Bioaccumulation and Toxicity of 13C-Skeleton Labeled Graphene


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Article 13

Bioaccumulation and Toxicity of C-skeleton Labeled Graphene Oxide in Wheat Lingyun Chen, Chenglong Wang, Hongliang Li, Xiulong Qu, Shengtao Yang, and Xue-Ling Chang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00822 • Publication Date (Web): 03 Aug 2017 Downloaded from http://pubs.acs.org on August 8, 2017

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Environmental Science & Technology

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Revised No. es-2017-008228

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Bioaccumulation and Toxicity of 13C-skeleton

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Labeled Graphene Oxide in Wheat

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Lingyun Chen1,§, Chenglong Wang2,§, Hongliang Li1,2, Xiulong Qu1, Sheng-Tao Yang1,*, and

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Xue-Ling Chang2,*

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1

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Chengdu 610041, P. R. China;

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2

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High Energy Physics, Chinese Academy of Sciences, Beijing 100049, P. R. China.

College of Chemistry and Environment Protection Engineering, Southwest Minzu University,

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of

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§

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KEYWORDS: Graphene oxide, Bioaccumulation, Toxicity, 13C-stable isotope, Oxidative stress

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*Corresponding Author

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Sheng-Tao

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[email protected]

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Xue-Ling

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[email protected]

These two authors contributed equally.

Yang,

Chang,

Tel:

+86-28-85522269,

Fax:

+86-10-88236456,

Email

address:

Tel:

+86-10-88236709,

Fax:

+86-10-88236456,

Email

address:

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ABSTRACT: Graphene nanomaterials have many diverse applications, but are considered to be

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emerging environmental pollutants. Thus, their potential environmental risks and biosafety are

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receiving increased attention. Bioaccumulation and toxicity evaluations in plants are essential for

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biosafety assessment. In this study, 13C-stable isotope labeling of the carbon skeleton of

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graphene oxide (GO) was applied to investigate the bioaccumulation and toxicity of GO in wheat.

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Bioaccumulation of GO was accurately quantified according to the 13C/12C ratio. Wheat

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seedlings were exposed to 13C-labeled GO at 1.0 mg/mL in nutrient solution for 15 d. 13C-GO

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accumulated predominantly in the root with a content of 112 µg/g at day 15, hindered the

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development and growth of wheat plants, disrupted root structure and cellular ultrastructure, and

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promoted oxidative stress. The GO that accumulated in the root showed extremely limited

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translocation to the stem and leaves. During the experimental period, GO was excreted slowly

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from the root. GO inhibited the germination of wheat seeds at high concentrations (≥0.4 mg/mL).

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The mechanism of GO toxicity to wheat may be associated with oxidative stress induced by GO

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bioaccumulation, reflected by the changes of malondialdehyde concentration, catalase activity

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and peroxidase activity. The results demonstrate that 13C labeling is a promising method to

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investigate environmental impacts and fates of carbon nanomaterials in biological systems.

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Table of Contents Graphic

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INTRODUCTION

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Graphene and its derivatives have attracted considerable recent interest owing to their unique

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structure and remarkable physical and chemical properties.1,2 Of particular interest, graphene

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oxide (GO) is a graphene sheet with carboxylic groups at the edges and phenol hydroxyl/epoxide

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groups on the basal plane, and exhibits excellent water dispersion and amphiphilic characteristics.

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Graphene materials have diverse applications in a wide variety of fields, including electronics,3

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energy,4 mechanics,5 advanced materials,6 biomedicine,7 environmental remediation8,

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biosensing,9 and agriculture.10 For example, graphene-based touch screen for cell phones is

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produced in Chongqing, China. Graphene rechargeable batteries are manufactured in several

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countries. Theranostic applications of GO are reported, such as drug/gene delivery, biosensing,

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bioimaging and a scaffold for cell growth.7 Graphene adsorbents have been developed for the

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remediation of polluted water.8,11,12 Graphene quantum dots stimulated the growth of coriander

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and garlic plants.10 On the basis of these innovations, there is growing demand for graphene

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products, which has stimulated the large-scale production of graphene for industrial applications.

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Several production lines are operational with an annual production capacity of several hundred

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tonnes. Generally, graphene nanomaterials are readily released into the environment and may

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lead to potential nanotoxicity and environmental risks.13-15 Potential exposure to graphene

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products continues to increase and the hazards of exposure to graphene require thorough

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investigation.16–18

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Graphene nanomaterials are considered to be emerging environmental pollutants, and their

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adverse effects on biota have received increasing attention.19–23 Recent studies reveal that

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graphene induces growth inhibition, cell death, oxidative stress and morphological changes in

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plants.24 Begum et al. observed that GO induces cell death in Arabidopsis thaliana, and water-

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soluble graphene is phytotoxic to cabbage (Brassica oleracea var. capitata), tomato

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(Lycopersicon esculentum), red spinach (Amaranthus tricolor L.), and lettuce (Lactuca sativa) at

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0.5~2.0 mg/mL.25,26 GO does not influence the germination and development of A. thaliana at

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concentrations lower than 1.0 mg/L.27 Graphene inhibits the germination and growth of faba

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bean (Vicia faba L.) via induction of oxidative damage at 0.1~1.6 mg/mL.28 In tomato, graphene

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penetrates the seed coat to accelerate germination and stimulates stem and root elongation of

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seedlings, but inhibits biomass accumulation at 0.04 mg/mL.29 GO inhibits root elongation of

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Brassica napus and regulates the concentrations of abscisic acid (ABA) and indole-3-acetic acid

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(IAA) at 0.025~0.1 mg/mL.30 Under co-exposure to graphene and pollutants, GO amplifies the

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toxicity of arsenic in wheat (Triticum aestivum) at GO concentrations of 0.1~10.0 mg/L.31

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Bioaccumulation of nanomaterials in plants is essential to fully understand their biological

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behavior and ecotoxicity.32 Bioaccumulation data guide nanotoxicity evaluations on plant tissues

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and shed light on the toxicological mechanisms. The bioaccumulation of diverse nanomaterials

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in plants has been widely investigated.33-35 The exposure concentration, particle size, surface

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charge, particle dissolution, surfactant/dissolved organic matter (DOM), and plant species are

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important factors that affect the bioaccumulation and translocation of nanomaterials in plant

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tissues.36-40 Nevertheless, quantification of nanomaterials in vivo remains a challenge.

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Quantification of graphene is more difficult than that of metal-containing nanoparticles, where

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the total element present can be digested and quantified by atomic emission spectrometry or

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inductively coupled plasma–mass spectrometry. Plant tissues contain high background quantities

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of carbon that interfere with direct quantification of graphene in biological samples.41 Several

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reports have used transmission electron microscopy (TEM) and Raman spectroscopy to detect

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the presence/absence of graphene in plant tissues. For example, Zhang et al. used TEM and

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Raman spectroscopy to confirm that graphene penetrates the seed coat and seedling root tip

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cells.29 Black spots in the TEM images were assigned as GO, and the D-band and G-band signals

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were observed in the seeds according to Raman spectra. Zhao et al. used TEM to investigate the

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accumulation and translocation of GO.27 GO was recognized as black spots in root, leaf and

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cotyledon cells.

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C-labeling of the carbon skeleton is a well-established approach for quantification of

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carbon nanomaterials in biological samples by analyzing the 13C/12C ratio with isotope ratio mass

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spectrometry (IRMS).41–45 The 13C-labeling of the carbon skeleton does not damage the stability

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and intrinsic structure of carbon nanomaterials 46–48 and enables the properties of carbon

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nanomaterials to be traced in biological systems. The technique has the advantage of overcoming

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the drawbacks of radioactive labeling, such as ready detachment, generation of radioactive waste,

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and requirement for specific experimental approval.42 Herein, we prepared 13C-labeled GO for

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bioaccumulation analysis and evaluated the toxicity of GO to wheat with reference to the

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bioaccumulation data. The bioaccumulation of 13C-GO was quantified by IRMS. The effects of

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GO on germination, growth, root elongation and oxidative stress of wheat were determined. The

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implications for quantification of graphene in biological systems and evaluation of the safety of

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graphene are discussed.

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MATERIALS AND METHODS Preparation of 13C-Labeled GO and Unlabeled GO. 13C-Labeled graphite was prepared

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by arc discharge method and oxidized by the modified Hummers method to produce 13C-GO.

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The details of preparation protocols were given in the Supporting Information. Unlabeled GO for

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toxicity evaluations was prepared by the modified Hummers method following our previous

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reports.21 The 13C content of 13C-GO was determined by IRMS (Delta V Advantage, Thermo,

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Bremen, Germany). Plant morphology was investigated by TEM (JEM-200CX, JEOL, Tokyo,

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Japan) and atomic force microscopy (AFM; SPM-9600, Shimadazu, Kyoto, Japan). The

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chemical states of elements were analyzed by X-ray photoelectron spectroscopy (XPS; Kratos,

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Manchester, UK). The functional groups were identified by infrared spectroscopy (IR; Nicolet

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Avatar 370, Thermo, Madison, WI, USA). The crystallinity was characterized by X-ray

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diffraction spectroscopy (XRD, D/MAX 2000, Rigaku, Tokyo, Japan). The hydrodynamic radii

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were measured on a nanosizer (Zetasizer 3000 HS, Malvern, Malvern, UK).

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Plant Cultivation and GO Exposure. Seeds of transgenic wheat ‘Lunxuan 987’ were

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obtained from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing.

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The cultivar ‘Lunxuan 987’ was chosen because it is high yielding, nutritious, and resistant to

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biotic/abiotic stresses. Seedlings were cultured in modified Hoagland nutrient solution as

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described in our previous report.42 The same protocol was adopted for plant culture here, because

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it allowed the direct comparison between fullerenol and GO and could reflect the regulative

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effects of size, shape and oxidation degree on the nano-biosafety. Wheat seeds were soaked in 15%

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NaCl for 30 min, then twice soaked for 15 min in water. For the germination assay, 30 seeds

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were placed on filter paper in a Petri dish (diameter 9 cm). Hoagland nutrient solution containing

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GO (0–2.0 mg/mL) was introduced to the Petri dish to moisten the filter paper. The seeds were

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incubated in an incubator in the dark at 25 °C and 80% relative humidity for 7 d. The germination

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frequency was determined daily. Germination potential was calculated with Equation 1.

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Germination potential=

(germinated seeds at day n+1)-(germinated seeds at day n) total seeds

(1)

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After germination, sets of three seedlings of uniform size were transferred to 100 mL

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beakers containing GO at 0, 0.04, 0.2, 0.4, 0.8, 2.0 mg/mL (three replicate beakers per

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concentration). The seedlings were cultivated under a day/night cycle of 12 h/12 h with

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temperatures of 25/20°C, illumination of 24 000 lx during the day cycle, and 80% relative

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humidity. Fresh Hoagland nutrient solution was added as necessary to maintain the volume of

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100 mL. The seedlings were harvested at 15 d for toxicity evaluation.

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Bioaccumulation of 13C-GO. To quantify the bioaccumulation of GO in wheat, wheat

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seedlings (3 d post-germination) were exposed to 13C-GO (1.0 mg/mL) in Hoagland nutrient

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solution. The seedlings were cultured by the aforementioned procedure and harvested at 7, 11,

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and 15 d for IRMS measurements. The sampled seedlings were divided into the roots, stems

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(here, stems referring to the whole seedlings except roots and leaves), and leaves, carefully

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washed with deionized water for three times, lyophilized and ground into powder. Before

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lyophilization, the root samples were examined with a scanning electron microscope (SEM, S-

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4800, Hitachi, Japan) and Raman spectrometer (inVia, Renishaw, Wotton-under-Edge, UK) to

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ensure the full removal of adsorbed 13C-GO. The 13C content in wheat samples was determined

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by IRMS and expressed as δ values relative to the 13C content of the Vienna Pee Dee Belemnite

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(VPDB) standard. The δ value was converted to GO concentration and percentage of exposed

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dose per gram (%ED/g) following our previous report.42

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Toxicity of GO to Wheat. During the toxicity evaluations, wheats were exposed to GO at

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concentrations of 0, 0.04, 0.2, 0.4, 0.8, 2 mg/mL, which covered the low and high concentrations

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used in the literature.25-31 After harvesting at 15 d, the root, stem, and leaf lengths were measured

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with a vernier caliper. The root, stem, and leaf fresh weights were recorded. After drying for 12 h

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at 90°C, the dry weight of the samples was recorded.

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Separately, fresh root samples harvested at 15 d were cut into small sections and fixed with

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formaldehyde-acetate-alcohol solution. The samples were embedded in paraffin and sections of

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10 µm thickness were stained with safranin and fast green. Images of the wheat paraffin sections

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were captured under a microscope (CAB-30PC, Cabontek Co., Chengdu, China). Additional sets

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of fresh root samples were embedded in tissue-freezing medium, frozen at −80 °C, cut into

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sections of 20–30 µm thickness with a cryomicrotome (Leica CM 1850, Nussloch, Germany).

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The sections were transferred onto cover glasses, stored at −20 °C until they were coated with

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gold for 5 s using a sputter coater (E-1045, Hitachi, Tokyo, Japan), and observed with a SEM (S-

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4800, Hitachi, Tokyo, Japan). A third set of samples was fixed with 3% glutaraldehyde, post-

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fixed in 1% osmium tetroxide, dehydrated in a graded alcohol series, and embedded in epoxy

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resin. Sections were cut with an ultramicrotome and post-stained with uranyl acetate and lead

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citrate for TEM examination.

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For oxidative stress assays, all kits were obtained from the Nanjing Jiancheng

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Bioengineering Institute, Nanjing, China. Root samples harvested at 15 d were homogenized in

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water (0.1 g tissue/1 mL water). The samples were centrifuged at 3000 rpm for 5 min to remove

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the residues. The protein concentration in the supernatant was determined by staining with

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Coomassie brilliant blue. The malondialdehyde (MDA) concentration and activities of

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peroxidase (POD) and catalase (CAT) were analyzed following the manufacturer’s instructions

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with an ultraviolet-visible spectrophotometer (UV-1800, Mapada, China). The protocols could

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be downloaded at http://elder.njjcbio.com/index_en.php.

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RESULTS AND DISCUSSION

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Characterization of 13C-Labeled GO. The AFM image of 13C-GO is shown in Figure 1A.

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The two-dimensional structure of the graphene sheets was identified and the height of the 13C-

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GO layer was about 0.9 nm, consistent with the typical layer height of GO. The TEM image

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further confirmed the sheet-like structure (Figure 1B). During the sampling, the 13C-GO sheets

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were slightly folded and stacked, and thus their appearance differed from that under AFM.

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According to the dynamic light scattering measurement, the hydrodynamic radius was 30 nm.

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The IR spectrum confirmed the abundance of oxygen-containing groups (Figure 1C). The peak at

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3382 cm−1 indicated the presence of –OH/-COOH groups. The C=O bonds were reflected by the

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peak at 1731 cm−1. C-O bonds were indicated by the peak at 1053 cm−1. The typical C=C signal

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was detected at 1623 cm−1. The XPS analysis indicated that the elemental contents of 13C-GO

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were 68.1 at% for C and 31.9 at% for O (Figure 1D). By analyzing the C1s XPS, the carbon

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atoms were divided into three components, namely C-C (27.8%), C-O (35.1%) and C=O (5.2%).

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The only 2θ angle of 10.4° for 13C-GO indicated the large layer distance of graphene layers and

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similar crystiline structure to 12C-GO, which had a 2θ angle of 10.5°. On the basis of IRMS

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analyses, about 7.1 at% of C atoms were 13C atoms, which was much higher than that observed

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in normal 12C-GO (1.1 at%), indicating the successful incorporation of 13C into the carbon

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skeleton of GO. Except for the elevated 13C content, the remaining characterization data were

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similar to those of normal 12C-GO sheets (Figure S1), indicating that 13C-GO was successfully

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prepared.

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Figure 1. Characterization of 13C-labeled GO. (A) AFM image; (B) TEM image; (C) IR

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spectrum; (D) C1s XPS spectrum. The inset in (B) shows the magnified edge of 13C-GO.

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Bioaccumulation of 13C-GO in wheat. The 13C contents of root, stem, and leaf samples

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were determined by IRMS after exposure of the wheat seedlings to 13C-GO during the growth

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period. Meaningful 13C abundance increases were observed in root samples comparing to the

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root samples of the control group. Instead, no meaningful increases were found in stems and

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leaves. The root uptake of 13C-GO was also supported by the SEM and TEM observations of

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xenobiotic materials in roots (Figure S2). In particular, the root samples were carefully washed to

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remove adsorbed GO before quantification. The SEM images and Raman spectra confirmed the

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complete removal of adhered sheets (Figure S3). These results suggested that 13C-GO was

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readily absorbed by the roots rather than attaching on the root surface, but the absorbed 13C-GO

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showed limited translocation to the stem and leaves. The 13C-GO concentration in the root at day

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7 was 283 µg/g, equivalent to 0.71% ED/g. The bioaccumulation of 13C-GO was much lower

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than that of fullerenol (C60-OH), which accumulated at ~7% ED/g in wheat roots. This difference

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might be due to that the large size of GO sheets hindered the penetration across biological

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barriers. The small size of the C60-OH molecule may have promoted its uptake and translocation

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in wheat. In addition, the high oxidation degree of GO might contributed to the root uptake.

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Larue et al. quantified the lowly oxidized 14C-carbon nanotubes (CNTs; containing only 1.64 at%

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oxygen) in wheat.49 Less than 0.0005% of applied CNTs were absorbed by the wheat root, and

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about 200 µg/kg of gum Arabic-suspended CNTs and 43 µg/kg of humic acid-suspended CNTs

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were detected in wheat leaves.

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Over the time-course of the experiment, a trend for decreasing 13C-GO concentration in the

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root was observed (112 µg/g at day 15), whereas the 13C-GO concentrations in the stem and

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leaves increased (Please note that the differences between exposed and control groups were not

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statistically significant; Figure 2). The mechanism for the decrease in 13C-GO concentration in

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the root was unclear. We speculated that there might be three possibilities. The first mechanism

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might be excretion directly via the root. As xenobiotic materials, the root might excrete 13C-GO

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in root exudates. The second mechanism may be translocation to the stem and leaf, which is

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predicted to be extremely slow on the basis of the present 13C measurements. The third

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possibility is that 13C-GO was metabolized into CO2 and released to the atmosphere. Degradation

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of GO is reported in the literature. For example, Lalwani et al. used lignin peroxidase to catalyze

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the decomposition of H2O2 for degradation of graphene.50 Similarly, a myeloperoxidase–H2O2

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system may also decompose GO.51 Girish et al. observed the in vivo degradation of graphene

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over a period of 3 months, in which a macrophage played an important role in the degradation.52

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Nevertheless, the degradation of GO in a biological system is expected to be slow, thus the total

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contribution of degradation to the decreased concentration of 13C-GO is likely to be small.

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Figure 2. Bioaccumulation of 13C-GO in wheat seedlings exposed to 13C-GO (1.0 mg/mL) in

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Hoagland nutrient solution for 15 days. * p < 0.05 compared with the control group (n=3).

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Toxicity of GO to wheat. In our study, GO showed toxicity to wheat seeds during the

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germination, reflected by the decreased germination frequency and germinability (Figure

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S4&S5). For normally germinated seedlings, GO also adversely affected biomass accumulation

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and elongation in wheat seedlings (Table S1). The gain in fresh and dry biomass of wheat

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seedlings was not significantly influenced at GO concentrations of 0.4 mg/mL and lower (Figure

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3 A&B). The dry weight was suppressed by GO to a greater degree than was fresh weight,

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implying that the water content of wheat seedlings was enhanced upon exposure to a high

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concentration of GO. Interestingly, seedling length showed a trend to increase with GO

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concentration from 0.04 to 0.4 mg/mL (although the differences were not statistically significant),

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but decreased at higher concentrations of GO (Figure 3C). Consequently, the seedling was longer

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and thinner with increasing GO concentration (Figure S6). Similarly, Zhang et al. reported that

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graphene enhanced the stem length of tomato seedlings, but fresh biomass accumulation was

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inhibited.29 These authors assigned the toxicity to penetration of vacuoles in root cells by

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graphene. Zhang et al. observed that root and leaf elongation in wheat was stimulated by

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graphene at concentrations from 250 to 1500 mg/L, but root hair development was suppressed.53

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After 30 d exposure, root elongation was still significantly enhanced, whereas shoot length was

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not. The shoot fresh weight was decreased in response to exposure to graphene for 30 d. The

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toxicity of GO is concentration dependent and also species dependent. Zhao et al. reported that

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the growth of A. thaliana was not influenced by GO at 1.0 mg/L and lower concentrations.27

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However, GO inhibits root and shoot growth of cabbage, tomato and red spinach.26

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Figure 3. Influence of GO on fresh weight (A), dry weight (B), seedling length (C) and root

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length (D) of wheat seedlings. * p < 0.05 compared with the control group (n=10).

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Damage to wheat root. The root number decreased (Figure S7) and the root elongation was

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inhibited upon exposure to GO (Figure 3D). Inhibition of root development might be associated

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with IAA and ABA.30 Cheng et al. reported that GO inhibited root growth of Brassica napus via

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reduction in IAA concentration, which is the best-characterized molecular signal for root system

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architecture and growth.30 Up-regulation of ABA also contributed, because a high concentration

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of ABA inhibited seedling growth. The inhibition of root development obviously would hinder

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the absorption of inorganic salts, water and other nutrients, and thereby affect seedling growth.

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Sections of wheat roots were examined to reveal structural changes. The intact structure of

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the root was observed in the control group. The epidermis, cortex, endodermis and xylem vessels

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were clearly observed (Figure S8 A&B). GO induced damage to the cortex at low and high

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concentrations (Figure S8 C–F). Aqtocytolysis occurred in the cortex to form aerenchyma-like

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tissue, which increased the porosity of the cortex. The GO-induced structural damage to the

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wheat root may render transportation of air to stems and leaves more difficult, thus increased

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porosity was required to maintain the breath of the roots.

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The ultrastructure of wheat roots was investigated by TEM to further examine the damage

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to root cells induced by GO. The control group showed typical cellular ultrastructure. The cell

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membrane was tightly appressed to the cell wall. The nucleus had a distinct nuclear membrane

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and nucleolus. Several empty vacuoles were observed. Upon exposure to 0.4 mg/mL GO, many

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vacuoles containing xenobiotic materials were observed, which we speculated to be GO or a GO-

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biomolecule complex. Slight detachment of the cell membrane and cell wall was observed, but

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the cell membrane and cell wall remained almost intact (Figure 4 A&B). These observations

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suggested that the uptake of GO at 0.4 mg/mL in the 13C quantification experiment was likely the

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result of endocytosis, rather than penetration via a damaged cell membrane and cell wall. Severe

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ultrastructural damage was observed in response to exposure to GO at 2.0 mg/mL. The typical

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nucleus structure was lost and detachment of the cell membrane and cell wall was obvious,

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indicating shrinkage of cells (Figure 4 C&D). Under higher magnification, many vacuoles were

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evident in the cell and it was impossible to distinguish the disrupted cellular components and GO

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particles. The cell membrane was also severely damaged. In one cell we observed chromatin

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condensation. The nuclear membrane was diffuse and the cytoplasm was divided into multiple

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components by membrane-like structures. Again, we could not distinguish GO particles from the

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disrupted cellular organelles. The structural damages induced by GO at high concentrations

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indicated the toxicity of GO to root cells. The structural changes of plant root induced by

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graphene were also reported in the literature.54

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Figure 4. Ultrastructural changes in wheat root upon exposure to GO. (A) Control group; (B)

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wheat root exposed to 0.4 mg/mL GO; (C, D) wheat root exposed to 2.0 mg/mL GO. The boxes

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represent higher magnification images of portions of images C and D.

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The possible mechanism of GO toxicity to roots might involve oxidative stress. The MDA

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concentration significantly decreased at GO concentrations of 0.04 and 0.2 mg/mL, indicating

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the absence of oxidative stress (Figure 5A). However, the MDA concentration markedly

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increased at GO concentration of 0.8 mg/mL, suggesting the oxidative stress in wheat root. The

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decline in MDA concentration at the GO concentration of 2.0 mg/mL may be attributable to

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acute toxicity and destruction of cellular function. Activity of CAT was significantly increased

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only with exposure to GO at 1.0 mg/mL (Figure 5B). Activity of POD was indicated to be more

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sensitive, with significantly increased activity observed at 0.04 and 0.2 mg/mL and decreasing

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activity at 0.4 mg/mL and higher concentrations (Figure 5C). Oxidative stress is widely

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acknowledged to be a toxicological mechanism for nanomaterials in diverse biological systems.

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Oxidative stress in plants in response to exposure to graphene has been observed previously. Ren

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et al. attributed the toxicity of sulfonated graphene to oxidative damage.54 GO induces an

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increase in reactive oxygen species concentration in leaves of cabbage, tomato and red spinach at

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0.5–2.0 mg/mL.26 Consistent with these changes, accumulation of H2O2 is observed in leaves.

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Oxidative damage leads to the death of root cells and membrane leakage in leaf cells. Anjum et

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al. reported that GO induced oxidative stress in faba bean at 0.2–1.6 mg/mL.28 Cheng et al.

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observed oxidative stress in B. napus exposed to GO at 0.025–0.1 mg/mL.30 No significant

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increase in MDA concentration was observed, but activities of superoxide dismutase (SOD),

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POD and CAT increased, indicating that exposure to GO represents a form of stress for plants.

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Zhang et al. reported oxidative stress in wheat induced by exposure to GO at 0.25–1.5 mg/mL

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for 48 h, with O2.− and MDA concentrations and SOD activity significantly increased. 53 Some

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exceptions have been reported. Zhao et al. did not observe generation of reactive oxygen species

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in A. thaliana seedlings, and the MDA and H2O2 concentrations and SOD and CAT activities

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remained unchanged, in response to GO exposure at 10–1000 µg/L.27 Hu et al. reported that 0.2

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mg/mL of hydrated graphene ribbon suppressed the oxidative stress of wheat seedlings.55

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However, overall, oxidative damage might be an important mechanism for GO-induced toxicity

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to wheat roots.

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Figure 5. Oxidative stress in wheat roots exposed to GO. (a) Malondialdehyde concentration; (b)

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catalase activity; (c) peroxidase activity. * p < 0.05 compared with the control group (n=10).

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In summary, we utilized stable isotope labeling for quantification of graphene in biological

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system, and demonstrated that 13C-GO accumulated in wheat roots at low concentrations. The

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large size of 13C-GO hinders its translocation to stem and leaves. The direct contact and

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bioaccumulation of GO in the root inhibited development of the root system, altered the root

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structure and cellular ultrastructure, and led to inhibition of seedling growth. The mechanism of

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GO toxicity to wheat roots may be oxidative stress. The present study provides a novel method

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for quantitatively tracing of GO in biosafety assessments and presents systematic information on

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the toxicity of GO to wheat. In future, this labeling technology could be used to investigate the

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nanotoxicity, the mechanisms, metabolism and transmission of graphene and its derivatives to

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evaluate long-term environmental health impacts. The quantifications and toxicity evaluations of

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different carbon nanomaterials using similar protocols would benefit the understanding of the

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unique nano-bioeffects, such as the shape effect, size effect and surface effect. We envisage that

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the results will benefit the the ongoing environmental risk assessments of graphene

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nanomaterials.

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ASSOCIATED CONTENT

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Supporting Information

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The Supporting Information includes the preparation protocol of 13C-GO, the characterization of

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unlabeled GO (Figure S1), the identification of GO in root by SEM and TEM (Figure S2), SEM

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images and Raman spectra of washed root surface (Figure S3), the influence of GO on

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germination (Figure S4), the photographs of germinated wheat seeds (Figure S5), the

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photographs of wheat seedlings (Figure S6), the root numbers of wheat seedlings (Figure S7), the

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structural changes of wheat roots (Figure S8), the detailed data of Figure 3 (Table S1).The

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Supporting Information is available free of charge on the ACS Publications website at DOI: .

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AUTHOR INFORMATION

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Corresponding Authors

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* Telephone: +86-28-85522269, Fax: +86-028-85524382, Email: [email protected] (Prof. S.-T.

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Yang)

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* Telephone: +86-10-88236709, Fax: +86-10-88236456, Email: [email protected] (Prof. X.-L.

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Chang)

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Xian Zhang at Key Lab of Urban Environment and Health, Institute of Urban

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Environment, CAS for IRMS analyses. This work was supported by the National Program for

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Support of Top-notch Young Professionals, the National Natural Science Foundation of China

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(Nos. 11475194 and 11675189), Beijing Natural Science Foundation (No. 2152038), the

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National Key Research and Development Program of China (2016YFA0201603), and the

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Fundamental Research Funds for the Central Universities, Southwest Minzu University (No.

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2016NZDFH01).

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REFERENCES

370

(1) Marchesan, S.; Melchionna, M.; Prato, M. Wire up on carbon nanostructures! How to play a

371

winning game. ASC Nano. 2015, 9(10), 9441–9450.

ACS Paragon Plus Environment

21

Environmental Science & Technology

372

(2) Zheng, L.; Zheng, L.; Sun, H. Y.; Gao, C. Superstructured assembly of nanocarbons:

373

Fullerenes, nanotubes, and graphene. Chem. Rev. 2015, 115(15), 7046−7117.

Page 22 of 29

374

(3) Li, N.; Yang, G. Z.; Sun, Y.; Song, H. W.; Cui, H.; Yang, G. W.; Wang, C. X. Free-standing

375

and transparent graphene membrane of polyhedron box-shaped basic building units directly

376

grown using a NaCl template for flexible transparent and stretchable solid-state

377

supercapacitors. Nano. Lett. 2015, 15(5), 3195−3203.

378

(4) Cui, S. M.; Mao, S.; Lu, G. H.; Chen, J. H. Graphene coupled with nanocrystals:

379

Opportunities and challenges for energy and sensing applications. J. Phys. Chem. Lett.

380

2013, 4(15), 2441−2454.

381 382 383 384 385 386

(5) Young, R. J.; Kinloch, I. A.; Gong, L.; Novoselov, K. S. The mechanics of graphene nanocomposites: A review. Compos. Sci Technol. 2012, 72(12), 1459–1476. (6) Wang, M.; Duan, X. D.; Xu, W. X.; Duan X. F. Functional three-dimensional graphene/polymer composites. ACS. Nano 2016, 10(8), 7231−7247. (7) Chung, C.; Kim, Y. K.; Shin, D.; Ryoo, S. R.; Hong, B. H.; Min, D. H. Biomedical applications of graphene and graphene oxide. Acc. Chem. Res. 2013, 46(10), 2211–2224.

387

(8) Zhao, L. Q.; Yu, B. W.; Xue, F. M.; Xie, J. R.; Zhang, X. L.; Wu, R. H.; Wang, R. J.; Hu, Z.

388

Y.; Yang, S. T.; Luo, J. B. Facile hydrothermal preparation of recyclable S-doped graphene

389

sponge for Cu2+ adsorption. J. Hazard. Mater. 2015, 286, 449–456.

ACS Paragon Plus Environment

22

Page 23 of 29

Environmental Science & Technology

390

(9) He, S. J.; Song, B.; Li, D.; Zhu, C. F.; Qi, W. P.; Wen, Y. Q.; Wang, L. H.; Song, S. P.; Fang,

391

H. P.; Fan, C. H. A graphene nanoprobe for rapid, sensitive, and multicolor fluorescent

392

DNA analysis. Adv. Funct. Mater. 2015, 20(3), 453–459.

393

(10) Chakravarty, D.; Erande, M. B.; Late, D. J. Graphene quantum dots as enhanced plant

394

growth regulators: Effects on coriander and garlic plants. J. Sci. Food. Agric. 2015, 95(13),

395

2772–2778.

396

(11) Zhao, L. Q.; Dong, P. J.; Xie, J. R.; Li, J. Y.; Wu, L. X.; Yang, S. T.; Luo, J. B. Porous

397

graphene oxide-chitosan aerogel for tetracycline removal. Mater. Res. Express. 2014, 1(1),

398

015601.

399

(12) Wu, R. H.; Yu, R. H.; Liu, X. Y.; Li, H. L.; Wang, W. X.; Chen, L. Y.; Bai, Y. T.; Ming, Z.;

400

Yang, S. T. One-pot hydrothermal preparation of graphene sponge for the removal of oils

401

and organic solvents. Appl. Surf. Sci. 2016, 362, 56–62.

402 403 404 405 406 407 408 409

(13) Teo, W. Z.; Sofer, Z.; Sembera, F.; Janousek, Z.; Pumera, M. Cytotoxicity of fluorographene. RSC Adv. 2015, 5, 107158–107165. (14) Teo, W. Z.; Chng, E. L. K.; Sofer, Z.; Pumera, M. Cytotoxicity of halogenated graphenes. Nanoscale 2014, 6, 1173–1180. (15) Chng, E. L. K.; Sofer, Z.; Pumera, M. Cytotoxicity profile of highly hydrogenated graphene. Chem. Eur. J. 2014, 20, 6366–6373. (16) Hu, X. G.; Zhou, Q. X. Health and ecosystem risks of graphene. Chem. Rev. 2013, 113(5), 3815−3835.

ACS Paragon Plus Environment

23

Environmental Science & Technology

Page 24 of 29

410

(17) Kang, S.; Mauter, M. S.; Elimelech, M. Microbial cytotoxicity of carbon-based

411

nanomaterials: Implications for river water and wastewater effluent. Environ. Sci. Technol.

412

2009, 43(7), 2648–2653.

413 414

(18) Seabra, A. B.; Paula, A. j.; Lima, R. D.; Alves, O. L.; Duran, N. Nanotoxicity of graphene and graphene oxide. Chem. Res. Toxicol. 2014, 27(2), 159−168.

415

(19) Mao, L.; Liu, C. L.; Lu, K.; Su, Y.; Gu, C.; Huang, Q. G.; Petersen, E. J. Exposure of few

416

layer graphene to limnodrilus hoffmeisteri modifies the graphene and changes its

417

bioaccumulation by other organisms. Carbon 2016, 109, 566−574.

418

(20) Xie, J. R.; Ming, Z.; Li, H. L.; Yang, H.; Yu, B. W.; Wu, R. H.; Liu, X. Y.; Bai, Y. T.;

419

Yang, S. T. Toxicity of graphene oxide to white rot fungus Phanerochaete chrysosporium.

420

Chemosphere 2016, 151, 324–331.

421

(21) Chang, Y. L.; Yang, S. T.; Liu, J. H.; Dong, E. Y.; Wang, Y. W.; Cao, A. N.; Liu, Y. F.;

422

Wang, H. F. In vitro toxicity evaluation of graphene oxide on A549 cells. Toxicol. Lett.

423

2011, 200(3), 201–210.

424 425 426 427

(22) Zou, X. F.; Zhang, L.; Wang, Z. W.; Luo, Y. Mechanisms of the antimicrobial activities of graphene materials. J. Am. Chem. Soc. 2016, 138(7), 2064−2077. (23) Hu, W. B.; Peng, C.; Luo, W. J.; Lv, M.; Li, X. M.; Li, D.; Huang, Q.; Fan, C. H. Graphenebased antibacterial paper. ACS Nano 2010, 4(7), 4317–4323.

ACS Paragon Plus Environment

24

Page 25 of 29

Environmental Science & Technology

428

(24) Lahiani, M. H.; Dervishi, E.; Ivanov, I.; Chen, J. H.; Khodakovskaya, M. Comparative study

429

of plant responses to carbon-based nanomaterials with different morphologies.

430

Nanotechnology 2016, 27(26), 0957–4484.

431

(25) Begum, P.; Fugetsu, B. Induction of cell death by graphene in Arabidopsis thaliana

432

(Columbia Ecotype) T87 cell suspensions. J. Hazard. Mater. 2013, 260(18), 1032–1041.

433

(26) Begum, P.; Ikhtiari, R.; Fugetsu, B. Graphene phytotoxicity in the seedling stage of

434 435 436

cabbage, tomato, red spinach, and lettuce. Carbon 2011, 49(12), 3907–3919. (27) Zhao, S. Q.; Wang, Q. Q.; Zhao, Y. L.; Rui, Q.; Wang, D. Y. Toxicity and translocation of graphene oxide in Arabidopsis thaliana. Environ. Toxicol. Pharm. 2015, 39(1), 145–156.

437

(28) Anjum, N. A.; Singh, N.; Singh, M. K.; Sayeed, I.; Duarte, A. C.; Pereira, E.; Ahmad, I.

438

Single-bilayer graphene oxide sheet impacts and underlying potential mechanism

439

assessment in germinating faba bean (Vicia faba L.). Sci. Total. Environ. 2014, 472(4), 834–

440

841.

441 442

(29) Zhang, M.; Gao, B.; Chen, J. J.; Li, Y. C. Effects of graphene on seed germination and seedling growth. J. Nanopart. Res. 2015, 17(2), 1–8.

443

(30) Cheng, F.; Liu, Y. F.; Lu, G. Y.; Zhang, Y. K.; Xie, L. L.; Yuan, Y. F.; Xu, B. B.

444

Graphene oxide modulates root growth of Brassica napus L. and regulates ABA and IAA

445

Concentration. J. Plant. Physiol. 2016, 193, 57–63.

446 447

(31) Hu, X. G.; Kang, J. Lu, K. H.; Zhou, R. R.; Mu, L.; Zhou, Q. X. Graphene oxide amplifies the phytotoxicity of arsenic in wheat. Sci. Rep. 2014, 4, 6122–6122.

ACS Paragon Plus Environment

25

Environmental Science & Technology

Page 26 of 29

448

(32) Zhao, J.; Wang, Z. Y.; White, J. C.; Xing, B. S. Graphene in the aquatic environment:

449

Adsorption, dispersion, toxicity and transformation. Environ. Sci. Technol. 2014, 48(17),

450

9995−10009.

451 452

(33) Miralles, P.; Church, T. L.; Harris, A. T. Toxicity, uptake, and translocation of engineered nanomaterials in vascular plants. Environ. Sci. Technol. 2012, 46(17), 9224−9239.

453

(34) Petersen, E. J.; Flores-Cervantes, D. X.; Bucheli, T. D.; Elliott, L. C. C.; Fagan, J. A.;

454

Gogos, A.; Hanna, S.; Kagi, R.; Mansfield, E.; Bustos, A. R. M.; Plata, D. L.; Reipa, V.;

455

Westerhoff, P.; Winchester, M. R. Quantification of carbon nanotubes in environmental

456

matrices: Current capabilities, case studies, and future prospects. Environ. Sci. Technol.

457

2016, 50 (9), 4587–4605.

458

(35) Tripathi, D. K.; Singh, S.; Singh, S.; Pandey, R.; Singh, V. P.; Sharma, N. C.; Prasadf, S.

459

M.; Dubeya, N. K.; Chauhan, D. K. An overview on manufactured nanoparticles in plants:

460

Uptake, translocation, accumulation and phytotoxicity. Plant Physiol. Biochem. 2017, 110,

461

2−12.

462

(36) Dimkpa, C. O.; McLean, J. E.; Martineau, N.; Britt, D. W.; Haverkamp, R.; Anderson, A. J.

463

Silver nanoparticles disrupt wheat (Triticum aestivum L.) growth in a sand matrix. Environ.

464

Sci. Technol. 2013, 47(2), 1082−1090.

465

(37) Zhai, G. S.; Gutowski, S. M.; Walters, K. S.; Yan, B.; Schnoor, J. L. Charge, size, and

466

cellular selectivity for multiwall carbon nanotubes by maize and soybean. Environ. Sci.

467

Technol. 2015, 49(12), 7380−7390.

ACS Paragon Plus Environment

26

Page 27 of 29

Environmental Science & Technology

468

(38) Xia, T.; Kovochich, M.; Liong, M.; Madler, L.; Gilbert, B.; Shi, H. B.; Yeh, J. I.; Zink, J. I.

469

Nel, A. E. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide

470

nanoparticles based on dissolution and oxidative stress properties. ACS Nano 2008, 2(10),

471

2121–2134.

472

(39) Wang, Z. Y.; Li, J.; Zhao, J.; Xing, B. S. Toxicity and internalization of CuO nanoparticles

473

to prokaryotic alga Microcystis aeruginosa as affected by dissolved organic matter.

474

Environ. Sci. Technol. 2011, 45(14), 6032–6040.

475

(40) Torre-Roche, R. D. L.; Hawthorne, J.; Deng, Y. Q.; Xing, B. S.; Cai, W. J.; Newman, L. A.;

476

Wang, Q.; Ma, X. M.; Hamdi, H.; White, J. C. Multiwalled carbon nanotubes and C60

477

fullerenes differentially impact the accumulation of weathered pesticides in four agricultural

478

plants. Environ. Sci. Technol. 2013, 47(21), 12539–12547.

479

(41) Du, M. M.; Zhang, H.; Li, J. X.; Yan, C. Z.; Zhang, X.; Chang, X.-L. Bioaccumulation,

480

depuration and transfer to offspring of 13C-labeled fullerenols by Daphnia magna. Environ.

481

Sci. Technol. 2016, 50(19), 10421–10427.

482

(42) Wang, C. L.; Zhang, H.; Ruan, L. F.; Chen, L. Y.; Li, H. L.; Chang, X. L.; Zhang, X.; Yang,

483

S. T. Bioaccumulation of 13C-fullerenol nanomaterials in wheat. Environ. Sci.: Nano 2016,

484

3(4), 799–805.

485

(43) Chang, X. L.; Ruan, L. F.; Yang, S. T.; Sun, B. Y.; Guo, C.; Zhou, L. J.; Dong, J. Q.; Yuan,

486

H.; Xing, G. M.; Zhao, Y. L.; Yang, M. Quantification of carbon nanomaterials in vivo:

487

Direct stable isotope labeling on the skeleton of fullerene C60. Environ. Sci.: Nano 2014,

488

1(1), 64–70.

ACS Paragon Plus Environment

27

Environmental Science & Technology

Page 28 of 29

489

(44) Yang, S. T.; Guo, W.; Lin, Y.; Deng, X. Y.; Wang, H. F.; Sun, H. F.; Liu, Y. F.; Wang, X.;

490

Wang, W.; Chen, M.; Huang, Y. P.; Sun, Y. P. Biodistribution of pristine single-walled

491

carbon nanotubes in vivo. J. Phys. Chem. C. 2007, 111(48), 17761–17764.

492

(45) Liu, J. H.; Yang, S. T.; Wang, X.; Wang, H. F.; Liu, Y. M.; Luo, P. G.; Liu, Y. F.; Sun, Y.

493

P. Carbon nanoparticles trapped in vivo-similar to carbon nanotubes in time-dependent

494

biodistribution. ACS Appl. Mater. Interfaces 2014, 6(16), 14672−14678.

495

(46) Wang, C. L.; Ruan, L. F.; Chang, X.-L.; Zhang, X. L; Yang, S.-T.; Guo, X. H.; Yuan, H.; 13

496

Guo, C. B.; Shi, W. Q.; Sun, B. Y.; Zhao, Y. L. The isotopic effects of

497

carbon cage (C70) fullerenes and their formation process. RSC Adv. 2015, 5(94), 76949–

498

76956.

499

(47) Ruan, L. F.; Chang, X. L.; Sun, B. Y.; Guo, C. B.; Dong, J. Q.; Yang, S. T.; Gao, X. F.;

500

Zhao, Y. L.; Yang, M. Preparation and spectra of

501

2014, 59(10), 905–912.

502 503

C-labeled large

13

C-enriched fullerene. Chin. Sci. Bull.

(48) Wang, Z. Z.; Chang, X. L.; Lu, Z. H.; Gu, M.; Zhao, Y. L.; Gao, X. F. A precision structural model for fullerenols. Chem. Sci. 2014, 5(8), 2940–2948.

504

(49) Larue, C.; Pinault, M.; Czarny, B.; Georgin, D.; Jaillard, D.; Bendiab, N.; Mayne-

505

L’Hermite, M.; Taran, F.; Dive, V.; Carrière, M. Quantitative evaluation of multi-walled

506

carbon nanotube uptake in wheat and rapeseed. J. Hazard. Mater. 2012, 227-228(43), 155–

507

163.

508

(50) Lalwani, G.; Xing, W. L.; Sitharaman, B. Enzymatic degradation of oxidized and reduced

509

graphene nanoribbons by lignin peroxidase. J. Mater. Chem. B. 2014, 2(37), 6354–6362.

ACS Paragon Plus Environment

28

Page 29 of 29

Environmental Science & Technology

510

(51) Kurapati, R.; Russier, J.; Squillaci, M. A.; Treossi, E.; Ménard-Moyon, C.; Rio-Castillo, A.

511

E. D.; Vazquez, E.; Samorì, P.; Palermo, V.; Bianco, A. Dispersibility-dependent

512

biodegradation of graphene oxide by myeloperoxidase. Small 2015, 11(32), 3985–3994.

513

(52) Girish, C. M.; Sasidharan, A.; G. Gowd, S.; Nair, S.; Koyakutty, M. Confocal raman

514

imaging study showing macrophage mediated biodegradation of graphene in vivo. Adv.

515

Healthcare Mater. 2013, 2(11), 1489–1500.

516

(53) Zhang, P.; Zhang, R. R.; Fang, X. Z.; Song, T. Q.; Cai, X. D.; Liu, X. J.; Du, X. T. Toxic

517

effects of graphene on the growth and nutritional levels of wheat (Triticum aestivum L.):

518

short- and long-term exposure studies. J. Hazard. Mater. 2016, 317, 543–551.

519

(54) Ren, W.; Chang, H. W.; Teng, Y. Sulfonated graphene-induced hormesis is mediated

520

through oxidative stress in the roots of maize seedlings. Sci. Total. Environ. 2016, 572,

521

926–934.

522 523

(55) Hu, X. G.; Zhou, Q. X. Novel hydrated graphene ribbon unexpectedly promotes aged seed germination and root differentiation. Sci. Rep. 2014, 4, 3782.

524

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