036 0.029
Mg 0.107 0.004 0.003 0.007 0.155 0.005 0.003
Na 0.034 0.022 0.010 0.026 0.062 0.049 0.009
P 0.213 0.028 0.014 0.029 0.486 0.032 0.015
Al 0.001 - - 0.001 0.002 - -
As - 0.002 0.001 - 0.002 0.002 0.009
B 0.661 0.185 0.202 0.031 0.403 0.134 0.131
Ba 0.004 0.002 0.002 - 0.005 0.002 0.002
Bi 0.004 0.011 0.014 0.005 0.005 0.019 0.013
Cd 0.001 0.005 0.003 - 0.001 0.006 0.003
Co 0.012 0.002 0.001 - 0.011 0.002 0.001
Cr 0.005 0.001 - - 0.005 0.001 -
Cs 0.010 0.001 0.001 0.002 0.011 0.001 0.001
Cu 0.185 - - - 0.389 - -
Fe 0.019 0.001 0.001 - 0.018 0.001 -
Hf - 0.016 0.012 0.003 - 0.016 0.012
Hg 0.006 0.003 0.003 - 0.007 0.005 0.003
In 0.018 - - 0.004 0.014 - -
Li 0.001 - - 0.002 0.002 - -
Mn 0.005 0.001 0.001 0.006 0.005 0.001 0.001
Mo 0.004 0.013 0.026 0.013 0.004 0.016 0.021
Ni 0.010 0.002 0.001 - 0.012 0.002 0.001
Pb 0.007 0.025 0.018 0.001 0.012 0.028 0.018
Rb 0.025 0.010 0.008 0.035 0.056 0.011 0.008
Re 0.032 0.083 0.093 0.235 0.046 0.096 0.063
Si 0.007 0.002 0.001 0.006 0.007 0.003 0.001
Sn - 0.004 0.004 0.001 0.000 0.004 0.003
Sr 0.230 0.009 0.007 0.041 0.320 0.009 0.006
Ta 0.097 0.094 0.011 0.046 0.126 0.093 0.045
Te 0.012 0.014 0.009 0.014 0.015 0.018 0.010
Th 0.012 0.001 0.002 0.003 0.016 0.003 0.002
W 0.033 0.038 0.017 - 0.051 0.045 0.023
Zn 0.025 0.024 0.015 - 0.038 0.023 0.016
Zr 0.002 0.001 0.001 - 0.002 0.001 0.001
NE 0.012 0.007 0.009 0.001 0.014 0.006 0.007
REEs 0.001 0.001 - 0.001 0.001 - -
PEN calculations for individual elements showed that for the extraction of 1.0 g of a
particular metal from the substrate a different number of plants was needed depending
on the experimental system. The lowest PEN values were calculated for Ca, K, Mg, Na, P
and Fe, which is a common observation due to the role of these metals in plant growth and
development (Table 5).

Plants 2021,10, 2049 16 of 25
Table 5.
Plant Effective Number (PEN) for major and trace elements determined in Paulownia plants growing under
particular experimental systems ("-“ means PEN value ≥107).
Element(s) 2018 2019
Control FT FT/BR MS/BR Control FT FT/BR
Ca1121123
K3233435
Mg 15 25 60 14 19 37 95
Na 76 33 184 30 74 43 305
P 6 64 115 52 7 71 190
Al 93 179 301 107 162 240 397
As 180,883 11,782 17,260 1038 41,915 11,700 25,006
B 397 985 1467 244 868 1294 2416
Ba 1067 503 721 395 1129 612 1173
Be - 1155,108 1831,804 1175,157 - 1023,751 2986,858
Bi 67,095 51,587 31,304 22,804 59,872 28,783 52,709
Cd 454,868 81,371 178,572 5798 343,407 85,800 258,478
Co 29,346 35,975 82,298 32,583 37,125 46,510 126,502
Cr 21,346 46,651 132,368 17,746 25,890 51,509 159,535
Cs 312 558 945 492 304 762 1487
Cu 522 271 310 194 435 369 557
Fe 12 100 177 69 12 141 286
Hf - 25,375 38,291 84,883 - 31,722 54,749
Hg 47,368 72,490 137,496 20,261 48,563 81,975 199,864
In 11,248 - - 34,008 13,581 - -
Li 147,995 87,681 148,266 20,714 116,128 96,560 211,251
Mn 874 742 1291 138 950 1044 2212
Mo 78,306 7309 7876 2938 67,619 7295 11,895
Ni 21,244 22,982 46,584 5283 20,741 27806 72,263
Pb 3252 351 591 607 2730 504 1051
Rb 4515 1325 1945 899 2988 1756 3403
Re 9345 5271 5818 1760 8067 4649 9661
Si 256 408 737 133 217 369 1310
Sn - 5271 8413 3693 6188 13,475
Sr 427 214 296 87 361 255 494
Ta 3539 3856 22,079 3148 3329 5347 14,337
Te 8114 4526 11,069 1843 7374 4767 13,965
Th 14,195 38,199 35,536 8784 10,207 22,912 49,506
Ti 10,980 11,834 16,860 2524 13,815 13,000 25,164
Tl - - - 1853 - - -
V 154,655 257,937 469,305 290,561 177,406 173,919 607,497
W 4152 3971 9218 2763 3596 4904 13,821
Zn 1279 434 709 220 1023 642 1146
Zr 62,235 64,720 75,715 32,245 55,774 60,544 118,032
NE 747 843 1154 698 791 1096 1844
REEs 13,095 17,124 26,265 6016 16,188 23,639 44,323
The highest PEN values were observed for Be, Li and V. Promising values regarding
practical aspects were noted for NE, Al, B, Ba, Cs, Cu, Mn, Pb, Si and Sr remediation,
where several dozen to several hundred plants are needed to extract 1.0 g of the mentioned
elements.
3.5. Pigment Content in Paulownia Leaves
Substrate modification did not cause any significant changes in chlorophyll content
(Table S4). Simultaneously, increased chlorophyll b was observed for plants cultivated in
mining sludge with the addition of biochar (MS/BR) compared to the control (~20%) and
biochar supplemented flotation tailings (FT/BR) (~44%), and relatively lowered for FT/BR
in comparison with pure FT (~20%). Carotenoid content increased for plants cultivated in

Plants 2021,10, 2049 17 of 25
waste materials, reaching the highest value for MS/BR (~180% compared to the control).
Subsequently, a significant drop of the chlorophyll-to-carotenoid ratio versus control was
noted, most pronounced for biochar enriched substrates (~32 and 47% for FT/BR and
MS/BR, respectively).
3.6. Phenolic Metabolites in Paulownia Leaves and Roots
Substrate modification led to a significant decrease of the phenolic fraction in Paulownia
leaves, clearly marked for plants cultivated in FT and MS/BR (by ~43 and 29%, respectively)
(Table S4). Further, the addition of BR to FT resulted in a significant increase of total phenolic
content compared to pure FT, although the value remained lower than in the leaves of the
control plants. Relative changes in phenolic accumulation in leaves were closely reflected
by total antioxidant capacity versus the DPPH radical (Table S4).
In the leaves and roots of control plants C6-C1 (hydroxybenzoic acids), C6-C3 (hydrox-
ycinnamic acids/phenylpropanoids) and C6-C3-C6 (flavonoids) were assessed (Figure 5,
Table S4).
Figure 5.
Percentage change of biochemical and physiological parameters of leaves (
A
) and roots (
B
,
C
) of Paulownia plants
cultivated in waste materials (FT—flotation tailings, FT/BR—flotation tailings supplemented with biochar, MS/BR—mining
sludge supplemented with biochar) in relation to control plants. For undetected compounds, the detection limit was
used for calculations as a minimal detectable amount. Only significant differences were present at
α
= 95% (2,5-DHBA—
2,5-dihydroxybenzoic acid, 4-HBA—4-hydroxybenzoic acid, C6-C1—hydroxybenzoic acids, C6-C3—phenylopropanoids,
C6-C3-C6—flavonoids, sum—a sum of low-molecular-weight organic acids (LMWOAs)).
In leaves, the dominant group was C6-C1 structures represented by
2,5-DHBA > 3,4-DHBA (protocatechuic) > 4-HBA > gallic acid. Other hydroxybenzoic
acids (vanillic, syringic and salicylic) were present in smaller amounts. The sum of C6-C1
was ~3-fold higher than the C6-C3 and C6-C3-C6 phenolics, and among phenylpropanoids,
ferulic acid was a dominant compound. Other hydroxycinnamic acids were detected in
lower amounts (trans-cinnamic, caffeic, p-coumaric, chlorogenic, ferulic, sinapic acids).
Among flavonoids, rutin, quercetin and catechin were detected at relatively lower levels.
The use of waste materials significantly affected the metabolic profile in leaves, and a
strong inhibition of phenolics accumulation was clearly noted (Figure 5). Among the C6-C1
compounds, 2,5-DHBA, salicylic and syringic acids were detected in significantly lower
amounts, while gallic, protocatechuic and 4-HBA were not detected in the case of the FT
substrate. The C6-C3 acids were accumulated in significantly lower amounts, while trans-
cinnamic acid was not detected, and only rutin was detected among flavonoids. For the
FT/BR substrate, an overall suppression of C6-C1 acids, caffeic, sinapic acids and catechin
was observed. The strongest inhibition of phenolic accumulation was noted for MS/BR,
with the exception of salicylic acid, for which a ~30% increase was observed in comparison
with the control. Considering C6-C3 structures, only a few acids were quantified at lower
levels, i.e., caffeic, chlorogenic and sinapic acids.
In roots, the dominant group was also C6-C1, with gallic and vanillic acids as the
main compounds, while quercetin was a major flavonoid (Table S4). Similarly to leaves,

Plants 2021,10, 2049 18 of 25
the addition of waste material led to quantitative changes in the phenolic profile. For FT
substrate, an increase in 2,5-DHBA, 4-HBA, caffeic, p-coumaric and sinapic acids, rutin
and catechin content was observed, while the synthesis of other compounds was inhibited
compared to the control (Figure 5). In contrast, the FT/BR substrate decreased the content
of phenolic compounds in comparison to the control and 2,5-DHBA and trans-cinnamic
acid were not detected. Also, for the MC/BR group a suppressed synthesis of phenolics
was observed, and 2,5-DHBA and caffeic acid were not present. Contrary to leaf tissue, the
relative changes in C6-C1, C6-C3, C6-C3-C6 content in roots shared a similar pattern with
the exception of 2,5-DHBA, caffeic and p-coumaric acids (Figure 5).
3.7. LMWOAs in Paulownia Roots
Considering all experimental groups, eight LMWOAs were detected in the roots
of Paulownia (Table S4). Roots of the control plants contained seven from eight of the
determined acids, and malic and acetic acids were dominant. The substrate modification
significantly affected the profile of organic acids (Figure 5). For FT substrate, malic and
succinic acids were dominant and a significant increase of succinic acids relative to the
control was noted (~270%). In the roots of FT/BR plants, a significant increase of malic
acid was observed compared to the control and FT plants (~560 and 470%, respectively).
In contrast, the accumulation of oxalic, fumaric and succinic acids was significantly
decreased compared to control and FT-cultivated plants (Figure 5). Further, citric acid was
detected only in the roots of plants cultivated in FT and FT/BR substrates at comparable
levels (Table S4). In the case of MS/BR substrate, malic and acetic acid were dominant, and
the sum of LMWOAs was assessed at a comparable level to the control plants.
4. Discussion
4.1. Biomass Yield and Phytoextraction Efficiency of Paulownia Hybrid
Generally, Paulownia, especially P. tomentosa prefers high acidity, although the best
growth is obtained at strongly acidic to mildly alkaline pH [
37
,
38
]. In general, the bioavail-
ability of elements is higher under acidic soil conditions and decreases with increasing
pH [
39
]. Under alkaline conditions, the mobility of some elements decreases, mainly due to
the ability of Ca compounds to absorb them [
40
]. In the present experiment, the control soil
reaction was acidic. At the same time, applied waste materials showed an alkaline reaction
with a pH range from 7.94 to 8.49 for MS/BR and FT/BR, respectively. The efficiency of
phytoextraction depends on many factors, among which the properties of the substrate
play an important role. The worst growth conditions for plants were determined for the MS
and MS/BR substrates, which resulted not only from the extreme content of the analyzed
elements, but also from their poor physicochemical parameters (Tables 1and 2). The
physical properties of these substrates, mainly determined by granulometric composition,
can fundamentally affect the availability of both nutrients and toxic elements [
41
]. Wa-
ter retention and water availability for plants also depend on the physical properties of
substrates which can directly determine the bioavailability of elements. In normal soil
conditions, fine particles usually show a higher water retention capacity, but in the case
of the substrates used in the experiment, especially the MS substrate, the predominance
of fine sized particles cause disturbed water infiltration, limited by the poor structural
properties of this kind of waste [
42
]. The predominance of fine fractions in these materials,
and especially in MS, leads to an increase in their density, which inhibits penetration by
plant roots, significantly hindering their proper growth and the ability to take up elements.
Moreover, excessive compaction of these wastes can also result in a disturbance of air
conditions, which in turn cause significant obstruction or inhibition of growth. In poor
ventilation conditions, plants may show a deficiency of nutrients, even with their high
concentration in the substrate. Additionally, more toxic elements like As, Pb and Cd are
strongly associated with fine fractions, which can also influence their bioavailability [
43
].
In the case of this type of waste, the application of porous materials, such as biochar, may
contribute to a significant loosening of their structure and an improvement of air-water

Plants 2021,10, 2049 19 of 25
properties, which directly affect the efficiency of phytoextraction processes. The use of
biochar due to the small specific surface area (50.1 m
2
g
−1
) aimed to increase the chances
of plants adapting to new, unfavorable growth conditions. Adsorption on biochar particles
because of the alkaline pH (10.0) was a factor limiting the rapid increase in the concentration
of toxic elements in the rhizosphere and ultimately facilitating the growth of seedlings. The
use of biochar with respect to active carbons seems to be more justified because it is not
about highly effective sorption but limiting the sudden increase in the concentration of
metals negatively affecting plant growth, especially at the beginning of the phytoextraction
process.
However, in our study, biochar did not significantly affect the investigated physic-
ochemical properties, disregarding the bioavailable forms of selected elements in the F1
fraction, which decreased in FT/BR and MS/BR substrates compared to pure waste materi-
als. The reduction of the F1 fraction of the analyzed elements resulted in a decrease in the
content of the analyzed elements in the Paulownia biomass, which was clearly observed
when comparing the FT and FT /BR substrate both in 2018 and 2019. Most of the analyzed
elements were significantly lower in the biomass of plants growing on FT / BR substrate
compared to the FT substrate, which clearly indicates the influence of biochar on the reduc-
tion of the bioavailability of elements. This fact may suggest that even a small addition of
biochar with a small specific surface may significantly affect the behavior of Paulownia.
The high efficiency of metal phytoextraction by Paulownia proved its high resistance
to elevated concentrations of toxic elements in cultivation substrates such as mining waste
materials. However, a drop in temperature below 0
◦
C for several days in January 2019
could have caused an aggravation of substrate-derived stress and consequently led to the
death of plants cultivated in the most polluted MS/BR. Moreover, seedlings exposed to MS
were unable to grow, and a possible cause of their withering could be an extremely high
concentration of DMA (42.4 mg kg
−1
) which has previously proved highly toxic to woody
plants [
44
,
45
]. The breakpoint of DMA concentration in modified Knop solution described
in the above mentioned papers was 41.4 mg kg
−1
, while higher concentrations resulted
in damage to young tree seedlings. This could explain why seedlings exposed to MS/BR
with a DMA concentration of 8.06 mg kg−1were able to grow.
Due to the effect of the chemical composition of the substrates, it is difficult to clearly
state to what extent the results obtained in this work rank the studied hybrid in a hierarchy
based on the potential of this species for effective metal phytoextraction. Oxytree could
be an effective and selective accumulator of NE, B, Hf, K, Mg, Mo, Na, P, Pb, Rb, Re, Sr,
Ta, Te, W and Zn as confirmed by a BCF > 1. Moreover, TF > 1 calculated for Ba, Be,
Bi, Cd, Cs, Cu, Hf, Hg, In, K, Mn, Mo, P, Rb, Re, Se, Sn, Sr, Ta, Te, Th, Tl, W and Zn
directly demonstrates the significant potential of this hybrid for the remediation of highly
contaminated wastes. Unfortunately, calculated MER values showed that extraction of the
studied elements was limited. All values of MER were lower than 1%, which is typical
for the majority of plants and suggests that treatment of the waste used in the experiment
will require the growth of Paulownia over several hundreds of years [
33
]. On the other
hand, MER values calculated for selected elements (B, Cu, Sr or Ta) in control plants in the
first or the second year of the experiment suggest that this process could be significantly
shortened. It should also be emphasized that the calculations performed relate to one plant.
Therefore, in the case of the practical use of the tested hybrid in the form of a larger number
of individuals, there may be an additional factor shortening the time of the remediation.
This is where the question of the number of plants is justified. For this reason, the PEN
was calculated for individual elements and their groups (NE and REEs). To extract 1 g of
elements such as NE, Al, B, Ba, Cs, Cu, Mn, Pb, Si and Sr from the substrate, the number
of plants (shoots) was lower than 2500, which confirms the potential of studied Paulownia
hybrid to remediate substrate polluted with selected elements only. PEN values calculated
previously for Cd in six Chinese cabbage cultivars were from 3745 to 53,433 [
33
], while for
Paulownia from 5800 (plants growing under MS/BR system) to 455,000 plants (control in the
first year of the study). On the other hand, PEN values for Pb and Zn were 351–3252 and

Plants 2021,10, 2049 20 of 25
220–1279 shoots
, respectively, while in the case of Piptatherum miliaceum (L.) Cosson they
were 22,000 and 4300 shoots, respectively [
36
]. It should be remembered that PEN values
are often calculated for whole plants, also taking into account the root that effectively
accumulates metals. This, of course, significantly reduces the number of plants needed
for effective remediation, although in our opinion, providing such results would be quite
debatable due to the possibility of growth of the studied Paulownia hybrid for many years.
The limited uptake of selected elements may be the effect of the high pH of the
applied substrates, which may be the next factor able to increase MER values. However,
limited translocation and metal accumulation in roots have previously been described for
Paulownia [
14
]. The authors studied two Paulownia lines of different leaf sizes, showing
varying efficiency in Pb and Zn phytoextraction. This suggests that the accumulation
of metals may be closely related to the size of plants. Any change in soil pH strongly
affects metal solubility and its bioavailability for plants [
46
]. In alkaline reaction, element
mobility generally decreases; this is usually associated with an extensive calcium level.
Excessive levels of calcium, especially in MS substrate can also affect nutrient (Mg, P, Mn,
Fe) bioavailability and uptake, which can explain the low content of this element in the
whole biomass of Paulownia compared to the control, especially for the FT and FT/BR
substrates, as well as the significant reduction of biomass. Under normal conditions, Ca
is a dominant component among base cation in the soil sorption complex and plays an
extremely important role, both in maintaining soil fertility and proper plant functioning.
In our study Ca
2+
concentration in the sorption complex of FT and MS substrates was
over 70 and 55 fold higher respectively, compared to the control. Despite the positive role
played by this cation, in such extremely high concentrations, it can disturb the proper
management of other plant nutrients present in the soil solution, such as K
+
and Mg
2+
[
47
].
An important parameter modifying plant growth conditions is the salinity of the substrate
caused by excessive salt concentration, including heavy metal salts. On the MS and MS/BR
substrates, the electrolytic conductivity (EC) was 6.75 and 6.53 mS cm
−1
respectively, which
was much higher in comparison with the other experimental substrates. In such conditions,
plants have a difficult uptake of water from the substrate, and as a result of osmotic stress,
their growth slows down [
48
]. This is additionally enhanced by the predominance of the
fine fraction in these materials.
4.2. Physiological Response
Paulownia, like other plants, has a characteristic physiological response [
49
–
51
]. The
photosynthetic activity and physiological state of Paulownia plants were indirectly mea-
sured by assessing pigment content in leaves [
52
]. However, waste materials did not cause
any negative changes in chlorophyll content despite triggering severe growth retardation.
The increase of carotenoid content without significant changes in the amount of chlorophyll
was manifested in a slightly decreased chlorophyll-to-carotenoid ratio. This may indicate
enhanced antioxidant activity to compensate for the inhibition of phenolic biosynthesis
showing high ROS-scavenging properties. During metal stress, the action of lipophilic
carotenoid is most likely targeted at the stabilization of membrane lipids from hydrophilic
oxidizers [53].
Phenolic compounds are secondary metabolites, playing numerous functions in plant
metabolism, including defense mechanisms against various stressors. They are recog-
nized as biomarkers of plant response in conditions of elevated metal concentration in
soil/substrate [
54
,
55
], and their enhanced biosynthesis under metal stress, along with their
antioxidant action, has been confirmed for many plants and various tissue types [
45
,
55
–
58
].
Surprisingly, in the present study the accumulation of phenolic metabolites was noted
neither in leaves nor roots of Paulownia elongata S.
×
Paulownia fortunei. The obtained results
clearly indicated that TPC and the biosynthesis of individual metabolites were inhibited,
as proved by the decreased total antioxidant activity values. In addition, hydroxybenzoic
acid biosynthesis was more susceptible to inhibition than hydroxybenzoic acids, especially
in leaves. In defense mechanisms under metal stress, phenolic compounds play the role

Plants 2021,10, 2049 21 of 25
of electron donors in the ROS scavenging system. However, even phenolic acids with
high antioxidant potentials such as caffeic, chlorogenic and ferulic were not accumulated
in Paulownia leaves and roots. The oxidative mechanisms resulting from metal stress are
represented by both cinnamic and benzoic phenolic acids by either chelating heavy metals
or removing free radicals [
59
]. However, these mechanisms were not observed in Paulownia
cultivated in waste materials. Considering the phenolic profile, breaks of the metabolic
pathway occurred at different stages depending on the waste material used, which is a
start point for future studies.
Plant stressors also activate other intercellular defense mechanisms in which low-
molecular-weight organic acids are involved. Their role is to maintain the homeostasis of
elements in cells [
60
], but they also participate in main metabolic processes such as photo-
synthesis, respiration and amino acid biosynthesis. However, their most important role is
the transport of cations and the detoxification of toxic elements (metals/metalloids) [
30
,
61
].
Thus, the presence of organic acids is a direct reflection of the physiological state of plants
under stress from the environment, including substrate properties [62].
In the present study, the sum of detected LMWOAs in Paulownia roots was similar
for the control and plants cultivated in FT, which was characterized by a high content
of K, Ca and Mg. However, a significant decrease of oxalic and acetic acids content was
observed, with a simultaneous increase in malic and citric acids. For biochar supplemented
FT, the sum of detected acids was more than two-fold higher, with preservation of the
tendency mentioned above. The observed changes may be related to the substrate charac-
teristics, where the soluble cations, predominantly Ca and Mg, may have been exchanged
with non-exchangeable and buffered soluble K as well as by organic acids produced by
plant roots into the rhizosphere [
63
]. Moreover, plants have developed several means
of metal detoxification, such as separation, chelation, avoidance and exclusion, which
involve LMWOAs via extra- and/or intracellular mechanisms [
64
,
65
]. Many studies are in
accordance with our results and have reported that plants are able to accumulate significant
amounts of heavy metals such as Cd, Cu, Pb and Zn, transfer them to the aerial parts
and store them in vacuoles as complexes with citric and malic ligands [
66
]. Increased
biosynthesis of LMWOAs under the FT/BR system in Paulownia roots could also be related
to the application of highly porous materials, such as biochar, which caused a significant
loosening of substrate structure and improvement of air-water properties, thus directly
affecting the defense mechanisms [
60
]. In the case of MS-cultivated Paulownia plants,
the accumulation of LMWOAs in roots decreased, while the addition of BR to MS again
stimulated their synthesis. The significant decrease in acid content may be related to the
elemental overload in the substrate, especially of Cu, Cd, Zn and Pb, with a concomitant
decrease of plant biomass. However, it is indisputable that increasing soil loosening with
BR again stimulated acid synthesis.
4.3. Paulownia as an Effective but Invasive Remediator
The expansion of invasive (alien) species is a significant ecological problem for a num-
ber of reasons, including the mixing of species ranges and ultimately the homogenization
of the environment. According to the Bern Convention on the Conservation of European
Wildlife and Natural Habitats, it is necessary to protect species and habitats through legisla-
tive and administrative measures [
67
]. For this reason, the practical use of invasive species
such as Paulownia, citing the Opinion of the Committee for Plants of the State Council for
Nature Conservation [68], for remediative purposes seems to be unreasonable.
Paulownia prefers higher temperatures and significant water requirements. Therefore
the first should be a proper selection of clones [
69
]. Clones with a greater resistance to
lower temperatures may be of particular importance in terms of their viability. This should
be considered in view of the progressive migration of invasive species due to climate
change, which may lead to a gradual shift of the current range. This phenomenon may
also be accompanied by the displacement of native species of trees and shrubs [
70
]. The
selection of clones capable of effective phytoextraction of toxic metals, but most of all

Plants 2021,10, 2049 22 of 25
characterized by high resistance and currently considered invasive species, requires the
implementation of specific procedures to limit their negative impact on the native fauna.
These climatic changes may, in the future, limit the regrowth of native and valued species
and promote those invasive species that are more responsive to growth in arid, polluted or
dry areas [71,72].
5. Conclusions
Paulownia showed a varying ability to uptake and translocate individual metals,
depending on the substrate on which it grew. The reduction of biomass compared to
the control plants was an obvious consequence of the presence of waste in the substrate.
On the other hand, the addition of biochar to highly polluted mining sludge suggests its
important role in increasing the adaptation of this plant to unfavorable growth conditions.
The obtained results suggest that growth on wastes with moderate concentrations of
metals could limit the physiological response of Paulownia and thus facilitate its growth in
subsequent years of the phytoextraction process.
The present studies clearly demonstrated that LMWOAs biosynthesis in plant roots
plays an important role in adaptation, tolerance and detoxification mechanisms under
conditions of excessive concentrations of toxic elements. Therefore, acid profiling may
be a key tool to elucidate the nature of plant acclimation and may ultimately aid the
development of new plant varieties that are tolerant to environmental changes. Thus,
plant metabolomics should be widely employed to evaluate stress biology by identifying
different compounds produced in response to environmental stressors and the role these
compounds play in acclimation or tolerance responses. Phenolic compounds play an
important role in response to metal stress in plants, which occurs as an increase in their
overall content or enhanced biosynthesis of particular metabolites. However, the observed
depletion in phenolic biosynthesis in Paulownia plants cultivated in heavily polluted waste
materials does not illustrate their protective role in defense and adaptation mechanisms
and this phenomenon requires further research.
Supplementary Materials:
The following are available online at https://www.mdpi.com/article/10
.3390/plants10102049/s1, Table S1: Concentration of major and trace element(s) [mg kg
−1
DW] in
particular fractions of used substrates, Table S2: Content of major and trace elements [
mg kg−1DW
]
in root, stem and leaves of Paulownia growing under particular experimental systems after the first
year of experiment, Table S3: Content of major and trace elements (mg kg
−1
DW) in root, stem
and leaves of Paulownia growing under particular experimental systems after the second year of
experiment, Table S4: Biochemical and physiological parameters of plants cultivated on soil (control)
and waste materials (FT—flotation tailings, FT/BR—flotation tailings supplemented with biochar,
MS/BR—mining sludge supplemented with biochar).
Author Contributions:
Conceptualization, K.D., S.B. and M.M.; Data Curation, K.D. and A.B.; Formal
Analysis, K.D., M.G. and Z.M.; Funding Acquisition, E.R. and M.G.; Investigation, K.D., M.G., Z.M.,
S.B., M.S., P.N. and B.D.; Methodology, P.N.; Resources, P.N.; Supervision, K.D. and M.M.; Validation,
P.N.; Visualization, K.D., A.B. and M.M.; Writing—Original Draft, K.D., M.G., Z.M., M.S., E.R., B.D.,
M.G. and M.M.; Writing—Review and Editing, K.D., M.S., M.G. and M.M. All authors have read and
agreed to the published version of the manuscript.
Funding:
This research was funded by the National Science Centre of Poland under grant code Opus
2014/15/B/NZ9/02172 for Piotr Goli´nski.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

Plants 2021,10, 2049 23 of 25
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