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RESEARCH ARTICLE
Uptake and translocation of metals and nutrients in tomato grown
in soil polluted with metal oxide (CeO
2
,Fe
3
O
4
,SnO
2
,TiO
2
)
or metallic (Ag, Co, Ni) engineered nanoparticles
Livia Vittori Antisari &Serena Carbone &
Antonietta Gatti &Gilmo Vianello &Paolo Nannipieri
Received: 8 October 2013 /Accepted: 24 August 2014
#Springer-Verlag Berlin Heidelberg 2014
Abstract The influence of exposure to engineered nanopar-
ticles (NPs) was studied in tomato plants, grown in a soil and
peat mixture and irrigated with metal oxides (CeO
2
,Fe
3
O
4
,
SnO
2
,TiO
2
) and metallic (Ag, Co, Ni) NPs. The morpholog-
ical parameters of the tomato organs, the amount of compo-
nent metals taken up by the tomato plants from NPs added to
the soil and the nutrient content in different tomato organs
were also investigated. The fate, transport and possible toxic-
ity of different NPs and nutrients in tomato tissues from soils
were determined by inductively coupled plasma-optical emis-
sion spectrometry (ICP-OES). The tomato yield depended on
the NPs: Fe
3
O
4
-NPs promoted the root growth, while SnO
2
-
NP exposure reduced it (i.e. +152.6 and −63.1 % of dry
matter, respectively). The NP component metal mainly accu-
mulated in the tomato roots; however, plants treated with Ag-,
Co- and Ni-NPs showed higher concentration of these ele-
ments in both above-ground and below-ground organs with
respect to the untreated plants, in addition Ag-NPs also con-
taminated the fruits. Moreover, an imbalance of K transloca-
tion was detected in some plants exposed to Ag-, Co- and
Fe
3
O
4
-NPs. The component metal concentration of soil rhi-
zosphere polluted with NPs significantly increased compared
to controls,and NPs were detected in the tissues of the tomato
roots using electron microscopy (ESEM-EDS).
Keywords Nanoparticles .Tomato (Lycopersicon esculentum
Mill.).Pollution .Translocation .Electron microscopy .
Inductively coupled plasma-optical emission spectrometry
Introduction
Nanotechnology is an emerging field with multiple potential-
ities and applications (Remédios et al. 2012). Engineered
nanomaterials (ENMs) are defined as materials with at least
one dimension under 100 nm (American Society for Testing
and Materials 2006; British Standards Institution 2007;Sci-
entific Committee on Emerging and Newly Identified Health
Risks 2007). Their small size provides a high surface/volume
ratio, leading to high reactivity, and both of these factors can
affect the strength and the electrical and optical properties of
the systems in which they are used (Farré et al. 2011). This
characteristic makes ENMs particularly suitable for use in the
cosmetics, medicine, pharmacy, food agriculture, high-tech,
aerospace, electronics and defence industries. ENMs can be
classified according to their size (e.g. with three dimensions
below 100 nm, they are named nanoparticles, NPs), morphol-
ogy (flatness, sphericity and aspect ratio) and composition
(made up of single material or several materials) (Remédios
et al. 2012). The production, use and disposal of NPs will
inevitably lead to their release into the air, water and soil (Lin
and Xing 2007; Vittori Antisari et al. 2011a) with a certain
amount of exposure to humans, and the risk posed by
NPs is currently subject to a robust debate (Ruffini
Castiglione et al. 2011).
Soil is rich in natural NPs, such as clay, iron oxides and
organic matter, although it could also be a preferential sink of
engineered NPs due to direct (e.g. agricultural practices) or
Responsible editor: Elena Maestri
L. Vittori Antisari :S. Carbone (*):G. Vianello
Dipartimento di Scienze Agrarie, Alma Mater Studiorum,
Università di Bologna, Viale Fanin 40 40127, Bologna, Italy
e-mail: serena.carbone2@unibo.it
A. Gatti
Nanodiagnostics, Srl Via Enrico Fermi 1/L, 41057 San Vito di
Spilamberto, Modena, Italy
P. Nannipieri
Dipartimento di Scienze Delle Produzioni Agroalimentari e
Dell’Ambiente, Università di Firenze, Piazzale delle Cascine,
28-50144 Florence, Italy
Environ Sci Pollut Res
DOI 10.1007/s11356-014-3509-0
indirect uses (e.g. via sewage treatments plants, aerial deposi-
tion or waste handling, Gottschalk et al. 2009). NPs can enter
the food chain through the soil in which crops are grown
(Pidgeon et al. 2009) and increase the risk of NP bioaccumu-
lation in the animal and human food chain (Zhu et al. 2008).
Rico et al. (2011) discussed the effects of the adsorption,
translocation and accumulation of NPs in food crops,
highlighting that these processes depend on the plant species
and the size, type, chemical composition and stability of NPs.
The accumulation of NPs and/or of component metal in plant
tissues is poorly known, and their effects on different crops
have been mainly studied in vitro: with aqueous suspensions
(Lin and Xing 2007;KumariandMukherjee2009;
Stampoulis et al. 2009; Yang and Watts 2009; El-Temsah
and Joner 2010; Lopez-Moreno et al. 2010), Hoagland solu-
tion (Schwabe et al. 2013) and agar culture media (Lee et al.
2008). Few experiments have been performed with soil (El-
Temsah and Joner 2010; Sheykhbaglou et al. 2010;Priester
et al. 2012;Wangetal.2012; Hernandez-Viezcas et al. 2013;
Servin et al. 2013). Toxicological experiments carried out in
hydroponic systems are very useful for understanding NP
behaviour in a standardized media; however, it could be
unrealistic since the physico-chemical and biological charac-
teristics of the soil can affect the amount of NPs available to
soil biota and crops (Vittori Antisari et al. 2011b,2012,2013;
Rico et al. 2011). For an excellent review on nanomaterials
and full life cycle of plant, see Gardea-Torresdey et al. (2014).
Tomato (L. esculentum Mill.) is one of the most important
vegetable crops worldwide because of its high consumption
(Ouzounidou et al. 2008). We therefore chose it as a model
plant to monitor the effects of different NPs added to a model
soil through the irrigation water.
The aim of this work was to evaluate the influence of
different metal oxide (CeO
2
,Fe
3
O
4
,SnO
2
,TiO
2
) and metallic
(Ag, Co, Ni) NPs on the following: (i) the morphological
parameters of tomato organs (e.g. dry weight and plant
height), (ii)NP component metal taken upby the tomato plant
(namely, Ag, Ce, Co, Fe, Ni, Sn and Ti) and (iii) the content of
nutrients (Ca, Mg, K, Na, P and S) in different tomato organs.
The fate, transport and possible accumulation in tomato or-
gans of different NPs as well as the nutrients were determined
by inductively coupled plasma-optical emission spectrometry
(ICP-OES).
Material and methods
NP characterization The metal oxide NPs (CeO
2
,Fe
3
O
4
and
SnO
2
) were provided by Nanostructured & Amorphous Ma-
terials, Inc (Houston, USA) and have a purity of at least 98 %.
According to the manufacturer, their specific surface area
(SSA) was 8–15, >40 and 14 m
2
/g for CeO
2
-, Fe
3
O
4
-and
SnO
2
-NPs, respectively; the particle sizes, calculated by the
SSA and transmission electron microscopy, were 50–105, 20–
30 and 61 nm, respectively.
The Ag-NPs were obtained from Polytech (Germany, type
WM 1000-c), supplied as a 1,000 mg L
−1
in a deionized water
suspension of metallic silver (Ag) with a NP size between 1
and 10 nm. Both Co- and Ni-NPs were obtained from Nano-
structured & Amorphous Materials, Inc. (USA) as a powder,
with a NP size of 28 and 62 nm, respectively. The TiO
2
-NPs
were supplied from Tal Materials, Inc. (USA), as a powder
with an average size between 20 and 160 nm.
The NPs were suspended in deionized water (solution of
1gL
−1
for each metal element of NPs) and dispersed by
ultrasonic vibration (100 W, 40 kHz) for 30 min, except for
the silver, which was used as is. Then, 20 μgmL
−1
of NP
elements was added to the soil near the plant root before
adding the irrigation water. Fertilizer was not used during
the experiment.
Experimental design The growth experiment was carried out
at the Agricultural Science Department of Bologna University
(Italy) in a climate-controlled greenhouse under full sunlight
from March 26 to August 4, 2012 (photoperiod 11.5/13 h
winter/summer); this period corresponds to the vegetative
cycle of tomato (L. esculentum cv. Cilao F1). The nominal
maximum temperature in the greenhouse was set at 28 °C. The
seedlings (about 10 cm high) were placed in pots (5 L each
with 5 kg of soil) containing a model soil made of natural soil
and peat (1:4 v/v) which represents an excellent growth me-
dium due to the high moisture and nutrient-holding capacity
with a uniform and slow breakdown rate of physical structure
(Ball et al. 2000).A quartz and feldspar sand layer (4 cm) was
placed at the bottom of the pot to maintain the drainage.
A total of 48 pots (6 pots for each NPs) were placed in a
randomized block; after 2 weeks of adaptation, the seedlings
were spiked with Ag-, CeO
2
-, Co-, Fe
3
O
4
-, Ni-, SnO
2
-and
TiO
2
-NP solutions at 20 μgmL
−1
of NP component metal
once per week, twice from the 13th week, to simulate a
chronic exposure to NPs supplied with irrigation. For the
control test, only water was supplied.
Plant sampling and vegetal tissues analysis At the end of the
growing cycle (130 days), each tomato plant was separated
into the aerial part (i.e. stem and leaves) and the below-ground
part. The above-ground plant was washed with deionized
water and oven-dried at 60 °C until constant weight to deter-
mine dry mass and water content. The fruits were also collect-
ed, washed, frozen at −80 °C and thenlyophilized. Dry tissues
of different organs of each tomato were ground and digested
using a nitric acid and oxygen peroxide solution in a micro-
wave oven (Start D 1200, Milestone) according to the US
Environmental Protection Agency (USEPA 2009) method,
modified by Vittori Antisari et al. (2011a). An approximately
0.4 g subsample of plant tissue was treated with 8 mL of
Environ Sci Pollut Res
concentrated plasma-pure nitric acid (65 % Suprapur grade,
Merck) plus 2 mL of hydrogen peroxide (30 % for electronic
use, Carlo Erba,). The mineralization was carried out in PTFE
bombs in the microwave oven, and the content of nutrients
(Ca, Mg, K, Na, P and S) and the component metal (Ag, Co,
Ni, Ce, Sn, Ti and Fe) in the leaves, stems, fruits and roots was
quantified by inductively coupled plasma spectrometry (ICP-
OES, Spectro Arcos, Ametek). Reagent blanks and interna-
tional reference materials (olive leaves BCR-CRM 062) were
analyzed to validate the method. In addition, standard solu-
tions (0.5 mg L
−1
Ag, Ce, Co, Ni and Sn) were analyzed every
ten samples for quality control/quality assurance purposes.
Soil sampling and analysis The soil of three tomato plants
was sampled as follows: A Plexiglas® cylinder was inserted in
the soil, and a column of 12 cm was sampled. The soilcolumn
was divided into four layers, each one 3 cm deep, and the
deepest sample was sand.
The root system of each plant was removed from the bulk
soil, and the rhizosphere soil samples were then obtained by
shaking the roots after soil drying (Turpault 2006) and care-
fully collecting the aggregate still attached to the roots. After-
wards, the roots were washed with distilled water and oven-
dried to perform dry mass and ICP-OES analysis (see “Plant
sampling and vegetal tissues analysis”section). No further
washing procedure (e.g. acidulated water) was carried out;
therefore, in ICP-OES analysis, both NP surface adsorbed
and taken up by the roots were determined.
The rhizosphere soil samples were air dried and finely
ground with an agate mill. The metal content was determined
according to Vittori Antisari et al. (2013). Briefly, the soil
(0.25 g) was treated with aqua regia (2 mL 65 % HNO
3
plus
6 mL 37 % HCl, Suprapur grade, Carlo Erba) in the micro-
wave oven, and the metal concentrations were deter-
mined by ICP-OES. The analysis of each sample was
replicated three times and compared with analyses of
the international reference materials (BCR 141) and
laboratory internal standards (MO and ML), which was
run after every ten samples to check changes in sensi-
tivity. Reagents blanks were also determined.
Translocation index The translocation index (TI) was calcu-
lated to synthesize the capability of the species to translocate
nutrients and pollutants from roots to shoots (Paiva et al.
2002), according to the following equations:
TI ¼DMLðÞ=DMR þDMS þDMLðÞ100 and
TI ¼DMSðÞ=DMR þDMS þDMLðÞ100
where DMR, DML and DMS are the element concentrations
as a function of dry matters of roots, leaves and stem,
respectively.
ESEM analysis Detection of NPs inside the tissues of tomato
organs was carried out by field emission gun-environmental
scanning electron microscope (FEG-ESEM) investigations
and energy-dispersive X-ray spectroscopy (EDS) analyses to
classify univocally the engineered NPs and differentiate them
from the natural particles. The electron microscope analyses
were performed in the secondary electron and backscattered
electron diffraction mode, in order to obtain information on
the morphology of the samples and on the chemical nature of
the NPs.
Statistical analysis Treatments were allocated in a completely
random design, and the data were reported as the mean of
three replicates (±standard deviations (SD)). Data were ana-
lyzed by variance and Tukey’s test with the statistical package
SPSS 15.0, unless otherwise stated.
Results
Concentration of NP component metal in rhizosphere and soil
column
The concentrations of NP component metals in treated soil
after dissolution in aqua regia are shown in Table 1.Generally,
the component metal concentration of soil rhizosphere pollut-
ed with NPs significantly increased (P<0.05) compared to
controls. With regard to the soil column, the concentration of
the surface layer (0–3 cm) increased significantly (P<0.05)
compared to the control, except for soil polluted with Fe
3
O
4
-
and TiO
2
-NPs. In the case of magnetite, the Fe concentration
increased by 75 % compared to the control in the 3–6-cm soil
layer, whereas in the case of the TiO
2
-NP treatment, the
deepest soil layers were more enriched (ca. 47 %) compared
to the control. In general, the amount of NP component metal
decreased with the depth, even if the lowest values were
generally determined in the 3 to 6-cm layer of the soil column.
Plantgrowthperformance
The plants exposed to NPs showed different vegetative
growth (Table 2). Magnetite promoted the root growth (4.8 g
dry weight (d.w.)) while SnO
2
-NP exposure (P<0.05) reduced
it (0.7 g d.w.) with respect to the control (1.9 g d.w.); the other
NP treatments showed no significant difference compared to
the control roots. Phenotype and dry matter of the above-
ground biomass varied among treatments: Ag-NP exposure
decreased the elongation of the stem (ca. −16 %) with respect
to the control but, at the same time, increased the dry matter of
thestem(ca.28%)(P<0.05). Conversely, in plants exposed
to Co-NPs, the reduction of the stem elongation (ca. −14 %)
was associated with a decrease of the above-ground dry
Environ Sci Pollut Res
biomass compared to the control (ca. −50 and −27 % for the
stem and the leaves, respectively).
CeO
2
treatments determined a slight increase of the stem
elongation, not statistically significant, associated to a de-
crease of the stem and leaves dry matter (ca. −36 and
−37 %, respectively).
Fe
3
O
4
NPs, although promoting the root growth for both
elongation and dry matter parameters, reduced significantly
the above-ground dry biomass (ca. −12 and −25 % for the
stem and the leaves, respectively).
The exposure to Ni-NPs determined a slight decrease of the
stem elongation, not statistically significant, associated to an
increase of the stem dry matter (5.6 %) as Ag-NP exposure
did, but in addition, it decreased significantly with respect to
the untreated plants the dry matter of the leaves (−13.1 %).
SnO
2
-NP exposure reduced significantly also the above-
ground dry matter (ca. −74 and −33 % for the stem and the
leaf, respectively).
Tomato plants exposed to TiO
2
-NPs did not show
significant changes in root and stem elongation but
Tabl e 1 Concentration of NP component metal in the soil rhizosphere and in the different layers of the soil column
Rhizosphere Soil column (cm)
0–33–66–99–12
Treatment SD SD SDSDSD
Ag-NPs 26.1*** 3.2 36.9*** 0.2 9.9*** 0.09 0.9* 0.04 0.03 ns –
Ag control 0.03 –0.03 –0.03 –0.03 –0.03 –
Co-NPs 45.6** 2.9 64.6** 0.1 8.8** 0.3 6.8 ns 0.1 7.6 ns 0.2
Co control 5.2 0.1 5.3 0.2 2.9 0.2 5.5 0.1 7.4 0.2
Ni-NPs 71.1** 5.0 32.6** 0.3 8.1 ns 0.2 23.4 ns 0.3 24.3 ns 0.9
Ni control 12.2 1.6 24.1 0.4 11.5 0.2 21.3 0.2 26.8 0.8
CeO
2
-NPs 37.7** 2.7 100.7** 4.1 9.2 0.7 14.6 ns 1.6 9.8 ns 5.8
Ce control 5.2 0.7 11.0 2.5 6.8 1.5 9.8 0.5 12.5 0.4
Fe
3
O
4
-NPs 5.6 ns 3.3 11.5 ns 0.4 10.4** 0.2 17.1 ns 0.3 16.5 ns 0.2
Fe control 5.6 0.8 12.1 0.3 5.9 0.2 12.2 0.2 16.1 0.4
SnO
2
-NPs 6.9** 8.3 3.0* 0.4 2.1* 0.3 1.1 ns 0.1 1.0 ns 0.2
Sn control 0.5 0.2 0.8 0.2 0.8 0.2 1.0 0.2 0.8 0.0
TiO
2
-NPs 95.2 ns 25.0 166.5 ns 18.8 104.6 ns 7.3 207.6* 7.3 206.5* 7.5
Ti control 138.6 8.9 198.2 112.7 138.6 6.9 142.2 0.0 140.2 6.8
One-way ANOVA and Tukey’stest(P< 0.05) were used to determine statistical significance of differences between the treatment and the control means.
The Ag value of the control and the 9–12-cm soil layer column was equal to the detection limit (DL). The data were expressed as microgram per gram
(μgg
−1
) d.w., except for Fe (mg g
−1
d.w.)
ns is not significant; SD is the standard deviation
***P<0.001; **P<0.01; *P<0.05
Tabl e 2 Effect of NPs on dry matter of roots, stems and leaves of Lycopersicon esculentum plants grown in pots
Root Stem Leaves Root elongation Plant height
Treatment g SD g SD g SD cm SD cm SD
Control 1.9 b 0.1 20.5 b 0.9 25.2 a 1.1 22 ab 1.3 98 a 3.8
Ag-NPs 1.6 b 3.3 26.2 a 1.2 24.2 a 0.9 19 b 1.5 82 b 5.3
Co-NPs 1.5 b 0.3 10.3 d 1.1 18.3 b 1.5 15 b 2.8 84 b 4.2
Ni-NPs 1.0 bc 0.3 26.1 a 1.2 12.1 d 0.9 15 b 3.2 93 ab 5.1
CeO
2
-NPs 2.2 ab 0.2 13.1 cd 1.4 15.7 c 0.7 23 ab 2.1 109 a 3.1
Fe
3
O
4
-NPs 4.8 a 0.2 18.1 c 0.8 18.9 b 1.3 25 a 2.3 106 a 3.5
SnO
2
-NPs 0.7 c 0.2 5.4 e 0.7 16.8 c 1.5 11 b 3.7 104 a 3.4
TiO
2
-NPs 1.4 b 0.1 19.2 b 1.1 18.8 b 0.8 17 b 2.1 110 a 4.1
Means followed by a different letter within a row are significantly different at P<0.05 according to the Duncan’s multiple range test
Environ Sci Pollut Res
showed a decrease of the leaf dry matter (−25.3 %)
with respect to the control.
Metal content in tomato organs
The NP component metals were accumulated in the tomato
roots (Table 3). Generally, the concentrations of these metals
in the control tissues were lower than the instrumental detec-
tion limit (DL, see values in Table 3). Organs of tomato grown
in soil irrigated with TiO
2
-NPs showed no difference com-
pared to the control, and this is also true for organs of tomato
exposed to Fe
3
O
4
-NPs, except for fruits in which the Fe
concentration was higher than the control (116.6 and
45.5 μgg
−1
, respectively). Generally, Ag, Co and Ni concen-
trations were higher than those of the control tomato organs,
except for fruits of plants subjected to Co-NPs and leaves and
roots of plants treated with Ni-NPs.
The amount of metals arising from NPs for each pot (μg
per pot) is shown in Table 4. The distribution of metals in the
above-ground organs of tomato changed as a function of the
NP exposure. Ag, Co and Ni accumulated in leaves with an
amount of 26.7, 22.0 and 24.6 μg per pot, respectively; Fe, Ce
and Sn accumulated in roots (2,087.0; 3.7 and 0.4 μg per pot,
respectively) and Ni in stems (16.3 μg per pot). The Ti did not
accumulate in the crop irrigated with TiO
2
-NPs compared to
the control. The translocation index (TI) showed that tomato
was able to translocate Ag and Co from root to above-ground
organs (Table 4), whereas the respectivetranslocation of Fe to
leaves was lower compared to the control (−30 and 69.5 % in
stem and leaves, respectively); Ni accumulated in stems. No
translocation of Sn, Ti and Ce was found either in the control
or in the treated tomatoes.
ESEM analysis of tomato roots
The examination of the organ tissues of the tomato
plants to find NPs was performed by ESEM-EDS. Ex-
amples of tomato roots exposed to NPs are shown in
Figs. 1,2and 3. Ag-NPs were detected within root
cells (Fig. 1) grouped as a large cluster (from 100 to
200 nm) but also individually dispersed. Cluster
Tabl e 3 Comparison between the concentration of NP component metal of stem, leaves, root and fruit of tomato grown with or without (control) NPs
Ag Co Ni Ce Fe Sn Ti
SD SD SD SD SD SD SD
Stem Treatment 0.2 0.01 0.4 0.001 0.9 0.1 DL 38.4 0.5 DL 1.4 0.1
Control DL DL DL DL 49.8 4.2 DL 1.7 0.1
** * ns ns
Leaves Treatment 1.1 0.05 1.2 0.05 1.3 0.3 DL 19.9 7.8 DL 3.3 0.8
Control DL DL 1.1 0.5 DL 20.5 8.6 DL 2.8 0.03
* * ns ns ns
Root Treatment 2.6 0.06 3.7 2.3 0.1 1.7 0.01 534.8 29.1 0.6 0.01 5.5 0.01
Control DL 0.2 3.2 0.3 DL 383.5 12.3 DL 7.9 0.2
** ns** *ns
Fruit Treatment 0.3 0.01 DL 0.8 0.1 DL 116.8 7.9 DL 2.9 0.4
Control DL DL DL DL 42.5 8.1 DL 5.5 0.1
***nsns
One-way ANOVA and Tukey’stest(P< 0.05) were used to determine statistical significance of the differences between treatment and the control means.
The data are expressed as μgg
−1
d.w.
***P<0.001;**P<0.01; *P<0.05
ns is not significant. DL was the instrumental detection limit of Ag (0.006 μgg
−1
), for Ce (0.01 μgg
−1
), for Co (0.0002 μgg
−1
), for Ni (0.01 μgg
−1
)
and for Sn (0.01 μgg
−1
). SD is the standard deviation
Tabl e 4 (A) Amount of NP component metal accumulated in stem,
leaves and root expressed as microgram per pot (referred to grams of
dry substance). (B) Translocation index (TI): calculated values expressed
as the percentage of NP component metal from above-ground to below-
ground organs
AAgCoNiCeFeSnTi
Stem Treatment 5.2 4.1 16.3 –998.4 –26.9
Control 1.2 –1.8 –944.2 –34.9
Leaves Treatment 26.7 22.0 24.6 –240.6 –62.0
Control 1.5 –27.8 –517.8 –70.7
Root Treatment 4.2 5.6 2.5 3.7 2,087.0 0.4 7.7
Control 0.1 0.4 6.1 –728.7 0.02 15.0
B Translocation index (TI; %)
TI NP Stem 14.4 12.9 37.6 –30.0 –27.8
Leaves 74.0 69.4 56.7 –7.2 –64.2
TI Ctr Stem ––5.0 –43.1 –28.9
Leaves ––55.4 –23.6 –58.6
Environ Sci Pollut Res
formationfromNPswasdeterminedintomatoroots
exposed to TiO
2
-andSnO
2
-NPs; TiO
2
-NPs were distributed
parallel to the longitudinal section of roots (Fig. 2), whereas
SnO
2
-NPs showed spherical clusters of different sizes (Fig. 3).
In all the spectra by EDS, the NPs were associated with soil
compounds (e.g. Al and Si).
Fig. 1 a–dESEM images of Ag nanoparticles in tomato roots of plants grown in soil amended with Ag-NPs. e,fEDS spectra of Ag-NPs and natural
nanoparticles
Fig. 2 a–cESEM images of TiO
2
nanoparticles detected in root tissues of tomato ofplants grown in soil amended with TiO
2
-NPs. dEDSspectrumof
TiO
2
-NPs and natural nanoparticles
Environ Sci Pollut Res
Nutrient content in tomato organs
The average concentrations of nutrients in tomato organs
grown in soil polluted with NPs are reported in Table 5.As
seen in the table, the nutrient content is influenced by the NP
treatments in different ways with respect to the control.
Ca content in leaves changed with the treatments and can
be ranked as follows: Ag> Ni > CeO
2
=Co=control (Ctr) =
TiO
2
=Fe
3
O
4
>SnO
2
NPs. The highest Ca concentration was
found in samples treated with Ag- and Ni-NPs, which had an
increase of 45.8 and 32.2 % with respect to the untreated
plants, while the lowest concentration was detected in plant
leaves exposed to SnO
2
(decrease of 55.1 %). Ca concentra-
tion in the stem was higher than the control for all treatments,
with a maximum increase of 54.4 % in plants treated with Co-
NPs and a minimum of 25.6 % in those exposed to SnO
2
-NPs.
The Ca content in root samples of different treatments can be
ranked as follows: Fe
3
O
4
>Ag= CeO
2
=Ni=SnO
2
>Ctr=Co=
TiO
2
NPs; notably, plants exposed to Fe
3
O
4
-NPs showed an
increase of 69.8 % with respect to the control.
The NP treatment scarcely affected the content of K in
tomato above-ground organs (leaves and stem tissues) with
the exception of plants treated with SnO
2
-NPs, where a 50 %
decrease of K concentration was found in leaves with respect
to the untreated plants. On the other hand, K content in the
roots was deeply influenced by NPs in the following order:
Co> Ag>CeO
2
>Ctr=TiO
2
=Fe
3
O
4
>Ni=SnO
2
NPs; in plants
treated with Co- and Ag-NPs, K content was markedly higher
than the control (114.3 and 82.1 % higher, respectively)
whereas in plants exposed to Ni- and SnO
2
-NPs, the element
content was significantly lower (−64.3 and −67.9 %, respec-
tively). Moreover, it should be noted that in some tomato
plants exposed to Ag-, Co- and Fe
3
O
4
-NPs, K content was
not detected in the leaves (data shown in Fig. 4and excluded
from the mean reported in Table 5) while a high K content was
stored in the roots.
Regarding Mg, its concentration in tomato above-ground
organs (leaves and stem) exhibited a behaviour similar to that
of Ca, showing the highest concentration in leaves, for sam-
ples treated with Ni- and Ag-NPs (increase of 54.2 and 37.5 %
whit respect to the control), the lowest for plants exposed to
SnO
2
-NPs (−54.2 %) and showing a higher concentration in
stems for all treatments compared to the control. Mg content
in the roots of plants exposed to different treatments can be
ranked as follows: Co> Ag>CeO
2
=Fe
3
O
4
>Ctr=TiO
2
>Ni=
SnO
2
NPs; in particular, plants exposed to Co-NPs showed a
marked increase (135.7 %) with respect to the untreated
plants, while both Ni- and SnO
2
-NPs decreased the element
content by 28.6 %.
Also with regard Na, as in Ca and Mg, Ag- and Ni-NP
treatments determined an increase of the element concentra-
tion in leaves with respect to the control (53.1 and 40.6 %,
respectively), whereas SnO
2
-NPs caused a marked decrease
(−46.9 %). Moreover, Ag- and Co-NP treatments were asso-
ciated with an increase of Na in stems compared to the control
(53.1 and 40.6 %, respectively), while the lowest value was
found in plants exposed to CeO
2
-NPs. The Na content was
higher in roots of plants treated with Ag- and Co-NPs (97.6
Fig. 3 a–dESEM images of
SnO
2
nanoparticles detected in
root tissues of tomato of plants
grown in soil amended with
SnO
2
-NPs. e,fEDS spectra of
SnO
2
-NPs and natural
nanoparticles
Environ Sci Pollut Res
and 57.1 %, respectively), whereas the lowest value was found
in plants exposed to Ni- and SnO
2
-NPs (−69.0 and −73.8 %,
respectively).
The average P content in leaves of different samples
can be ranked as follows: TiO
2
>Ctr>CeO
2
=Co=
Fe
3
O
4
=Ni>Ag>SnO
2
NPs; remarkably, only plants
treated with TiO
2
-NPs showed an increase of P
(15.4 %) with respect to the untreated plants. P concen-
tration in stems reached the highest value compared to
the control in plants treated with Ag-NPs (65.2 %),
while the lowest was found in plants exposed to Co-
NPs (−8.7 %). Contrary to what was observed in leaves,
root tissues exposed to Ag-NPs showed an increase in P
concentration (55.6 %), whereas a notable decrease in
this element was detected in plants treated with Ni- and
SnO
2
-NPs (−22.2 and −33.3 %, respectively).
Leaves of plants exposed to Ag-NPs showed an increase of
S content with respect to the control, whereas a marked
decrease was detected in plants treated with SnO
2
-NPs
(−60.7 %). As noted for Ca concentration in stems, S content
was higher with respect to the control for all treatments
with a maximum increase of 51.4 % in plants treated
with Co-NPs and a minimum of 9.9 % in those exposed
to CeO
2
-NPs. As already observed for K, Mg and Na,
roots of plants treated with Ag- and Co-NPs showed the
highest amount of S compared to the untreated plants
(21.4 and 25 %, respectively), while the lowest content
was determined in plants treated with Ni- and SnO
2
-NPs
(−42.9 and −50 %, respectively).
The nutrient concentration in tomato fruits showed a high
K content (from 59 % for CeO
2
-andFe
3
O
4
-NPs to 63 % for
Ag-NPs) and a low level of Mg (from −33 % for SnO
2
-NPs to
−47 % for CeO
2
-andTiO
2
-NPs), P (from ca. −51 % for Ag-,
Ni- and SnO
2
-NPs to ca. −63 % for Fe
3
O
4
-NPs) and S (from
ca. −41 % for Ag- and Ni-NPs and −53 % for CeO
2
-andTiO
2
-
NPs) after irrigation with NPs compared to the control
(Fig. 5), while no significant differences were found for Ca
and Na.
Tabl e 5 Nutrient concentration (g kg
−1
d.w.) in tomato organs cultivated in control soil (Ctr) and soil polluted with NPs
Ca K Mg Na P S
Leaves SD SD SD SD SD SD
Control 11.8 c 0.2 8.4 a 0.2 2.4 b 0.0 3.2 b 0.0 1.3 b 0.0 2.8 a 0.1
Ag-NPs 17.2 a 0.1 8.7 a 0.1 3.3 a 0.1 4.9 a 0.1 1.0 cd 0.0 3.2 a 0.1
Co-NPs 11.9 c 0.1 7.8 ab 0.1 2.7 b 0.0 3.4 b 0.0 0.9 d 0.0 1.9 b 0.0
Ni-NPs 15.6 b 0.3 8.5 a 0.1 3.7 a 0.1 4.5 a 0.1 1.1 c 0.0 2.8 a 0.0
CeO
2
-NPs 10.5 cd 0.2 7.5 b 0.0 2.2 b 0.0 2.9 c 0.0 1.2 c 0.0 1.8 b 0.0
Fe
3
O
4
-NPs 12.0 c 0.0 8.3 a 0.1 2.5 b 0.0 2.3 c 0.0 1.0 cd 0.0 1.91 b 0.0
SnO
2
-NPs 5.3 e 0.6 4.2 c 0.1 1.1 c 0.2 1.7 d 0.1 0.8 d 0.1 1.1 c 0.2
TiO
2
-NPs 10.7 cd 0.1 8.0 a 0.1 2.5 b 0.0 3.1 b 0.0 1.5 a 0.0 1.9 b 0.0
Stem
Control 19.5 c 0.2 8.7 a 0.1 3.1 b 0.0 2.2 b 0.0 2.3 c 0.0 11.1 c 0.1
Ag-NPs 28.5 ab 1.9 8.3 a 0.7 4.9 a 0.3 3.5 a 0.2 3.8 a 0.3 16.7 ab 0.9
Co-NPs 30.1 a 0.3 8.7 a 0.1 4.6 a 0.0 2.9 a 0.0 2.1 c 0.0 16.8 a 0.1
Ni-NPs 24.9 b 0.3 8.6 a 0.1 4.1 a 0.0 2.5 b 0.0 2.6 b 0.0 13.1 b 0.2
CeO
2
-NPs 25.1 b 0.5 8.8 a 0.0 3.9 a 0.1 1.9 c 0.0 2.4 bc 0.1 12.2 bc 0.5
Fe
3
O
4
-NPs 27.2 b 0.2 8.5 a 0.2 5.0 a 0.1 2.5 b 0.0 2.3 c 0.1 13.3 b 0.1
SnO
2
-NPs 24.5 b 0.1 8.5 a 0.0 4.2 a 0.0 2.1 b 0.0 2.9 ab 0.0 12.7 b 0.1
TiO
2
-NPs 26.1 b 0.2 8.7 a 0.1 4.1 a 0.1 2.1 b 0.0 2.6 b 0.0 13.2 b 0.3
Root
Control 12.9 cd 0.3 2.8 d 0.1 1.4 d 0.1 4.2 cd 0.4 0.9 b 0.1 2.8 b 0.1
Ag-NPs 16.8 b 0.5 5.1 b 0.1 2.3 b 0.1 8.3 a 0.4 1.4 a 0.1 3.4 a 0.1
Co-NPs 14.6 c 0.1 6.0 a 0.0 3.3 a 0.1 6.6 b 0.0 1.5 a 0.0 3.5 a 0.0
Ni-NPs 12.0 d 0.1 1.0 e 0.0 1.0 e 0.1 1.3 e 0.0 0.7 c 0.0 1.6 c 0.0
CeO
2
-NPs 16.9 b 0.1 3.5 c 0.0 1.9 c 0.0 4.0 d 0.0 1.4 a 0.0 2.6 b 0.0
Fe
3
O
4
-NPs 21.9 a 0.3 2.7 d 0.0 2.0 c 0.0 4.3 d 0.1 1.2 ab 0.0 2.4 b 0.1
SnO
2
-NPs 10.8 d 0.3 0.9 e 0.0 1.0 e 0.1 1.1 e 0.0 0.6 c 0.0 1.4 c 0.0
TiO
2
-NPs 13.3 c 0.3 3.0 d 0.0 1.6 d 0.1 5.4 c 0.0 1.3 ab 0.0 2.6 b 0.0
Means followed by a different letter within a row are significantly different at P<0.05 according to one-way ANOVA Tukey’stest
SD is the standard deviation
Environ Sci Pollut Res
Discussion
We showed via ICP-OES that, with the exception of Fe
3
O
4
-
and TiO
2
-NPs, a high amount of NP component metal
remained in the rhizosphere soil and/or in the superficial
layers (0–3, 3–6 cm) of bulk soils as compared to the deeper
ones, thereby highlighting a low mobility of NPs in the soil.
Only in the case of TiO
2
-NPs, high values were found in the
deepest soil layers, suggesting a greater mobility of these
particles along the soil column than the other NPs
(Ben-Moshe et al. 2010).
Several studies to assess the bioavailability of NPs have
been conducted in hydroponic solution (Rico et al. 2011),
even though the growth medium is important since surface-
reactive particles, such as clays and organic matter-coated
particles in soil (Lee et al. 2012;Dimkpaetal.2012;Du
et al. 2011), can affect the behaviour of these NPs, favouring
their aggregation and thus decreasing the risk of toxicity
(Dinesh et al. 2012).
Chronic exposure to NPs affects plant growth, and in fact,
both the above- and below-ground growth was stunted in
tomato exposed to Ag-NPs, as already observed in Sorghum
bicolor and Triticum aestivum (Lee et al. 2012; Dimkpa et al.
2013), and atthe same time, we detected a significant increase
in the stem dry weight. This could be due to oxidative stress
and membrane damage, as observed in many
nanophytotoxicity studies (Wang et al. 2011), where the in-
crease of lignification observed in transgenic tobacco could be
Fig. 4 Concentration (mg g
−1
)of
major nutrients in some
specimens oftomato plants grown
in soil amended with Ag-, Co-,
and Fe
3
O
4
-NPs
Fig. 5 Concentration (mg g
−1
)of
nutrients in tomato fruits of plants
grown in soil amended with metal
oxide and metallic NPs
Environ Sci Pollut Res
due to the over-expression of the peroxidase gene, thus en-
hancing the generation of H
2
O
2
(Kim et al. 2008). However, a
recent study conducted in rice plants showed that CeO
2
-NPs
decreased the lignifications in rice despite the enhanced per-
oxidase activity and H
2
O
2
content (Rico et al. 2013a). In this
experiment, CeO
2
-NP treatment did not affect stem and root
elongation, while Wang et al. (2012) observed that the above-
ground part oftomato plants treated with CeO
2
-NPs showed a
slight improvement in the average height of stems. Moreover,
we observed that CeO
2
-NP treatment caused a decrease in the
dry biomass, as previously reported by Priester et al. (2012)in
soybean plants exposed to a comparable dose (0.1 g CeO
2
kg
−1
dry soil).
Magnetite promoted root growth for both elongation and
dry matter parameters since iron is one of the essential ele-
ments for plant growth, and thus, the application of nano-iron
on soybean leaf increased the dry pod weight, leaf plus dry
pod weight and yield (Sheykhbaglou et al. 2010). However,
these results contradict what Lee et al. (2010) reported: the
inhibition of root elongation on Arabidopsis thaliana after
exposure to Fe
3
O
4
-NPs in agar medium. Contrasting results
were also found for TiO
2
-NP treatment, which reduced the
leaf dry matter of tomatoes, whereas Song et al. (2013)re-
ported that the biomass of plants exposed to TiO
2
did not
significantly vary among 1,000–5,000 mg L
−1
treatments.
These differences may be due to the fact that plants were
grown in agar medium and in Hoagland solution in the
studies by Lee et al. (2010) and Song et al. (2013), respec-
tively; indeed, differences in species and medium might ac-
count for our differing results (Anjum et al. 2013).
To the best of our knowledge, this is the first report about
the interaction between Co-, Ni- and SnO
2
-NPs and
L. esculentum. Our work showed that Co-, Ni- and SnO
2
-NP
treatments have a detrimental effect on plant growth, decreas-
ing the above-ground dry biomass compared to the control.
In this study, we employed ICP-OES to assess the compo-
nent metal concentration in tomato organs and ESEM to
investigate the amount of root-associated NPs that could be
available for translocation (Schwabe et al. 2013). A previous
study showed that roots could be the main route of the plant’s
exposure to NPs (Anjum et al. 2013). In this present study, for
all NPs, except Ni, an increase was found in the component
metal in the root tissues, which was accompanied by an
element translocation only for Ag (in stem, leaves and fruit),
Co (stem and leaves), Fe (fruit) and Ni (stem and fruit). While
no references were found regarding the impact of Co- and Ni-
NPs on plant species, generally, many studies on Ag-NPs
highlighted the possibility of silver transport in plant shoots
(Anjum et al. 2013). Moreover, Ag distribution in shoot
organs is species-specific and is correlated with the Ag-NP
size; in fact, smaller NPs showed a faster uptake in Populus
deltoides × nigra (Wang et al. 2013). Geisler-Lee et al. (2013)
found that Ag-NPs in A. thaliana are accumulated
progressively in roots in this sequence: border cells, root
cap, columella and columella initials.
Ce and Sn did not translocate in the control nor in the
treated tomato (CeO
2
- and SnO
2
-NPs, respectively). Sn con-
centration in soil and root tissues was lower than expected,
probably because of an underestimation of Sn concentration
due to the incomplete dissolution of SnO
2
-NPs in the acid
mixture used for the mineralization of soil and plants (Vittori
Antisari et al. 2013). As for CeO
2
-NPs, the literature is con-
tradictory. In research conducted on maize plants, cerium was
absent (Birbaum et al. 2010) or found at low concentrations in
the shoots of plants grown in low organic matter soil (Zhao
et al. 2012), while it was found in rice grains (Rico et al.
2013b). Moreover, a study conducted on tomato plants grown
in potting mix and treated with CeO
2
-NPs (10–30 nm) at
increasing doses assessed the presence of Ce in the following
order: root>stem> leaf>fruit (Wang et al. 2012). These con-
trasting results could be due to the different size of the CeO
2
-
NPs employed, namely, 50–105 nm in our experiment and
10–30 nm in that of Wang et al. (2012). In fact, translocation
to the shoot is generally limited and depends on the NPs’
primary diameter, as reported by Zhang et al. (2011).
The accumulation of iron in tomato roots growing in the
presence of magnetite was not accompanied by Fe transloca-
tion into shoots (Zhu et al. 2008; Trujillo-Reyes et al. 2014)
but only into the fruit, probably because the Fe was retained in
the roots similarly to low soluble compounds (e.g. oxides and
organic acids) (Taiz and Zeiger 1998).
In contrast to previously published data by Servin et al.
(2012,2013) regarding the TiO
2
-NP uptake by Cucumis
sativus, in our study, we did not assess Ti translocation in
the above-ground organs. However, the ESEM images
showed the presence of TiO
2
-NPs distributed parallel to the
longitudinal section of roots. Probably, the translocation of
NPs is associated with the absorption patterns of water and
nutrients within the root (Lee et al. 2010). Moreover, the
translocation of NPs, or of their component metal, could be
enhanced in crops with higher water transpiration (Schwabe
et al. 2013). According to Du et al. (2011), the impact of the
TiO
2
-NPs might result from the mere presence of the NPs in
cells or accumulated on the cell walls, which might cause
changes in the soil microenvironment.
To the best of our knowledge, studies have been carried out
on the impact of NPs on accumulation and translocation of
macro- and micro-elements in various plants, but not in toma-
to. However, the imbalance of nutrient absorption in the soil-
tomato system has been investigated (Carvalho Bertoli et al.
2012), and different physiological disorders related to heavy
metals pollution (Larbi et al. 2002; Dong et al. 2006)or
salinity (Carvajal et al. 2000; Levent Tuna et al. 2007)were
investigated. For example, Ca and K concentrations were seen
to decrease under stressful growing conditions determined by
salinity (Levent Tuna et al. 2007). Although the assessment of
Environ Sci Pollut Res
the impact of NPs on plant nutrient content is in its infancy, it
is highly possible that NPs could alter the nutrient profile of
food crops (Gardea-Torresdey et al. 2014).
Considering that accumulations of Ca, K, Mg, Na, P and S
in roots are significantly higher with respect to the control,
CeO
2
-, Co- and Fe
3
O
4
-NPs can be considered the NPs that
inhibit K translocation to stem and leaves. Moreover, CeO
2
-
NPs do not allow Ca, Mg and P to be translocated to leaves;
the same effect was exerted by Co-NPs on P and S and by
Fe
3
O
4
-NPs on Na, P and S. This evidence suggests a compet-
itive inhibition between K and other cations, e.g. NP compo-
nent metal, as highlighted in tomato stressed with high Cd
concentration by Carvalho Bertoli et al. (2012).
SnO
2
- and Ni-NPs determined a lower element concentra-
tion, linked to a lower plant growth (root and leaf dry biomass
significantly lower with respect to the control); however, no
data are available about this aspect; therefore, further investi-
gations are needed.
Tomato is a fruit generally rich in K and mineral salts, thus
representing an important source of elements for human be-
ings. In this study, the fruits of plants exposed to NPs showed
a general enrichment in K and depletion of Mg, P and S with
respect to the control.
Generally, little is known about the influence of NPs on
plant mineral composition or on the macro- and micro-
element accumulation in different plant organs. A study con-
ducted on Lactuca sativa exposed to nano-Fe/Fe
3
O
4
and
nano-Cu/CuO treatments showed changes in the nutrient up-
take. A decrease in Mg, P and Ca was detected in leaves of
plants exposed to Cu treatments, while an increase in S and Ca
was found in the roots (Trujillo-Reyes et al. 2014); probably,
the component metal damage to root cellular membranes
could alter the plant’s capacity to absorb and transport some
nutrients (Fernandes and Henriques 1991;Wangetal.2011).
The effect of TiO
2
-NPs on plant mineral composition in
cucumber fruits was investigated by Servin et al. (2013)who
found a significant increase in K and P in this organ and
suggest that NPs have an effect similar to that of plant hor-
mones like cytokinins and gibberellins (Mandeh et al. 2012),
given that cytokinins havebeen found to affect P and K uptake
(Wu et al. 2003). Moreover, a study on the quality of rice
grains from plants grown in soil amended with CeO
2
-NPs
indicated that the exposure to these NPs could compromise
the quality of rice grain (Rico et al. 2013b).
Conclusion
The question of the uptake, bioaccumulation, biotransforma-
tion and risk of NPs in food crops is still not well understood.
The metal oxides and metallic engineered NPs were found to
have differing effects on the morphological parameters, the
uptake and the translocation of NP component metals in the
various tomato organs. The determination of the component
metal in tomato organs by ICP-OES showed that the tomato
plants exposed to Ag-, Co- and Ni-NPs have metal concen-
trations higher than the controls and that the fruits of plants
treated with Ag-NPs were contaminated. Moreover, changes
in the tomato plant mineral composition were assessed, and
the physiological disorders could be directly correlated to the
exposure to NPs. Further studies are required to evaluate the
behaviour ofthese NPs under field conditions and their fate in
the food chain.
Acknowledgments This study was supported by the INESE project
funded by the Italian Institute of Technology (IIT, Genoa, Italy).
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