Risks of Single-Walled Carbon Nanotubes Acting as Contaminants

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Risks of Single-Walled Carbon Nanotubes Acting as Contaminants-Carriers: Potential Release of Phenanthrene in Japanese Medaka (Oryzias Latipes) Yu Su, Xiaomin Yan, Yubing Pu, Feng Xiao, Dongsheng Wang, and Min Yang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es304479w • Publication Date (Web): 11 Apr 2013 Downloaded from http://pubs.acs.org on April 14, 2013

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Environmental Science & Technology

Risks of Single-Walled Carbon Nanotubes Acting as Contaminants-Carriers: Potential Release of Phenanthrene in Japanese Medaka (Oryzias Latipes) Yu Su, Xiaomin Yan, Yubing Pu, Feng Xiao, Dongsheng Wang*, Min Yang

State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, People's Republic of China

*Corresponding author: Tel.: +86 10 6284 9138; Fax: +86 10 6292 3541; Email: [email protected]

TOC / Abstract Art

Abstract

The performance of carbon nanotubes (CNTs) acting as contaminants-carriers in vivo is critical for understanding the environmental risks of CNTs. In this study, the whole body accumulation and tissue distribution of phenanthrene in Japanese medaka was examined in the presence of single-walled carbon nanotubes (SWCNTs) and the potential release of phenanthrene was investigated from two types of SWCNTs suspensions differed in surface charge and stability. The 1

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results showed that the coexistence of SWCNTs facilitated the accumulation of phenanthrene in the digestive track of fish and therefore enhanced the whole body phenanthrene concentration by 2.1 fold after exposure for 72 h. Meanwhile, 6.4-48 and 20-34 times higher phenanthrene concentrations were measured in the liver and brain of fish exposure to the two mixtures, respectively, when comparing with the phenanthrene alone treatment with equal concentration of soluble phenanthrene. The extra phenanthrene was from the SWCNTs-associated phenanthrene that accumulated in the digestive track, indicating the release of phenanthrene from SWCNTs did occur in fish. Moreover, the neutrally charged SWCNTs showed different agglomeration behaviors from the negatively charged SWCNTs, which could affect the accumulation of SWCNTs in the digestive track of fish and subsequently influence the retention of phenanthrene associated with the carbon nanotubes. Introduction With the rise of nanotechnology, the unique physic-chemical properties of carbon nanotubes (CNTs) have expanded their application in industrial and consumer products.1 The surge in production of CNTs will increase the accessibility of nanoparticles (NPs) to the environment. The safety issues of CNTs have aroused great attention among researchers, from human health related studies2-3 to concerns on the environmental organisms.4-6 One of the environmental risks of CNTs is their potential ingestion by the ecological receptors,7,8 and the surface properties might have impacts on the biological uptake and depuration of NPs.9-11 Regardless of the environmental behaviors of CNTs themselves, CNTs with large surface area would affect the transport, fate and bioavailability of organic contaminants in the environment,12-14 such as polycyclic aromatic hydrocarbons (PAHs).15,16 Previous studies have shown reversible adsorption of PAHs on CNTs17 and the presence of biomolecules in the simulated digestive fluids could enhance the desorption of phenanthrene on CNTs,18 implying the CNTs-associated pollutants 2


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could be bioavailable. However, little is known about the release of organic contaminants from CNTs in vivo. Unlike CNTs, the desorption hysteresis of PAHs17 and 17α-ethinylestradiol19 was observed for fullerene, due to the rearrangement of fullerene aggregates and the penetration of sorbate into the closed interstitial spaces.17 Recently, Park et al. reported that the association with fullerene could reduce the bioavailability of 17α-ethinylestradiol in zebrafish.20,21 These studies indicated that NPs might have impacts on the bioavailability of NPs-associated contaminants, and the extent to which would partly depend on the adsorption-desorption. Therefore, bridging adsorption of organic contaminants on CNTs and the bioavailability of CNTs-associated organic contaminants are critical for understanding the environmental risks of CNTs. In the aquatic environment, ubiquitous PAHs could be taken up by fish and the distribution of PAHs was different among tissues.22,23 CNTs are supposed to interact with PAHs and then be ingested by fish. It is possible that CNTs-associated PAHs primarily present in certain tissues and then will be distributed to other tissues if PAHs are released from CNTs with their digestion by fish. In the present study, two types of SWCNTs suspensions with different surface charge and stability were prepared; phenanthrene and Japanese medaka (Oryzias latipes) were selected as a model organic contaminant and test organism, respectively. The adsorption-desorption of phenanthrene on SWCNTs was examined. Moreover, the effects of SWCNTs on the whole body accumulation and the tissue distributions of phenanthrene were assessed, and the release of phenanthrene from SWCNTs in fish was tested and verified. Materials and Methods Synthesis and Characterization of SWCNTs. SWCNTs synthesized by the CVD method were purchased from Shenzhen Nanotech Port Co.Ltd of China. The raw SWCNTs (R-S) were purified by using a modified method described by Liu et al..24 Briefly, a raw sample of nanotubes (5 g) was 3



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refluxed in 600 mL of 2.6 M nitric acid for 11 h, filtered, and washed with deionized water. Standard suspensions (1mg/ml) of SWCNTs were prepared by bath sonicating 1 mg of P-S with 1 mL of 25 mg/mL PEG solution for 30 min. Bath and probe sonication was performed to prepare two types stock suspensions of purified SWCNTs (15 mg/L) respectively. For preparation the P-S suspensions, nanotubes were bath sonicated for 30 min with standard medium (290 mg/L CaCl2, 120 mg/L MgSO4·7H2O, 65 mg/L NaHCO3 and 6 mg/L KCl). For preparation better dispersions of P-S, nanotubes were probe-sonicated for 3 h (ensuring all precipitates became suspended) with the standard medium by using an ultrasonic processor (Shangchao FC-450V, China) operated at 400 W. Probe sonication was conducted by using a 20 s “on”/ 60 s “off” pulse sequence with a 20 mm diameter probe tip. The prolonged and intensive probe sonication made P-S present in smaller suspended SWCNTs, which were denoted as SP-S. The solid SWCNTs samples were observed directly by using a scanning electron microscope (SEM) (Leo 1530, Germany). SWCNTs were dispersed and then were dropped on the copper grids and dried for transmission electron microscope (TEM) observation (Hitachi 7500, Japan). Raman spectra of SWCNTs were obtained by using a Renishaw RM 1000 microscopic confocal Raman spectroscope equipped with a laser excitation of 633 nm (He–Ne laser). Thermal gravimetric analysis (TGA) (Netzsch STA 409 C131F, Germany) was conducted under a flow of air and heated to 1000 ºC at a rate of 10 ºC /min. Surface acidic groups of SWCNTs were measured by Boehm titration method25 using a 716 DMS automatic potential titrator (Metrohm, Switzerland). Zeta potential of SWCNTs suspensions was determined by using a Malvern Zetasizer 2000 instrument. Nitrogen adsorption-desorption isotherms of SWCNTs (Figure S5 of the Supporting Information, SI) were measured by using a Micromeritics ASAP 2000 instrument. Surface area was calculated by multi-point Brunauer-Emmett-Teller (BET) method, the average mesopore diameter and volume 4


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were calculated from the desorption branches of the isotherms by Barrett-Joyner-Halenda (BJH) method. Chemicals. Phenanthrene (99.8% purity, Sigma–Aldrich) was dissolved in DMSO to prepare a stock solution (1 g/L). All chemicals needed to prepare a SDS-PAGE gel were purchased from Amresco Inc. Test Organism. Japanese medaka (Oryzias latipes) was selected from a brood stock that originated from the Laboratory of Freshwater Fish at the Bioscience Center of Nagoya University of Japan. Fish were cultured in charcoal-dechlorinated tap water (pH 7.2~7.6) at 25±1 ºC (16:8 h light:dark photoperiod) and were fed with brine shrimp once a day. The four months-old fish with an average length of 38±2 mm and weighing 600±100 mg (n=200) were used. Prior to the exposure experiment, selected fish were cultured in the standard medium overnight without feeding. Exposure Assay. Prior to the start of exposure, adsorption and desorption of phenanthrene on the two types of SWCNTs suspensions was investigated at 25 ºC by batch experiments (procedures are described in SI). Also, preadsorption of phenanthrene on SWCNTs suspensions was conducted. Briefly, following pH adjustment to 7 by 0.1 M NaOH, 15 mg/L SWCNTs stock suspensions and 60 µg/L phenanthrene solution were mixed in aluminum foil wrapped glass bottles. Then, the bottles were sealed with aluminum foil-lined screw caps and shaken at 150 rpm (25 ºC) for five days to reach adsorption equilibrium. After centrifugation (3,000 g for 5 min), the concentrations of soluble phenanthrene were measured (procedures are described in SI). The bottles without addition of carbon nanotubes were used for phenanthrene loss assessment, which showed that the loss was less than 3% of the initial concentration. Therefore, phenanthrene removed from the aqueous phase was adsorbed by SWCNTs.

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When the adsorption equilibrium was reached, 200 mL a mixture of SWCNTs and phenanthrene was added in an amber bottle (250 mL), one fish was transferred into it, and then the bottle was covered with a glass stopper and stored at 25±1 ºC (16:8 h light:dark photoperiod). Fish were exposed to solutions proximately containing 3 and 60 µg/L phenanthrene (prepared with the standard medium) as controls. In total, there were four treatments: 1. 3 µg/L phenanthrene alone, Phe(3); 2. 60 µg/L phenanthrene alone, Phe(60); 3. Phe(60)+P-S; 4. Phe(60)+SP-S. The concentrations of soluble phenanthrene in each treatment are listed in Table 2. Ten fish were exposed for each treatment at each exposure time. The mixtures or the phenanthrene solutions were not renewed during the whole experiment. The SWCNTs settling experiments were conducted (procedures are described in SI) to evaluate the stabilities of the two mixtures during the three days. Moreover, the bottles merely containing phenanthrene solution without fish were used for phenanthrene loss assessment, which showed that the loss was less than 8% of the initial concentrations at 72 h. At each time point (1, 4, 12, 24 and 72 h), ten bottles in each treatment were removed, all fish were sacrificed in ice-water bath and then placed on filter papers to get rid of the surface water on their body and weighted. Five fish were stored at -80 ºC for measurement the whole body phenanthrene levels. The remaining five fish were dissected; the main tissues (liver, digestive track, gill and brain) were weighted and then stored at -80 ºC for measurement the tissue contents of phenanthrene. The presence of black substance was checked via an optical microscope, and SWCNTs were mainly found in the digestive track of fish, which were confirmed by the Raman spectroscope afterwards. In order to minimize the fluorescence interference originated from the tissue, the black substance in the digestive track was squeezed out and dried. The Raman spectra were excited with a 514 nm laser line.

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Extraction of Phenanthrene and SWCNTs. The sampled whole body and tissues of fish were homogenized with 400 µL of 0.1 M PBS buffer (pH 7.4) in ice-water bath, and then were mixed with 200 µL of 1 mM CaCl2, 200 µL of 1 mM MgCl2 and 100 µL of 10% SDS to break down the tissues26 and to ensure cell lysis.27 Then, 1 mL of acetonitrile (HPLC grade) was added into the homogenates. After bath sonication for 45 min at 20 ºC, the samples were stored at 4 ºC for 24 h. Following phenanthrene extraction, the samples were centrifuged for 10 min at 10,000 g and the supernatants were used for HPLC analysis the concentration of phenanthrene. The pellets of the digestive track were kept for quantification of SWCNTs. The content of phenanthrene was calculated by the wet weight of tissue dividing the mass of phenanthrene in the tissue. Recovery checks were performed to validate the procedure for phenanthrene extaction. The sampled tissues were spiked with 100 µL of phenanthrene stock solution and then were treated in the same manner. The calculated extraction recovery was 63-70% for phenanthrene. Quantification of SWCNTs in the Digestive Track of Fish. The mass of SWCNTs in the digestive track of fish was quantified by using a modified method described by Wang et al..27 A 30% polyacrylamide gel stock solution was prepared by mixing acrylamide and bisacrylamide in a ratio of 29:1. Each gel had two layers: an upper larger pore stacking gel (6% polyacrylamide, pH 6.8) and a lower smaller pore resolving gel (10% polyacrylamide, pH 8.8). Following extraction of phenanthrene, the pellets of the digestive track were homogenized with 100 µL of 5% sulfosalicylic acid and diluted 2 fold with 20% glycerol, and then 10 µL of samples were loaded into wells of the gel. After electrophoresis at 100 V for 30 min, the optical images of the gel were acquired in “Epi White” mode by using a Gel Doc XR imaging system (Bio-Rad, U.S.A). The pixel intensities of the digitized gels were quantified by using the Quantity One 1-D software. The intensities of all samples were background-subtracted from the intensities of control gels that did not contain SWCNTs. The 7



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mass of SWCNTs in samples was quantified by SWCNTs calibration curves (Figure S8 of the SI) prepared with the SWCNTs standard suspensions. The content of SWCNTs was calculated by the wet weight of tissue dividing the mass of SWCNTs in the tissue. For preparation the SWCNTs calibration curves, the SWCNTs standard suspensions were diluted 20 fold with 20% glycerol and were subsequently loaded into the wells of the gel with various volumes. The SWCNTs-containing samples should be diluted to meet the limit of detection and should be bath sonicated before being loaded to each well of the gel. Statistical Analysis. All data are expressed as means and standard deviations. One-way ANOVA with Tukey’s multiple comparison test was used to determine the statistical differences in phenanthrene content among treatments. Statistical difference was set at p

Risks of Single-Walled Carbon Nanotubes Acting as Contaminants

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