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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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┴
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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 (
