Photochemical Production of Reactive Oxygen Species by C60 in the

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Environ. Sci. Technol. 2007, 41, 2529-2535

Photochemical Production of Reactive Oxygen Species by C60 in the Aqueous Phase During UV Irradiation JAESANG LEE, JOHN D. FORTNER, JOSEPH B. HUGHES, AND JAE-HONG KIM* School of Civil and Environmental Engineering, Georgia Institute of Technology, 200 Bobby Dodd Way, Atlanta, Georgia 30332-0373

The objective of this study was to investigate photochemical production of singlet oxygen (1O2) and superoxide radical anion (O2•-) by C60 in water. It was demonstrated that photoexcited C60 in the aqueous phase efficiently mediated transfer of absorbed energy to oxygen and produced singlet oxygen when associated with surfactant (Triton X100 and Brij 78) or polymer (polyvinylpyrrolidone), which is consistent with previously observed behavior in organic solvents. However, when C60 was present as colloidal aggregate suspension, prepared through solvent exchange or sonication, this intrinsic character was lost. Similarly, C60 associated with surfactant mediated electron transfer from electron donor (triethylamine) to oxygen producing superoxide radical, while C60 aggregates and C60 associated with polymer did not. These results suggest that the ability of C60 to mediate energy and electron transfer may be affected by the degree of C60 aggregation in the aqueous phase as well as characteristics of associated stabilizing molecules. Dependence of photochemical reactivity of C60 on its dispersion status in the aqueous phase is critical in assessing environmental impact and cytotoxicity of this material, as C60 associated with model natural organic matter was found to exist in aggregate form and did not produce reactive oxygen species under UV irradiation.

Introduction Carbon fullerenes represent a third allotrope of carbon different from both diamond and graphite in their physical and chemical properties (1). Among the fullerenes, C60, a truncated icosahedron, has been studied extensively as it is available in both high purity and relatively large quantities. As a result, the physical and chemical properties of C60 are extremely well characterized (2). With increasing commercial interest in its unique properties, the manufacture and application of C60 are expected to grow rapidly over the next decade. For example, the Mitsubishi Corporation plans on producing multi-ton quantities of C60 starting in 2007 (3). Projected uses of C60 are rapidly expanding and currently include superconductivity devices (4), biomedical agents (5), and high-temperature lubricants (6) among others. Unfortunately, information regarding the impact of C60 on natural environment and human health is limited. * Corresponding author phone: (404) 894-2265; fax: (404) 3857087; e-mail: [email protected]. 10.1021/es062066l CCC: $37.00 Published on Web 03/01/2007

 2007 American Chemical Society

Concerns regarding the aqueous phase behavior and reactivity of this material have been aggravated by recent findings which demonstrated that C60 can form water stable, negatively charged aggregates upon exposure to water (7-10), despite being exceedingly hydrophobic and virtually non-wettable (solubility of C60 in water 18.2 MΩ) produced by a Milli-Q water purification system (Millipore, Billerica, MA) was used for the preparation of all solutions. Preparation of Aqueous Stable C60. Aqueous stable C60 aggregates were prepared according to Fortner et al. (8). C60 sample prepared according to this specific method is herein referred to as nC60. The physical and chemical characteristics of nC60 have been reported in the literature (8, 28). The second preparation method involved application of ultrasound to a heterogeneous mixture of toluene containing C60 and ultrapure water in a sealed bottle (26). Aqueous suspensions of C60 prepared according to this specific method is herein referred to as son/C60. C60 was also stabilized in water using polymer (PVP) and surfactants (TX and Brij 78 (Brij)) following the methods by Yamakoshi et al. (27) and Beeby et al. (25), respectively. These C60 samples are referred to as C60/PVP, C60/TX, and C60/Brij in this study. Detailed descriptions of these preparation methods are available in the Supporting Information. C60 associated with model NOM was prepared by applying an ultrasound (50/60 Hz, 125 W) on the mixture of toluene containing C60 and Milli-Q water containing SRNOM for 24 h. Size Determination. When applicable, aggregate size measurements were taken by dynamic light scattering (DLS) in suspension using a Zetasizer Nano ZS90 with a low end detection limit of ca. 5 nm for these studies (Malvern Instruments, Worcestershire, OK). In triplicate, aqueous C60 samples were diluted accordingly, filtered with a 0.45 µm hydrophilic PES syringe filter (Millipore Corp.), and assigned a refractive index of 2.2 (29). Photochemical Experiments. Photochemical experiments were carried out using a magnetically stirred 60 mL cylindrical quartz reactor surrounded by six 4-W black light blue lamp (BLB lamp, Philips TL4W) at ambient temperature (22 °C). Emission wavelength region of 350-400 nm was confirmed by a Spectropro-500 spectrophotometer (Acton Research Co., USA). The incident light intensity in this active wavelength region was measured at 3.33 × 10-4 Einstein‚ min-1L-1 by ferrioxalate actinometry (30). Reaction solutions contained 5 mg/L C60 and indicator chemicals discussed below and were buffered at pH 7 using phosphate (10 mM for 1O2 experiments and 50 mM for O2•- experiments). As the photochemical reaction proceeded, sample aliquots of 1 mL were withdrawn from the reactor using a syringe, filtered through a 0.45-µm PTFE filter (Millipore), and injected into a 2-mL amber glass vial for further analyses. All experiments were run in duplicate. Concentrations of 1O2 were measured using furfuryl alcohol (FFA) as an indicator (k(FFA + 1O2) ) 1.2 × 108 M-1s-1 (31)). Productions of O2•- were estimated by the spectrophotometric methods using nitro blue tetrazolium (NBT2+) (32) and tetranitromethane (TNM) (33) (k(NBT2+ + O2•-) ) 5.88 × 104 M-1s-1 (34); k(TNM + O2•-) ) 1.9 × 109 M-1s-1 (34)). Concentrations of O2•- were alternatively measured by an indirect indicator (H2O2) formed in the presence of superoxide dismutase (SOD), in which concentrations of H2O2 were measured by DMP (2,9-dimethyl-1,10-phenanthroline) method (35). Detailed descriptions of these methods are available in the Supporting Information.

Results and Discussion Comparing Photochemical Production of 1O2 by C60 in Organic Solvent and Water-Stable C60 Aggregates. It is well2530

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FIGURE 1. (a) FFA degradation in UV-irradiated suspensions of nC60, son/C60, and fullerol ([FFA]0 ) 0.85 mM; [phosphate]0 ) 10 mM; pHi ) 7) and (b) UV-vis absorption spectra of nC60, son/C60, and fullerol in water, and C60 in toluene. known that ground-state 1C60 excites to 1C60* under UV irradiation, which subsequently converts to triplet state 3C60* through an intersystem crossing pathway (ISC) (17-19). 3C60* is efficiently quenched by ground-state triplet oxygen to generate 1O2 via energy transfer with high quantum yield (e.g., Φ(1O2) (355 nm) ) 0.76 in benzene (17)). This sequence of photochemical reaction is summarized below:

Contrary to pristine-C60 in organic solvent (Supporting Information, Figure S4), 1O2 was not generated by nC60 and son/C60 in water under the same UV irradiation condition as shown in Figure 1a. UV-vis absorption spectra of nC60 and son/C60 presented in Figure 1b showed appearance of a new broad band UV absorption in the wavelength region from 400 to 500 nm compared to C60 in toluene. This broad band absorption is known to occur in C60 in solid-state film and aggregate due to symmetry forbidden transitions, hence indicating solid-state C60-C60 interactions (8, 28, 36, 37). Accordingly, average population diameters, taken by DLS, were 100.1 and 86.2 nm for nC60 and son/C60, respectively. Under the same conditions, derivatized C60 (fullerol, polyhydroxyfullerene) induced photochemical degradation of FFA, albeit the kinetics was much slower as compared to C60

FIGURE 2. Comparison of the FFA degradation rates in UV-irradiated aqueous suspension of C60/PVP, prepared at different weight ratios ([C60]/[PVP])(w/w) of 0.008, 0.004, and 0.0008 ([C60/PVP]0 ) 5 mg/L; [FFA]0 ) 0.85 mM; [azide ion]0 ) 5 mM; [D2O] ) 70%(v/v); [phosphate]0 ) 10 mM; pHi ) 7). in organic solvent, consistent with the earlier findings (38). This result was unexpected since (1) nC60 and son/C60 absorbed UV light in the active wavelength region much more efficiently than fullerol (Figure 1b) and (2) nC60 would not be chemically modified according to recent 13C NMR analysis (8). This result suggests that colloidal aggregation of C60 in the aqueous phase may affect photochemical properties. Photochemical Production of 1O2 by C60 associated with Polymer and Surfactant in Aqueous Phase. C60/PVP at weight ratio of C60 to PVP at 0.008 and 0.004 did not produce any measurable amount of 1O2 under UV irradiation in water, consistent with the finding by ref 22 in which the same C60/ PVP ratio was examined (Figure 2). However, when the ratio was decreased to 0.0008 (i.e., a higher amount of PVP), at equivalent C60 concentrations, 1O2 production occurred at a rate corresponding to ca. 5% of that in toluene (estimated according to ref 31). Production of 1O2 by C60 in this case was confirmed as (1) FFA degraded at ca. twice faster rate in 70% (v/v) D2O, which is a less effective 1O2 quenching solvent than H2O (kd (H2O) ) 2.4 × 105 s-1; kd (D2O) ) 1.6 × 104 s-1 (31, 39)); (2) presence of excess azide ion as 1O2 scavenger completely suppressed FFA decay; and (3) PVP solution in the absence of C60 did not produce 1O2 under identical conditions (data not shown). Effect of dispersion status of C60 in the aqueous phase on its photochemical reactivity was further examined using C60/ TX prepared at two different weight ratios. It has been reported that C60 tends to disperse as aggregates at lower TX concentration, while it is molecularly solubilized within micelles at higher TX concentration above c.m.c. (25). Consistently, C60/TX at [C60]/[TX](w/w) of 0.00094 (i.e., higher TX concentration) exhibited a sharp absorption centered at 330 nm, similar to C60 in organic solvent (8) and no broad band absorption in 400-500 nm (Figure 3a). In contrast, C60/ TX at [C60]/[TX](w/w) of 0.015 (i.e., higher ratio, lower TX concentration with the same C60 concentration), showed a slightly red-shifted, blunt specific peak at ca. 340 nm and

FIGURE 3. a) UV-vis absorption spectra of C60 stabilized in water, using TX at two different weight ratios (([C60]/[TX])(w/w) ) 0.015 and 0.00094) and Brij78 (([C60]/[Brij78])(w/w) ) 0.002) (b) photochemical degradation of FFA in aqueous suspension of C60/TX and C60/Brij78. ([C60/TX]0 ) [C60/Brij]0 ) 5 mg/L; [FFA]0 ) 0.85 mM; [azide ion]0 ) 50 mM; [D2O] ) 70%(v/v); [phosphate]0 ) 10 mM; pHi is 7). a broad band absorption over the wavelength region between 400 and 500 nm, suggesting the presence of aggregate forms of C60 (8, 28, 36). DLS analysis supported C60/TX spectral observations, as higher TX concentration suspensions were monodispersed with an average diameter below 10 nm which approaches the instrument detection limit. Lower TX concentrations gave rise to a polydispersed C60/TX population with both small (50 nm) fractions presumably due to partial C60 aggregation. Under UV irradiation, C60/TX at lower ratio (more molecularly dispersed) produced 1O2 much more efficiently than that at higher ratio (more aggregated) (Figure 3b). Production of 1O by C /TX was further confirmed as (1) addition of D O 2 60 2 increased FFA degradation rate, (2) azide ion inhibited FFA degradation, and (3) no FFA degradation was observed by TX alone under UV irradiation (data not shown). A few other types of surfactants were also examined using the preparation method suggested by Beeby et al. (25). A VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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cationic surfactant, cetyltrimethylammonium bromide (CTAB), stabilized C60 at a relatively high concentration (i.e., 2 g CTAB per 5 mg C60), but the resulting suspension precipitated overnight perhaps due to Coulombic interaction between cationic CTAB and negatively charged C60 aggregates (9). Sodium dodecyl sulfate (SDS) is an anionic surfactant that is frequently used to disperse carbon nanotubes in the aqueous phase (40). SDS applied directly into water (SDS is insoluble in organic solvent and the method by Beeby et al. (25) was not applicable) at concentration 5 g/L (above c.m.c.) did not stabilize C60. While non-ionic Tween 65 did not stabilize C60 in the aqueous phase, another non-ionic surfactant, Brij 78 efficiently dispersed C60 in water, producing a yellowish suspension. Accordingly, C60/Brij did not show a broad band in the wavelength region from 400 to 500 nm (Figure 3a) and effectively produced 1O2 under UV irradiation (Figure 3b). Through these studies, it is demonstrated that 1O2 was produced efficiently by molecularly dispersed C60 in toluene and less efficiently by C60 dispersed and stabilized in the aqueous phase through association with TX and Brij. In contrast, no measurable amount of 1O2 was produced when C60 was present as aggregate (nC60 and son/C60). When C60 was present with PVP, 1O2 was produced only when PVP concentration was high, eliminating C60 aggregation. These observations suggest that inhibition of 1O2 production by C60 under UV irradiation may result from C60 aggregation and the degree of C60 aggregation is influenced by the type of solvent and dispersion method (41). The effect of C60 aggregation on its differential photoactivity may have resulted because the sites that are available for energy transfer to oxygen are greatly reduced when C60 is present as aggregates as compared to individual molecules. Alternatively, it can be hypothesized that photoexcited triplet C60 (3C60*) at the aggregate surface may be effectively quenched by surrounding ground state C60 (self-quenching) and another triplet C60 (triplet-triplet annihilation) within the aggregate structure and dissipated as heat (42) according to the following scheme: hν

nC60 ((C60)n) 98 n3C60* ((3C60*)m/(C60)n-m) (photoexcitation) (2) n3C60* ((3C60*)m/(C60)n-m) + m3O2 f nC60 ((C60)n) + m1O2 (1O2 production) (3) n3C60* ((3C60*)m/(C60)n-m) f nC60 ((C60)n) + heat (self-quenching or triplet-triplet annihilation) (4) Such a transfer of absorbed photoenergy to ground or excited state C60 in direct contact with 3C60* (reaction 4) may be favored to energy transfer to oxygen (reaction 3). At present, the quantitative evaluation of the contribution of either mechanism is not complete and requires further study. Photoactivity of C60 Associated with NOM. NOM has been reported to enhance the availability of hydrophobic organic pollutants (e.g., polycycloaromatics) in the aqueous phase (43, 44). Similar to surfactants that promote hydrophobic environment within the micelle structure, NOM may facilitate C60 stabilization in the aqueous phase. UV-vis spectra of 20 mg/L C60 associated with 40, 100, and 200 mg/L of SRNOM and diluted four times (Figure 4a) showed a presence of broad band absorption over 400-500 nm, although absorption was weaker than nC60, and spectrum patterns were slightly modified (e.g., blue-shifted specific peak at 350 nm and stronger absorption at 262 nm). Accordingly, the average population diameters of 103.8 and 104.7 nm were measured for 100 mg/L and 200 mg/L NOM samples, respectively, by DLS, indicating C60 aggregation occurred. The absorption 2532

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FIGURE 4. (a) UV-vis absorption spectra of 20 mg/L C60 stabilized in water with 40, 100, and 200 mg/L SRNOM after diluting four times, and (b) photochemical degradation of FFA in aqueous suspension of C60/SRNOM. ([C60/SRNOM]0 ) 5 mg/L; [FFA]0 ) 0.85 mM; [phosphate]0 ) 10 mM; pHi is 7). band at 400-500 nm became more evident at higher concentrations of C60/NOM, further confirming aggregation (data not shown). Unlike polymer and surfactant, NOM did not inhibit the aggregation even at higher concentration (up to 200 mg/L per 5 mg/L of C60). Results from photochemical experiments shown in Figure 4b suggests that 1O2 production by C60/NOM is negligible, after taking into consideration FFA degradation by SRNOM solution (prepared by the same procedure without C60) (31). This result suggests that C60 existing in natural water as aggregates may not exhibit intrinsic photochemical reactivity. However, a more detailed study needs to be conducted over a wider range of conditions to verify this conclusion. Photochemical Production of Superoxide Radical Anion. Photoexcited C60 accepts one electron from electron donors (EDs) such as dimethylaniline (45), triethylamine (TEA) (46), ascorbate (47), and photoexcited TiO2 (48) more efficiently than ground state C60 (E0(3C60*/3C60•-) ) +1.1 VNHE vs E0(1C60/1C60•-) ) -0.2 VNHE, ref 23). Subsequently, 3C60•- transfers electrons to easily reducible species (22, 23) such as oxygen, producing O2•-. Overall, C60 mediates electron transfer from ED to oxygen under UV irradiation as follows:

Experimental results examining O2•- generation by 5 mg/L of C60 with 5 mM TEA as ED and 0.1 mM NBT2+ as O2•indicator are shown in Figure 5. Changes in absorption spectra indicate production of purple-colored monoformazan (MF+) with λmax ) 530 nm (i.e., product of NBT2+ reduction by O2•-) and consequently, O2•-. Control tests performed with

FIGURE 5. UV-vis absorption spectra of aqueous suspensions of (a) nC60, (b) C60/PVP (([C60]/[PVP])(w/w) ) 0.0008), and (c) C60/TX (([C60]/ [TX])(w/w) ) 0.00094) in the presence of NBT2+ and TEA, as a function of irradiation time ([C60]0 ) [C60/PVP]0 ) [C60/TX]0 ) 5 mg/L; [NBT2+]0 ) 1 mM; [TEA]0 ) 5 mM; [phosphate]0 ) 50 mM; pHi is 7).

FIGURE 6. Generation of H2O2 in UV-irradiated suspension of (a) nC60, (b) C60/PVP (([C60]/[PVP])(w/w) ) 0.0008), and (c) C60/TX (([C60]/ [TX])(w/w) ) 0.00094) with SOD and TEA. ([nC60]0 ) [C60/PVP]0 ) [C60/TX]0 ) 5 mg/L; [SOD]0 ) 20,000 unit/L; [TEA]0 ) 5 mM; [phosphate]0 ) 50 mM; pHi is 7). (1) NBT2+ alone, (2) NBT2+ with TEA, and (3) NBT2+ with C60/TX did not produce O2•- photochemically (results not shown). Consistent with 1O2 results, nC60 did not produce any measurable amount of O2•- (Figure 5a). A minor change in the spectra of nC60 during initial 20 min of reaction was also observed in the dark and was not the result of photoinduced O2•- production. The absence of nC60 photoactivity might be due to a decrease in available reaction sites and/or deactivation of 3C60* by neighboring C60 molecules within the aggregate as previously discussed. Also consistent with the observation made for production of 1O2, C60/TX (([C60]/[TX])0 ) 0.00094) generated O2•- at an estimated concentration of ca. 50 µM after 60 min of UV irradiation (Figure 5c). A much lower, yet measurable level of O2•- (4 µM after 120 min) was produced by C60/Brij (results not shown). However, C60/PVP (([C60]/[PVP])0 ) 0.0008) was not able to produce O2•- (Figure 5b), even though 3C60* should be available for further reaction (i.e., accept electron) as evidenced by 1O2 production. A separate set of experiments was performed to measure production of H2O2 in the presence of SOD as an indirect

measurement of O2•- production. For these systems, a commonly used DPD (N,N-diethyl-p-phenylenediamine) colorimetric method was not appropriate for H2O2 measurement (49), as nC60 caused colorization of the mixture of DPD and horseradish peroxidase, thus interfering with detection of trace concentrations of H2O2. Therefore, a DMP method (35) was used instead. The method of using H2O2 as indirect indicator was verified by independent control experiments that showed (1) production of O2•- (measured by TNM method) and H2O2 (measured by DMP method) by xanthine and xanthine oxidase (i.e., O2•- precursors) and (2) quenching of O2•- and enhanced production of H2O2 in the presence of SOD (results not shown). Consistent with the previous results, negligible amount of H2O2 was produced by nC60 and C60/ PVP ([C60]/[PVP])0 ) 0.0008) (Figure 6a and b), while a significant amount of H2O2 (about 14 µM H2O2 after 60 min of UV irradiation) was generated by C60/TX (([C60]/[TX])0 ) 0.00094) (Figure 6c). This production rate of H2O2 as indirect probe (ca. 0.23 µM/min) would correspond to ca. 0.83 µM O2•-/min, measured using NBT method, based on the observation that initial generation rate of O2•- in xanthine/ VOL. 41, NO. 7, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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xanthine oxidase with SOD was estimated as ca. 4 times faster than that measured indirectly through H2O2. Negligible production of O2•- by C60/PVP can be attributed to relatively efficient recombination of C60•- and TEA•+ in this system which would prevent the electron transfer to oxygen. For example, the lifetime of C60•- produced photochemically in the presence of 1,4-diazabicyclo[2,2,2]octane as an ED was reported to be much shorter in organic C60 solution than in C60/TX micellar solution, despite identical quantum yields for the production of 3C60* (50, 51). When C60 is present within TX micelles, these ionic radicals (C60•-, ED•+) can be separated between TX micelle core (i.e., organic phase) and bulk aqueous phase thus stabilizing C60•- by retarding their recombination with oxidized ED (50). It has also been recognized that more hydrophobic ED shortens the lifetime of C60•- to a greater extent due to easier access to C60•- located at hydrophobic core (51). In conclusion, it is demonstrated that the aggregation of C60 in the aqueous phase resulted in loss of intrinsic photochemical reactivity by pristine C60 with respect to production of 1O2 and O2•-. This study further highlights the difference between 1O2 production versus O2•- production by photoinduced C60 in the aqueous phase, as the latter is affected not only by its aggregation status but also the nature of stabilizing molecules. These findings are significant considering toxicological effects of nC60 which have been hypothesized by some to result from production of these ROS. While our results suggests that C60 in a water stable aggregate might not trigger cytotoxicity through ROS production, as observed for nC60 and son/nC60, it may very well be possible, however, for C60 to produce ROS under specific environments, e.g., within lipid bilayers, a condition similar to surfactant micelles. Future studies, therefore, should be focused on understanding the exact dispersion status of C60 inside biological receptors.

Acknowledgments This work was funded by the United States Environmental Protection Agency (USEPA) STAR grant no. D832526. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. The scientific views expressed are solely those of the authors and do not necessarily reflect those of USEPA. Support for J.S.L. was partially provided by Korea Research Foundation grant no. KRF-2006-214-D00082. We thank Dr. Min Cho at Korea Interfacial Science and Engineering Institute, Korea, for assistance with construction of photochemical reactor used in this study.

Supporting Information Available Descriptions of the chemical used, method for the aqueous suspension preparation, and analytical procedures for monitoring reactive oxygen species and H2O2. A control test to verfify FFA degradation by 1O2 performed with UV-irradiated rose-bengal solution is presented. Production of 1O2 pristine C60 in toluene was confirmed. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review August 29, 2006. Revised manuscript received January 25, 2007. Accepted January 26, 2007. ES062066L

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