Cerium Oxide Nanoparticles and Bulk Cerium Oxide Leading to

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Cerium Oxide Nanoparticles and Bulk Cerium Oxide Lead to Different Physiological and Biochemical Responses in Brassica rapa Xingmao Ma, Qiang Wang, Lorenzo Rossi, and Weilan Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b04111 • Publication Date (Web): 21 Dec 2015 Downloaded from http://pubs.acs.org on December 27, 2015

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Environmental Science & Technology

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Cerium Oxide Nanoparticles and Bulk Cerium Oxide Lead to Different Physiological and

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Biochemical Responses in Brassica rapa

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Xingmao Ma1*, Qiang Wang2, Lorenzo Rossi1, Weilan Zhang1

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Station, TX, 77843-3136, USA

Zachry Department of Civil Engineering, Texas A&M University, 3136 TAMU, College

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IL, 62901, USA

Department of Civil and Environmental Engineering, Southern Illinois University Carbondale,

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*Corresponding author

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Xingmao Ma

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Zachry Department of Civil Engineering

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Texas A&M University

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3136 TAMU College Station, TX, USA, 77843

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Email: [email protected]

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Ph: 979-862-1772

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Fax: 979-862-1542

ACS Paragon Plus Environment

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Abstract

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Cerium oxide nanoparticles (CeO2NPs) have been incorporated into many commercial

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products and their potential release into the environment through the use and disposal of these

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products has caused serious concerns. Despite the previous efforts and rapid progress on

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elucidating the environmental impact of CeO2NPs, the long term impact of CeO2NPs to plants, a

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key component of the ecosystem, is still not well understood. The potentially different impact of

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CeO2NPs and their bulk counterparts to plants is also unclear. The main objectives of this study

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were (1) to investigate whether continued irrigation with solutions containing different

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concentrations of CeO2NPs (0, 10 and 100 mg/L) would induce physiological and biochemical

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adjustments in Brassica rapa in soil growing conditions and (2) to determine whether CeO2NPs

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and bulk CeO2 particles exert different impacts on plants. The results indicated that bulk CeO2 at

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10 and 100 mg/L enhanced plant biomass by 28% and 35% respectively while CeO2NPs at

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equivalent concentrations did not. While the bulk CeO2 treatment resulted in significantly higher

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hydrogen peroxide (H2O2) in plant tissues at the vegetative stage, CeO2NPs led to significantly

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higher H2O2 in plant tissues at the floral stage. The activity of superoxide dismutase (SOD) in

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Brassica rapa also displayed a growth-stage dependent response to different sizes of CeO2 while

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catalyse (CAT) activity was not affected by either size of CeO2 throughout the life cycle of

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Brassica rapa. Altogether, the results demonstrated that plant responses to CeO2 exposure varied

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with the particle sizes and the growth stages of plants.

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Keywords

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Oxidative Stress, Cerium Oxide Nanoparticles, Bulk Particle, Brassica rapa

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TOC Art

CeO2NPs or Bulk CeO2

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Introduction

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Rapid increase in the manufacturing and use of engineered nanoparticles (ENPs) is

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expected to result in significant accumulation of ENPs in the environment 1-5. With an estimated

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annual global production of 100-1000 tons/yr 6, cerium oxide nanoparticles (CeO2NPs) were

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shown to affect plant growth and cause oxidative stress in plants under different exposure

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conditions. For example, Lopez-Moreno et al.

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tomato was significantly reduced by CeO2NPs (7 nm) at 4000 mg/L. Ma et al. 8 also reported that

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exposure to 2000 mg/L of CeO2NPs of the same size for five days inhibited the root elongation

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of lettuce. Both studies were conducted hydroponically. Zhao et al.

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CeO2NPs at 400 and 800 mg/kg dry soil for ten days led to significant increases in H2O2 content,

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as well as significantly higher catalase (CAT) and ascorbate peroxidase (APX) activities in corn.

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In a study with Arabidopsis seedlings in MS (Murashige and Skoog) media, CeO2NPs (10-30

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nm) were shown to significantly reduce plant growth and chlorophyll content and increase root

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membrane lipid peroxidation at concentrations greater than 1000 mg/L

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demonstrated that 1000-2000 mg/L of CeO2NPs affected the expression of some stress response

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genes in sulfur assimilation and glutathione (GSH) biosynthesis pathway. Interestingly, the same

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study showed that the growth of Arabidopsis was improved by CeO2NPs lower than 500 mg/L 10.

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Wang et al.

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showed no adverse or slightly beneficial effect on tomato growth and yield. The enhancive effect

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of CeO2NPs may be attributed to the antioxidant properties of CeO2NPs at low concentrations. A

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recent study found that 250 mg/kg of CeO2NPs increased the total antioxidant capacity of radish

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by 30% while 500 mg/kg of CeO2NPs reduced plant seed germination 12. With the recognition of

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the potential effect of CeO2NPs in plants, several long term studies concerning the physiological

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reported that root elongation of alfalfa and

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noticed that exposure to

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. The study also

also found that irrigation with CeO2NPs at concentrations lower than 10 mg/L


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and biochemical impact of CeO2NPs and their uptake and accumulation by agricultural crops

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have emerged. Zhao et al. 13 assessed the impact of CeO2NPs (8 nm, primary particle size) on the

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photosynthesis of corns grown in enriched soil and found that CeO2NPs up to 800 mg/kg did not

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affect the gas exchange or stomata conductance of corn leaves throughout the life cycle of the

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plants. However, the fully developed cobs was significantly reduced and the corn yield was

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reduced by 38%

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reproductive stage. In a field growing condition, CeO2NPs up to 400 mg/kg did not affect the

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plant yield of wheat, however, a series of physiological parameters such as the relative

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chlorophyll content of wheat leaves and biochemical parameters such as the activity of CAT

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were affected

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impact of CeO2NPs and other ENPs on plants is greatly needed. With the continued emphasis on

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particle size reduction to enhance reactivity, detailed understanding on the size effect of particles

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, suggesting that CeO2NPs might have affected corn flowering in the

. These new findings indicate that mechanistic understanding on the long term

on plants is also imperative. ENPs are designed to have higher reactivity due to a greater proportion of atoms on the 15

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surface compared with bulk particles

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properties compared with their bulk counterparts. For example, AgNPs (99.5%) for no longer than ten minutes

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until chlorophyll was completely removed

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Kruss MBL3000 light microscope (Hamburg, Germany) equipped with Kruss digital camera

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(Hamburg, Germany).

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Root Imaging with Scanning Electron Microscope/Energy Dispersive X-ray Spectroscopy

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(SEM/EDS)

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. The cleared leaves were then examined using a

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Ten-mm segments of the root tip tissue were sectioned and mounted onto a SEM stub

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with a carbon type. Imaging and X-ray analysis were performed using a FEI Quanta 450-FEG

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SEM (Eindhoven, The Netherlands) with an attached 50 mm2 Oxford detector (Oxford

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Instrument, Abingdon, England) in the low vacuum mode. Secondary electron (SE) images were

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acquired at 10 kV. EDS and backscatter analysis were operated at 30 kV.

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Statistical analysis

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Two-way ANOVA analysis with two main factors (size and concentration) followed by

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Tukey’s post-hoc test was performed. One-way ANOVA analysis followed by Tukey’s post-hoc

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test was conducted to determine the effects of CeO2NPs or bulk CeO2 on the plant growth and

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oxidative stress compared with the controls. The results are mean ± standard error (SE). All

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statistical tests were processed with SAS 9.3 for windows software package (SAS Institute Inc.,

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Cary, NC).

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Results


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Effects of CeO2NPs and bulk CeO2 on plant growth

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Shoot height was not sensitive to the treatment of CeO2NPs or bulk CeO2 and all plants

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reached similar height on day 17 at the seed ripening stage (Supplementary Figure 2). For plant

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biomass, neither particle size nor exposure concentration affected the total biomass during the

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vegetative and floral stage (Figure 1). However, the irrigation with bulk CeO2 resulted in

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significantly greater total biomass in the seed ripening stage compared with the controls (two-

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way ANOVA; p = 0.030). Treatment with 10 and 100 mg/L of bulk CeO2 increased the total

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biomass by 28% and 35% compared with the controls in the seed ripening stage (one-way

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ANOVA; p = 0.007). In contrast, the total biomass of CeO2NPs treated plants was statistically

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the same as that of the controls in the seed ripening stage.

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The relative chlorophyll content of Brassica rapa was similar between plants exposed to

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CeO2NPs and same concentrations of bulk CeO2 in the entire exposure period (two-way

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ANOVA; p > 0.05 for both NP type and concentration factors) (Table 1). There was also no

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statistical differences between treated plants and control plants in the vegetative stage. However,

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CeO2NPs at 10 and 100 mg/L significantly increased the relative chlorophyll content by 13% and

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10% compared with the controls in the floral stage (p = 0.003). Exposure to 100 mg/L of bulk

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CeO2 also significantly increased the relative chlorophyll content by 10% compared with the

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controls in the floral stage (p = 0.022). In the seed ripening stage, exposure to CeO2NPs at 10

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and 100 mg/L increased the relative chlorophyll content of plants by 13% and 16% compared

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with the controls (p = 0.004). The plants exposed to bulk CeO2 at 10 and 100 mg/L also showed

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10% and 15% higher relative chlorophyll content compared with the controls (p < 0.001).

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Estimation of SOD and CAT activities


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The impact of CeO2 on SOD activity varied with the growth stages and concentrations

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(Figure 2). Two-way ANOVA analysis suggested a significant effect of particle size on SOD

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activity during the vegetative stage (p = 0.017) (Figure 2), but the concentration was not a

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significant factor influencing SOD activity. The plants exposed to bulk CeO2 exhibited

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significantly higher SOD activity than the ones exposed to CeO2NPs or controls at the vegetative

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stage (Figure 2). During the floral and seed ripening stage, neither particle size nor concentration

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was found to be a significant factor for the SOD activity. CAT activity was not affected by either

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CeO2NPs or bulk CeO2 exposure at both test concentrations throughout the entire exposure

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period (Figure 3).

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H2O2 quantification and visualization

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Bulk CeO2 resulted in higher H2O2 content in Brassica rapa in the vegetative stage than

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CeO2NPs did (two-way ANOVA; p = 0.006) (Figure 4). Bulk CeO2 at concentrations of 10 and

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100 mg/L significantly increased the H2O2 content by 54% and 42% during the vegetative stage

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compared with the controls (one-way ANOVA; p = 0.049). In contrast, CeO2NPs at equivalent

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concentrations did not change the H2O2 content compared with the controls at this stage.

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However, CeO2NPs induced significantly higher H2O2 production compared with bulk CeO2 at

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equivalent concentrations (two-way ANOVA; p = 0.003) in the floral stage. H2O2 content in 10

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and 100 mg/L CeO2NPs treated plants was significantly increased by 30% and 52% compared

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with the controls (one-way ANOVA; p < 0.002). On the contrary, bulk CeO2 treated plants

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showed a similar level of H2O2 compared with the controls during the floral stage. In the seeds

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ripening stage. Neither CeO2NPs nor the bulk CeO2 treated plants displayed significantly

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different H2O2 concentrations compared with control plants. However, the H2O2 content of the


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plants exposed to 100 mg/L of bulk CeO2 was significantly lower than that of the plants exposed

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to 10 mg/L of bulk CeO2 or 100 mg/L of CeO2NPs (one-way ANOVA; p = 0.025).

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DAB staining illustrated different patterns of H2O2 accumulation in plants treated with

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different particle sizes of CeO2 (Supplementary Figure 3). Larger DAB stained area was

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observed on both the leaves and roots of plants treated with CeO2NPs than plants treated with

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bulk CeO2. H2O2 was spotted on both the blade and the margin of the leaves exposed to

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CeO2NPs at concentration of 100 mg/L. Stained veins were observed on the leaves under both

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CeO2NPs and bulk CeO2 treatment. Stained root epidermis, cortex cells and vascular tissues

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were observed on all plants including the control plants.

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Root SEM/EDS Analysis

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SEM/EDS analysis (Figure 5) did not detect any CeO2 particles on control root surface.

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Some deposition was observed on roots exposed to both the CeO2NPs (Figure 6A) and the bulk

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CeO2 (Figure 7A). No significant aggregation was observed on the root surface treated by

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CeO2NPs (Figure 6B). The SE image (Figure 7A) illustrated rod shaped particles on the

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epidermis of the bulk CeO2 treated roots. The size and shape of the reflections matched the

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morphology of the bulk CeO2 particles used in this study. A few large rod-shaped reflections

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with approximately 5 µm in length were also found on the bulk BSE image (Figure 7C), EDS

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analysis confirmed that the rod shaped deposition was bulk CeO2.

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Discussion

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This study investigated the impact of CeO2NPs and bulk CeO2 irrigation at

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concentrations of 10 and 100 mg/L on the plant growth and oxidative stress of the soil-cultivated

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Brassica rapa. Despite the vast information available on the toxicity of nanoparticles to plants,


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few studies have been conducted to evaluate the underlying mechanisms by which nanoparticles

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exert their effect on plant growth and development. To our knowledge, this is one of the first

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studies to elucidate the differential long term impact of CeO2NPs and bulk CeO2 particles on the

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plant growth and oxidative stress responses in soil grown plants through root exposure. A

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general observation was that indeed CeO2NPs and bulk CeO2 demonstrated different impacts on

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plant biomass development, relative chlorophyll content and other physiological and biochemical

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parameters. H2O2 quantification/visualization and the antioxidant enzymes assays illustrated that

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both sizes of CeO2 caused oxidative stress to Brassica rapa. However, the differences of the

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H2O2 concentration and antioxidant proteins activities caused by the exposure to different

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particle sizes of CeO2 suggested that CeO2 at different size scales induced oxidative stress

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through different mechanisms. One possible reason is the different concentrations of Ce3+ on the

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surface of CeO2NPs and bulk CeO2. Their different shapes as well as their different uptake and

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accumulation patterns by plants may also contribute to the different impact of bulk and nano-

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sized CeO2. Another general observation was that the response of Brassica rapa to CeO2NPs and

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bulk CeO2 varied greatly at different growth stages.

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It has been reported that the effects of CeO2 on plant biomass depend on the exposure

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conditions and plant species. One previous study in soil growth condition demonstrated that

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CeO2NPs at low concentrations (up to 130 mg/kg soil) did not exhibit any harmful effect on

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tomato plant growth throughout the life cycle of the tomato plants

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observed that 53 days of exposure to CeO2NPs dispersed in soil at 400 mg/kg notably increased

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magnesium (Mg2+) uptake of cucumber plant, the central ion of the chlorophyll pigment. Similar

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result was also reported in wheat after 94 days of exposure to CeO2NPs amended soil at 250

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mg/kg 31. Some previous studies also reported that bulk CeO2 may facilitate the uptake of Mg2+


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. Moreover, Zhao et al.

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or other central metal ions of chlorophyll molecule such as Fe2+, Cu2+, Ni2+ and Co2+

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Interestingly, significantly higher relative chlorophyll contents were observed in plants treated by

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both the CeO2NPs and the bulk CeO2 during the floral and seed ripening stages in this study.

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However, higher relative chlorophyll content was not observed in the vegetative stage in plants

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exposed to either CeO2NPs or bulk CeO2, which might be attributed to the relatively short

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exposure of plants to CeO2 at this stage. After 28 days of exposure to 1,228 mg/kg of either bulk

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CeO2 or CeO2NPs, the biomass of zucchini root, stem and leaf did not show any differences from

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control plants. However, the biomass of flowers was significantly reduced by both sizes of CeO2,

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with the CeO2NPs resulting in the smallest flower biomass 23.

.

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The significantly higher plant biomass caused by the exposure to bulk CeO2 may be

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attributed to the higher relative chlorophyll content and enhanced photosynthesis. The unique

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physiological effects of bulk and nano-sized CeO2 (e.g. weaker oxidative stress caused by bulk

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CeO2 than CeO2NPs) may account for the larger biomass observed on the plants exposed to bulk

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CeO2 than the ones exposed to CeO2NPs at equivalent concentrations in the seed ripening stage.

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Herein, the term “oxidative stress” is used to describe the “oxidative damage” to cellular

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components

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species, ROS) and antioxidative reactions occurs 34. Higher oxidative stress caused by CeO2NPs

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may inhibit the biomass production of the Brassica rapa plants at tested concentrations.

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However, the oxidative stress caused by CeO2NPs irrigation at low concentrations (i.e. 10 and

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100 mg/L) was not strong enough to result in noticeable biomass and shoot height reduction of

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Brassica rapa plants compared with the controls.

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, where a shift of the balance between prooxidative (lead by reactive oxygen

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The redox homeostasis in Brassica rapa was determined through the quantification of

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H2O2 in plant tissues. H2O2 was measured as a representative ROS because of its stability and


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sensitivity to environmental stresses. H2O2 is predominantly produced in plant cells during

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photosynthesis and photorespiration, and plays a crucial role in signaling

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finding in this study is that the levels of H2O2 in plants in response to the treatment of CeO2NPs

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and bulk CeO2 varied at different growth stages. For the bulk CeO2, we postulate that the

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deposition of particles on root surface and physical damage caused by the bulk CeO2 deposition

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or its interference in the plant root nutrient uptake were the primary reasons for the toxicity of

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bulk CeO2. Due to their heavy size, the deposition could occur must faster than CeO2NPs.

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According to the SEM images of the bulk CeO2 treated Brassica rapa root, it appeared that some

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needlelike particles penetrated into the root epidermis (Figure 7B). It is possible that the sharp

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edges of bulk CeO2 have damaged cell wall and membranes, causing a shift between

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prooxidative and antioxidative balance as confirmed in rice cells exposed to single-walled carbon

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nanotubes (SWCNT) at 20 mg/L which report significantly increased H2O2 production 36. On the

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other hand, even though CeO2NPs may also deposit on root surface, the smaller size and

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spherical shape of CeO2NPs might cause much less damage on plant root tissues or have a

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significantly smaller effect on plant nutrient uptake at short exposure. However, with the

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extension of exposure time, more CeO2NPs could be taken up by plants and CeO2NPs would

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display a stronger effect than the bulk due to their enzyme mimetic property. Future studies

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should include the quantification of Ce element in plant tissues at different growth stages. The

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observation that short term exposure to CeO2NPs generally had less effect on plants agrees with

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previous studies

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to CeO2NPs suspension at concentrations from 62.5 to 250 mg/L were either unaffected or had

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lower H2O2 production and unaffected antioxidant protein activities within 10 days of exposure.

30, 37

35

. An important

. Those studies revealed that both rice and cucumber seedlings in exposure


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To better understand the biochemical adjustment and H2O2 formation at the cellular level,

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SOD and CAT enzymatic activities, and DAB staining were performed. SOD is a metal-

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containing enzyme that catalyzes the dismutation of superoxide radicals to O2 and H2O2. SOD

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activity is also a good indicator for plant stress tolerance 38. In this study, the SOD activity was

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not altered by CeO2NPs exposure in all three growth stages, yet higher concentrations of H2O2

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were observed. The results may be attributed to the SOD mimetic activity of CeO2NPs as

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previous reported by Heckert et al.

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exposure to 2,000 mg/L CeO2NPs, Ce presented in the roots of cucumber as CeO2 and CePO4,

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while in shoots as CeO2 and cerium carboxylates. The biotransformation of Ce4+to Ce3+ has been

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reported as a cause of H2O2 production 42. Thus, it is possible that as the exposure lengthens, the

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biotransformation of Ce4+/Ce3+ significantly induced H2O2 production without significantly

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altering the SOD activity. The SOD mimetic activity observed on CeO2NPs is absent on bulk

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CeO2 due to the high concentrations of Ce4+ on the surface of bulk CeO2 39. Thus, consistent with

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the increase of H2O2 in bulk CeO2 treated plants, SOD activity of those plants was also

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increased. The results agree with a previous report that high SOD activity was associated with

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increasing H2O2 level

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concentrations in plants treated with bulk CeO2 and control plants in the seed ripening stage,

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exposure to 100 mg/L of bulk CeO2 significantly lowered the H2O2 production compared with

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plants treated with 10 mg/L of bulk CeO2. The reason for the concentration-dependent effect of

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bulk CeO2 on H2O2 concentration in the seed ripening stage is unclear. However, the SEM and

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TEM images illustrated that a small fraction of the bulk CeO2 particles was in nanoscale, which

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probably possessed similar properties as CeO2NPs. Previous studies indicated that CeO2NPs

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could function as a radical scavenger at very low concentration while induce H2O2 production at

37, 43

39

and Rico et al. 40. Zhang et al.

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found that after 21 days

. Even though no statistical differences were observed for the H2O2


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high concentrations 44. Other studies indicated that low concentrations of CeO2NPs enhanced the

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total antioxidant capacity of plants

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probably worked as a radical scavenger, but the concentration of nano-sized CeO2 particles

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provided in 10 mg/L bulk CeO2 irrigation was too low to exhibit any effect on H2O2

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concentration. Among the enzymes involved in the removal of the excess H2O2 generated

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spontaneously or by O2– dismutation via SOD, CAT and other antioxidant proteins such as APX

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and GPX play a key role. The unaffected CAT activity may suggest an increased activity of other

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antioxidant proteins or nonenzymatic antioxidants

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of both enzymatic and non-enzymatic activities in plants is needed to provide a more

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comprehensive understanding on the antioxidant activities in plants as a response to the exposure

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of bulk and nanosized CeO2 particles.

12

. It is postulated that the nanosized CeO2 in the bulk

45

. Further evaluation on a broader spectrum

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The DAB technique was commonly used to detect the presence and distribution of H2O2

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in plant cells. DAB is oxidized by H2O2 in the presence of heme- proteins, such as peroxidases,

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to generate a dark brown precipitate 46. DAB staining reveals the accumulation site of H2O2. In

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combination with the spectrophotometric measurement, it is possible that CeO2NPs might have

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been transported to the blade and leaf margins via veins

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visibly smaller and lighter stained area on the root tip and leaf of plants exposed to 100 mg/L

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bulk CeO2 than to CeO2NPs at the same concentration, consistent with the spectrophotometric

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measurement in the floral stage. The DAB analysis was not performed on seedlings at other

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growth stages due to time and resource limitations, however, it is reasonable to assume that H2O2

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distribution in plants exposed to different sizes of CeO2 particles would vary based on other

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physiological and biochemical measurements. This is the first study to report that the toxicity

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exerted by root exposure to CeO2 at different sizes varied at different growth stages in soil

9, 47

. The DAB staining results showed


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growing conditions. The findings of the differential toxicity and behavior of different sizes of

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CeO2 call for more investigation on the potential long term impact of engineered nanoparticles

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and their food chain accumulation.

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Acknowledgement

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The authors acknowledge the financial support of the USDA-AFRI (#2011-67006-30183) and

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Dr. Stephen Ebbs at Southern Illinois University Carbondale for his assistance with the

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measurement of SOD and CAT activities.

414 415

Supplemental Information Available

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The SEM and TEM images of bulk cerium oxide particles (Supplementary Figure 1) used in this

417

study; the shoot height of plants exposed to different sizes of CeO2 at three growth stages

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(Supplementary Figure 2) and DAB staining of leaves and roots from plants exposed to

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difference sizes of cerium oxide on day 12 (Supplementary Figure 3) are available. The

420

supporting information is free of charge on the ACS publications site.

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Reference 1. Schwabe, F.; Tanner, S.; Schulin, R.; Rotzetter, A.; Stark, W.; von Quadt, A.; Nowack, B., Dissolved cerium contributes to uptake of Ce in the presence of differently sized CeO2nanoparticles by three crop plants. Metallomics 2015, 7, (3), 466-77. 2. Rogers, N. J.; Franklin, N. M.; Apte, S. C.; Batley, G. E.; Angel, B. M.; Lead, J. R.; Baalousha, M., Physico-chemical behaviour and algal toxicity of nanoparticulate CeO2 in freshwater. Environmental Chemistry 2010, 7, (1), 50-60. 3. Zhang, H.; He, X.; Zhang, Z.; Zhang, P.; Li, Y.; Ma, Y.; Kuang, Y.; Zhao, Y.; Chai, Z., Nano-CeO2 exhibits adverse effects at environmental relevant concentrations. Environ Sci Technol 2011, 45, (8), 3725-30. 4. Ma, X., Geisler-Lee, J., Interactions between engineered nanoparticles (ENPs) and plants. Science of the Total Environment 2010, 408, (16), 3053-61.


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Figure 1. Biomass of Brassica rapa growing in the presence of 10 and 100 mg/L of CeO2NPs or bulk CeO2 in vegetative, floral and seed ripening growth stages (n=5). B: bulk and N: nanoparticles. Error bars represent standard error. Significant differences calculated by one-way ANOVA followed by Tukey’s post-hoc test (p < 0.05) are indicated by letters.

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Figure 2. SOD activity in Brassica rapa growing in the presence of 10 and 100 mg/L of CeO2NPs or bulk CeO2 in vegetative, floral and seed ripening growth stages (n=5). B: bulk and N: nanoparticles. Error bars represent standard error. Significant differences calculated by oneway ANOVA followed by Tukey’s post-hoc test (p < 0.05) are indicated by letters.

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Figure 3. CAT activity in Brassica rapa growing in the presence of 10 and 100 mg/L of CeO2NPs or bulk CeO2 in vegetative, floral and seed ripening growth stages (n=5). B: bulk and N: nanoparticles. Error bars represent standard error. Significant differences calculated by oneway ANOVA followed by Tukey’s post-hoc test (p < 0.05) are indicated by letters.

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Figure 4. Concentration of H2O2 in Brassica rapa treated with 10 and 100 mg/L of CeO2NPs or bulk CeO2 in vegetative, floral and seed ripening growth stages (n=5). B: bulk and N: nanoparticles. Error bars represent standard error. Significant differences calculated by one-way ANOVA followed by Tukey’s post-hoc test (p < 0.05) are indicated by letters.

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Figure 5. Secondary electron image (A), backscatter electron image (B), and EDS analysis (C) for control Brassica rapa root at floral stage (day 12). EDS analysis was performed in red rectangular area.


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Figure 6. Secondary electron image (A), backscatter electron image (B), and EDS analysis (C) for CeO2NPs at 100 mg/L treated Brassica rapa root at floral stage (day 12). EDS analysis was performed in red rectangular area. Red arrow shows Ce peak.


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Figure 7. Secondary electron image (A), backscatter electron image (B), and EDS analysis (C) for bulk CeO2 at 100 mg/L treated Brassica rapa root at floral stage (day 12). EDS analysis was performed in red rectangular area. Red arrow shows Ce peak.


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Table 1. Relative chlorophyll content in the Brassica rapa plants exposed to CeO2NPs and bulk CeO2 at 10 and 100 mg/L during the vegetative, floral and seed ripening stages. The data was reported as percentage of the control plants at each growth stage. Data shown in the Table are in means ± standard error. Letters (a and b) behind the values marked the statistical significance based on the one-way ANOVA analysis followed by Tukey’s post-hoc test (n=5). Vegetative stage Floral stage Seed ripening stage Day 7 Day 12 Day 17 Treatment a a Control 100±0.0 100±0.0 100±0.0a NP 10 101.7±0.9a 113.1±2.6b 112.5±3.4b NP 100 105.8±2.4a 110.5±1.6b 116.4±1.9b Bulk 10 101.5±1.8a 105.6±2.4ab 110.4±2.2b a b Bulk 100 102.2±1.5 110.7±2.2 115.5±1.3b

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Cerium Oxide Nanoparticles and Bulk Cerium Oxide Leading to

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