Ambient Water and Visible-Light Irradiation Drive Changes in

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Ambient water and visible-light irradiation drive changes in graphene morphology, structure, surface chemistry, aggregation and toxicity Xiangang Hu, Ming Zhou, and Qixing Zhou Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es503003y • Publication Date (Web): 16 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Environmental Science & Technology

ACS Paragon Plus Environment


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Ambient water and visible-light irradiation drive changes in graphene

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morphology, structure, surface chemistry, aggregation and toxicity

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Xiangang Hu, Ming Zhou, Qixing Zhou*

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Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education),

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Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of

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Environmental Science and Engineering, Nankai University, Tianjin 300071, China.

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ABSTRACT

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The environmental behaviors and risks of graphene have attracted considerable

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attention. However, the fundamental effects of ambient water and visible-light

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irradiation on the properties and toxicity of graphene remain unknown. This work

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revealed that hydration and irradiation result in the transformation of large-sheet

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graphene to long-ribbon graphene. The thickness of the treated graphene decreased,

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and oxides were formed through the generation of singlet oxygen. In addition,

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hydration and irradiation resulted in greater disorder in the graphene structure and in

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the expansion of the d-spacing of the structure due to the introduction of water

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molecules and modifications of the functional groups. Oxidative modifications with

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two-stage (fast and low) kinetics enhanced the number of negative surface charges on

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the graphene and enhanced graphene aggregation. The above property alterations

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reduced the nanotoxicity of graphene to algal cells by reducing the generation of

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reactive oxygen species, diminishing protein carbonylation and decreasing tail DNA.

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A comparative study using graphene oxide suggested that oxidative modifications

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could play an important role in inhibiting toxicological activity. This study provides a

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preliminary approach for understanding the environmental behaviors of graphene and

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avoids overestimating the risks of graphene in the natural environment.

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Keywords: Nanotoxicity, Graphene, Visible light, Transformation, Algae

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INTRODUCTION

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Graphene-related research has rapidly grown in a wide range of disciplines because of

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its broad applications in physics, chemistry, biology, medicine, environmental

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protection and manufacturing.1-4 Given the potential of environmental and human

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exposure to graphene due to its versatile applications, scientists are directing more

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attention towards its environmental fate and biosafety.5-8 Generally, the

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physicochemical properties of graphene determine its environmental fate and risks.9,10

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A fundamental understanding of how graphene properties are altered under

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environmentally relevant conditions is important for scientifically evaluating

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ecological risks and designing safe graphene products.8,11 However, the effects of

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ambient water and visible-light irradiation on the morphology, structure, surface

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chemistry and toxicity of graphene remain unknown. This lack of information can

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lead to over- or under-estimations of graphene risks in the natural environment.

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Water is ubiquitous in the natural environment. For example, surface water,

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underground water and atmospheric water (humidity) exist in the environment. In

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addition, water is commonly used in scientific research and industry. Therefore, the

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interactions of nanomaterials with water (hydration) comprise a fundamental issue

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that links various fields of research related to nanomaterials and their applications.

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During long-term stirring and trace levels of ozone in water, sufficient C60O was

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produced at the surfaces of the nC60 particles to allow the formation of a stable

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suspension in water.12 At room temperature, water has been observed to

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spontaneously induce phase and morphology transformations of nanotubes.13

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However, the interactions of water with two-dimensional graphene under

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environmentally relevant conditions remain largely unknown.

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Sunlight-catalyzed redox reactions (photooxidation and photoreduction) may be

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critical transformation processes that affect nanomaterial coatings, oxidation states,

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the generation of reactive oxygen species (ROS), and persistence.14-17 The oxidation

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and mineralization of fullerenes dispersed in water by natural sunlight may result in

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the attenuation of carbon-based nanomaterials.14 Many nanomaterials are innately

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photoactive (e.g., fullerenes and carbon nanotubes), potentially producing ROS when 2



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exposed to sunlight.15,16 In addition, oxidation and carboxylation of carbon nanotubes

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by OH radicals have been observed.17 Nanotube oxidation increases the surface

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charge of the carbon nanotubes and their stability against aggregation while

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decreasing the hydrophobicity.17 The effects of natural visible-light irradiation on the

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morphology, structure, surface chemistry and aggregation of graphene are unclear and

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worthy of additional study.

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The physico-chemical features (e.g., morphology, structure, functional group, size

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distribution) of nanomaterials are relevant for their toxicity. Coatings and

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functionalization reduced the in vivo toxicity of carbon nanotubes.18 The

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shape-dependent toxicity of carbon nanotubes was reported, which affects their

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bioavailability.19 The surface defects of carbon nanotubes can result in the generation

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of ROS and induce toxic effects in vivo or in vitro.20 In addition, the aggregation state

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can affect the shape and surface area of carbon nanotubes and is related to the

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inhibition of algal growth.21 However, the influences of physico-chemical features on

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graphene toxicity are poorly understood. Compacted graphene sheets are more

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damaging to mammalian fibroblasts than less densely packed graphene oxides,

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depending on the nanomaterial aggregation.22 When hydration or visible-light

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irradiation alters the properties of graphene, the corresponding nanotoxicity likely

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differs from that of the pristine graphene.

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The primary objectives of this study were to determine how water and

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visible-light irradiation alter the morphology, structure, surface chemistry,

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aggregation and toxicity of graphene. Specifically, we determined the following: (i)

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the morphology, structure, surface chemistry and aggregation of graphene before and

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after treatment using various optical and microscopic techniques; (ii) their

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nanotoxicities (oxidative stress, protein carbonylation, tail DNA, development

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inhibition and ultrastructure damage) to a model species (C. Vulgaris) before and after

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these treatments; and (iii) the correlations between the graphene properties and

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nanotoxicity.

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MATERIALS and METHODS 3


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Water and visible-light irradiation treatments

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Graphene nanosheets were obtained from the Nanjing XFNANO Materials Tech Co.,

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Ltd., China. Single-layer graphene was prepared using thermal exfoliation and

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hydrogen reduction. Similar to other carbon nanomaterials, one of the principal

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challenges associated with testing the environmental fate or ecotoxicological effects

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of graphene is that carbon nanomaterials are not readily dispersed at detectable

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concentrations without adding a surfactant and/or a dispersion process. Sonication is a

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well-reported and relatively acceptable method for dispersing nanomaterials.23

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Compared with probe sonication, ice-bath sonication at low temperatures and less

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than 150 W has been widely used for graphene, carbon nanotubes and fullerene.23-25

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In this study, graphene (0.02 g) was suspended in 200 mL of pure water (18.2 Ω/cm)

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and subjected to ice-bath sonication for 40 min at 120 W. The same sonication

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treatment was used for all treatments to minimize the occurrence of artifacts between

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the treatments due to sonication. To prepare the irradiated graphene, the suspended

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graphene was placed in a visible-light incubator maintained with an irradiation of 34

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W/m2, a temperature of 24°C, and a relative humidity of 80%. To prepare the hydrated

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graphene, the graphene suspension was covered with aluminum foil and placed in the

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same incubator. Hydration and irradiation treatments were imposed for 120 days.

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During treatment, pure water was added to maintain a constant volume of 150–200

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mL. To stimulate perturbation, the treated samples were gently stirred for 10 min

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twice each day at 150 rpm. To collect a sufficient amount of the treated graphene, ten

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identical samples (100 mg/L, 200 mL per sample) were prepared. After hydration and

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irradiation, the graphene suspensions were filtered through 0.1 µm polyether sulfone

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resin membranes, and the materials on the membranes were lyophilized to form

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powders for X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS)

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and other analyses.

115 116

Characterization of graphene before and after treatment (see Supporting

117

Information)

118 4



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Generation of free radicals and modification kinetics

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The concentrations of furfuryl alcohol (FFA), which is used as an 1O2 trapping reagent,

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were determined using high performance liquid chromatography (Waters, 2695, USA)

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with ultraviolet detection (Waters, 2487, USA), as previously reported.14 The kinetics

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of graphene photomodification were measured using the time profile of damage to the

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pristine sp2 structure. The sp2 structure was indirectly quantized using the specific

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adsorption of graphene at 270 nm and detected using a TU-1901 spectrophotometer

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with UVWin5 software.

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C. vulgaris cultivation

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C. vulgaris and its culture medium (BG-11) were purchased from the Freshwater

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Algae Culture Collection at the Institute of Hydrobiology in China. All materials that

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came into contact with the microalgae were sterilized before use. The current

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environmental concentrations of graphene remain unknown. To directly compare the

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reported doses of carbon nanomaterials for the toxicity tests,26,27 graphene suspensions

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were prepared using several different concentrations (0.1, 1.0 and 10 mg/L) in BG-11

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culture medium adjusted to pH = 7.0. To avoid the effects of nanomaterial

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aggregation and prepare an exact concentration, a 10 mg/L stock solution was

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dispersed in BG-11 culture medium under ice-bath sonication for 40 min at 120 W

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before immediately diluting to 1 mg/L. Similarly, the prepared 1 mg/L suspensions

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were dispersed using sonication before immediately diluting to 0.1 mg/L. The

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concentration of algal cells at the beginning of the test was 0.5 × 106 cells/mL. The

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suspensions were shaken once every 8 h (150 rmp for 10 min per time) and placed in

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a light incubator under 34 W/m2 irradiation and 80% humidity for 5 days at 24°C.

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Reactive oxygen species (ROS)

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To determine the generation of ROS in cells, an intracellular ROS indicator,

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2′,7′-dichlorofluorescin diacetate (DCFH-DA), was used. After 5 days of exposure to

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graphene, the algal cells were washed twice with medium and then incubated in 2 mL

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of 10 mM DCFH-DA at 37°C for 30 min. The cells were subsequently washed three 5


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times with PBS buffer at pH = 7.4, and their fluorescence was determined at an

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excitation of 485 nm and emission of 520 nm using a fluorescence spectrometer

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(PerkinElmer LS 55, USA).

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Cellular development and malondialdehyde content

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The number and diameter of cells were counted using a cell analyzer

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CASY-TT (Innovatis, Germany). The amounts of chlorophyll a and chlorophyll b

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were quantified to determine algal photosynthesis. The lipid peroxide concentration

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was determined in terms of the malondialdehyde (MDA) content, and the chlorophyll

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a, chlorophyll b and MDA contents were analyzed using a TU-1901

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spectrophotometer, as described elsewhere.28

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Comet assay

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The damage level of the DNA strands in the algal cells was determined using a comet

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assay and the method described in our previous study.29 After electrophoresis, the

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slides were washed three times with 0.5 M Tris buffer (pH 7.5) and the DNA was

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stained using ethidium bromide. The stained slides were examined using a

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fluorescence microscope (Zeiss, Axio Imager Z1, Germany) equipped with a CCD

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camera. Three slides were prepared per group, and 50 randomly selected and

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non-overlapping cells were analyzed on each slide. The images were analyzed using

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the CASP software, and the percentage of tail DNA (% DNA) was measured as an

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indicator of DNA damage.

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Protein carbonyls and cellular ultrastructure measurements (see Supporting

173

Information)

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Statistical analysis

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All treatments included three replicates, and the error bars on the results represented

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the mean ± SD (standard deviation). Differences were regarded as statistically

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significant at P < 0.05. The data were analyzed using a one-way analysis of variance 6



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(ANOVA) and were compared using a post hoc Tukey’s test. All analyses were

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performed using the IBM Statistics SPSS 19 software.

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RESULTS and DISCUSSION

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Morphological alterations

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In this study, atomic force microscopy (AFM) and field-emission transmission

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electron microscopy (FETEM) were conducted to study the morphology of the

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graphene before and after treatment. As shown in Figure 1a, the pristine graphene had

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a nanosheet morphology and was 0.872 nm thick (average 0.812 ± 0.071 nm, n = 6),

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which was consistent with the 0.5–1.0 nm thickness reported for single-layer

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graphene.30 Moreover, abnormal pores and dentate edges were observed on and

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around the nanosheets, which represented the inherent defects that formed during their

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synthesis.31 After treatment, the large-sheet morphology transformed into a

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long-ribbon morphology. After treatment, the thicknesses of the hydrated and

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visible-light-irradiated graphene decreased to 0.672 (average 0.652 ± 0.057 nm, n = 6)

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and 0.356 nm (average 0.366 ± 0.028 nm, n = 6), respectively. Raised dots were

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distributed on the treated graphene, as indicated by the black arrows shown in Figure

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1a. The thicknesses of the raised dots reached approximately 12 and 4 µm for the

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hydrated and irradiated graphene, respectively. As shown in Figure 1b, the EDX

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analyses revealed that the chemical compositions of the dots included oxides, nitrides

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and sulfides, which originated from the atmosphere during treatment. During

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long-term hydration and irradiation, the carbon atoms located in the pores and on the

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edges of the graphene defects possessed dangling bonds with unpaired electrons that

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could drive the formation of oxides, nitrides and sulfides.32 Furthermore, transmission

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electron microscopy (TEM) showed the morphological alterations of the graphene

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before and after hydration and irradiation treatments. As shown in the left-hand

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images of Figure 2, the treated graphene exhibited more wrinkles than the pristine

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graphene. Graphene is known to exhibit severely wrinkled or folded structures on its

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edges and planes due to a large number of defects and a high surface-to-volume ratio.

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Scrolls and multiple folds can result in a number of dark lines, even in monolayer 7


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graphene (as observed on the graphene shown in the images on the right side of

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Figure 2).33 The scrolls and multiple folds of pristine graphene exhibited an ordered

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morphology. However, this ordered morphology became disordered in the hydrated

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and irradiated graphene, as indicated by the red arrows in Figure 2. This result

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suggested that the initial π-π stacking interactions were degraded due to hydration and

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irradiation.

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Structural alterations

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Next, Raman spectrometry (RS) was used to analyze the structure of the graphene

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before and after the hydration and irradiation treatments, as shown in Figure 3a.

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Typical D and G bands of graphene were detected at approximately 1350 and 1594

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cm-1. These bands reflect the disordered structure and ordered sp2 carbon system of

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graphene, respectively.34 Other features, such as the 2D band and the combined-mode

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D + G bands located at approximately 2689 and 2937 cm-1, respectively,35 were also

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observed in the RS spectra. No obvious shifts in any of the aforementioned peaks

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were observed after the treatments. The ratios of the D-band intensity to the G-band

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intensity were 1.08, 1.39, and 1.24 for the graphene, hydrated graphene and irradiated

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graphene, respectively, indicating that hydration and visible-light irradiation enhance

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the disordered structure of graphene. These results are consistent with the TEM

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images presented in Figure 2.

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The structural alterations of the graphene were further investigated using XRD, as

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shown in Figure 3b. The widths of the peaks were very large, and the half-peak widths

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and crystalline grain sizes of the XRD peaks were 9.1° and 0.9 nm, respectively, in all

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samples. The XRD patterns of the graphene, hydrated graphene and

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visible-light-irradiated graphene contained peaks at 2θ = 22.9°, 20.2°, and 19.4°,

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respectively. In contrast with the low-concentration single-layer graphene on the mica

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plates of AFM or the copper meshes of TEM, graphene nanosheets (graphene

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powders) interact with one another through intrinsic π electrons in XRD patterns.

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Based on Braggs law, the d-spacing of graphene should be slightly larger than that

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(0.34 nm) of graphite.36 The d-spacings of graphene, hydrated graphene and 8



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visible-light-irradiated graphene were calculated to be 0.39, 0.43 and 0.46 nm,

240

respectively. These data suggest that hydration and visible-light irradiation increased

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the d-spacing of the graphene. According to a previous report, the relative humidity

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and stacking configuration of graphene sheets are the primary factors that govern the

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d-spacings of graphene-based nanomaterials.13 The hydrodynamic diameter of a water

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molecule is 0.27 nm, and the introduction of a water monolayer results in 0.22–0.25

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nm of lattice expansion,37 which is significantly larger than the difference between the

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pristine and treated graphene. However, the wide peaks could be differentiated into

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more than one small peak. For example, the peak from the pristine graphene consisted

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of three small peaks at 2θ = 17.3°, 22.9° and 29.5°, with calculated d-spacings of 0.51,

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0.39 and 0.30 nm, respectively. The peak from the hydrated graphene consisted of

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three small peaks at 2θ = 12.2°, 17.0°, and 26.0°, with calculated d-spacings of 0.73,

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0.52, and 0.34 nm, respectively. Given the changes in the d-spacings of the

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differentiated peaks, a few water molecules most likely became incorporated between

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the carbon atoms of the graphene. However, water molecules were not the only

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factors that affected the lattice expansion. The lattice defects and the oxygen/nitrogen

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functional groups reduced the d-spacings of the crystalline peaks.38,39 Therefore, the

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crystal structures were a systemic result of the incorporation of water-molecules and

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the introduction of defects and functional groups.

258 259

Alterations in surface chemistry

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The Fourier-transform infrared (FTIR) spectra of the graphene before and after the

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hydration and irradiation treatments are presented in Figure S1. In the pristine

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graphene spectrum, the C=C band at 1600-1700 cm-1 was assigned to the skeletal

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vibrations of un-oxidized graphene. This peak disappeared in the spectra of the treated

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graphene. In addition, C=O, C-OH and C-O-C vibrations all contributed to the broad

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peaks at 600-1100 cm-1,38,40 which suggested that the graphene was oxygenated after

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the treatments. The peaks at approximately 1380 and 3290 cm-1 were assigned to C-N

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and N-H vibrations,38,39 which indicated that the nitrogen groups were modified.

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Changes in the surface chemistry of the graphene were further confirmed by XPS. 9


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The full spectra of the graphene before and after treatments are presented in Figure S2.

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The pristine graphene spectrum consisted of 94.0% C1s and 6.0% O1s. The small

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amount of oxygen in the pristine graphene resulted from the synthesis procedure.41

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The spectrum of the hydrated graphene contained 92.7% C1s, 5.8% O1s, 1.3% N1s

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and 0.2% S2p, and the spectrum of the irradiated graphene contained 90.2% C1s,

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8.9% O1s, 0.7% N1s and 0.2% S2p. The EDX results reflected the presence of a small

275

amount of nitrogen and sulfur in the raised dots of the hydrated and irradiated

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graphene. Considering that nitrogen and sulfur were not observed in the pristine

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graphene, both elements most likely resulted from the ambient atmosphere. The

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remarkable increase of O1s in the irradiated graphene implied that graphene can be

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gradually oxidized using visible-light irradiation under an ambient atmosphere. The

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specific compositions of the C1s and O1s signals were determined, as shown in

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Figure S3. The C1s signal consisted of C=O and C-C, which was consistent with the

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FTIR results presented in Figure S2. Unlike the O1s for the other graphene samples,

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the O1s signal in the hydrated graphene had multiple components, including -OH

284

(37.7%), C-O (8.9%) and C-O-C (53.4%).

285

The UV-vis absorption spectra of the graphene before and after the hydration and

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irradiation treatments are presented in Figure S4. The peak in the spectrum of the

287

pristine graphene at approximately 270 nm reflected the concentration of π electrons

288

and a structural ordering that was consistent with the presence of sp2 carbon.42 In

289

addition, the pristine graphene resulted in a peak at 230 nm due to the presence of

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6.0% oxygen, as shown by the XPS results. After all treatments, the peak at 270 nm

291

shifted to approximately 230 nm, which is characteristic of the π–π* transition of the

292

C=C in graphene oxide.43 These results are consistent with graphene oxygenation

293

during treatment. The UV-vis absorption decreased remarkably between 250 and 800

294

nm in the treated graphene, which suggested that the electronic conjugation within the

295

pristine graphene sheets was disrupted.44 In addition, the modifications of oxygen and

296

nitrogen altered the d-spacings in the graphene.45 The d-spacing expanded in the

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treated graphene, as shown in Figure 3b, and was linked to alterations in the surface

298

chemistry, as shown in Figure S3. In addition, the disordered structures evident in the 10



299

FETEM images and RS spectra were consistent with the introduction of oxygen and

300

nitrogen groups. These results demonstrated that graphene can be oxidized, and its

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aromatic structure can be rearranged in the natural environment.

302 303

Alteration of the dispersion state

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The initial aggregation period was defined as the period between experimental

305

initiation and when the hydrodynamic diameter (Dh) was 1.5-fold less than its initial

306

value (1.5Dih).46 In this study, the initial aggregation rate was calculated for the period

307

between 0 and 300 s because the Dh of graphene exceeds 1.5Dih after 300 s. The initial

308

aggregation rates were obtained from Dh data fitting using a one-dimensional linear

309

regression equation and Origin 9.0 software.46 As shown in Figure S5, graphene was

310

unstable, and its size increased with time, yielding an initial aggregation rate constant

311

of 1.43 nm/s. The hydrated and irradiated graphene exhibited lower initial aggregation

312

rates (0.53 and 0.38 nm/s, respectively). During the nanomaterial agglomeration

313

period, the nanomaterials undergo rapid agglomeration and moderate agglomeration

314

before remaining relatively stable.47 However, the initial aggregation rate of graphene

315

was fast. Given that graphene suspensions were exposed to C. vulgaris for 5 days

316

(shaken once every 8 h at 150 rmp for 10 min) in the toxicological experiments, the

317

Dh values of the materials were detected on the 5th day under the shaking conditions

318

described above. The final Dh values of the graphene, hydrated graphene, and

319

irradiated graphene, were 2132, 1245 and 836 nm on the 5th day, respectively. The

320

aggregation results demonstrated that irradiation enhanced graphene aggregation,

321

which agreed with the increasing concentrations of oxygen-containing groups.

322

Furthermore, the ζ-potential results confirmed that irradiation improved the dispersion

323

of graphene. As shown in Figure S6, the ζ-potential became negative as the pH

324

increased, which suggested that the surfaces of the three materials exhibited negative

325

charges. Irradiation caused the graphene surface to become more negative, with

326

ζ-potentials ranging from −27 to −38 mV at pH 7−10.

327 328

Generation of free radicals and the kinetics of modification 11


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Previous research showed that aqueous colloidal dispersions of carbon nanotubes and

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nC60 can generate ROS intermediates, including singlet oxygen (1O2), when exposed

331

to solar light.48 In theory, pristine graphene is chemically inert due to the giant

332

π-conjugation system. However, defects and impurities could occur during fabrication,

333

including the formation of nanopores and oxygen-containing groups, as shown in

334

Figures 1 and S3. These defects and impurities are highly photochemical active, likely

335

form free radicals and result in the photomodification of graphene.49 In this study, the

336

generation of 1O2 was investigated using the probe compound, FFA, as shown in

337

Figure S7. A remarkably higher FFA decay rate was obtained for the graphene under

338

irradiation relative to the graphene that was not irradiated. The FFA decay rate

339

constants from hydrated (without light) graphene and irradiated graphene were

340

0.0019/min and 0.022/min, respectively, according to common pseudo first-order

341

kinetics fitting for 1O2,48 which suggested that the irradiation triggered the generation

342

of 1O2.

343

Direct photoreactions generally exhibit first order kinetics.50 However, the kinetics

344

of photomodification exhibit two different stages before and after 24 h, fast and slow

345

photomodification, as shown in Figure S8. The two stages were fit using first order

346

kinetics and resulted in R2 values greater than 0.9. The fast and slow kinetics

347

constants were 6.9×10-3/h and 5.6×10-4/h for hydrated graphene, respectively. For

348

irradiated graphene, the fast and slow kinetic rate constants were 22.3×10-3/h and

349

13.5×10-4/h, respectively. These values implied the presence of at least two

350

components with different photoreactivities. Furthermore, XPS was used to detect

351

alterations of the chemical components during the fast and slow stages. The ratios of

352

C=O to C-OH during the fast (12 h irradiation) and slow stages (120 h irradiation)

353

were 0.71 and 0.12, respectively. These results demonstrated that the rate of graphene

354

photomodification decreased as the ratio of C=O to C-OH decreased.

355 356

Reduction of oxidative stress and DNA damage

357

Excessive ROS generation is a general phenomenon and the primary mechanism of

358

nanotoxicity.51-53 Excessive ROS generation will alter cellular membrane properties, 12



359

damage DNA and reduce enzyme activity.54,55 As shown in Figure 4a, pristine

360

graphene resulted in the generation of more ROS than was observed in the control

361

without graphene exposure. However, hydration and irradiation inhibited the

362

generation of ROS by graphene. The concentrations of MDA reflected the lipid

363

peroxide concentrations.56 Hydration and irradiation reduced the enhancements of the

364

MDA content that were caused by graphene. The carbonyl-protein content is another

365

important indicator of oxidative stress.55 As illustrated in Figure 4, pristine graphene

366

increased the concentration of carbonyl proteins, and hydration and irradiation

367

reduced the protein carbonylation. The proportion of tail DNA reflects the level of

368

DNA damage. The pristine graphene triggered a significant increase in tail DNA

369

relative to the control, and the hydrated and irradiated graphene resulted in less tail

370

DNA. These results demonstrated that ambient hydration and visible-light irradiation

371

reduce the oxidative stress and DNA damage induced by graphene in algal cells.

372 373

Reductions of reproduction inhibition and damage to cellular structures

374

The effects of the hydration and irradiation treatments on algal reproduction and

375

development were investigated, as shown in Figure S9. Compared with the control,

376

the pristine graphene significantly reduced the reproduction of algal cells and the

377

concentrations of chlorophyll a and b, which are indicators of photosynthesis. The

378

hydration and irradiation of graphene reversed these adverse effects. Neither the

379

pristine graphene nor the treated graphene induced any changes in the diameters of the

380

algal cells, which indirectly indicated the agglomeration of the cells, as shown in

381

Figure S9. To explore the roles of chemical compositions on retarding toxicological

382

activities, a comparative study using surface modified graphene (graphene oxide) was

383

conducted. The properties of used graphene oxide are presented in detail in our

384

previous work.57 As shown in Figure S9, graphene oxides remarkably reduced the

385

adverse effects on the number of cells, cell diameter and chlorophyll content,

386

suggesting that oxidative modification may play an important role in decreasing

387

toxicological activity. 13


Page 14 of 29

Page 15 of 29


388

The TEM images of the algal cells confirmed the positive effects of hydration and

389

irradiation, as shown in Figure S10. In the control cells, the cytoplasm membrane was

390

near the intact cell wall. A layered structure of thylakoids was visible in the

391

chloroplasts. In the cells that were exposed to pristine graphene, plasmolysis occurred

392

and the cell wall was ruptured, as indicated by double and single red arrows,

393

respectively. The structure of thylakoids was obscured, as indicated by the pink

394

arrows. In the cells that were exposed to the treated graphene, the cell walls remained

395

intact, and the occurrence of plasmolysis was reduced, especially in the case of the

396

irradiated graphene. Given that the effective diameter of the pores in the cell walls of

397

the algae did not exceed 3 nm, the large graphene nanosheets could not directly cross

398

the cell walls.58 However, the adsorption of graphene onto cell walls or its

399

incorporation into cell walls was observed, as indicated by the yellow arrows. The

400

observed alterations in the cell walls and chloroplasts are consistent with the

401

aforementioned results related to oxidative stress, chlorophyll content and cellular

402

reproduction. These data demonstrate that ambient hydration and visible-light

403

irradiation reduce the graphene-induced inhibition of reproduction and

404

cellular-structure damage of algae.

405 406

Correlations between nanomaterial properties and nanotoxicity

407

The structural defects of carbon nanotubes play a major role in causing toxicity, even

408

genotoxicity,20,59 largely because of their high activity. For pristine graphene,

409

numerous defects (pores and dentate edges) were observed on the nanosheets, as

410

displayed in the AFM image presented in Figure 1a. Generally, the graphene edges

411

are sharp with dangling bonds and are highly reactive to guest atoms or molecules.54

412

Reportedly, these sharp edges can directly rupture the membranes of cells, bacteria

413

and viruses, thereby inducing physical damage to living organisms.55,60,61 Thus, the

414

sharp edges of the pristine graphene caused damage to the cell walls, as illustrated in

415

Figure S10. By contrast, for the treated graphene, the sharp and dentate edges

416

disappeared, and the cell walls that were exposed to the treated graphene remained

417

intact. 14



418

Morphology is another factor that affects graphene nanotoxicity.62 The nanosheets

419

observed in the pristine graphene were transformed into nanoribbons in the treated

420

graphene, as described in Figure 1a. Previous studies showed that graphene with a

421

nanoribbon morphology exhibits a lower phytotoxicity to wheat relative to graphene

422

nanosheets.28 Compared with the thickness of graphene, the thicknesses of the

423

hydrated and irradiated graphene decreased by 19.7% and 56.2%, respectively. This

424

decreased thickness reduced the stiffness and rigidity of the graphene, and the thinner

425

materials were readily deformed by weak forces, such as the water surface tension.33

426

The pathological response to fibers and carbon nanotubes increased with their

427

stiffness,63 and a similar phenomenon was observed for plate-like graphene in this

428

study. The UV-vis absorption spectra reflected the effects of light irradiation, and the

429

reduced visible-light absorption (as shown in Figure S4) suggested that the

430

photoreactivity of the treated graphene was diminished. These results suggest that

431

hydration and irradiation reduced the structural defects, rigidity, photoreactivity and

432

nanotoxicity of the graphene.

433

The irradiated graphene presented more surface negative charges and was more

434

stable than graphene and hydrated graphene, as shown in Figure S5. The negative

435

charges reduce the direct interactions with algal cells because the surfaces of the algal

436

cells are charged.64 In addition, irradiation reduced the aggregation of nanomaterials

437

due to electrostatic repulsion. The aggregation rate of the irradiated graphene was

438

smaller than that of the non-irradiated graphene. The aggregation state of graphene

439

can affect the shape, size and surface area of carbon nanomaterials. This effect is

440

more pronounced for highly agglomerated carbon nanotubes, which results in a

441

greater decrease in the overall DNA content than the better-dispersed carbon nanotube

442

bundles.65 Similarly, the irradiated graphene exhibited a lower toxicity than the

443

graphene that was not irradiated. One possible explanation for this result is that the

444

larger agglomerated materials were stiffer and more rigid, which could result in

445

greater physical damage to organisms.66

446 447

The inhibition of the growth of algal cells exposed to carbon nanotubes was strongly correlated with the shading of the nanomaterials,26 and similar indirect 15


Page 16 of 29

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448

effects were observed for the cells exposed to graphene in this study. Different

449

graphene suspensions (10 mg/L) were placed in a constant-temperature incubator at

450

24°C for 5 days and were stirred once every 8 h. Black layers of graphene formed on

451

the surfaces of the pristine and hydrated graphene suspensions, as indicated by the

452

yellow arrows shown in Figure S11. The suspension of irradiated graphene was more

453

transparent than the other graphene suspensions, and no black layer was observed on

454

its surface. The transparency and presence or absence of a graphene layers on the

455

surfaces of each suspension are consistent with the hypothesis that the observed

456

variations in reproduction and chlorophyll concentrations could be attributed to the

457

shading effects of the graphene, as shown in Figure S9. Considering that transparency

458

was dependent on the level of graphene oxidation,67 the oxygen groups provided a

459

connection between the transparency of the graphene suspension and the inhibition of

460

algal reproduction. Notably, the various physicochemical properties (morphology,

461

defects, structure, surface chemistry, etc.) of nanomaterials were interdependent for

462

determining the nanotoxicity of such materials.8 It is important to consider the

463

systematic effects of these properties when analyzing the nanotoxicity of graphene.

464 465

ASSOCIATED CONTENT

466

Supporting Information Available

467

Additional descriptions of the graphene characterization, protein carbonyls and cellular

468

ultrastructure measurements, figures regarding the characteristics of graphene before

469

and after treatments, and the effects of hydration and irradiation on cellular number,

470

diameter and chlorophyll contents. This information is available free of charge via the

471

Internet at http://pubs.acs.org/.

472 473

AUTHOR INFORMATION

474

Corresponding author

475

* E-mail: [email protected] (Q.Z). Phone: +86-022-23507800; fax:

476

+86-022-66229562.

477 16



478

Notes

479

The authors declare no competing financial interest.

480 481

ACKNOWLEDGMENTS

482

This work was financially supported by the National Natural Science Foundation of

483

China as a general project (grant Nos. 31170473 and 21307061), a joint project (grant

484

No. U1133006) and a key project (grant No. 21037002), the Ministry of Education of

485

China as an innovative team project (grant no. IRT 13024), Tianjin Natural Science

486

Foundation (grant No. 14JCQNJC08900), the Specialized Research Fund for the

487

Doctoral Program of Higher Education of China (grant No. 2013003112016), the

488

Postdoctoral Science Foundation of China (grant No. 2014M550138) and the

489

Fundamental Research Funds for the Central Universities (grant No. 65121006).

490 491

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675 676 677 678 679 680 681 682 683 684 685 686 687 23


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Figure Legends

689

Figure 1. Atomic force microscope images of graphene before and after treatment (a)

690

and the energy-dispersive spectra of the raised dots on the treated graphene (b).

691 692

Figure 2. Field-emission transmission electron microscopy images of pristine

693

graphene, hydrated graphene and visible-light-irradiated graphene. The scale bars of

694

the left- and right-hand images represent 0.2 µm and 10 nm, respectively.

695 696

Figure 3. Raman spectra (a) and X-ray powder diffraction patterns (b) of pristine

697

graphene, hydrated graphene and irradiated graphene. The labels D, G, 2D and D + G

698

denote the typical Raman spectral peaks of graphene. The main peaks in the X-ray

699

diffraction pattern can be subdivided into multiple individual subpeaks (d1, d2, d3 and

700

d4).

701 702

Figure 4. Effects of hydration and visible-light irradiation on the reduction of

703

oxidative stress and DNA damage induced by pristine graphene.

704 705 706 707 708 709 710 711 712 713 714 715 716 717 24



Figures

718

719 720 721

Figure 1. ---------------------

722 723 724 725 726 727 728 729 730 731 732 25


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733 734 735

Figure 2. ------------------

736 737 738 739 740 741 742 743 744 745 26



746 747 748

Figure 3. -----------------

749 750 751 752 753 754 755

27


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756 757 758

Figure 4. -----------------

759

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