Delineating Oxidative Processes of Aqueous C60 Preparations: Role

Nov 19, 2008 - ... Rice University, Houston, Texas 77005, and Department of Civil and Environmental Engineering, Rice University, Houston, Texas 77005...
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Environ. Sci. Technol. 2009, 43, 108–113

Delineating Oxidative Processes of Aqueous C60 Preparations: Role of THF Peroxide BO ZHANG,† MIN CHO,† JOHN D. FORTNER,‡ JAESANG LEE,§ CHING-HUA HUANG,† JOSEPH B. HUGHES,† AND J A E - H O N G K I M * ,† School of Civil and Environmental Engineering, Georgia Institute of Technology, 200 Bobby Dodd Way, Atlanta, Georgia 30332, Department of Chemistry, Rice University, Houston, Texas 77005, and Department of Civil and Environmental Engineering, Rice University, Houston, Texas 77005

Received July 10, 2008. Revised manuscript received October 21, 2008. Accepted October 23, 2008.

The oxidative reactivity of THF derivatives formed during THF/ nC60 synthesis was evaluated with indigo dye as a model compound. The results showed that the formation of previously undetected oxidizing agents during THF/nC60 synthesis accounted for the degradation of indigo dye by THF/nC60 (THF/ nC60/unwashed), while THF/nC60 after vigorous washing (THF/ nC60/washed) and nC60 prepared without the use of THF were not reactive. γ-Butyrolactone (GBL) was detected by GC-MS in the THF/nC60/unwashed as one of THF derivatives, but showed no reactivity with indigo dye. An organic peroxide was detected in the THF/nC60/unwashed by HPLC,and was reactive with indigo dye. This compound was found to also account for the elevated antibacterial and bactericidal activities of THF/ nC60/unwashed on E. coli. Analysis by LC/(+ESI)MS and 1H NMR showed that the detected THF peroxide was tetrahydro2-(tetrahydrofuran-2-ylperoxy)furan. The formation of THF peroxide during the preparation of aqueous stable C60 aggregates provides another potential explanation for the reactivity and oxidative stress mechanisms of THF/nC60 system reported in the literature, although it does not exclude the potential reactivity and toxicity of nC60 itself.

Introduction The toxicity and reactivity of C60 in aquatic systems are of interest due to facile formation of stable C60 colloidal suspension (nC60) in water and potential environmental risk(s). Recent reports on the toxicity and reactivity of the nC60 colloidal suspensions are, however, somewhat controversial and appear to be dependent upon the preparation method (1-6). Aqueous stable C60 aggregates can be formed via a solvent exchange protocol in which C60 is first dissolved in a water miscible, polar organic solvent such as tetrahydrofuran (THF) and mixed with water, with the transfer organic solvent subsequently removed via distillation (7). * Corresponding author phone: (404) 894-2216; fax: (404) 3857087; e-mail: [email protected]. † Georgia Institute of Technology. ‡ Department of Chemistry, Rice University. § Department of Civil and Environmental Engineering, Rice University. 108

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This method has been widely applied to test the toxicity and reactivity of nC60 due to high yield of nC60 formation and ease of controlling aggregate size (1, 2, 8, 9). Alternatively, nC60 can be formed by dissolving C60 in a water immiscible organic solvent such as toluene and applying ultrasound to gradually transfer C60 into water as the organic solvent is evaporated (10). Simply adding C60 to distilled water and vigorously mixing for an extended period of time also leads to the formation of nC60 (11). The nC60 prepared using THF as a transfer solvent (THF/ nC60) has consistently been reported to be more toxic than nC60 aggregates obtained by the sonication (son/nC60) or stirring in water (aqua/nC60) (Supporting Information, Table S1), which is speculated to be, in part, due to the presence of trace THF or THF derivatives (3, 8, 12). Some studies have suggested the potential interference of residual THF that was not effectively removed during the preparation process (12, 13), while others have reported that the concentration of residual THF was negligible, and even if present at trace level, too low to exert any measurable toxicological effect (3). In another recent study, a THF derivative, γ-butyrolactone (GBL), formed during preparation of THF/nC60 has been found to exert oxidative damage to the genes of larval zebrafish (14). Work presented herein examines THF derivatives formed during the THF/nC60 synthesis, with oxidative reactivity evaluated using a model dye chemical, indigo trisulphonate, in the presence or absence of light. The THF peroxide, identified as a major reactive THF derivative in the THF/ nC60 system, was demonstrated to play a significant role in the oxidative reactivity of THF/nC60. In addition, this THF derivative was found to play a major role in the antibacterial and bactericidal activity of THF/nC60 on Escherichia coli (E. coli), which has been widely used for demonstration of nC60 toxicity in previous studies (8, 15).

Experimental Section nC60 Preparation. THF/nC60 at a typical concentration of 6 mg/L was prepared according to the method described by Fortner et al. (8) and is referred to as THF/nC60/unwashed in this study. To prepare THF/nC60/washed, a 100 mL aliquot of THF/nC60/unwashed was collected on a ultrafiltration membrane (YM10, molecular weight cutoff ) 10 000, Millipore, U.S.) using a stirred cell (model 8200, Millipore, U.S.) and washed using 50 mL of ultrapure water 12 times (16). Son/nC60 was prepared by applying ultrasound (50/60 Hz, 125 W) to the binary mixture of toluene containing C60 and ultrapure water at an ambient temperature until the toluene was completely evaporated (17). Details of the preparation methods are provided in Supporting Information Text S1. Batch Experiments. Batch kinetic experiments were performed both in the presence and absence of light under anoxic condition. For the experiments performed under light, six 4 W black light blue lamps (BLB lamps, Philips TL4W), placed inside a black cube, were used as a light source. The emission wavelength region of 350-400 nm was monitored by a Spectropro-500 spectrophotometer (Acton Research Co., U.S.). The incident light intensity in this active wavelength region was measured as 3.33 × 10-4 Einstein/min/L by ferrioxalate actinometry (18). The reaction solution contained 1.5 mL nC60 solution (or other test solutions as discussed below) and 1.5 mL indigo dye stock (32 µM, 5, 5′, 7-indigotrisulfonic acid tripotassium salt, Sigma-Aldrich) buffered at pH 7.0 using 10 mM phosphate. These solutions were individually purged with high purity (99.999%) nitrogen for 30 min before mixing. The mixture was transferred to a 3.5 10.1021/es8019066 CCC: $40.75

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FIGURE 1. Kinetics of indigo degradation by nC60 prepared by different methods. [nC60] ) 3 mg/L; [indigo] ) 16 µM; pH 7.0; temperature ) 22 °C. Control tests were performed without nC60. Each data point in this Figure and Figures 2, 3, and 7 was presented with an error bar. mL quartz cell reactor, purged with nitrogen for an additional 3 min, and closed with a screw cap. All the experiments were performed six times (i.e., three sets of duplicates) at room temperature (22 °C). Indigo concentration was determined via UV-vis analysis at 600 nm (Varian Cary 50 spectrophotometer). Characterization of THF Derivatives. THF derivatives in the THF/nC60/unwashed were analyzed using an Agilent 6890/5793 GC/MS equipped with an HP-5MS column (30 m × 0.25 mm i.d.) after extraction (1 mL of dichloromethane added into 1 mL of sample). Helium was the carrier gas at a constant flow rate of 1.0 mL/min. Splitless injection was conducted at 280 °C. The GC oven temperature was initially held at 40 °C for 4 min, increased to 100 at 5 °C/min, and then ramped to 300 at 50 °C/min with a final hold at 300 °C for 3 min. Electron impact ionization at positive mode with a mass scan range of m/z 25-100 was conducted. THF derivatives were also analyzed by an Agilent 1100 series HPLC/DAD/MS with a Zorbax SB-C18 reverse phase column (4.6 × 150 mm, 5 µm) and an isocratic eluent (10% methanol and 90% water) at a flow rate of 1.0 mL/min. C60 aggregates in THF/nC60/unwashed were removed by filtering with a Whatman Anotop 25 plus 0.02 µm filter prior to injection. For MS, 95% 10 mM ammonium acetate and 5% methanol at a flow rate of 0.25 mL/min were used as the mobile phase. Electrospray ionization (ESI) was conducted at positive mode with a mass scan range of m/z 50-500. THF derivatives were also separated by an Agilent 1100 HPLC/DAD system; the peak at retention time of 4.4 min was collected by a Foxy Jr. fraction collector, dried using nitrogen in the dark, redissolved in D2O, and analyzed using an AMX 400 1H NMR at 400 MHz and a QNP 5 mm probe. Antibacterial and Bactericidal Activity. Antibacterial activity of nC60 was assayed by evaluating the minimal inhibitory concentration (MIC) for E. coli (ATCC strain DH5R) grown in Luria-Bertani (LB, Difco Co., U.S.) media (8, 15). The nC60 samples tested were prepared by washing the THF/ nC60/unwashed sample different times (from 0 to 12 times) following the procedure described above. Various concentrations of these nC60 samples were added to Minimal Davis media (i.e., Davis media with potassium phosphate reduced by 90%) after purging with nitrogen under heat. Both negative and positive control tests were performed using distilled water and son/nC60 (instead of THF/nC60), respectively, added to Minimal Davis media. After inoculation and incubation overnight at 37 °C, E. coli was enumerated by the spread plate method (triplicate) and the optical density method at 600 nm (OD600). The concentration of nC60 resulting in no growth of E. coli after 18 h of incubation was denoted as the MIC. Bactericidal activity of THF/nC60/unwashed and THF derivative (a fraction collected from HPLC at 4.4 min retention time) was assessed by performing disinfection experiments in the dark (19). THF/nC60/unwashed (5 mg/L after mixing)

FIGURE 2. Kinetics of indigo degradation with samples obtained after membrane filtration or centrifugation of THF/nC60/ unwashed. [nC60] ) 3 mg/L (concentration in THF/nC60/unwashed before membrane filtration or centrifugation); [indigo] ) 16 µM; pH 7.0; temperature ) 22 °C; under light, no oxygen. Resuspended pellet: nC60 aggregates collected after centrifugation were resuspended in water. or THF derivative was added to 20 mM phosphate buffer at pH 7.0 containing 105 cfu/mL of E. coli. Viability after different exposure times was assessed by the spread plate method (triplicate). Disinfection experiments were repeated three times and average values were reported.

Results and Discussion nC60 Reactivity with Indigo Dye. Indigo degradation kinetics obtained from batch experiments are shown in Figure 1. These experiments were performed in the absence of oxygen, and thus any change in the degradation kinetics should be related to the reaction directly involving nC60. Results of control tests performed without nC60 showed that indigo dye degraded by about 6% after UV irradiation for 5 h. In the dark, there was no indigo degradation. The degradation kinetics of indigo were not changed by the addition of THF/ nC60/washed and son/nC60 with or without light, in the absence of oxygen. Even in the presence of oxygen, the indigo degradation kinetics were not changed by the addition of THF/nC60/washed or son/nC60 (results not shown). These observations are consistent with an earlier finding that the intrinsic photochemical reactivity of C60 is lost when C60 forms colloidal aggregates in the aqueous phase and no reactive oxygen species are generated (20-22). In contrast, THF/nC60/unwashed notably enhanced indigo degradation kinetics. Indigo concentration decreased by 50% within 5 h in the presence of light. Degradation of indigo dye under this condition conforms to first-order kinetics with k ) 2.4 ( 0.14 × 10-3 min-1. Even in the dark, the indigo dye was degraded by THF/nC60/unwashed, albeit to a lesser extent VOL. 43, NO. 1, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Kinetics of indigo degradation with THF (1.5 g/L), GBL (1.9 g/L), samples obtained from refluxing the mixture of THF and water (refluxed THF/water and condensate) or the mixture of THF/nC60/washed and the fraction (4.4 min retention time) collected from HPLC separation of THF/nC60/unwashed. [nC60] ) 3 mg/L; [indigo] ) 16 µM; pH 7.0; temperature ) 22 °C; under light, no oxygen.

FIGURE 4. GC chromatogram and MS spectra of THF/nC60/ unwashed. (6%). UV-vis spectral changes of the indigo dye as a function of irradiation time are shown in Supporting Information Figure S1. The characteristic absorption of indigo at 600 nm gradually decreased upon degradation with a concurrent increase in 250 nm absorption peak. The above results highlight the difference in the (photo)chemical reactivity between THF/nC60/unwashed and 110

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FIGURE 5. HPLC chromatogram of refluxed THF/water, THF/nC60/ unwashed, THF/nC60/washed, and son/nC60. Peak at 4.4 min corresponded to THF-peroxide. THF/nC60/washed. As far as the preparation method is concerned, the only difference is the separation of nC60 from the preparation solution using ultrafiltration membrane and washing the latter with ultrapure water. Consistent with the above observation, the filtrate obtained during this washing step showed an obvious reactivity toward the indigo dye with the first-order rate constant k ) 2.6 ( 0.18 × 10-3 min-1 in the presence of light, which was similar to that of THF/ nC60/unwashed (Figure 2). A similar result was obtained when nC60 was separated using centrifugation instead of membrane filtration. THF/nC60/unwashed sample was centrifuged at 9300g for 1 h and the resulting supernatant and resuspended pellet (nC60) were reacted with indigo dye, respectively. The supernatant was reactive with indigo dye with the first-order reaction rate constant k ) 2.6 ( 0.21 × 10-3 min-1. In contrast, the resuspended aggregates showed no reactivity (Figure 2) (i.e., no reactivity compared to control test results shown in Figure 1). Additional experiments were performed using the samples prepared by refluxing the 1:1 mixture of THF and ultrapure water without C60 following the same procedure used for THF/nC60 preparation. The water remaining after THF evaporation (refluxed THF/water in Figure 3) rapidly degraded indigo dye, with the concentration of indigo dye decreased by 90% within 2 h under experimental conditions. The indigo degradation kinetics with refluxed THF/water were much faster than those with THF/nC60/unwashed. The condensed liquid, which consisted mostly of THF, was only mildly reactive with indigo (Figure 3). Residual THF concentration in the THF/nC60/unwashed sample was below the GC-MS detection limit of approximately sub-ppb. Even if a trace amount below the detection limit had been present, it should have a negligible influence on indigo degradation as shown in Figure 3. Only a slight increase in indigo degradation kinetics was observed even though a large amount of fresh THF was added. Collectively, the above results suggest that derivatives of THF, much less volatile and more reactive than THF, accumulate in the THF/

FIGURE 6. MS and 1H NMR spectra of a fraction (4.4 min retention time) collected after liquid chromatographic separation of THF/ nC60/unwashed.

FIGURE 7. Kinetics of E. coli inactivation by THF/nC60 unwashed and THF-peroxide. [nC60] ) 5 mg/L; [THF-peroxide] ) the same as that present in 5 mg/L THF/nC60/unwashed sample, collected from HPLC; temperature ) 22 °C.

TABLE 1. Minimal Inhibitory Concentration (MIC) for E. coli by nC60 Obtained after Washing THF/nC60/Unwashed Different Times (- indicates no growth and + indicates growth) number of repeated washing nC60 concentration (mg/L)

1

2

3

4

5

8

12

0.03 0.10 0.25 0.50 1.00 2.00

-

-

+ -

+ -

+ + -

+ + + + -

+ + + + + -

nC60/unwashed sample (i.e., water phase) during the evaporation procedure. Reactivity and Identification of THF Derivative. The GC analysis of THF/nC60/unwashed (Figure 4a) suggested the presence of at least two major THF derivatized products, consistent with the findings by Henry et al. (14). They have assigned the first peak at 7 min and the second peak at 10.1 min as tetrahydro-2-furanol and γ-butyrolactone (GBL), respectively. The mass spectrum analysis of the second peak (Figure 4b) and the comparison to an authentic standard confirmed that the second peak was GBL. The GBL was of particular interest as a potential oxidant for indigo dye since

it had been shown to be toxic to larval zebrafish (14). However, a test performed with the standard GBL at excessive concentrations suggested that it was not responsible for indigo degradation under the experimental conditions investigated (Figure 3). The tetrahydro-2-furanol might not have played a significant role in indigo degradation, even though the reactivity with indigo was not independently tested due to the lack of an authentic standard. Based on the chemical structure of tetrahydro-2-furanol, it is not expected to act as an oxidant. Interestingly, peroxide oxygen of approximately 12 mg/L was detected in the THF/nC60/unwashed sample by potassium iodide titration (23). A large amount of peroxide oxygen (224 mg/L) was also detected in the refluxed THF/water, which might account for its higher reactivity compared to THF/nC60/unwashed (Figure 3). No peroxide was detected for the freshly opened THF (without further purification (24)) and the freshly prepared solution of THF and C60. As most organic peroxides are not stable (25) and subject to pyrolysis during GC analysis, the THF/nC60/unwashed was further analyzed using HPLC. A peak appeared at 4.4 min in both THF/nC60/unwashed and refluxed THF/water samples (Figure 5). Subsequent MS and 1H NMR analyses shown in Figure 6 suggested that this peak corresponded to tetrahydro-2(tetrahydrofuran-2-ylperoxy)furan (referred to as THFperoxide below). An ammonium adduct of the parent compound, [M+NH4]+, appeared as m/z 192.1. The m/z 122.1 and 104.1 corresponded to the NH4+ and H+ adduct of the fragmentation product after the parent compound lost a tetrahydrofuran-fragment, respectively. The m/z 71.1 corresponded to the ionized tetrahydrofuran moiety. The 1H NMR spectrum contained β-CH2 at 2.00 ppm, γ- CH2 at 1.85 ppm, R-CH2 at 3.75 ppm, and CH group at 5.48 ppm. Both MS and NMR results agreed with each other and supported the hypothesized structure. The THF-peroxide was effectively removed during the nC60 washing procedure (i.e., THF/nC60/washed) nor was present in the son/nC60 sample (Figure 5). Note that these nC60 samples were not reactive toward indigo dye. When THF-peroxide (peak at 4.4 min) was fraction collected during HPLC separation and added to nonreactive THF/nC60/ washed, indigo dye degradation kinetics were comparable to those with THF/nC60/unwashed (Figure 3). For this experiment, the fraction from HPLC eluent was repeatedly collected until the amount of THF-peroxide, as measured based on peak area, was equal to the amount that existed in VOL. 43, NO. 1, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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original THF/nC60/unwashed sample. This result suggests that most of THF/nC60/washed reactivity toward indigo dye resulted from peroxide derivative of THF which formed during THF reflux process and accumulated in the water phase. During the preparation of THF/nC60, the THF is exposed to light, oxygen, and heat which collectively provide a favorable condition of peroxide formation from alkyl ethers (25). Consequently, the hypothesized structure of THFperoxide was likely to have formed from THF autoxidation by a free radical chain mechanism (25). This dialkyl peroxide decomposes homolytically when irradiated (25) and resulting in RO• and ROO• that are much stronger oxidants than molecular oxygen. The decolorization of the indigo dye by THF-peroxide was likely induced by the breakage of the double-bond conjugation system of the chromophoric group of indigo by RO• and ROO•. The THF peroxide in the THF/ nC60/unwashed sample identified in this study was relatively stable with the concentration decreasing only by 25% after 33 days of storage (Supporting Information Figure S2). It suggests that THF/nC60/unwashed sample could contain appreciable amount of THF-peroxide even after some storage time. Antibacterial and Bactericidal Activity of THF Derivative. Organic peroxides have been reported to have toxicological effect on bacteria, degrading DNA, and inducing lipid peroxidation (26-28). THF-peroxides can decompose to GBL (29, 30) which has also been shown to be toxic. Past studies have suggested that THF/nC60 is toxic to E. coli with estimated MIC ranging between 0.5 and 1.0 mg/L (15). Table 1 shows the inhibition of E. coli growth by THF/nC60 as a funcion of washing times. It is interesting to note that MIC gradually increased with washing times, reaching the value between 1 and 2 mg/L, the range also observed for son/nC60 in a control test. When E. coli were exposed to 5 mg/L of THF/nC60 for 6 h, the level of bactericidal effect also appeared dependent on the number of washings. More than 4 logs (99.99%) of E. coli was inactivated by THF/nC60 with less than three times of washing. After 4, 8, and 12 washings, E. coli was inactivated at 2.9 (99.9%), 0.6 (74.9%), and 0.02 logs (4.5%), respectively. The kinetics of E. coli inactivation by the water phase of the THF/water reflux mixture, obtained by collecting the 4.4 min fraction during HPLC separation, were almost identical to those by THF/nC60/unwashed (Figure 7) when the same amount of THF-peroxide was added. This result suggests that the increased bactericidal effect of THF/nC60/unwashed was majorly due to the presence of the THF-peroxide, in line with the above finding that THF-peroxide was primarily responsible for the reactivity of THF/nC60/unwashed with indigo dye. Importance of THF Derivative Formation during THF/ nC60 Synthesis. This research is the first to demonstrate that a THF-peroxide derivative may play a prominent role for the (photo)chemical reactivity observed in THF/nC60 systems. This THF-peroxide also appeared to be responsible for significantly increasing the growth inhibition and bactericidal effect of THF/nC60 on E. coli. Such findings should prove valuable as THF/nC60 systems have demonstrated increased toxicity when compared to other aggregated C60 systems (3, 8, 12). It should be noted that this finding does not exclude the potential reactivity and toxicity of nC60 itself. Even though indigo degradation was negligible with THF/nC60/washed and son/nC60, past studies as well as our own experimental results showed the growth inhibition of E. coli in the absence of THF derivatives. While further study is necessary for an accurate assessment of the reactivity and toxicity involved with nC60 systems, this study suggests that without complete understanding of all reactive species present (i.e., impurities and derivatives) prudence should be used when interpreting toxicity data. 112

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Acknowledgments We thank Dr. Leslie Gelbaum at the NMR Facilities of Department of Chemistry and Biochemistry, Dr. Mei Zheng at Department of Earth and Atmospheric Sciences, and Dr. Guangxuan Zhu at School of Civil and Environmental Engineering, Georgia Institute of Technology, for assistance in instrumental analyses. This study was supported by U.S. Environmental Protection Agency (STAR Grant no. D832526) and Georgia Institute of Technology.

Supporting Information Available Additional experimental details. This material is available free of charge via the Internet at http://pubs.acs.org.

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(17) Andrievsky, G. V.; Kosevich, M. V.; Vovk, O. M.; Shelkovsky, V. S.; Vashchenko, L. A. On the production of an aqueous colloidal solution of fullerenes. J. Chem. Soc., Chem. Commun. 1995, 1281–1282. (18) Hatchard, C. G.; Parker, C. A. A new sensitive chemical actinometer. 2. Potassium ferrioxalate as a standard chemical actinometer. Proc. R. Soc. A 1956, 235, 518–536. (19) Cho, M.; Lee, Y.; Choi, W.; Chung, H.; Yoon, J. Study on Fe(VI) species as a disinfectant: quantitative evaluation and modeling for inactivating Escherichia coli. Water Res. 2006, 40, 3580–3586. (20) Lee, J.; Fortner, J. D.; Kim, J. H. Photochemical production of reactive oxygen species by C60 in the aqueous phase durig UV irradiation. Environ. Sci. Technol. 2007, 41, 2529–2535. (21) Lee, J.; Kim, J. H. Effect of encapsulating agents on dispersion status and photochemical reactivity of C60 in the aqueous phase Environ. Sci. Technol. 2008, in press. (22) Lee, J.; Yamakoshi Y.; Hughes, J. B.; Kim, J. H., Mechanism of C60 photochemistry in the aqueous phase: fate of triplet state and radical anion and production of reactive oxygen species. Environ. Sci. Technol. 2008, in press. (23) Johnson, R. M.; Siddiqi, I. W., The Determination of Organic Peroxides; Wiley: London, 1970.

(24) Deguchi, S.; Algrgova, R. G.; Tsujii, K. Stable dispersions of fullerenes, C60 and C70 in water. Preparation and characterization. Languir 2001, 17, 6013–6017. (25) Swern, D. Organic Peroxide; Wiley-Interscience: New York, 1970. (26) Carlsson, J.; Nyberg, G.; Wrethen, J. Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmosphere oxygen. Appl. Environ. Microbiol. 1978, 36, 223. (27) Mahmood, N.; Khan, S. G.; Athar, M.; Rahman, Q. Differential role of hydrogen peroxide and organic peroxides in augmenting asbestos-mediated DNA damage: implications for asbestos induced carcinogenesis. Biochem. Biophys. Res. Commun. 1994, 200, 687–694. (28) Koster, J. F.; Slee, R. G. Lipid peroxidation of human erythrocyte ghosts induced by organic hydroperoxides. Biochim. Biophys, Acta. 1983, 752, 233–239. (29) Robertson, A. Tetrahydrofuran hydroperoxide. Nature 1948, 162, 153–153. (30) Murai, S.; Sonoda, N.; Tsutsumi, S. Redox reaction of tetrahydrofuran hydroperoxide. Bull. Chem. Soc. Jpn. 1963, 36, 527–530.

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