Environ. Sci. Technol. 1996, 30, 1409-1410
Response to Comment on “Solubility Enhancement and Fluorescence Quenching of Pyrene by Humic Substance: The Effects of Dissolved Oxygen on Quenching Processes” SIR: The comments on our paper (1) by Green and Blough (2) are insightful and well-taken. Nonetheless, we believe that our observations do quantify processes at the molecular level that are not artifactial in nature. Green and Blough (2) noted that our lifetime data could be caused by environmental factors such as the undersaturation of dissolved oxygen (DO) prior to the inception of an experiment or fluctuations in temperature (which would invariably increase or decrease DO). With respect to being undersaturated with dissolved oxygen, our samples were small (approximately 3 mL), made 2 days in advance of the analysis, and stored in a refrigerator. Prior to the experiments, the samples were brought up to the system temperature. We believe that this provided a sufficient amount of time for the solution phase to preequilibrate with atmospheric oxygen. With respect to the later comment, all the experiments were conducted in a temperaturecontrolled lab (where temperature fluctuations were anticipated to be small). Even if one assumes that the temperature in our lab decreased by 10 °C, the increase in oxygen’s solubility to 329 µM (assuming that ∆H° remains constant over this range) (3) would translate into a decrease of τ by only 9 ns (using a kq of 1010 M-1s-1) (2). We, however, observed a consistent decrease in pyrene lifetime with increasing fulvic acid concentration (ref 1, Figure 2). Had this been attributable to a temperature-related increase in DO, it would imply that the temperature of the solution in the cuvette consistently decreased at higher fulvic acid concentrations. Indeed at the highest fulvic acid concentration, we observed a 17% decrease (∼22 ns) in pyrene’s lifetime. Green and Blough’s comment (2) regarding our reported observations of a 8-10% decrease in the probe lifetime is reflected at only one of the lower fulvic acid concentrations. Finally, if temperatures did indeed fluctuate (i.e., increased and decreased) through the time course of the experiments, one would anticipate random measured lifetimes. In addition to decreases in pyrene’s lifetime as a function of Suwannee River fulvic acid concentration, we also observed decreases in τ with increasing concentrations of Suwannee River humic acid (Figure 1) as well as for other humic materials (Lake Fryxell and Aldrich humic acid). Thus, it is highly improbable that our observations are caused by environmentally generated fluctuations in dissolved oxygen as a result of temperature variations. With respect to the precision of our data, we had the most variability for the “air-saturated” pyrene in the absence of FA, where the lifetime was 141 ( 8 ns. Measurements in the presence of humic substances were significantly more precise, and we never observed error bars more severe than (4 ns (most were near (0.5 ns). In this regard, we believe that Green and Blough are correct in attributing this magnitude of variability to temperature-induced changes (which would be small in our lab) in the dissolved oxygen concentration.
0013-936X/96/0930-1409$12.00/0
1996 American Chemical Society
FIGURE 1. Fluorescence lifetimes of pyrene in the absence and presence of Suwannee River humic acid. Note that the concentrations of humic acid do not exceed 15 mg/L organic carbon.
We are in partial agreement with Green and Blough in the inherent dangers of fitting our data to the later half of our decay curves. We were forced to analyze our data in this manner to circumvent the interfering fluorescence originating from our fulvic and humic acids. Nonetheless, despite the fact that 59% of the photons are emitted before 115 ns, we did collect more than 10 000 counts in the channel corresponding to 115 ns for that very reason. Furthermore, we weighted our fits using Poisson statistics, so we do not think the tails of the curves were overemphasized in the fitting procedure. The standard deviations “of the fits” were never high, and none were more than (0.5 ns. The arguments put forth by Green and Blough are logical and consistent with oxygen’s perceived behavior in aqueous systems in the presence of humic materials. In light of this, it would seem improbable that our original mechanism (1) can explain all the discrepancies between fluorescence quenching and solubility enhancement derived partition coefficients (Koc). Nonetheless, we still believe that the presence of oxygen in or near humic material microenvironments can play a significant role. To illustrate this point, we calculated a Koc value of 23.2 L/kg of OC for oxygen using a “super heated” aqueous liquid solubility value of 1.48 × 10-1 M, (a vapor pressure of 552 atm at 25 °C was calculated from temperature-vapor pressure data and the Antoine equation) (4) and the relationships found in Schwarzenbach et al. (5) (we assumed Koc ) 2 × Kom). By assuming that the partitioned oxygen is in equilibrium with the its “pure water” saturation value of 266 µM, the amount associated with the humic material becomes 6.1 × 10-3 mol of O2/kg of OC. For pyrene, we calculated a value of 6.91 × 10-3 mol/kg of OC based upon our measured Koc value of 10 230, and the assumption that the amount sorbed is in equilibrium with its “pure water” solubility value of 0.676 µM (1, 5). Thus, within these “microenvironments”, there are essentially equal numbers of oxygen and probe molecules per unit mass of humic substance expressed as organic carbon, and it is probable that any fluorescing pyrene molecule (bound or free) may be subject to dynamic quenching by DO.
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The comments provided by Green and Blough (2) did further alert us to the existence of other photochemical processes that we did not consider before. Foremost are the formation of phototransients (e.g., singlet oxygen, carbon-centered radicals, hydroxide radicals, etc.) that may influence both the lifetime and stability of pyrene in the presence of humic materials. Indeed it is highly probable that these complex parallel and competing reactions may be responsible for many of the discrepancies that one observes between fluorescence quenching derived Koc values and those measured using other techniques. In the absence of oxygen, the formation of photo transients is sharply reduced (carbon-centered radicals and solvated electrons can still be formed but they have exceedingly low quantum yields), and one would see no effect on the quenching of pyrene by these species. In air-saturated solutions, however, oxygen-derived reactive species can be formed from the quenching of the excited triplet state humic materials by molecular oxygen, and these transients may exert influences on the fluorescence of the probe compound. Thus, any decreases in the fluorescence of pyrene caused by its interaction with these photooxidants could be interpreted as static quenching, which would result in higher than anticipated Koc values.
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Literature Cited (1) Danielsen, K. M.; Chin, Y. P.; Buterbaugh, J. S.; Gustafson, T. L.; Traina, S. J. Environ. Sci. Technol. 1995, 29, 2162. (2) Green, S. A.; Blough, N. V. Environ. Sci. Technol. 1996, 30, 14071408. (3) Stumm, W.; Morgan, J. J. Aquatic Chemistry, 3rd ed.; J. Wiley and Sons: New York, 1995. (4) CRC Handbook of Chemistry and Physics, 60th ed; CRC Press: Cleveland, OH, 1980; p D-203. (5) Schwarzenbach, R. P.; Gschwend, P. M.; Imboden, D. Environmental Organic Chemistry; J. Wiley & Sons: New York, 1993; p 273.
Yu-Ping Chin,* Jeffrey M. Buterbaugh, Terry J. Gustafson, and Samuel J. Traina The Ohio State University Columbus, Ohio 43210
Karlin M. Danielsen CH2MHILL Sacramento, California 95833 ES951009H
