Synchrotron Verification of TiO2 Accumulation in Cucumber Fruit: A

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Synchrotron verification of TiO2 accumulation in cucumber fruit: A possible pathway of TiO2 nanoparticle transfer from soil into the food chain Alia Servin, Maria I Morales, Hiram Castillo-Michel, Jose Hernandez-Viezcas, Berenice Munoz, Lijuan Zhao, Jose E. Nunez, Jose R Peralta-Videa, and Jorge L Gardea-Torresdey Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es403368j • Publication Date (Web): 16 Sep 2013 Downloaded from http://pubs.acs.org on September 22, 2013

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Environmental Science & Technology

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Synchrotron verification of TiO2 accumulation in cucumber fruit: A possible

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pathway of TiO2 nanoparticle transfer from soil into the food chain

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Alia D. Servin†, ‡,§, Maria Isabel Morales†,‡, Hiram Castillo-Michel£, Jose Angel

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Hernandez-Viezcas†,§, Berenice Munoz£, Lijuan Zhao†, Jose E. Nunez†, Jose R. Peralta-

6

Videa†,‡,§, Jorge L. Gardea-Torresdey†,¶,‡,§*

7 †

8

Department of Chemistry, The University of Texas at El Paso, 500 W. University Avenue, El Paso Texas 79968, United States

9 ¶

Environmental Science and Engineering PhD Program, The University of Texas at El

10

Paso, 500 W. University Avenue, El Paso Texas 79968, United States

11 12

£

Environmental Science program, The University of Texas at El Paso, 500 W. University Avenue, El Paso Texas 79968, United States

13 ‡

14

USDA Center for Education and Training in Agriculture and Related Sciences

15

(CETARS), The University of Texas at El Paso, El Paso,

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Texas 79968, United States §

17

UC Center for Environmental Implications of Nanotechnology (UC CEIN), United States

18 19

£

European Synchrotron Radiation Facility, B.P.220 - 38043 Grenoble, Cedex, France

20 21

*Corresponding author (J.L Gardea-Torresdey) [email protected]; phone (915) 747-

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5359, fax (915) 747-5748

23

ACS Paragon Plus Environment


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Abstract

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The transfer of nanoparticles (NPs) into the food chain through edible plants is of great

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concern. Cucumis sativus L. is a freshly consumed garden vegetable that could be in

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contact with NPs through biosolids and direct agrichemical application. In this research,

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cucumber plants were cultivated for 150 days in sandy loam soil treated with 0 to 750 mg

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TiO2 NPs kg−1. Fruits were analyzed using synchrotron µ-XRF and µ-XANES, ICP-OES,

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and biochemical assays. Results showed that catalase in leaves increased (U mg-1 protein)

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from 58.8 in control to 78.8 in 750 mg kg-1 treatment; while ascorbate peroxidase

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decreased from 21.9 to 14.1 in 500 mg kg-1 treatment. Moreover, total chlorophyll

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content in leaves increased in the 750 mg kg-1 treatment. Compared to control, FTIR

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spectra of fruit from TiO2 NP treated plants showed significant differences (p ≤ 0.05) in

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band areas of amide, lignin, and carbohydrates suggesting macromolecule modification

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of cucumber fruit. In addition, compared with control, plants treated with 500 mg kg-1

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had 35% more potassium and 34% more phosphorous. For the first time, µ-XRF and µ-

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XANES showed root-to-fruit translocation of TiO2 in cucumber without

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biotransformation. This suggests TiO2 could be introduced into the food chain with

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unknown consequences.

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Keywords: TiO2 NPs, µ-XRF, µ-XANES, Translocation, Cucumber fruit.

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TOC art

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Introduction

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Multiple applications of nanotechnology have generated a high demand for

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engineered nanoparticles (NPs). It is estimated that by 2020, the worldwide value of

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products for nanotechnologies will reach US $3 trillion.1 Recent reports indicate that the

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most widely used metal oxide nanoparticle is titanium dioxide (TiO2) with up to 10,000 t

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per year of global production.2 Approximately 50-80% of the total production of TiO2

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NPs is used in the cosmetic and sunscreen industries.3,4 Among other uses, TiO2 NPs

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have been used in coatings, plastics, paints, cement,4 and as a photo-catalyst.5 As a result

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of the high production and uses of TiO2 NPs, environmental release is inevitable.

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Previous reports have shown that TiO2 NPs can be correlated with major

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toxicological problems.6 For example, In vitro experiments conducted on human lung

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cells, have shown that TiO2 NPs in their anatase crystalline phase inhibited cell growth,

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induced oxidative stress,7 increased intracellular cytokine contents,8 and caused cell death

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by an intrinsic apoptotic pathway.9 In vitro studies on nervous cells reported induction of

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apoptosis in embryonic striatum cells, as well as the internalization of TiO2 in BV2 and

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N27 cytoplasm cells;10 while necrotic and apoptotic cells were observed in studies with

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white human blood cells.11 Other studies have shown that TiO2 NPs also can affect

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bacteria and plant species. For example, studies in Escherichia coli showed that TiO2 NPs

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induced significant oxidative stress, resulting in DNA damage and cell death.12 In

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addition, TiO2 NPs increased the level of malondialdehyde, an indicator of lipid

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peroxidation in Allium cepa and Nicotiana tabacum, suggesting DNA damage could

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ocurr.13 In contrast, other reports have indicated minimal toxicity of TiO2 NPs in plants.

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For example, studies in Arabidopsis thaliana roots showed relatively weak impact on


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genes involved in responses to TiO2 NPs exposure.14 Previous studies have used micro X- ray fluorescence (µ-XRF) and micro X-ray

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absorption spectroscopy (µ-XAS) analyses to study the accumulation and translocation of

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NPs in plants. Larue et al.,15 reported that TiO2 NPs were accumulated in roots and

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shoots in hydroponically grown wheat (Triticum aestivum). Similarly, in a previous

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study, we demonstrated that cucumber (Cucumis sativus) plants take up and transport

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TiO2 NPs from the roots to leaf trichomes.16 However, there are no previous studies on

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the translocation of TiO2 NPs into the cucumber fruit and the effects of these NPs on the

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cucumber quality. Cucumber consumption in the United States has increased since the

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1960’s, reaching 10.3 pounds per capita; 60% consumed as fresh produce and 40% as

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pickled products.17 If these NPs reach the cucumber fruit, they could be introduced into

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the food chain with unknown consequences.

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In the present study, cucumber plants were grown until fruit production in soil

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treated with TiO2 NPs. The total chlorophyll content and activity of antioxidant enzymes

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catalase (CAT) and ascorbate peroxidase (APX) were measured in mature plants. At

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harvest, the fruit was analyzed by synchrotron µ-XRF and FTIR techniques in order to

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determine the possible presence of TiO2 NPs, as well as induced changes in key

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macromolecules, respectively. In addition, changes in nutrient accumulation in the fruit

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were determined by using inductively coupled plasma-optical emission spectroscopy

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(ICP-OES).

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Materials and Methods

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TiO2 NP suspensions. Semispherical TiO2 NPs (27 ± 4 nm, Evonik Degussa, Nippon



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Aerosil) were provided by the University of California Center for Environmental

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Implications of Nanotechnology (UC-CEIN). These NPs were previously characterized

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by Keller at al.18 and were found to have a surface area of 51.5 m2 g-1 and both anatase

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(82%) and rutile (18%) crystalline phases were present. For the present study, the NPs

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were suspended in Millipore water (MPW) and diluted to have 0, 250, 500 and 750 mg

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TiO2 NPs kg-1 soil. In order to avoid precipitation, the suspensions were stirred for 5 min,

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and sonicated in a water bath before mixing with the soil (Crest Ultrasonics, Trenton, NJ)

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at 10°C for 30 min (120 V, 3 Amps, 50/60 Hz).

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Soil Preparation. The soil for this experiment was collected from the top 50 cm of

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irrigated cotton field near Fabens, TX and previously characterized as sandy loam soil.19

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The soil was sieved through a 2 mm mesh prior to experimental use. After sieving,

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portions of 4000 g were put in plastic trays and mixed with the TiO2 NP suspensions.

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The soil with NPs was placed into pots of 24 cm diameter x 23 cm high. Each treatment

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(suspension) was replicated three times.

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Cucumber Growth. Cucumber seeds (Westar Seeds International, El Centro, CA) were

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stirred for 15 min in a 4% NaClO solution, followed by a rinsing with sterilized MPW.

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Then, seeds were stirred for one hour in sterilized MPW. Subsequently, triplicate sets of

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ten cucumber seeds per pot (120 plants per experiment) were planted at 2cm depth from

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the surface and placed into the growth chamber (Environmental Growth Chamber,

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Chagrin Falls, OH) at a controlled temperature of 25/20°C day/night, 65% relative

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humidity, light intensity of 340 µmole m-2s-1, and a 14 h photoperiod. After one month of


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growth under the TiO2 NP treatments, plants were thinned to have only five plants per

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pot. Control and TiO2 NP treated plants were watered with MPW every day and no

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fertilizer was added. The experiment lasted 150 days. Hand-pollination was accomplished

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by transferring pollen with an artist brush.

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Chlorophyll Content. To investigate the impact of TiO2 NP exposure on chlorophyll

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production in 120-day old plants, five leaves per replicate were analyzed using the

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Minolta Chlorophyll Meter SPAD- 502 (Minolta, Japan) as previously described in

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literature. 20

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CAT/APX assays. To determine cucumber stress response to TiO2 NP exposure, the

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activity of catalase and ascorbate-peroxidase were determined. Control and TiO2 NP

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treated plants were sampled prior to fruit formation (120 days after exposure to TiO2 NP

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treatments). Fresh true leaves were washed with 0.01 M HNO3 solution followed by a

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rinse with MPW in order to remove leaf surface adhering NPs. Extracts of leaves of three

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plants per pot were used to determine the activity of CAT and APX, as it has been

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previously described in literature, 21 with minimal modifications.22 A ratio of 10% w/v of

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cucumber leaves was homogenized with 25 mM monopotassium phosphate buffer

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(KH2PO4) at a pH of 7.4. The extracts were centrifuged for 5 min at a temperature of - 4

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°C at 10,000 rpm (Eppendorf AG bench centrifuge 5417 R, Hamburg, Germany).

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For the CAT assay, 50 µL of the sample were mixed with 950 µL of 10 mM H2O2 in a

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quartz cuvette, having a final volume of 1 mL. The absorbance at 240 nm was recorded

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for 3 min in a Perkin Elmer Lambda 14 UV/Vis Spectrometer (single-beam mode,



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Perkin-Elmer, Uberlinger, Germany). For the APX assay, the activity was evaluated

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according to Murguia et al.23 with minor modifications.22 A volume of 100 µL of the

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sample was placed in a quartz cuvette and mixed with 886 µL of 0.1 M KH2PO4 buffer at

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pH 7.4, 4 µL of a 25 mM ascorbate solution, and 10 µL of 17 mM H2O2. The absorbance

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was recorded at 265 nm for 3 min in a Perkin Elmer Lambda 14 UV/Vis Spectrometer.

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The linear slope at 3 minutes recording was used to calculate the specific activity for the

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H2O2 decomposition, by using an extinction coefficient of H2O2 and corrected by protein

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content determined by Bradford assay.

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Elemental Analysis. For macro and microelements quantitation, triplicates samples per

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treatment of cucumber fruit were washed with 0.01 M HNO3 and MPW to remove

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surface adhering NPs. Subsequently, cucumber fruit were dried at 60 °C for 48 h. The

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dried samples were microwave-assisted acid digested using a CEM microwave (CEM

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Marsx, Mathews, NC) following the US-EPA 3051 method (1200 W, 5 min ramping to

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175 for 10 min at a pressure of 350 PSI). Samples were digested with 3 mL plasma pure

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HNO3 (SCP Science, New York) and the digest were adjusted to a volume of 30 mL

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using MPW. Concentrations of macro and micronutrients (Ca, Mg, K, P, Fe, Mn, Na, Cu,

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Zn, B, and Ni) in cucumber fruit were determined using ICP-OES (Perkin-Elmer Optima

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4300 DV). Ti concentrations were not determined in plant samples due to difficulties to

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fulfill the requirements for the digestion of TiO2 NPs. A certified standard reference

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material (NIST-SRF 1570A, Metuchen, NJ) was used for calibration and quality

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assurance/quality control. In addition, an external certified standard of each element was

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used after every 10 samples to monitor the matrix effect on the analytes.


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FTIR Analysis. Three week-old cucumber fruit harvested from plants cultivated for 150

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days in soil treated with the TiO2 NPs were lyophilized for 48 h, as it has been previously

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reported in literature.24 The dried cucumber fruits were grinded and placed on a sample

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holder plate. Then, samples were analyzed in a FT-IR Spectroscopy (Perkin Elmer,

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Spectrum 100, Universal ATR Sampling Accessory) with a range of 650-3950 cm-1.

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Micro-XRF and Micro-XANES Data Acquisition. µ-XRF and µ-XANES were used to

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analyze three week-old cucumber fruit harvested from plants cultivated for 150 days in

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soil treated with TiO2 NPs. The fruits were frozen in liquid nitrogen and stored at -80 ºC

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until analysis. Fruit of cucumber plants exposed to 750 mg TiO2 kg-1 and control were

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sent to the European Synchrotron Radiation Facility (ESRF, Grenoble, France) under

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cryogenic conditions. Once at the beamline, portions of fruit were immersed in Tissue

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Tek resin and plunged freeze in liquid nitrogen. Samples were axially sectioned at 40 µm

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thickness using a cryomicrotome. Micro-XRF mapping of Ti K-edge was performed

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under cryogenic conditions with a 5.1 KeV incident beam at beamline ID21 of the ESRF.

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The beam was focused with the use of KB mirrors to a size of 0.300 x 0.700 µm² (VxH).

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The fluorescence signal was detected by a Si drift detector. Two photodiodes were used

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to measure the incident and transmitted beam intensities. Dwell time and distance of the

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detector were optimized for each XRF map keeping the dead time below 15%. The XRF

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data was processed using the PyMCA software.25 For micro-XANES data acquisition, the

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energy was selected using a Si111 monochromator and scanned from 4950 to 5100 eV.

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The final Ti K-edge spectra were the sum of 10-30 individual scans with 0.1 s integration

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time and 0.5 eV resolution step. Reference materials were TiO2 (100% anatase) spherical



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NPs of 4 nm nominal diameter and TiO2 (100% rutile) spherical/elongated 137 nm

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particles analysed as powder pellets in transmission and fluorescence mode.16

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Statistical Analysis. The data reported for chlorophyll content was an average of five

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replicates; the rest of the data were averages of three replicates. A one-way ANOVA, and

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Duncan test was used to determine statistical significance for enzyme assays, while

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Tukey’s HSD test was used for the remainder analysis. The significance was determined

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with p ≤ 0.05.

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Results and Discussion

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Chlorophyll Content. The chlorophyll content in leaves of 120 day-old cucumber plants

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was determined using a SPAD- 502 chlorophyll meter (Figure 1). In the TiO2 NP treated

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plants, the chlorophyll content in leaves increased as the external concentration of NPs

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increased. The highest chlorophyll content was recorded in leaves of plants treated with

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750 mg TiO2 kg-1. This concentration was significantly higher compared with control

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leaves (p≤ 0.05). Chen et al.26 reported that TiO2 anatase-rutile NPs (21 nm particle size)

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increased the contents of chlorophyll b on a unicellular green alga Chlamydomonas

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reinhardtii.26 Other reports indicate that TiO2 NPs can affect chlorophyll content of

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diverse plant species. For example, Sadiq et al.27 reported the reduction of total

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chlorophyll content in algal species as the concentration of TiO2-anatase (25 nm particle

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size) NPs increased. Song et al. 28 reported no differences in chlorophyll content in

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tomato upon exposure to 1000 and 5000 mg TiO2 NPs/L (anatase: rutile 80:20, 27 nm

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particle size). The effect of TiO2 NPs on pigments is still an open question.


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Catalase and Ascorbate Peroxidase Activity. The effects of TiO2 NPs on CAT and

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APX specific activities are shown in Figure 2. As seen in this figure, compared to control,

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all TiO2 NP treatments increased CAT activity in leaves (Figure 2A). This suggests that

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TiO2 exposure increased generation of H2O2 and, consequently, a higher activity of CAT

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was observed. Previous studies have suggested that TiO2 NPs (rutile) increase the activity

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of CAT in plants; a response to protect chloroplast membranes from ROS.29 Recent

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studies have also reported an increase in CAT activity in spinach chloroplasts treated

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with TiO2-anatase NPs.30 As it can be seen in Figure 2B, compared to control, APX

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activity in leaves decreased at 500 and 750 mg TiO2 NPs kg-1. These results suggest that

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TiO2 NPs produce stress in cucumber plants; since CAT was increased at all TiO2 NP

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concentrations and APX decreased at high TiO2 NP concentrations.

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Effect of TiO2 in macro and micro-elements accumulation in cucumber fruit. In the

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present study, macro and micronutrients (Ca, Mg, K, P, Fe, Mn, Na, Cu, Zn, B, and Ni)

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were determined in three week-old cucumber fruit harvested from plants treated with 0-

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750 mg TiO2 NPs kg-1 (Table 1). As seen in this table, relative to control, fruit from

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plants treated with 500 mg TiO2 kg-1 had significantly more K and P, compared to the

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other treatments. At this time we cannot explain why only the uptake of these two

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elements was increased. However, previous studies have shown that TiO2 NPs have

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similar effects than some plant hormones like cytokinins and gibberellins31 and

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cytokinins have been found to affect P and K uptake.32 Phosphorus is essential in plant’s

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development processes which require energy transfer, it is also found in large quantities

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in fruit and seeds.33 Previous studies have shown that in Capsicum annuum L., there was



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an increase in P uptake associated with the presence of Ti. The authors also reported an

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increase in photosynthetic activity associated with the Ti.34 Therefore, changes in nutrient

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content can impact the nutritional quality and taste of the crop.35 The role of K in plants is

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essential for many biochemical and physiological processes. For example, in controlling

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the opening/closing of stomata, the activation of enzymes such as the starch synthetase,

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and for water/nutrient transport.36 Therefore, high concentration of K can improve the

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physical quality and nutritional value of fruit.36

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FTIR Analysis. FTIR spectra of macromolecules from the cucumber fruit are displayed

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in Figure 3A, and the FTIR band areas of plants are shown in Figure 3B. As seen in this

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figure, the band area of the amide I group (U) of the cucumber fruit treated with 500 and

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750 mg TiO2 NPs kg-1 decreased compared with the control. Furthermore, the lignin I

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band area (V) of the cucumber fruit increased in plants treated with 250 and 500 mg TiO2

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NPs kg-1, compared with the control. A previous report indicates that the dissolution of

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TiO2 is very low; thus, it is assumed that the changes observed in cucumber plant were

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produced by the TiO2 NPs.37 Previous studies with mangosteen fruit (Garcinia

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mangostana L.) have shown correlation of an increase in lignin with impact damage.38 As

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it can be observed in Figure 3A, there is a reduction in the carbohydrate band areas (X) of

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fruit from plants treated with TiO2. The peak height of the carbohydrate group decreased

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as the concentration of TiO2 NPs increased. The decrease in area of the affected groups is

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likely due to partial decomposition of these groups, which in turn causes environmental

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changes in the area for the amide I (U), lignin I (V), and carbohydrate groups (X), which

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are not chemically affected. The apparent increase and decrease of the area of these


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groups (U, V, and X) upon changes in NP concentrations suggests an environmental

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change around these groups, as a result of chemical changes within the affected

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functionalities (S, T, W, Y, and Z). Changes of the area corresponding to the aromatic

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region of the lignins (V) and those of the amide groups (U) may also be a result of

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coordination interactions of the amide’s carbonyl and nitrogen groups with the TiO2

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nanoparticles, which induce a change in the chemical environment of the plant. These

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types of metal-lignin complexes have been previously reported, and are well known to

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change the chemical environment of the plant. Morales et al.21 reported that at 125 and

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500 mg kg-1 CeO2 NPs changed the chemical environment of carbohydrates in cilantro

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(Coriandrum sativum L.) shoots, suggesting a change in the nutritional properties of

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cilantro. The results of the present study indicate that TiO2 NPs have an effect in the

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chemical environment of macromolecules in cucumber fruit, which could alter nutritional

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value and quality.

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Micro-XRF and micro-XANES Analysis. Figure 4A shows the tricolor micro-XRF

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map of the cucumber fruit cross-section treated with 750 mg TiO2 NPs kg-1 soil. The red

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color denotes titanium (Ti), blue calcium (Ca), and green potassium (K). The micro-XRF

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image corroborates the presence of Ti inside the cucumber fruit. Moreover, the

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temperature map in Figure 4B shows the distribution of Ti within the cucumber fruit, in

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which the color scale units represent the Ti intensity regions. This indicates that the TiO2

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NP treated plants absorbed the Ti from soil through the roots and translocated them to the

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edible portion. The micro-XANES technique was used to determine the chemical form of

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Ti within the fruit. Figure 4C displays the micro-XANES spectra of reference materials



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(1 and 2) and selected spots (3 and 4) where Ti was located in the cucumber fruit. TiO2

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anatase and rutile were used as reference materials for the present study. As one can see

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in Figure 4C, spectrum three acquired from Ti in cucumber fruit resembles the reference

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material TiO2 rutile (spectrum two). Moreover, spectrum four resembles the reference

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material anatase (spectrum one). This indicates the cucumber plants take up both anatase

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and rutile crystalline phases from the soil and transport them through the tissues without

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crystal phase modification. In previous investigation, we reported the translocation of

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TiO2 NPs from root to the cucumber shoot.16 We reported that TiO2 rutile was mainly

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found in the central vein and trichomes of cucumber leaf, whereas TiO2 anatase

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crystalline phase was mainly observed in the root of cucumber plants. The linear

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combination of spectra obtained from cucumber leaf and trichomes, showed a higher

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percentage resembling the rutile crystalline phase than the anatase form (70:30, rutile:

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anatase in leaf; 85:15, rutile: anatase in trichomes).16 The results of the present study

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corroborate that both crystalline phases are transported from the roots to the cucumber

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fruit. These results agree with those reported by Larue et al.15 who found TiO2 NPs in the

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roots and other vegetative parts of wheat plants without any biotransformation or crystal

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phase modification.

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In summary, the ICP-OES analysis showed that cucumber fruit from plants treated with

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500 mg TiO2 kg-1 NPs have a higher content of primary macronutrients P and K.

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Moreover, FTIR analysis demonstrated that at all concentrations (250-750 mg kg-1) TiO2

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NPs could modify the chemical environment of macromolecules in cucumber fruit. The

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FTIR analyses suggest significant chemical changes in lipids, amide, lignin and

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carbohydrates. However, a decrease in nutritional value would be directly correlated to


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the extent of decomposition of the macromolecules, which is not quantified by these IR

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experiments. Furthermore, the present study suggests that at 750 mg TiO2 NPs kg-1

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exposure results in an increase of chlorophyll content. In addition, all TiO2 NP

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concentrations increased catalase activity cucumber leaves. Results from synchrotron

306

techniques have shown that TiO2 was translocated without biotransformation or

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crystalline phase modification to the edible part of cucumber plants, suggesting that TiO2

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NPs could be introduced into the food chain with unknown consequences.

309 310

Acknowledgments

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The authors acknowledge the USDA grant number 2011-38422- 30835 and the NSF

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Grant # CHE-0840525. This material is based upon work supported by the National

313

Science Foundation and the Environmental Protection Agency under Cooperative

314

Agreement Number DBI-0830117. Any opinions, findings, and conclusions or

315

recommendations expressed in this material are those of the author(s) and do not

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necessarily reflect the views of the National Science Foundation or the Environmental

317

Protection Agency. This work has not been subjected to EPA review and no official

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endorsement should be inferred. J. L. Gardea-Torresdey acknowledges the Dudley family

319

for the Endowed Research Professorship, the STARs programs of the University of Texas

320

System, and the Academy of Applied Science/US Army Research Office, Research and

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Engineering Apprenticeship program (REAP) at UTEP, grant # W11NF-10-2-0076, sub-

322

grant 13-7. A. D. Servin acknowledges the GK-12 program (NSF Grant # 0947992).

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These X-ray spectromicroscopy experiments were performed at the ID21 beamline in the

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European Synchrotron Radiation Facility (ESRF), Grenoble, France.



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Table 1. Phosphorus and potassium concentration in cucumber fruit of plants treated for

456

150 days in sandy loam soil treated with 0 to 750 mg TiO2 NPs kg-1. Data represent

457

means of three replicates ± SE (standard error). Different letters indicate statistically

458

significant difference between treatments at p ≤ 0.05.

mg kg-1 d wt biomass

TiO2 NPs in soil (mg kg-1)

Phosphorus

Potassium

0

6.0 ± 0.3 b

22.4 ± 3.6 b

250

5.4 ± 0.3 b

21.0 ± 1.0 b

500

9.1 ± 0.4 a

34.6 ± 1.8 a

750

4.9 ± 0.1 b

18.4 ± 1.9 b

459 460 461 462 463 464 465 466 467 468 469 470 471


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23 472

Figure Legends

473 474

Figure 1. Total chlorophyll content of cucumber leaves treated for 120 days in inorganic

475

soil with TiO2 NPs up to 750 mg kg-1. Plants were set in the growth chamber at a

476

temperature of 25/20°C day/night, 65% relative humidity, 14 hour photoperiod and 340

477

µmole m-2s-1. Lowercase letters represent statistically significant differences between

478

treatments. Error bars stand for standard error (α ≤ 0.05).

479

Figure 2. Activity of catalase (A) and ascorbate peroxidase (B) in leaves from cucumber

480

plants treated for 120 days in inorganic soil with 0-750 mg TiO2 NPs kg-1. Data are

481

means of five replicates ±SE (standard error). Lowercase letters among columns indicate

482

statistically significant differences between TiO2 NPs treatments. Error bars stand for

483

standard error (α ≤ 0.05).

484

Figure 3. (A) FTIR spectra of cucumber fruit treated with 0-750 mg TiO2 NPs kg-1. Data

485

are means of three replicates. Uppercase letters above spectra indicate the band area from

486

each frequency range, S and T represent lipids, U and W represent amide, V, Y, and Z

487

represent lignin band area. (B) FTIR absorption band frequencies of functional group

488

components in plants.39

489 490

Figure 4. (A) Tricolor micro-XRF images of the cross sections of cucumber fruit treated

491

with 750 mg TiO2 NPs kg-1, red color stands for titanium, green for potassium, and blue

492

for calcium. (B) Ti temperature map, color scale units are counts per second. (C) Micro-

493

XANES spectra of reference materials (1-2) and spots of interest (3-4) from Figure 4A.



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24

Figure 1.


Page 25 of 27


25

Figure 2.



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26

A

B S

Frequency range (cm-1) 2840-2960

T

1720-1740

Lipids II

U V W X Y

1650 1635 1550 900-1200 1515

Amide I Lignin I Amide II Carbohydrate Lignin II

Z

845

Lignin III

Band

Assignment

Type

Lipids I

C-H Symmetric/asymmetric stretch C=O stretching of carboxylic/phenolic ester Amide: C=O and C-N stretch Aromatic C=C stretch N-H deformation and C-N stretch Carbohydrate fingerprint region C=C phenolic stretch Aromatic C-H wag of aromatic ring associated with lignin

Figure 3.


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27

Figure 4.


Synchrotron Verification of TiO2 Accumulation in Cucumber Fruit: A

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