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Environmental Pollution 174 (2013) 150e156

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Environmental Pollution

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Bioavailability of nanoparticulate hematite to Arabidopsis thaliana

Yevgeniy Marusenko a,*, Jessie Shipp b, George A. Hamilton b, Jennifer L.L. Morgan b, Michael Keebaugh b,Hansina Hill b, Arnab Dutta b, Xiaoding Zhuo b, Nabin Upadhyay b, James Hutchings b, Pierre Herckes b,Ariel D. Anbar b,c,d, Everett Shock b,c,d, Hilairy E. Hartnett b,c,d

a School of Life Sciences, Arizona State University, Tempe, AZ 85287, USAbDepartment of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USAc School of Earth and Space Exploration, Arizona State University, Tempe, AZ 85287, USAd School of Sustainability, Arizona State University, Tempe, AZ 85287, USA

a r t i c l e i n f o

Article history:Received 11 July 2012Received in revised form20 October 2012Accepted 9 November 2012

Keywords:Arabidopsis thalianaBioavailabilityIron limitationNanomaterialsNanoparticles

* Corresponding author.E-mail address: ymarusen@asu.edu (Y. Marusenko

0269-7491/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.envpol.2012.11.020

a b s t r a c t

The environmental effects and bioavailability of nanoparticulate iron (Fe) to plants are currentlyunknown. Here, plant bioavailability of synthesized hematite Fe nanoparticles was evaluated usingArabidopsis thaliana (A. thaliana) as a model. Over 56-days of growing wild-type A. thaliana, thenanoparticle-Fe and no-Fe treatments had lower plant biomass, lower chlorophyll concentrations, andlower internal Fe concentrations than the Fe-treatment. Results for the no-Fe and nanoparticle-Fetreatments were consistently similar throughout the experiment. These results suggest that nano-particles (mean diameter 40.9 nm, range 22.3e67.0 nm) were not taken up and therefore not bioavailableto A. thaliana. Over 14-days growing wild-type and transgenic (Type I/II proton pump overexpression)A. thaliana, the Type I plant grew more than the wild-type in the nanoparticle-Fe treatment, suggestingType I plants cope better with Fe limitation; however, the nanoparticle-Fe and no-Fe treatments hadsimilar growth for all plant types.

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1. Introduction

Widespread interest in the industrial use of nanoparticles (NPs)has spurred research into the occurrence of NPs in the environment(Raiswell et al., 2008; Mirme et al., 2010; Qafoku, 2010). Nano-particles may exist in various forms andmorphologies (Buzea et al.,2007) and can be readily disseminated throughout the environ-ment where they interact with both biotic and abiotic components(Das et al., 2012; Reed et al., 2012). The nature of these interactionsvaries greatly with the type of biotic or abiotic factor involved andoften depends on NP size, shape, and composition (Nowack andBucheli, 2007; Silva and da Boit, 2011; Xu et al., 2011; Pandey andPandey, 2009). The diversity in NP structures and reactivity callsfor systematic investigations of their fate in the environment andthe effects of NPs on natural biogeochemical processes.

Nanoparticles in the environment have both anthropogenic (i.e.,purposely engineered as well as incidental NPs, such as from fossilfuel combustion) and natural sources (Nowack and Bucheli, 2007;Xu et al., 2011). Naturally occurring NPs originate as weathering

).

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byproducts of minerals, as biogenic products of microbial activity,or as precipitation nuclei in super-saturated fluids (Wigginton et al.,2007; Buzea et al., 2007). Natural iron (Fe) oxide NPs are among themost common types of NPs found in the environment (He et al.,2008). Fe oxide NPs are produced by the oxidation of dissolvedFe(II)-bearing species (Waychunas et al., 2005). The unique physicaland chemical characteristics of nano-scale materials have led toa recent expansion in the production of engineered nanomaterials.For example, synthetic Fe oxide NPs have a variety of industrialapplications (Beker et al., 2010; Pham et al., 2008; Khurshid et al.,2009), with hematite NPs in particular used in pigments,magnetic bioseparation in biotechnology, and lithium-ion batteries(Wang et al., 2005; Park et al., 2007; Zeng et al., 2008). Conse-quently, anthropogenic NPs are released into the environmentduring production, handling, use, and disposal; there has beena concomitant increase of interest in the effects of NPs on theenvironment and the pollution risks to environmental quality(Darlington et al., 2009; Kaegi et al., 2010; Nowack et al., 2012).

The ecological effect of Fe NPs on photosynthesis is particularlyintriguing because Fe is an essential element for structures sup-porting biophysical and electrochemical processes like chlorophyllsynthesis and photophosphorylation (e.g., cytochromes; Spiller andTerry, 1980), but the extent of NP bioavailability and uptake in

Y. Marusenko et al. / Environmental Pollution 174 (2013) 150e156 151

plants is generally, poorly known. Colloidal Fe NPs (one dimension<100 nm) have been shown to be bio-accessible to marine cya-nobacteria, likely playing an important role in phytoplanktonproductivity (Wang and Dei, 2003). Pumpkin plants (Cucurbitamaxima) have been grown in aqueous media containing magnetite(Fe3O4) NPs (individual NPs of w20 nm and agglomerates up to2 mm) and were shown to absorb, translocate, and accumulate theparticles in the plant tissue, but no NP uptake occurred when limabeans (Phaseolus limensis) were grown under similar conditions(Zhu et al., 2008). Furthermore, inhibition of seed germination androot growth may vary with different types of engineered NPs andplant species (Lin and Xing, 2007), highlighting the importance ofvegetationmanagement practices and data assessment for NP risks.Taken together, these findings suggest plants have a wide variety ofresponses to NPs, including a potential role as biological indicatorsfor NP exposure; but additional research is needed to gain a betterunderstanding of the bioavailability of NPs to plants (Navarro et al.,2008; Klaine et al., 2012).

We evaluate the bioavailability of Fe NPs, specifically hematite(Fe2O3), as an Fe source for the plant species Arabidopsis thaliana(A. thaliana). A. thaliana is a well-studied model plant with well-defined growth stages; it has survival mechanisms for copingwith Fe deficiency, and has been genetically-altered for generegulation and environmental adaptation experiments (Boyes et al.,2001; Jeong and Guerinot, 2009; Gaxiola et al., 2001). Previousstudies have demonstrated that titanium dioxide NPs (TiO2; meandiameter 2.8 nm), can be taken up by A. thaliana (Kurepa et al.,2010); the titanium NPs were shown to traverse cell walls whenplants were grown on TiO2-containing agar media (Wang et al.,2011). In the present study, A. thaliana was grown on agar mediawith three different Fe treatments: (1) media amended with Fe2O3NPs (NPeFe), (2) media amended with ethylenediaminetetraaceticacid bound Fe (EDTAeFe), and (3) media with no added Fe (NoeFe).If NPs are found intracellularly in plants, their presence alone doesnot indicate that the NP Fe is available to be used. Therefore,biomass, chlorophyll concentrations, and total internal Fe concen-tration of the plants were measured over a 56-day period todetermine Fe uptake and plant growth responses. Additionally, thethree treatments were used to evaluate growth of both wild-typeand transgenic A. thaliana plants over a 14-day period. Based onA. thaliana literature, we hypothesized that plants will employsurvival strategies to utilize Fe from NP sources when other Fesources are unavailable.

2. Methods and materials

2.1. Synthesis of Fe nanoparticles

Conventional heating, microwave heating and solegel processes are welldocumented methods for synthesizing nano-sized hematite (a-Fe2O3) particles(Torres et al., 1990; Katsuki and Komarneni, 2001; Lam et al., 2008). In the presentstudy, a conventional heating technique was used to produce hematite NPs.Approximately 50 mg/mL of FeCl3$6H2O (EM Science, Gibbstown, NJ) was dissolvedin 4 mM HCl (EMD, Darmstadt, Germany). This solution was filtered using a 0.8/0.2 mm Acrodisc syringe filter (Pall Corporation, Life Sciences, Port Washington, NY)to remove particles (mainly larger Fe2O3 particles) from the solution. The filtrate wasboiled and stirred for 20 min then placed in an oven at 100 �C overnight. To removelarger hematite particles, the solution was centrifuged for 15 min (3500 rpm). Thesupernatant was collected, washed with 4 mM HCl and centrifuged again. This stepwas repeated five times to yield a supernatant containing NPs of the desired sizerange (<50 nm).

2.2. Characterization of Fe nanoparticles

Total Fe concentration of the NP solution (pH ¼ 4.3) was measured by induc-tively coupled plasma-mass spectrometry (ICP-MS). Nanoparticle images were ob-tained using a Philips CM200-FEG high-resolution transmission electronmicroscope(TEM) by dispensing a droplet of NP solution onto a TEM-copper grid and air-dryingbefore imaging. Low cost copper grids were used because no copper was added in

the synthesis of the NPs, thus no Cu signal was expected from the sample. Miner-alogy of the NPs was determined via X-ray diffraction (XRD) analysis. The NPsolution was loaded on a glass plate in a thin layer and scanned with a Rigaku D/Max-IIB Standard Powder Diffractometer. The sample was scanned from 5 to 90�

2-theta (�2Q), with a step size of 0.02� . The size distribution of the NPs was alsodetermined by dynamic light scattering (DLS, Brookhaven Instrument Corp; 6 runs,1 min/run, 25 �C). Energy dispersive X-ray spectroscopy (EDS) was used to charac-terize the elemental composition of the NPs.

2.3. Growing and harvesting Arabidopsis thaliana

The three media treatments, NoeFe, NPeFe and EDTAeFe, were made frommodified BG-11 Fe-free stockmediawith the following composition: 1.25mMKNO3,1.50 mM Ca(NO3)2, 0.75 mM MgSO4, 0.5 mM KH2PO4, 0.05 mM KCl, 0.05 mM H3BO3

(anhydrous), 0.01 mM MnSO4 (anhydrous), 2.0 mM ZnSO4 (heptahydrate), 1.5 mMCuSO4 (pentahydrate), 0.2 mM Na2MoO4 (dihydrate), 0.1 mM Na2SO3, and 1.2% agar.The EDTA-bound Fe stock solution was made by adding EDTA in excess; specifically,249 mg of EDTA disodium salt dihydrate (Martell and Smith, 1989) was added to248 mg of FeCl3 and diluted to a final volume of 50 mL with deionized water(18.2 MU$cm). To make EDTAeFemedia, the EDTA/FeCl3 stock solutionwas added tothe stock media for a targeted final Fe concentration of 4 ppm (72 mM). EDTAeFemedia and Fe-free stock media solutions were autoclaved prior to pouring intopetri dishes. The NPeFe media were made by adding 246 mL of the NP solution to300 mL of the autoclaved stock media to yield a targeted Fe concentration similar tothe EDTAeFemedia of 4 ppm (72 mM). The final Fe concentration used here is knownto be sufficient for A. thaliana growth (Laganowsky et al., 2009). The NPs were addedimmediately before the media was poured and began to solidify. This addition of FeNPs at pH 4.3 did not affect the pH of the media (all treatments had a pH of 6.1). TheNoeFe media utilized autoclaved Fe-free stock media. The final Fe concentration insamples from random areas and depths of the media in the 3 treatments wasdetermined using ICP-MS.

A. thaliana (ecotype Columbia) seeds were stored at 4 �C and washed in 0.05%polysorbate-20 prior to planting. Seeds were planted on the surface of the agarmedia in a class-10 laminar flow exhaust hood in a class-100 trace metal-free cleanlab. Forty seeds were planted in each petri dish and the dishes were sealed withparafilm. Plants were grown in an incubator at 18 �C with a 16 h light/8 h dark cycle.One dish (40 plants) from each type of Fe treatment was harvested on days 0, 7, 12,18, and 56 (600 total plants across 3 treatments, 1 replicate plate, and 5 time points).Measurements were duplicated on day 18 (120 additional plants across 3 treat-ments). During harvest, plants and roots were removed from the media, and rinsedwith 18.2 MU$cm water. Harvested plants were either sealed in a micro centrifugetube to await chlorophyll measurement (additional plate on day 18), or dried forweighing andmetal analysis. Plants from each platewere composited andmeasuredas one sample. An analysis of variance (ANOVA) statistical test was used to explorethe effects of Fe treatment on plant Fe concentration over time.

2.4. Chlorophyll analysis

Chlorophyll from the harvested A. thaliana plants was extracted using thestandard acetone extraction method described below (AOAC International, 1995;Goodwin, 1965). Briefly, excess water was removed from the vial containingA. thaliana and replaced with 1.5 mL of an acetone:water (85:15) solution. Thesolution and plants were agitated for 30 s in a vortexer and stored in the dark atroom temperature for 12 h. The resulting acetone/water/chlorophyll mixture wasfiltered (0.45 mm nylon syringe filter; MicroLiter Analytical Supplies, Inc.) into cleanvials. The chlorophyll extract was transferred into a 1.5mL cuvette (1 cm pathlength)and the chlorophyll concentrations were compared amongst different samplesbased on the absorption spectrum generated using a UV-1700 Shimadzu UVeVisScanning Spectrophotometer (scanning parameters: 700 nme400 nm, 1 nm scaninterval).

2.5. Metal analysis via ICP-MS

For each harvest, 40 plants were collected and placed in a pre-weighed microcentrifuge tube. The cap was left open and the samples were dried in an oven at60 �C for 4e8 weeks. The dried samples were weighed and then removed by rinsingthe micro centrifuge tubes with 15.8 M ultra-clean nitric acid (Fisher Scientific). Thesamples were transferred to Teflon� bombs, sealed and heated at 160 �C overnight.Alternating rounds of 5 mL 1:1 ultra-clean nitric acid and ultra-clean hydrogenperoxide (Fisher Scientific) and 5 mL of 1:1 concentrated ultra-clean hydrochloricacid (Fisher Scientific) and ultra-clean nitric acid were added to the sample. Thedigestion continued until the samples were clear and free of organic material. Mediasamples were digested using the same method. Digested samples were dried anddiluted in 0.32 M ultra-clean nitric acid prior to quadrupole ICP-MS (ThermoScientific X Series) analysis. Samples and standards were introduced in parallel withan internal standard composed of germanium, yttrium and indium to allow fornormalization of plasma variations. Analytical replicate analyses of selected samplesyielded a 2s error of �5%.

Y. Marusenko et al. / Environmental Pollution 174 (2013) 150e156152

2.6. Transgenic Arabidopsis thaliana growth experiment

The EDTAeFe, NPeFe, and NoeFe treatments were used to test the effects on 3different A. thaliana seed types: (1) ecotype Columbia (wild-type), (2) AVP1-1 (TypeI), and (3) AVP1-2 (Type II). Type I and Type II seeds were genetically modified toexpress more of the AVP1 protein that is responsible for vacuolar Hþ-pyrophos-phatase production. The Type II mutant overexpresses the protein at a higher ratethan the Type I mutant. The mutants have higher salt tolerance, better cationretention, enhanced Hþ-pumping capability, drought tolerance, solute and waterretention, acidification, and absorptive area through denser root hairs relative to thewild-type (Gaxiola et al., 2001, 2011). Growth methods and media treatments werefollowed as described earlier, with the exception that 3 replicate plates were usedper treatment type, with 10 seeds per plant type in each plate (270 total seeds across3 treatments, 3 seed types, and 3 replicate plates). Plant growth was determinednon-destructively at days 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, and 14 using total counts ofplants that have reached a certain phenotypic growth stage as identified by Boyeset al. (2001). Each growth stage was given a numerical value (0.10 ¼ 0, 0.50 ¼ 1,etc.) to assess the mean and variation of growth counts across the 10 replicates ineach combination of plant type and treatment type. The effect of Fe treatment onplant growthwas statistically analyzed using two-way ANOVA, followed by post-hocTukey HSD multiple comparison separately for the 3 seed types over time (SPSS,version 20). Two-way ANOVA and Tukey HSDwere also used to analyze the effects ofplant type on plant growth over time separately for the 3 seed types.

3. Results and discussion

3.1. Nanoparticle characterization

Various types of natural and anthropogenic metal oxide NPs arefound in the environment (Nowack and Bucheli, 2007; Klaine et al.,2008). Since Fe is a necessary nutrient for plants, we chose toexamine Fe NPs that can be expected to be particularly susceptibleto plant uptake. Total Fe concentration of the synthetic Fe NPsolution, based on ICP-MS, was 4882 ppm. A transmission electron

Fig. 1. TEM images of Fe2O3 nanoparticles: a) in concentrated solution, b) the same particlea result of the TEM grid. The size distribution of NPs in the TEM is consistent with data fro

microscope (TEM) image of NP clusters shows individual NPs witha diameter of <50 nm, without obvious NP clumping in the media(Fig. 1a, b). The energy dispersive X-ray spectroscopy (EDS) analysisof a selected spot on the NP surface showed that O and Fe are thedominant elements in the NPs (Fig. 1c). The X-ray diffraction (XRD)spectrum of the nanoparticles shows the characteristic pattern ofpeaks for hematite (Fe2O3), with no detectable peaks correspond-ing to other minerals (Fig. 2). Particle-size measurements based ondynamic light scattering (DLS) analysis showed that the NPs hadamean diameter of 40.9 nmwith a log-normal distribution range of22.3e67.0 nm (geometric standard deviation of 1.38) and poly-dispersity index value of 0.109, consistent with the sizes observedby TEM. These results confirm that chemically pure, nanometer-sized hematite (Fe2O3) was synthesized. Hematite is a naturaloccurring mineral and this type of NP could be formed naturally orvia anthropogenic processes, thus the successfully synthesized NPspresented here are a reasonable representation of Fe NPs that couldbe found in the environment (Handy et al., 2008).

3.2. Arabidopsis thaliana growth

The target Fe concentration in the growth media was 4 ppmfor the EDTAeFe and NPeFe treatments, and less than thedetection limit in the NoeFe media. Actual Fe concentrationswere 2.7 ppm (0.05 mM) for the EDTAeFe, 4.6 ppm (0.08 mM) forthe NPeFe, and 0.1 ppm (0.002 mM) for the NoeFe media (ICP-MS). Different regions of the media were sampled and themeasured Fe concentration was consistent across multiple areasand depths for each petri dish. The nanoparticles appeared to besuspended with an even and random distribution throughout the

s at 10� higher resolution, and c) EDS spectrum of Fe2O3 nanoparticles. The Cu peak ism the DLS analysis.

Fig. 3. Dry weight (mg) of 10 plants combined into one sample for each sampling day(NPeFe:,; NoeFe:C; EDTA-Fe:A). The scatter on day 18 represents variability fromtwo different plates (10 plants each). Note break on y-axis scale.

Fig. 2. X-ray diffraction pattern from synthesized Fe NPs; lower panel is a modelspectrum for hematite.

Y. Marusenko et al. / Environmental Pollution 174 (2013) 150e156 153

agar media, and not settled at the bottom of the dishes, allowingthe roots to be in near proximity to the NPs. Even though the Feconcentration in the EDTAeFe media was less than the targetconcentration, there was still ample Fe in the media to sustainA. thaliana growth (Laganowsky et al., 2009). If the Fe from thehematite NPs is bioavailable, the NPeFe media should alsocontain sufficient Fe for A. thaliana growth. The chemicalcomposition of Fe and homogeneous distribution in the solutionwithout aggregation (Figs. 1 and 2), confirmed hematite was innanoparticulate form as intended for the experiment. Eventhough we used growth characteristics instead of microscopymethods to evaluate NP uptake after treatment, hematite NPslikely did not aggregate during the experiment due to mediaconditions used (pH <8, salt concentration <30 mM; He et al.,2008). The Fe concentration in the NoeFe treatment is likelydue to contamination, but the concentration is still quite low andlikely to result in Fe limitation.

Plants in all three media treatments germinated at the sametime, reached the identical growth stage at the same rate, andremained alive for the duration of the experiment. Our intentionwas to take the last sample prior to death of Fe-limited plants e

predicted to be day 24 (Boyes et al., 2001). However, the plantsremained relatively healthy, and we were able to extend theexperiment to day 56, measuring dry weights of 10 combinedplants from each treatment throughout the duration of theexperiment (Fig. 3). Duplicate plates were harvested on day 18 toshow variation between plates; the deviation between plants wasless than the variability in weight increase over time in all threetreatments. Plant size differences in the three treatments werenoticed visually after day 18. In the first 18 days of the experiment,there is no discernable difference in weight between the three Fetreatments. Between days 18 and 56, the EDTAeFe treated plantsappeared to grow larger than those in the other two treatments.There was no difference between the weights of NPeFe and NoeFe treated plants throughout the experiment. Based on resultsfrom day 56, plants treated with EDTAeFe are likely to producemore biomass than plants with NoeFe or with NPeFe. The NPtreated plants had more Fe in the media than the EDTAeFe treatedplants (4.64 ppm vs. 2.68 ppm, respectively) but even with moreFe they appeared to grow at the same rate and to the same extentas the NoeFe treatment, suggesting plants were not able to usethe Fe present in the NPs.

3.3. Fe uptake measurements

To compare Fe uptake between plants grown in the threetreatments, UVeVisible spectroscopy was used to compare chlo-rophyll concentration (chlorophyll a and b). Since plants utilize Fefor chlorophyll biosynthesis, plants deficient in Fe will not be ableto produce as much chlorophyll as plants that are receivingadequate Fe. The effect of insufficient Fe, and hence insufficientchlorophyll production, leads to a yellowing of the leaves known aschlorosis (Varotto et al., 2002). Chlorophyll typically exhibitsa broad absorption band (Soret band) in the 400e500 nm range anda sharp absorbance feature around 640e670 nm (Q-band; Marquezand Sinnecker, 2007). Since absorption is proportional to concen-tration, peak heights of the Q-band at 663 nm can be used tocompare plant chlorophyll production in different samples. Chlo-rophyll measurements taken on day 18, when dry weights for theEDTAeFe treatment were higher than those of the other twotreatments, reveal that spectra for the NoeFe and NPeFe plantswere similar to each other, and generally had lower absorbancethan those from the FeeEDTA treatments (Fig. 4). Specifically, thepeak heights at 663 nm qualitatively show that the NoeFe and NPeFe treated plants have less chlorophyll than the EDTAeFe treat-ment. This is consistent with a visual observation of yellow leaves in95% of plants in the NoeFe and NPeFe treatments, but green leavesin 100% of plants in the EDTAeFe treatment, suggesting the Fe NPswere not utilized for chlorophyll production.

As a second method for evaluating Fe uptake, the total amountof Fe in the plant tissue was determined by digesting the plants andmeasuring Fe concentration using ICP-MS. Iron concentrationsnormalized to plant dry weights for the three treatments show thatplants grown with EDTAeFe media contained more Fe than plantsgrown on media with NoeFe or NPeFe (Fig. 5; ANOVA: P ¼ 0.016).Fe concentration in all three treatments was greatest on day 7(ANOVA: P¼ 0.010) and decreased throughout the remainder of theexperiment. The largest difference between the three treatments isalso on day 7, where the EDTAeFe treated plants contained642 ppm Fe, the NPeFe plants contained 236 ppm Fe, and the NoeFe plants contained 336 ppm Fe. The multiple points on day 18

Fig. 4. Absorbance spectra from chlorophyll extracts. Data shown are from samplescollected on day 18. The peak at 663 nm corresponds to the chlorophyll a plus chlo-rophyll b absorption band.

Y. Marusenko et al. / Environmental Pollution 174 (2013) 150e156154

show the concentration for two replicate plates for each Fe treat-ment (Fig. 5). The decrease in Fe concentration through time for alltreatments reflects a progressive dilution of plant-Fe as plantbiomass increased. The Fe source for the NPeFe and NoeFe plants isthus likely due to Fe initially present in the seed. A sampling of 10seeds had approximately 109 ppm Fe that is presumably availableto germinating plants. This minimum Fe concentration in the seedcannot be directly compared to the Fe concentration in a germi-nated plant because the biomass of the plant is smaller than theseed immediately following germination. We did not measure Fe

Fig. 5. Fe concentration in A. thaliana plants as a function of time (NPeFe: ,; NoeFe:C; EDTA-Fe: A). Error bars represent analytical standard error of the ICP-MSmeasurements (�5%); error bars that are not visible are smaller than the symbols.On day 18, lines are run through the average of measurements from duplicate plates.

concentration in roots and shoots independently; but, the Feconcentration data for the media, seeds, and in whole-plantssuggest that NP Fe did not adsorb to the root surface. Throughoutthe experiment, the consistently higher Fe concentrations in theEDTAeFe treated plants relative to the NPeFe treated plants, andthe similar internal Fe concentrations between the NoeFe and NPeFe treated plants, confirms that A. thaliana was able to take upEDTAeFe but not NPeFe. Due to these results suggesting that NPswere not bioavailable, microscopic approaches were not used forvisualization of where NPs may have traveled within the plant(Corredor et al., 2009).

The results above are somewhat surprising given the multiplestrategies A. thaliana can use to facilitate Fe uptake. Non-graminaceous plants, such as A. thaliana, release protons into thesoil to increase Fe solubility and through a reduction strategyconvert Fe3þ to Fe2þ via membrane-bound ferric reductase oxidaseenzymes (Jeong and Guerinot, 2009). In times of Fe deficiency,A. thaliana plants have also been shown to increase activity ofproteins for Fe-regulated transporters, to manage intraplant Feallocation, and to stimulate expression of enzymes that acidify theenvironment to mobilize Fe (Puig et al., 2007; Varotto et al., 2002;Vert et al., 2002). The multiple pathways for Fe acquisition did notappear to be successful for A. thalianawhen the Fe was provided innanoparticulate hematite form. These strategies are plausibleregardless of whether the Fe is in bulk EDTA form, or in the sizefraction of fine particulates or nanoparticles (e.g., Wang and Dei,2003; Zhu et al., 2008; Harris and Bali, 2008; Ma et al., 2010).However, one mechanism we could not test is the possibility thatNPs enter the plant directly; the NPs in this study (>22 nm) may belarger than the openings available for particles to penetrate phys-ical barriers (Asli and Neumann, 2009). The mechanism of NPbioavailability needs to be further explored as it is possible thatA. thaliana did not recognize NP Fe, that transporter proteins werenot compatible with NP Fe, or that chelation and reduction strat-egies did not affect NP Fe. Since NPs may induce toxicity or otherinhibitory plant responses (Navarro et al., 2008), the failure toobtain NP-derived metals may be an indicator of other plantsurvival adaptations to prevent unintentional internal NP exposure(Ma et al., 2010). Aside from agar media, the fate of NP Fe and typeof plant response may be different if the experiment is carried outunder other conditions, such as the soil environment or in liquidmedia (e.g., Gangopadhyay et al., 2009).

3.4. Transgenic A. thaliana growth

Wild-type A. thaliana growth over time was affected differentlyby the Fe treatments (Table 1; treatment*time, P < 0.001; time,P < 0.001; treatment, P < 0.001). Due to the significant interactioneffect of treatment and time, the main effect of each Fe treatmentwas analyzed separately at each level of the variable time andinterpreted using SPSS profile plots (Tybout et al., 2001). Specifi-cally, growth was lower in both the NPeFe and NoeFe treatmentscompared to EDTAeFe during days 12, 13, and 14 (Fig. 6). Incontrast, the Type I and Type II plants exhibited different patternsover time.

The Fe concentration in the seeds of the three plant types didnot differ, while the Fe concentration in the three plant types at day14 was lower in the NPeFe and NoeFe treatments compared to theEDTAeFe treatment. NPeFe treatments had the same effect as NoeFe treatments on plant growth in all plant types. The Type II plantshowed a growth response pattern similar to that of the wild-type(treatment*time, P < 0.001; time, P < 0.001; treatment, P < 0.001).Interestingly, the Type I plant also exhibited a significant interac-tion between treatment and time (treatment*time, P< 0.001; time,P < 0.001; treatment, P ¼ 0.795). In Type I plants, however, these

Table 1Significance values (P) for the overall ANOVA model at each day and multiple comparisons of treatment at each day analyzed separately for plant type.

Plant type Treatment comparison Growth time (days)

3 4 5 6 7 8 9 11 12 13 14

Wild-type ANOVA **<0.01 *0.05 0.15 0.21 0.10 **0.01 **<0.01 *0.04 **<0.01 **<0.01 **<0.01NPeFe vs. NoeFe **<0.01 0.36 0.20 0.73 0.86 *0.02 **<0.01 0.98 0.80 0.19 0.77

NPeFe vs. EDTAeFe 0.15 *0.04 0.21 0.18 0.27 *0.03 **<0.01 *0.05 **<0.01 **<0.01 **<0.01NoeFe vs. EDTAeFe **<0.01 0.51 1.00 0.54 0.10 1.00 0.87 0.07 **<0.01 **<0.01 **<0.01

ANOVA 0.33 0.62 0.23 *0.05 0.33 **<0.01 **<0.01 0.35 **<0.01 0.19 0.91Type I NPeFe vs. NoeFe 0.91 1.00 0.75 0.99 0.97 0.88 1.00 0.64 0.88 0.68 0.99

NPeFe vs. EDTAeFe 0.32 0.67 0.59 0.07 0.35 **<0.01 **<0.01 0.82 **<0.01 0.16 0.96NoeFe vs. EDTAeFe 0.55 0.67 0.20 0.09 0.47 **<0.01 **<0.01 0.34 **<0.01 0.51 0.90

ANOVA 0.20 0.06 0.14 0.63 0.68 0.28 0.07 0.17 **<0.01 **<0.01 **<0.01Type II NPeFe vs. NoeFe 0.19 0.76 0.66 0.90 0.66 0.47 *0.05 0.91 0.95 0.15 0.39

NPeFe vs. EDTAeFe 0.86 0.25 0.52 0.60 0.85 0.90 0.68 0.16 **<0.01 **<0.01 **<0.01NoeFe vs. EDTAeFe 0.44 0.06 0.12 0.85 0.94 0.30 0.43 0.30 **<0.01 *0.04 **<0.01

* Significance at a ¼ 0.05. ** Significance at a ¼ 0.01.

Y. Marusenko et al. / Environmental Pollution 174 (2013) 150e156 155

interaction effects revealed that the NPeFe, NoeFe, and EDTAeFetreatments were not different during days 13 and 14 (Fig. 6). Thephysiological advantages of Type I plants allow growth during Fedeficiency and in the presence of Fe NPs. Furthermore, growth inboth the NPeFe (plant*time, P < 0.001; time, P < 0.001; plant,P ¼ 0.08) and the NoeFe (plant*time, P < 0.001; time, P < 0.001;plant, P ¼ 0.07) treatments was higher for Type I plants comparedto both Type II and wild-type plants. In contrast, growth in EDTA-Fetreatment (plant*time, P ¼ 0.19; time, P < 0.001; plant, P < 0.001)was higher for wild-type plants compared to both Type II (TukeyHSD: P < 0.001) and Type I (Tukey HSD: P < 0.001) plants over thecourse of the experiment.

The gene for the AVP1 protein has been engineered to improveplant growth responses under environmental Fe limitation and ithas been successfully expressed in plants with similar functions asType I A. thaliana (specifically, tomato and rice; Gaxiola et al., 2001,

Fig. 6. Growth stage of A. thaliana plants as a function of time (NPeFe: ,; NoeFe: C;EDTA-Fe: A); the left-hand panel is the wild-type, the right-hand panel is thetransgenic Type I. Error bars represent standard error (natural variability) for 10replicate plants. Vertical dashed lines are drawn where Fe treatment effects areobserved in wild-type but not in Type I plant.

2011). Our results indicate the Type I transgenic plants grow betterthan the wild-type in the absence of EDTA-Fe, but that NPeFe wasnot bioavailable to any Arabidopsis plant type in our experiments.

4. Conclusions

In summary, A. thaliana plants grownwith nanoparticulate (NP)hematite exhibited visible signs of Fe deficiency. Plants grownwithNPeFe did not grow as large, contained less chlorophyll, and hadlower internal Fe concentrations than plants grown with EDTA-bound Fe. These patterns suggest that Fe acquisition strategies inA. thaliana were ineffective in obtaining Fe in a nanoparticulateform, particularly for the size and size distribution of NPs used inthis study (mean diameter 40.9 nm, range 22.3e67.0 nm). The sameconclusion is drawn for transgenic A. thaliana, even for Type I plantsthat grew the same amount regardless of Fe treatment (NPeFe,EDTAeFe, or NoeFe). Further research could determine why someplants (Zhu et al., 2008) are more effective than others in theirability to utilize nanoparticulate metals and it remains unclear whythese NPs were resistant to the iron acquisition strategies inA. thaliana. Future work could also investigate whether the size ofNPs or the addition of ligands can have an effect on theirbioavailability, whichmay be dependent upon the type of plant andtype of NP under investigation. The aim of this research was toinvestigate the bioavailability of NPs in the environment; ourresults show that hematite NPs were not a bioavailable source of Feto wild-type or transgenic A. thaliana.

Acknowledgments

We gratefully acknowledge the use of the facilities within theLeRoy Eyring Center for Solid State Science and the W. M. KeckFoundation Laboratory for Environmental Biogeochemistry at Ari-zona State University. We thank Dr. Roberto Gaxiola for providingA. thaliana seeds and for his guidance about A. thaliana, Karl Weissfor assistance with TEM analysis, and Kiril Hristovski and PaulWesterhoff for assistance with the DLS analysis. We also acknowl-edge Chenge Weng for help with NP synthesis.

This project was completed as part of a graduate seminar class inGeo/Environmental Chemistry attended by graduate students andfaculty with broad and diverse backgrounds. The aim was todevelop an environmental chemistry project that incorporated theuse of facilities and resources available at Arizona State University.The completion of this project was dependent on interdisciplinarycollaboration among chemistry, geology and biology students. Wethank all of the students from the Environmental Geochemistry 501

Y. Marusenko et al. / Environmental Pollution 174 (2013) 150e156156

seminar who were involved in the experiments and contributedideas for this project from Fall 2007-Spring 2011.

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