Aqueously Released Graphene Oxide Embedded in Epoxy Resin

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Aqueously released graphene oxide embedded in epoxy resin exhibits different characteristics and phytotoxicity of Chlorella vulgaris from the pristine form Xiangang Hu, Weilu Kang, and Li Mu Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017 Downloaded from http://pubs.acs.org on April 24, 2017

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Environmental Science & Technology

Differences of Properties & Toxicity Pristine graphene oxide

Environmentally released graphene oxide

Differences of Environmental Implications

ACS Paragon Plus Environment


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Aqueously released graphene oxide embedded in epoxy resin exhibits different

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characteristics and phytotoxicity of Chlorella vulgaris from the pristine form

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Xiangang Hu†,*, Weilu Kang†, Li Mu‡

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†

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Education), Tianjin Key Laboratory of Environmental Remediation and Pollution

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Control, College of Environmental Science and Engineering, Nankai University,

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Tianjin 300071, China.

Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of

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‡

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China

Institute of Agro-environmental Protection, Ministry of Agriculture, Tianjin 300191,

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*Corresponding author, Xiangang Hu

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College of Environmental Science and Engineering, Nankai University, Tianjin

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300071, China. Phone: 86-22-23507800; fax: 86-22-66229562; E-mail:

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[email protected]

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ABSTRACT

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The environmental release of nanoparticles is attracting increasing attention.

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Graphene oxide (GO) embedded in epoxy resin (ER) is a popular composite that has

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been used in various fields, but the environmental release of GO-ER composites and

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the effects on organisms in the environment remain unknown. The present work found

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that GO-ER composites in water for 2-7 days resulted in the release of 0.3-2.1%

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GO-ER at nanoscale (2-3 nm thickness and approximately 70-130 nm lateral length).

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Interestingly, pristine GO quenched 30-45% hydroxyl and 12% nitroxide free radicals,

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whereas this capacity was not observed for the released particles from GO-ER. At

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environmentally relevant concentrations (µg/L), released GO-ER particles, but not

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GO or ER matrix, promoted algal reproduction by 34% and chlorophyll biosynthesis

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by 65-127% at 96 h. Released GO-ER entered algal cells and induced a slight increase

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in reactive oxygen species but did not elicit notable cell structure damage. The

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upregulated amino acids and phenylalanine metabolism, and the downregulated fatty

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acid biosynthesis contributed to algal growth promoted by released GO-ER. Previous

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studies of pristine nanoparticles were unable to reflect the environmental effects of

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released nanoparticles into the environment, and our research on the

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exposure-toxicological continuum adds important contributions to this field.

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Keywords: Environmental release; Environmental transformation; Graphene oxide;

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Chlorella vulgaris; Phytotoxicity; Metabolism

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INTRODUCTION

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Great interest in graphene-based materials has recently arisen, due to their 2D

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structure, high chemical stability, and unique electronic and mechanical properties.1,2

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In graphene-based nanomaterials, graphene oxide (GO) can be dispersed in many

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solvents, particularly in water, due to its oxygen functional groups. GO attracts much

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attention for large-scale production and multiple applications in energy storage,

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electrocatalysis, biosensors, biomedicine and the removal of environmental

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pollutants.3,4 In general, GO-based products or composites, rather than pristine GO,

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are released into the environment during their life cycle, because they are

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incorporated into a matrix or present on the surface of a matrix during their

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manufacture.5 Therefore, studying the environmental implications of the GO released

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from composites or products is more meaningful than studying the effects of pristine

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GO.

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Epoxy resin (ER) has been widely used as a matrix for polymer-based

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nanocomposites due to its unique stiffness, dimensional stability, chemical resistance

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and strong adhesion.6 An increasing number of reports discuss the release of

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nanoparticles from composites into the environment, but these have mainly focused

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on a few specific nanomaterials, such as nano-Ag, nano-TiO2, nano-SiO2 and carbon

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nanotubes.5,7 Although the deposition and release of GO on mineral or silica surfaces

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were reported,8 information about the released particles from GO-ER composites

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remains unclear.

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Currently, the studies on nanoparticle release involve the effects of various 3


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scenarios (e.g., nanomaterial synthesis, use and disposal types, and leaching solution)

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on nanoparticle release and the preliminary characterization of released

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nanoparticles.7-12 Ion strength, organic matters, coagulants, conductivity and

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irradiation influenced the deposition and release of GO on solids.8 Physicochemical

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characterizations (e.g., size, morphology and quantified mass release) of nanoparticles

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released from products or composites are necessary, and comparisons of the

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characteristics of released and pristine nanoparticles are more valuable for

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understanding what occurs during release. Notably, characterizations of size and

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morphology and quantitative data on mass release are not sufficient to elucidate

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nanoparticle release. We wondered whether the environmentally or biologically

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relevant activities of released nanoparticles differ from those of the pristine forms.

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Unpaired electrons are a remarkable characteristic of nanoparticles. Their interactions

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with free radicals are related to their environmental transformation and biological

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toxicity.4,13 However, information about released nanoparticles is lacking.

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Although the nanoparticles released from simulated products or composites may be

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found at concentrations up to several to hundreds of parts per million (ppm) under

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acidic corrosion and intensive sanding conditions, such high concentrations are not

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possible in a real environment.7,14,15 According to estimates derived from

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mathematical models, environmentally relevant concentrations of engineered

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nanomaterials (e.g., Ag nanoparticles, carbon nanotube and fullerenes) should be in

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the parts per billion (ppb) or parts per trillion (ppt) ranges in surface water (data for

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GO are not currently available).16,17 Frequent reports have indicated that pristine 4



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nanoparticles including GO at ppm levels could induce plasma membrane damage,

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impairment of mitochondrial activity, oxidative stress increase, and protein/gene

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DNA/RNA damage, eventually leading to inhibition of biological development,4,18

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although there were a few reports where GO improved the growth of some

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microorganism, plants or cells.8,18 In contrast, the effects of nanoparticles released at

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trace concentrations from composites on organisms in the environment are rarely

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reported.

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To address these questions, the released particles from a GO-ER composite were

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characterized in detail (e.g., size, morphology, quantified mass release, unpaired

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electrons and interactions with free radicals) for comparison with pristine GO.

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Subsequently, the biological effects of both released and pristine GO at trace

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concentrations (1-100 µg/L) on a single-cell algal species, Chlorella vulgaris as a

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typical aquatic organism, were investigated. Finally, the specific algal responses to

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both released and pristine GO, and the related mechanisms were explored. The

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present study covers the release-toxicological continuum of nanoparticles and

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provides insight into the specific properties and effects of released nanoparticles on

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organisms in the environment with comparison to the effects of the pristine forms.

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MATERIALS AND METHODS

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Preparation of the GO-ER composite and nanoparticle release

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GO nanosheets (production number: XF002-2) were prepared by the classic Hummers’

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method and obtained from Nanjing XFNANO Materials Tech Co., Ltd, China. The 5


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GO-ER composite was prepared with modifications of procedures described in the

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literature.19 Briefly, 0.3 g GO was grinded into a homogeneous paste in 5 mL acetone.

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Then, the GO suspension was dispersed in 60 mL acetone and sonicated at 100 W for

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30 min in an ice-water bath. Next, 1.8 g ER was added to the GO suspension, which

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was then stirred and sonicated at 60℃ to remove acetone. Subsequently, 0.9 mL

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methyltetrahydrophthalic anhydride and 0.03 g 1,2-dimethylimidazole were added

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and stirred for 1 h at 60℃. Finally, the paste was solidified in a cylindrical stainless

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steel groove (1.0 cm diameter and 0.22 cm height) at 100℃ for 12 h. The effects of

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leaching solution chemistry on nanoparticle release have been widely studied,7-12 and

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similar investigations were not repeated in the present study. Next, 0.2 g solidified

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composition was added to a 15 mL brown vial (diameter, 20.6 mm; height, 71 mm)

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with 5 mL deionized water, as illustrated in Figure S1. The diameter and height of

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bulk GO-ER composites were approximately 1.0 cm and 0.22 cm, respectively. For

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comparison with the release duration (24 h-7 days) of other carbon-based

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references,5,20,21 a release experiment was conducted for 7 days at 25℃ with gentle

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shaking at 150 r/min. As a control experiment, an ER composite without GO was

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prepared following the same process, and the release experiment for the ER

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composite alone was performed similarly to the GO-ER composite experiment. To

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determine the released GO concentrations, a subexperiment with palladium-labeled

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GO was used. The preparation of palladium-labeled GO was conducted based on

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procedures described in a previous study,22 and the labeled palladium accounted for

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5.7% of the total mass. Preparation of the palladium-labeled GO-ER composite and 6



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the release processes were conducted in the same manner as the GO-ER experiments.

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The released GO was indirectly quantified using the labeled palladium and

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inductively coupled plasma optical emission spectrometry (700-ES, Varian, USA).

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The routine methods for the digestion and detection of palladium have been described

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in a previous work.23 To avoid the influence of palladium on biological effects, GO

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without palladium was used in the test of biological effects, such as algal growth,

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oxidative stress and metabolism analysis. To determine the effects of shear stress on

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the release of GO-ER, GO-ER composites were placed in glass vials (one composite

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per vial) for shaking at 0, 50 and 150 r/min in a sub-experiment, respectively. To

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analyze the effects of particle surface on the release of GO-ER, GO-ER composites

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with 0.74, 1.56 and 2.26 cm2 surface areas (corresponding diameters 0.5, 0.8 and 1.0

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cm with the same height 0.22 cm, respectively) were prepared and analyzed in a

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sub-experiment.

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Nanoparticle characterization

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The characterized particles included the pristine GO and the particles released from

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the GO-ER or ER compositions. Details of the nanoparticle characterization are

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included in our previous studies.24 The hydrodynamic diameters of the released

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particles for release durations of 2, 4 and 7 days were measured using dynamic light

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scattering (Nano-ZS90, Malvern, England). The zeta potentials of pristine GO and

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released particles at 7th day were obtained by performing dynamic light scattering

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using a ZetaPALS instrument that was equipped with a 30 mW 635 nm laser

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(BI-200SM, Brookhaven, USA). To study the nanoparticle morphology, atomic force 7


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microscopy (AFM) was conducted using a Nanoscope IV (VEECO, USA). The

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reactions between the free radicals and nanoparticles were detected using an electron

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paramagnetic resonance (EPR) spectrometer (MagnetTech MiniScope 400, Germany)

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with MiniScope Control software. 5,5-Dimethyl-1-pyrroline (DMPO) (25 µL, 0.5 M)

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was used as an unpaired electron probe to detect the unpaired electrons on the

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nanoparticles. Then, 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) was employed as

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the source of nitroxide free radicals. A typical Fenton reaction was used as the source

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of hydroxyl radicals. The EPR spectrometer was operated at a microwave frequency

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of 9.4 GHz and a magnetic field modulation frequency of 100 kHz at 296 K. Raman

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spectra were obtained using the 780 nm laser of a DXR Microscope (Thermo

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Scientific, USA). Fourier transform infrared (FTIR) spectra were recorded on a

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Bruker Tensor 27 infrared spectrometer with a resolution of 2 cm−1 from 4000 to 400

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cm−1.

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Chlorella vulgaris cultivation

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Chlorella vulgaris and Blue-Green culture medium (BG-11) were purchased from the

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Freshwater Algae Culture Collection at the Institute of Hydrobiology, China. All

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vessels used to contain the microalgae were sterilized first. Based on the estimated

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concentrations (ng-µg/L) of carbon nanotubes or fullerenes in surface water (data for

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GO were not reported),16,17 the released nanoparticles at trace concentrations (1, 10

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and 100 µg/L) were prepared in BG-11 culture medium for the toxicological

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experiments. The initial concentration of the used particles released from GO-ER 8



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composite was 1.3 mg/L, which was diluted to 1, 10 and 100 µg/L directly by once

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dilution using BG-11, respectively. To make the suspension uniform, the suspension

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at low concentrations was sonicated for 5 min at 100 W before and after dilution. In

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addition, 100 µg/L of the matrix released from ER composites on the 7th day was used

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a matrix control. Pristine GO at 100 µg/L was tested as a nanomaterial control. Prior

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to exposure experiments, the initial concentration of algal cells was approximately

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6×104 cells/mL. Previous study showed that GO could be adsorbed on algae.25 To

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mitigate the settling of released particles with algae, algal suspensions with and

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without released particles were shaken once every 8 h and placed in a light incubator

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with 566 µmol/m2/s irradiation and 80% humidity.

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Algal growth and chlorophyll assay

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Cell number was determined at 0, 24, 48, 72 and 96 h. After 96 h, the density of cells

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in the control had increased to 1.65×105 cells/mL. To avoid the influence of high cell

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density on cell reproduction and allow comparison with the exposure times of other

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reports,26,27 the algal exposure experiment was conducted up to 96 h. Direct

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microscopic counts were performed on the samples of the algal cell suspension using

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a counting chamber (Qiujing, XB-K-25, China) and an inverted fluorescence

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microscope (Olympus, CKX41, Japan). To measure the contents of chlorophyll a and

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chlorophyll b, 5 mL of the algal cell suspension was centrifuged at 3600 g for 15 min.

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Subsequently, 5 mL of methanol was added to the suspended cells and incubated in

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the dark for 30 min at 45℃. The mixture was centrifuged at 3600 g for 15 min. The

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supernatants were measured at 652 nm and 665 nm using a UV-vis spectrophotometer 9


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(TU-1900, Persee, China), according to procedures described in the literature.28

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Chlorophyll a (µg/mL)=-8.096A652+16.52A665. Chlorophyll b

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(µg/mL)=27.44A652-12.17A665.

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Reactive oxygen species and transmission electron microscopy imaging

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These are routine methods; thus, more information about these methods is presented

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in the Supporting Information.

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Metabolic analysis

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Details about the metabolic analysis can be found in our previous study,24 as well as

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in the Supporting Information.

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Statistical analysis

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Five independent replications for each group and their error bars are presented as the

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

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at the level of P4)

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layer graphene sheets are typically >1.6, ~ 0.8, ~ 0.30 and ~ 0.07, respectively.31 The

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2D/G ratios of pristine GO and released GO-ER particles were 0.08 and 0.04,

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respectively, which was consistent with the few-layer graphene structure in GO

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(approximately1.5 nm thickness) and released GO-ER particles (approximately 3.0

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nm thickness) in the AFM images. The surface charges of particles were detected by

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zeta potentials with various pH values. As shown in Figure S2, pristine GO and

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released particles from GO-ER composite exhibited negative charges at the pH

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(approximately 7.4) of algal culture medium. The released GO-ER particles presented

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more negative charges than pristine GO. Given the negative charges on algal cells,25

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the negative charges on the released particles did not facilitate their direct interactions

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with algal cells. FTIR was used to analyze the interactions between GO and ER in the

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released particles from GO-ER composites. As shown in Figure S3, there was no C=O

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peak at 1510 cm-1 for the pristine GO sample, while C=O peak was observed in the

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released GO-ER particles due to the ring opening of immobilized ER. The C-OH peak

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in the released GO-ER particles was obviously weaker than that in both of ER and

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GO samples, implying that GO and ER bounded together in the released GO-ER

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particles probably through C-OH groups. 12



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The released GO concentrations were quantified using palladium-labeled GO

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(transmission electron microscopy and element analysis of palladium-labeled GO are

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presented in Figure S4 and Table S1, respectively, of the Supporting Information).

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The released concentrations of GO at 2, 4 and 7 days were 0.16, 0.85 and 1.3 mg/L,

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respectively, which accounted for 0.3%, 1.4% and 2.1% of the total GO, respectively.

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The release of particles from GO-ER composites reduced with the decrease of shaking

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speed. At static, 50 r/min and 150 r/min conditions, the concentrations of released

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particles were 0.14, 0.32 and 1.3 mg/L (Figure S5a), respectively, implying that the

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shear stress could promote particle release. Similarly, the release of particles increased

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by 238% and 287% when the surface areas of original bulk GO-ER composites

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increased from 0.74 cm2 to 1.56 cm2 and 2.66 cm2, respectively, as shown in Figure

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S5b. A composite of carbon nanotubes in polyurethane that was immersed in fresh

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water for 24 h released 0.7% carbon nanotubes,32 which was comparable to the

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present result. In contrast, most materials released from the carbon nanotube

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composites via sanding, sawing and abrasion processes for 24-48 h were usually on

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the micrometer scale, and the concentrations in the leaching solution were up to

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hundreds of mg/L.33,34 In general, the released concentrations depended on solution

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chemistry, polymer type and use scenarios.7,11 Ions easily released into the water

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through diffusion, desorption and dissolution; for example, 7%-75% of the total Ag

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imbedded within a textile fiber was released into artificial sweat solutions after 24 h

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of migration.35,36 In contrast, matrix degradation may be the main pathway for the

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release of carbon-based nanomaterials,37 as confirmed by the released matrix of ER in 13


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Figure 1c. In other words, the particles that were released to the environment during

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use or after their disposal were composites of the matrix (e.g., ER) with nanoparticles

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(e.g., GO) rather than the pristine nanoparticles. However, the environmentally

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relevant activity and toxicity of nanoparticles embedded in a matrix are rarely

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studied.7,11 The next section addresses this knowledge gap.

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Environmentally relevant activity of released nanoparticles

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Unpaired electrons on nanoparticles and their interactions with free radicals are

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closely related to the nanoparticle environmental transformation and biological

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toxicity.4,13 As shown in Figure 2a, with DMPO as a free radical probe, matrix ER

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alone induced an EPR signal at g= 2.1060. GO-ER produced a stronger EPR signal

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than GO at g=2.1064, which was associated with carbon-related dangling bonds in

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graphene planes and edge.38 These carbon-related dangling bonds were the active sites

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of the reaction of graphene-based nanomaterials in environmental and biological

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matrices.39,40 Hydroxyl radicals are important signaling molecules in plants and were

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extremely reactive with DNA, RNA, lipids, and proteins.41 As shown in Figure 2b,

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GO inhibited 30-45% of the hydroxyl radical signals (peak height), while the GO-ER

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and matrix ER had no obvious effect on the hydroxyl radical signals. The capacity to

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quench hydroxyl radical signals was most likely due to the sp2 structure of GO, in

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which the unpaired electron could reside on the oxygen atom, or on ortho or para

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carbons on the adjacent aromatic rings,42 but this capacity was suppressed when GO

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was imbedded in the ER matrix (i.e., released GO-ER particles). In addition, there

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were some reports regarding the generation of ROS by GO. On one hand, excited 14



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electron transferred to GO, which reduced electron-hole recombination and improved

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the generation of ROS in environmental matrix.43 On the other hand, GO damaged

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cell structures (e.g., mitochondrion), and promoted oxidative stress and the generation

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of ROS in cells.24 Symbiotic interactions occurred between reactive oxygen species

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(ROS) and nitroxide free species (NFS), and both are critical for plant defense and

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development.44 Therefore, the interactions between nanoparticles and NFS (TEMPO

315

as a typical NFS source) were studied. As shown in Figure 2c, GO and ER

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significantly inhibited approximately 12% and 25% of the NFS signals (peak height),

317

respectively, while GO-ER had limited influence on the NFS signals. These

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alterations may be related to changes in the carbon-related dangling bonds in the

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graphene planes and edge of GO-ER.38 Furthermore, the main carbon groups of

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nanoparticles were analyzed using FTIR, and the specific groups were identified using

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procedures described in the literature.45 As shown in Figure 2d, compared with those

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of GO and ER, the particles released from GO-ER showed weak C-OH and C-H

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bands. In contrast, compared with GO, GO-ER exhibited a strong C=O band and 50

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cm-1 bathochromic-shift of the C=C bands (denoted by the green arrow in Figure 2d).

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Released particles at trace concentrations promote algal growth

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Figure 3a does not obviously show growth inhibition (cell density). In contrary, the

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slightly activation of algal growth under GO and released GO-ER particle exposure

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was observed, especially after 72 h. Pristine GO slightly enhanced algal growth by 9.8%

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at 96 h compared with the control. Compared with control, the alterations in GO

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exposure group before and after 72 h implied the tolerance and adaptive responses of 15


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the algae to stress.24 Compared with control, the alterations in GO exposure groups

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were not significant (P>0.05) probably due to the short exposure time (up to 96 h),

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while the cell density in the released GO-ER particle group was 34.3% higher than

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that in the control group at 96 h. Nanoparticles improved plant growth most likely by

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improving photosynthetic efficiency.46 Furthermore, biosynthesis of chlorophyll a and

336

chlorophyll b was detected, as shown in Figure 3b. The contents of chlorophyll a and

337

b were decreased by 8.5-16% and 8.8-8.9% via released ER and pristine GO,

338

respectively. In contrast, the contents of chlorophyll a and chlorophyll b were

339

increased by 68-102% and 65-127%, respectively, by GO-ER. The chlorophyll

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contents were lower for 10 µg/L GO-ER than for both 1 µg/L and 100 µg/L GO-ER.

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Similar phenomena were observed for zebrafish exposed to trace pristine GO, and

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these varying responses were most likely due to nanoparticle aggregation with

343

increasing concentrations.47 As verified in Figure S6, the hydrodynamic diameters of

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GO-ER (released at 7th day) at 1, 10 and 100 µg/L were 296, 323 and 379 nm,

345

respectively. Biological responses were affected by the structure, composition and

346

sizes of nanoparticles together,18,48 and the released particle characterizations are

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presented in Figures 1 and 2. Together, the remarkably different responses of the algae

348

to pristine and released GO demonstrates that the pristine forms may not exactly

349

reflect the potential environmental risks of nanoparticles.

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Algal uptake of released nanoparticles with increasing ROS

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Our recent work confirmed that pristine GO could enter algal cells and induce

352

obvious plasmolysis and cell wall damage at 100 µg/L.49 ER was also highly 16



353

biocompatible with algal cells.50 However, the algal uptake and ultrastructural

354

responses to the released GO with ER matrix remain unclear. For comparison with

355

previous studies,49 transmission electron microscope (TEM) and Raman spectra were

356

performed to analyze the uptake of released GO-ER at 100 µg/L. In the present work,

357

The algal cells in the control (Figures 4a) and GO-ER (Figures 4b) groups exhibited

358

an intact structure, except for cell wall damage (denoted by red arrows). The pore

359

sizes of plant cell walls are approximately 20 nm, and the pierced cell walls are a

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pathway for the uptake of large nanoparticles.29,48,51 The nanotoxicological

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mechanisms of GO-based nanomaterials probably linked to cell wall membrane

362

damage by their sharp edges and wrapping/trapping cells by their aggregation,52-55

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although the latter phenomenon was not obvious in the present work. Some large,

364

black dots (denoted by green arrows) were observed in the cells exposed to GO-ER

365

(Figure 4b). To determine whether these dots contained GO, Raman spectrum

366

experiments were conducted. As shown in Figure 4c, the typical D and G bands were

367

detected at approximately 1360 and 1590 cm-1, respectively, which confirmed that the

368

released GO entered algal cells. The redox state of graphene-based structure could be

369

detected by the ratios of D to G peaks from Raman spectrum.56 The ratios of D to G

370

peaks of the released GO-ER particles before and after entered algal cells were 0.94

371

and 0.68, respectively, suggesting that graphene-based structure was oxidized

372

furthermore by cell matrix. In general, the oxidation of graphene-based structure as a

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passivation could mitigate its toxicity.17,57 The chloroplast structure in the GO-ER

374

groups was denser than that in the control group (denoted by yellow arrows in Figures 17


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4a and 4b), which was consistent with the chlorophyll results in Figure 3b. Our

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previous work showed that the plasmolysis areas due to the pristine GO exposure at

377

100 µg/L accounted for more than 10% of the whole cells,49 while GO-ER did not

378

induce obvious plasmolysis at the same concentration (Figure 4b). Moreover, the

379

uptake of graphene-based materials with small lateral sizes could induce genotoxicity,

380

such as DNA fragmentations and chromosomal aberrations,58 which deserves

381

attention.

382

There were no obvious differences in the ROS levels between GO and control

383

groups (Figure 4d). The slightly lower ROS level may be due to the capacity of GO to

384

quench free radicals, as shown in Figures 2b and 2c. However, GO-ER promoted the

385

39-65% increase in the relative ROS levels. ER also induced a 28% increase in ROS

386

levels. Increased ROS levels are considered the primary mechanism by which

387

nanoparticles induce adverse effects.59 This conclusion deserves reconsideration,

388

especially for plants. ROS are toxic byproducts of aerobic metabolism but are also

389

required for many important signaling reactions, including the cellular proliferation

390

and differentiation of plants.41 Increased ROS (but not too much) is beneficial as a

391

plant defense against external stress.41,60 These theories are consistent with the results

392

of increases in both algal growth and ROS levels in Figures 3 and 4d, respectively. In

393

addition, ROS have a role in stress perception and protection as a result of triggering

394

secondary metabolisms.61 These metabolic responses are analyzed and discussed in

395

the next section.

396

Released nanoparticles mediate algal growth via metabolism changes 18



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The untargeted metabolomics assay enables the discovery of novel modes of

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nanoparticle-biological interactions and provides a representation of the global

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biological response to stress.62 As presented in Figure S7a, nanoparticles altered the

400

metabolisms of amino acids, carbohydrates, alkanes, fatty acids and other small

401

molecular acids. Through HCL analysis, samples were divided into two clusters:

402

released GO-ER and others (control, GO and ER). A similar result was observed using

403

PCA (Figure S7b). There were 14 metabolites (e.g., sucrose, alanine, serine and

404

phenylalanine) that were obviously upregulated by exposure to ER, GO and GO-ER

405

(Figure S7a). The main metabolic pathways of these upregulated metabolites are

406

indicated in Figure S7c. The pathways for glycine, serine, threonine and

407

phenylalanine metabolisms were remarkably impacted (pathway impact more than

408

0.4). Phenylalanine metabolism is an important source of secondary metabolites in

409

plant cells. Multi-walled carbon nanotubes trigged biosynthesis of secondary

410

metabolites in Satureja khuzestanica with increased ROS levels,61 which was

411

consistent with the work presented in Figure 4d. The pathways of aminoacyl-tRNA

412

biosynthesis and glutathione metabolism exhibited obvious differences (-log(p) more

413

than 8) compared with the control. Palmitoylglycerol alone was downregulated by ER,

414

GO and GO-ER together. Five metabolites, such as dodecanoic acid, palmitic acid and

415

monopalmitin, were downregulated by the released GO-ER but upregulated by both

416

GO and ER. Metabolic pathway analysis showed that these metabolites were mainly

417

related to fatty acid biosynthesis and metabolism. CeO2 nanoparticles have also

418

affected fatty acid metabolism and promoted wheat yield.63 The toxic effects of GO 19


Page 20 of 38

Page 21 of 38


419

were typically observed at mg/L levels.56 In the present work, the adverse effects of

420

GO and released GO-ER particles at trace concentrations were observed, probably

421

due to the non-monotonic dose-response relationships, as proposed in the previous

422

work.57,64

423

To explain the specific mechanisms of algal growth induced by GO-ER, the

424

connections between algal metabolites and algal cell density were analyzed using

425

OPLS-DA. Algal metabolites (peak abundance) and algal cell density were set as the

426

X and Y variables, respectively. Variable importance in projection (VIP) plots are

427

presented in Figure S8. VIP values greater than 1 for some metabolites (e.g., sucrose,

428

alanine, glycerol monostearate) suggest that these metabolites make substantial

429

contributions to algal growth. To distinguish the negative and positive contributions of

430

metabolites to algal growth, coefficient CS (coeffCS) was analyzed, and the

431

metabolites with VIP values greater than 1 were labeled with asterisks, as shown in

432

Figure 6a. The metabolites (e.g., glycerol monostearate, monopalmitin and stearic

433

acid) with a negative coeffCS provided negative contributions to algal growth. In

434

contrast, other metabolites (e.g., alanine, glycine and sucrose) played positive roles in

435

algal growth. Furthermore, the linear correlations between the abundance of

436

metabolites (VIP>1.3) and algal cell density are plotted in Figure 6b. Alanine

437

biosynthesis had a positive correlation with algal cell density at R2=0.86. In contrast,

438

glycerol monostearate had a negative correlation with algal cell density at R2=0.73.

439

This analysis provides insight into the mechanisms of algal growth promotion and can

440

be used to screen the specific metabolites correlated with the growth induced by 20



441 442

released nanoparticles. The environmental release of nanoparticles from products or composites is

443

attracting increasing attention. As a popular composite, GO-ER has been used in

444

various fields, but the environmental release of GO-ER composites and the effects of

445

released particles on organisms in the environment remain unknown. We first

446

illustrated the environmental release of GO-ER composite and then explored its

447

specific effects on green algae. For the environmental release of GO-ER composite,

448

we discovered that the released particles present remarkably different characteristics

449

(e.g., size, morphology, quantified mass release, unpaired electrons and interactions

450

with free radicals) from those of the pristine form. However, it is clear that the release

451

of materials depends on their immobilization ways.7,10,11 Moreover, in contrast to the

452

released matrix or pristine GO, the released GO-ER at trace concentrations promoted

453

algal growth via metabolism regulation. In the past decades, attention has been

454

focused on pristine nanoparticles.4,59 However, there are obviously different properties

455

and biological responses between released and pristine nanoparticles. In the future,

456

more attention will be needed in regards to environmentally released nanoparticles,

457

especially particles at environmentally relevant activity and concentrations.

458 459

ASSOCIATED CONTENT

460

Supporting Information

461

Method of reactive oxygen species, transmission electron microscopy imaging,

462

metabolic analysis and the related figures. 21


Page 22 of 38

Page 23 of 38


463

AUTHOR INFORMATION

464

Corresponding Author

465

*E-mail: [email protected]

466

Notes

467

The authors declare no competing financial interest.

468

Acknowledgment

469

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

470

China (grant nos. 21577070, 21307061 and 21407085), the Tianjin Natural Science

471

Foundation (grant nos. 14JCQNJC08900 and 16JCQNJC08400) and the State Key

472

Laboratory of Pollution Control and Resource Reuse Foundation (grant no.

473

PCRRF16023).

474

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Figure Captions

661 662

Figure 1. Characterization of released particles from composites. a, Hydrodynamic

663

diameters of released particles; b, atomic force microscope images of pristine

664

graphene oxide (GO); c, particles released from the ER composite without GO at the

665

7th day; d, particles released from the graphene oxide-epoxy resin (GO-ER) composite

666

at the 7th day; and e, Raman spectra of pristine GO and released particles at the 7th

667

day.

668 669

Figure 2. Unpaired electrons on nanoparticles and their interactions with free radicals.

670

a, Electron paramagnetic resonance (EPR) signals with DMPO as an unpaired

671

electron probe; b, EPR signals of hydroxyl radicals from the typical Fenton reaction; c,

672

EPR signals of nitroxide radicals from TEMPO; d, Fourier transform infrared spectra.

673

DMPO, 5,5-Dimethyl-1-pyrroline; TEMPO, 2,2,6,6-Tetramethyl-1-piperidinyloxy.

674

The tested concentrations of released GO-ER or ER and of pristine GO were 100

675

µg/L.

676 677

Figure 3. Released particles at trace concentrations promoting algal growth. a,

678

Increase in algal cell density with time; and b, contents of chlorophyll a and b. The

679

GO-ER exposures at 1, 10 and 100 µg/L are labeled GO/ER-1, GO/ER-10 and

680

GO/ER-100, respectively.

681

Figure 4. Algal uptake of released nanoparticles with increasing reactive oxygen

682

species (ROS) levels. a, Transmission electron microscopy imaging of control algae 31


Page 32 of 38

Page 33 of 38


683

without nanoparticle exposure; b, transmission electron microscopy imaging of algae

684

exposed to released GO-ER at 100 µg/L for 96 h; c, Raman spectra of the dark dots in

685

the transmission electron microscope sections; and d, relative levels of ROS in algal

686

cells. The GO-ER exposures at 1, 10 and 100 µg/L are labeled GO/ER-1, GO/ER-10

687

and GO/ER-100, respectively.

688 689

Figure 5. Connections between algal metabolism and cell density under nanoparticle

690

exposure. a, Connections between algal metabolism and cell density analyzed by

691

OPLS-DA (* denotes the metabolites with VIP>1.0); and b, linearity correlations

692

between the abundance of algal metabolites (VIP>1.3) and cell density. OPLS-DA is

693

orthogonal projections to latent structures discriminant analysis; VIP is variable

694

importance in projection.

695

32



696 697

Figure 1.

698

33


Page 34 of 38

Page 35 of 38


400

GO ER GO/ER

-100

Signal amplitude (a.u.)


0

-200 -300

GO

•OH

ER

GO/ER

200 0 -200 -400 -600 -800

-400

-1000 316

TEMPO

GO

324

ER

318 1.04

GO/ER

6000

Transmission (a.u.)


9000

318 320 322 Magnetic field (mT)

3000 0 -3000 -6000

GO

ER

322

GO/ER

1.00

0.96

0.92

C-OH

C=C CH

C=O 0.88

-9000 318

320 Magnetic field (mT)

322

0.84

699 700

320 Magnetic field (mT)

Figure 2.

701

34


C-O; C-O-C 1000

2000

3000

Wavenumber (cm-1)

4000


b

Concentrations (µg/mL)

Cell density (105cells/mL)

a

3.5 2.5 2.0 1.5 1.0 0.5 0

3.0 2.5 2.0 1.5 1.0 0.5 0.0

702 703

Control GO/ER-100 GO/ER-10 GO/ER-1 ER GO

3.0

24

48 72 Time (hour)

Chlorophyll a

trol -100ER-10/ER-1 ConO/ERG O / GO G

96

Chlorophyll b

ER GO

Figure 3.

704 705

35


Page 36 of 38

Page 37 of 38


706 707

Figure 4.

708

36



Urea Glycerol monostearate * Monopalmitin * Ethanolamine Putrescine Phenylalanine Ornithine 5-Oxoproline Lysine Threonine Serine Proline Glycine Alanine Palmitoylglycerol Inositol Stearic acid Palmitic Acid Boric acid Dodecanoic acid Glutamic acid Aminobutanoic acid Hexanoic acid Phosphoric acid Alkane Sucrose

*

* * *

* * * *

* * *

Relative abundance of metabolites (a.u.)

-0.15 -0.10 -0.05 0.00 0.05 CoeffCS

3.0 2.5 2.0 1.5

0.10

0.15

Alanine Sucrose Glycerol monostearate Monopalmitin

R2=0.56 R2=0.68

1.0 R2=0.86 0.5 0.0 1.5

R2=0.73 2.0 2.5 Cell density (105cells/mL)

3.0

709 710

Figure 5.

37


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Aqueously Released Graphene Oxide Embedded in Epoxy Resin

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