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Science of the Total Environment 515516 (2015) 6069

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Comparative phytotoxicity of ZnO NPs, bulk ZnO, and ionic zinc onto the
alfalfa plants symbiotically associated with Sinorhizobium meliloti in soil
Susmita Bandyopadhyay a,d, Germn Plascencia-Villa c, Arnab Mukherjee a,d, Cyren M. Rico b,d,
Miguel Jos-Yacamn c, Jose R. Peralta-Videa a,b,d, Jorge L. Gardea-Torresdey a,b,d,

Environmental Science and Engineering PhD program, The University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, United States
Department of Chemistry, The University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, United States
Department of Physics and Astronomy, The University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249, United States
University of California Center for Environmental Implications of Nanotechnology (UC CEIN), The University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, United States




ZnO NPs, bulk ZnO and ZnCl2 differentially affected alfalfaS. meliloti symbiosis.
ZnO NPs and ionic Zn reduced root
and shoot biomass by 80% and 25%,
Ionic treatments showed a signicant
reduction in leaf protein content.
Both ZnO NP and bulk ZnO treatments
altered CAT levels in roots, stems, and

a r t i c l e

i n f o

Article history:
Received 18 December 2014
Received in revised form 4 February 2015
Accepted 4 February 2015
Available online xxxx
Editor: D. Barcelo
ZnO nanoparticles
Bulk ZnO
Ionic Zn
General plant behavior
Zn uptake

a b s t r a c t
ZnO nanoparticles (NPs) are reported as potentially phytotoxic in hydroponic and soil media. However, studies
on ZnO NPs toxicity in a plant inoculated with bacterium in soil are limited. In this study, ZnO NPs, bulk ZnO,
and ZnCl2 were exposed to the symbiotic alfalfa (Medicago sativa L.)Sinorhizobium meliloti association at concentrations ranging from 0 to 750 mg/kg soil. Plant growth, Zn bioaccumulation, dry biomass, leaf area, total protein,
and catalase (CAT) activity were measured in 30 day-old plants. Results showed 50% germination reduction by
bulk ZnO at 500 and 750 mg/kg and all ZnCl2 concentrations. ZnO NPs and ionic Zn reduced root and shoot biomass by 80% and 25%, respectively. Conversely, bulk ZnO at 750 mg/kg increased shoot and root biomass by 225%
and 10%, respectively, compared to control. At 500 and 750 mg/kg, ZnCl2 reduced CAT activity in stems and
leaves. Total leaf protein signicantly decreased as external ZnCl2 concentration increased. STEM-EDX imaging
revealed the presence of ZnO particles in the root, stem, leaf, and nodule tissues. ZnO NPs showed less toxicity
compared to ZnCl2 and bulk ZnO found to be growth enhancing on measured traits. These ndings are signicant
to reveal the toxicity effects of different Zn species (NPs, bulk, and ionic Zn) into environmentally important
plant-bacterial system in soil.
2015 Elsevier B.V. All rights reserved.

Corresponding author at: The University of Texas at El Paso, 500 West University Ave., El Paso, TX 79968, United States.
E-mail address: (J.L. Gardea-Torresdey).
0048-9697/ 2015 Elsevier B.V. All rights reserved.

S. Bandyopadhyay et al. / Science of the Total Environment 515516 (2015) 6069

1. Introduction
Engineered nanoparticles (ENPs) have been dened as fabricated
particles that have a characteristic dimension from 1 to 100 nm and
have properties that are not shared by non-nanoscale particles with
the same chemical composition (Auffan et al., 2009). Due to their
novel and remarkable structural and physicochemical properties,
these materials showed enhanced physical, chemical, optical, and biomedical properties (Nel et al., 2006; Sahoo et al., 2007) with widespread
applications in industrial and household sectors (Manzo et al., 2011).
ZnO NPs are one of the most widely used metal oxide NPs with
photocatalyzing and photo-oxidizing abilities (Szabo et al., 2003). However, inappropriate handling, incidental and/or accidental release of NPs
could result in environmental contamination (Dimkpa et al., 2012). This
raises the immediate need to assess the potential toxicological impacts
of NPs on human health and the environment (Nel et al., 2006; Auffan
et al., 2009; Ge et al., 2011; Bandyopadhyay et al., 2012a,b). However,
it is largely unknown whether the toxicity is due to the exposure of
NPs or the released ions (Parsons et al., 2010). Besides, the quantication of NPs in the environmental matrix is also very challenging
(Bandyopadhyay et al., 2012a,b, 2013). In the past few years, several reports have suggested that ENPs produce adverse effects in terrestrial
plants (Lin and Xing, 2007, 2008; Zhao et al., 2013; Mukherjee et al.,
2014a,b; Rico et al., 2014). These studies reported that plants can accumulate high amounts of metals in their tissues when exposed to ENPs.
This not only impacts their physiological and biochemical responses
(Lin and Xing, 2007; Zhao et al., 2013; Rico et al., 2014; Mukherjee
et al., 2014b) but could be the possible path of alteration and contamination to the food chain (Asli and Neumann, 2009; Dimkpa et al., 2012).
Reports indicate that ZnO NPs adversely affect plant growth of green
pea, corn, cucumber, rye, zucchini, soybean, and wheat, in a dosedependent manner (Lin and Xing, 2007, 2008; Dimkpa et al., 2012;
Bandyopadhyay et al., 2012a,b; Mukherjee et al., 2014a,b). For example,
Dimkpa et al. (2012) reported that ZnO NPs reduced wheat plant
growth with an increased production of reactive oxygen species
(ROS). Lin and Xing (2007, 2008) reported that ZnO NPs affected root
elongation in ryegrass (Lolium perenne), radish (Raphanus sativus) and
rape (Brassica napus). The reported phytotoxicity was due to a disruption in the water and nutrient pathways in plants (Lin and Xing,
2007). Authors also conrmed the adsorption and aggregation of ZnO
NPs to the root surface of ryegrass, whereas high magnication TEM images showed the presence of NPs in the apoplast, cytoplasm, and nuclei
of the endodermal cells and the vascular cylinder (Lin and Xing, 2008).
However, X-ray absorption spectroscopy conrmed the non-existence
of the NPs. Kim et al. (2011) reported the phytotoxic effects of ZnO
NPs in Cucumis sativus caused by excess Zn bioaccumulation; whereas,
Lpez-Moreno et al. (2010) reported genotoxic effects of ZnO NPs to
soybean (Glycine max). Recently, Mukherjee et al. (2014b) showed
that ZnO NPs imposed higher toxicity than bulk ZnO onto green peas
due to higher ROS production in different plant tissues. Nevertheless,
to the best of authors' knowledge, only one report was found where
the interaction of NPs on symbiotic association of plant-bacterium was
reported (Priester et al., 2012). By using inductively coupled plasmaoptical spectrometry (ICP-OES) analysis and electron microscopy imaging, Priester et al. (2012) reported higher Zn accumulation in above and
below ground parts of soybean plants, including the nodules. This study
showed that ZnO NPs adversely affect soybean growth; however, the
effect on nodulation/nitrogen xation remained unaltered. Zn ion
accumulation in different parts of plants was conrmed through
environmental scanning electron microscopy (ESEM) and scanning
transmission electron microscopy (STEM) (Priester et al., 2012). However, to the best of the authors' knowledge, there are no reports on
the comparative toxicological studies of ZnO NP and its bulk/ionic counterparts on alfalfaS. meliloti symbiotic association. Therefore, studies
exploring this knowledge gap would be useful for a better understanding of the plantNP interactions in soil.


Alfalfa (Medicago sativa L.) is one of the world's major forage crops
(Barnes et al., 1988; Gonzalez et al., 1996). The symbiotic association
of alfalfa and S. meliloti is of great signicance in terms of soil fertility,
crop production, and balance of the global nitrogen cycle. Therefore,
toxicological effects of ZnO NPs and comparative studies of bulk and
ionic Zn species on this plant-bacterium system are critical to explore.
Previously we have reported that ZnO NPs were toxic to the bacterium S. meliloti isolated from the root nodules of alfalfa in a concentration
dependent manner (Bandyopadhyay et al., 2012a,b). The present study
aimed to investigate the potential phytotoxic effects of ZnO NPs towards
alfalfaS. meliloti association in soil. In order to compare the effects of
particle size on ecotoxicity, micron-sized bulk ZnO and soluble Zn salt
(ZnCl2 as positive control) were tested simultaneously with ZnO NPs.
Plants were exposed for 30 days to treatment concentrations ranging
from 0750 mg/kg. Upon harvest Zn bioaccumulation, plant growth,
biomass, leaf area, leaf protein content, and catalase (CAT) activity
were determined. Low Voltage Scanning Transmission Electron Microscopy (STEM) coupled with energy dispersive X-ray spectroscopy (EDX)
was used to conrm the presence and aggregation of ZnO NPs in different plant tissues.

2. Materials and methods

2.1. Characterization of ZnO NPs
ZnO NPs (Meliorum Technologies, Rochester, NY) of 10 nm mean diameter were obtained from the University of California Center for Environmental Implications of Nanotechnology (UC-CEIN). The NPs were
characterized by TEM, pXRD, and other techniques (Keller et al., 2010;
Bandyopadhyay et al., 2012a,b). Fig. S1 (Supplementary material)
shows the TEM images of aggregated ZnO NPs with an average size of
322 187 nm, and with size range from 94 to 1127 nm when dispersed
in Millipore Water (MPW) water. Bulk ZnO at 250 and 750 mg/kg concentrations showed average sizes of 1585 208.9 nm and 8354
275.8 nm in MPW.
The desired amounts of ZnO NPs were suspended in MPW and sonicated for 15 min in a water bath (Crest Ultrasonics, Trenton, NJ). The NP
suspensions were then applied to the soil to achieve concentrations of
250, 500, and 750 mg/kg. Control (without NPs) treatments were set
up for all three different set of treatments.

2.2. Ionic and bulk zinc

Three concentrations (250, 500, and 750 mg/kg of soil) of bulk ZnO
and ZnCl2 (Sigma-Aldrich) were prepared simultaneously with the
NPs in MPW and mixed with the soil.

2.3. Soil sampling, characterization, and pot preparation

The top 20 cm soil was collected from Fabens, Texas and mixed in a
ratio of 2:1 (v/v) with organic potting soil (Scotts) to enhance fertility.
The soil was air dried and sieved through 2 mm mesh prior to characterization. Based on the USDA soil classication scheme, the original soil
type (without organic potting mix and NPs) was loam in texture, although very close to the sandy loam (Sperazza et al., 2004). The soil
pH was 7.8 0.4, cation exchange capacity (CEC) was 8.2 0.2 and
Zn content was 28 0.5 mg/kg. Zn content was reduced to 18.6
0.5 mg/kg after 30 days of cultivation.
The pots were lled with 300 g of soil amended with desired
amounts of nano ZnO, bulk ZnO, and ZnCl2. A control treatment (no
Zn amendment), with its respective replicates, was also set up. The
seeds were sown 24 h after soil amendment. Three replicates were prepared for each treatment.


S. Bandyopadhyay et al. / Science of the Total Environment 515516 (2015) 6069

2.4. Particle dissolution experiments

Dissolution of Zn was measured by dispersing ZnO (bulk and NPs)
amended soils in MPW water. Zinc chloride was excluded due to its
complete solubility in water. 5 g of soil was suspended in 20 mL of
Millipore Water (MPW) in 50 mL centrifuge tubes and shaken overnight. After shaking, the tubes were allowed to settle for 4 h. The supernatant was collected and was centrifuged at 25,000 g (Eppendorf AG
bench centrifuge 5417R, Hamburg, Germany) for 30 min. Then, the
supernatant was transferred in a separate centrifuge tube and recentrifuged at 25,000 g for another 30 min. The process of centrifugation was repeated for three consecutive times to avoid any particulate/
suspended ultrane/nanometer size particles. Then, 1 mL of 2% HNO3
was added to the transparent supernatant and elemental Zn was measured using ICP-OES as described below. Each measurement was done
in three replicates.
2.5. Seed germination
Alfalfa (M. saliva L.) seeds (Del Norte Seed & Feed, Vinton, TX, USA)
were sterilized in 4% sodium hypochlorite solution for 10 min and
rinsed with MPW four times. Seeds were inoculated with S. melilotisuspension for 2 h before sowing into the soil. Approximately 60 seeds
of the same size were selected and sown in each pot. Seeds were placed
about 1 cm deep in the soil and covered with thin layer of soil. The pots
with alfalfa seeds were placed in a growth chamber (Environmental
Growth Chamber, Chagrin Falls, OH, USA) with a 14 h photoperiod,
25/20 C day/night temperature, 65% relative humidity, and light intensity of 340 mol s 1 m2 for 30 days for optimal plant growth
(Mukherjee et al., 2014b). The pots were watered with approximately
50 mL of MPW every day. The alfalfa sprouts appeared after one day
of sowing, and after ve days, the number of germinated seeds was

recorded in a Perkin Elmer Lambda 14 UV/Vis Spectrometer (singlebeam mode, Perkin-Elmer, Uberlinger, Germany).
The catalase (CAT) activity was investigated by observing the degradation of H2O2 (extinction coefcient 39.4 mM1 cm 1) at 240 nm
(Aebi, 1974). 20 L crude fresh enzyme extract was mixed with 980 L
10 mM H2O2 and the amount of enzyme necessary to decompose
1 mol of H2O2 per minute was considered as one unit of CAT (Aebi,
2.9. Electron microscope imaging
The ZnO NP treated alfalfa plants (along with controls) were used for
EM imaging in order to see the aggregation of ZnO NPs within the plant
tissues. Plant tissues were harvested (nodules, roots, stems, and leaves)
to determine the location and accumulation of ZnO NPs. Samples were
xed with phosphate buffered with 4% formaldehyde/3% glutaraldehyde at pH 7.2 for 24 h. Fixed tissues were rinsed with PBS and postxed with PBS + 1% OsO4 in 0.12 M phosphate buffer for 1 h at room
temperature. After dehydration with increasing series of ethanol (75,
95, and 100%), samples were inltrated with the Poly/Bed 812 plastic
resin (Luft formulations; purchased from Polysciences, Inc.) prepared
in acetone and cured for 24 h at 68 C. 90 nm ultra-thin sections were
cut with Leica Ultracut ultramicrotome using a 45 diamond knife. Sections were mounted on 200 mesh copper grids (2 PSI) and dried at
37 C.
Tissue sections were imaged with a HITACHI S-5500 In-Lens Ultrahigh Resolution Field Emission Scanning Electron Microscope (UHR
FE-SEM) coupled with BF/DF Duo-STEM, Low angle backscattered
(LABE) and YAG-BSE detectors, and solid state EDX detector (Bruker),
and the microscope was operated with an accelerated voltage of
30 kV. Electron microscope images were analyzed with QUARTZ PCI
and X-ray microanalysis acquired with QUANTAX-EDS.
2.10. Statistical analysis

2.6. Plant growth

The plants were grown for 30 days. At harvest, the root and shoot
length were recorded for each plant separately (total of 10 plants).
The plants were washed with 4% HNO3, followed by triple rinsing
with MPW. Shoots and roots were separated and dried at 80 C for
72 h and the biomass was recorded for each treatment.
2.7. Quantication of Zn in dry plant tissues
Dried root, stem, and leaf tissues were acid digested with concentrated plasma-pure HNO3 and H2O2 (30%) (1:4) mixture in a microwave
acceleration reaction system (CEM, Mathews, NC) and analyzed for
Zn content by ICP-OES (Perkin-Elmer Optima 4300 DV, Shelton, CT
equipped with a Burgener PEEK MiraMist nebulizer with argon ow).
For Quality Control (QC) check, Certied Reference Material (spinach
leaves, NIST 1547) was analyzed and the Continuing Calibration Verication (CCV) was set at 1 ppm. Zn recovery was found to be 99.82
2.8. Protein and enzymatic assays
Thirty-day-old fresh alfalfa plants were washed with 4% HNO3
solution and three times with MPW to remove any external contaminant. Samples of 0.1 g of fresh roots, stems, and leaves were extracted
using extraction buffer of 25 mM KH2PO4 at pH 7.4 and centrifuged
for 8 min at 4 C at 9600 rpm (Eppendorf AG bench centrifuge
5417 R, Hamburg, Germany). The supernatants were collected to
microcentrifuge tubes and stored at 20 C until further analysis (Lee
and Lee, 2000). Total leaf protein content was determined according
to the method of Bradford (1976) using bovine serum albumin as standard. For protein and enzyme estimation, the absorbance kinetics was

Each concentration was set in triplicate including the control in a

completely random design. Separate sets of plants were used for Zn concentration determination, physiological measurements, and microscopy
imaging. The data (means SD) were reported as averages of three
replicates. One-way ANOVA test was performed and Tukey-HSD multiple comparisons test performed using the statistical package SPSS version 12.0 (SPSS, Chicago, IL). Statistical signicance was based on
probabilities of p 0.05.
3. Results and discussion
3.1. NP characterization
The ZnO NPs were characterized as previously reported in the literature (Keller et al., 2010; Bandyopadhyay et al., 2012a,b). The primary
size was of 10 nm diameter with a purity of 99%. The electron microscope images of aggregated ZnO NPs (Fig. S1, Supplementary material
(SM)) showed a size ranging from 94 to 1127 nm, with an average of
322 187 nm. Higher standard deviation could be due to the larger aggregation and/or precipitations in MPW. It has been evident that ZnO
with nanometric size has higher dissolution rate in comparison with
bulk ZnO due to their smaller size (Born et al., 2006; Young and Xie,
2006). However, our results (Fig. S2 SM) are not in agreement with previously reported results (Born et al., 2006; Young and Xie, 2006). We
observed higher dissolution rates in bulk ZnO, compared to ZnO NPs
(Fig. S2 SM). This could be due to the fact that ZnO NPs aggregated
under our experimental procedures and the formation of larger aggregates might hinder the dissolution kinetics by reducing the equilibrium
solubility (Franklin et al., 2007). When NPs enter in contact with environmental media like soil, the physico-chemical behavior might suffer
changes, which might affect the toxicological characteristics of ZnO NPs.

S. Bandyopadhyay et al. / Science of the Total Environment 515516 (2015) 6069

3.2. Effect on seed germination

Seed germination was affected differently by different concentrations of ZnO NPs, bulk ZnO, and ZnCl2. ZnO NPs did not affect germination rates at 250 and 500 mg/kg (Figs. 1 and S3 SM), which followed the
previously reported data on mung beans (Patra et al., 2013). However,
the germination rate increased to 23% at 750 mg/kg ZnO NP treatment,
compared to control. Bulk ZnO treatments showed 50% lower germination rate at 500 and 750 mg/kg compared to control. The ionic treatments were found to be germination inhibitors with higher ZnCl2
concentrations. At 500 and 750 mg/kg ZnCl2 treatments, germination
rate reduced by 50% and 80%, respectively. The ionic treatments showed
lower germination rates in all three concentrations compared to control,
which clearly signies the higher toxicity of ionic zinc. Although, Zn is
an essential element for plant growth, excess Zn might result in growth
inhibition and can produce phytotoxic symptoms (Rout and Das, 2003).
In addition, it should be pointed out that NP-stress response could be
different for different plant species depending on physical and biochemical conditions.
3.3. Zn uptake and translocation in alfalfa
Concentrations of Zn in one month-old alfalfa plants are exhibited in
Fig. 2(AC). In each treatment, Zn levels increased in alfalfa roots along
with increased concentrations of NPs, bulk ZnO, and ionic Zn, compared
to control. The highest accumulation was observed at 750 mg of ZnCl2
treatments with an average of 382 mg/kg of Zn in roots (Fig. 2C). The
higher root uptake can be due to the fact that ZnCl2 is fully ionized in
the soil and thus plants uptake more Zn and bio-accumulate it in root
tissues. However, in comparison to bulk ZnO, higher root uptake was
observed in NP treatments, even though the dissolution of bulk ZnO
was higher than ZnO NPs in soil (Fig. S2 SM). This suggests that the


smaller size of the NPs induces more uptake than the bulk and hence
more Zn bioaccumulated in alfalfa roots. In stem, the accumulation
was higher at 250 and 500 mg/kg of ZnO NP treatments, compared to
bulk ZnO. Zn uptake increased, respectively, to 106% and 166% at
500 mg/kg and 250750 mg/kg treatments of ZnO NPs, compared to
control. In stems, Zn accumulation was similar in bulk ZnO and ZnO
NP treatments. However, ionic treatments showed a different trend
as: control b250 mg/kg, b 500 mg/kg, and b750 mg/kg, with signicantly higher amounts of Zn (~3 times) at 750 mg/kg treatment. Leaves accumulated more Zn (~2 times) in NP treated plants compared to bulk,
ionic, and control. This might suggest a possible uptake of small size
ZnO NPs and release of Zn2 + from ZnO within the tissue (Dimkpa
et al., 2012).
The translocation factor (TF; SM Table S1ab) calculation showed
that more Zn was translocated to alfalfa leaves at 500 and 750 mg/kg
ZnO NP treatments (TF: 0.94 0.30 and 1.37 0.22) than from ZnCl2
treatments (TF: 0.78 0.20 and 0.86 0.23). Higher TF signies
more Zn in the leaves of NP treated plants compared to ionic treatments
at 500 and 750 mg/kg (Jiang and Wang, 2008). The lower TF for ionic
treatments explains the fact that possibly Zn2+ complexes with organic
acids and Zn is sequestered in root vacuoles and become less available
for xylem transport (Jiang and Wang, 2008; Straczek et al., 2008). On
the other hand, free smaller size ZnO NPs were available for transport
in the xylem; hence, higher translocation was observed in the 500 and
750 mg/kg ZnO NP treatments. More elaborated studies are required
in order to explain the higher bioaccumulation of Zn2 + and/or ZnO
NPs and related mechanisms in leaves, compared to bulk and ionic
treatments. In spite of higher Zn accumulation in plant leaves, no visual
sign of toxicity was observed in NP treated plant leaves; however, the
ionic treated plants turned yellow after three weeks of germination
(SM Fig. S7).
3.4. Effects of ZnO NPs, bulk ZnO, and ZnCl2 on biomass production, root and
shoot length

Fig. 1. Germination of alfalfa seeds (for 5 days) in soil treated with ZnO, bulk ZnO, and
ZnCl2 at 0, 250, 500, and 750 mg/kg. Means with the same letters are not signicantly different at p 0.05.

The dry biomass and plant elongation were quantied as indicators

of plant health. Dry root biomass signicantly decreased (80%) in all
ZnO NP treated plants (Fig. 3A). These results are in accordance with
Priester et al. (2012) who reported decreased root biomass in soybean
plants with higher Zn accumulation. The dry root biomass was reduced
by 70%, 72%, and 87% in plants exposed to 250, 500, and 750 mg/kg
of ZnCl2, respectively (Fig. 3A). On the other hand, the 250 and
500 mg/kg ZnO NP treatments imposed a higher shoot biomass reduction than the same ionic treatments. These results conrmed the phytotoxicity of ZnO NPs and ionic Zn to alfalfa plants.
In the aboveground plant portion, ZnCl2 at 750 mg/kg reduced shoot
biomass by 43% (Fig. 3B), compared to control. Conversely, bulk ZnO at
250, 500, and 750 mg/kg increased shoot biomass by 27%, 179%, and
223%, respectively, compared to control (Fig. 3B), while all NP treatments reduced shoot biomass by 35% (Fig. 3B). At the highest concentration of bulk ZnO (750 mg/kg), alfalfa plants had more than twice
shoot biomass (p 0.05), compared to control. Therefore, both ZnO
NPs and ionic Zn are considered to be phytotoxic to alfalfa plants in
terms of reduction of root and shoot biomass.
Fig. S4-A (SM) shows the changes in root length. As seen in this gure, 250 mg/kg ZnO NP treatment increased the root length by 67%,
while at 500 and 750 mg/kg no signicant changes were observed, compared to control. Exposure to bulk ZnO treatments produced contrasting
results. The root elongation showed no changes with 250 mg/kg bulk
ZnO but 50% size reduction (root length) was observed in 500 and
750 mg/kg treatments, compared to control. However, the plant roots
exposed to the ionic treatments remained statistically unaltered.
Fig. S4-B (SM) shows the effects of Zn treatments in alfalfa shoot
growth. As shown in this gure, bulk ZnO at 500 mg/kg signicantly increased shoot length, compared to the other treatments, while ionic Zn
signicantly reduced shoot length.


S. Bandyopadhyay et al. / Science of the Total Environment 515516 (2015) 6069

Fig. 2. Bioaccumulation of Zn in roots, stems, and leaves of alfalfa plants grown for 30 days in soil treated with 0 (control), 250, 500, and 750 mg/kg of (A) ZnO NPs, (B) bulk ZnO, and
(C) ZnCl2. Error bars stand for standard deviations. Bars with the same capital letter show no statistically signicant difference at p 0.05. Comparisons were made between same
plant tissues of different treatments.

Reduction in root length and biomass by ZnO NP is in good agreement with previously reported data. (Lin and Xing, 2007; Dimkpa
et al., 2012; Priester et al., 2012). But, toxicity was much higher in
ionic treatments compared to the NP treatments. ZnCl2 is highly soluble
and a portion of the amended ZnCl2 to the soil is washed out.

Nevertheless zinc amended as ZnCl2 affected plant behavior to a higher

extent than zinc applied as ZnO NP or bulk ZnO. On the other hand,
plants were healthy in bulk treatments in terms of root and shoot biomass production. It can be hypothesized that the amount of accumulated Zn might be crucial for alfalfa growth in bulk treatments and hence,

Fig. 3. (A) Root and (B) shoot biomass (dry weight) of alfalfa plants treated with 0750 mg/kg of ZnO NPs, bulk ZnO, and ZnCl2 after 30 days of treatment. Bars with the same letters/
symbols show no statistically signicant difference at p 0.05.

S. Bandyopadhyay et al. / Science of the Total Environment 515516 (2015) 6069

at certain concentration, it acts as a growth promoting factor for alfalfa

plants (Skoog, 1940; Straczek et al., 2008). This corroborates that, at
the concentrations tested, ionic Zn was more toxic than ZnO NPs and,
at certain concentrations, bulk ZnO acted as a growth-enhancing factor
for alfalfa.
3.5. Changes in leaf area
Leaf area can be a good indicator of plant health (Gutierrez-Boem
and Thomas, 2001; Weryszko-Chmielewska and Chwil, 2005). In this
study, alfalfa leaf area signicantly increased (p 0.05) in plants treated
with 500 mg bulk ZnO/kg, compared to NP, ionic, and control treatments (Fig. 4 and SM Fig. S6). These results concur with other results reported recently (Namasivayam and Chitrakala, 2011; Salama, 2012). Zn
bioaccumulation in leaves of alfalfa plants treated with 500 mg/kg
followed the order ZnO NPs N bulk ZnO N ionic Zn (37.65, 25.42, and
18.34 mg of Zn per kg dry weight). However, the leaf area index was
in order: bulk ZnO N ionic Zn N ZnO NPs. This suggests that at
500 mg/kg, the bulk form/species impacted the meristematic tissue; increasing cell division, resulting in increased leaf area (Patra et al., 2013).
We also observed that the bulk treatments triggered the early owering
of alfalfa in 2 months, which suggests that at 500 mg/kg, bulk ZnO acted
as growth enhancer for this particular alfalfa genotype (Skoog, 1940).
3.6. Total protein content in leaves
Alfalfa leaves have been widely used as forage crop and high source
of proteins in human diet (Xie et al., 2008). In this study, total leaf protein content was measured to nd out the impact of ZnO NPs and corresponding bulk and ionic Zn species on this particular trait. Fig. 5 shows
that ZnO NP treatments caused a numerical reduction in total leaf protein of alfalfa; however, the reduction was not high enough to reach statistical signicance at a p value of 0.05. A similar result was observed
with bulk ZnO at 750 mg/kg. But, the ionic treatments showed a significant reduction in leaf protein content of ~17%, 28%, and 71% at 250, 500,
and 750 mg/kg ZnCl2 treatments, respectively. A decrease in protein by
ionic treatments suggests the phytotoxic effect of Zn2+. Our results are
in accordance with the previously reported studies where ionic zinc was
found to be phytotoxic at higher concentration (Rout and Das, 2003;
Salama, 2012). High Zn concentrations in leaves can displace essential
elements in functional sites (Patra et al., 2013). In addition, ionic/dissolved Zn2+ from ZnO NPs/ZnCl2 could hinder many biochemical function/s comprising proteins which are involved in different structural,
transport, and/or catalytic (enzymes) functions (Rout and Das, 2003).


A decrease in leaf protein will directly affect the food value of alfalfa
(as a protein source) and could serve as an indicator of phytotoxicity.
3.7. Catalase activity
The main function of catalase (CAT) within cells is to prevent the accumulation of toxic levels of hydrogen peroxide, generated as a byproduct of plant's metabolic processes and it can serve as a good indicator of plant's physiological responses (Panda and Choudhury, 2005).
CAT activity is shown in Fig. 6AC. None of the ZnO NP and bulk ZnO
treatments signicantly altered CAT levels in roots, stems, and leaves
of alfalfa. However, the ionic treatments showed decreasing trends of
CAT in stems and leaves at 500 and 750 mg/kg treatments, compared
to control (about 2 times less). In addition, ionic treatments produced
yellow leaves (SM Fig. S7-C). Decreased plant size (shoot length,
Fig. 4B) implies lower cellular activity and higher H2O2 accumulation/
generation. This is in accordance with other published results (Zhao
et al., 2013; Mukherjee et al., 2014b) where lower CAT activity was reported with higher ZnO NP/Zn concentrations. These studies demonstrated that the relation between CAT activity and ZnO NPs is also
related with the increased release of Zn2+ at high ZnO NP concentration. Mukherjee et al. (2014b) reported a concentration-dependent reduction of CAT activity in green pea plant leaves treated with ZnO
NPs. This inhibition of CAT activity might result in an increase in H2O2
and hence plant's defense system might be affected (Mukherjee et al.,
2014b). Nonetheless, Hernandez-Viezcas et al. (2011) reported an increase in CAT activity with increasing ZnO NP concentration in mesquite
(Prosopis juliora-velutina) plant. This suggests that CAT activity depends on the treatment (NP, bulk, and/or ionic Zn), growth condition,
and plant species.
3.8. Electron microscopy imaging
STEM imaging of root nodules (Fig. 7) conrmed the bioaccumulation of ZnO nanoparticle aggregates. Low magnication DF-STEM
helped to locate electrodense regions in the samples corresponding to
metal oxide particles owning high Z-contrast, which is directly related
to their atomic number (Fig. 7A). ZnO NPs seemed to be accumulated
primarily on cell periphery. High magnication BF-STEM of the area
selected in panel A revealed that the ZnO aggregates were located on
cell walls and membranes (Fig. 7B), and in some cases, nuclear invasion
occurred, as some particles were observed on nuclear membrane
and inside nucleus. Chemical composition was conrmed by X-ray microanalysis. EDX spectra (Fig. 7D) showed characteristic peaks of Zn

Fig. 4. Total leaf surface area (cm2/trifoliate leaves) of alfalfa plants treated for 30 days with 500 mg/kg of ZnO NPs, bulk ZnO, and ZnCl2. Error bars stand for standard deviations and bars
with different letters show statistically signicant difference at p 0.05.


S. Bandyopadhyay et al. / Science of the Total Environment 515516 (2015) 6069

Fig. 5. Total leaf protein content (expressed in mg/g fresh weight) of alfalfa plants treated with ZnO NPs, bulk ZnO, and ZnCl2 at 0 (control), 250, 500, and 750 mg/kg concentrations. Bars
with different letters stand for statistically signicant differences at p 0.05.

centered at 1.012 and 8.637 keV, whereas, mapping of Zn (Fig. 7C) conrmed the distribution and sites of accumulation of particles observed
by DF and BF-STEM. Backscattered electron imaging with Low Angle
Backscattered (LABE) and high sensitivity YAG-BSE detectors, and EDX



mapping of Zn and O conrmed the observations by DF/BF-STEM (SM

Fig. S9).
In alfalfa roots, ZnO NPs appeared mainly concentrated along cell
walls forming aggregates that were clearly seen with DF-STEM imaging


Fig. 6. CAT activity in roots, stems, and leaves of alfalfa plants grown for 30 days in soil amended with 0 (control), 250, 500, and 750 mg/kg of ZnO NPs, bulk ZnO, and ZnCl2. One unit of CAT
is the amount of enzyme necessary to decompose 1 mol of H2O2 per minute. Error bars stand for standard deviations and bars with the same letters/symbols show no statistically signicant difference at p 0.05.

S. Bandyopadhyay et al. / Science of the Total Environment 515516 (2015) 6069


Fig. 7. Electron microscope imaging of alfalfa nodules from plants treated for 30 days with 500 mg ZnO NPs/kg soil. (A) DF-STEM, (B) BF-STEM selected area in (A), (C) EDX mapping Zn
(blue) and (D) EDX spectra showing the presence of Zn and O. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)

(Fig. 8A). High resolution BF-STEM and EDX mapping conrmed that
these high contrast aggregates corresponded to ZnO (Fig. 8B, C, E). Zn
signal was also obtained in the intra and extracellular regions, indicating
that ZnO could be suffering dissolution into single particles and nally
into ionic Zn form that interact with cellular components (especially
proteins and nucleic acids). No evidence or signs of apoptosis like DNA
condensation, damaged organelles or compromised cell membrane
were observed. Additionally, backscattered electron imaging (LABE
and YAG-BSE) and EDX mapping of Zn and O are in agreement with
DF/BF-STEM data (SM Fig. S10).
Imaging of alfalfa stem tissue revealed that Zn accumulated along
the cell walls, forming aggregates of ZnO particles (Fig. 9AC).

DF-STEM, which is sensitive to atomic number due to Z-contrast,

showed that these striations are formed by small particles of
912 nm (Fig. 9D and E). Punctual EDX spectroscopy conrmed
that these high contrast structures corresponded to Zn (Fig. 9F).
The other peaks observed in spectra corresponded to osmium
(1.91 and 8.9 a keV) and copper (0.93 and 8.04 keV). STEM imaging
of leaves showed regions of high contrast with electrodense compounds located around the cell walls. High resolution STEM (bright
eld) imaging showed bright nano-sized spots in leaf tissue epidermis. Similar STEM imaging of soybean leaves treated with ZnO
NPs was shown by Priester et al. (2012). However, EDX spectroscopy
did not reveal Zn peaks, probably due to very low concentration, but

Fig. 8. Electron microscope imaging of alfalfa roots treated for 30 days with 500 mg ZnO NPs/kg of soil. (A) DF-STEM, (B) BF-STEM selected area in (A), (C) EDX mapping Zn (blue), (D) EDX
mapping O (red), (E) EDX mapping of Zn and O combined showing the aggregation of ZnO NPs, (F) EDX spectra showing Zn and O. (For interpretation of the references to color in this
gure legend, the reader is referred to the web version of this article.)


S. Bandyopadhyay et al. / Science of the Total Environment 515516 (2015) 6069

Fig. 9. STEM imaging of alfalfa stem treated for 30 days with 500 mg ZnO NPs/kg soil conrming the presence of ZnO aggregates. (A) ZnO nano-aggregates in cell walls of alfalfa stem cells.
(B) BF-STEM of ZnO aggregates in cell wall. (C) BF-STEM selected area in (B), (D) DF-STEM selected area in (C), (E) High magnication DF-STEM selected area in (D), (F) EDX spectra of
selected area in (E) showing Zn and O.

DF and BSE imaging conrmed that accumulations observed with BFSTEM had high z-contrast (SM Fig. S8A to F). Conversely, Fig. S11
shows images of control tissues where high-contrast regions or accumulations were absent.
Electron microscopy results corroborate our Zn uptake data
(Fig. 2A) where Zn accumulation was observed in root, stem, and
leaves of alfalfa treated with ZnO NPs. Lin and Xing (2008) also conrmed the aggregation of ZnO NPs in root tissues of ryegrass and
their cell organelles. However, it is important to know if those aggregations are ZnO NPs or some other Zn species. Dissolution data (SM
Fig. S2) showed that ZnO NPs release Zn 2 + in soil, which can be
biotransformed in stems and leaves of alfalfa. However, the accumulation was high in ZnO NP treatments compared to bulk and ionic Zn.
Consequently, it can be hypothesized that root exudates could dissolve a portion of the ZnO NPs on the root surface and both ZnO
NPs and Zn 2 + were translocated to stem and leaf tissues. It could
be also possibly that the ZnO NPs incorporated into the roots released ionic Zn within the root nodules.
The aggregation of ZnO/Zn species was conrmed by STEM imaging (and coexistence of Zn and O). Nonetheless, additional research
is needed in order to conrm the biotransformation of the ZnO NPs
and the associated mechanism. It is also necessary to conrm
other factors affecting the ionization. ZnO NPs were phytotoxic in
terms of reduction in plant growth. However, no other phytotoxic
effects were conrmed notwithstanding Zn accumulation in different parts of alfalfa.

hence, affecting soil fertility and plant growth. However, the toxicity
of ZnO depends on speciation, size, agglomeration, environmental
conditions, and the plant type.
This material is based upon work supported by the National Science
Foundation and the Environmental Protection Agency under Cooperative Agreement Number DBI-0830117. Any opinions, ndings, and
conclusions or recommendations expressed in this material are those
of the author(s) and do not necessarily reect the views of the National
Science Foundation or the Environmental Protection Agency. This work
has not been subjected to EPA review and no ofcial endorsement should
be inferred. This work was supported by Grant 2G12MD007592 from
the National Institutes on Minority Health and Health Disparities
(NIMHD), a component of the National Institutes of Health (NIH). The
authors also acknowledge USDA grant number 2011-38422-30835,
and NSF Grant # CHE-0840525. J. L. Gardea-Torresdey acknowledges
the Dudley family for the Endowed Research Professorship in Chemistry. The authors acknowledge Chuan Xiao, for using his spectroscopy
facility. We also acknowledge the Facilities of Kleberg Advanced Microscopy Center (KAMiC) and NIH RCMI Nanotechnology and Human
Health Core NIH RCMI Biophotonics Core (Grant 5G12RR013646-12
and Grant G12MD007591) at The University of Texas at San Antonio
Appendix A. Supplementary data

4. Conclusion
In this study we compared the phytotoxicity of ZnO NPs, bulk
ZnO, and ionic Zn (ZnCl2 ) in soil grown alfalfa plants associated
with S. meliloti. Plant growth, Zn uptake, dry biomass production,
leaf area index, and changes in different biochemical parameters
were measured. Results showed that ZnCl2 was phytotoxic at all concentrations, whereas ZnO NPs at 500 and 750 mg/kg treatments reduced plant growth and dry biomass production. Conversely, at
500 mg/kg bulk ZnO acted as alfalfa's growth promoter, as ZnO triggered the shoot dry biomass production and enhanced leaf area
index. Our results also showed the aggregation of ZnO NPs in different parts of alfalfa plants along with root nodules, the active site for
nitrogen xation. It is likely that in long term exposure, these particles/dissolved Zn species might affect the nitrogen xation process;

Supplementary data to this article can be found online at http://dx.
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