plants

Article
Assisted Phytostabilization of Mine-Tailings with
Prosopis laevigata (Fabaceae) and Biochar
Juan Ramírez-Zamora 1 , Patricia Mussali-Galante 2, *, Alexis Rodríguez 2 , María Luisa Castrejón-Godínez 3 ,
Leticia Valencia-Cuevas 3 and Efraín Tovar-Sánchez 4, *

1 Doctorado en Ciencias Naturales, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001,
Col. Chamilpa, Cuernavaca CP 62209, Mexico
2 Laboratorio de Investigaciones Ambientales, Centro de Investigación en Biotecnología, Universidad
Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, Cuernavaca CP 62209, Mexico
3 Facultad de Ciencias Biológicas, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001,
Col. Chamilpa, Cuernavaca CP 62209, Mexico
4 Centro de Investigación en Biodiversidad y Conservación, Universidad Autónoma del Estado de Morelos,
Av. Universidad 1001, Col. Chamilpa, Cuernavaca CP 62209, Mexico
* Correspondence: [email protected] (P.M.-G.); [email protected] (E.T.-S.);
Tel.: +52-(777)-3297-000 (P.M.-G.); +52-(777)-3297-019 (ext. 3277) (E.T.-S.)

Abstract: Phytoremediation is a cost-effective technique to remediate heavy metal (HM) polluted

sites. However, the toxic effects of HM can limit plant establishment and development, reducing
phytoremediation effectiveness. Therefore, the addition of organic amendments to mine wastes, such
as biochar, improves the establishment of plants and reduces the bioavailability of toxic HM and
its subsequent absorption by plants. Prosopis laevigata can establish naturally in mine tailings and
accumulate different HM; however, these individuals show morphological and genetic damage. In
this study, the effect of biochar on HM bioaccumulation in roots and aerial tissues, HM translocation,
Citation: Ramírez-Zamora, J.;
morphological characters and plant growth were evaluated, after three and six months of exposure.
Mussali-Galante, P.; Rodríguez, A.;
Plants grown on mine tailings with biochar presented significantly higher values for most of the
Castrejón-Godínez, M.L.;
evaluated characters, in respect to plants that grew on mine tailing substrate. Biochar addition
Valencia-Cuevas, L.; Tovar-Sánchez,
E. Assisted Phytostabilization of
reduced the bioaccumulation and translocation of Cu, Pb, and Cd, while it favored the translocation
Mine-Tailings with Prosopis laevigata of essential metals such as Fe and Mn. The addition of biochar from agro-industrial residues to mine
(Fabaceae) and Biochar. Plants 2022, tailings improves the establishment of plants with potential to phytoextract and phytostabilize metals
11, 3441. https://doi.org/10.3390/ from polluted soils. Using biochar and heavy metal accumulating plants constitutes an assisted
plants11243441 phytostabilization strategy with great potential for HM polluted sites such as Cd and Pb.

Academic Editor: Jaco Vangronsveld

Keywords: biochar; phytostabilization; mine tailings; heavy metals; Prosopis laevigata
Received: 24 September 2022
Accepted: 7 December 2022
Published: 9 December 2022

Publisher’s Note: MDPI stays neutral

1. Introduction
with regard to jurisdictional claims in Worldwide, mining is among the most important productive activities which pro-
published maps and institutional affil- vides strategic factors for economic development and wealth generation [1,2]. Mexico is
iations. among the main global producers of 17 different minerals and has been the first in silver
production for more than 12 consecutive years. However, despite its economic and social
benefits, mining is recognized as one of the most environmentally disturbing activities,
due to the great amount of hazardous waste that it generates, mainly in the processes
Copyright: © 2022 by the authors.
related to the extraction of valuable ores from non-economic value materials [3,4]. Mining
Licensee MDPI, Basel, Switzerland.
releases to the environment diverse pollutants, including several gases, wastewater, and
This article is an open access article
mine tailings [5]. These wastes contain several toxic HM, which are non-degradable and
distributed under the terms and
persistent in the environment, causing soil and water pollution and threatening wildlife
conditions of the Creative Commons
and human health [6,7].
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
Mine tailings are finely ground solid waste generated because of rock trituration
4.0/).
processes in the recovery of the economic value metals. These materials are commonly

Plants 2022, 11, 3441. https://doi.org/10.3390/plants11243441 https://www.mdpi.com/journal/plants

Plants 2022, 11, 3441 2 of 18

deposited without treatment as mounds on the side of mines or submerged as sludge in

tailing dams [3]. Mine tailings are commonly found in the surroundings of abandoned
mining districts and small-scale or artisanal mining operations. Contact of mine tailings,
with rain and winds, favors pollutant release and dispersion in the surrounding areas [8].
Therefore, active or abandoned mine tailings are recognized as one of the primary sources
of HM pollution [9].
The presence of metals in the environment can exert adverse effects on the ecosystems
because of their bioaccumulation in different organisms. Plants can uptake HM from the
soil through the root system and some metals may be translocated to the aerial tissues [10].
Then, these plants are consumed by different herbivores resulting in metal entrance and
transfer along the trophic webs causing HM biomagnification [11]. Hence, the remediation
of HM polluted sites has been of great relevance in the last decades [12–15].
In this context, bioremediation has been proposed as an effective alternative for
metal cleaning, using different organisms such as bacteria, yeasts, fungi, algae, and
plants [16,17]. The use of plants for removing pollutants from the environment is known as
phytoremediation [18–20], which is an economical, efficient, and environmentally friendly
approach [21,22].
For successful phytoremediation, plants must be capable of tolerating HM exposure
by exclusion, stabilization, and by accumulating them in the aerial tissues [23–25]. More-
over, mine-tailing phytoremediation can be carried out in situ, thus reducing the risks of
secondary contamination in the handling and transporting of polluted materials. However,
there are some limitations for the application of phytoremediation approaches; the physical,
chemical, and biological properties of these wastes affect the establishment and growth
of plants [21,26], metal toxicity causes a reduction in size and other morphological and
physiological characters [23,27], it is restricted to the surface and depth of the root [28],
and phytoremediation is a slow process, that could take several years, in which favorable
climatic conditions for plant development are required [20].
To overcome such phytoremediation disadvantages, several assisted phytostabiliza-
tion strategies have been proposed, including the addition of different organic amendments
to the polluted substrates [29,30]. These amendments modify the physicochemical proper-
ties of the substrates, improve their structure, provide nutrients, and lead to a greater water
retention capacity [31–33]. Among the amendments used for this purpose, biochar is partic-
ularly effective [34]. Biochar is the product obtained from thermal degradation at low levels
or in the absence of oxygen (pyrolysis) of different organic materials [35,36]. In addition,
biochar reduces the bioavailability of HM and metalloids and their absorption by plants
because they can interact with metals through various mechanisms, such as electrostatic
interactions, ion exchange, sorption via π electron displaced carbon, surface precipitation or
co-precipitation, and adsorption [37]. These interactions allow the biochar to retain metals
in the soil, considerably reducing their concentration in plant tissues [35,36]. In addition,
the alkaline pH of biochar increases the pH of the soil into which it is incorporated [34],
reducing the solubility and bioavailability of metals [38].
Prosopis laevigata (Humb. et Bonpl. ex Willd) M.C. Johnston (Fabaceae) is an arboreal
species with a broad worldwide distribution in arid and semi-arid zones. In addition, this
plant can establish itself in variable temperature and moisture conditions and grow in
nutrient-poor soils [39,40]. Some studies show that it settles in mine tailings with high
HM and metalloid contents and is capable of accumulating Al, As, Cd, Cr, Cu, Fe, Mg,
Ni, Pb, V, and Zn [23,41–45]. However, P. laevigata shows significant adverse effects due
to the bioaccumulation of several HM in root and leaf tissues, such as genetic damage
and reductions in several morphological and physiological characters [23]. Therefore, the
aim of this study was to evaluate the effect of biochar when mixed with mine tailings to
(1) evaluate the establishment of P. laevigata in tailing substrates (in situ establishment); (2) to
evaluate if this assisted phytostabilization can reduce the accumulation and translocation of
metals in plant tissues; (3) to evaluate if exposure to mine tailing and mine tailing/biochar
substrates cause changes in size and biomass of P. laevigata individuals. This approach will
 proach will allow us to determine if biochar from coconut fiber and corn cob can be
appropriate methodology to facilitate the establishment of this particular HM accum
tor plant for phytostabilization of polluted soils, especially those polluted with the n
essential and most toxic HM.
Plants 2022, 11, 3441 3 of 18

2. Results
2.1.
allowPhysicochemical
us to determineCharacterization of the Corncobs/Coconut
if biochar from coconut fiber and corn cob Fiber
can beBiochar
an appropriate
methodology
Accordingto facilitate the establishment of
to the physicochemical this particular HM
characterization, theaccumulator plant
biochar (50% for cobs/co
corn
phytostabilization of polluted soils, especially those polluted with the non-essential and
nut fiber mixture), employed in the present research as an organic amendment, show
most toxic HM.
pH value of 10, 4.4% of OM, 10.5 mg/kg of phosphorous, 0.033 mg/kg of nitrogen,
mg/kg of carbon, and C/N ratio of 78.8. According to the reference values establishe
2. Results
the
2.1. Mexican standard,
Physicochemical NOM-021-SEMARNAT-2000,
Characterization theBiochar
of the Corncobs/Coconut Fiber corn cobs/coconut fiber bioc
is a strongly
According alkaline substrate with
to the physicochemical a high OMtheproportion.
characterization, biochar (50% In contrast,
corn concerning
cobs/coconut
fiber mixture), employed in the present research as an organic amendment,
C/N ratio and phosphorous content the observed values were situated in the med showed
a pH value of 10, 4.4% of OM, 10.5 mg/kg of phosphorous, 0.033 mg/kg of nitrogen,
range.
2.6 mg/kg of carbon, and C/N ratio of 78.8. According to the reference values established
in the Mexican standard, NOM-021-SEMARNAT-2000, the corn cobs/coconut fiber biochar
2.2. Effect onalkaline
is a strongly the Mine Tailing
substrate Exposure
with andproportion.
a high OM the BiocharIn Addition over the Size
contrast, concerning the Characters
C/N o
laevigata
ratio and phosphorous content the observed values were situated in the medium range.
After three months of exposure, P. laevigata plants growing on mine tailing–bioc
2.2. Effect on the Mine Tailing Exposure and the Biochar Addition over the Size Characters of
substrate
P. laevigata showed significantly higher values in the basal diameter and fresh biomas
the aerial tissues
After three withof
months respect
exposure,to the plants plants
P. laevigata growing on mine
growing tailing
on mine substrate. Similar
tailing–biochar
sults were
substrate observed
showed after sixhigher
significantly months of exposure,
values in the basalthe plantsand
diameter thatfresh
were grownofon the m
biomass
tailing–biochar
the aerial tissues substrate
with respect showed higher
to the plants valuesoninmine
growing the basal
tailing diameter
substrate. and the fresh
Similar
results were observed after six months of exposure, the plants that were grown
dry biomass of the roots in comparison to the plants growing on the mine tailing subst on the mine
tailing–biochar substrate showed higher values in the basal diameter and the fresh and
(Figure 1, Table S1). Because of the heavy metal exposure, the plants growing on m
dry biomass of the roots in comparison to the plants growing on the mine tailing substrate
tailing
(Figure substrate
1, Table S1).showed
Becauseaofsignificant reduction
the heavy metal in the
exposure, thefresh
plantsbiomass
growing of onthe
mineroot and
aerial
tailing substrate showed a significant reduction in the fresh biomass of the root and the subst
tissues through exposure time, while the plants growing on mine tailing
supplemented with exposure
aerial tissues through biochar didtime,not show
while the significant
plants growingchanges
on mineintailing
their size characters o
substrate
supplemented with biochar did
the exposure time (Figure 1, Table S1).not show significant changes in their size characters over
the exposure time (Figure 1, Table S1).

Figure 1. Cont.
Plants 2022, 11, x FOR PEER REVIEW 4 o
Plants 2022, 11, 3441 4 of 18

Figure 1. Size and biomass characters for P. laevigata growing on tailing (T) and tailing/biochar
Figure 1. Size and biomass characters for P. laevigata growing on tailing (T) and tailing/biochar (
(T/B) in in situ conditions during 90 and 180 days. APL = aerial part length, RL = root length,
in in situ conditions during 90 and 180 days. APL = aerial part length, RL = root length, BD = b
BD = basal diameter, FRB = fresh root biomass, FAPB = fresh aerial part biomass, DRB = dry root
diameter, FRB = fresh root biomass, FAPB = fresh aerial part biomass, DRB = dry root biomass, DA
biomass, DAPB = dry aerial part biomass. Different lowercase letters denote significant differences
=among
dry aerial part biomass. Different lowercase letters denote significant differences among tr
treatments after 90 days of the exposure (Tukey p < 0.05). Different uppercase letters denote
ments after 90 days of the exposure (Tukey p < 0.05). Different uppercase letters denote signifi
significant differences among treatments after 180 days of the exposure (Tukey p < 0.05). * = statistical
differences among treatments
differences between exposure timesafter 180character.
for each days of the exposure (Tukey p < 0.05). * = statistical di
ences between exposure times for each character.
2.3. Bioaccumulation and Translocation Profiles of Nonessential Heavy Metals in P. laevigata
The bioaccumulation
2.3. Bioaccumulation and of two non-essential
Translocation heavy
profiles metals, Cd and
of Nonessential Pb, was
Heavy evaluated
Metals in P. laevigata
in the roots and aerial tissues of P. laevigata plants exposed to two treatments: individuals
The bioaccumulation of two non-essential heavy metals, Cd and Pb, was evalua
growing on mine tailings and mine tailings supplemented with 12% of corncobs/coconut
in thebiochar.
fiber roots and
Two aerial
samples tissues of P. laevigata
were obtained plants
to evaluate exposed
the effects torespective
of the two treatments:
treatmentindividu
growing on mine
and the exposure timetailings
of three andandmine tailings supplemented with 12% of corncobs/coco
six months.
fiber After
biochar.
three Two
months samples
of exposure,werenoobtained
significantto evaluate
effect the effects
of the treatments of the
(plants respective tr
growing
on mine tailings and mine tailings supplemented
ment and the exposure time of three and six months. with biochar) over the bioaccumulation
of Cd in the roots of P. laevigata individuals was observed. However, after six months of
After three months of exposure, no significant effect of the treatments (plants gr
exposure to the mine tailing substrate, P. laevigata individuals bioaccumulated a higher
ing on mine tailings
Cd concentration andThe
in roots. mine tailings
exposure timesupplemented with biochar)
showed a significant effect on Cdover the bioaccum
bioaccu-
lation
mulation of in
Cdtheinroots
theofrootsplantsofgrowing
P. laevigata
on theindividuals was observed.
mine tailing substrate. However, However,
the Cd after
months of exposure
concentration in the roots to ofthe mine tailing
P. laevigata plants substrate,
growing on P. thelaevigata
substrate individuals
supplementedbioaccumula
with
abiochar
higherdid Cdnot increase over time.
concentration in roots. The exposure time showed a significant effect on
The plants exposed
bioaccumulation in the to roots
the mine of tailing
plantssubstrate
growing bioaccumulated
on the mine significantly higher Howe
tailing substrate.
Cd in the aerial tissues at three and six months of exposure, compared to Cd bioaccu-
the Cd concentration in the roots of P. laevigata plants growing on the substrate sup
mulation in the aerial tissue of plants exposed to the biochar supplemented mine tailing
mented
substrate.with biochar
Moreover, Cddid not increase
concentration over
in the time.
aerial tissues of P. laevigata growing on the
The plants exposed to the mine
mine tailing substrate significantly increased over tailing substrate bioaccumulated
time. In contrast, the plantssignificantly
growing hig
Cd in presence
in the the aerial of tissues at three andbiochar,
the coconut/corncob six months
showed of exposure,
a reduction compared to Cd bioaccum
in Cd concentration
over time, in the same tissue (Figure 2, Table S2). However,
lation in the aerial tissue of plants exposed to the biochar supplemented mine Cd translocation factors (TF)tailing s
were lower than one for both treatments (plants growing on mine tailings
strate. Moreover, Cd concentration in the aerial tissues of P. laevigata growing on the m and mine tailings
supplemented with biochar).
tailing substrate significantly increased over time. In contrast, the plants growing in
presence of the coconut/corncob biochar, showed a reduction in Cd concentration o
time, in the same tissue (Figure 2, Table S2). However, Cd translocation factors (TF) w
lower than one for both treatments (plants growing on mine tailings and mine taili
supplemented with biochar).
Plants 2022, 11, x FOR PEER REVIEW 5 of 19
Plants 2022, 11, 3441 5 of 18

Figure
Figure 2. Average
2. Average ± standard
± standard deviation
deviation andand two-wayANOVA
two-way ANOVA results resultsfor
forheavy
heavy metal concentration
metal concentration
(mg−1)Kg
(mg Kg in−1roots
) in roots
(a,b)(a,b)
andand leaves
leaves (c,d) P. laevigata
(c,d)ofofP. laevigata growing
growingonontailingtailing(T)(T)
andand
tailing/biochar
tailing/biochar
(T/B)(T/B)
in ininsitu
in situ conditions
conditions during
during 9090and
and180180 days.
days. Different
Different lowercase
lowercaseletters denote
letters denotesignificant
significant
differences among treatments in heavy metal root concentration (Tukey p < 0.05). Different upper-
differences among treatments in heavy metal root concentration (Tukey p < 0.05). Different uppercase
case letters
lettersdenote
denotesignificant
significant differences
differences among among treatments
treatments in heavy inmetal
heavy metal
leaves leaves concentration
concentration (Tukey
(Tukey
p <p0.05),
<0.05),
n.s.n.s.
= not= not significant
significant differences,
differences, ** = p <**0.01,
= p <***0.01,
= p <*** = p < 0.001.
0.001.

respect to Pb bioaccumulation in the roots of P. laevigata, no significant effect of

WithWith
respect to Pb bioaccumulation in the roots of P. laevigata, no significant effect of
the treatments was observed after three months of exposure. However, Pb concentration in
the treatments was observed after three months of exposure. However, Pb concentration
roots increased significantly in P. laevigata individuals after six months of exposure to the
in roots
mineincreased significantly
tailing substrate. in P.
The plants laevigata
exposed individuals
to the mine tailingafter six months
substrate showed of exposure to
a significant
the mine tailing substrate. The plants exposed to the mine tailing substrate showed
increase in Pb concentration in the roots over time. In contrast, exposure time did not show a sig-
nificant increaseeffect
a significant in Pbonconcentration in thein
Pb bioaccumulation roots overof
the roots time. In contrast,
the plants exposure
established in the time
mine did
not show
tailingasubstrate
significant
witheffect on Pb bioaccumulation
the presence in the roots
of coconut fiber/corncobs of the
biochar plants
(Figure established
2, Table S2).
Three months after the establishment of P. laevigata in the treatments,
in the mine tailing substrate with the presence of coconut fiber/corncobs biochar (Figure no significant
effectS2).
2, Table was detected on the bioaccumulation of Pb in the aerial tissues of the plants. While
Three months after the establishment of P. laevigata in the treatments, no significant
effect was detected on the bioaccumulation of Pb in the aerial tissues of the plants. While
after six months of exposure, it was documented that the plants established in the mine
tailing substrate, bioaccumulated a significantly higher Pb concentration in the aerial tis-
Plants 2022, 11, 3441 6 of 18

after six months of exposure, it was documented that the plants established in the mine
tailing substrate, bioaccumulated a significantly higher Pb concentration in the aerial tissues.
Plants of P. laevigata established in the mine tailing substrate showed a significant increase
in Pb concentration in the aerial part over time. In contrast, the exposure time did not
significantly affect Pb bioaccumulation in the aerial tissues of the plants growing in the
presence of coconut/corncobs biochar (Figure 2, Table S2). After three and six months of
exposure, the TF values observed in plants growing in mine tailing substrate were higher
than one, while in the plants growing in the presence of corncobs/coconut fiber biochar,
the TF showed values lower than one in both sampling times, three and six months.

2.4. Bioaccumulation and Translocation Profiles of Essential Heavy Metals in P. laevigata

The bioaccumulation of four essential heavy metals, Cu, Fe, Mn, and Zn was evaluated
in the roots and aerial tissues of P. laevigata plants exposed to mine tailing substrate and
mine tailings supplemented with 12% of corncobs/coconut fiber biochar. Roots and leaf
samples were obtained after three and six months of exposure.
After three months, the plants growing on mine tailing substrate bioaccumulated
significantly higher Cu in roots than Cu concentrations in the roots of those plants growing
on the presence of the corncobs/coconut fiber biochar. However, after six months, Cu
bioaccumulation in the roots of plants growing on both treatments did not show significant
differences. Hence, the exposure time did not affect Cu bioaccumulation in P. laevigata roots
regardless of the treatment (Figure 2, Table S2).
Copper bioaccumulation in aerial tissues of P. laevigata was significantly higher in
the plants exposed to the mine tailing substrate at two sampling times, three and six
months, when compared to Cu bioaccumulation in the aerial tissues of the plants growing
on the presence of corncobs/coconut fiber biochar. However, Cu bioaccumulation in the
aerial tissue of the exposed plants to mine tailing, decreased from three to six months,
while in the plants exposed to biochar supplemented with mine tailing substrate, Cu
bioaccumulation increased over time. Finally, the TF determined at both sampling times in
the plants growing on mine tailing substrate showed values higher than one, indicating the
translocation of Cu from roots to the aerial tissues, while in the plants growing on biochar
supplemented with mine tailing substrate, Cu translocation was reduced, resulting in TF
values lower than one (Figure 2, Table S2).
Iron bioaccumulation in the aerial tissues of the plants exposed to mine tailing sub-
strate was significantly higher in comparison to Fe bioaccumulation in the aerial tissues
of plants growing on the mine tailing substrate with biochar at two sampling times. Iron
bioaccumulation in aerial tissues did not increase over time in the plants growing on
both treatments. With respect to Fe translocation, in the plants exposed to mine tailing
substrate, TF values were higher than one at the two sampling times, while in the plants
growing in the presence of corncobs/coconut fiber biochar, TF values were lower than one
(Figure 2, Table S2).
After three months of exposure, the bioaccumulation of Mn in the roots of P. laevigata
plants was not affected by the treatment, while after six months of exposure, the plants
growing on the presence of biochar bioaccumulated higher Mn concentrations in the roots.
However, in both treatments, the plants showed significant increases in Mn concentration
in the roots over time (Figure 2, Table S2).
Concerning Mn bioaccumulation in the aerial tissues of P. laevigata, the plants growing
on both treatments increased Mn concentrations over time. However, Mn concentrations in
the aerial tissues were similar in the plants of both treatments after three and six months
of exposure. TF values in the plants exposed to mine tailing substrate were higher than
one at the two sampling times, while in the treatment with biochar, after three months of
exposure the plants showed TF values lower than one, but after six months of exposure,
the TF increased to greater than one (Figure 2, Table S2).
The plants growing on mine tailing substrate and those growing on the substrate
with biochar bioaccumulated similar Zn concentrations in the roots after three months of
Plants 2022, 11, x FOR PEER REVIEW 7 of 19

Plants 2022, 11, 3441 7 of 18

roots, in respect to those growing on mine tailings. Over exposure time, Zn concentration
in the roots
exposure. of the plants
However, growing
after six monthson of the mine tailing
exposure, showed
P. laevigata a significant
individuals growingincrease.
on mine How-
tailings
ever, forwith biochargrowing
the plants bioaccumulated a significantly
on biochar, higher Znin
Zn concentrations concentration in the roots,
roots were similar at the two
in respect to those growing on
sampling times (Figure 2, Table S2). mine tailings. Over exposure time, Zn concentration in the
rootsWith
of therespect
plants growing on the mine tailing showed a significant increase. However,
to Zn bioaccumulation in the aerial tissues of P. laevigata, the treatments for
the plants growing on biochar, Zn concentrations in roots were similar at the two sampling
did not show a significant effect. Plants bioaccumulated similar concentrations of Zn in
times (Figure 2, Table S2).
the aerial tissues at two sampling times regardless of the treatment. Zinc concentration in
With respect to Zn bioaccumulation in the aerial tissues of P. laevigata, the treatments
the
did aerial
not showtissues of plants
a significant growing
effect. Plantson mine tailing similar
bioaccumulated substrate increased over
concentrations of Znexposure
in
time, while in the plants growing biochar, Zn concentrations did not increase
the aerial tissues at two sampling times regardless of the treatment. Zinc concentration over
in time.
The
the aerial tissues of plants growing on mine tailing substrate increased over exposure time,the P.
treatment and exposure time did not significantly affect Zn translocation in
laevigata
while in theindividuals. TF values
plants growing were
biochar, Znhigher than onedid
concentrations in both treatments
not increase overattime.
two The
sampling
times (Figure
treatment and 2, Table S2).
exposure time did not significantly affect Zn translocation in the P. laevigata
individuals. TF values were higher than one in both treatments at two sampling times
(Figure
2.5. 2, Table S2).
P. laevigata Total Heavy Metals Phytoextraction
2.5. P.After 180 Total
laevigata daysHeavy
of exposure, P. laevigata plants growing in the mine tailing treatment
Metals Phytoextraction
showed total
After 180phytoextraction
days of exposure,values for non-essential
P. laevigata plants growing metals
in theof 75.07
mine ± 31.5
tailing mg/ha for Cd
treatment
and
showed total phytoextraction values for non-essential metals of 75.07 ± 31.5 mg/ha for Cdmetals
374.40 ± 181.0 mg/ha for Pb. While the total phytoextraction values for essential
were 14.52±± 181.0
and 374.40 8.3 mg/ha
mg/hafor for Mn, 29.25the
Pb. While ± 12.7
totalmg/ha for Cu, 40.80
phytoextraction values± for
24.5 mg/hametals
essential for Zn, and
664.22 ± 311.1
were 14.52 ± 8.3mg/ha
mg/hafor Fe. Concerning
for Mn, the plants
29.25 ± 12.7 mg/ha growing
for Cu, in the
40.80 ± 24.5 minefortailing/biochar
mg/ha Zn, and
664.22 ±treatment,
mixture 311.1 mg/hathefortotal phytoextraction
Fe. Concerning values
the plants increased
growing formine
in the all metals evaluated, be-
tailing/biochar
mixture
ing 34.24treatment, the for
± 17.4 mg/ha total phytoextraction
Mn, valuesfor
46.96 ± 17.1 mg/ha increased
Cu, 113.19for±all metals
41.2 mg/ha evaluated,
for Cd, 466.37
being 34.24 ± 17.4 mg/ha for Mn, 46.96 ± 17.1 mg/ha for Cu, 113.19
± 190.8 mg/ha for Pb and 1986.48 ± 965.5 mg/ha for Fe. Both treatments observed ± 41.2 mg/ha for Cd,
the high-
466.37 ± 190.8 mg/ha for Pb and 1986.48 ± 965.5 mg/ha
est total phytoextraction for Pb and Fe (Figure 3, Table S3). for Fe. Both treatments observed
the highest total phytoextraction for Pb and Fe (Figure 3, Table S3).

Figure 3. Cont.
Plants 2022, 11, x FOR PEER REVIEW 8 of 19
Plants 2022, 11, 3441 8 of 18

.
Figure 3. Total heavy metals phytoextraction in P. laevigata growing on tailing (T) and tailing/biochar
Figure 3. Total heavy metals phytoextraction in P. laevigata growing on tailing (T) and tailing/bio-
(T/B) in in situ conditions for 180 days. The plant arrangement in the field plot was 2 × 2 m,
char (T/B) in in situ conditions for 180 days. The plant arrangement in the field plot was 2 × 2 m, BC:
BC: heavy metal bioconcentration per hectare after 180 days, TPE: total phytoextraction per hectare
heavy metal bioconcentration per hectare after 180 days, TPE: total phytoextraction per hectare after
after
180 180 days.
days.
3. Discussion
3.3.1.
Discussion
Physicochemical Properties of Biochar
3.1. Physicochemical Properties
According to the of Biocharanalysis, the coconut fiber/corn cob biochar has
physiochemical
a strong alkalineto
According pHthevalue. In previous reports,
physiochemical the pH
analysis, the values
coconut of corn cob biochar
fiber/corn showed has a
cob biochar
a highly alkaline profile reporting value of 8.7 [46] and 10.3 [47]. Meanwhile, the biochar
strong alkaline pH value. In previous reports, the pH values of corn cob biochar showed
produced from coconut fiber at 500 and 700 ◦ C showed pH values of 10.3 and 10.5, respec-
atively,
highlywhile
alkaline profile reporting value of 8.7 [46] and 10.3 [47]. Meanwhile, the biochar
in the reports of Li et al. [48] the pH of the coconut fiber biochar was of 10.3.
produced
These high pH values arefiber
from coconut at 500toand
attributed the 700 °C showed
presence of metals pHsuch
valuesas K,ofCa,
10.3 and
and Mg10.5, respec-
in the
tively,
biochar while in the reports
composition [46,47].ofHowever,
Li et al. [48]
otherthe pH ofmention
authors the coconut fiber
that the biochar
biochar was of 10.3.
alkalinity
These high pH values are attributed to the presence of metals
is influenced by the presence of organic functional groups (COOH and OH), carbonates such as K, Ca, and Mg in
the biochar
(CaCO 3 andcomposition
MgCO3 ), and[46,47].alkalineHowever,
salts [32,49].other authors mention that the biochar alkalin-
ity is The coconut fiber/corn
influenced by the presence cob biochar evaluated
of organic in this report
functional groups showed
(COOH a high
andcarbon
OH), car-
content, while the nitrogen content was
bonates (CaCO3 and MgCO3), and alkaline salts [32,49]. low, and had a concentration of 10.5 mg/kg for
phosphorous. So, the C/N ratio is high (78.8), similarly to the report
The coconut fiber/corn cob biochar evaluated in this report showed a high carbon by Guarnieri et al. [32],
where the C/N ratio in coconut fiber biochar was 72.4. Prakongkep et al. [49] reported
content, while the nitrogen content was low, and had a concentration of 10.5 mg/kg for
the chemical characteristics of the biochar were produced by employing different agro-
phosphorous. So, the C/N ratio is high (78.8), similarly to the report by Guarnieri et al.
industrial wastes as raw material, including coconut fiber and corn cobs; overall, the
[32], where
carbon the C/Nin
percentages ratio
the in coconut
biochar fiber biochar
composition rangedwas 72.4.
from 46Prakongkep
to 80%, whileetthe al.nitrogen
[49] reported
the
percentages ranged from 0.3 to 2.3%. The biochar application as an amendment in soils agro-
chemical characteristics of the biochar were produced by employing different
industrial
increases thewastes
organicas carbon
raw material,
percentage including coconut
[50], which fiber and
is important corn cobs;
to increase overall, the car-
the productivity
bon percentages
of crop in the in
fields, especially biochar composition
poor-nutrient ranged from
and overexploited 46 However,
soils. to 80%, while
some the nitrogen
studies
percentages
report that the ranged from of
application 0.3biochar
to 2.3%.with The biochar
a high C/N application as an amendment
ratio to soil reduces the availabilityin soils
of nitrogen forms such as NO 3− and NH4+ [51], as an adverse effect. Moreover, it has been
increases the organic carbon percentage [50], which is important to increase the produc-
reported
tivity that biochar
of crop retains a significant
fields, especially proportionand
in poor-nutrient of the phosphorus present
overexploited in the raw some
soils. However,
material employed in its production; after application to the soil,
studies report that the application of biochar with a high C/N ratio to soil reduces the the biochar dissolution
availability of nitrogen forms such as NO3− and NH4+ [51], as an adverse effect. Moreover,
it has been reported that biochar retains a significant proportion of the phosphorus pre-
Plants 2022, 11, 3441 9 of 18

makes phosphorus available as nutrients for plants [52]. Furthermore, the addition of
biochar to soil increases phosphorus availability by up to 45% [51]. It has been documented
that biochar addition to soil increases the abundance, speciation, and availability of P in
soils, while reducing the P leaching loss, such beneficial effects on P availability can be due
to: (1) the P input to the soil from biochar composition, in which soluble and exchangeable
P is present; (2) the enhancement of the endogenous P availability in soils by biochar, due to
P complexation and by promoting the phosphate-solubilizing microorganism metabolism;
and (3) biochar reduces the leaching P losses from the soil through absorption due to
its porous structure, large internal surface area, and high water retention capacity, thus
increasing the P retaining in soil and favoring plant P uptake. Hence, the use of biochar as
an amendment in phytostabilization approaches may be beneficial for plant development,
due to its positive effect of increasing the soil P availability and favoring P plant uptake [53].

3.2. Size Characters of P. laevigata

P. laevigata plants growing on the mine tailing with biochar showed higher values for
the basal diameter and the fresh and dry biomass of roots compared to the plants growing
on the mine tailing substrate. Similar results show that the addition of biochar in soils
contaminated with HM has positive effects on the growth and on biomass development of
plants distributed in these sites concerning those plants grown on the substrate without
biochar [54,55]. These findings highlight that the addition of biochar on polluted substrates
enhances plant grow and biomass development which facilitates plants to cope with HM
exposure and therefore use for phytostabilization strategies.
The exposure of P. laevigata individuals to mine tailing substrate caused a significant
reduction in the FRB and FAB values over time. In contrast, this effect was not documented
in plants grown on biochar. The biomass reduction observed in the P. laevigata individuals
exposed to mine tailings can be attributed to the presence of HM in the substrate. Similar
results were achieved by Muro-González et al. [23] and Buendía et al. [42]. The reduction
in size characters observed in the mine tailing exposed plants can be related to HM bioac-
cumulation, since they interfere with important metabolic processes in plants, resulting
in growth anomalies [56]. Maldonado-Magaña et al. [57] observed that the absorption of
Pb causes a decrease in the growth rate of the roots and affects the branching pattern in
Acacia farnesiana L. Willd. Additionally, Shanker et al. [58] mentioned that Cr bioaccumu-
lation inhibited root growth in Caesalpinia pulcherrima (L.) Sw., Triticum aestivum L., and
Vigna radiata (L.) R. Wilczek. These findings are important because they highlight that even
though P. laevigata individuals are visibly affected by HM bioaccumulation, these native
plants can remain useful for phytoremediation strategies without causing their death.
On the other side, the reduction in the fresh biomass values of root and aerial tissues of
P. laevigata plants exposed to mine tailings could also be attributed to an effect of the hydric
stress caused by drought season at the time of the second sampling. In the plant growing on
the presence of biochar, the biomass values in roots and aerial tissues were not affected by
the drought conditions; this could indicate that biochar adding to the mine tailing substrate
retains the soil moisture minimizing the hydric stress. Saffari et al. [59] observed that the
presence of biochar from corn residues in soils improved water retention in the substrate
and increased its availability. Similarly, Dhar et al. [60] suggested that biochar produced
from coconut fiber through a pyrolysis process (500–600 ◦ C), has employment potential as
an organic soil amendment for water retention.

3.3. Bioaccumulation and Translocation of Nonessential Heavy Metals in P. laevigata

Cadmium bioaccumulation in roots and aerial tissues was higher in plants exposed
to mine tailing substrate, Cd concentration in both tissues also increased over time, while
the plants growing on biochar showed a contrary effect. According to these findings,
the addition of biochar to mine tailings reduces the bioaccumulation of Cd in P. laevigata
tissues. It is important to highlight that even that the tailing/biochar substrate had 12%
less Cd than the tailing substrate, the bioaccumulation in roots and leaves was less than the
Plants 2022, 11, 3441 10 of 18

expected proportion without biochar. Hence, this type of biochar contributes to cadmium
phytostabilization in soils. Different studies document a reduction in Cd bioaccumulation
in plants when biochar is added to the substrate [35,56]. In general, the addition of biochar
to the soil increases the overall pH in soil, alkaline pH conditions conduct to the Cd
ions precipitation as Cd(OH)2 , reducing its bioavailability in soils [61]. In the study by
Muro-González et al. [23], P. laevigata plants exposed ex situ to mine tailings from the
Huautla mining district, did not show Cd bioaccumulation in their tissues, thus contrasting
to the findings of the present study. Hence, this discrepancy can be attributed to the
different experimental conditions used (ex situ vs. in situ). In in situ studies, the tailing
samples are homogenized, resulting in more consistent HM concentration across samples,
contrasting to in situ studies where the individuals grow directly on the tailing, which
can have different HMs, resulting in some plants showing more Cd bioaccumulation
than others.
With respect to Pb bioaccumulation, the P. laevigata plants exposed to mine tailing
substrate showed higher Pb concentrations in both root and aerial tissues with respect
to plants growing in the presence of biochar, Pb concentration increased over exposure
time, as well as the registered TF which was higher than 1. It is important to highlight
that even though the tailing/biochar substrate had 12% less Pb than the tailing substrate,
the bioaccumulation in roots and leaves was less than the expected proportion with-
out biochar. Hence, this type of biochar contributes to lead phytostabilization in soils.
Muro-González et al. [23], observed that Pb bioaccumulation in P. laevigata plants exposed
ex situ to mine tailings from Huautla, Morelos, increased according to the exposure time
(2, 4, and 6 months). Although Pb is not an essential element for plant nutrition and
development, when it is bioavailable in the soil, it is commonly absorbed by roots through
the apoplastic pathway or the employment of ion channels and then translocated to aerial
tissues of plants [62]. It has been documented that the presence of biochar in HM polluted
substrates reduces Pb bioaccumulation in plant tissues (roots and leaves). Li et al. [63],
observed a reduction in the bioaccumulation of Pb in rice, when plants were grown on
a Pb-enriched soil supplemented with coconut fiber–biochar, the effect was explained
by a reduced Pb bioavailability which generated a strong interaction between Pb ions,
the biochar surface, and the formation of chemical compounds such as Pb3 (PO4 ) and Pb
carboxylates [64].

3.4. Bioaccumulation and Translocation of Essential Heavy Metals in P. laevigata

P. laevigata plants established in the mine tailing substrate bioaccumulated higher Cu
concentration in the aerial tissues compared to those plants exposed to the mine tailing–
biochar substrate. However, Cu concentrations were reduced in the aerial tissues of the
plants of both treatments. These findings contrast with those of Muro-González et al. [23],
in which an increase in Cu bioaccumulation was observed in the aerial tissues of P. laevigata
growing on mine tailing substrate from Huautla, Morelos, according to the exposure time,
while Cu bioconcentration was reduced in the roots of these plants. Lu et al. [35] reported
that the presence of bamboo and rice straw biochar in soils reduces the bioavailability of Cu
ions. Eissa [65] observed a lower Cu bioaccumulation in different tissues of Cucurbita pepo L.
growing on soils supplemented with cow manure biochar compared to Cu bioaccumulation
in plants growing on a substrate without biochar. In another study, Zong et al. [66] observed
a reduction in the mobility and bioavailability of the Cu fraction in soils as a result of the
application of biochar generated from different sources (wheat straw, corn stalk, and rice
husk), as a consequence, Cu bioaccumulation was reduced in rice, corn, and radish plants.
Biochar presents a great number of C=O functional groups that can interact with Cu ion
reducing its bioavailability in soils [66].
In respect to Fe bioaccumulation, plants exposed to mine tailing substrate bioaccu-
mulated higher concentrations in the roots, while the plants growing on biochar bioac-
cumulated more Fe in the aerial tissues. Muro-González et al. [23] reported higher Fe
concentration in the roots of plant exposed to mine tailings from Huautla, Morelos. Sim-
Plants 2022, 11, 3441 11 of 18

ilarly, Santoyo-Martínez et al. [27] reported higher Fe concentrations in the roots of the
arboreal plant Vachellia campechiana (Mill Seigler and Ebinger) exposed to mine tailings
from the same site. The lower Fe concentrations observed in the roots of P. laevigata plants
growing in the presence of biochar could be related to an increase in the pH of the sub-
strate as a result of the addition of this organic amendment of alkaline nature. Moreover,
Fe bioavailability decreases in the presence of alkaline substrates [32], while at lower
pH values, Fe solubility and bioavailability increases, due to a reduction from Fe III to
Fe II [61,67]. Once Fe is absorbed in the roots, it is translocated to aerial tissues, such as
leaves, in which Fe demand is higher. On deficiency, Fe translocation is preferential from
roots to young leaves [67]. This fact could explain the higher Fe concentration observed in
aerial tissues of plants exposed to mine tailing/biochar, in which the Fe bioavailability is
lower compared to the mine tailing substrate, thereby increasing the translocation rates.
The healthy growth of plants requires small amounts of Mn, however, an excess
of this element may cause toxic effects over plants [68]. During exposure, P. laevigata
plants growing on both treatments increased their Mn concentrations in roots and aerial
tissues. After six months of exposure, the plants growing on biochar showed higher
Mn concentrations in the roots, while Mn bioaccumulation in the aerial tissues was not
affected by any of the treatments, similar Mn concentrations were observed in the plants
growing on both treatments. Similarly, Abbas et al. [69] observed an increase in Mn
concentrations in roots, shoots, and grains of wheat plants established in HM polluted soils
supplemented with rice straw biochar as organic amendment. Although alkaline pH in
soils reduces HM mobility and availability, including Mn, in this study the addition of
coconut fiber/corn cob biochar with a pH value around 10 to the mine tailing substrate,
favored Mn bioaccumulation in roots of P. laevigata.
Zinc is an essential micronutrient involved in a wide variety of plant physiological
processes, although Zn in excess is toxic for plants [70]. In this study, plants exposed to the
mine tailing substrate showed a lower Zn concentration in roots and aerial tissues over
exposure time, contrary to the plants growing on mine tailing/biochar substrate. In the
study by Muro-González et al. [23], a decrease in Zn bioaccumulation was observed in
P. laevigata roots during exposure time to mine tailings from Huautla, Morelos, as well
as the Zn concentration in these plants was higher in the roots than in the leaves, while
in the present research, Zn bioaccumulation was higher in the aerial tissues of plants
exposed to mine tailing substrate. In this study, the treatment did not show a significant
effect over Zn bioaccumulation in P. laevigata plants, contrary to what was expected at
the beginning of this research. Different researchers mention that the addition of biochar
from natural sources such as sugarcane grass, rice husks, and bamboo to soils reduces the
overall mobility and bioavailability of Zn [35,54]. However, Fellet et al. [71] did not observe
significant differences in Zn bioaccumulation between plants growing on mine tailings, and
those growing on the presence of different proportions of biochar from pruning residues,
spruce trees, and manure in the substrate.
Concerning the total HM phytoextraction, the corncob/coconut biochar addition to
mine tailing substrate causes a positive effect in biomass production in P. laevigata, resulting
in higher phytoextraction values for all the analyzed HM, Fe and Pb being the metals that
showed the higher phytoextraction values after 180 days of exposure in both treatments.
In the present study, for total heavy metal phytoextraction determinations, a 2 × 2 m
plant arrangement was considered for the field plot, equivalent to 2500 plants per hectare.
However, in phytostabilization studies, arrangements with closer distances between plants
have been tested, for example, a 0.4 × 0.4 m (60,000 plants·ha−1 ) arrangement was consid-
ered for the determination of Cd, Cu, Ni, Pb, and Zn phytoextraction by the plant species
Brassica rapa L., Cannabis sativa L., Helianthus annuus L., and Zea mays Gaertn, in the presence
of soil amendments such as ethylenediamine tetra-acetic acid (EDTA) or ethylenediamine
di-succinic acid (EDDS). The evaluated plants can produce 10–20 ton/ha of dry weigh
biomass, the highest total phytoextraction was observed for Zn removal with a range of
0.3–1.1 mg·kg−1 of soil [72]. In another study, using a 0.2 × 0.2 m (250,000 plant·ha−1 )
Plants 2022, 11, 3441 12 of 18

arrangement for Cd, Pb, and Zn phytoextraction by the plant species Dianthus chinensis L.,
Rumex acetosa L., Rumex crispus L., Sedum alfredii Hance, Vetiveria zizanioides (L.) Nash, and
Viola baoshanensis W.S.Shu, W.Liu and C.Y.Lan in the presence of EDTA as amendment,
the dry weigh biomass production by hectare ranged from 2.5 to 30 ton·ha−1 , the highest
HM phytoextraction values were observed for Zn, 37.2 kg ha−1 without soil amendment
and around to 50 kg ha−1 in presence of EDTA [73]. Felker et al. [74] determined the dry
weight biomass production in different Prosopis sp. after a year from germination in
a 1.5 × 1.5 m arrangement in field plots, the biomass production ranged from 7 to
14.5 ton·ha−1 . Hence, arrangements with a higher number of plants in field plots can
increase the total HM phytoextraction and phytostabilization by P. laevigata in phytoreme-
diation field experiments.
Phytostabilization strategies can have a positive effect on stabilizing HM, which are
potentially toxic. It reduces contaminants’ bioavailability using plants, often in combination
with soil amendments [75,76]. Soil amendments strongly reduce the availability of HM to
plant uptake limiting eventual toxicity to plants, allowing revegetation of polluted soils.
Establishment of a vegetative cover significantly reduces HM leaching to groundwater and
prevents the dispersal of these toxic elements through wind and water erosion from less
vegetated sites [76,77], reducing potential risks to ecosystem health.

4. Materials and Methods

4.1. Study Site
The present study was carried out in an abandoned mine district located in the
community of Huautla, in the state of Morelos, Mexico (8◦ 260 36.3700 N and 99◦ 010 26.7100 W,
at an altitude of 974 m). In this area, because of mining activities, around 780 thousand
tons of mining wastes rich in HM and metalloids, such as As, Cd, Cr, Cu, Fe Pb, Mn, and
Zn, were disposed without any environmental attention. Additionally, this polluted site is
Plants 2022, 11, x FOR PEER REVIEW 13 of 19
in the vicinity of Huautla town, a fact that poses a human and environmental risk for this
region (Figure 4).

Figure 4.4. Geographical

Figure Geographical distribution
distribution of the study
study site
site at
at the
the Sierra
Sierra de
de Huautla
Huautla Biosphere
Biosphere Reserve,
Reserve,
Morelos,Mexico.
Morelos, Mexico.

4.2. Plant Species

Prosopis laevigata (Humb. et Bonpl. ex Willd) M.C. Johnston (Fabaceae), is an arborous
species with natural distribution and dominance in the polluted sites of Huautla, Morelos.
This plant is considered as an HM accumulator species [23,41–45]. The P. laevigata seeds
were collected in the site of Quilamula, Morelos (18°30′52″ N and 98°59′59″ W, at an alti-
Plants 2022, 11, 3441 13 of 18

4.2. Plant Species

Prosopis laevigata (Humb. et Bonpl. ex Willd) M.C. Johnston (Fabaceae), is an arborous
species with natural distribution and dominance in the polluted sites of Huautla, Morelos.
This plant is considered as an HM accumulator species [23,41–45]. The P. laevigata seeds
were collected in the site of Quilamula, Morelos (18◦ 300 5200 N and 98◦ 590 5900 W, at an
altitude of 1070 m); this site shares geographical and ecological characteristics with the
Huautla site, where mine tailings were deposited [72], but without records of mining
activities or HM pollution associated to human activities [11]. For germination, seeds were
mechanically scarified to break their physical dormancy, planted in plastic seedbeds with
128 cavities (53 × 27 × 4.4 cm, 19 mL cavity volume), and kept in greenhouse-controlled
conditions. After three months, the plants were translated to the experimental plots located
on Huautla mine tailings.

4.3. Biochar Production and Physicochemical Characterization

In this study, biochar was produced from coconut fiber and corncobs from agro-
industrial wastes at 600 ◦ C without oxygen through a pyrolysis process. In the in situ
experimental procedures, a 50% mixture of both biochar types was employed. For physico-
chemical characterization, biochar was considered as soil, pH, organic matter percentage,
total carbon, and nitrogen determinations were carried out according to the Mexican stan-
dard NOM-021-SEMARNAT-2000. In the present study biochar was produced at 600 ◦ C
through a pyrolysis process, using corncobs and coconut fiber as feedstocks, according to
the concentrations of total polycyclic aromatic hydrocarbons (PAHs) in similar feedstocks of
biochar and to the pyrolysis temperature employed, the concentration of PAHs could range
from 0.16 to 1.9 mg·Kg−1 , concentrations below the maximum permissible concentration
(4 mg·Kg−1 ) for premium grade biochar [78].

4.4. In Situ Phytostabilization Design

For the in situ phytostabilization strategy, two experimental treatments were eval-
uated. In the first treatment, 50 plants (5 cm height) were transplanted to 20 cm depth
holes in the experimental plots directly on the mine tailing substrate (2 m distance between
them). In contrast, in the second treatment, 50 plants (5 cm height) were transplanted
to 20 cm depth holes in the experimental plots adding 10% of the biochar mixture, in
a completely randomized design, ten plants of each treatment were randomly sampled
at three and six months after experimental plots transplantation. Sampling times were
established to avoid the roots of the plants exceeding the depth of the mine tailing and
biochar mixture, guaranteeing the contact between the roots and the mixture during all
experimental evaluations. Metal concentrations in mine tailing substrate were (mg/kg):
Fe (80.67), Pb (67.4), Cd (23.78), Cu (7.78), Zn (2.14), and the HM concentration in the sub-
strate mine tailing/biochar were Fe (82.93), Pb (59.31), Cd (20.92), Cu (7.49), and Zn (10.59).
In particular, we did not detect the presence of non-essential HM in corncobs/coconut
fiber biochar.

4.5. Plants size and Biomass Determinations

To evaluate the effect of biochar addition over the establishment and development
of plants, seven size and biomass characters were evaluated: aerial part length (AL), root
length (LR), basal diameter (BD), fresh aerial part biomass (FAB), fresh root biomass (FRB),
dry aerial part biomass (DAB), and dry root biomass (DRB).

4.6. Heavy Metal Determination

For HM concentration determinations in P. laevigata roots and leaves, 20 plants were
randomly selected (10 per treatment/5 plants per exposition time). After sampling, total
roots and leaves were separated and washed twice with distilled water to remove substrate
and dust remnants. Subsequently, clean tissues were oven-dried (60 ◦ C), the dry tissues
were ground, and 250 mg samples of each tissue were acid-digested, adding 10 mL of
Plants 2022, 11, 3441 14 of 18

aqueous HNO3 (70% v/v) in a Microwave Accelerated Reaction System (CEM® MARS-5);
an additional sample without tissue was simultaneously processed in the same conditions
as a control. Once digested, samples were solubilized in distilled water to 50 mL of final
volume, the tissue concentration of six heavy metals was calculated (Cd, Cu, Fe, Mn, Pb, and
Zn) through Atomic Absorption Spectrophotometry (GBC-908-AA, Scientific equipment).
According to the manufacturer, the equipment detection limits for each metallic element in
mg·kg−1 were Cd (0.0004), Cu (0.001), Fe (0.005), Mn (0.0015), Pb (0.001), and Zn (0.0005).
For concentration determinations, calibration curves for each heavy metal were generated
employing known concentration standard solutions for each evaluated metal, and prepared
with pure metal ions (ULTRA Scientific, North Kingstown). All standard calibration curves
showed correlation coefficients (R2 ) between 0.99 and 1.0. In each sample, the heavy
metal concentrations were determined by triplicate and reported as the average of such
determinations in milligrams/kilogram dry weight (mg/kg DW).

4.7. Heavy Metal Translocation Index and Total Phytoextraction

The translocation of HM from roots to leaves was evaluated through the translo-
cation factor (TF) according to Equation (1), TF values higher than 1 indicate heavy
metal translocation.
TF = [HM]LT/[HM]RT (1)
where: TF = translocation factor; [HM]LT = heavy metal concentration in leaf tissue
[mg·kg−1 DW]; [HM]RT = heavy metal concentration in root tissue [mg·kg−1 DW].
The total phytoextraction potential per hectare of P. laevigata for each heavy metal
tested after 180 days of treatment, was calculated according to Equation (2), for TPE
determinations, the plant arrangement in field plots was 2 × 2 m, equivalent to 2500 plants
per hectare.
TPE = [BAroot × BDWroot ] + [BAleaf × BDWleaf ] (2)
where TPE is the total phytoextraction for each heavy metal [mg·ha−1 ]; BCroot is the heavy
metal bioconcentration in root tissues [mg·kg−1 DW]; BDWroot is the dry weight of root
biomass per hectare [kg·ha−1 ]; BCleaf is the heavy metal bioconcentration in leaf tissues
[mg·kg−1 DW]; and BDWleaf is the dry weight of leaf biomass per hectare [kg·ha−1 ].

4.8. Statistical Analysis

For statistical analysis, the normality of the experimental data set was evaluated
through the Shapiro–Wilk “W” test [79]. Subsequently, two-way analysis of variance
(ANOVA) was utilized to evaluate the treatment effect (mine-tailing substrate and 10%
biochar addition) and the exposure time (3 and 6 months) over the heavy metal bioaccu-
mulation and the size characters and biomass of the P. laevigata individuals. Afterward,
a post-hoc Tukey test (p < 0.05) was conducted to determine significant differences between
pairs of average values among the exposure times for each treatment [79]. The statistical
analyses were carried out in the R platform [80]. Finally, regression analyses were carried
out to test the relationship between the bioaccumulation of each evaluated heavy metal and
the root length development over time in both treatments. The software used for statistical
analysis were STATISTICA 8.0 [81].

5. Conclusions
The in situ application of coconut fiber/corn cob biochar to mine tailings favored the
establishment of P. laevigata plants in the HM polluted site by increasing their biomass and
basal diameter. In addition, biochar retained the moisture in the substrate, favoring the
development of plant fresh biomass. Concerning the HM bioaccumulation, the biochar
addition increased the bioaccumulation of essential metals such as Fe and Mn in P. laevigata
tissues while reducing the bioaccumulation and translocation of Cu and the non-essential
and most toxic metals, Pb and Cd. Therefore, it is suggested that the use of biochar
from coconut fiber and corn cob can be an appropriate methodology to facilitate the
Plants 2022, 11, 3441 15 of 18

establishment of plants for phytostabilization of contaminated soils, especially those with

non-essential heavy metals such as Pb and Cd. Moreover, the addition of biochar improves
HM phytostabilization in soils in comparison with other approaches such as compost which
improves phytoextraction efficiency
However, it would be convenient to carry out more in situ studies to prove that
P. laevigata is applicable for phytoremediation processes (it is capable of accumulating
heavy metals) when the roots of the plants exceed the depth of the biochar mixture. Ad-
ditionally, it would be interesting to analyze the benefits of adding the biochar gener-
ated from other plant sources to mine tailings in order to enhance phytoextraction and
phytoremediation processes.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/plants11243441/s1, Table S1: Mean ± standard deviation and
Two way ANOVA results for size and biomass characters for P. laevigata growing on tailing and
tailing/biochar in in situ conditions.; Table S2: Average ± standard deviation and Two way ANOVA
results for heavy metal concentration (mg·Kg−1 ) in roots and leaves of P. laevigata growing on
tailing and tailing/biochar in in situ conditions; Table S3: Total heavy metal phytoextraction in
Prosopis laevigata.
Author Contributions: Conceptualization, E.T.-S. and P.M-G.; performed the experiments, J.R.-Z.;
validation, E.T.-S. and P.M.-G.; methodology, J.R.-Z. and A.R.; formal analysis, J.R.-Z., A.R., M.L.C.-G.
and E.T.-S.; writing—original draft preparation, J.R.-Z., A.R., M.L.C.-G. and L.V.-C.; writing—review
and editing, E.T.-S. and P.M.-G.; supervision, E.T.-S. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was supported in part by CONACyT scholarship grant to J.R.Z.
(Grant 686476).
Data Availability Statement: Data recorded in the current study are available in all tables of
the manuscript.
Acknowledgments: The authors thank Marcos Rosas Ramírez, Janeth Esteves Aguilar, Miriam
Serrano Muñoz, and Miguel Ángel Galván Ramírez for their help in field. We also thank the
Doctorado en Ciencias Naturales (UAEM).
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Carvalho, F.P. Mining industry and sustainable development: Time for change. Food Energy Secur. 2017, 6, 61–77. [CrossRef]
2. Gabarrón, M.; Zornoza, R.; Acosta, J.A.; Faz, Á.; Martínez-Martínez, S. Mining environments. Adv. Chem. Pollut. Environ. Manag.
Prot. 2019, 4, 157–205. [CrossRef]
3. Artiola, J.F.; Walworth, J.L.; Musil, S.A.; Crimmins, M.A. Soil and land pollution. In Environmental and Pollution Science; Elsevier:
Amsterdam, The Netherlands, 2019; pp. 219–235. [CrossRef]
4. Farjana, S.H.; Huda, N.; Parvez Mahmud, M.A.; Saidur, R.A. Review on the impact of mining and mineral processing industries
through life cycle assessment. J. Clean. Prod. 2019, 231, 1200–1217. [CrossRef]
5. Matinde, E.; Simate, G.S.; Ndlovu, S. Mining and Metallurgical Wastes: A Review of Recycling and Re-Use Practices. J. S. Afr. Inst.
Min. Metall. 2018, 118, 825–844. [CrossRef]
6. Vardhan, K.H.; Kumar, P.S.; Panda, R.C. A Review on Heavy Metal Pollution, Toxicity and Remedial Measures: Current Trends
and Future Perspectives. J. Mol. Liq. 2019, 290, 111197. [CrossRef]
7. Briffa, J.; Sinagra, E.; Blundell, R. Heavy Metal Pollution in the Environment and Their Toxicological Effects on Humans. Heliyon
2020, 6, e04691. [CrossRef] [PubMed]
8. Cleaver, A.E.; Jamieson, H.E.; Rickwood, C.J.; Huntsman, P. Tailings dust characterization and impacts on surface water chemistry
at an abandoned Zn-Pb-Cu-Au-Ag deposit. Appl. Geochem. 2021, 128, 104927. [CrossRef]
9. Del Rio-Salas, R.; Ayala-Ramírez, Y.; Loredo-Portales, R.; Romero, F.; Molina-Freaner, F.; Minjarez-Osorio, C.; Pi-Puig, T.; Ochoa–
Landín, L.; Moreno-Rodríguez, V. Mineralogy and geochemistry of rural road dust and nearby mine tailings: A case of ignored
pollution hazard from an abandoned mining site in semi-arid zone. Nat. Resour. Res. 2019, 28, 1485–1503. [CrossRef]
10. Dinu, C.; Vasile, G.G.; Buleandra, M.; Popa, D.E.; Gheorghe, S.; Ungureanu, E.M. Translocation and accumulation of heavy metals
in Ocimum basilicum L. plants grown in a mining-contaminated soil. J. Soils Sediments 2020, 20, 2141–2154. [CrossRef]
11. Mussali-Galante, P.; Tovar-Sánchez, E.; Valverde, M.; Rojas del Castillo, E. Biomarkers of exposure for assessing environmental
metal pollution: From molecules to ecosystems. Rev. Int. Contam. Ambient. 2013, 29, 117–140. [CrossRef]
Plants 2022, 11, 3441 16 of 18

12. Sun, W.; Ji, B.; Khoso, S.A.; Tang, H.; Liu, R.; Wang, L.; Hu, Y. An extensive review on restoration technologies for mining tailings.
Environ. Sci. Pollut. Res. 2018, 25, 33911–33925. [CrossRef] [PubMed]
13. Qayyum, S.; Khan, I.; Meng, K.; Zhao, Y.; Peng, C. A review on remediation technologies for heavy metals contaminated soil.
Cent. Asian J. Environ. Sci. Technol. Innov. 2020, 1, 21–29. [CrossRef]
14. Asadollahfardi, G.; Sarmadi, M.S.; Rezaee, M.; Khodadadi-Darban, A.; Yazdani, M.; Paz-Garcia, J.M. Comparison of Different
Extracting agents for the recovery of Pb and Zn through electrokinetic remediation of mine tailings. J. Environ. Manag. 2021,
279, 111728. [CrossRef] [PubMed]
15. Manca, P.P.; Massacci, G.; Pintus, D.; Sogos, G. The flotation of sphalerite mine tailings as a remediation method. Miner. Eng.
2021, 165, 106862. [CrossRef]
16. Verma, N.; Sharma, R. Bioremediation of toxic heavy metals: A patent review. Recent Pat. Biotechnol. 2017, 11, 171–187. [CrossRef]
[PubMed]
17. Singh, R.; Ahirwar, N.K.; Tiwari, J.; Pathak, J. Review on sources and effect of heavy metal in soil: Its bioremediation. Int. J. Res.
Appl. Nat. Soc. Sci. 2018, 2008, 1–22.
18. Ashraf, S.; Ali, Q.; Zahir, Z.A.; Ashraf, S.; Asghar, H.N. Phytoremediation: Environmentally sustainable way for reclamation of
heavy metal polluted soils. Ecotoxicol. Environ. Saf. 2019, 174, 714–727. [CrossRef]
19. DalCorso, G.; Fasani, E.; Manara, A.; Visioli, G.; Furini, A. Heavy metal pollutions: State of the art and innovation in phytoreme-
diation. Int. J. Mol. Sci. 2019, 20, 3412. [CrossRef]
20. Shah, V.; Daverey, A. Phytoremediation: A multidisciplinary approach to clean up heavy metal contaminated soil. Environ.
Technol. Innov. 2020, 18, 100774. [CrossRef]
21. Wang, L.; Ji, B.; Hu, Y.; Liu, R.; Sun, W. A Review on in situ phytoremediation of mine tailings. Chemosphere 2017, 184, 594–600.
[CrossRef]
22. Nedjimi, B. Phytoremediation: A sustainable environmental technology for heavy metals decontamination. SN Appl. Sci. 2021,
3, 286. [CrossRef]
23. Muro-González, D.A.; Mussali-Galante, P.; Valencia-Cuevas, L.; Flores-Trujillo, K.; Tovar-Sánchez, E. Morphological, physiological,
and genotoxic effects of heavy metal bioaccumulation in Prosopis laevigata reveal its potential for phytoremediation. Environ. Sci.
Pollut. Res. 2020, 27, 40187–40204. [CrossRef]
24. Singh, B.; Kaur, B.; Singh, D. Assessment of different multipurpose tree species for phytoextraction of lead from lead-contaminated
soils. Bioremediat. J. 2020, 24, 215–230. [CrossRef]
25. Sharma, P.; Ngo, H.H.; Khanal, S.; Larroche, C.; Kim, S.H.; Pandey, A. Efficiency of transporter genes and proteins in hyperaccu-
mulator plants for metals tolerance in wastewater treatment: Sustainable technique for metal detoxification. Environ. Technol.
Innov. 2021, 23, 101725. [CrossRef]
26. Cameselle, C.; Gouveia, S.; Urréjola, S. Benefits of phytoremediation amended with DC electric field. Application to soils
contaminated with heavy metals. Chemosphere 2019, 229, 481–488. [CrossRef] [PubMed]
27. Santoyo-Martínez, M.; Mussali-Galante, P.; Hernández-Plata, I.; Valencia-Cuevas, L.; Flores-Morales, A.; Ortiz-Hernández, L.;
Flores-Trujillo, K.; Ramos-Quintana, F.; Tovar-Sánchez, E. Heavy metal bioaccumulation and morphological changes in Vachellia
Campechiana (Fabaceae) reveal its potential for phytoextraction of Cr, Cu, and Pb in mine tailings. Environ. Sci. Pollut. Res. 2020,
27, 11260–11276. [CrossRef] [PubMed]
28. Sharma, P. Efficiency of bacteria and bacterial assisted phytoremediation of heavy metals: An update. Bioresour. Technol. 2021,
328, 124835. [CrossRef]
29. Grobelak, A. Organic soil amendments in the phytoremediation process. Phytoremediation Manag. Environ. Contam. 2016, 4, 21–39.
[CrossRef]
30. Wiszniewska, A.; Hanus-Fajerska, E.; MuszyŃska, E.; Ciarkowska, K. Natural organic amendments for improved phytoremedia-
tion of polluted soils: A review of recent progress. Pedosphere 2016, 26, 1–12. [CrossRef]
31. Urra, J.; Alkorta, I.; Garbisu, C. Potential benefits and risks for soil health derived from the use of organic amendments in
agriculture. Agronomy 2019, 9, 542. [CrossRef]
32. Guarnieri, S.F.; do Nascimento, E.C.; Costa Junior, R.F.; de Faria, J.L.B.; de Almeida Lobo, F. Coconut fiber biochar alters physical
and chemical properties in sandy soils. Acta Sci. Agron. 2021, 43, e51801. [CrossRef]
33. Siedt, M.; Schäffer, A.; Smith, K.E.C.; Nabel, M.; Roß-Nickoll, M.; van Dongen, J.T. Comparing straw, compost, and biochar
regarding their suitability as agricultural soil amendments to affect soil structure, nutrient leaching, microbial communities, and
the fate of pesticides. Sci. Total Environ. 2021, 751, 141607. [CrossRef] [PubMed]
34. Hui, D. Effects of biochar application on soil properties, plant biomass production, and soil greenhouse gas emissions: A
mini-review. Agric. Sci. 2021, 12, 213–236. [CrossRef]
35. Lu, K.; Yang, X.; Gielen, G.; Bolan, N.; Ok, Y.S.; Niazi, N.K.; Xu, S.; Yuan, G.; Chen, X.; Zhang, X.; et al. Effect of bamboo and rice
straw biochars on the mobility and redistribution of heavy metals (Cd, Cu, Pb and Zn) in contaminated soil. J. Environ. Manag.
2017, 186, 285–292. [CrossRef] [PubMed]
36. Chen, D.; Liu, X.; Bian, R.; Cheng, K.; Zhang, X.; Zheng, J.; Joseph, S.; Crowley, D.; Pan, G.; Li, L. Effects of biochar on availability
and plant uptake of heavy metals—A meta- analysis. J. Environ. Manag. 2018, 222, 76–85. [CrossRef]
37. Puga, A.P.; Melo, L.C.A.; de Abreu, C.A.; Coscione, A.R.; Paz-Ferreiro, J. Leaching and fractionation of heavy metals in mining
soils amended with biochar. Soil Tillage Res. 2016, 164, 25–33. [CrossRef]
Plants 2022, 11, 3441 17 of 18

38. Keshavarzifard, M.; Moore, F.; Sharifi, R. The Influence of physicochemical parameters on bioavailability and bioaccessibility of
heavy metals in sediments of the intertidal zone of Asaluyeh region, Persian Gulf, Iran. Geochemistry 2019, 79, 178–187. [CrossRef]
39. Palacios, A.; Rodríguez, R.; Hernández, M.D.L.L.; Jiménez, E.; Tirado, D. Potential distribution of Prosopis laevigata (Humb. et
Bonpl. Ex Willd) M. C. Johnston Based on an ecological niche model. Rev. Mex. Ciencias For. 2016, 7, 35–36. [CrossRef]
40. Hernández-Madrigal, F.; Contreras-Negrete, G.; Aguilar-Romero, R.; Pineda-García, F.; González-Rodríguez, A. Differentiation in
seed mass and seedling biomass allocation in Prosopis laevigata throughout its distribution range in mexico is associated to water
availability. Bot. Sci. 2020, 100, 274–290. [CrossRef]
41. Buendía, L.; Orozco, J.; Cruz, F.; Barrera, C.E.; Vernon, E.J. Prosopis laevigata a potential chromium (VI) and cadmium (II)
hyperaccumulator desert plant. Bioresour. Technol. 2010, 101, 5862–5867. [CrossRef]
42. Buendía, L.; Orozco, J.; Estrada, M.; Barrera, C.; Vernon, E.; Cruz, F. In vitro lead and nickel accumulation in mesquite (Prosopis
laevigata) seedlings. Rev. Mex. Ing. Química 2012, 9, 1–9.
43. Alcalá-Jauregui, J.; Rodriguez-Ortiz, J.C.; Hernández-Montoya, A.; Filippini, M.F.R.; Martinez-Carretero, E.E.; Diaz-Flores, P.E.
Capacity of Two vegetative species of heavy metal accumulation. Rev. FCA UNCUYO 2018, 50, 1853–8665.
44. Ramírez, V.; Baez, A.; López, P.; Bustillos, R.; Villalobos, M.Á.; Carreño, R.; Contreras, J.L.; Muñoz-Rojas, J.; Fuentes, L.E.;
Martínez, J.; et al. Chromium hyper-tolerant Bacillus sp. Mh778713 assists phytoremediation of heavy metals by mesquite trees
(Prosopis laevigata). Front. Microbiol. 2019, 10, 1833. [CrossRef] [PubMed]
45. Buendía, L.; Cruz, F.; Rodríguez, M.E.; Barrera, C.E.; Hernández, C.; Orozco, J. In vitro simultaneous accumulation of multiple
heavy metals by Prosopis laevigata seedlings cultures. Rev. Mex. Ing. Química 2019, 18, 1167–1177. [CrossRef]
46. Pipíška, M.; Krajčíková, E.K.; Hvostik, M.; Frišták, V.; Ďuriška, L.; Černičková, I.; Kaňuchová, M.; Conte, P.; Soja, G. Biochar from
wood chips and corn cobs for adsorption of thioflavin T and erythrosine B. Materials 2022, 15, 1492. [CrossRef]
47. Oluk, I.; Nagawa, C.B.; Banadda, N.; Tumutegyereize, P.; Owusu, P.A. Development of maize cob-based biochar filter for water
purification. Water Environ. J. 2021, 35, 349–358. [CrossRef]
48. Li, J.; Wang, S.L.; Zhang, J.; Zheng, L.; Chen, D.; Shaheen, S.M.; Rinklebe, J.; Ok, Y.S.; Wang, H.; Wu, W. Coconut-fiber biochar
reduced the bioavailability of lead but increased its translocation rate in rice plants: Elucidation of immobilization mechanisms
and significance of iron plaque barrier on roots using spectroscopic techniques. J. Hazard. Mater. 2020, 389, 122117. [CrossRef]
[PubMed]
49. Prakongkep, N.; Gilkes, R.J.; Wiriyakitnateekul, W. Forms and solubility of plant nutrient elements in tropical plant waste
biochars. J. Plant Nutr. Soil Sci. 2015, 178, 732–740. [CrossRef]
50. Kimetu, J.M.; Lehmann, J.; Ngoze, S.O.; Mugendi, D.N.; Kinyangi, J.M.; Riha, S.; Verchot, L.; Recha, J.W.; Pell, A.N. Reversibility
of soil productivity decline with organic matter of differing quality along a degradation gradient. Ecosystems 2008, 11, 726–739.
[CrossRef]
51. Gao, S.; DeLuca, T.H.; Cleveland, C.C. Biochar additions alter phosphorus and nitrogen availability in agricultural ecosystems: A
meta-analysis. Sci. Total Environ. 2019, 654, 463–472. [CrossRef] [PubMed]
52. Mašek, O.; Brownsort, P. Biochar production. In An Assessment of the Benefits and Issues Associated with the Application of Biochar to
Soil; Shackley, S., Sohi, S., Eds.; UK Biochar Research Centre: Edinburgh, UK, 2011; pp. 37–44.
53. Yang, L.; Wu, Y.; Wang, Y.; An, W.; Jin, J.; Sun, K.; Wang, X. Effects of biochar addition on the abundance, speciation, availability,
and leaching loss of soil phosphorus. Sci. Total Environ. 2021, 758, 143657. [CrossRef] [PubMed]
54. Puga, A.P.; Abreu, C.A.; Melo, L.C.A.; Beesley, L. Biochar application to a contaminated soil reduces the availability and plant
uptake of zinc, lead and cadmium. J. Environ. Manag. 2015, 159, 86–93. [CrossRef] [PubMed]
55. Lebrun, M.; Macri, C.; Miard, F.; Hattab-Hambli, N.; Motelica-Heino, M.; Morabito, D.; Bourgerie, S. Effect of Biochar amendments
on As and Pb mobility and phytoavailability in contaminated mine technosols phytoremediated by Salix. J. Geochem. Explor. 2017,
182, 149–156. [CrossRef]
56. Bini, C.; Wahsha, M.; Fontana, S.; Maleci, L. Effects of heavy metals on morphological characteristics of Taraxacum officinale Web
growing on mine soils in NE Italy. J. Geochemical Explor. 2012, 123, 101–108. [CrossRef]
57. Maldonado-Magaña, A.; Favela-Torres, E.; Rivera-Cabrera, F.; Volke-Sepulveda, T.L. Lead bioaccumulation in Acacia farnesiana
and Its effect on lipid peroxidation and glutathione production. Plant Soil 2011, 339, 377–389. [CrossRef]
58. Shanker, A.K.; Cervantes, C.; Loza-Tavera, H.; Avudainayagam, S. chromium toxicity in plants. Environ. Int. 2005, 31, 739–753.
[CrossRef]
59. Saffari, N.; Hajabbasi, M.A.; Shirani, H.; Mosaddeghi, M.R.; Owens, G. Influence of corn residue biochar on water retention and
penetration resistance in a calcareous sandy loam soil. Geoderma 2021, 383, 114734. [CrossRef]
60. Dhar, S.A.; Sakib, T.U.; Hilary, L.N. Effects of Pyrolysis temperature on production and physicochemical characterization of
biochar derived from coconut fiber biomass through slow pyrolysis process. Biomass Convers. Biorefinery 2020, 12, 2631–2647.
[CrossRef]
61. Li, S.; Zhou, X.; Chen, J.; Chen, R. Is There a strategy i iron uptake mechanism in maize? Plant Signal. Behav. 2018, 13, e1161877.
[CrossRef]
62. Pourrut, B.; Shahid, M.; Dumat, C.; Winterton, P.; Pinelli, E. Lead uptake, toxicity, and detoxification in plants. Rev. Environ.
Contam. Toxicol. 2011, 213, 113–136. [CrossRef]
63. Li, J.; Zheng, L.; Wang, S.L.; Wu, Z.; Wu, W.; Niazi, N.K.; Shaheen, S.M.; Rinklebe, J.; Bolan, N.; Ok, Y.S. Sorption mechanisms of
lead on silicon-rich biochar in aqueous solution: Spectroscopic investigation. Sci. Total Environ. 2019, 672, 572–582. [CrossRef]
Plants 2022, 11, 3441 18 of 18

64. Li, J.; Wang, S.L.; Zheng, L.; Chen, D.; Wu, Z.; Xie, Y.; Wu, W.; Niazi, N.K.; Ok, Y.S.; Rinklebe, J. Sorption of lead in soil amended
with coconut fiber biochar: Geochemical and spectroscopic investigations. Geoderma 2019, 350, 52–60. [CrossRef]
65. Eissa, M.A. Effect of Cow manure biochar on heavy metals uptake and translocation by zucchini (Cucurbita pepo L). Arab. J. Geosci.
2019, 12, 48. [CrossRef]
66. Rechberger, M.V.; Kloss, S.; Wang, S.L.; Lehmann, J.; Rennhofer, H.; Ottner, F.; Wriessnig, K.; Daudin, G.; Lichtenegger, H.; Soja, G.
Enhanced Cu and Cd sorption after soil aging of woodchip-derived biochar: What were the driving factors? Chemosphere 2019,
216, 463–471. [CrossRef] [PubMed]
67. Kobayashi, T.; Nozoye, T.; Nishizawa, N.K. Iron transport and its regulation in plants. Free Radic. Biol. Med. 2019, 133, 11–20.
[CrossRef] [PubMed]
68. Shao, J.F.; Yamaji, N.; Shen, R.F.; Ma, J.F. The key to Mn homeostasis in plants: Regulation of Mn Transporters. Trends Plant Sci.
2017, 22, 215–224. [CrossRef] [PubMed]
69. Abbas, T.; Rizwan, M.; Ali, S.; Zia-ur-Rehman, M.; Farooq Qayyum, M.; Abbas, F.; Hannan, F.; Rinklebe, J.; Sik Ok, Y. Effect
of Biochar on cadmium bioavailability and uptake in wheat (Triticum aestivum L.) grown in a soil with aged contamination.
Ecotoxicol. Environ. Saf. 2017, 140, 37–47. [CrossRef]
70. Balafrej, H.; Bogusz, D.; Abidine Triqui, Z.E.; Guedira, A.; Bendaou, N.; Smouni, A.; Fahr, M. Zinc hyperaccumulation in plants:
A Review. Plants 2020, 9, 562. [CrossRef]
71. Fellet, G.; Marmiroli, M.; Marchiol, L. Elements uptake by metal accumulator species grown on mine tailings amended with three
types of biochar. Sci. Total Environ. 2014, 468, 598–608. [CrossRef]
72. Meers, E.; Ruttens, A.; Hopgood, M.; Lesage, E.; Tack, F.M.G. Potential of Brassic rapa, Cannabis sativa, Helianthus annuus and
Zea mays for phytoextraction of heavy metals from calcareous dredged sediment derived soils. Chemosphere 2005, 61, 561–572.
[CrossRef]
73. Zhuang, P.; Yang, Q.W.; Wang, H.B.; Shu, W.S. Phytoextraction of heavy metals by eight plant species in the field. Water Air Soil
Pollut. 2007, 184, 235–242. [CrossRef]
74. Felker, P.; Cannell, G.H.; Clark, P.R.; Osborn, J.F.; Nash, P. Biomass production of Prosopis species (mesquite), Leucaena, and other
leguminous trees grown under heat/drought stress. For. Sci. 1983, 29, 592–606.
75. Vangronsveld, J.; Cunningham, S.D. Introduction to the concepts. In Metal-Contaminated Soils: In-Situ Inactivation and Phytorestora-
tion; Vangronsveld, J., Cunningham, S.D., Eds.; Springer: Berlin/Heidelberg, Germany, 1998; pp. 1–15.
76. Vangronsveld, J.; Herzig, R.; Weyens, N.; Boulet, J.; Adriaensen, K.; Ruttens, A.; Thewys, T.; Vassilev, A.; Meers, E.;
Nehnevajova, E.; et al. Phytoremediation of contaminated soils and groundwater: Lessons from the field. Environ. Sci. Pollut. Res.
2009, 16, 765–794. [CrossRef]
77. Vangronsveld, J.; Colpaert, J.; Van Tichelen, K. Reclamation of a bare industrial area contaminated by non-ferrous metals:
Physicochemical and biological evaluation of the durability of soil treatment and revegetation. Environ Pollut. 1996, 94, 131–140.
[CrossRef]
78. IBI. International Biochar Initiative, Comparison of European Biochar Certificate Version 4. 8 and IBI Biochar Standards Version 2.0.
2014. Available online: https://www.biochar-international.org/wp-content/uploads/2018/04/IBI-EBC_comparison_Oct2014.
pdf (accessed on 1 December 2022).
79. Zar, J.H. Biostatistical Analysis, 5th ed.; Prentice Hall: Hoboken, NJ, USA, 2010.
80. R Core Team. R: A Language and environment for statistical computing. R Found. Stat. Comput. 2020. Available online:
https://www.R-project.org (accessed on 3 June 2022).
81. Statsoft, Inc. Statistica for Windows; Statsoft, Inc.: Tulsa, OK, USA, 2007.