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Plant Physiology and Biochemistry 80 (2014) 128e135

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Cerium dioxide and zinc oxide nanoparticles alter the nutritionalvalue of soil cultivated soybean plants

Jose R. Peralta-Videa a,b,c, Jose A. Hernandez-Viezcas a,c, Lijuan Zhao a,c,Baltazar Corral Diaz e, Yuan Ge c,d, John H. Priester c,d, Patricia Ann Holden c,d,Jorge L. Gardea-Torresdey a,b,c,*

aChemistry Department, The University of Texas at El Paso, 500 W. Univ. Avenue, El Paso, TX 79968, United Statesb Environmental Science and Engineering PhD Program, The University of Texas at El Paso, 500 W. Univ. Avenue, El Paso, TX 79968, United StatescUniversity of California Center for Environmental Implications of Nanotechnology (UC CEIN), United StatesdBren School of Environmental Science and Management, and Earth Research Institute, University of California, Santa Barbara, CA 93106, United StateseUniversidad Autónoma de Ciudad Juarez, Departamento de Química y Biología, Instituto de Ciencias Biomédicas, Anillo envolvente PRONAF y Estocolmo,Ciudad Juarez, Chih 32310, Mexico

a r t i c l e i n f o

Article history:Received 21 January 2014Accepted 28 March 2014Available online 5 April 2014

Keywords:NanoparticlesSoybeanMineral elementsBioaccumulationFood quality

* Corresponding author. Chemistry Department, TPaso, 500 W. Univ. Avenue, El Paso, TX 79968, Unitedfax: þ1 915 747 5748.

E-mail address: jgardea@utep.edu (J.L. Gardea-Tor

http://dx.doi.org/10.1016/j.plaphy.2014.03.0280981-9428/� 2014 Published by Elsevier Masson SAS

a b s t r a c t

The aim of this study was to determine nutrient elements in soybean (Glycine max) plants cultivated infarm soil amended with nCeO2 at 0e1000 mg kg�1 and nZnO at 0e500 mg kg�1. Digested samples wereanalyzed by ICP-OES/MS. Compared to control, pods from nCeO2 at 1000 mg kg�1 had significantly lessCa but more P and Cu, while pods from 100 mg kg�1 nZnO had more Zn, Mn, and Cu. Plants treated withnZnO showed significant correlations among Zn, P, and S in pods with Zn in roots. Correlations amongpod Zn/root Zn was r ¼ 0.808 (p � 0.01) and pod P/root P was r ¼ 0.541 (p � 0.05). The correlation amongpod S/root S was r ¼ �0.65 (p � 0.01). While nCeO2 treatments exhibited significant correlations be-tween pod Ca/root Ca (r ¼ 0.645, p � 0.05). The data suggest that nCeO2 and nZnO alter the nutritionalvalue of soybean, which could affect the health of plants, humans, and animals.

� 2014 Published by Elsevier Masson SAS.

1. Introduction

Roots of soil cultivated plants indiscriminately absorb essentialand non-essential elements from the soil solution (Epstein, 1994;Peralta-Videa et al., 2009). This enhances the law of naturalgradient, which is obeyed by soil chemical elements (Brady andWeil, 1999). The uptake of nutrient elements by plants isgoverned by the availability of elements, the ability of the plant toaccumulate the elements, and the competition among elements(Kabata-Pendias and Pendias, 2001). Absorption by roots is affectedby the interaction of elements that can be antagonistic, synergisticormultiplicative (Peralta-Videa et al., 2003). Under nowater deficit,ion absorption by roots is also affected by several factors includingplant species, soil type, soil pH, and organic matter (Brady andWeil,1999; Lavado et al., 2001; Maathuis, 2009). In natural environ-ments, a combination of these factors can determine the uptake

he University of Texas at ElStates. Tel.: þ1 915 747 5359;

resdey).

.

and accumulation of elements in plant tissues. It has been reportedthat, during the late period of growth, soybean plants cultivated inacid soil accumulated more cationic elements in stems and leavesthan plants cultivated in neutral soil (Wang et al., 2000). Murakamiand Ae (2009) reported that soybean shoots (Suzuyutaka cultivar),accumulated less Zn in an andosol (pH 6.1) than in a fluvisol (pH5.3).

A few reports have described the effects of some lanthanideelements in plants. Most of these reports indicate that lanthanum(La) negatively affects several growth parameters in plants. Forinstance, in wheat, the addition of La (0.5e25 mg L�1) to the hy-droponic medium inhibited primary root elongation, reduced rootsand shoots dry weight, and the content of Ca, Mg, K, Cu, Zn (Huet al., 2002). In Juglands nigra, La reduced fine root growth,photosynthesis, the content of chlorophyll a, and the concentrationof Mg, Ca, Ni, and P in roots and shoots (Nicodemus et al., 2009).Lanthanum also decreased the main root length, plant height, leafarea, and the dry weight of roots, stems and leaves of hydroponi-cally grown soybean seedlings (Wen et al., 2011). Conversely, re-ports indicate that cerium (Ce), another member of the lanthanideseries, stimulates root growth and other plant functions. For

J.R. Peralta-Videa et al. / Plant Physiology and Biochemistry 80 (2014) 128e135 129

example, Yuan et al. (2001) reported that “Changle”, a fertilizerwith 50.2% of Ce, increased root growth in rice (Oryza sativa)seedlings. In cowpea plants (Vigna unguiculata), low levels of Ce(0.713e17.841 mM) were positively associated with foliar chloro-phyll content, dry matter production, and nitrate reductase activity(Shyam and Aery, 2012). Conversely, Diatloff et al. (2008) reportedthat 5$0 mm Ce decreased corn shoots dry weight by 32%. However,to the authors’ knowledge, there are no reports on the effects ofrare earth element oxide nanoparticles (NPs) on the uptake andaccumulation of macro and micro elements in crop plants.

The effects of NPs on plants will depend, among others, on thetype of soil and the ability of plants to adjust to the toxicity effects.Zhao et al. (2012a,b) also reported that the mobility and bioavail-ability of NPs in soil is strongly affected by soil organic matter,which also increased the concentration of Ce in corn (Zea mays)roots. This suggests that the type of NP and soil conditions willaffect the accumulation of nutritional elements in plants grown inNP impacted soils. Recently, Priester et al. (2012) reported the up-take and distribution of Ce and Zn in soybean tissues grown in farmsoil impacted with low, medium and high concentrations of eithernCeO2 or nZnO. The data showed that both the nCeO2 and nZnOaffected soybean growth parameters; however, the effects of bothnanomaterials in nutrient accumulation have yet to be reported. Inthe present manuscript we report a study aimed to determine theeffects of both the nCeO2 and nZnO on macro and micro nutrientaccumulation in different organs of soybean plants, particularly inpods. While there are increasingly insights into when and hownanomaterials affect plant growth and yield, this research ad-dresses a gap toward understanding the implications of nano-materials in soil on plant nutrient uptake, and thus, food cropnutritional quality.

2. Materials and methods

2.1. Soil source and characteristics

As described in Priester et al. (2012), the soil for this researchwas obtained from an organic farm in Carpinteria, CA (N 34� 230

4000, W 119� 280 4000). Prior to use, sieved soil samples (2 mm) wereair-dried at room temperature, and stored at 4 �C. Before treatment,the samples were analyzed for texture, pH, saturation, cation ex-change capacity, soluble salts, organic matter, total nutrients (C, Cu,Fe, Mn, N, Zn), extractable nutrients (B, Ca, Cl, Cu, Fe, Mg, Mn, Na, P,Zn, HCO3, CO3, NH4, NO3) and exchangeable nutrients (Ca, K, Mg,Na) by the UC Davis Analytical Laboratory (Davis, CA; http://anlab.ucdavis.edu/). This datawas included in the supporting informationof Priester et al. (2012). In the present manuscript, we included theICP data for macro and micro elements after treatment application.

2.2. Nanoparticles and addition to soil

Ten nm nZnO and eight nm nCeO2, both from Meliorum Tech-nologies, Rochester, NY, were added to the soil approximately 24 hbefore planting. The NPs were added as a powder to soil to obtainfinal concentrations of 50, 100 and 500 mg nZnO kg�1 and 100, 500and 1000 mg nCeO2 kg�1. The procedure was previously describedin Priester et al. (2012).

2.3. Planting

The planting protocol was described in Priester et al. (2012).Briefly, control and NP treated soil samples of 2.4 kg each, weredeposited in polyethylene bags and placedwithin 4-L polyethylene/polypropylene blend garden pots previously lined at the base (in-ner) with polyethylene mesh (Easy Gardener, Waco, TX.) and

bottom-filled with 400 g of washed gravel (1.25e2.5 cm). All bagshad 20 holes of 5 mm for drainage. The entire root system waswithin the bags and allowed for easier removal from the pots atharvest time. Dwarf soybean seeds (Early Hakucho, variety product#5555) purchased from Park Seed Company (Greenwood, SC) weregerminated in peat pellets and transplanted to the pot soil whenthe true leaves emerged (18 days after planting). At the center ofeach pot, a hole of 3.8 cm diameter and 5 cm deep was drilled and aseedling was then transplanted. Each treatment had four replicates.The experiment was conducted in a climate-controlled greenhouseunder full sunlight and a temperature range of 31 �C max and12 �C min. For ten pots, sensors (model 5TE, Decagon) wereinstalled into the soil for periodic direct measurement of watercontent, temperature, and conductivity.

2.4. Watering

During the whole growth period, the pots were watered every72 h to reach a total of 150 L of water per m3 soil. During the firstweek, the pots received 100 mL at each watering time. In thesubsequent irrigation events, the amount of water/pot increased asfollows: to 200 mL from days 9e21, 250 mL from days 21e27, and300mL fromday 27 until the completion of the experiment. At eachwatering, a sub-sample of H2O was measured for Zn and Ce con-centrations by ICP-AES (atomic emission spectroscopy). Ceriumwas not detected; Zn was present in concentrations ranging from0 to 0.17 mg L�1, and accounted for a cumulative addition of<0.1 mg per pot. Water content, conductivity, and temperatureweremeasured immediately before and after each watering using aDecagon ProCheck data recorder connected to the buried sensors(Pullman, WA).

2.5. Harvesting description

The plants were harvested after w48 d of growth in the NPtreated soil (Priester et al., 2012). Roots and shoots were severed atthe crown of the plant with a razor blade and weighed. The shootswere separated by stem, leaves, and pods and weighed separately.The separate tissues were oven dried (70 �C/72 h) in paper bagsprior to digestion for ICP analysis. The root system of each plant wasremoved by first carefully breaking apart the soil with a metalscoopula, followed by rinsing (1 min, 3 times) in deionized water(DI) H2O. The root systemwas allowed to air dry (w15 min) beforeweighing. Nodules for ICP analysis were massed, oven dried (70 �C/72 h), and massed a second time to determine water content. Roottissues for ICP analysis were dried and massed as described above.After harvesting, soil samples were stored (4 �C, and �80 �C) forfuture analysis.

2.6. ICP analysis

For the ICP analysis, the oven dried samples were acid digestedassisted by a microwave oven (CEM acceleration reaction system;Mathews, NC). Plant samples from the nZnO treatments weredigested following the EPA 3051 method. Soil samples weredigested with a mixture of concentrated plasma-pure HNO3 andHCl (1:3) (Aqua regia). Plant samples from the nCeO2 treatmentswere digested with concentrated plasma-pure HNO3 and H2O2

(30%) (1:4) as described by Packer et al. (2007) with slightmodifications.

To validate the digestion and analytical method, standardreferencematerials fromNIST 1547, 1570a and 2709awere digestedand read as samples, obtaining recoveries between 90 and 99%. Tovalidate the ICP readings, every ten samples, the blank and samplesspikedwith Ce and Zn at 10mg L�1 were read. The average readings

Table 2Bioaccumulation factors (plant/soil ratios) of cerium and zinc in soybean plantsgrown fully into bean production in a farm soil amended with nCeO2 and nZnO.Calculated from values reported in Priester et al. (2012).

(mg kg�1)b Root Nodule Stem Leaf (�1E3)a Pod

CeriumLow (100) 3.7 � 10�3 3.2 � 10�3 1.3 � 10�3 8.4 � 10�4 7.6 � 10�4

Medium(500)

0.39 0.04 4.6 � 10�4 5.5 � 10�4 3 � 10�4

Zincb

Low (50) 0.40 0.07 0.28 1.10 0.33Medium

(100)0.32 0.17 0.33 1.17 0.40

a Indices in the leaf column for the Ce data were obtained from original datamultiplied by 1000.

b Bioconcentration factors for control and the highest nCeO2 and nZnO concen-trations were already shown in Hernandez-Viezcas et al. (2013).

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for Ce and Zn in the spiked samples were, respectively,10.1 � 0.30 mg L�1 and 9.9 � 0.20 mg L�1.

Total Ce determination in root tissues was achieved by usingICP-OES (PerkineElmer Optima 4300 DV), while Ce in aerialbiomass was determined by ICP-MS (PerkineElmer ELAN DRC II,Shelton, CT). Zinc and other nutrient element determinations inplant samples were performed using ICP-OES. Operation parame-ters for the ICP-OES were as follows: nebulizer flow, 0.80 L min�1;power, 1450 W; peristaltic pump rate, 1.5 mL min�1; flush time,15 s; delay time, 20 s; read time, 10 s; wash time, 60 s; and everysample was read in triplicate. The ICP-MS was operated with an RFpower of 1400 W, plasma gas flow (Ar) 18 L min�1, scanning modepeak hopping, Nebulizer flow 0.95 L min�1, dwell time 35 ms,sweeps 30, replicates 3.

2.7. Statistical analyses

Statistical analyses of data were performed using the StatisticalAnalysis System (SAS, version 9.0). Means were compared usingTukey's HSD test, assuming a two-tailed distribution and equalvariance. A Student's t test was used to compare treatment meansfrom plants grown with 500 mg kg�1 of either nCeO2 or nZnO. Thenumber of replicates were n¼ 4, except for the low nZnO treatmentwhere n ¼ 3.

3. Results and discussion

3.1. Concentration of cerium and zinc in soil and plants andbioaccumulation factors in pods

Cerium and zinc concentrations in soil after treatment applica-tion are shown in Table 1. Concentration of Ce in control soil wassimilar to the Ce concentration in pristine soils, which are on theorder of 10e100 mg g�1 (Tyler and Olsson, 2001). At the 100, 500,and 1000 mg kg�1 treatments, the soil contained approximately 4,14, and 28 times more Ce than control, but most of it was in theform of nCeO2, as these NPs are rather insoluble (Zhao et al., 2012b).In the case of Zn, the background concentration in un-amendedsoils is in the range of 17e125 mg kg�1 (Kabata-Pendias andPendias, 2001). Therefore, Zn in control soil was in the normalrange and in the soil treated with 50, 100 and 500 mg nZnO kg�1, itwas 1.4, 1.7, and 5 times over the control. As the nZnO dissolved (Liet al., 2011), most of the Zn in soil solution was ionic Zn.

Concentrations of both elements, Ce and Zn, in soybean plantparts of this experiment were previously reported (Priester et al.,2012) and were included in this manuscript as SupportingInformation (SI, Table S1) in order to support the calculation ofthe bioaccumulation factors (BAFs) in pods. (Except for the BAF instems of nZnO treaded plants, only BAFs in pods were analyzed,because only soybean seeds are consumed as food). BAFs for Ce andZn in the edible portion of soybean (Table 2) were calculated bydividing the concentration of element in pods/concentration ofelement in the soil. Table 2 does not show BAFs for control and thehighest NP concentrations because these were previously shown inHernandez-Viezcas et al. (2013). In Table 2, the BAFs for Ce in

Table 1Concentration of cerium and zinc in soil after treatment application. The data represent

Control (mg kg�1) nCeO2 (mg kg�1)

Treatments 0 100 500Cerium 32�2a 133 � 11 449 � 71Zinc 95 � 16 NA NA

NA ¼ not analyzed.a mg kg�1.

soybean pods, at all nCeO2 concentrations, were very low. However,as previously reported, most of the nCeO2 are stored in soybeangrains as NPs (Hernandez-Viezcas et al., 2013); thus, it is predictedthat soybean plants grown in soil spiked with 500 mg nCeO2 kg�1

accumulated nCeO2 in pods with a BAF of 3 � 10�4. Currently, thereis no way to evaluate the connotation of this amount of Ce insoybean pods, but very likely, the nCeO2may be in the next soybeangeneration. In addition, to the best of the authors’ knowledge, thereare no references about bioconcentration of Ce in the seeds of cropsgrown in nCeO2 treated medium; thus, it was not possible tocompare the obtained data.

The BAFs for Zn in soybean pods were very high compared to CeBAFs. Interestingly, the BAF was lower (0.16) (Hernandez-Viezcaset al., 2013) for the highest nZnO treatment (500 mg kg�1) andhighest (0.40) for the medium (100 mg kg�1) nZnO treatment.Previous studies have shown that in a 500mg nZnO L�1 suspension,the ionic Zn was 8 mg L�1 (de la Rosa et al., 2011). At lower con-centrations (50 and 100mg nZnO L�1), the dissolutionwas to 10mgZn L�1 (unpublished data); perhaps the enhanced solubility of thelower concentration of nZnO was the reason for the high Zn accu-mulation in pods. Barzini et al. (2012) found that in Sesbania virgata(Cav.) Poir., a tropical legume pertaining to the same family ofsoybean, the BAF in the shoot was 0.32 for an available Zn con-centration in soil of 377�46 mg kg�1. The BAF in stem found in thepresent study, for a Zn concentration of 424 � 27 mg kg�1 (fromnZnO) was 0.30. This could be due to differences in varietal abilityfor Zn uptake/translocation or that Zn in nZnO treated soil was lessavailable for plant uptake.

3.2. Micronutrient accumulation

Accumulation of micronutrients under the presence of nCeO2

and nZnO is shown in Figs. 1 and 3, respectively, and Tables S2 andS3. Tables S2 and S3 show the averages and standard errors of fourreplicates for nCeO2 data and for medium and high nZnO; threereplicates for low nZnO data. Tables S4 and S5 show statisticaldifferences among averages according to the Tukey’s test.

averages of three replicates �standard errors. Data were rounded to the next digit.

nZnO (mg kg�1)

1000 50 100 500907 � 111 NA NA NANA 118 � 4 142 � 8 424 � 27

Fig. 1. Concentration of zinc (A), iron (B), molybdenum (C), manganese (D), copper (E), and sodium (F) in soybean plant tissues grown in soil treated with nCeO2 at 100 (low), 500(medium), and 1000 mg kg�1 (high concentration). *Stands for statistical differences at p � 0.05 and ** at p � 0.01 according to the Tukey’s procedure for pairwise comparisons ofmeans (Table S4).

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As seen Figs. 1 and 3, both nCeO2 and nZnO differentiallyaffected accumulation of microelements in soybean plant tissues.Micronutrients participate in a variety of functions in plant life.Most micronutrients are involved in electron transfer and enzy-matic activities in different plant organs (Taiz and Zeiger, 1998).Thus, some of them, like Mo, are retained in roots for nitrogenreduction, while others such as Zn, Cu, and Fe are fundamental inthe photosynthesis process (Taiz and Zeiger, 1998). In this study,nCeO2 did not affect Zn accumulation and, as expected, nZnOincreased Zn in all tissues. Interestingly, at low nZnO, Zn in tissueswas similar than in control tissues and at high nZnO concentration,therewas a significant accumulation of Zn, compared to control andthe other nZnO treatments (Fig. 3). There was a significant increaseof Zn in stems (541%) and pods (155%) in plants treated with500 mg nZnO kg�1, compared to control (Table S5).

Only nZnO interfered with Fe accumulation, which was signifi-cantly reduced in leaves (p � 0.01) at all NP concentrations (Fig. 3and Table S5). Iron is mobilized to chloroplasts, where it is uti-lized in electron transport or stored complexed with ferritin(Grusak et al., 1999). Zinc in leaves is also required for chlorophyllbiosynthesis, and most of it is localized in the cytoplasm (Arrivaultet al., 2006). However, excess Zn2þ becomes toxic because it bindsto proteins, displacing other metal ions, such as Fe2þ, from theirbinding sites (Sinclair and Kramer, 2012; Hille et al., 2011).

nCeO2 and nZnO differentially affected the accumulation of Mo.While medium and high nZnO treatments increased Mo in nodules(134% and 164%, respectively), medium and high nCeO2 decreased

Mo in stems by 75% and 85%, respectively. There was also areduction of Mo in roots. At high nCeO2, Mo was not detected inroots, but the response among the treatments was not consistent(Table S4 and S5, Figs. 1 and 3). Molybdenum is a component ofplant sulfite oxidases, and nitrate reductases (Hille et al., 2011). It isalso commonly used in bacterial nitrogenase, used in dinitrogenfixation (Seefeldt et al., 2009). This suggests that nCeO2 couldreduce nitrate assimilation, while nZnO could improve nitrogenassimilation in soybean plants. However, there is lack of informa-tion about the possible deficiency/toxicity of insufficiency/excessMo on the nutritional value of soybean seeds harvested from NPexposed plants.

nCeO2 and nZnO affected the accumulation of Mn in differentparts of soybean plants. However, the data did not show ten-dencies; thus, we cannot conclude about the effects of these NPs onMn accumulation in soybean. At high concentration, both thenCeO2 and nZnO reduced Cu accumulation. In roots, reductionwithnCeO2 reached 71% and with nZnO, reduction was by 59% (Figs. 1and 3, Tables S4 and S5). However, Cu in pods increased by 371%with the high nCeO2 treatment and by 386%with themedium nZnOtreatment (Figs.1 and 3, Tables S4 and S5). Only nCeO2 disturbed Naaccumulation. All nCeO2 concentrations significantly reduced Naaccumulation in soybeanpods and at low concentration reduced Nain leaves (Fig. 1, Table S4). nCeO2 also significantly reduced Al insoybean pods (Fig. 2, Table S4). As previously mentioned, the bio-logical importance of excess/deficiency of micronutrient accumu-lation in pods has yet to be studied.

Fig. 2. Concentration of magnesium (A), calcium (B), phosphorus (C), potassium (D), sulfur (E) and aluminum (F) in soybean plant tissues grown in soil treated with nCeO2 at 100(low), 500 (medium), and 1000 mg kg�1 (high concentration). *Stands for statistical differences at p � 0.05 and ** at p � 0.01 according to the Tukey’s procedure for pairwisecomparisons of means (Table S4).

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3.3. Macronutrient accumulation

Concentrations of Ca, Mg, P, K, and S found in soybean tissuesgrown to maturity in soil treated with nCeO2 and nZnO are shownin Figs. 2 and 4, and Tables S4 and S5. The nCeO2 treatmentsdisturbed the accumulation of Ca, Mg, P, K, and S (Fig. 2); while thenZnO interfered with the accumulation of K and Mg (Fig. 4).Respect to control, accumulation of Mg was significantly reducedin roots by nZnO and nCeO2 at high concentration (p � 0.05)(Figs. 2 and 4; Tables S4 and S5). At the rootesoil interface, Mg isabsorbed through transporters similar to bacterial transportersCorA Mg2þ, which form selective ion channels (Maathuis, 2009).Thus, it is possible that NPs stuck on the root surface block thechannels, reducing the absorption of Mg. Soybean grains are asource of Mg for human nutrition (Moraghan et al., 2006); hence,this suggests nCeO2 and nZnO could reduce the nutritional valueof soybean.

Calcium accumulation was significantly reduced in pods bynCeO2 at high concentration (p � 0.05) (Fig. 2, Table S4). Calcium isa messenger that mediates several responses in plants, such asnodulation, hormone production, biotic and abiotic environmentalstressors (Hashimoto and Kudla, 2011), and development (self-in-compatibility) (Reddy and Reddy, 2004). Thus, nCeO2 may interferewith the nodulation and other physiological functions.

Potassium was another element reduced by nCeO2 and nZnO athigh concentrations. The highest amendment of nCeO2 significantlyreduced K accumulation in nodules (Fig. 2, Table S4), while nZnOreduced K in roots (Fig. 4 and Table S5). Potassium is usuallyaccumulated at high concentration in plant cells; even if it is at lowconcentration in soil (Hedrich and Kudla, 2006). Results for K in

soybean tissues suggest that neither nCeO2 nor the nZnO representa problem for K transport to the upper soybean plant parts. How-ever, the reduction of K accumulation in nodules and roots couldresound in soybean quality (Better Crops, 1998). Potassium is animportant element for N2 fixation (McLean et al., 1983), whichsuggests that CeO2 NPs could be detrimental for soybean produc-tion. nCeO2 also reduced P in roots but increased it in pods for thehighest NP amendment (p � 0.05). Although there was no clearanswer for S, the medium nCeO2 treatment showed increase,compared to low amendment (Table S4 and Fig. 2).

Although the concentration of Ca, K, andMg vary with soil types,the soil used in the present study (Table 3) had comparableamounts of total Ca and Mg to that of some agricultural soils fromKansas (Sparks, 2001; Matz, 2012), similar amounts of K comparedto that of a Delaware ultisol, and three and seven fold to that ofVirginia and Florida ultisols (Sparks, 2001), respectively. Conse-quently, the amounts of macronutrients found in control plantswere similar to previously reported data for soybean grown insoutheast Kansas (Matz, 2012). However, the data from pods sug-gest that, at high concentration, the nCeO2 amendment woulddisturb the balance of some essential elements. Imbalance innutritional elements such as Ca, P and Na could have serious con-sequences for the nutrition and health of humans and animals(White et al., 2012).

3.4. Comparison of element accumulation with both nanoparticles

To measure the impact of element accumulation in roots onthe element accumulation in pods, correlation analyses for ele-ments in pods vs. elements in roots were calculated (Table 4). This

Fig. 3. Concentration of zinc (A), iron (B), molybdenum (C), manganese (D), copper (E), and sodium (F) in soybean plant tissues grown in soil treated with nZnO at 50 (low), 100(medium), and 500 mg kg�1 (high). *Stands for statistical differences at p � 0.05 and ** at p � 0.01 according to the Tukey’s procedure for pairwise comparisons of means (Table S5).

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table shows that for the nZnO, there was a positive significantcorrelation between pod Zn/root Zn (0.808, p � 0.01), pod P/root P(0.541, p � 0.05) and a highly significant negative correlation in Saccumulation (�0.65, p � 0.01). These correlation values areclosely related to the mobility of elements in tissues. Zinc isconsidered one of the most mobile elements and it is remobilizedfrom leaves, in this case to the pods (Grusak et al., 1999). As forthe nCeO2, the ratio pod Ca/root Ca was positively correlated(0.645, p � 0.05), while the ratio pod Cu/root Cu was negativelycorrelated (�0.537, p � 0.07). Previous work has shown that Cuaccumulated in roots requires nicotianamine (a metal chelatingmolecule) for its transport to the upper plant parts (Grusak et al.,1999). Thus, the negative correlation for pod Cu/root Cu (Table 4),suggests that nCeO2 could decrease the production of nicotian-amine, which could contribute to a reduction in the transport ofCu to the pods.

It has been suggested that the Ca/P ratio in food must be 1.0 andthat a low dietary Ca/P ratio may interfere with the homeostasis ofCa metabolism in humans (Kemi, 2010). In the present study, theCa/P ratio for podswaswell above 1.0 in all treatments (Table S6). Incontrol, the ratio was 33; while in nCeO2 treatments, the ratiovaried from 10 (high concentration treatment) to 22 (low andmedium). In nZnO treatments, the ratio varied from 27(100 mg kg�1 treatment) to 70 at the low concentration treatment(50 mg kg�1). These ratios were higher (more than 4 times) thanconcentrations reported for field grown soybean plants (Jurgonskiet al., 1997). However, compared to control, the Ca/P ratio in soy-bean pods was reduced approximately 33% by nCeO2 at low andmedium concentrations and by w60% at high concentration

(1000 mg kg�1) (Table S6). On the other hand, 50 mg nZnO kg�1

doubled the Ca/P ratio, but the other treatments did not affect theratio. Although the meaning of such high ratios is currently un-known, the results suggest that nCeO2 could reduce the nutritionalvalue of soybean.

In summary, the results of this analysis indicate that both thenCeO2 and nZnO, even at concentrations as low as 100 mg kg�1 and50 mg kg�1, respectively, have the potential to disturb the accu-mulation of some nutritional elements in soybean plants grown inNP amended farm soil. In the experimental conditions of this study,the nZnO impacted the most the accumulation of essential ele-ments (Table S4 and S5). As expected, in plants exposed to nZnO, Znaccumulation was significantly enhanced in all analyzed organs,including pods. The meaning of excess Zn in reproductive organshas yet to be evaluated. On the other hand, the results suggest that,at high concentration, nCeO2 interfere with the uptake of elementsinvolved in nitrogen metabolism and photosynthesis. In addition,as shown in the previous publication (Priester et al., 2012), lowamounts of Ce were found in the soybean pods. The X-ray analysesof soybean grains (Hernandez-Viezcas et al., 2013), suggests thatsome nCeO2 would be in soybean derived food and in the nextsoybean plant generation.

Contribution

J.R. Peralta-Videa gathered all the results and wrote the firstdraft of this manuscript. J.A. Hernandez-Viezcas and L. Zhao pro-cessed the samples and performed the analytical determinations. B.Corral Diaz performed all the statistical analysis and made the

Fig. 4. Concentration of magnesium (A), calcium (B), phosphorus (C), potassium (D), sulfur (E) and aluminum (F) in soybean plant tissues grown in soil treated with nZnO 50 (low),100 (medium), and 500 mg kg�1 (high). *Stands for statistical differences at p � 0.05 and ** at p � 0.01 according to the Tukey’s procedure for pairwise comparisons of means(Table S3).

Table 3Total concentration of nutrient elements in the farm soilfrom Carpinteria, California, used in the soybean experiment.Data are average of four replicates �standard deviation.

Element Average (mg kg�1)

Zn 84.00 � 15.88Al 18,867.50 � 811.65Fe 16,649.17 � 676.36Cu 24.92 � 1.58Mn 324.42 � 9.25Na 203.67 � 30.38

P 85.92 � 4.04K 7486.67 � 332.31Ca 7594.17 � 435.86Mg 6099.17 � 390.93S 247.92 � 54.78

Table 4Correlation between element concentration (mg kg�1 dry weight) in pods and roots of snZnO.

Zn Fe Mo Mn Cu M

nZnOr 0.808 �0.414 0.344 �0.247 �0.449 �p Values 0.000 0.125 0.209 0.375 0.094 0.Significance ** n.s. n.s. n.s. n.s. n.

nCeO2

r �0.072 0.190 �0.285 0.432 �0.537 0.p Values 0.593 0.553 0.369 0.161 0.072 0.Significance n.s. n.s. n.s. n.s. ns n.

J.R. Peralta-Videa et al. / Plant Physiology and Biochemistry 80 (2014) 128e135134

graphs. Y. Ge, J.H. Priester, and P.A. Holden designed and performedthe experiment, harvested the plants, and measured all agronomicparameters. J. Gardea-Torresdey participated in the design ofexperiment, supervised all analytical determinations and contrib-uted in the preparation of the final manuscript. All of the authorsparticipated in the writing of the manuscript.

Acknowledgments

This material is based upon work supported by the NationalScience Foundation and the Environmental Protection Agency un-der Cooperative Agreement Number DBI-0830117. Any opinions,findings, and conclusions or recommendations expressed in thismaterial are those of the author(s) and do not necessarily reflect theviews of the National Science Foundation or the Environmental

oybean plants grown to maturity in soil treated with 500 mg/kg of either nCeO2 or

g Ca P K Na Al S

0.301 0.133 0.541 �0.344 0.149 �0.138 �0.65276 0.636 0.037 0.209 0.597 0.624 0.009s. n.s. * n.s. n.s. n.s. **

487 0.645 �0.169 �0.227 0.071 �0.541 �0.413108 0.023 0.598 0.479 0.826 0.069 0.182s. * n.s. n.s. n.s. n.s. n.s.

J.R. Peralta-Videa et al. / Plant Physiology and Biochemistry 80 (2014) 128e135 135

Protection Agency. This work has not been subjected to EPA reviewand no official endorsement should be inferred. The authors alsoacknowledge the U.S. Department of Agriculture (USDA) grantnumber 2011-38422-30835 and the National Science Foundation(NSF) Grant # CHE-0840525. Gardea-Torresdey acknowledges theDudley family for the Endowed Research Professorship and theAcademy of Applied Science/US Army Research Office, Research andEngineering Apprenticeship Program (REAP) at UTEP, grant#W11NF-10-2-0076, sub-grant 13-7.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2014.03.028.

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