Science of the Total Environment 452–453 (2013) 321–332

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Evaluation of developmental responses of two crop plants exposed to silver and zincoxide nanoparticles

Lok R. Pokhrel a, Brajesh Dubey b,⁎a Department of Environmental Health, College of Public Health, East Tennessee State University, Johnson City, TN 37614–1700, USAb Environmental Engineering, School of Engineering, University of Guelph, 50 Stone Road East, Guelph, Ontario, Canada

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Citrate–nAg and nZnO were evaluatedfor potential developmental phytotox-icity.

• Differential alterations in cellularstructures were observed uponexposure to nanoparticles or ions.

• While nZnO caused tunneling-likeeffect, AgNO3 eroded apicalmeristem in maize.

• Metaxylem count was altered withCitrate–nAg, AgNO3, and ZnSO4,unlike with nZnO, in maize.

• Metal-based nanoparticles(Citrate–nAg and nZnO) or ions mayimpair plant growth and development.

⁎ Corresponding author. Tel.: +1 519 824 4120x5250E-mail address: bdubey@uoguelph.ca (B. Dubey).

0048-9697/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.scitotenv.2013.02.059

a b s t r a c t

a r t i c l e i n f o

Article history:Received 6 January 2013Received in revised form 21 February 2013Accepted 21 February 2013Available online 24 March 2013

Keywords:Metal nanoparticlesPhytotoxicityMetaxylemZone of elongationGerminationWater stress

The increasing applications of different nanomaterials in themyriadof nano-enabledproducts and their potential forleaching have raised considerable environmental, health and safety (EHS) concerns. As systematic studies investi-gating potential anomalies in themorphology and anatomy of crop plants are scarce, hereinwe report on the devel-opmental responses of two agriculturally significant crop plants, maize (Zea mays L.) and cabbage (Brassicaoleracea var. capitata L.), upon in vitro exposure to nanoparticles of citrate-coated silver (Citrate–nAg) and zincoxide (nZnO). Analyses involve histology of the primary root morphology and anatomy using light microscopy,metal biouptake, moisture content, rate of germination, and root elongation. Comparative toxicity profiles of theionic salts (AgNO3 and ZnSO4) are developed. Notably, we uncover structural changes in maize primary root cellsupon exposure to Citrate–nAg, nZnO, AgNO3, and ZnSO4, possibly due to metal biouptake, suggesting potentialfor functional impairments in the plant growth and development. Citrate–nAg exposure results in lower Agbiouptake compared to AgNO3 treatment in maize. Microscopic evidence reveals ‘tunneling-like effect’ withnZnO treatment, while exposure to AgNO3 leads to cell erosion in maize root apical meristem. In maize, a signif-icant change in metaxylem count is evident with Citrate–nAg, AgNO3, and ZnSO4 treatment, but not with nZnOtreatment (p > 0.1). In both maize and cabbage, measures of germination and root elongation reveallower nanoparticle toxicity compared to free ions. As moisture data do not support osmotically-induced water

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stress hypothesis for explaining toxicity, we discuss other proximate mechanisms including the poten-tial role of growth hormones and transcription factors. These findings highlight previously overlooked,anatomically significant effects of metal nanoparticles, and recommend considering detailed anatomi-cal investigations in tandem with the standard developmental phytotoxicity assays (germination androot elongation) as the latter ones appear less sensitive for screening plant responses to nanomaterialinsults.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Novel functionalities (e.g., catalytic, electrical,mechanical, optical, andelectromagnetic) that emerge at nanoscale (1–100 nm in at least one di-mension; NNI, 2006; Maynard, 2011) have allowed for manufacturingnano-enabled products, which are being commercialized in the myriadof sectors including electronics, therapeutics, medical diagnostics, cloth-ing, and personal care (www.nanotechproject.org). Among the manytypes of nanomaterials are the silver- and zinc-based nanoparticles(NP) that are increasingly used (www.nanowerk.com) for their promi-nent antibacterial, plasmonic, and opto-electrical properties (Pokhrelet al., 2012). The same particle-intrinsic novel properties that enabledwide applications of nanomaterials have, on the other hand, raised con-siderable environmental, health and safety (EHS) concerns (Polandet al., 2008; Auffan et al., 2009; FIFRA, 2009; Pokhrel et al., 2012; Pokhreland Dubey, 2012a). Despite particle size, surface charge/ligands, and sur-face area are generally identified as factors for NP toxicity (El Badawyet al., 2011; Maynard, 2011; Silva et al., submitted for publication), ithas remained unclear how NPs impart toxicity to the biotic receptors(European Commission, 2008), including to the terrestrial plant species(NNI, 2006; Yin et al., 2011).

Terrestrial plants can be exposed to a multitude of nanomaterialsthrough soil inmanyways: potential leaching fromnano-enabled prod-ucts, intentional sub-surface release for environmental remediation(e.g., nano zero valent iron for trichloroethylene removal; Zhang,2003), surface run-off, irrigation using contaminated surface water,land applications of contaminated biosolids, or wastewater effluent dis-charge. Although some progress toward understanding nanotoxicologyhas been achieved studying microbial populations, algae, animalmodels, or mammalian cell lines (Navarro et al., 2008; Yu et al., 2009;El Badawy et al., 2011; Pokhrel et al., 2012; Pokhrel and Dubey, 2012a,b; Silva et al., submitted for publication), yet understanding ofnanotoxicity in higher plants is limited to few studies, which evaluatedbiouptake/bioaccumulation (Gardea-Torresdey et al., 2003; Lin andXing, 2007, 2008; Khodakovskaya et al., 2009; Judy et al., 2011; Ricoet al., 2011; Yin et al., 2011), phenotypic changes (e.g., root/shootlength, biomass; Lin and Xing, 2007; Khodakovskaya et al., 2009; Ricoet al., 2011), biodistribution (Lin and Xing, 2008; Khodakovskayaet al., 2009), or the DNAdamage (Atha et al., 2012).More recently, mea-suring the seedling growth in Phaseolus radiatus and Sorghum bicolor,Lee et al. (2012) reported particle-mediated toxicity of citrate-coatedsilver nanoparticles (Citrate–nAg) in soil, while free Ag+ toxicity wasfound to be more pronounced when tested in agar medium. Aconcentration-dependent inhibition of two different sized (20 nm vs.100 nmdiameter) AgNPs on the biomass growth and frond number, in-cluding the greater free Ag+ effects compared to AgNPs, were observedin Lemna minor (Gubbins et al., 2011). However, the toxicity of gumArabic-coated AgNPs was higher compared to the same concentrationof dissolved Ag+ in Lolium multiflorum, with greater bioaccumulationoccurred with AgNP treatment than with Ag+ (Yin et al., 2011). Study-ing Zea mays, Zhao et al. (2012) analyzed multiple variables related tostress upon exposure to CeO2 (cerium dioxide) NPs. Interestingly, theauthors found no lipid peroxidation (despite higher H2O2 in leaves) orion leakage, and that the physiological measures (transpiration, photo-synthesis, and stomatal conductance) also remained unchanged. In thesame study, the authors, however, found increased enzyme activity(catalase and ascorbate peroxidase) and upregulation of heat shock

protein (Hsp70), suggestive of protective effects of CeO2 NPs in maize(Zhao et al., 2012). In Lolium perenne, considerable effects of ZnO NPsin the root epidermis and cortex, with internalization of NPs in the en-dodermal and vascular tissues were observed (Lin and Xing, 2008).Studies have also shown plant potential to biotransform metallic ionsinto NPs during their growth and development; although the mecha-nism of such in vivo transformation is not known (Gardea-Torresdeyet al., 2003).

Given the notable inconsistency among the studies assessing phyto-toxicity of NPs (Stampoulis et al., 2009; Yin et al., 2011; Lee et al., 2012)and the lack of understanding of whether alterationsmight occur in thecellularmorphology of the vital structures (e.g., in the root tip, the zoneof elongation, and themetaxylem vessels) during plant development, acomprehensive study of the root anatomywould then allowuncoveringany cellular anomalies following exposure to metal-based NPs. Alter-ations in the shape and size of the cells at earlier stages of growth anddevelopment are known to cause severe functional impairments associ-ated with tissue differentiation and solute transport in young plants(Puertas-Mejia et al., 2010; Delmail et al., 2011). Hence, it becomes crit-ically important to address these knowledge gaps and explore the prox-imate mechanisms of the observed toxicity of NPs.

In this study, we investigate the potential developmental phytotox-icity of citrate-coated silver (Citrate–nAg) and zinc oxide (nZnO)nanoparticles against two agriculturally significant crop plants, maize(Zeamays L.) and cabbage (Brassica oleracea var. capitata L.), by system-atically providing complimentary information through histological ex-aminations of the primary root morphology and anatomy, andanalyses of metal biouptake, moisture content, germination rate, androot elongation. Toxicity profiles of the ionic salts (i.e., AgNO3 andZnSO4), including of the untreated controls (moderately hard water),are developed for comparison purposes.Whilewe determine variabilityinmoisture content andmetal biouptake, other proximatemechanisms(involving growth hormones and transcription factors) explaining theobserved toxicity are discussed.

2. Materials and methods

2.1. Materials characterization

As previously stated, NPs of citrate-coated silver (Citrate–nAg)and zinc oxide (nZnO) were tested for potential developmentalphytotoxicity against maize (Z. mays L. cv. NK-199) and cabbage(B. o. var. capitata L. cv. Golden Acre). The synthesis procedurefor Citrate–nAg is presented in the Supplementary information. Citrate–nAgwas purified using a tangential flow filtration (TFF) process (detailedin the following section). nZnO was procured as an aqueous suspensionfrom Meliorum Technologies, Inc., NY, USA (purity — 99.9%) and wasused as-obtained. For comparison, AgNO3 and ZnSO4 were tested fortheir potential toxicities. The ionic salts were purchased from Fisher Sci-entific, Inc., USA (AgNO3, USP grade; ZnSO4, ACS grade, purity: 99–102%).

NPs were characterized using UV–vis spectroscopy, dynamic lightscattering (DLS), and transmission electronmicroscopy (TEM). The sur-face Plasmon resonance (SPR) spectra were recorded using a HACHDR5000 spectrophotometer (HACH Company, CO, USA). A PSS Nicomp380ZLS particle sizer/zeta potential unit (Particle Sizing Systems, CA,USA) was calibrated at 23 °C with a Duke 500 (491 nm) NIST 3490Astandard (PSS Nicomp, FL, USA) before measuring hydrodynamic

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diameters (HDD) and zeta (ζ) potentials. The Smoluchowski equationwas used to estimate ζ potential from electrophoretic mobility of theparticles. A Philips EM 420 transmission electron microscope (TEM)was used to image NPs by dropping an aliquot of sample onto acarbon-coated copper formvar grid (Ted Pella, Cat # 01813-F) and,allowing it to air dry before recording images, operating TEM at 120 kVin the bright-field mode. By importing representative TEM images intoImageJ 1.44 program (www.rsb.info.nih.gov/ij/), particle size distribu-tions (PSD)were determined. An inductively coupledplasma-mass spec-trometer (Bruker 820-ICP-MS) was used to quantify total metalconcentrations in the test suspensions, prior to which samples weredigested using nitric acid (trace metal grade) following the standardUSEPA method 3050B.

2.2. Purification of Citrate–nAg

As-synthesized Citrate–nAg was cleaned using polysulfone (PS)10 kD hollow fiber diafiltration membranes (P/N: X31S-300-02P, sur-face area = 145 cm2) connectedwith a Kros FloResearch IIi TFF Systemand controlled via KF COMMsoftware (ver. S16; Spectrum Laboratories,CA). A suitable shear pressure generated by pump head enabledmaintaining a tangential peristaltic flowof aqueous suspension throughthe hollow fiber membranes. These membranes are designed to permitlesser than 10 kD (~1–2 nm) sized particles including dissolved ionsand residual impurities to pass across the membranes and formingthe permeate, while allowing greater than 10 kD sized particles toflow through the membrane’s lumen and forming the retentate. Thisprocess involved buffer exchange of the residual impurities and ionspresent in the as-synthesized Citrate–nAg suspension with nanopurewater (electrical conductivity = 2 μS/cm). A measure of electrical con-ductivity was used as an established surrogate (El Badawy et al., 2010;Kanel and Al-Abed, 2011; Pokhrel et al., 2012), indicating likely absence

Fig. 1. Representative transmission electron microscopy (TEM) images of Citrate–nAg (scalenZnO (d) were estimated using the ImageJ 1.44 program and are provided in Table 1. Num

of ions and/or impurities accounting for suspension conductance (Sup-plementary Table S1). Characterization of permeate and cleaned cit-rate–nAg was explained earlier (Fig. 1, Table 1; Supplementary Fig. S1).

2.3. Seed preparation and exposure

Untreated seeds of maize and cabbage were purchased from EdenBrothers, Dahlonega, GA, USA. Seeds were visually inspected for anymorphological damage or discoloration, cleaned three timeswith sterilewater, followed by immersion in 70% ethanol for 2 min to ensure seedsurface sterility. Soon the seeds were washed several times with sterilewater to remove any left-over ethanol from the surface. Onto each Petridish (100 mm × 15 mm) was placed a filter paper (Yang and Watts,2005; Atha et al., 2012) moistened with 3 mL of sterile nanopurewater to prevent it from soaking the test chemical, while, at the sametime, allowing the seeds to be thoroughly exposed to the test chemical.

Ten seeds were randomly assigned onto each Petri dish, and weremanually spaced apart from each other by at least a cm. Each dish re-ceived 5 ml of the test chemical, or a 5 ml of sterile moderately hardwater (MHW) as an untreated control (Yang and Watts, 2005). NPs ortheir ionic formswere diluted in sterileMHW to obtain the desired con-centrations. The suspended NP stability or the state of aggregation inMHW (as a diluent) was evaluated using the DLS method (Supplemen-tary Table S2). All test runswere conducted in triplicates, including thatof the controls, unless noted otherwise. Seeds were allowed to germi-nate in dark for about a week at 23 ± 1 °C to offer optimal growth con-dition, prior to which seeds were soaked in sterile MHW for 24 h toallow water imbibition. Seed preparation and assignment into thedish were performed under the laminar flow hood to maintain sterileworking environment. The pH of all the test chemicals was maintainedwithin a narrow range (6.8–7.2).

50 nm; a), and nZnO (scale 20 nm; c). Particle size distributions of Citrate–nAg (b) andber of particles analyzed for both types of nanoparticles = 215.

Table 1Characteristics of nanoparticles evaluated for developmental phytotoxicity in maize and cabbage.

Material pH Particle size distribution (PSD) Average zeta potential⁎⁎ (mV) Plasmon resonancespectra

Manufacturer reportedmean particle size (nm)

Hydrodynamic diameter⁎

(Mean ± S.D.) nmTEM diameter(Mean ± S.D.) nm

λmax

(nm)absorbance(au)

Citrate–nAg 7.24 na 11.0 ± 0.7 56.1 ± 13.8 −25.13 425 2.38(n = 215)

nZnOa 7.03 10 11.0 ± 0.7 17.4 ± 4.9 −13.21 340 3.64(n = 215)

na, data not available (lab. synthesized); n = number of particles measured for sizing from TEM images using ImageJ 1.44 program.⁎ Volume weighted particle size distribution.

⁎⁎ Average zeta potential approximated using Smoluchowski equation by NICOMP 380 ZLS Zeta Potential unit; λmax represents maximum wavelength at which surface Plasmonpeak was observed.

a Purchased from Meliorum Technologies, Inc., NY, USA.

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2.4. Developmental phytotoxicity assessment

The seeds were exposed for six (cabbage) or seven (maize) days toeach test chemical, following which experiments were ended whenthe controls displayed over 80% germination; the root length wasmeasured (in mm) and germination rate was recorded for each repli-cate. The USEPA OPPTS 850.4200 guideline that seeds are consideredgerminated when the root measures at least 20 mm in length for con-trols was followed (USEPA, 1996). Relative seed germination inhibi-tion and relative root growth inhibition were calculated using thefollowing standard equations (USEPA, 1996).

Relative Seed Germination Inhibition %ð Þ¼ GerminationControl−GerminationTreat

GerminationControl

� �� 100 ðiÞ

Relative Root Growth Inhibition %ð Þ¼ REControl−RETreat

REControl

� �� 100

ðiiÞ

where RE denotes root elongation, and Treat indicates treatmentgroup.

As shown in Fig. 3, the concentrations at which significant effectwas observed in root elongation measure were chosen for each testchemical for comprehensive microscopic studies in maize. Maizewas chosen because of its sturdy nature of the root, which enabledfree-hand sectioning. Detailed investigations of the root morphologyand anatomy were conducted focusing on potential alterations thatwould occur in the cellular structure and alignment in the root tipand zone of elongation, including the metaxylem vessel counts. Thesectioned root piece was stained using Toluidine Blue O (0.05%) for30 s, washed with running water for 1 min, and temporarily mountedon the glass slide with glycerin. Both longitudinal and transverse sec-tions of fresh young roots were observed under microscope within3 h of slide preparation. Samples were analyzed using an OlympusBX41 system microscope and images were acquired using an OlympusMicroFire™ color digital camera and PictureFrame imaging software(Olympus America, Inc. NY).

2.5. Moisture analysis

To quantifymoisture content in the seedlings and that retained in thefilter papers, separate experiments were conducted. For this, each Petridishwas assigned one filter paper and ninemaize seeds. The experimentcomprised of treatments with Citrate–nAg (73.4 μg total Ag/mL) orAgNO3 (127 μg total Ag/mL), and comparisons were made withuntreated control. To analyze the moisture content that retained in thefilter papers, after 7 days of the experimental period, each (wet) filterpaper was weighed and oven-dried at 105 °C overnight (Gubbins et al.,2011). The difference between the wet weight and the dry weight of

filter paper was calculated as themoisture content; the data were aver-aged for two replicates and expressed as % moisture per dry weight offilter paper. After 7 days, the seedlings (whole seedlings analyzed)were weighed (initial fresh weight), then oven-dried at 105 °C over-night to estimate dry weight (Gubbins et al., 2011). The difference be-tween the wet- and dry weight of nine seedlings was calculated asmoisture content, and the values were averaged for two replicates(n = 18 seedlings) and expressed as % moisture per dry weight of theseedlings.

2.6. Biouptake of silver

Nine oven-dried seedlings of maize from each replicate wereashed in the Muffle furnace at 550 °C for 30 min and digested follow-ing the USEPA method 3050B (using ultrapure HNO3). The sampleswere analyzed using an ICP-MS (Bruker 820-ICP-MS) to determinetotal Ag concentration in each replicate; data were averaged for tworeplicates (n = 18 seedlings).

2.7. Data analysis

Our data were normally distributed (Kolmogorov–Smirnov (K–S)test: p > 0.1 in all cases); except for a measure of root elongation incabbage with nZnO treatment (K–S test: Z = 1.644, p b 0.01), whichwere then log-transformed for use in further analyses as they satisfiednormality (K–S test: Z = 1.194, p > 0.1; Pokhrel et al., 2013). Datawere analyzed for significant differences in the variances andmeans be-tween the treatment and the control. When sample variances were sig-nificantly different from the control, the t- and p-values were adjustedfor independent samples t-test, which tested the null hypothesis thatthe treatment means are not significantly different from the control atthe p ≤ 0.05 level. The Chi-square goodness-of-fit test was used totest if all treatments, including the control, contained the same propor-tion of themetaxylem counts. Data for Citrate–nAg are reported as totalAg, while other test chemicals are reported as the respective com-pounds, unless otherwise noted. Data were plotted as the concentra-tion–response curves for the measures of root elongation and seedgermination (%). EC50 (i.e., effective concentrations for 50% inhibition)values were estimated using the linear regression analysis.

3. Results and discussion

3.1. Characteristics of nanoparticles

Both types of NPs had the same HDD (11 ± 0.7 nm) and their char-acteristic SPR spectra are presented in Supplementary Fig. S1. On aver-age, TEMdiameterwas larger for Citrate–nAg than nZnO (Table 1),withparticles appearing polymorphic for Citrate–nAg, and roughly oval fornZnO (Fig. 1). Detailed particle characteristics are presented in Fig. 1,Table 1, and Supplementary Table S2. Evaluation of HDD and ζ potential

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values revealed that both types of NPs were fairly stable in MHW (Sup-plementary Table S2).

3.2. Impact of metal nanoparticles on the primary root tip in maize

Exposure to nZnO (1000 μg/mL) caused ‘tunneling-like effect’, char-acterized by a deep invagination in the primary root tip in maize(Fig. 2b; Supplementary Fig. S2). It is likely that the tender cells of apicalmeristem upon contact with nZnO suspension led to cell dissolutionfrom cellular structural disintegration; this, however, was not observedwith dissolved Zn+2 treatment (Fig. 2b and c). Because the frequency of‘tunneling-like effect’was low (one out of total four samples analyzed),it is possible that the effect could be a rare occurrence as no significantimpact on the physical root growth of the seedlings was observed(Fig. 3a). Alternately, the variability in individual sensitivity to nZnOmight explain this discrepancy. In maize, an erosion of apical meristemcells was observed upon exposure to free Ag+ (as AgNO3; 200 μg/mL;Fig. 2e), which led to reduced root growth (Fig. 3c; SupplementaryTable S4).

Fig. 2. Impacts of metal nanoparticles or their corresponding ionic salts on the primary root tip incell erosion of apical meristem occurred with AgNO3 (200 μg/mL) treatment leading to reduced(73.4 μg/mL; d). Magnifications are shown at the bottom right corner of each panel. Arrows ind

Root hair density, however, appearedunaffectedwithNPs orwith cor-responding dissolved ions at the evaluated concentrations (SupplementaryFig. S3). On exposure to 100 μg AgNO3/mL, maize roots were generallythinner with tips dried out and red pigmented that severely progressedas concentration increased to 200 μg AgNO3/mL, followed by significantgermination and root growth inhibitions at 500 μgAgNO3/mL (Fig. 3c; Sup-plementary Fig. S4, Tables S3 and S4).

3.3. Biouptake of Ag and analysis of moisture content

Quantification of silver in maize seedlings (whole seedlings ana-lyzed) showed more than five order of magnitude (55×) higher Ag(as total Ag) uptake with AgNO3 (200 μg/mL) treatment than thebaseline control. In contrast, this biouptake was 4.5 times higher withCitrate–nAg treatment than the control. The biouptake concentrationswere 22 and 1.8 ng Ag/mg dry weight of seedlings for AgNO3 and cit-rate–nAg treatments, respectively (Fig. 4; Supplementary Table S8).This was 12.2-fold lower Ag uptake with Citrate–nAg treatment com-pared to AgNO3 treatment. When exposure concentrations were nor-malized, Ag biouptake was 7.06-fold lower (as total Ag) with Citrate–

maize (Zea mays). Exposure to nZnO (1000 μg/mL) caused ‘tunneling-like effect’ (b), whileroot elongation in maize seedlings (e). Control (a), ZnSO4 (1000 μg/mL; c), and Citrate–nAgicate areas of potential impacts.

Fig. 3. Concentration–response curves showing effects of nanoparticles or their corresponding ionic salts on the root growth and development in Zea mays (top panels a–c) and Brassicaoleracea var. capitata (bottom panels d–e). Error bars represent ± 1 standard error (SE) of the means. Significant difference between the treatment means and control was tested usingthe independent samples t-test at the 0.05 level (*, p b 0.05). nAg denotes Citrate–nAg; C denotes control.

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nAg compared to AgNO3 treatment, suggesting that minimal dissolu-tion of Ag might have emanated of Citrate–nAg surface during the testperiod.

Analysis of averagemoisture content in the seedlings revealed greatermoisture content with AgNO3 (168.7% — dry wt.) or Citrate–nAg(163.9% — dry wt.) treatment compared to the control (147.3% — drywt.). A similar trend was observed for moisture content that wasretained in the growth substrate (Fig. 5; Supplementary Table S8).This is consistent with a previous study that showed greater seedmois-ture content in tomato treatedwithmulti-walled carbon nanotubes in atwo day experiment, while themechanism behind enhanced water up-take by the seeds remained unclear (Khodakovskaya et al., 2009). It ispossible that increased water uptake by the seedlings might have facil-itated greater Ag biouptake with AgNO3 treatment compared to Cit-rate–nAg treatment in the current study.

Fig. 4. Increased silver biouptake occurred with AgNO3 (exposed concentration = 127 μgtotal Ag/mL) treatment compared to Citrate–nAg (exposed conc. = 73.4 μg total Ag/mL)treatment in Zea mays seedlings. When exposure concentrations were normalized, Agbiouptake (as total Ag) was 7.06-fold greater for AgNO3 compared to Citrate–nAg treat-ment. Two replicates were analyzed (n = 18), each containing 9 seedlings, to determinetotal Ag concentrations using an ICP-MS; data representmeans of two replicates. Ag recov-ery was 103%.

3.4. Impact of metal nanoparticles on root elongation

In maize, the primary root cells were structurally modified in shapeand/or size at the zone of elongation upon exposure to metal NPs or thedissolved ions evaluated (Fig. 6). Cells were elongated with both NPtreatments (Fig. 6b and d); they appeared thinner and irregular withAgNO3 treatment (Fig. 6e); while with ZnSO4 treatment cells exhibitedshorter and wider morphology, compared to the control (Fig. 6a and c).These contrasting observations for metal NPs versus correspondingionic salts suggested that the samemechanism(s) could not be respon-sible for the observed toxicity pattern, and/or this effectmight be due todifferential metal uptake as observed in maize seedlings. Phenotypicroot elongation measurement (Fig. 3a–c) was in good agreement withthe microscopic observations of the cells at the zone of elongation(Fig. 6). In maize, unlike with AgNO3 or ZnSO4 treatment that resultedin significant dose-dependent effects, Citrate–nAg or nZnO treatment

Fig. 5. Variation in averagemoisture content in Zea mays seedlings and that retained in thefilter paper with Citrate–nAg or AgNO3 treatment. Each data point represents an average of% moisture content (dry weight basis) pooled from two replicates, with each replicateconsisting of nine seedlings or a filter paper used as growth substrate.

Fig. 6. Effects of metal nanoparticles and their corresponding ionic salt in the zone of elongation in Zea mays. Control (a), nZnO (1000 μg/mL; b), ZnSO4 (1000 μg/mL; c), Citrate–nAg(73.4 μg/mL; d), and AgNO3 (200 μg/mL; e). Note elongated cells with nanoparticles treatments (b, d), but cells appeared thinner and irregular upon Ag+ exposure (e), and showedshorter and wider morphology with Zn+2 treatment (c), compared to control (a).

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had little to no effect on root growth (p > 0.05 in all cases; Fig. 3a–c;Supplementary Table S4).

In cabbage, root elongation was not affected by nZnO or ZnSO4 in anexcess of 100 μg/mL exposure level (Fig. 3d). However, root develop-ment was completely halted as seeds were unable to germinate athigher ZnSO4 concentrations (t = 10.288, p b 0.001; SupplementaryTables S5 and S6), unlike nZnO treatment that showed only 40.6%growth inhibition at the highest concentration tested (i.e., 1000 μgnZnO/mL; p b 0.005; Fig. 3d; Supplementary Table S6). At concentra-tions greater than 10 μg/mL, root growth was significantly inhibitedby AgNO3 in a dose-dependentmanner (p b 0.05 in all cases), while cit-rate–nAg inhibited root growth by only 24.1% at the highest tested con-centration (73.4 μg/mL as total Ag) compared to the control in cabbage(t = 2.648, p b 0.05; Fig. 3e and f; Supplementary Table S6). These datacoupled with their EC50 values (Table 2), defined as an effective

concentration at which 50% inhibition is observed, revealed lowertoxicity of metal NPs than their corresponding free ions on theprimary root growth and development in maize and cabbage. More-over, Ag+ was found more toxic than Zn+2 as shown by the rootelongation measurements (Fig. 3), or their corresponding EC50 values(Table 2).

3.5. Impact of metal nanoparticles on metaxylem vessel count

Since metaxylem vessels are not transient as the protoxylems are,the former is retained throughout plant life representing permanenttransportation conduits in mature plants (Esau, 1977). It is knownthat the xylem vessel number will increase when plants are subjectedto water-stress and would decrease upon supply of adequate water(Zimmermann et al., 1993; Holbrook et al., 2001; Schneider et al.,

Table 2Comparison of effective concentration for 50% inhibition (EC50) in the rate of germination (%) and root elongation in maize and cabbage.

Plant type EC50 for germination rate (μg/mL) EC50 for root elongation (μg/mL)

nZnO Zn+2 Citrate–nAg Ag+ nZnO Zn+2 Citrate–nAg Ag+

Zea mays na 0.05 na 192 na na na 53.2Brassica oleacea var. capitata 136 9.72 na 1.42 na 78.2 na 8.89

‘na’ indicates data not available as EC50 was not estimated due to less than 50% inhibition at the highest concentration tested; ZnSO4 was used as a source of Zn+2 and AgNO3 wasused as a source of Ag+; the EC50 values for ionic salts are presented for Zn+2 and Ag+.

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2007). Hence, the measurement of metaxylem number in plants haswell served to understand osmotically-induced stress by water loss(Zimmermann et al., 1993; Holbrook et al., 2001; Schneider et al.,2007). Growth-hormones such as auxin and cytokinin are also knownto influence xylem vessel formation (Fukuda, 2004; Kubo et al., 2005).Although genetic mechanism of xylem formation has been poorlyunderstood, plant specific transcription factors such as VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 have been recently attrib-uted to induction of trans-differentiation of various cell types intometaxylem- and protoxylem-like vessel elements (Kubo et al., 2005).To date, whether exposure to metal-based NPs could modulate the fre-quency of metaxylem vessel has not been documented in plants, butcontextual evidence from studies involving heavymetal stress onplantsstimulated this part of research (Mufarrege et al., 2010; Delmail et al.,2011).

Differential metaxylem counts were observed upon exposure tometal NPs or their respective ionic salts (Table 3). The metaxylemcounts ranged from 5 to 6 in the control, which increased to 7 upon ex-posure to ZnSO4 (1000 μg/mL) or AgNO3 (200 μg/mL; Fig. 7; Table 3).While with either type of NP treatment (Citrate–nAg: 73.4 μg Ag/mL;nZnO: 1000 μg/mL), metaxylem counts fluctuated in the range of 5–6(Fig. 7). The Chi-square test showed Citrate–nAg, ZnSO4, and AgNO3

treatment led to significantly different proportion of metaxylem fre-quency in maize primary root; however, with nZnO treatment the pro-portion of metaxylem count was not statistically different (p > 0.1;Table 3).

Because with either ionic salt treatment, formation of seven meta-xylem vessels was observed in maize (Table 3), this is indicative ofhigher stress exerted by biolabile metal ions than the evaluated NPs.These results suggested that the evaluated NPs had lower effects onthe primary root anatomy, possibly due to lower metal biouptake(as shown by total Ag analysis for maize seedlings; Fig. 4), or perhapsdue to different mode of action between NPs and the respective ionicsalts. Given the significance of growth hormones and plant specifictranscription factors in metaxylem vessel formation (Fukuda, 2004;Kubo et al., 2005), it remained to be understood if enhanced metal(e.g., Ag, and perhaps Zn) uptake in this study (Supplementary TableS8) promoted formation of additional metaxylem vessels. Becausewater stress did not likely occur under our experimental scenario asdemonstrated by the moisture analysis data (Fig. 5), involvement of

Table 3Effect on metaxylem count in the primary root of Zea mays upon exposure to metal nanopa

Treatment (μg/mL) Metaxylem count

#5 (%) #6 (%) #7 (%) Samp

Control 5 3 0 8(62.5) (37.5)

nZnO (1000) 4 4 0 8(50) (50)

Citrate–nAg (73.4) 6 7 0 13(46.1) (53.8)

ZnSO4 (1000) 6 0 1 7(85.7) (14.3)

AgNO3 (200) 2 15 10 27(7.4) (55.5) (37.0)

The Chi-square (χ2) goodness-of-fit test was used to test if all treatments, including the con

some other mechanisms such as the stimulation of the growth hor-mones (e.g., auxin or cytokinin) or the transcription factors (suchas VND6, VND7) in inducing additional metaxylem vessels in maizecannot be ruled out. Thus, studies designed to address putativesignaling mechanisms, which might be involved in differential in-crease in metaxylem frequency under metal/NM stress, at physiolog-ical and biomolecular levels may provide further understanding ofthe observed toxicity.

3.6. Impact of metal nanoparticles on seed germination

In maize, exposure to a wider range of nZnO concentrations(0.01–1000 μg/mL) did not inhibit seed germination (p > 0.1 in allcases; Fig. 8a; Supplementary Table S3), unlike in cabbage a dose-dependent germination inhibition occurred (Fig. 8d; SupplementaryTable S5). These data showing little to no inhibition of germinationwith nZnO treatment inmaize are consistentwith the findings of a previ-ous study that used the same plant species and similar experimental con-ditions (El-Temsah and Joner, 2012). In contrast, exposure to ZnSO4

caused significant germination inhibition in both maize (p b 0.05) andcabbage (p b 0.05; Fig. 8a and d; Supplementary Tables S3 and S5).While complete germination inhibition occurred at 500 μg ZnSO4/mL incabbage, it only led to ca. 50% germination inhibition in maize at thesame concentration (Supplementary Tables S3 and S5).

Citrate–nAg showed some potential to inhibit germination inmaize;this inhibition was, however, always less than 35% even at the highestconcentration tested (73.4 μg/mL; p > 0.1 compared to the control;Fig. 8b; Supplementary Table S3). Germination was not significantly af-fected in maize upon exposure to an excess of 200 μg AgNO3/mL(p > 0.1), while at 500 μg AgNO3/mL germination was significantlyinhibited by 84.4% (p b 0.05; Fig. 8c; Supplementary Fig. S4, Table S3).In cabbage, a concentration-dependent germination inhibition was ob-served with AgNO3 treatment, and this was greater than the effectcaused by Citrate–nAg at comparable concentrations (Fig. 8e and f; Sup-plementary Table S5).While both theNPs caused lower germination in-hibition in maize, Ag+ ions also exhibited lower toxicity at ≤200 μgAgNO3/mL; whereas cabbage displayed higher sensitivity toward boththe NPs and their ionic salts (Fig. 8). The corresponding EC50 valuesfor NPs and their ions against seed germination are presented in Table 2.

rticles or their corresponding ionic salts.

les observed (n) Remarks χ2, p-value

Number varied between 5 and 6 4.750, >0.05

Number varied between 5 and 6 4.0, >0.1

Number varied between 5 and 6 6.615, b0.05

Number varied between 5 and 7 8.857, b0.05

Number varied between 5, 6 or 7 9.556, b0.01

trol, contained the same proportion of metaxylem vessel number at the p b 0.05 level.

Fig. 7. Metaxylem vessel count varied with treatments of metal nanoparticles and corresponding ionic salts in Zea mays. Control (a, b), nZnO (1000 μg/mL; c), ZnSO4 (1000 μg/mL;d), Citrate–nAg (73.4 μg/mL; e), and AgNO3 (200 μg/mL; f). Note, induction of 7 metaxylem vessels upon free Ag+ (f) or Zn+2 treatment (d). mx, metaxylem. Metaxylem count dataare presented in Table 3 and Supplementary Fig. S7.

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Although characterization of NP morphology via TEM revealed vari-ability in average particle diameter, the DLSmeasurements revealed sim-ilar average HDDs for both types of NPs and were smaller than the TEMvalues for particle diameter (Fig. 1; Table 1; Supplementary Table S2). Lit-erature suggests that different sizing techniques, such as TEM and DLS,may offer some level of bias, and should be used as complementary toeach other (Ito et al., 2004; Pokhrel et al., in press). Given that averageHDDs of both types of NPs were similar in aqueous suspension (Table 1;Supplementary Table S2), considering contrasting seed sizes of the testedplant species indicated that the potential interactions of similar-sized NPsmight be greater for the smaller seeds of cabbage compared to the largerseeds of maize, largely due to greater surface-to-volume ratio of thesmall-sized seeds to that of the large-sized seeds (Lin and Xing, 2008).These results corroborate the findings of an earlier study reporting highertoxicity of the rare earth oxide NPs to the smaller seeds of rape, radish,and cabbage compared to the larger seeds of wheat (Ma et al., 2009).Alternately, this may indicate plant species-specific toxic response as

reported forCitrate–nAg (particle diameter = 2–20 nm) showinggermi-nation inhibition in ryegrass and barley (El-Temsah and Joner, 2012), butnot in flax (El-Temsah and Joner, 2012) or lettuce at as high as 100 μg/mLlevels (Barrena et al., 2009).

Nanomaterials are emerging environmental contaminants forwhichpotential impacts on the terrestrial crop plants are far from clear(Stampoulis et al., 2009; Yin et al., 2011; Yin et al., 2012). In nature,plants are exposed to various stressors, including that of chemicals, atdifferent stages of their life cycles. Exposure to engineered NMs hasshown to inhibit germination, seedling growth, and development inplants in the laboratory experiments (Gubbins et al., Lin and Xing,2007, 2008; Stampoulis et al., 2009; Yin et al., 2011; Yin et al., 2012).The inhibition may arise due to several reasons; however, specific iontoxicity, osmotically-induced stress due to water loss, or hormonal im-balance are among the predominant factors being explored to explainobserved toxicity in many plant species (Khodakovskaya et al., 2009;Stampoulis et al., 2009; Nawaz et al., 2010). In the current study,

Fig. 8. Concentration–response curves showing effects of metal nanoparticles and their ionic salts on seed germination in Zea mays (top panels a–c) and Brassica oleracea var. capitata(bottom panels d–f). Error bars represent ± 1 standard error (SE) of the means. Significant difference between the treatment means and the control was tested using Independent sam-ples t-test at the 0.05 level (*, p b 0.05). nAg, Citrate–nAg; C, control.

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development of toxicity profiles of two predominantly used metal NPs,including their ionic salts (AgNO3 and ZnSO4), against two agriculturallysignificant plant species (maize and cabbage) was achieved by evalua-tion of moisture content in the 7 day old seedlings and the substrateused, andmore importantly, the histological details revealing anomaliesin maize root anatomy provided information on the potential toxic re-sponses of the maize and cabbage seedlings upon in vitro exposure toa wide exposure range (0.01–1000 μg/mL) of metal-based NPs andions. As novel properties can be acquired by manipulating surface moi-eties, surface charge, and size of the nanoscalematerials, any changes inNP properties could trigger different biological responses, and thusmaylead to unanticipated toxicity (Williams et al., 2010). Our observationsof ‘tunneling-life effect’ in maize apical meristem with nZnO treatmentand elongated cellmorphology at the zone of elongationwith nZnO andCitrate–nAg treatments are consistentwith the fact that NMsmay causedifferent biological interactions and thus different toxic outcomes com-pared to their specified ion toxicity. A study by Stampoulis et al. (2009)has recently shown higher toxicity of Ag (particle diameter = 100 nm)and ZnO (particle diameter = 5 nm) NPs to the seedlings of Cucurbitapepo compared to their bulk powder form evaluated or the controls. Al-though reports on plant species-specific toxicity (Lin and Xing, 2008;Ma et al., 2010) and assay-dependent toxicity (Stampoulis et al., 2009;Lee et al., 2012) tend to complicate the limited understanding of phyto-toxicity of NMs, it nonetheless highlights the need to explore variouscomplimentary and measureable endpoints to capture enough informa-tion explaining the observed differences in toxicity for metal-basednanomaterials versus their free ions.

Studies have shown that reduced vacuole size could result in de-creased cell turgidity, which could subsequently cause cell walls to de-form and thus irregular cell alignments as observed in maize leaf or inbarley (Hordeum vulgare) root with cadmium treatment (Puertas-Mejiaet al., 2010). In addition, binding of metals onto the cell walls and toother sub-cellular structures could lead to a loss in cell wall elasticity(Sieghardt, 1984; Barceló et al., 1986). Our results showing cellular alter-ations in the zone of elongation, especially by ionic Ag+ and Zn+2,possibly due to higher metal uptake, suggest that similar mechanisms

might be responsible for such altered cellular phenotype. Studiesshow plant hormones, especially gibberellins, can induce seed ger-mination and cell elongation (Celik et al., 2008; Finch-Savage andLeubner-Metzger, 2006). This research provides a basis to testwhether our observation of elongated cellular phenotype in maizeunder metal-based NP treatments (Fig. 6) might be associated withmetal NP-mediated synthesis of plant hormones, such as gibberel-lins, causing cells to grow longer than the control.

Inclusion of several anatomical features as examined using lightmicroscopy, which were in good agreement with the plant phenotypicresponses (i.e., corresponding germination and root elongation mea-surements; Figs. 3 and 8), should offer a comprehensive understandingof the potential toxic responses of the crop plants at early developmen-tal stage on exposure to metal-based NPs or their corresponding ioniccounterparts. Although silver biouptake by maize seedlings in ourexperimental period was lower (ng/mg dry weight), AgNO3 treatmentresulted in 7.06-fold higher uptake than with Citrate–nAg treatmentwhen exposure concentrations were normalized (Fig. 4; SupplementaryTable S8). It is important to note that these anatomical anomalieswere observed at ng/mg level of biouptake (measured for Ag), whichcorresponded to higher exposure concentrations (≥73.4 μg/mL) thatwere chosen based on the root elongation measurements as shown inFig. 3. Although growing use of NMs and nano-enabled products partic-ularly associated with metal NPs could inevitably lead to environmentalcontamination, our observations of cellular alterations in differentregions and structures in maize primary root offer valuable informa-tion on potential hazard of metal-based NPs to the crop plants.The prospective anatomical impairments that could occur at envi-ronmentally realistic concentrations (sub μg/L level) may not bedirectly emulated from our histological evidence of altered cellularphenotype, but should provide a basis of what could happen if expo-sure occurs at the tested levels. Statistical analysis of the phenotypicresponses as exhibited by both crop species offers an understandingof potential phytotoxicity in a wide range of exposure concentra-tions (μg/L–mg/L; Figs. 3 and 8). More research is needed to under-stand how these observed morphological and anatomical alterations

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would affect plant development as (or should) they grow to maturityunder field environments.

In summary, these results show that NPs of Ag and ZnO are poten-tially toxic to the early development and growth in maize and cabbage,albeit their toxic responses were generally lower for NPs than theirspecified ionic salts. Notably, the strategic investigations of root anato-my revealing cellular alterations in apical meristem, zone of elongation,andmetaxylem count, coupledwithmetal biouptake,moisture content,germination, and root elongation under NPs versus ionic salt treatmentsshowed differential potential of metal-based NPs and their ions for de-velopmental toxicity in agriculturally important crop plants. Generally,the standard USEPA OPPTS 850.4200 bioassay for measuring germina-tion rate and root growth appeared to be not sensitive enough to cap-ture the effects (Stampoulis et al., 2009) that we observed at theanatomical level, underscoring the need to consider detailed histologi-cal studies involving various anatomical structures as investigatedhere. Taken together, our observations of ‘tunneling-like effect’ uponnZnO treatment, elongated cellswith bothNPs treatments versus differ-entially altered cells with free ions treatments, and varied metaxylemfrequency with NPs or free ions treatments suggest potential risks ofmetal NPs, including of their free ions, on the growth and developmentof agriculturally significant plant species.

Conflict of interest

There is no conflict of interest.

Acknowledgements

The authors would like to thank M. Zavada and Y. Liu for providingaccess to the light microscopy facility at the East Tennessee State Uni-versity (ETSU). Thanks also to M. Coviello, TEM Analysis Services Lab,TX for support with NP characterization. This research was partiallyfunded by the ETSU Research Development Committee Grant # 82064and the Research Grant # 83003 from the Office of Research and Spon-sored Program, ETSU.

Appendix A. Supplementary data

Citrate–nAg synthesis protocol; Characteristics of nanoparticles;Ultraviolet–visible spectra of nanoparticles; Figure showing tunneling-like effect in maize root; Photographs of maize root hairs, metaxylem,and seed germination in cabbage; TFF protocol; Impact of dilution onnanoparticle characteristics; Statistical analyses; Analysis of moisturecontent and Ag biouptake. Supplementary data associated with this arti-cle can be found online at http://dx.doi.org/10.1016/j.scitotenv.2013.02.059.

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