Environ. Sci. Technol. 2009, 43, 6208–6213
Evaluation of the Oxidation of Organic Compounds by Aqueous Suspensions of Photosensitized Hydroxylated-C60 Fullerene Aggregates SO-RYONG CHAE, ERNEST M. HOTZE, AND MARK R. WIESNER* Department of Civil and Environmental Engineering, Pratt School of Engineering, Duke University, Durham, North Carolina 27708
Received April 17, 2009. Revised manuscript received June 1, 2009. Accepted June 30, 2009.
Ultraviolet (UV) irradiated polyhydroxylated fullerene (fullerol) nanomaterials are examined for their potential to degrade organic compounds via reactive oxygen species (ROS) mediated by a photosensitization process. Organic compounds were selected for their sensitivity to individual species of reactive oxygen (hydroxyl radical ( · OH-) for degradation of salicylic acid (SA); singlet oxygen (1O2) for degradation of 2-chlorophenol (2CP), and superoxide (O2 · -) for oxidation of ethanol) and were monitored over time in aqueous suspensions of fullerol aggregates. Only the 2CP showed significant degradation underscoring the specificity of the fullerol in producing singlet oxygen in these conditions. Monitoring these processes via high performance liquid chromatography (HPLC) confirmed that organic compounds degraded primarily by ROS over a range of fullerol concentrations, pH values, and temperatures.
1. Introduction An interesting and potentially useful characteristic of some nanomaterials is their ability to catalyze reactions, including photocatalytic and redox-reactions that produce oxidizing species that may degrade compounds or damage cells (1, 2). In particular, aqueous suspensions of some fullerene aggregates have been observed to produce reactive oxygen species (ROS) such as singlet oxygen and peroxide radicals in the presence of ultraviolet (UV) or visible light (3-5). Fullerenes owe their photochemical activity to their strong absorbance throughout the UV spectrum, and to their conjugated molecular structure (6). Fullerol, a polyhydroxylated form of C60, readily forms stable suspensions of aggregates in water that are efficient producers of ROS (5). In previous studies, we have shown that fullerol aggregates produce ROS via UV sensitization when measured by a variety of means: furfuryl alcohol degradation (5), electron paramagnetic resonance (EPR) (7), XTT reduction (7), and bacteriophage inactivation (8). Singlet oxygen was the primary ROS molecule detected but superoxide was also observed when an appropriate electron donor was present with the fullerol (7, 8). In the latter study photosensitized fullerol aggregates were capable of inactivating MS2 bacteriophage at a rate twice that produced by UV light alone (8). * Corresponding author phone: 919-660-5292; fax: 919-660-5219; e-mail:
[email protected]. 6208
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Moreover, data from the medical literature detail the ability of C60 and C70 fullerenes to cleave deoxyribonucleic acid (DNA) and kill viruses, bacteria, and tumor cells (9-12). Lastly, based on our previous experiments hydroxyl radical production by sensitized fullerol is not anticipated, although this powerful form of ROS may result from secondary products (3, 4). These properties suggest important medical and environmental engineering applications as well as possible toxic effects if released to the environment. While direct toxicity of fullerenes has been studied over the past decade, very little consideration has been given to the impacts that nanomaterials might have on aquatic environments through their ability to transform other molecules in solution. In this work, we examine the potential for oxidation of three organic compounds by ROS produced by fullerol suspensions. Three compounds were selected based on their known relative affinity for oxidation by three species of reactive oxygen (i.e., singlet oxygen, superoxide, and the hydroxyl radical). The compound 2-chlorophenol (2CP) was selected for its sensitivity to degradation by singlet oxygen (13). Ethanol was selected based on its sensitivity to oxidation by superoxide (O2 · -) (14) while salicylic acid (SA) is known to react with the hydroxyl radical ( · OH-) (15). Thus, the reactivity of these compounds serves as a probe for the dominant species of reactive oxygen present under given conditions of fullerol photosensitization. Rose Bengal (RB), a well-known photosensitizing compound that produces singlet oxygen (16), serves as a positive control for experiments.
2. Materials and Methods 2.1. Chemicals. Fullerol (C60(OH)24)) was purchased from MER (Tucson, AZ). Superoxide dismutase (SOD), SA, 2,3dihydroxy benzoic acid (2,3-DHBA), 2,5-dihydroxy benzoic acid (2,5-DHBA), pyrocatechol, 2CP, ethanol (200 proof), 2-chloro-1,4-benzoquinone, and maleic acid were obtained from Sigma-Aldrich (St. Louis, MO). Detailed chemical information of the “probe” organic compounds (i.e., SA, 2CP, and ethanol) is available in Supporting Information (SI) Table S1. Monobasic potassium phosphate (KH2PO4) and dibasic potassium phosphate (K2HPO4) were purchased from EMD Chemicals (Gibbstown, NJ). Rose Bengal (RB) and sodium azide (NaN3) were purchased from Acros Organics (Fairlawn, NJ). All reaction solutions were prepared in deionized (DI) water (NANOpure, Barnstead, Dubuque, IA) that had a minimum resistivity of 18.2 MΩ-cm and dissolved organic carbon concentration was less than 0.05 ppm. 2.2. Preparation and Characterization of Fullerol Suspensions. Stock fullerol suspensions were prepared by adding fullerol in powder form to DI water as described previously (7). Total carbon (TC) concentrations of fullerol suspensions were measured by a total organic carbon (TOC) analyzer (TOC-5050A, Shimadzu, Columbia, MD). Analysis of the stock suspension showed that it contained 58 mg/L as TC (excluding CO2). The resultant suspension was then added to solutions containing each organic compound at an initial concentration of 50 µM. This mixed suspension was added to 50 mM potassium phosphate buffered at pH values of 4.5, 6.9, and 9.0 (Table 1). Size distributions were measured by dynamic light scattering (DLS) using an ALV/CGS-3 Compact Goniometer System (ALV-GmbH, Germany). Electrophoretic mobility and conductivity were determined by a Zetasizer Nano ZS (Malvern Instrument, Bedford, MA). Molecular weight distributions of dissolved carbon in fullerol suspensions were determined by liquid chromatography (LC) (Hitachi, Japan) as described elsewhere (17) with UV (Hitachi 10.1021/es901165q CCC: $40.75
2009 American Chemical Society
Published on Web 07/17/2009
TABLE 1. Effects of Ion Strengths on Aggregation of Fullerol Nanoparticlesa fullerol (10 µM)
volume composition pH
KH2PO4
K2HPO4
dp ζ potential conductivity (nm) (mV)
In DI water 5.4 4.5 in buffer solutions 6.9 9.0
0% 100% 50% 0%
0% 0% 50% 100%
98.6 3.56 5.50 7.66
µS/cm mS/cm mS/cm mS/cm
102 228 248 276
sitizing dye such as RB (19). Assuming that the probe substance reacts primarily with singlet oxygen (1O2) and negligibly with the excited sensitizers, a simplified reaction scheme can be used to describe this system where we substitute fullerol, F, for photosensitizers originally described (19):
-32.4 -21.9 -18.4 -14.2
hv
F 98 1(F)∗ (F)∗ f 3(F)∗
1
a
dp: hydrodynamic diameter of aggregates measured by DLS.
(2)
Φ
3
L-4000, Japan) and online organic carbon detectors (Sievers 810 turbo portable TOC analyzer, General Electric, Boulder, CO) following two size exclusion chromatography (SEC) columns in order to separate organic molecules by size. 2.3. Experimental Conditions. All experiments were performed in glass beakers (90, o.d. × 115 mm, H) with a water jacket connected to a water circulator for temperature control (i.e., 10, 20, and 30 °C). Two 15 W fluorescent ultraviolet bulbs (Philips TLD 15W/08) in an UV/Cryo chamber (Electron Microscopy Science, Hatfield, PA) were used as a light source. These bulbs had an output spectrum ranging from 310 to 400 nm and a total irradiance of 24.1 W/m2 with a peak at 365 nm. Water samples were collected from the suspension every five minutes for 30 min for further analyses. All experiments were performed in triplicate. Student’s t test was used to assess the significance of the results employing a 95% confidence interval. 2.4. Analysis of Probe Organic Compounds. The concentrations of SA, 2CP, as well as oxidation products of SA (i.e., 2,3-DHBA, 2,5-DHBA, pyrocatechol) (15) and 2CP (i.e., pyrocatechol, 2-chloro-1,4-benzoquinone, 2-chlorohydroquinone, and maleic acid) (18) were measured using a highperformance liquid chromatography (HPLC) (ProStar, Varian, Palo Alto, CA) equipped with a reverse phase column (Ultra aqueous C18, 5 µm, 150 × 4.6 mm, RESTEK, Bellefonte, PA) and a photodiode array (PDA) detector at a wavelength of 210 nm. For the analysis, the flow rate of the mobile phase consisting of 25 mM KH2PO4 and HPLC-grade acetonitrile was maintained at 1 mL/min. The composition of mobile phases (KH2PO4/acetonitrile) was 80:20 for the first 2 min, it was then adjusted at rate of 10%/min to 50:50 which was maintained for 7 min. The pH in the mobile phase was adjusted to 2.5 using 6N HCl solution. Under these conditions, the retention times of pyrocatechol, 2,5-DHBA, 2,3-DHBA, SA, and 2CP were 5.6, 6.1, 6.6, 8.5, and 9.0 min, respectively. Ethanol concentration was determined by gas chromatography (GC) with a flame ionization detector (GCMS-QP5050, Shimadzu, Columbia, MD) equipped with a fused silica capillary column (DB-1, 5 µm, 30 m × 0.32 mm, J&W Scientific, Folsom, CA) using diethyl ether as an internal standard. The oven temperature was ramped from 40 to 160 °C at a rate of 10 °C/min. The injector temperature and detector temperature were 250 and 300 °C, respectively. The flow rate of the carrier gas (He) was 16.2 mL/min. Under these conditions, the retention times of ethanol and diethyl ether were 2.7 and 3.8 min, respectively. 2.5. Fullerol Separation from the Suspension. The removal of probe organic compounds by adsorption on fullerol aggregates, was evaluated by dialysis using a dialysis tube of molecular weight cut off (MWCO) 100 Da (Float-ALyzer, Spectrum Laboratories, Inc., Rancho Dominguez, CA) to separate fullerol from the dialysate. However, this method was revealed to yield imperfect separations of the fullerol as discussed in Section 3. 2.6. Kinetics. Photo oxidation of phenolic compounds has been widely studied by continuous irradiations of solutions containing a probe substrate (P) and a photosen-
(1)
Wa
(F)* + 3O2 98 F + 1O2
(3)
kq
1
O2 + P 98 3O2 + P
(4)
kr
1
O2 + P 98 products 1
(5)
kd
O2 98 3O2
(6)
where F is the ground-state fullerol (sensitizer), F* is the excited sensitizer, 1F is the singlet-sate fullerol, 3F is the tripletsate fullerol, 3O2 is ground-state O2, 1O2 is singlet O2, P is the probe compound, Wa is the rate of light absorption by the sensitizer, Φ is the quantum efficiency of the photosensitized production of 1O2, kq is the second-order rate constant of physical quenching of 1O2 by the probe compound, kr is the second-order rate constant of chemical reaction between 1 O2 and the probe compound, kd is the first-order rate constant of physical quenching of 1O2 by water. When irradiation is continuous (Wa ) constant), and concentration of F is constant, the steady-state approximation for 1O2 can be applied and the following rate law is derived for a well-mixed system. -d[P]/dt ) Wa*Φ* kr[P]/{kd + (kr + kq)[P]}
(7)
In this work, the initial concentration of the probe compound is 50 µM (5 × 10-5 M) and the kd value is taken as 4.4 × 105 s-1 (19). In this case kd . (kr + kq)[P] and eq 7 can be simplified as first order in concentration of P ([P]). -d[P]/dt ) (Wa*Φ* kr/kd)[P] ) kobs[P]
(8)
where kobs is the pseudo-first-order rate constant (sec-1) of chemical reaction between 1O2 and the probe compound. The rate of photochemical transformation of the compound P by direct photolysis under illumination at a wavelength λ, depends on the absorption coefficient at that wavelength (ελ), the light intensity flux of incident light (Io,λ) and quantum yield of the reaction (φλ) (20). -d[P]/dt ∝ (ελ)(Io,λ)(φλ)[P] ) kdp[P]
(9)
where kdp is the pseudo-first-order rate constant (s-1) of chemical degradation by direct photolysis from the experimental results. The net pseudo-first-order rate constant, knet (s-1) is calculated by subtracting kdp from kobs. knet ) kobs - kdp
(10)
3. Results and Discussion 3.1. Characteristics of Fullerol in Potassium Buffer Solutions. Size and electrophoretic mobility (EPM) of the fullerol aggregates were characterized over a range of ionic strengths in potassium buffer solutions. As shown in Table 1, it was VOL. 43, NO. 16, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. Oxidation of 2-chlorophenol by the photosensitized-fullerol (10 µM) with various pHs at 20 °C (A) and various temperatures at pH 6.9 (B). FIGURE 1. Oxidation of model organic compounds by the photosensitized-fullerol with various fullerol concentrations at 20 °C and pH 6.9. observed that the diameters of the fullerol aggregates in DI water ranged from 30 to 140 nm with a number mean diameter of 102 nm. The ζ potential (calculated from EMP using Henry’s equation) of the fullerol suspension was -32.4 mV. Upon introduction of the stock fullerol to potassium buffer solutions showing various pHs (i.e., 4.5, 6.9, and 9.0), the fullerol nanoparticles likely form larger aggregates. As ionic strength increased the zeta potential of the fullerol suspensions became less negative, increasing from -21.9 to -14.2 mV. Concurrently, the size of fullerol aggregates increased from 228 to 276 nm likely due to the decrease in charge repulsions between the molecules, an observation we have previously made for fullerol (21). 3.2. Effects of Fullerol Concentration, Temperature, and pH on Oxidation of Organic Compounds. To determine the effects of fullerol concentration, temperature, and pH on organic degradation, various batch tests were conducted under UV irradiation. First, the effect of fullerol loading on the degradation of probe organic compounds was studied. In these experiments, temperature and pH were maintained at 20 °C and 6.9 (buffered at 50% KH2PO4 and 50% K2HPO4), respectively. For degradation experiments, SA concentration remained roughly constant over 30 min of irradiation (Figure 1) while 2CP degradation could be directly correlated with the concentration of fullerol present in suspension. Approximately 9, 18, 23, and 38% of initial 2CP concentration (50 µM) was degraded at concentrations of 1, 10, 25, and 50 µM fullerol, respectively. The 2CP degradation efficiency by 10 µM RB was slightly higher than that observed for 25 µM fullerol. In order to control for any nonsinglet oxygen reactions with 2CP, NaN3 (100 µM) was introduced as a scavenger of singlet oxygen with 10 µM fullerol, and 6210
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consequently degradation of 2CP was not observed (data not shown). In the case of SA and ethanol, the concentrations of the initial SA and ethanol concentrations decreased by approximately 7% in the presence of 50 µM fullerol. Because the molecular structure of SA is similar to that of 2CP it is important to understand why the degradation efficiency of 2CP was much higher than that of SA in these experiments. Evaluating the hydrogen dissociation energies of the three compounds reveals that the oxygen-hydrogen bond on 2CP is most easily dissociated (
