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Oct 7, 2013 - Graphene layers are potential candidates in a large number of applications. However, little is known about their ecotoxicological risks ...
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Biological Uptake and Depuration of Radiolabeled Graphene by Daphnia magna Xiangke Guo, Shipeng Dong, Elijah J Petersen, Shixiang Gao, Qingguo Huang, and Liang Mao Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es403230u • Publication Date (Web): 07 Oct 2013 Downloaded from http://pubs.acs.org on October 13, 2013

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Biological Uptake and Depuration of Radio-labeled Graphene by

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Daphnia magna

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Xiangke Guo†,‡,# , Shipeng Dong†,# , Elijah J. Petersen§, Shixiang Gao†, Qingguo Huang┴,

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Liang Mao†,*

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State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment;

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Lab of Mesoscopic Chemistry, School of Chemistry and Chemical Engineering, Nanjing

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University, Nanjing 210093, P. R. China;

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§

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of Standards and Technology, 100 Bureau Drive, Stop 8311, Gaithersburg, MD 20899-0001,

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USA;

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States

Material Measurement Laboratory, Biosystems and Biomaterials Division, National Institute

Department of Crop and Soil Sciences, University of Georgia, Griffin, GA 30223, United

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Address correspondence to L. Mao, State Key Laboratory of Pollution Control and Resource

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Reuse, School of the Environment, Nanjing University, Nanjing 210093, P. R. China.

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Telephone: (86)25-89680393. Fax: (86)25-89680393. E-mail: [email protected].

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Author contributions: # These authors contributed equally to this work.

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ABSTRACT

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Graphene layers are potential candidates in a large number of applications. However, little is

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known about their ecotoxicological risks largely as a result of a lack of quantification

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techniques in complex environmental matrices. In this study, graphene was synthesized by

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means of graphitization and exfoliation of sandwich-like FePO4/dodecylamine hybrid

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nanosheets, and

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artificial freshwater and the uptake and depuration of graphene by Daphnia magna were

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assessed. After exposure for 24 h to a 250 µg/L solution of graphene, the graphene

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concentration in the organism was nearly 1% of the organism dry mass. These organisms

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excreted the graphene to clean artificial freshwater and achieved roughly constant body

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burdens after 24 h depuration periods regardless of the initial graphene exposure

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concentration. Addition of algae and humic acid to water during the depuration period

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resulted in release of a significant fraction (~90%) of the accumulated graphene, but some

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still remained in the organism. Accumulated graphene in adult Daphnia was likely transferred

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to the neonates. The uptake and elimination results provided here support the environmental

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risk assessment of graphene and the graphene quantification method is a powerful tool for

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additional studies.

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C was incorporated in the synthesis.

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C-labeled graphene was spiked to

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INTRODUCTION

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Graphene, first isolated in 2004, is a flat monolayer of carbon atoms tightly packed into

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a two-dimensional lattice.1,2 Two-dimensional graphene consists of a single layer or up to 10

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layers,1,3 exhibiting exceptionally high crystal and electronic quality that has a number of

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potential applications.4-7 It is inevitable that graphene will enter the environment in increasing

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amounts with usage in consumer products, but their ecological risks are largely unknown.

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Thus far, it has been documented that graphene induces cytotoxic effects such cell membrane

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damage and bacterial toxicity.8-10 Furthermore, it was also demonstrated recently that

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graphene can even show some genotoxic effects on human stem cells like DNA

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fragmentations and chromosomal aberrations.11,12

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One critical factor is the extent to which graphene bioaccumulates in organisms and

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spreads through food chains.13 However, this topic has not been investigated yet, largely

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because methods have not been available to readily quantify it in complex environmental or

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biological systems. Techniques that can be used to measure graphene in relatively pristine

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samples such as scanning probe microscopy, Raman spectroscopy and X-ray diffraction are

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severely limited by the presence of other carbonaceous materials.2 An imaging technique

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based on Fourier transform infrared spectroscopy mapping was developed to analyze the

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graphene spatial distribution inside nematodes,14 but this approach does not provide

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quantitative results. Similarly, light microscopy has been used to qualitatively detect

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graphene in cells.8 Thus, a quantitative method that can be used in complex environmental

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and biological matrices is clearly needed.

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To overcome this challenge, we here report the synthesis of carbon-14 labeled graphene

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for the first time to our knowledge, by means of graphitization and exfoliation of

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sandwich-like FePO4/dodecylamine hybrid nanosheets. A similar approach has recently been

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successfully employed to quantify carbon-14 labeled carbon nanotubes and fullerenes in

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complex biological samples by detecting Beta emissions from the C-14 isotope.15-19

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Carbon-14 graphene was dispersed in water and its uptake and depuration behaviors

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measured using aquatic organism Daphnia magna, which is a standard US EPA test

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organism.20 D. magna are filter feeders and have a central position in freshwater food web

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dynamics.21 Once accumulation occurs, organisms at higher trophic levels that consume filter

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feeders might be exposed to elevated concentrations.

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EXPERIMENTAL SECTION

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Materials. All reagents used are of analytical grade without further purification. The

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carbon-14

ring-labeled phenol was purchased from Moravek Biochemicals and

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Radiochemicals (CA, USA).

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dated HPLC radiochromatogram) and was stored in an ethanol solution. Ferrous chloride

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tetrahydrate (FeCl2•4H2O), ammonium dihydrogenphosphate (NH4H2PO4) and hydrochloric

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acid (HCl, 37%) were purchased from Nanjing Chemical Reagent Co., Ltd. Dodecylamine

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and other chemicals were purchased from J & K Technology Co., Ltd. Suwannee River

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humic acid (HA) was obtained from the International Humic Substances Society.

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C(U)-phenol had a chemical purity of ≥ 97% (analyzed with

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Graphene Synthesis and Characterization. Twenty mL of an ethanol solution

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containing dodecylamine (5.000 g) was added into 80 mL of a mixed aqueous solution of

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FeCl2·4H2O (1.590 g) and NH4H2PO4 (0.900 g) at 50°C. The solid product was collected by

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centrifugation and dried under vacuum at 40°C for 12 h. Eight hundred µL of carbon-14

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phenol ethanol solution was added into the dry powder and dried at 40°C for 12 h. The

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mixture was heated at 700°C for 12 h under argon. After cooling, the black powder and 40

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mL of 37% hydrochloric acid were transferred into a Teflon-lined autoclave and sealed before

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being heated in an oven at 180°C for 24 h. The black solid product was collected by

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centrifugation, and then washed by water and anhydrous ethanol over ten additional times to

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ensure the purity of the graphene. The powder was then dried under vacuum at 40°C for 12 h.

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To disperse the carbon-14 graphene in water, a 500-mL beaker containing graphene and 300

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mL of artificial freshwater (AF) (CaCl2·2H2O, 58.8 mg/L; MgSO4·2H2O, 24.7 mg/L;

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NaHCO3, 13.0 mg/L; KCl, 1.2 mg/L; hardness [Ca2+] + [Mg2+]=0.5 mmol/L)17,22 was seated

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in ice-water bath. The solution was sonicated for 6 h with the probe tip of ultrasonic

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processor (100 W, P=7.52 J/s)23 approximately 0.4 cm from the bottom of the beaker.

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The Fe content in triplicate samples of the graphene powder was analyzed using

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inductively coupled plasma optical emission spectroscopy (ICP-OES, Optima 5300DV)

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under the following parameters: 0.55 L/min nebulizer flow; 1.3 mL/min sample pump flow;

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cyclonic spray chamber; 2 mm alumina injector; and 238 nm wavelength. The minimum

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detection limit of Fe for ICP analysis was 0.005 mg/L.

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To assess potential carbon-14 byproducts from the synthesis procedure, carbon-14

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graphene (0.3 mg) was extracted in sequence using dichloromethane (5 mL), n-hexane (5

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mL), and dichloromethane (5 mL). These solutions were recombined, and subjected to

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anhydrous sodium sulfate to remove water. After that, the sample was dried using a gentle

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nitrogen stream and reconstituted in methanol and dichloromethane (4:1, v/v) for HPLC and

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GC-MS analysis, (see Part I in Supporting Information (SI) for additional details). Filtrate

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from the sonicated graphene samples were similarly analyzed (see Part I in SI for details). In

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addition, the reconstituted solution for the graphene powder and a filtrate sample from the

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sonicated graphene were added to Gold Star scintillation cocktail (Meridian) and their

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radioactivities were measured in a liquid scintillation counter (LSC) (LS6500; Beckman

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Coulter; U.S.).

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Transmission electron microscopy (TEM) and High-resolution TEM (HRTEM)

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measurements were conducted with a JEM-2010 electron microscope, using an accelerating

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voltage of 200 kV. Raman spectroscopic analysis was performed with a Renishaw InVia

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system utilizing a 514 nm incident radiation. Scanning electron microscopy (SEM)

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measurements were conducted with a Hitachi S4800 instrument, using an accelerating

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voltage of 10 kV. TEM, HRTEM, Raman spectroscopy, and SEM analyses were performed

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for both the graphene powder and the sonicated graphene. Graphene solution (100 and 250

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µg/L) was filtered using 0.22 µm filter to obtain the above sonicated graphene. X-ray

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Photoelectron Spectroscopy (XPS) measurements of graphene powder were performed on a

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PHI 5000 VersaProbe with a monochromatic Al Ka X-ray source. Nitrogen sorption

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isotherms of the graphene powder were collected at 77 K using Micromeritics ASAP2020

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equipment. Brunauer-Emmett-Teller (BET) and BJH models are respectively used for

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specific surface area and porosity evaluation.

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Carbon-14 Labeling Quantification. The purified graphene powder was dispersed in

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the solution by sonication as described above and the radioactivity of the purified graphene

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solution was quantitatively measured in a LSC following combustion in a biological oxidizer

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(BO) or direct addition to scintillation cocktail (Gold Star, Meridian). The BO (OX-500;

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Zinsser Analytic, Germany) was used to burn the graphene at 900°C for 4 min under a stream

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of oxygen gas running at 360 mL/min. The

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was captured in alkaline carbon-14 scintillation cocktail (Zinsser Analytic, Germany) and

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then counted by LSC. The direct addition of the above graphene solution to scintillation

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cocktail (Gold Star, Meridian) followed by scintillation counting was found to consistently

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underestimate the radioactivity of the graphene relative to burning the graphene in the BO.

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The direct addition method yielded only 48% of the radioactivity measured using the BO

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method (10.9 ± 0.07 mCi/g for direct method and 22.71 ± 1.78 mCi/g for BO method; errors

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always represent standard deviation values; n=3). However, since the coefficient of variation

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of the BO method for triplicate sample (7.86%) was inferior to that of the direct addition

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method (1.37%), the direct addition method was predominately used. The radioactivity

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response detected using the direct addition method was linear (R2=0.99) to the graphene

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concentration.

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CO2 released during the combustion process

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Test Organisms. Daphnia magna were cultured in AF aerating for more than 3 d (20±

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1°C, 16:8 h light:dark photoperiod), as performed using a standard method.22 D. magna were

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fed three to five times a week with a culture of green algae (mainly Scenedesmus sp.).

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Daphnia neonates (