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Chemosphere 159 (2016) 326e334
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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Uptake, translocation and physiological effects of magnetic iron oxide
(g-Fe2O3) nanoparticles in corn (Zea mays L.)
Junli Li a, Jing Hu a, Chuanxin Ma b, Yunqiang Wang c, Chan Wu a, Jin Huang a, d, *,
Baoshan Xing b
a
School of Chemistry, Chemical Engineering and Life Science, Wuhan University of Technology, Wuhan 430070, PR China
Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, United States
Institute of Economic Crops, Hubei Academy of Agricultural Science, Wuhan 430064, PR China
d
School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
b
c
h i g h l i g h t s
The lower concentration of g-Fe2O3 NPs had more positive effects on corn seeding growth.
The higher concentration of g-Fe2O3 NPs stress induced modulation of antioxidant enzymes and relevant mechanisms were studied.
g-Fe2O3 NPs mostly existed around epidermis of root and could not translocate from roots to shoots.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 February 2016
Received in revised form
23 May 2016
Accepted 28 May 2016
Iron oxide nanoparticles (g-Fe2O3 NPs) have emerged as an innovative and promising method of iron
application in agricultural systems. However, the possible toxicity of g-Fe2O3 NPs and its uptake and
translocation require further study prior to large-scale field application. In this study, we investigated
uptake and distribution of g-Fe2O3 NPs in corn (Zea mays L.) and its impacts on seed germination,
antioxidant enzyme activity, malondialdehyde (MDA) content, and chlorophyll content were determined.
20 mg/L of g-Fe2O3 NPs significantly promoted root elongation by 11.5%, and increased germination index
and vigor index by 27.2% and 39.6%, respectively. However, 50 and 100 mg/L g-Fe2O3 NPs remarkably
decreased root length by 13.5% and 12.5%, respectively. Additionally, evidence for g-Fe2O3 NPs induced
oxidative stress was exclusively found in the root. Exposures of different concentrations of NPs induced
notably high levels of MDA in corn roots, and the MDA levels of corn roots treated by g-Fe2O3 NPs (20
e100 mg/L) were 5e7-fold higher than that observed in the control plants. Meanwhile, the chlorophyll
contents were decreased by 11.6%, 39.9% and 19.6%, respectively, upon NPs treatment relative to the
control group. Images from fluorescence and transmission electron microscopy (TEM) indicated that gFe2O3 NPs could enter plant roots and migrate apoplastically from the epidermis to the endodermis and
accumulate the vacuole. Furthermore, we found that NPs mostly existed around the epidermis of root
and no translocation of NPs from roots to shoots was observed. Our results will be highly meaningful on
understanding the fate and physiological effects of g-Fe2O3 NPs in plants.
© 2016 Elsevier Ltd. All rights reserved.
Handling Editor: Tamara S. Galloway
Keywords:
g-Fe2O3 NPs
Physiological effect
Uptake
Translocation
1. Introduction
With the rapid development of nanotechnology, nanoparticles
(NPs) have been widely applied in various fields including environmental remediation, energy, biosensors, food industry and
* Corresponding author. School of Chemistry, Chemical Engineering and Life
Science, Wuhan University of Technology, Wuhan 430070, PR China.
E-mail address: [email protected] (J. Huang).
http://dx.doi.org/10.1016/j.chemosphere.2016.05.083
0045-6535/© 2016 Elsevier Ltd. All rights reserved.
medicine (Casero et al., 2014; Guo et al., 2014; Lu et al., 2014;
Martins et al., 2014). Currently, nanotechnology application aiming at crop protection and improvements of crop agronomic traits
engenders considerable interests. Nanotechnology-based fertilizer
and other agro-chemicals are promising to promote sustainable
development of agriculture. Evidence exists for NPs uptake and
localization of NPs inside of plant cells were reported in previous
studies (Khodakovskaya et al., 2009; Lin and Xing, 2008; Wang
et al., 2012; Zhu et al., 2008). Further studies have been
J. Li et al. / Chemosphere 159 (2016) 326e334
conducted to investigate impacts of NPs on plants from the aspects
of morphology and physiology. For example, carbon nanotubes,
gold (Au) nano-rods and titanium dioxide (TiO2) NPs could enhance
seed germination, root elongation as well as seedling growth
(Khodakovskaya et al., 2009; Mondal et al., 2011; Servin et al., 2012;
Wan et al., 2014; Yan et al., 2013). In contrast, other studies have
reported that NPs, such as TiO2, cerium oxide (CeO2), zinc oxide
(ZnO), g-Fe2O3, could also cause damage in plants, including decreases in photosynthesis efficiency (Lei et al., 2007; Ze et al., 2011),
chlorophyll degradation, total protein reduction (Krystofova et al.,
2013; Zhao et al., 2013), as well as micro and macro-nutrient
displacement (Servin et al., 2013; Zhao et al., 2014a). Oxidative
stresses caused by NPs could activate defense mechanisms to
counteract nanotoxicity in plants (Morales et al., 2013; Rico et al.,
2013; Servin et al., 2013). Many publications have reported the
main reason of toxicity upon NPs exposure, but results were contradictory. Kim et al. (2009) suggested that the toxicity of Ag NPs is
mainly due to oxidative stress and released silver ions. However,
Qian et al. (2013) compared the toxicity of silver nanoparticles and
silver ions on the growth of terrestrial plant model Arabidopsis
thaliana and found that Ag NPs showed stronger toxicity to
A. Thaliana compared with Agþ, demonstrating that growth inhibition and cell damage of plants can be directly attributed to the
nanoparticles themselves, not only the dissolved silver released
from Ag NPs. And other authors reported that both silver ions and
Ag NPs contribute to the toxicity (Kawata et al., 2009; Li et al., 2015;
Navarro et al., 2008).
Iron plays an essential role in plants at the physiological level,
especially in the process of photosynthesis. Iron deficiency in the
plants directly results in chlorophyll decrease and subsequently
lowers net photosynthesis efficiency. Iron deficiency is usually
recognized by chlorotic or yellowed interveinal areas in new leaves,
and could severely cause reduction in crop yields and even complete crop failure (Cuerinot and Yi, 1994). Due to the quick conversion of Fe into plant-unavailable Fe (III) forms and poor mobility
of Fe in phloem, soil application of inorganic Fe fertilizers to Fedeficient soils is usually ineffective (Rengel et al., 1999). In order
to address the shortcomings in the traditional iron-supplementing
methods, iron oxide nanoparticles are being tested as a new iron
delivery method. g-Fe2O3 NPs have many advantages, such as
biocompatibility, structural stability as well as magnetic properties.
The mechanism of uptake and translocation of g-Fe2O3 NPs and
their potential toxicity require further study prior to large-scale use
in agriculture. Li et al. (2013) and Ren et al. (2011) studied the
physiological effects of g-Fe2O3 NPs on watermelon (Citrullus
lanatus) planted in quartz sand and Chinese Mung Bean (Vigna
radiata L.) grown in silica sediment, respectively, and found that gFe2O3 NPs can physiologically enhance seed germination, root
growth, chlorophyll content in watermelon and mung bean,
although induced oxidative stress was also evident in plants upon
NPs exposure. Besides, root lengths in soybean (Glycine max (L.)
Merr.) (Alidoust and Isoda, 2013) and rice (Oryza sativa L. var.
Koshihikari) (Alidoust and Isoda, 2014) were increased in the presents of g-Fe2O3 NPs.
However, a comprehensive study focusing on mechanisms of
uptake and translocation of g-Fe2O3 NPs has not yet been reported.
Therefore, in our study, g-Fe2O3 NPs, as an iron source, were used to
investigate the effects of g-Fe2O3 NPs on corn (Zea mays L.) at
physiological level. Seed germination, activities of antioxidant enzymes, malondialdehyde (MDA) level and chlorophyll content were
tested. In addition, we examined the uptake and translocation of gFe2O3 NPs in corn, and analyzed the relationship between in-vivo
distribution of g-Fe2O3 NPs and corresponding physiological
changes in plant. This study could further fill up the gaps of g-Fe2O3
NPs use in agricultural crops from the aspect of nutrient
327
supplement and figure out the mechanism of uptake and translocation of g-Fe2O3 NPs in corn plants by using fluorescence labeled
NPs.
2. Materials and methods
2.1. Preparation of g-Fe2O3 NPs and fluorescent g-Fe2O3 NPs
Fe3O4 NPs were synthesized followed by a traditional coprecipitation method as described in Wang et al. (2015). Briefly,
2.7 g of FeCl3$6H2O and 1.4 g of FeCl2$4H2O were dissolved in
200 mL distilled water in a three-neck round-bottom flask equipped with a mechanical stirrer, reflux condenser and an inlet of nitrogen. The mixture was stirred vigorously for 15 min under
nitrogen flow, followed by heating up the reaction system to 60 C
for another 30 min. A volume of 200 mL of 1 M aqueous ammonia
solutions was added into the mixture, then pH was adjusted to
10e11. Fe3O4 precipitation was collected via magnet and further
purified with ethanol washing for five times. The collected precipitation was re-suspended in distilled water to make a suspension
of 2 g/L Fe3O4, which was stirred at 90 C for 6 h with a pH of 2e3.
The mixture was separated through magnetic separation, washing
with absolute ethanol for three times, vacuum-dried at 60 C for
12 h. As shown in Fig. S1A, g-Fe2O3 NPs presented a spherical shape
with a uniform size of 17.7 ± 3.9 nm under transmission electron
microscopy (TEM).
In order to observe g-Fe2O3 NPs distribution in the corn roots,
fluorescent dye labeled g-Fe2O3 NPs was applied (Fig. S2). The
fluorescent g-Fe2O3 NPs with an average diameter of 21.2 ± 2.9 nm
were prepared as follows (Fig. S1B). The image of fluorescent gFe2O3 NPs suspended in 1 phosphate buffered saline (PBS) was
shown in Fig. 5C. Briefly, 10 mg g-Fe2O3 NPs were suspended in a
mixture of 80 mL cyclohexane and 3 mL Nonidet P 40. The fluorescent dye was prepared by dissolving 3.75 mL fluorescein isothiocyanate (FITC) into 18 mL (3-aminopropyl) triethoxysilane
(APTES) and 0.65 mL aqueous ammonium hydroxide solution and
added into the g-Fe2O3 NPs suspension drop by drop. After 24 h
stirring, 0.3 mL tetraethyl orthosilicate (TEOS) was used to hydrolyze the bond between FITC and APTES for another 24 h. The
fluorescent g-Fe2O3 NPs (FITC-conjugated g-Fe2O3 nanoparticles)
were collected in menthol and purified by ethanol washing. The
purified NPs were centrifuged at 8000 rpm for 8 min and this
process was repeated 5 times. Finally, the synthesized FITCconjugated g-Fe2O3 NPs was vacuum-dried at 60 C for 12 h. The
related results of characterization of g-Fe2O3 NPs and fluorescent gFe2O3 NPs are present in supporting information, which could
provide solid evidence that FITC was conjugated to g-Fe2O3 NPs and
FITC labeled g-Fe2O3 NPs were stable and had fluorescent properties (Fig. S3e5).
2.2. Seed germination trial
Corn seeds were purchased from Hubei Academy of Agriculture
Sciences, China, and thoroughly washed with reverse osmosis (RO)
water prior to use. The seeds were soaked in Erlenmeyer flask
containing g-Fe2O3 NPs with concentrations of 0, 20, 50 and
100 mg/L for 3 h, and then germinated on moist filter paper in Petri
dishes at 28 C. Ten seeds were placed on each Petri dish; there
were three replicate dishes for each NPs concentration. After 36 h,
germination rate and root growth were measured.
2.3. Seedling trial
Corn seeds were soaked with deionized water in Erlenmeyer
flask, and then germinated on moist filter paper in Petri dishes at
328
J. Li et al. / Chemosphere 159 (2016) 326e334
28 C. Uniform seedlings were transferred to hydroponic system
and exposed to 0, 20, 50 and 100 mg/L of g-Fe2O3 NPs amended
nutrient solution [in 1 L solution (unit: mmol L1): K2SO4, 0.1875;
Ca(NO3)2, 0.5; KCl, 0.025; KH2PO4, 0.0625; MgSO4$7H2O, 0.1625;
H3BO3, 0.25 103; MnSO4$H2O, 0.25 103; ZnSO4$7H2O,
0.25 103; CuSO4$5H2O, 0.25 104; (NH4)6Mo7O24, 1.25 106,
pH 6.8]. The plants were grown in an environmentally controlled
growth chamber at 28/18 C with a 16 h/8 h light/dark cycle; the
light intensity was 2000 l. And the air was pump into the hydroponic system every 3 h for 30 min. Three replicates were applied
in each treatment. Activities of antioxidant enzymes, including
superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT),
malondialdehyde (MDA) and chlorophyll content were measured
after exposure for two weeks.
2.4. Malondialdehyde (MDA) content assay
A sample of 0.35 g root or 0.25 g leaf tissues was grounded in
2.0 mL of 10% trichloroacetic acid (TCA), and the mixture was
centrifuged at 1000 rpm for 10 min. A volume of 2 mL supernatant
was added into 2.0 mL of 0.6% thiobarbituric acid (TBA) and the
mixture was boiled in water bath for 15 min and then cooled down
at ambient temperature. The mixture was centrifuged at 4000 rpm
for 10 min. MDA content was measured by a UV752N spectrophotometer (Shanghai Jinke, China) at 450, 532 and 600 nm (Heath
and Packer, 1968).
absorbance at the wavelengths of 645 and 663 nm with 80%
acetone as a reference. Evaluation for total chlorophyll content was
determined by the following formula:
Chl a ¼ 12:72A663 2:59A645
Chl b ¼ 22:88A645 4:67A663
Chl T ¼ Chl a þ Chl b
2.7. Fluorescent observation of fluorescent g-Fe2O3 NPs in plant
root
Corn seedlings were cultivated in 20 mg/L FITC-conjugated gFe2O3 NPs suspensions in a growth chamber at 28/18 C with a 16 h/
8 h light/dark cycle; the light intensity was 2000 l, and the suspension was changed once a day. After 5 days of growth in the FITCconjugated g-Fe2O3 NPs suspensions, roots were sampled from the
corn seedlings after PBS washing, followed by sample fixation in
4 vol% paraformaldehyde for 6 h. Root sections of corn were
observed on an Olympus BX60 fluorescence microscope equipped
with a cooled CCD camera (1410E B0, Sensys Photometrics) using
Meta Morph software (Universal Imaging, version 4.5).
2.8. TEM observation of plant roots
2.5. Antioxidant enzyme activities
The roots (0.4 g) and leaves (0.3 g) of corn were separately homogenized in 10 mL of 0.05 M phosphate buffer (pH 7.8). The
mixture was centrifuged at 1000 rpm for 20 min, and stored at 4 C
until analysis.
2.5.1. Superoxide dismutase (SOD) activity assay
SOD activity was determined by the ability to inhibit photochemical reduction of nitrobluetetrazolium (NBT). The reaction
mixture consisted of 0.5 mL supernatant, 0.5 mL of 130 mM
methionine, 0.5 mL of 750 mM NBT, 0.5 mL of 100 mM EDTA-Na,
0.5 ml of 20 mM lactochrome and 3.5 mL of 0.05 M phosphate
buffer (pH 7.8). The thoroughly mixed sample was illuminated for
20 min, while the control sample prepared in distilled water were
placed in the dark. The absorbance was measured at 560 nm (Wang
et al., 2004).
2.5.2. Catalase (CAT) activity assay
CAT activity was measured by adding 0.5 mL of supernatant into
3 mL of 100 mM phosphate buffer (pH 7.0) containing 0.01% H2O2.
CAT activity was recorded at 240 nm every 30 s for 4.5 min (Gallego
et al., 1996).
2.5.3. Peroxidase (POD) activity assay
POD activity was measured based on the oxidization of guaiacol
catalyzed by peroxidase (Zhang et al., 1995). Briefly, 0.5 mL of crude
extract, 28 mL of 0.05 M guaiacol and 19 mL of 30% H2O2 were added
into 100 mM phosphate buffer (pH 7.0), to form a 3 mL testing
mixture. The reaction was measured at 470 nm (△A470) every 30 s
for 4.5 min. POD activity was expressed as the change of absorbance
per minute (△A470/min/g FW).
2.6. Chlorophyll content assay
Fresh leaves of corn (0.3 g) were ground in 10 mL of 80% acetone
and then the obtained homogenates were centrifuged at 1000 rpm
for 10 min. The supernatant was collected to determine the
Corn seedlings were cultivated in 20 mg/L g-Fe2O3 NPs suspension for 12 days. Upon harvest, roots were sampled from the
corn seedlings after PBS washing and fixed in 0.1 M PBS (pH 7.4)
containing 2.5% glutaraldehyde for 4 h at 4 C and then post-fixed in
1% osmium tetraoxide for another 2 h at room temperature. Next,
root samples were immersed in gradient ethanol for dehydration
and acetone embedding and root samples were sectioned on Leica
EM UC6 ultramicrotome for TEM observation. Ultrathin roots
samples of corn were observed on a FEI Tecnai G2 20 TWIN
transmission electron microscope.
2.9. Iron content in plant tissues
Root and shoot were separately harvested and dried at 70 C for
48 h. Oven-dried root and shoots were digested with 10 mL
perchloric acid (50%) in porcelain crucible for 30 min and then
filtered through 0.45 mm millipore-filter. Fe content in samples was
determined using Atomic Absorption Spectrometry (AAS).
2.10. Statistical analysis
Each treatment was conducted with three replicates, and the
results were presented as mean ± SD (standard deviation). The
statistical analysis of experimental data was verified with one-way
ANOVA followed by Duncan multiple comparison in the statistical
package IBM SPSS Version 22.
3. Results and discussion
3.1. Seed germination assays
The data of the seed germination test are presented in Table 1. As
shown in Table 1, germination rate and germination energy were
not significantly different among all treatments. However, germination index in the treatments with 20 and 50 mg/L g-Fe2O3 NPs
was 27.2% and 18.9% higher than the control, respectively. Vigor
index in 20 mg/L g-Fe2O3 NPs treatment was 39.6% higher than the
J. Li et al. / Chemosphere 159 (2016) 326e334
329
Table 1
Effect of g-Fe2O3 NPs on germination rate, germination energy, germination index, and vigor index measured 3 days after incubation.
Concentration of g-Fe2O3 NPs (mg/L)
Germination rate (%)
0
20
50
100
86.7
87.5
87.5
70.0
±
±
±
±
5.7a
5.0a
15.0a
14.0a
Germination energy (%)
76.6
85.0
70.0
62.5
±
±
±
±
5.8a
5.8a
18.3a
15.0a
Germination index
18.50
23.53
21.99
18.26
±
±
±
±
0.21c
0.31a
0.95b
1.08c
Vigor index
390.0
544.5
404.8
341.3
±
±
±
±
4.45b
7.19a
17.6b
20.3c
The different letters signify the value P 0.05.
control, while that of 100 mg/L g-Fe2O3 NPs was significantly lower
(12.5%) than the control. Overall, g-Fe2O3 NPs at lower concentration (20 mg/L) might enhance plant growth at early stage of
germination.
Dose-dependent experiments were conducted to investigate the
effects of g-Fe2O3 NPs on corn seeds germination. After 2 days incubation, root length was measured every 3 h for successive 12 h.
As shown in Fig. 1A, exposure to 20 mg/L g-Fe2O3 NPs notably
increased root elongation compared with the control group. At the
last record, 20 mg/L of g-Fe2O3 NPs significantly promoted root
elongation by 11.5% compared with control. Phenotypic difference
of root elongation at 20 mg/L was observed in Fig. 1B. Wang et al.
(2012) found that multi-walled carbon nanotubes could enhance
root elongation of wheat (Triticum aestivum), and they observed
that root cell lengths in wheat seedlings upon exposure to 80 mg/mL
o-MWCNTs were increased by 1.4-fold, which suggested that cell
elongation in the root system could lead to faster root growth. Kim
et al. (2014) reported that nano zero valent iron enhanced root
elongation by inducing OH radical-induced cell wall loosening. In
order to figure out the mechanism of plants root elongation upon
exposure to g-Fe2O3 NPs, further studies need to be conducted. In
addition, NPs inhibited root elongation as exposure doses
increased, especially at 100 mg/L g-Fe2O3 NPs treatment. This
might be attributed to the fact that NPs in high concentration could
form clusters and tend to block the pathways of nutrition uptake
(Ren et al., 2011).
3.2. MDA content
MDA content is a significant parameter to assess cell membrane
integrity and its content represents plant senescence and injury in
the presence of pollutants (Nair et al., 2014; Zhang et al., 2007; Song
et al., 2012). Fig. 2A shows MDA content in root and leaf tissues
treated with different concentrations of g-Fe2O3 NPs. Excess
amounts of MDA production were evident in root tissues among all
treatments relative to the control group. The MDA levels of corn
roots treated by g-Fe2O3 NPs (20e100 mg/L) were 5e7-fold higher
than that observed in the control plants, indicating that oxidative
stress occurred in plants. The common findings in the previous
studies indicated metal-based NPs could elevate the MDA content
and subsequently result in oxidative stresses in plants. Upon
exposure to both 200 and 250 mg/L, silver nanoparticles (Ag NPs)
could significantly elevate MDA content in shoot and root of wild
type (WT) Crambe abyssinica (Ma et al., 2015a). At an exposure of
1000 ppm CeO2 NPs, the MDA levels of Arabidopsis thaliana (L.)
Heynh. were 4-fold higher than that observed in the control plants
(Ma et al., 2013). However, in our study, g-Fe2O3 NPs have no
positive impact on MDA production in corn leaves as compared
with the control. According to the result of Fe distribution (Fig. 3B,
as will be discussed later), none of the g-Fe2O3 NPs increased the
level of Fe concentration in shoots, which could be the reason why
no lipid peroxidation occurred in corn leaves.
3.3. Antioxidant enzyme activities
Reactive oxygen species (ROS), especially for hydrogen peroxide
(H2O2) and superoxide (O
2 ), induced by NPs could cause oxidative
stress in plants. In order to counteract such toxicity, plant could
alter activities of antioxidant enzymes such as SOD, CAT, and POD,
all of which are able to scavenge ROS and further lower their
toxicity. Fig. 2BeD shows the enzyme activity in corn roots and
leaves treated with different concentrations of g-Fe2O3 NPs. In plant
cells, three types of SOD, containing Fe-SOD, Mn-SOD, and Cu-ZnSOD, are able to rapidly convert O
2 to H2O2 (Ma et al., 2015b).
CAT is one of the common antioxidant enzymes that convert H2O2
to H2O and O2 (Ma et al., 2015b). As shown in Fig. 2B, no difference
of SOD activity in corn roots was statistically observed as compared
to control or among treatments. In contrast, inhibition of SOD activity only happened in leaves treated with 20 mg/L g-Fe2O3 NPs.
No difference of CAT activity in roots was found until exposure dose
Fig. 1. (A) Root length of corn seeds incubated in the solution with 0 mg/L, 20 mg/L, 50 mg/L, 100 mg/L g-Fe2O3 NPs for successive 12 h (Triplicate groups with 10 seeds each). Values
of root length followed by different letters are significantly different at p 0.05. (B) Photograph of corn seedlings incubated in the solution amended with 0 mg/L, 20 mg/L, 50 mg/L,
100 mg/L g-Fe2O3 NPs after 2.5 days incubation.
330
J. Li et al. / Chemosphere 159 (2016) 326e334
Fig. 2. (A) MDA content and enzyme activities containing (B) activity of superoxide dismutase (SOD); (C) activity of catalase (CAT); (D) activity of peroxidase (POD) in root and leaf
tissues of corn exposed to 0, 20, 50 and 100 mg/L g-Fe2O3 NPs. Data are mean ± standard error of three replicates. Values followed by different lowercase and uppercase letters,
respectively, are significantly different at p 0.05.
Fig. 3. (A) Chorophyll content and (B, C) Fe uptake and distribution in corn treated with 0, 20, 50, 100 mg/L g-Fe2O3 NPs. Panel B and C represent Fe content in shoots and roots, TFs
(shoots/roots), respectively. Data are mean ± standard error of three replicates. Values followed by different letters are significantly different at p 0.05.
increased to 100 mg/L (Fig. 2C). Our results suggest that treatment
of g-Fe2O3 NPs could lead to the accumulation of H2O2 in corn roots,
which might stimulate up-regulation of CAT enzyme activity to
break down the excessive amounts of H2O2. However, g-Fe2O3 NPs
treatment did not show any inhibition effect on CAT activity in the
corn leaves, indicating oxidative stress did not happen in leaf tissues. Fig. 2D shows POD activity decreased in corn roots treated
with 20 mg/L g-Fe2O3 NPs and increased at the 100 mg/L of g-Fe2O3
NP concentration. In corn leaves, POD activity was not inhibited in
contrast with the control, except at the concentration of 50 mg/L.
Our results of antioxidant enzyme activities suggest that g-Fe2O3
NPs could activate an antioxidant defense in corn root through
inducing the oxidative stress, and plant, itself, could regulate SOD,
CAT and POD activities to break down excess ROS. Furthermore,
comparing with the previous studies, ROS generation and antioxidant enzyme activities may vary with exposure conditions, NPs
type, and plant species (Ma et al., 2015b). For example, SOD activities in ryegrass (Lolium perenne L.) and pumpkin (Cucurbita mixta)
exhibited different responses upon exposure to Fe3O4 NPs (Wang
et al., 2011). In ryegrass, SOD activity in the roots was elevated in
the treatments of 30 and 100 mg/L Fe3O4 NPs, however, this
elevation was only evident in pumpkin root treated with 30 mg/L
Fe3O4 NPs. Similarly, the decrease in SOD activity was found in
100 mg/L Fe3O4 NPs treated ryegrass shoots, while Fe3O4 NPs had
no impact on the SOD activity on pumpkin shoots.
3.4. Chlorophyll content
Chlorophyll is the critical photosynthetic pigment, chlorophyll
levels can be a significant indicator of toxicity to plants (Ma et al.,
2015a). As shown in Fig. 3A, for g-Fe2O3 NPs treatment, the chlorophyll contents were decreased by 11.6%, 39.9% and 19.6%,
J. Li et al. / Chemosphere 159 (2016) 326e334
respectively, relative to the control group. Similarly, a decrease in
chlorophyll content upon exposure to metal and metal oxide
nanoparticles has been reported by Nair et al. (2014), Nair and
Chung (2014), Barhoumi et al. (2015). Oxidative damage could
happen in chloroplast within plant cells through interactions with
metal-based NPs, ultimately disrupt the synthesis of chlorophyll or
cause the degradation of chlorophyll in leaves (Ma et al., 2015b).
However, in our study, no lipid peroxidation occurred in corn
leaves, suggesting that the decrease of chlorophyll content could be
an alternative mechanism. Besides, Fe content in shoots was in a
low level, and shoot-to-root translocation factor (TF; Fe concentration in shoot divided Fe concentration in root) among all g-Fe2O3
NPs treatments were significantly lower than the control
(Fig. 3BeC). The inefficient translocation of iron in the plant shoots
might result in the decrease in chlorophyll content.
3.5. Uptake pathway of g-Fe2O3 NPs in corn plants
In order to figure out the pattern of uptake and distribution of gFe2O3 NPs in plants, FITC-labeled NPs were applied to corn for
short-term exposure. Fluorescent images of root epidermis and
cross-section are shown in Fig. 4. The cyan bright dots in the image
represented the FITC-conjugated g-Fe2O3 NPs. In both Fig. 4A and B,
FITC-conjugated g-Fe2O3 NPs were observed in the epidermis.
Previous studies reported that the surface of NPs could be covered
by various macromolecules, and the chemical surface property and
the ligand density of NPs could strongly influence the interaction
between NP-bound ligands and cellular receptors (Chithrani et al.,
2006; Mahmoudi and Serpooshan, 2011; Rauch et al., 2013). This
could be due to the fact that macromolecules in root exudate
altered physicochemical properties of the NPs surface and resulted
in NPs accumulation in the root epidermis (Ghafariyan et al., 2013;
Judy et al., 2012). Fig. 4C and D depict that a small fraction of FITCconjugated g-Fe2O3 NPs can move into endodermis and cortex of
corn root, and a small portion were also located close to xylem.
331
Most of NPs were located in the intercellular space among the cell
walls of cortex tissues, which suggested that the g-Fe2O3 NPs could
penetrate corn roots and migrate between cells from the epidermis
to the endodermis via the apoplastic pathway.
In order to figure out the NPs distribution at cellular level, TEM
images of corn roots are shown in Fig. 5. Dark spots in Fig. 5B were
observed in comparison to the control (Fig. 5A). In Fig. 5E, the size
of these spots was approximately 19 nm, which was similar to the
size of g-Fe2O3 NPs, indicating the NPs could penetrate into the
corn cells. As shown in Fig. 5C and D, g-Fe2O3 NPs mainly accumulated in the vacuole. Almost all mature plant cells have large
vacuoles that occupy a large part of the cell volume contained both
hydrolytic enzymes and a variety of defense proteins (HaraNishimura and Hatsugai, 2011). Larue et al. (2012) reported the
common finding that TiO2 nanoparticles (anatase, 14 nm or rutile,
22 nm) were observed in the vacuoles of wheat (Triticum aestivum
spp.) roots.
3.6. Translocation of Fe from roots to shoots
As seen in Fig. 3B, the iron content in the roots exposed to
20 mg/L, 50 mg/L of g-Fe2O3 NPs was 9.9 and 18.6 times of the
control group. However, at the treatment of 100 mg/L g-Fe2O3 NPs,
Fe content in roots dramatically decreased in contrast with 50 mg/L
g-Fe2O3 NPs treatment. In Fig. 3C, notably, the TFs among all gFe2O3 NPs treatments were significantly lower than the control,
implying that low amounts of iron could translocate to the shoot
parts (TF < 0.05 in this case). The sizes of aggregated NPs adsorbed
on the root surface were larger than the size of porous space in the
cell wall, only a few individual ones could move into the endodermis and cortex of corn root and were available for the upward
transport. Another possible reason for the low TF could be attributed to the magnetism of g-Fe2O3 NPs. Hong et al. (2009) concluded
that g-Fe2O3 NPs transport was influenced by a combination of
electrostatic and magnetic interactions. The accumulation of large
Fig. 4. (A) Fluorescence image of roots epidermis, and (B, C) cross-sections of roots from corn seedlings that were treated with 20 mg/L FITC-conjugated g-Fe2O3 NPs for 5 days; (D)
is magnified view of the squared region in (C). (The cyan bright dots (NPs) in the photographs represented the FITC-conjugated g-Fe2O3 NPs.).
332
J. Li et al. / Chemosphere 159 (2016) 326e334
Fig. 5. Root cell TEM image of corn seedlings exposed with or without g-Fe2O3 NPs exposure for 2 weeks: (A) control group and (B) treated group with 20 mg/L g-Fe2O3 NPs
exposure, (C) magnified view of the squared region in (B), magnified views (D, E) of the squared regions in (C).
amount of NPs in roots indicated that magnetically induced aggregation and subsequent straining resulted in greater retention.
Given the evidence for the absence of g-Fe2O3 NPs in the NPs
treated corn shoot, we further investigated g-Fe2O3 NPs distribution in shoot by fluorescence microscope (Fig. 6). The corn seedlings
were exposed to FITC-conjugated g-Fe2O3 NPs for 5 days. The cyan
fluorescence signals of the FITC-conjugated g-Fe2O3 NPs were
exclusively found in the root epidermis, while no presence of
fluorescent signal was observed in shoot cross-section. Therefore,
most g-Fe2O3 NPs adhered to the surface of corn roots, and could
not transport to aboveground part in spite of iron element uptake.
Lack of translocation of g-Fe2O3 NPs in corn plants might explain
that oxidative stress and plant membrane impairment only
happened in the roots rather than in the leaves. Furthermore, high
accumulation of g-Fe2O3 NPs in the epidermis of corn roots may
have led to the shortage of the iron element in the corn leaves, and
Fig. 6. Fluorescence image of (A) corn roots cross-sections and (B) shoots. (The cyan bright dots (NPs) in the image represented FITC-conjugated g-Fe2O3 NPs.).
J. Li et al. / Chemosphere 159 (2016) 326e334
caused the decrease in chlorophyll content.
4. Conclusions
The comprehensive physiological effects of g-Fe2O3 NPs on corn
plants, including seed germination tests, activities of antioxidant
enzymes in plant defense system, the MDA contents as well as the
chlorophyll contents, have been investigated in the present work.
The images from fluorescence microscope and TEM demonstrated
that g-Fe2O3 NPs were mainly accumulated in the epidermis of corn
roots and migrated among cells from epidermis to endodermis by
apoplastic pathway, and could further penetrate into the corn cells
and localize in the vacuole. Moreover, the translocation of g-Fe2O3
NPs in corn plants revealed that high concentrations of NPs
distributed on the surface of corn roots, whereas the presence of gFe2O3 NPs in the shoot was not evident. And this phenomenon
could further explain the reason that oxidative stress induced by gFe2O3 NPs exclusively happened in the corn roots. The inefficient
translocation of iron in the plant shoots might result in the decrease
in chlorophyll content. The FITC labeled g-Fe2O3 NPs is an original
attempt and our results confirmed that tracking behavior of fluorescent g-Fe2O3 NPs in plants under fluorescence microscope could
be more visualized and accurate to reflect the fate of NPs after
entering root cells. There are several papers that have already reported the effects of NPs on crops over full growth cycle. For
example, CeO2 NPs could affect the yield and quality of cucumber
(Zhao et al., 2013, 2014b) and corn (Zhao et al., 2015), but had no
impact on the final biomass and yield, Ce concentration in shoots,
as well as sugar and starch contents in wheat grains (Du et al.,
2015). The investigation of uptake, translocation and physiological effects of g-Fe2O3 NPs in corn seedlings could lay a foundation
for NPs potential impact in the whole life circle of corn. In
conclusion, our study could help us better understand how NPs
impact crop from the aspects of the physiological and biochemical
levels and provide the useful information for the safe application of
g-Fe2O3 NPs in agriculture.
Acknowledgments
This work was supported by the International Science & Technology Cooperation Program of China, Ministry of Science and
Technology of China (Grant No. 2014DFG52500); National Natural
Science Foundation of China (Grant No. 31301735); the Fundamental Research Funds for the Central Universities (WUT:
2016IB006).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.chemosphere.2016.05.083.
References
Alidoust, D., Isoda, A., 2013. Effect of gFe2O3 nanoparticles on photosynthetic
characteristic of soybean (glycine max (L.) merr.): foliar spray versus soil
amendment. Acta. Physiol. Plant 35 (12), 3365e3375.
Alidoust, D., Isoda, A., 2014. Phytotoxicity assessment of g-Fe2O3 nanoparticles on
root elongation and growth of rice plant. Environ. Earth Sci. 71 (12), 5173e5182.
Barhoumi, L., Oukarroum, A., Ben Taher, L., Smiri, L., Abdelmelek, H., Dewez, D.,
2015. Effects of superparamagnetic iron oxide nanoparticles on photosynthesis
and growth of the aquatic plant lemna gibba. Arch. Environ. Contam. Toxicol. 68
(3), 510e520.
Cuerinot, M.L., Yi, Y., 1994. Iron: nutritious, noxious, and not readily available. Plant
Physiol. 104, 815e820.
Chithrani, B.D., Ghazani, A.A., Chan, W.C., 2006. Determining the size and shape
dependence of gold nanoparticle uptake into mammalian cells. Nano Lett. 6 (4),
662e668.
zquez, L., Parra-Alfambra, A.M.,
Casero, E., Alonso, C., Petit-Domínguez, M.D., Va
333
Merino, P., et al., 2014. Lactate biosensor based on a bionanocomposite
composed of titanium oxide nanoparticles, photocatalytically reduced graphene, and lactate oxidase. Microchim. Acta 181 (1), 79e87.
Du, W., Gardea-Torresdey, J., Ji, R., Yin, Y., Zhu, J., Peralta-Videa, J., Guo, H., 2015.
Physiological and biochemical changes imposed by CeO2 nanoparticles on
wheat: a life cycle field study. Environ. Sci. Technol. 49 (19), 11884e11893.
Gallego, S.M., Benavídes, M.P., Tomaro, M.L., 1996. Effect of heavy metal ion excess
on sunflower leaves: evidence for involvement of oxidative stress. Plant. Sci. 121
(2), 151e159.
Ghafariyan, M., Malakouti, M., Dadpour, M., Stroeve, P., Mahmoudi, M., 2013. Effects
of magnetite nanoparticles on soybean chlorophyll. Environ. Sci. Technol. 47
(18), 10645e10652.
Guo, X., Liang, B., Jian, J., Zhang, Y., Ye, X., 2014. Glucose biosensor based on a
platinum electrode modified with rhodium nanoparticles and with glucose
oxidase immobilized on gold nanoparticles. Microchim. Acta 181 (5), 519e525.
Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. I. kinetics
and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125 (1),
189e198.
Hong, Y., Honda, R., Myung, N., Walker, S., 2009. Transport of iron-based nanoparticles: role of magnetic properties. Environ. Sci. Technol. 43 (23),
8834e8839.
Hara-Nishimura, I., Hatsugai, N., 2011. The role of vacuole in plant cell death. Cell
death Differ. 18 (8), 1298e1304.
Judy, J., Unrine, J., Rao, W., Wirick, S., Bertsch, P., 2012. Bioavailability of gold
nanomaterials to plants: importance of particle size and surface coating. Environ. Sci. Technol. 46 (15), 8467e8474.
Kim, S., Choi, J.E., Choi, J., Chung, K.H., Park, K., Yi, J., Ryu, D.Y., 2009. Oxidative stress
dependent toxicity of silver nanoparticles in human hepatoma cells. Toxicol.
Vitro 23, 1076e1084.
Kawata, K., Osawa, M., Okabe, S., 2009. In vitro toxicity of silver nanoparticles at
noncytotoxic doses to HepG2 human hepatoma cells. Environ. Sci. Technol. 43,
6046e6051.
Khodakovskaya, M., Dervishi, E., Mahmood, M., Xu, Y., Li, Z., Watanabe, F., Biris, A.,
2009. Carbon nanotubes are able to penetrate plant seed coat and dramatically
affect seed germination and plant growth (retracted article. see vol. 6, pg. 7541,
2012). ACS Nano 3 (10), 3221e3227.
Krystofova, O., Sochor, J., Zitka, O., Babula, P., Kudrle, V., Adam, V., Kizek, R., 2013.
Effect of magnetic nanoparticles on tobacco BY-2 cell suspension culture. Int. J.
Environ. Res. Public. Health 10 (1), 47e71.
Kim, J., Lee, Y., Kim, E., Gu, S., Sohn, E.J., Seo, Y.S., et al., 2014. Exposure of iron
nanoparticles to Arabidopsis thaliana enhances root elongation by triggering
cell wall loosening. Environ. Sci. Technol. 48 (6), 3477.
Lei, Z., Mingyu, S., Chao, L., Liang, C., Hao, H., Xiao, W., et al., 2007. Effects of
nanoanatase TiO2 on photosynthesis of spinach chloroplasts under different
light illumination. Biol. Trace Elem. Res. 119 (1), 68e76.
Lin, D., Xing, B., 2008. Root uptake and phytotoxicity of ZnO nanoparticles. Environ.
Sci. Technol. 42 (15), 5580e5585.
Larue, C., Laurette, J., Herlin-Boime, N., Khodja, H., Fayard, B., Flank, A., et al., 2012.
Accumulation, translocation and impact of TiO2 nanoparticles in wheat (triticum aestivum spp.): influence of diameter and crystal phase. Sci. Total Environ.
431, 197e208.
Li, J., Chang, P., Huang, J., Wang, Y., Yuan, H., Ren, H., 2013. Physiological effects of
magnetic iron oxide nanoparticles towards watermelon. J. Nanosci. Nanotechnol. 13 (8), 5561e5567.
Lu, W., Ling, M., Jia, M., Huang, P., Li, C., Yan, B., 2014. Facile synthesis and characterization of polyethylenimine- coated Fe3O4 superparamagnetic nanoparticles
for cancer cell separation. Mol. Med. Rep. 9 (3), 1080e1084.
Li, L., Wu, H., Peijnenburg, W., van Gestel, C., 2015. Both released silver ions and
particulate ag contribute to the toxicity of AgNPs to earthworm eisenia fetida.
Nanotoxicology 9 (6), 792e801.
Mahmoudi, M., Serpooshan, V., 2011. Large protein absorptions from small changes
on the surface of nanoparticles. J. Phys. Chem. C 115 (37), 18275e18283.
Mondal, A., Basu, R., Das, S., Nandy, P., 2011. Beneficial role of carbon nanotubes on
mustard plant growth: an agricultural prospect. J. Nanopart. Res. 13 (10),
4519e4528.
Morales, M., Rico, C., Hernandez-Viezcas, J., Nunez, J., Barrios, A., Tafoya, A., et al.,
2013. Toxicity assessment of cerium oxide nanoparticles in cilantro (coriandrum
sativum L.) plants grown in organic soil. J. Agric. Food. Chem. 61 (26),
6224e6230.
Ma, C., Chhikara, S., Xing, B., Musante, C., White, J., Dhankher, O., 2013. Physiological
and molecular response of arabidopsis thaliana (L.) to nanoparticle cerium and
indium oxide exposure. ACS Sustain. Chem. Eng. 1 (7), 768e778.
ndez, P.S., Troiani, H.E., Martins, M.E., Arenillas, A., Camara, G.A.,
Martins, C.A., Ferna
2014. Agglomeration and cleaning of carbon supported palladium nanoparticles
in electrochemical environment. Electrocatalysis 5 (2), 204e212.
Ma, C., Chhikara, S., Minocha, R., Long, S., Musante, C., White, J.C., et al., 2015a.
Reduced silver nanoparticle phytotoxicity in crambe abyssinica with enhanced
glutathione production by overexpressing bacterial g-glutamylcysteine synthase. Environ. Sci. Technol. 49 (16), 10117.
Ma, C., White, J., Dhankher, O., Xing, B., 2015b. Metal-based nanotoxicity and
detoxification pathways in higher plants. Environ. Sci. Technol. 49 (12),
7109e7122.
Navarro, E., Piccapietra, F., Wagner, B., Marconi, F., Kaegi, R., Odzak, N., Sigg, L.,
Behra, R., 2008. Toxicity of silver nanoparticles to Chlamydomonas reinhardtii.
Environ. Sci. Technol. 42, 8959e8964.
334
J. Li et al. / Chemosphere 159 (2016) 326e334
Nair, P.M., Kim, S., Chung, I.M., 2014. Copper oxide nanoparticle toxicity in mung
bean (vigna radiata L.) seedlings: physiological and molecular level responses of
in vitro grown plants. Acta. Physiol. Plant 36 (11), 2947e2958.
Nair, P., Chung, I., 2014. Physiological and molecular level effects of silver nanoparticles exposure in rice (oryza sativa L.) seedlings. Chemosphere 112, 105e113.
Qian, H.F., Peng, X.F., Han, X., Ren, J., Sun, L.W., Fu, Z.W., 2013. Comparison of the
toxicity of silver nanoparticles and silver ions on the growth of terrestrial plant
model arabidopsis thaliana. J. Environ. Sci. (China) 25 (9), 1947e1955.
Rengel, Z., Batten, G.D., Crowley, D.E., 1999. Agronomic approaches for improving
teh micronutrient density inedible portions of field crops. Field. Crop. Res. 60,
27e40.
Ren, H., Liu, L., Liu, C., He, S., Huang, J., Li, J., et al., 2011. Physiological investigation of
magnetic iron oxide nanoparticles towards chinese mung bean. J. Biomed.
Nanotechnol. 7 (5), 677e684.
Rico, C., Hong, J., Morales, M., Zhao, L., Barrios, A., Zhang, J., et al., 2013. Effect of
cerium oxide nanoparticles on rice: a study involving the antioxidant defense
system and in vivo fluorescence imaging. Environ. Sci. Technol. 47 (11),
5635e5642.
Rauch, J., Kolch, W., Laurent, S., 2013. Mahmoudi, M. Big signals from small particles: regulation of cell signaling pathways by nanoparticles. Chem. Rev. 113 (5),
3391e3406.
Song, H., Wang, Y., Sun, C., Wang, Y., Peng, Y., Cheng, H., 2012. Effects of pyrene on
antioxidant systems and lipid peroxidation level in mangrove plants, bruguiera
gymnorrhiza. Ecotoxicology 21 (6), 1625e1632.
Servin, A.D., Castillo-Michel, H., Hernandez-Viezcas, J.A., Diaz, B.C., PeraltaVidea, J.R., Gardea-Torresdey, J.L., 2012. Synchrotron micro-XRF and microXANES confirmation of the uptake and translocation of TiO2 nanoparticles in
cucumber (cucumis sativus) plants. Environ. Sci. Technol. 46 (14), 7637e7643.
Servin, A., Morales, M., Castillo-Michel, H., Hemandez-Viezcas, J., Munoz, B., Zhao, L.,
et al., 2013. Synchrotron verification of TiO2 accumulation in cucumber fruit: a
possible pathway of TiO2 nanoparticle transfer from soil into the food chain.
Environ. Sci. Technol. 47 (20), 11592e11598.
Wang, Y., Wang, X., Ying, Y., Chen, J., 2004. Transgenic arabidopsis overexpressing
mn-SOD enhanced salt-tolerance. Plant Sci. 167 (4), 671e677.
Wang, H., Kou, X., Pei, Z., Xiao, J., Shan, X., Xing, B., 2011. Physiological effects of
magnetite (Fe3O4) nanoparticles on perennial ryegrass (lolium perenne L.) and
pumpkin (cucurbita mixta) plants. Nanotoxicology 5 (1), 30e42.
Wang, X., Han, H., Liu, X., Gu, X., Chen, K., Lu, D., 2012. Multi-walled carbon
nanotubes can enhance root elongation of wheat (triticum aestivum) plants.
J. Nanopart. Res. 14 (6), 1e10.
Wan, Y., Li, J., Ren, H., Huang, J., Yuan, H., 2014. Physiological investigation of gold
nanorods toward watermelon. J. Nanosci. Nanotechnol. 14 (8), 6089e6094.
Wang, M., Liu, X., Hu, J., Li, J., Huang, J., 2015. Nano-ferric oxide promotes watermelon growth. J. Biomater. Nanobiotech. 6 (3), 160e167.
Yan, S., Zhao, L., Li, H., Zhang, Q., Tan, J., Huang, M., , et al.Li, L., 2013. Single-walled
carbon nanotubes selectively influence maize root tissue development
accompanied by the change in the related gene expression. J. Hazard Mater.
246, 110e118.
Zhang, J., Cui, S., Li, J., Kirkham, M.B., 1995. Protoplasmic factors, antoxidant responses, and chilling resistance in maize. Plant Physiol. Biochem. 33, 567e575.
Zhang, F., Wang, Y., Lou, Z., Dong, J., 2007. Effect of heavy metal stress on antioxidative enzymes and lipid peroxidation in leaves and roots of two mangrove
plant seedlings (kandelia candel and bruguiera gymnorrhiza). Chemosphere 67
(1), 44e50.
Zhu, H., Han, J., Xiao, J., Jin, Y., 2008. Uptake, translocation, and accumulation of
manufactured iron oxide nanoparticles by pumpkin plants. J. Environ. Monit. 10
(6), 713e717.
Ze, Y., Liu, C., Wang, L., Hong, M., Hong, F., 2011. The regulation of TiO2 nanoparticles
on the expression of light-harvesting complex II and photosynthesis of chloroplasts of arabidopsis thaliana. Biol. Trace Elem. Res. 143 (2), 1131e1141.
Zhao, L., Sun, Y., Hernandez-Viezcas, J., Servin, A., Hong, J., Niu, G., et al., 2013. Influence of CeO2 and ZnO nanoparticles on cucumber physiological markers and
bioaccumulation of Ce and Zn: a life cycle study. J. Agric. Food. Chem. 61 (49),
11945e11951.
Zhao, L., Peralta-Videa, J.R., Peng, B., Bandyopadhyay, S., Corral-Diaz, B., Osuna, P.,
Montes, M.O., Keller, A.A., Gardea-Torresdey, J.L., 2014a. Alginate modifies the
physiological impact of CeO2 nanoparticles in corn seedlings cultivated in soil.
J. Environ. Sci. 26 (2), 382e389.
Zhao, L., Peralta-Videa, J., Rico, C., Hernandez-Viezcas, J., Sun, Y., Niu, G., et al., 2014b.
CeO2 and ZnO nanoparticles change the nutritional qualities of cucumber
(cucumis sativus). J. Agric. Food. Chem. 62 (13), 2752e2759.
Zhao, L., Sun, Y., Hernandez-Viezcas, J.A., Hong, J., Majumdar, S., Niu, G., et al., 2015.
Monitoring the environmental effects of CeO2 and ZnO nanoparticles through
the life cycle of corn (zea mays) plants and in situ m-XRF mapping of nutrients in
kernels. Environ. Sci. Technol. 49 (5), 2921e2928.