Heavy metal accumulation in leaves and beans of cacao ( Theobroma cacao L.) in major cacao growing regions in Peru

Abstract

Peru is one of the leading exporters of organic cacao beans in the world. However, the accumulation of heavy metals in cacao beans represents a problem for cocoa bean export and chocolate quality. The aim of this study was to investigate the distribution and accumulation of heavy metals in cacao leaves and cocoa beans in three major cacao growing regions of Peru. The study was conducted in cacao plantations of 10 to 15 years old in three regions of Peru: North (Regions of Tumbes, Piura, Cajamarca, and Amazonas); Center (Regions of Huánuco and San Martin) and South (Junin and Cuzco). Samples of leaf and cacao beans were collected from 70 cacao plantations, and the nature of cacao clone or genotype sampled was recorded. The concentrations of heavy metals such as Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in leaves and beans were determined using atomic absorption spectrophotometer. Overall, concentrations of heavy metals were below the critical limits; however, the presence of high levels of Cd in cacao grown in Amazonas, Piura, and Tumbes regions is of primary concern. Plantations of cacao with different cacao clones show differences in Cd accumulation both in leaves and cocoa beans. Therefore, it is promising to screen low Cd accumulator cacao genotypes for safe production of cacao on lightly to moderately Cd contaminated soils. Also, synergism between Zn and Cd present both in plant and soil suggests that Zn has a direct effect on Cd accumulation in cacao.

Full text

Heavy metal accumulation in leaves and beans of cacao (Theobroma
cacao L.) in major cacao growing regions in Peru
Enrique Arévalo-Gardini
a,
⁎, Cesar O. Arévalo-Hernández
a
, Virupax C. Baligar
b
, Zhenli L. He
c
a
Instituto de Cultivos Tropicales (ICT),Tarapoto, Peru
b
U.S. Department of Agriculture/Agricultural Research Service, Beltsville Agricultural Research Center, Beltsville, MD 20705, USA
c
Department of Soil and Water Sciences, Indian River Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, Fort Pierce, FL 34945, USA
HIGHLIGHTS
•From 2019, the European Community
will begin to limit levels of cadmium
for chocolate affecting the Cocoa econo-
my of Peru
•N60% of the cacao beans studied had Cd
content bellow the critical level
•Concentration of Cd, Cr, Cu, Mn, Ni and
Zn in leaves were correlated with the
accumulation in cocoa beans
•The accumulation of heavy metal in
leaves and beans of cacao, are different
by genotypescomposition in the planta-
tions
•bCd and Pb accumulation in leaves and
cacao beans were in plantations with
more clonesand with natives genotypes
GRAPHICAL ABSTRACT
abstractarticle info
Article history:
Received 4 April 2017
Received in revised form 14 June 2017
Accepted 15 June 2017
Available online xxxx
Editor: D. Barcelo
Peru is one of the leading exporters of organic cacao beans in the world. However, the accumulation of heavy
metals in cacao beans represents a problem for cocoa bean export and chocolate quality. The aim of this study
was to investigate the distribution and accumulation of heavy metals in cacao leaves and cocoa beans in three
major cacao growing regions of Peru. The study was conducted in cacao plantations of 10 to 15 years old in
three regions of Peru: North (Regions of Tumbes, Piura, Cajamarca, and Amazonas); Center (Regions of Huánuco
and San Martin) and South(Junin and Cuzco).Samples of leaf and cacaobeans were collected from 70 cacaoplan-
tations, and the nature of cacao clone or genotype sampled was recorded. The concentrations of heavy metals
such as Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in leaves and beans were determined using atomic absorption spectro-
photometer. Overall,concentrationsof heavy metals were belowthe critical limits;however, the presenceof high
levels of Cd in cacao grown in Amazonas, Piura, and Tumbes regions is of primary concern. Plantations of cacao
with different cacao clones show differences in Cd accumulation both in leaves and cocoa beans. Therefore, it
is promisingto screen low Cd accumulator cacao genotypes for safe production of cacao on lightly to moderately
Cd contaminated soils. Also, synergism between Zn and Cd present both in plant and soil suggests that Zn has a
direct effect on Cd accumulation in cacao.
© 2016 Elsevier B.V. All rights reserved.
Keywords:
Cadmium
Lead
Zinc
Cacao beans
Food quality
Trace elements
Science of the Total Environment 605–606 (2017) 792–800
⁎Corresponding author.
E-mail addresses: e.arevalo@ict-peru.org (E. Arévalo-Gardini), v.c.baligar@ars.usda.gov (V.C. Baligar).
http://dx.doi.org/10.1016/j.scitotenv.2017.06.122
0048-9697/© 2016 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv

1. Introduction
In 2014, the European Union announced, by Regulation (EU) No
488/2014, plans to implement regulations governing chocolate and
cocoa products containing excessive levels ofCd, which will bein effect
on January 1, 2019 (EU, 2014). Currently, the Codex Alimentarius Com-
mission of the Joint FAO/WHO Food Standards Programme is in the pro-
cess of soliciting comments on the proposed draft maximum levels for
Cd in chocolate and cocoa derived products (EU, 2014). Non-
compliance with the Regulationswill bear significant economic and so-
cial consequences for cocoa producing countries from 2019. Cadmium
has received attention in the last decade due to its importance in food
quality and security, and human health, since consumption of food
with high Cd content could produce renal tubular dysfunction, forma-
tion of kidney stones, disturbance of calcium metabolism and skeletal,
endocrine, reproductive and respiratory defects (Järup and Åkesson,
2009; Tripathi et al., 2007; Salem et al., 2000; WHO, 2010).
In general, Latin America possess higher levels of heavy metals in
cacao beans, especially Cd and Pb (Bertoldi et al., 2016), as compared
with other producers in the world (West Africa) (Takrama et al., 2015).
The presence of heavy metals in cocoa beans poses a threat to cacao pro-
ducers, since high contents of heavy metals could affect the exportation of
cocoa beans. This crop is in high demand for manufacturing cacao liquor,
cacao butter, and chocolate, etc. Chocolate has been attributed to attain-
ment of optimal human health and development due to its high content
of flavonoids that are crucial in reducing the risk or delaying the develop-
ment of cardiovascular disease, cancer and other age-related diseases
(Cooper et al., 2008; Afoakwa, 2008). Heavy metals are defined as ele-
ments with a density exceeding 5 g cm
−3
(Ali et al., 2013). Copper, Fe,
Mn and Zn at low concentrations are essential for biochemical and phys-
iological processes in plants, while As, Cd, and Pb have no known role in
plants (Benavides et al., 2005). Accumulation of heavy metals in plants
is affected by several factors such as pH, organic matter content and tex-
ture of soil, plant genotype and heavy metal content in growth medium
(Businelli et al., 2009; Alloway, 2013). Heavy metal accumulation in
plants varies between elements and plant species (Vaculík et al., 2012;
Jan and Parray, 2016). Cadmium is one of the most mobile elements
with a bioconcentration factor in plants greater than some essential nutri-
ents, and mostly accumulates in shoots while Pb mainly accumulates in
roots (Kabata-Pendias, 2011; Li et al., 2014). Elemental interactions influ-
ence heavy metal accumulation in shoots and fruits, and plants with ade-
quate levels of essential nutrients usually contain less heavy metal such as
Cd (Sarwar et al., 2010). Nevertheless, interactions among these elements
could lead to a higher accumulation of heavy metals in plants (Nan et al.,
2002); Zinc and Cd interactions are controversial, both synergism and an-
tagonism being reported, while P and As interactions were shown to be
antagonistic (Kabata-Pendias, 2011; Alloway, 2013).
Since high concentration of heavy metals in cacao beans threatens
food security and economic development, there is a great need for infor-
mation regarding concentrations of heavy metals in this crop. In Peru,
cacao plantations have increased considerably; and the cultivated area
currently has reached N107,000 ha with a total cocoa bean production
of 82,000 mt/year (MINAGRI, 2016), the export of cocoa beans is vital
to its international trade. With the impending implementation of
these Regulations (EC Regulation No 1881/2006) on Maximum Permis-
sible levels for Cd, it is crucial that all cocoa producing countries under-
take this type of research as shown in this study, which was aimed to
investigate the accumulation of heavy metals in leaves and cocoa
beans in the major cacao production regions of Peru.
2. Material and methods
2.1. Location and cacao plants
In Peru, cacao is mainly propagated by seeds (for rootstock) and af-
terwards, grafted with the desired clone, most of cacao plantations in
Peru are dominated by CCN-51.The study was conducted in 70 cacao
plantations between 10 and 15 years of age, located in North (Amazo-
nas, Cajamarca, Piura and Tumbes Regions); Center (Huánuco, San Mar-
tin and Junin Regions) and South (Cuzco Region), the main cacao
growing regions of Peru (Fig. 1). In each sampled plantation, the genetic
material of planted cacao was characterized. The Table 1,showsthege-
notypes and genotypes associations identified in each region.
2.2. Sampling of soil
Soil samples were collected at the depth (0–20 cm), where the roots
of cacao have more intense activity of nutrients absorption (Wood and
Lass, 1985), in the cacao plantations selected in each region between
Februaryand April of 2014. The collected soils were air dried and sieved
prior to analysis. The results of soil analysis and content of heavy metals
in each selected region were reported earlier by Arévalo-Gardini et al.
(2016).
2.3. Sampling of leaves and fruits
Samples of leaves and fruits were collected between February and
April of 2014, the samples were collected from the central part of the
trees located near soil sampling sites described by Arévalo-Gardini
et al. (2016). For each plantation, three plots were randomly selected
for sampling. Within each plot, 10 cacao trees were selected, and one
middle-aged leave was sampled in each cardinal point of the tree.
Fruits were sampled according to its availability in each tree (at
least one). After collection, leaves and fruits samples were properly
labeled and transported to Instituto de Cultivos Tropicales (ICT) Lab-
oratory in Tarapoto-Peru, where they were thoroughly rinsed with
distilled water. Beans were extracted from each fruit and mixed for
a composite sample. Separately, leaves and beans were oven dried
at 60 °C, mixed and milled (20 mesh) and stored in plastic bag
prior to analysis.
2.4. Determination of heavy metals in leaves and beans
Oven dried leaf or bean samples were weighed (500 mg each) and
digested with 10 ml of a mixture of HNO
3
(65%) and HClO
4
(98%)
from Merck® in 4:1 ratio respectively (Nogueira et al., 2005). The diges-
tion was conducted on a block at 120 °C for 3 h and then at 200 °C for 2 h
(Güldaş,2008). The digested solution was filtered through a Whatman
N°42 filter paper, and diluted prior to analysis. Concentrations of Cd, Cu,
Cr, Fe, Mn, Ni, Pb and Zn in the filtrate were determined using atomic
absorption spectrophotometer (SAA; Varian model “Spectra 55B”
Made in Australia). In order to achieve analytical quality, three repeti-
tions were carried for each sample and the means were used for statis-
tical analysis.
2.5. Statistical analysis
All the statistical analyses wereperformed in R, ver 3.2 (RCoreTeam,
2014). Concentrations of Cd, Cu, Cr, Fe, Mn, Ni, Pb, and Zn in leaves and
beans were submitted to analysis of variance (ANOVA) for each studied
region; the same analysis was conducted for genotype from each region
sampled to assess genotypic differences in heavy metal accumulation.
Region wide means were compared by the DGC (Di Rienzo et al.,
2014) test (P≤0.05). A Pearson's correlation was carried out between
concentrations of Cd, Cu, Cr, Fe, Mn, Ni, Pb and Zn in leaves and beans
in cacao. Finally, soil attributes were correlated with concentrations of
the metals in beans, and a cluster analysis was performed using
ClustofVar R package (Chavent et al., 2010).
793E. Arévalo-Gardini et al. / Science of the Total Environment 605–606 (2017) 792–800

3. Results and discussion
3.1. Regional variation in metal concentrations of cacao leaves and beans
Mean concentrationsof Cd, Cu, Cr, Fe, Mn, Ni, Pb and Zn in leaves of
cacao, per region stud ied is presented in Fig. 2.Significant differences
(P≤0.05) for Cu, Cr, Fe, Mn, and Zn were observed, except for Cd, Ni
and Pb (PN0.05)(Table S1).
Concentrations of Cd in leaves ranged from 0.23 ± 0.62 μgg
−1
in
Cuzco to 2.50 ± 0.62 μgg
−1
in Tumbes; Cr of 1.0 ± 0.73 μgg
−1
in
Huanuco to 2.86 ± 0.31 μgg
−1
in Amazonas; Cu showed a range of
7.17 ± 0.62 μgg
−1
in Piura to 10.25 ± 1.08 μgg
−1
in Tumbes; Fe of
50.5 ± 24.75 μgg
−1
in Junin to 275.77 ± 13.73 μgg
−1
in Piura; Mn of
88.20 ± 58.05 μgg
−1
in Cajamarca to 347.38 ± 36.00 μgg
−1
in San
Martin; Ni from 2.25 ± 3.28 μgg
−1
Cajamarca and 12.20 ± 2.94 in
Cuzco; Pb of 1.20 ± 0.32 μgg
−1
in Cajamarca to 2.50 ± 0.36 μgg
−1
in
Junin; Zn of 43.80 ± 12.17 μgg
−1
in Cuzco to 103.95 ± 4.36 μgg
−1
in
Amazonas. In leaves, the highest mean values of these elements were
observed in Tumbes (Cd, Cu), Amazonas (Cr, Zn), San Martin (Mn),
Piura (Fe), Junin (Pb), and Cuzco (Ni).
In Peru, Huamani-Yupanqui et al. (2012) reported elemental con-
centration values in cacao leaves,which were within the concentration
ranges reported in this study, except that the means of Cd, Mn, Pb and
Zn were lower. This could be explained by the higher concentrations
of these elements in the north region; for the same locations mean
values were similar. In cacao leaves, Aikpokpodion (2010), in Nigeria,
reported higher elemental concentration for Fe and lower for Mn and
Zn, even though soil pH values of the soils assessed was N6.5, indicating
that probably these types of soils had higher background levels of these
heavy metals. Sodré et al. (2001) in Bahía, Brazil reported higher values
of Cu and Zn concentrations in cacao leaves while lower for Fe and Mn.
In Trinidad and Tobago, Ramtahal et al. (2016) reported higher concen-
tration values for Cd in leaves. In Bolivia, Gramlich et al. (2017) ob-
served, in general, lower values for Cd, higher for Fe, and within the
range of this study for Zn.
In cacao beans, the mean concentration of Cd, Cu, Cr, Fe, Mn, Ni, Pb
and Zn in cacao beans per region studied are presented in Fig. 2.Signif-
icant differences (P≤0.05) in concentrations of Cu, Cr, Mn, Pb and Zn
were observed, while insignificant differences (PN0.05) were noted
for Cd, Fe and Ni (Table S1).
Fig. 1. Location of cacao plantations sampled for heavy metalanalysis in Peru. (Google earth*). Theplantation stretched from the northeast point (3.10°S; 77.83°W) to the southeastpoint
(12.41°S; 72.52°W).
Table 1
Cacao genotypes sampled for determinations of heavy metals in leaf andcacao beans in
each region of Peru.
Region Department Cacao genotypes
North Tumbes CCN51, Spontaneous hybrids
Piura Blanco Piurano, Spontaneous hybrids
Cajamarca CCN51, Native from Marañon
Amazonas CCN51/ICS95
Center San Martín CCN51, CCN51/ICS95, CCN51/ICS95/ICS39
Huánuco CCN51
Junín CCN51, CCN51/ICS95/ICS39, Native from Satipo
South Cuzco CCN51/ICS95/ICS39, Chuncho
794 E. Arévalo-Gardini et al. / Science ofthe Total Environment 605–606 (2017) 792–800

Mean concentrations of Cd in beans ranged from 0.17 ± 0.41 μgg
−1
in
Cuzcoto1.78±0.35μgg
−1
in Tumbes. Concentration of Cu of 18.75 ±
3.91 μgg
−1
in Cuzco to 30.41 ± 1.41 μgg
−1
in Amazonas. Chromium
concentration ranged from 1.00 ± 0.87 μgg
−1
inSanMartinto4.83±
0.71 μgg
−1
in Amazonas. Concentrations of Fe were from 34.00 ±
5.95 μgg
−1
in Cuzco to 53.55 ± 3.59 μgg
−1
in Piura. Concentration
of Mn ranged from 13.33 ± 3.76 μgg
−1
in Junin to 28.5 ± 3.25 μgg
−1
in Huanuco. Nickel concentration values ranged from 3.5 ± 2.00 μgg
−1
in Tumbes to 9.25 ± 2.00 μgg
−1
in Huanuco. Concentrations of Pb
were between 1.00 ± 0.67 μgg
−1
in Cajamarca and Cuzco to 3.78 ±
0.39 μgg
−1
in Piura. Zinc varied from 37.25 ± 2.70 μgg
−1
in Huanuco
to 59.17 ± 1.56 μgg
−1
in Piura. Nearly 57% of all collected samples
exceeded this critical limit (0.8 μgg
−1
for Cd), indicating that it is neces-
sary to execute a national plan in Peru to prevent Cd contamination in
cacao production systems.
Lee and Low (1985), in Malaysia, reported concentration within
the range observed in this study for Cu and Cd while it was higher
for Pb. In Nigeria, Aikpokpodion (2010) reported higher concentra-
tions of Fe, Mn and Zn in cacao beans. Afoakwa et al. (2013),in
Ghana, reported lower cacao bean concentrations of Cu, Fe and Zn
in comparison with our study. In Brazil, Loureiro (2014) reported
lower concentration values in cacao beans for Cu, Zn and Cd, higher
for Fe and similar for Mn as compared with the results from this
study. Chavez et al. (2015), studied Cd in soils and plants of
Ecuador, and Ramtahal et al. (2016), in Trinidad and Tobago, report-
ed higher concentration values of Cd in comparison to our research,
however our values were within the ranges reported in those stud-
ies. In Bolivia, Gramlich et al. (2017), observed, in general, lower
values for Cd and Fe and similar values for Zn.
3.2. Concentrations of metals in leaves and beans of different cacao
accessions
Mean concentrations (±SE) of Cd, Cu, Cr, Fe, Mn, Ni, Pb and Zn from
cacao leaves and beans of genotypes invarious regions of Peru, are pre-
sented in Fig. 3. In this study significant differences (P≤0.05) in leaf and
bean concentrations were found for Cd, Cr, Cu, Fe and Zn. Concentra-
tions of Cd,Cr, Cu, Fe, Mn, Pb and Zn in cacao beans were statistically sig-
nificant among the different genotypes (P≤0.05) (Table S2).
Mean concentrations of Cd in leaves was in the range of 0.10 ± 0.9
μgg
−1
(Native from Marañon) to 2.6 ± 0.7 μgg
−1
(Spontaneous Hy-
brids). Chromium had a low concentration of 0.7 ± 0.8 μgg
−1
(Chuncho) with maximum value of 4.4 ± 0.3 μgg
−1
(CCN51/ICS95/
ICS39). Mean Cu concentration ranged from low of 7.00 ± 0.6 μgg
−1
(Blanco Piurano) to high of 11.3 ± 0.5 μgg
−1
(CCN51/ICS95/ICS39).
Mean Fe concentrations ranged from 76.3 ± 29.9 μgg
−1
(Chuncho) to
243 ± 16.4 μgg
−1
(Blanco Piurano). Manganese concentrations were
in the range of 72.3 ± 75.5 μgg
−1
(Spontaneous Hybrids) to 230.0 ±
38.0 μgg
−1
(CCN51). Mean concentrations of Ni ranged from 3.0 ±
4.7 μgg
−1
(Native from Marañon) to 15.0 ± 3.8 μgg
−1
(Chuncho).
Lead had a low concentration of 0.94 ± 0.3 μgg
−1
(CCN51/ICS95/
ICS39) to 3.0 ± 1.0 μgg
−1
(Native from Satipo). Mean concentrations
of Zn ranged from low of 44.7 ± 18.4 μgg
−1
(Chuncho) to 105.0 ±
31.8 μgg
−1
(Native from Satipo).
Similar trends occurred to metal concentrations in beans of different
cacao genotypes. Mean concentration of Cd in cacao beans ranged from
0.15 ± 0.8 μgg
−1
(Native from Satipo) to 1.8 ± 0.4 μgg
−1
(Spontane-
ous Hybrids). Chromium mean concentrations in Chuncho and Native
from Satipo were below the limit of detection, also low values were
Fig. 2. Meanconcentrationsof Cd, Cr, Cu, Fe, Mn, Ni,Pb and Zn in cacao leavesand beans per regionin Peru, i.e. Tumbes(TUM), Piura (PIU),Cajamarca (CAJ),Amazonas (AMA), SanMartín
(SMA), Huánuco (HUA), Junin (JUN), Cuzco (CUZ).
795E. Arévalo-Gardini et al. / Science of the Total Environment 605–606 (2017) 792–800

found from 0.2 ± 0.4 μgg
−1
(CCN51/ICS95) to 5.1 ± 0.5 μgg
−1
(CCN51/ICS95/ICS39). Mean concentrations for Cu ranged from
19.7 ± 4.3 μgg
−1
(Chuncho) to 38.1 ± 2.0 μgg
−1
(CCN51/ICS95/ICS39).
Concentrations of Fe were from 34.0 ± 6.5 μgg
−1
(Chuncho) and
63.0 ± 8.0 μgg
−1
(Native from Marañon). Concentrations of Mn were
from 14.5 ± 4.3 μgg
−1
(Spontaneous hybrids) to 32.0 ± 8.5μgg
−1
(Na-
tive from Satipo). Concentrations of Ni were from 3.3 ± 2.1 μgg
−1
(Spontaneous hybrids) to 10.0 ± 4.2 μgg
−1
(Native from Satipo). Con-
centrations of Pb were from 0.67 ± 0.7 μgg
−1
(Chuncho) to 3.0 ± 1.3
μgg
−1
(Native from Satipo). Concentrations of Zn were from 34.7 ±
3.4 μgg
−1
(Chuncho) to 59.0 ± 1.8 μgg
−1
(Blanco Piurano). Highest
mean values of heavy metal concentrations in cacao beans were record-
ed in genotypes Blanco Piurano (Zn); CCN51/ICS95/ICS39 (Cr, Cu);
Spontaneous hybrids (Cd); Native from Marañon (Fe) and Native from
Satipo (Mn, Ni, Pb).
Plants have differential responses to soil contamination with heavy
metals (Kabata-Pendias, 2011). Some plant species or genotypes can
tolerate high concentration of heavy metals in soil by excluding these
elements from entering into root or by volatilization through stomata
(Peer et al., 2005; He et al., 2015). For perennial plants, Zacchini et al.
(2009) reported differential accumulation patterns of Cd in Poplar
clones; while Willow clones showed higher tolerance to Cd toxicity.
For cacao, literature that describes the accumulation of heavy metals
by different genotypes is limited. However, Castro et al. (2015) and
Reis et al. (2015), in Bahia-Brazil, worked with cacao seedlings from a
self-fertilization of Catongo and a cross of CCN51 × SCA6, and assessed
the effects of Cd and Pb, respectively. They reported that the progeny
of the cross of CCN51 × SCA6 was more tolerant to highCd and Pb stress,
as comparedwith the progeny of Catongo, indicatingthat there is differ-
ential response of the plant species/genotypes to heavy metal stress.
Also, associated to genotype differences, production systems and soil
management could have an important effect onheavy metal absorption
in cacao; Gramlich et al. (2017), studied cacao cropping systems associ-
ated to organic and conventional managementin agroforestry or mono-
culture; the authors report that even though no significant differences
were found for Cd and Zn in leaves and beans, TSH 565 absorbed
more Cd in relation to ICS 1 in monoculture. Nevertheless, Fe in leaves
was more accumulated in the conventional system in relation to the or-
ganic system and TSH 565 absorbed more Fe in relation to ICS 1, no sig-
nificant differences were reported for beans. These results indicate that
the genetic factor could be an important strategy in agriculture systems
with excess heavy metals in plants.
3.3. Critical limits of heavy metals in cacao leaves and cacao beans
Since there is no consensus in critical limits for Cd concentration
levels in cacao leaves and beans, we used optimal values and critical
limits for cacao leaves established by Sodré et al. (2001);Broadley
et al. (2012) and Kabata-Pendias (2011) to evaluate the data from the
present study. For bean Cd concentration, the critical value of 0.8
μgg
−1
, established by the European community and becoming effective
on the first of January of 2019, as maximum permissible for chocolate
containing ≥50% of cacao beans solids will be used for assessment. For
the other elements, the ranges established by Kabata-Pendias (2011)
will be employed as a reference. For optimal limits in cacao beans, the
values established by Araujo et al. (2014) were adopted for comparison.
In cacao leaves, all the elements studied, in general, presented con-
centrations below the maximum limits regardless of regions assessed.
However, for Cd, the values per region were above the normal concen-
tration (0.2 μgg
−1
) for most plants (Kabata-Pendias, 2011). The mean
Fig. 3. Mean concentrations of Cd, Cr, Cu, Fe, Mn, Ni, Pb y Zn in cacao leaves and beans per genotype found in the regions studied in Peru.
796 E. Arévalo-Gardini et al. / Science ofthe Total Environment 605–606 (2017) 792–800

Ni concentration in Cuzco was higher that the tolerable crop value
(10 μgg
−1
) established by Kabata-Pendias (2011).Pbconcentra-
tions in leaves at all regions were below the value of b4.3 μgg
−1
sug-
gested by Araujo et al. (2014).
For beans,concentrations of Cr, Cu, Fe, Mn, Ni, Pb, and Zn per region
were in general, higher than the optimal values established by Araujo
et al. (2014), but below the values reported as tolerable in agronomic
crops by Kabata-Pendias (2011) of 2 μgg
−1
;50μgg
−1
; 300 μgg
−1
;
50 μgg
−1
; 300 μgg
−1
for Cr, Cu, Mn, Ni and Zn, respectively. Bean Cd
concentrations in Amazonas, Piura and Tumbes exceed the maximum
permissible limit of 0.80 μgg
−1
. Cuzco reported bean Cd concentration
above the normal value of 0.20 μgg
−1
(Araujo et al., 2014). Cuzco was
the only region that presented normal values for this element, indicat-
ing that Cd contamination of cacao beans may be a problem to other re-
gions. Further studies are needed to confirm the potential risk of Cd
contamination in these regions including Amazonas, Piura and Tumbes.
Overall, concentrations of heavy metals in leaves of various geno-
types (Fig. 3) were below the critical limits established for agronomic
crops (Kabata-Pendias, 2011). However, in cacaoplantations withgeno-
types CCN51 and ICS95, Cd exceeded the tolerable value (3.0 μgg
−1
)
proposed for agronomic crops Kabata-Pendias (2011),indicatinga
higher accumulation of Cd in these plantations probably dueto grafting
combination, where the plants from spontaneous hybrids were used as
rootstock, since CCN51 clone alone had a leaf Cd concentration 50%
lower than that of the combination of clones (CCN51/ICS95). Further-
more, the combination in the plantation of clones CCN51/ICS95/ICS39,
where the plants from spontaneoushybrids were usedas a rootstock ac-
cumulated less Cd in relation to the combination of CCN51/ICS95 and
CCN51 itself. This implies that the combination of more clones may re-
duce Cd concentration in leaves and the rootstock could be an efficient
alternative for preventing Cd accumulation in shoots. Further studies
are needed to assess the genotypic differences for Cd uptake efficiency
in soils from various cacao growing regions.
The assessed genotypes had Cd concentrations in beans (Fig. 3)
mostly between the normal levels (Araujo et al., 2014) and critical limits
proposed by Kabata-Pendias (2011). However, the clones and combina-
tions of Blanco Piurano, CCN51, CCN51/ICS95 and spontaneous hybrids
had bean Cd concentrations exceeding the critical limit of 0.80 μgg
−1
established by Commission of Regulation (EU) (EU, 2014), implying
that the use of these combinations should be used with caution in the
strategies of diminishing Cd accumulation in cacao beans, especially in
regions with soil Cd enrichment. On the other hand, the combination
of clones CCN51/ICS95/ICS39 could lead to low Cd concentration in
beans and merit attention for futurestrategy in diminishing Cd accumu-
lation. Chuncho and Native Marañon should be considered high in soil
Cd. However more controlled research is needed to confirm their traits
related to Cd uptake and accumulation in beans. Effects of rootstocks
and scion on the accumulation and translocation of heavy metals have
been reported by Arao et al. (2008) working with Solanum melongena
for Cd and Rouphael et al. (2008) in Cucumis sativus for Cu. This ap-
proach needs to be considered in the assessment of various cacao clones
for their uptake and translocation efficiency for Cd either as single clone
or as grafted material, since genetic differences and their interaction
could be an efficient strategy in Cd absorption in cacao cropping
systems.
3.4. Relationship between concentrations of heavy elements in cacao leaves
and beans
Complex interactions between heavy metals occur in plants that
could affect their absorption, levels of toxicity and regulatory mecha-
nisms. The interactions can be additive, synergistic or antagonistic de-
pending on environmental conditions and plant species (Siedlecka,
1995; Kabata-Pendias, 2011). These relationships could give an overall
idea of the possible practices that can lead to efficient management of
heavy metals in cacao production regions.
The correlation matrix of Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn concentra-
tions in cacao beans and leaves are presented in the Table 2. Significant
(P≤0.05) correlations were observed for concentrations of Cd, Cr, Cu,
Mn, Ni, andZn while non-significant correlations occurred forFe and Pb.
Cadmium in cacao beans had a significant (P≤0.05) positive correla-
tion (0.21)with Zn in leaves which is contradictory to some previous re-
ports that Zn is antagonistic with Cd uptake (Kabata-Pendias, 2011;
Alloway, 2013). Cadmium toxicity may, in part, result from its interac-
tions with mineral nutrients such as Zn, Fe, Ca, K, Mn, Cu and Mg
(Wang et al., 2007; Nedjimi and Daoud, 2009). Metals that can increase
or decrease uptake of other elements are readily absorbed and
translocated to plant shoots, and these processes are competitive be-
tween elements, especially between Cd and Zn (Mengal et al., 2001).
However, Zn and Cd could be simultaneously absorbed due to their
ionic similarities; therefore, Cd is mainly absorbed by the same carriers
as Ca
+2
,Fe
+2
,Mg
+2
,Cu
+2
and Zn
+2
(Papoyan et al., 2007); principally
in sites where Zn has a crucial catalytic or structural function (Tang
et al., 2014). Other reports indicated that Zn favored Cd absorption
(Synergistic effect) in tobacco (Nicotiana tabacum)(Tkalec et al.,
2014); maize (Zea mays)(Nan et al., 2002); barley (Hordeum vulgare)
(Nan et al., 2002) and spring wheat (Triticum spp.) (Piotrowoska et al.,
1994).
The concentrations of Ni in beans had significant (P≤0.05) correla-
tions with Mn (0.31) and Ni (0.68) in leaves. Chen et al., 2009 reported
inhibitory effects of high concentrations of Ni on Mn uptake in Oryza
sativa.Cataldo et al. (1978) reported inhibitory effects of Ni on Mn in
soybean (Glycine max) seedlings; Heale and Ormrod (1982) reported
the same results in Acer rubrum,Cornus stolonifer,Lonicera tatarica and
Pinus resinosa. This behavior can be explained by the similar character-
istics of Ni in relation to Mn, resulting in competition for uptake. How-
ever since concentrations of Ni in cacao leaves were mostly below the
tolerable levels for agronomical crops (50 μgg
−1
)(Kabata-Pendias,
2011), and thus, competition in uptake between these metals may not
occur in cacao.
Bean Zn concentration had significant (P≤0.05) correlations with
concentration of Zn (0.26), Mn (−0.33), Fe (0.50), Ni (−0.30) and Cd
(0.21) in leaves. Zinc has a significant role in the translocation of Fe
from roots to aerial parts; therefore, plants with higher contents of Zn
have a tendency to accumulate higher concentrations of Fe (Rengel
and Graham, 1995; Rengel et al., 1998). Imtiaz et al. (2003) reported
negative effects of Mn concentrations in soil solution on Zn nutrition
in wheat plants; without application of Zn, the Mn concentration in
plants was high. Due to similar characteristics of Zn and Ni, competition
of these sitesis expected (Cataldoet al., 1978; Heale andOrmrod, 1982).
The positive correlation between Zn concentration in beans and Cd con-
centration in leaves confirms the synergism of these elements in cacao
plants sampled, indicating that interactions with other metals should
be considered in an efficient management plan for Cd absorption in
cacao plants.
Table 2
Correlation matrixof Cd, Cu, Cr, Fe, Mn, Ni,Pb y Zn in leaves and beansof cacao plants sam-
pled in different regions of Peru.
Elements in
cacao beans
(n= 72)
Elements in cacao leaves (n= 72)
Cu Zn Mn Fe Pb Ni Cd Cr
Cu 0.51⁎0.20 −0.12 0.03 −0.07 −0.19 0.12 0.05
Zn 0.02 0.26 −0.33 0.50 −0.14 −0.30 0.21 0.05
Mn −0.01 −0.03 0.58 −0.11 0.05 0.19 0.04 0.00
Fe −0.04 0.12 −0.09 0.12 −0.19 −0.06 0.06 0.14
Pb −0.06 −0.13 0.05 0.10 0.12 −0.02 −0.15 −0.01
Ni 0.00 0.19 0.31 0.00 −0.05 0.68 0.16 −0.16
Cd 0.07 0.21 −0.05 0.21 −0.08 −0.09 0.74 0.07
Cr 0.07 −0.15 −0.10 0.03 0.04 −0.12 −0.06 0.21
⁎Values in bold are significant at P≤0.05.
797E. Arévalo-Gardini et al. / Science of the Total Environment 605–606 (2017) 792–800

3.5. Soil attributes and heavy metals in cacao beans
The relationships of soil attributes and concentration of heavy
metals (Cd, Cr, Cu, Fe, Mn,Ni, Pb and Zn)in cacao beans are represented
in Fig. 4. These results confirmed the observed correlations between
beans and leaves of cacao, where synergy of these elements and their
joint absorption occurred. The ratio of Cd over Fe or Zn in beans was
synergistic, this may be due to the use of the same transporters for
their entry into the cells (Broadley et al., 2012). Nan et al. (2002) report-
ed significant positive correlations between total Cd in soil and Cd in the
plant of wheat. Chavez et al. (2015) reported highly significant correla-
tions between the concentrations of extractable Cd in the soil (Melich-3
and HCl) and the Cd content in cacao beans, indicating that the method-
ologies used in the study are appropriate for the prediction of available
content of Cd on the soil. Ramtahal et al. (2016) indicated that the
concentration of Cd is mainly distributed in leaves and in a lower concen-
tration in grains and soil; these authors reported significant correlations
(P≤0.05) between the concentration of Cd in soil, leaves and cacao beans.
These results confirm the hypothesis proposed by Nan et al. (2002) that,
in general, significant correlations between Cd and Zn are observed in the
field, while the opposite occurred under nursery conditions.
Concentration of Pb in beans was related to the presence of silt,
sand, Fe, Zn and Cu in the soil. This can be attributed to the interac-
tions of Fe, Zn and Cu with Pb in competing for absorption by the
plant (Alloway, 2013). Nan et al. (2002) reported positive correla-
tions between Pb concentration in beans and Zn concentration in
the soil. The concentration of Ni in beans was related to the availabil-
ity of Cr, Ni, S and P in the soil.
On the other hand, the concentration of Cu in beans was mainly re-
lated to the concentrations of Pb and Mn in the soil. Manganese concen-
tration in beans was related to pH, since it is the main soil factor that
determines Mn availability (Alloway, 2013). Finally, the concentration
of Cr in beans was related to available K, B, Mg and electrical conductiv-
ity of the soil (CE).
4. Conclusions
The European Union has announced that the addition to Regulation
1881 of 2006 begins to be mandatory from January 2019, as this may af-
fect the Cocoa economy of Peru. From 2019, the European Community
will begin to demand maximum levels of Cd for cocoa and its deriva-
tives. The European Union notified the Committee on Sanitary and
Phytosanitary Measures of the World Trade Organization (EC) No
1881/2006. Three major cacao-producing regions (North, Center and
South) of Peru were selected for assessing the uptake and accumulation
of heavy metals in cacao leaves and beans. The results indicated that
concentrations of Cu, Fe, Mn, and Zn in cacao leaves were adequate
and beans concentrations of Cr, Cu, Fe, Mn, Ni, Pb, and Zn were below
the critical limits. However high levels of Cd occurred in cacao beans
in Piura and Tumbes, of Peru. This may pose a threat to cacao production
in these regions. There were genotypic differences in Cd uptake and ac-
cumulation in cacao leaves and beans. Some clones or combinations ac-
cumulated less Cd in leaves and beans under the same soil Cd
conditions, indicating that this may represent an opportunity to
exploiting low Cd accumulators for future breeding and production of
cacao in the high Cd background regions. Cadmium and Zn interactions
were foundin both plant and soil, indicating that Zn mayhave direct im-
plications in Cd accumulation in cocoa beans.
Conflict of interest
The authors declare that they have no conflicted of interest.
Acknowledgments
We are grateful to “Programa Nacional de Innovación para la
Competitividad y Productividad (Innóvate Perú)”,forfinancial support,
Contract No 167-FINCyT-IB-2013. Special deference to Velia Maruxie
Yufra Picardo and Carmen Rosa Maza Córdova for opportune advisor
Fig. 4. Dendrogram withsoil attributes and concentration of Cd, Cr,Cu, Fe, Mn, Ni, Pb and Zn in cacao beans for all the studied areas. The mean concentrations for these elements are
represented by BCd, BCr, BCu, BFe, BMn, BNi, BPb and BZn, respectively.
798 E. Arévalo-Gardini et al. / Science ofthe Total Environment 605–606 (2017) 792–800

to develop this work. To United State Department of Agriculture –Agri-
cultural Research Service (USDA-ARS) for technical and financial sup-
port. To Indian River Research and Education Center (IRREC),
University of Florida, USA for technical support (Cooperative Agreement
ICT-IRREC/UF). To US Embassy in Lima-Perú for partial financial support
to this study. To “Instituto de Cultivos Tropicales –ICT”for facilities of
infrastructure and laboratories. To all Cacao Organizations of Peru for fa-
cilities of samples obtained. We thank Marshall Elson for review of the
manuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the
online version, at doi: http://dx.doi.org/10.1016/j.scitotenv.2017.06.
122. Thesedata include the Google map of the most important areas de-
scribed in this article.
References
Afoakwa, E.O., 2008. Cocoa and chocolate consumption —are there aphrodisiac and other
benefits for human health. S. Afr. J. Clin. Nutr. 21 (3), 107–113.
Afoakwa,E.O., Quao, J., Takrama, J., Budu,A.S., Saalia, F.K.,2013. Chemical composition and
physical quality characteristics of Ghanaian cocoa beans as affected by pulp pre-
conditioning and fermentation. J. Food Sci. Technol. 50:1097–1105. http://dx.doi.
org/10.1007/s13197-011-0446-5.
Aikpokpodion, P., 2010. Nutrients dynamics in cocoa soils, leaf and beans in Ondo State,
Nigeria. J. Agric. Sci. 1 (1), 1–9.
Ali, H., Khan,E., Sajad, M.A., 2013. Phytoremediation of heavy metals.Concepts and appli-
cations. Chemosphere 91, 869–881.
Alloway, B.J., 2013. Heavy Metals in Soils: Trace Metals and Metallloids in Soils and their
Bioavailability. 3rd ed. Environmental Pollution Vol. 22. Springer, p. 614p.
Arao, T., Takeda, H., Nishihara, E., 2008. Reduction of Cadmium translocation from root to
shoots in eggplant (Solanum melongena) by grafting onto Solanum torvum rootstock.
Soil Sci. Plant Nutr. 54, 555–559.
Araujo, Q.R., Fernandes, C.A.F., Ribeiro, D.O., Efraim, P., Steinmacher, D., Lieberei, R.,
Bastide, P., Araujo, T.G., 2014. Cocoa quality index —a proposal. Food Control 46,
49–54.
Arévalo-Gardini, E., Obando-Cerpa, M.E., Zúñiga-Cernades, L.B., Arévalo-Hernández, C.O.,
Baligar, V., He, Z., 2016. Heavy metals in soils of cocoa plantations (Theobroma
cacao l.) in three regions of Peru. Ecol. Apl. 15, 81–89.
Benavides, M.P., Gallego, S.M., Tomaro, M.L., 2005. Cadmium toxicity in plants. Braz. J. Plant
Physiol. 17 (1):21–34. http://dx.doi.org/10.1590/S1677-04202005000100003.
Bertoldi, D., Barbero, A., Camin, F., Caligiani, A., Larcher, R., 2016. Multielemental finger-
printing and geographic traceability of Theobroma cacao beans and cocoa products.
Food Control 65, 46–53.
Broadley, M., Brown, P., Cakmak, I., Rengel, Z., Zhao, F., 2012. Function of nutrients:
micronutrients. In: Marschner (Ed.), Marschner's Mineral Nutrition of Higher Plants,
3rd edn Elsevier, pp. 191–248.
Businelli, D., Massaccesi, L., Said-Pullicino, D., Gigliotti, G., 2009. Long-term distribution,
mobility and plant availability of compost-derived heavy metals in a landfill covering
soil. Sci. Total Environ. 407, 1426–1435.
Castro, A.V., Almedia, A.A.F., Pirovani, C.P., Reis, G.S.M., Almeida, N.M., Mangabeira, P.A.O.,
2015. Morphological, biochemical, molecular and ultrastructural changes induced by
Cd toxicity in seedlings of Theobroma cacao L. Ecotoxicol. Environ. Saf. 115, 174–186.
Cataldo, D.A., Garland, T.R., Wildung, R.E., 1978. I: uptake kinetics using intact soybean
seedlings. Plant Physiol. 62, 563–565.
Chavent, M., Kuentz, V., Liquet, B., Saracco, J., 2010. The Clust of Var R Package. The CRAN
Project.
Chavez, E., He, Z.L., Stofella, P.J., Mylavarapu, R.S., Li, Y.C., Moyano, B., Baligar, V.C., 2015.
Concentration of cadmium in cacao beans and its relationship with soil cadmium in
southern Ecuador. Sci. Total Environ. 533, 205–214.
Chen, C., Huang, D., Liu,J., 2009. Functionsand toxicity of nickel in plants: recent advances
and future prospects. Clean J. 37, 304–313.
Cooper, A.K., Donovan, J.L., Waterhouse,A.L., Williamson,G., 2008. Cocoa and health:a de-
cade of research. Br. J. Nutr. 99, 1–11.
Di Rienzo, J.A., Casanoves, F., Balzarin, M.G., Gonzalez, L., Tablada, M., Robledo, C.W., 2014.
InfoStat versión 2014. Grupo InfoStat, FCA, Universidad Nacional de Córdoba,
Argentina http://www.infostat.com.ar.
EU, 2014. COMMISSION REGULATION (EU) No 488/2014 amending regulation (EC) No
1881/2006 as regards maximum levels of cadmium in foodstuffs. Off. J. Eur. Union
138, 75.
Gramlich, A., Tandy, S., Andres, C., Paniagua, J.C., Armengot, L., Schneider, M., Schulin, R.,
2017. Cadmium uptake by cocoa trees in agroforestry and monoculture systems
under conventional and organic management. Sci. Total Environ. 580, 677–686.
Güldaş, M., 2008. COMPARISON of digestion methods and trace elements determination
in chocolate with pistachio using absorption spectrometry. J. Food Nutr. Res. 47 (2),
92–99.
He, S., He, Z., Yang, X., Stoffella, P.J., Baligar, V.C., 2015. Soil biogeochemistry, plant physi-
ology and phytoremediation of cadmium contaminated soils. In: Sparks, D.L. (Ed.),
Advances in Agronomy. 134:pp. 135–225. http://dx.doi.org/10.1016/bs.agron.2015.
06.005 (ISSN 0065-2113).
Heale, E.L., Ormrod, D.P., 1982. Effects of nickel and cooper on Acer rubrum and
Cornus stolonifers,Lonicera tatarica,andPinus resinosa.Can.J.Bot.60,
2674–2681.
Huamani-Yupanqui, H.A., Huauya-Rojas, M.A., Mansilla-Minaya, L.G., Florida-Rofner, N.,
Neira-Trujillo, G.M., 2012. Presence of heavy metal in organic cacao (Theobroma
cacao L.). Acta Agron. 61 (4), 339–344.
Imtiaz, M.,Alloway, B.J., Shah, K.H., Siddiqui, S.H., Memon, M.Y., Aslam, M., Khan, P., 2003.
Zinc nutrition of wheat:II: interaction of zinc with other trace elements. Asian J. Plant
Sci. 2 (2), 156–160.
Jan, S., Parray, J.A., 2016. Approaches to Heavy Metal Tolerance in Plants. Springer http://
dx.doi.org/10.1007/978-981-10-1693-6_1.
Järup, L., Åkesson, A., 2009. Current status of cadmium as an environmental health prob-
lem. Toxicol. Appl. Pharmacol. 238 (3), 201–208.
Kabata-Pendias, A., 2011. Trace Elements in Soils and Plants. 4th ed. CRC Press (505p).
Lee, C.K., Low, K.S., 1985. Determination of cadmium, lead, copper and arsenic in raw
cocoa, semi-finished and finished chocolate products. Pertanika 8 (2), 243–248.
Li, W., Xu, B., Song, Q., Liu, X., Xu, J., Brookes, P.C., 2014. The identification of ́hotspots ́of
heavy metal pollution in soil-rice systems at regional scale in eastern China. Sci. Total
Environ. 472, 407–420.
Loureiro, G.A.H.A., 2014. Qualidade de solo e qualidade de cacau. (Tesis de Maestria).
Universidade Estadual de Santa Cruz, Ilhéus, Bahia, Brazil.
Mengal, K., Kirkby, E.A., Kosegarten, H., Appel, T., 2001. Principles of Plant Nutrition.
Kluwer Academic Publishers Dordrecht, The Netherland. Springer (849p).
MINAGRI, 2016. Sistema Integrado de Estadísticas Agrarias. Series Históricas de
Producción Agrícola —Compendio Estadístico (SISCA). http://frenteweb.minag.gob.
pe/sisca/ (Revised 20/04/2016).
Nan, Z., Li, J., Zhang, J., Cheng, G., 2002. Cadmium and zinc interactions and their transfer
in soil-crop system under actual field conditions. Sci. Total Environ. 285, 187–195.
Nedjimi, B., Daoud, Y., 2009. Cadmium accumulation in Atriplex halimus subsp.
schweinfurthii and its influence on growth, proline, root hydraulic conductivity and
nutrient uptake. Flora 204, 316–324.
Nogueira, A.R.A., Matos, A.O., Carmo, C.A.F.S., Silva, D.J., Monteiro, F.L., Souza, G.B., Pita,
G.V.E., Carlos, G.M., Oliveira, H., Comastri Filho, J.A., Miyazawa, M., Oliveira Neto,
W., 2005. Tecido vegetal. In: Nogueira, A.R.A., Souza, G.B. (Eds.), Manual de
laboratórios: solo, água, nutrição vegetal, nutrição animal e alimentos. Embrapa
Pecuária Sudeste, São Carlos, pp. 145–199.
Papoyan, A., Piñeros, M., Kochian, L.V., 2007. The effect of plant cadmium and zinc status
on root and shoot heavy metal accumulation in the heavy metal hyper accumulator,
Thlaspi caerulescens. New Phytol. 175, 51–58.
Peer, W.A., Baxter, I.R.,Richards, E.L., Freeman, J.L., Murphy, A.S., 2005. Phytoremediation
and hyper accumulator plants. In: Tamás, M.J., Martinoia, E. (Eds.), Molecular Biology
of Metal Homeostasis and Detoxification; From Microbes to Man-Topics in Current
Genetics. Springer, New York, USA (509 p).
Piotrowoska, M., Dudka, S., Chilopecka, A., 1994. Effect of elevated concentrations of Cd
and Zn in soil on spring wheat yield and metal contents in plants. Water Air Soil
Pollut. 76, 333–341.
R Core Team, 2014. R: A Language and Environment for Statistical Computing. R Founda-
tion for Statistical Computing, Vienna, Austria http://www.R-project.org/.
Ramtahal, G., Yen, I.C., Bekele, I., Bekele, F., Wilson, L., Maharaj, K., Harrynanan, L., 2016.
Relationships between cadmium in tissues of cacao trees and soils in plantations of
Trinidad and Tobago. Food Nutr. Sci. 7:37–43. http://dx.doi.org/10.4236/fns.2016.
71005.
Reis, G.S.M., Almeida, A.A.F., Almeida, N.M., Castro, A.V., Mangabeira, P.A.O., Pirovani, C.P.,
2015. Molecular, biochemical and ultrastructural changes induced by Pb toxicity in
seedlings of Theobroma cacao L. PLoS One 10 (7), e0129696. http://dx.doi.org/10.
1371/journal.pone.0129696.
Rengel, Z.,Graham, R.D., 1995. Importance of seed Zn content for wheat grown on Zn de-
fiant soil grain yield. Plant Soil 176, 317–324.
Rengel, Z., Romheld, V., Marschner, H., 1998. Uptake of zincand iron by wheat genotypes
differing in tolerance to zinc deficiency. J. Plant Physiol. 142, 433–438.
Rouphael, Y., Cardarelli, M., Rea, E., Colla, G., 2008. Grafting of cucumber as a means to
minimize copper toxicity. Environ. Exp. Bot. 63, 49–58.
Salem, H.M., Eweida, E.A., Farag, A., 2000. Heavy Metals in Drinking Water and Their En-
vironmental Impact on Human Health. ICEHM2000, Cairo University, Egypt,
pp. 542–556.
Sarwar, N., Saifullah, Malhi S.S., Zia, M.H., Naeem, A., Bibi, S., Farid, G., 2010. Role of min-
eral nutrition in minimizing cadmium accumulation in plants. J. Sci. Food Agric. 90:
925–937. http://dx.doi.org/10.1002/jsfa.3916.
Siedlecka, A., 1995. Some aspects of interactions between heavy metals and plantmineral
nutrients. Acta Soc. Bot. Pol. 64, 265–272.
Sodré, G.A., Marrocos, P.C.L., Chepote, R.E., Pacheco, R.G., 2001. Use of standard error for
estimation of the sample size of cocoa plants (Theobroma cacao L.) in nutrition stud-
ies. Agrotrópica 13 (3), 145–150.
Takrama, J., Afrifa, A.A., Ofori-Frimpong, K., Jonfia-Essien, W.A., Agyemang, P., Galyuon, I.,
2015. Cadmium contamination of cocoa beansand cocoa growing agricultural soilsof
Ghana: there is no cause for public alarm. Peak J. Public Health Manag. 3 (4):56–61.
http://www.peakjournals.org/sub-journals-PJPHM.html.
Tang, L., Qiu, R., Tang, Y., Wang, S., 2014. Cadmium-zinc exchange and their binary rela-
tionship in the structure of Zn-related proteins: a mini review. Metallomics http://
dx.doi.org/10.1039/c4mt00080c.
Tkalec, M., Štefanić, P.P., Cvjetko, P., Šikić,S.,Pavlica,M.,Balen,B.,2014.Theeffectsof
cadmium-zinc interactions on biochemical responses in tobacco seedlings and
adult plants. PLoS One 9 (1), e87582. http://dx.doi.org/10.1371/journal.pone.
0087582.
799E. Arévalo-Gardini et al. / Science of the Total Environment 605–606 (2017) 792–800

Tripathi, R.D., Srivastava, S., Mishra, S., Singh, N., Tuli, R., Gupta, D.K., Maathuis, F.J.M.,
2007. Arsenic hazards: strategies for tolerance and remediation by plants. Trends
Biotechnol. 25, 158–165.
Vaculík, M., Konlechner, C., Langer, I., Adlassnig, W., Puschenreiter, M., Lux, A., Hauser,
M.T., 2012. Root anatomy and element distribution vary between two Salix caprea
isolates with different Cd accumulation capacities. Environmental Pollution 163
(1):117–126. http://dx.doi.org/10.1016/j.envpol.2011.12.031 (Barking, Essex: 1987).
Wang, M., Zou, J.H., Duan, X.C., Jiang, W.S., Liu, D.H., 2007. Cadmium accumulation and its
effects on metal uptake in maize (Zea mays L.). Bioresour. Technol. 98, 82–88.
WHO, 2010. Exposure to Cadmium a Major Health Concern. WHO Document Produc-
tion Services, Geneva, Switzerland http://www.who.int/ipcs/features/cadmium.
pdf?ua=1.
Wood, G.A.R., Lass, R.A., 1985. Cocoa. 4th Edition. Longman, London (648 p).
Zacchini, M., Pietrini, F., Mugnozza, G.S., Iori, V., Pietrosanti, L., Massacci, A., 2009. Metal
tolerance, accumulation and translocation in poplar and willow clones treated with
cadmium in hydroponics. Water Air Soil Pollut. 197, 23–34.
800 E. Arévalo-Gardini et al. / Science ofthe Total Environment 605–606 (2017) 792–800