Environ. Sci. Technol. 1997, 31, 424-429
Effects of Dissolved Oxygen and Light Exposure on Determination of KOC Values for PAHs Using Fluorescence Quenching CHRISTINE L. TILLER* AND KIM D. JONES School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332
The fluorescence of some polycyclic aromatic hydrocarbons (PAHs) has been observed to decline over time, potentially leading to overestimation of PAH-humic binding coefficients (KOC) determined by fluorescence quenching. This phenomenon was investigated using phenanthrene, anthracene, pyrene, triphenylene, and a soil humic acid. Fluorescence quenching experiments were conducted under ambient conditions and under conditions that minimized exposure of the samples to UV radiation and dissolved oxygen. All four compounds experienced decays in fluorescence intensity when exposed to UV radiation and dissolved oxygen, but anthracene and pyrene were affected to a much larger extent than phenanthrene and triphenylene. Baseline decays in fluorescence intensity were reflected in inflated apparent KOC values, particularly for anthracene and pyrene. For example, the apparent KOC for anthracene when no attempt was made to limit dissolved oxygen or exposure to UV radiation was 10.9 × 104 mL/g of C. When exposure to UV radiation alone was minimized, the apparent KOC decreased by a factor of 2. A similar result was found when exposure to dissolved oxygen alone was minimized. When exposure to both UV radiation and dissolved oxygen was minimized, KOC was 3.69 × 104 mL/g of C. Photooxidation processes may explain these observations; however, more work is needed to confirm this.
Introduction The behavior and fate of hydrophobic organic contaminants in groundwater and surface waters are significantly influenced by the interactions of these compounds with natural organic matter, a large fraction of which is comprised of humic substances. Many recent studies have evaluated the extent of binding of pesticides (1-4), polycyclic aromatic hydrocarbons (2, 5-15), and other organic compounds (2-4, 1620) by natural organic materials from a variety of sources. Several methods have been used to evaluate the binding coefficient, KOC, of these organic contaminants with humic substances, including dialysis (1, 5, 12), reverse-phase separation (2, 6, 10), solubility enhancement (3, 4, 15), and fluorescence quenching (6-15, 20). Each method has some limitations. In some studies that have used phase separation techniques, for example, the experimental binding coefficients have been observed to decrease with increasing humic concentrations (1, 2, 5, 6). The solubility enhancement method is usually considered reliable, but it typically requires * Corresponding author telephone: (404) 894-9723; fax: (404) 8949724; e-mail address:
[email protected].
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high concentrations of humic substances (at least 10-50 mg of C/L and as much as 1000 mg of C/L) and may be insensitive when used for compounds whose solubility exceeds about 10-5 M (3, 4, 15). Fluorescence quenching does not rely on phase separation and can be conducted using low concentrations of target compounds and humic substances that are more relevant to environmental conditions. This method too is susceptible to certain experimental artifacts. For example, frequently noted in the literature has been the possibility of solute sorption to the reactor walls (6, 10, 13, 14). Some investigators have also noted that for some compounds care should be taken to prevent photodegradation (6, 10, 14). One class of organic contaminants particularly suited to fluorescence quenching is polycyclic aromatic hydrocarbons (PAHs). PAHs are commonly found in areas contaminated by creosote or coal tar and locations where crude oil spillage has occurred. They also can be produced as a result of incomplete combustion in incinerators, power plants, etc. Some PAHs are known carcinogens while many others are still undergoing toxicity evaluation (21). Because it is a fast and efficient technique, fluorescence quenching has been a popular method for studying the interaction of PAHs with natural organic materials (6-15). The objective of this work was to investigate experimental artifacts associated with sample exposure to UV radiation and oxygen during the determination of KOC values for PAHs using fluorescence quenching. The motivation for this study is illustrated in Figure 1 which shows that the baseline fluorescence intensity of four PAH compounds decreased over time when the samples were continuously monitored. For reasons that will be detailed later in this paper, these fluorescence losses are not explained by sorption to the walls of the fluorescence cell. Even if the observation is restricted to the first 5 min, representing the minimum total time a sample is exposed to UV radiation during a typical fluorescence quenching experiment, the baseline fluorescence decay can be substantial for some compounds. In order to evaluate the effect of this phenomenon on KOC determination, binding coefficients were determined under ambient conditions and under conditions designed to limit the presence of oxygen in the samples and their exposure to UV radiation. Four PAHs representing a range of characteristics were studied (phenanthrene, anthracene, pyrene, and triphenylene) along with a standard soil humic acid. Some properties of these PAHs are summarized in Table 1. While some consideration is given in this paper to the possible origins of the observed effects, our emphasis is on their consequences for studies of KOC.
Fluorescence Quenching Stern-Volmer Equation. The binding coefficient, KOC (mL/g of C), between humic substances and a PAH in aqueous solution can be defined as the ratio of the concentration of PAH bound to humic material, COC (mg/g of C), and the concentration of PAH in solution, CW (mg/mL):
KOC )
COC CW
(1)
The concentration of a fluorescent PAH is directly proportional to its fluorescence intensity. Since binding by humic material quenches PAH fluorescence, KOC can be determined by measuring the decrease in fluorescence intensity of a PAH solution caused by the addition of humic substances. Three types of fluorescence quenching have been described: static, dynamic, and apparent (6). In the case of static quenching, the complex that is created by the interaction
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1997 American Chemical Society
FIGURE 1. Baseline PAH fluorescence intensity vs time measured under ambient conditions with the shutter open. Nominal concentrations were 13 µg/L for pyrene, 56 µg/L for phenanthrene, 6.7 µg/L for anthracene, and 3.6 µg/L for triphenylene. No humic acid was present in these samples.
TABLE 1. Properties of Selected PAH Compounds phenanthrene anthracene pyrene triphenylene
formula
log KOWa
log Sa
τb
C14H10 C14H10 C16H10 C18H12
4.63 4.63 5.22 5.45
-5.15 -6.377 -6.176 -6.726
56.0 4.9 450 36.6
aK OW, octanol-water partition coefficient; S, solubility (M/M) in water at 25 °C (22). b τ, fluorescence lifetime (ns) in cyclohexane (23).
of the fluorophore and the quencher is nonfluorescent. Dynamic quenching occurs when the quencher diffuses to the fluorophore during the lifetime of its excited state and then deactivates that state without fluorescence. Apparent quenching is not a true quenching process but is the decrease in measured fluorescence caused by partial absorption of the excitation beam due to the optical density of material in the fluorescence cell (the inner filter effect). When static quenching by the humic material is predominant, a simple equation that relates KOC to fluorescence quenching can be derived from the mass balance equation for the PAH:
Ctotal ) CW + CQCOC
(2)
where Ctotal (mg/mL) is the total concentration of the PAH, and CQ is the concentration of the humic material (g of C/mL). Substituting for COC using eq 1 and rearranging yields the following expression:
Ctotal ) 1 + CQKOC CW
(3)
If there is no interference and the fluorescence of bound PAH is completely quenched, aqueous PAH concentration is proportional to the fluorescence intensity of the sample, and eq 3 can be rewritten as
F0 ) 1 + CQKOC F
(4)
where F is the fluorescence intensity of the sample in the
presence of humic concentration CQ, and F0 is the fluorescence intensity of the unquenched sample in the absence of humic material. Equation 4 is the Stern-Volmer equation for static quenching. A plot of F0/F versus CQ should yield a straight line with an intercept of 1 and a slope of KOC if the assumptions in the analysis are valid. If dynamic quenching by the humic material is important, a slightly more complex version of the Stern-Volmer equation can be applied (24). Gauthier et al. (6) analyzed data for interactions of pyrene, phenanthrene, and anthracene with six different humic materials and concluded that dynamic quenching was an insignificant contribution in these systems. Typically the static quenching form of the equation has been used for PAHs and natural organic matter (6-15). Similarly, the assumption of complete quenching of the fluorescence of bound PAH appears to have been made in all of these studies, although Backhus and Gschwend (10) and Schlautman and Morgan (14) have noted that this is not necessarily true for all quenchers. For example, although the quantum yield of perylene bound to Aldrich humic acid was found to be close to zero (indicating complete quenching), that of perylene bound to bovine serum albumin was found to be close to 0.6 (10). Potential Experimental Artifacts. Many factors that can affect the applicability of eq 4 are related to interferences of various types. Any effect that might reduce the observed fluorescence intensity of the sample, if unaccounted for, would increase the apparent quenching and yield an apparent KOC value larger than the actual value. Some potential artifacts are easily and routinely accounted for. The inner filter effect, which is unavoidable, can be accounted for using a correction factor based on cell geometry and the absorbance characteristics of the solution (6). When the fluorescence quenching measurements are performed by adding incremental amounts of humic material to a sample, dilution effects must also be taken into account (6, 11, 15). Fluorescence that may be contributed by compounds other than the PAH can be accounted for based on measurements of the background fluorescence (6, 8, 10-15). More complicated sources of error that have been mentioned in the literature include sorption losses and photodegradation (6, 8, 10, 13, 14). When natural water samples are used, the possibility of the presence of quenchers other than the natural organic material of interest must also be considered (10). Sorption of the target PAH to the walls of the reaction vessel or other surfaces (such as a stir bar) would decrease the measured fluorescence intensity and increase the apparent quenching. Since the Stern-Volmer equation is based on a ratio between the unquenched and quenched fluorescence and does not depend on precise knowledge of the PAH concentration, Gauthier et al. (6) suggested that sorption is of little concern as long as the PAH is allowed to equilibrate with the vessel surfaces prior to recording the unquenched fluorescence. Backhus and Gschwend (10) pointed out that this assumes that no desorption occurs later in the experiment as the concentration of free aqueous PAH decreases due to binding with humic material. Suggestions for altering the Stern-Volmer analysis to account for sorption are available in the literature (10, 13, 14). In an attempt to minimize photodegradation, a commonly used procedure is to keep the fluorometer lamp’s shutter closed between measurements (6, 8, 10, 14). To our knowledge, no systematic study of the effect of photodegradation on the determination of KOC values of PAHs has been reported. Given the sensitivity of many PAHs to photocatalyzed reactions that has been documented in many texts and reviews (23, 25-28), it is expected that photoreactions may occur during fluorescence quenching experiments for some PAHs. Dissolved oxygen is present during most determinations of KOC using fluorescence quenching since experiments
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typically are conducted under ambient atmospheric conditions, but it generally has not been mentioned as a potential source of interference. Backhus and Gschwend (10) accounted for quenching by components other than organic colloids present in their natural water samples (including oxygen) using control experiments. Danielsen et al. (15) cautioned that dynamic quenching by O2 may be an important process and that it may be influenced by interactions between molecular oxygen and humic material when the humic concentration is high. Oxygen can also play an important role in photoreactions with PAHs (23, 26, 29-31). Effects of Oxygen and Light on Fluorescence. Dynamic quenching by O2 reduces the fluorescence of virtually all organic compounds, and aromatic hydrocarbons are especially affected (25). For this reason, fluorescence lifetime measurements are routinely conducted on de-aerated samples (20, 23, 25-27). Oxygen is believed to quench fluorescence by promoting the intersystem crossing of the excited species, 1 A*, to its triplet state, 3A*, where in this case 1A* represents an excited PAH molecule; from the excited triplet state, the molecule can return to the ground state through the radiationless transition of 3A* to 0A (25). Since dynamic quenching involves interaction with excited PAH molecules, it occurs only when a sample is being exposed to UV radiation and cannot reach a steady-state condition until after the shutter is open. Photodegradation is not the only pathway by which light can affect PAH fluorescence. Under some conditions, an excited PAH molecule can combine with a ground-state PAH molecule to form an excited dimer. In many cases, the dimers that are formed are stable only in the excited state and decompose to two ground-state molecules, resulting in some quenching of fluorescence (27). Pyrene is particularly susceptible to this process (25, 32, 33), but it has also been observed for many derivatives of anthracene (25, 29). In the presence of oxygen, photooxidation of PAH molecules is expected to compete with photodimerization (23). Detailed studies of photoreactions of PAHs in aqueous solution are rare. In aqueous solutions that also contained a polystyrene sulfonate-type polymer, irradiation in the absence of oxygen was observed to result in photosensitized dimerization of anthracene (29). In the presence of oxygen, photooxidation of the anthracene occurred to a larger extent than photodimerization. Similar observations were reported for anthracene (31) and acenaphthylene (30) adsorbed on dry silica surfaces in the absence and presence of atmospheric oxygen. In the presence of oxygen, irradiation at 350 nm for 15 min resulted in a 73% loss of anthracene in these experiments (31). Observations of photodegradation have been reported for a variety of PAHs under a variety of conditions (28, 34-36). Photodimerization is unlikely to have a significant effect on determination of KOC using fluorescence quenching since it has only been observed at concentrations much higher than typically used in these types of experiments (25). Photodegradation, on the other hand, may be particularly important at low concentrations (26).
Materials and Methods Phenanthrene, anthracene, pyrene, and triphenylene were 98+% pure. Phenanthrene was obtained from Aldrich; the others were from Fluka. Concentrated stock solutions of the PAHs were prepared in methanol unless otherwise stated. Representative microliter quantities of organic solvent were checked for background fluorescence and found to cause insignificant interference at the wavelengths used in these experiments. The soil humic acid was obtained from the International Humic Substances Society (IHSS Soil Reference humic acid 1R102H). Elemental analysis data were provided by the IHSS: 58.90% C, 3.38% H, 33.46% O, 4.31% N, 0.41% S, and 0.42% P on a dry, ash-free basis. The humic stock
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solution was prepared by dissolving the humic acid in ultrafiltered distilled water with the aid of sodium hydroxide. All experiments described in this paper were conducted at pH 4 and ionic strength of 0.001. pH was adjusted using nitric acid. No buffer was used since it was not needed and could complicate data interpretation. pH varied no more than 0.02 unit within a particular experiment and was between 3.9 and 4.1 for all samples. Unless otherwise stated, ionic strength was adjusted using NaNO3. Fluorescence measurements were performed with a Shimadzu Model RF-1501 recording spectrofluorophotometer. Excitation wavelength, emission wavelength pairs (in nm) used for the PAHs were 288/364 (phenanthrene), 252/380 (anthracene), 334/390 (pyrene), and 250/361 (triphenylene). For each fluorescence quenching experiment, 3 mL of aqueous solution (adjusted for pH and ionic strength) was placed in a quartz cuvette. The sample was spiked with the desired PAH using a glass syringe. The nominal PAH concentrations were 41 µg/L phenanthrene, 12 µg/L anthracene, 13 µg/L pyrene, and 3.6 µg/L triphenylene. The sample was mixed thoroughly by capping and inverting the cuvette. Samples were allowed to equilibrate for 15 min (20 min for anthracene) before the unquenched fluorescence intensity was recorded. An aliquot of humic acid was added to the sample, and the cuvette was capped and inverted five times, which provided consistent mixing of the sample. Comparison testing using caps wrapped with aluminum foil showed that there was no noticeable sorption of the PAHs to the cuvette cap. The fluorescence intensity stabilized (except for the baseline decay) in 1-3 min and was recorded after 5 min. Measurements were based on the mean of data continuously collected during 1 min of monitoring. This procedure was repeated until a total of six aliquots of humic stock had been added. A maximum concentration of 5.3 mg of C/L of soil humic acid was achieved in the phenanthrene and anthracene experiments and 1.9 mg of C/L in the pyrene and triphenylene experiments. Typically at least three replicate experiments were done for each KOC determination. UV-vis absorbance measurements of the samples were made to correct for the inner filter effect as recommended by Gauthier et al. (6). The correction factor used in this work typically ranged from 1.01 to 1.8, which is comparable to that of other investigators that have reported this information (6, 10, 12, 14). (For triphenylene, the correction factor ranged up to 2.8.) Fluorescence intensity measurements were also corrected for dilution. Control experiments without added PAH were conducted to determine the background fluorescence in each sample as a function of humic concentration. Background fluorescence accounted for up to 5.7% of the total fluorescence intensity in pyrene experiments, up to 12% for anthracene and phenanthrene, and up to 17% for triphenylene. Gauthier et al. (6) and Magee et al. (8) stated that it did not exceed 3% in their studies, but background fluorescence up to 80% of the total has also been reported (12). Although it depends on the wavelengths used, substantial background fluorescence is difficult to avoid when the humic concentration exceeds about 2 mg of C/L. In this paper, Fcorr represents the fluorescence intensity of the sample corrected for background, dilution, and the inner filter effect. Fluorescence quenching experiments were conducted under four different conditions: ambient atmospheric conditions with the shutter open and closed; low O2 conditions with the shutter open and closed. In open shutter experiments, the sample was continuously exposed to the fluorometer lamp, including during the initial equilibration period. In closed shutter experiments, the shutter was opened only to make measurements, resulting in a total exposure time of about 7 min. For experiments conducted under ambient conditions, the samples were allowed to equilibrate with the ambient atmosphere, yielding dissolved oxygen concentrations of about 8 mg/L. For experiments conducted
TABLE 2. Partition Coefficients for PAHs with Soil Humic Acida ambient conditions KOC (104 mL/g of C)
phenanthrene
low O2 conditions KOC (104 mL/g of C)
A open shutter
B closed shutter
C open shutter
D closed shutter
baseline-adjusted KOC,adj (104 mL/g of C) open shutter, ambient
4.39 (0.66)
3.79 (1.24) p ) 0.50 5.68 (0.66) p ) 1.8 × 10-4 32.8 (2.0) p ) 0.27 76.7 (7.6) p ) 0.81
3.72 (0.40) p ) 0.30 4.81 (0.69) p ) 4.6 × 10-4 31.0 (1.1) p ) 0.011 76.6 (7.6) p ) 0.82
3.04 (0.50) p ) 0.027 3.69 (0.49) p ) 2.6 × 10-6 24.7 (1.4) p ) 0.0010 78.5 (5.6) p ) 0.98
3.09 (0.59) p ) 0.91 3.87 (0.50) p ) 0.63 26.9 (0.42) p ) 0.075 71.9 (6.9) p ) 0.21
anthracene
10.9 (0.74)
pyrene
34.3 (0.46)
triphenylene
78.2 (7.3)
a Standard deviation is given in parentheses. First four columns of data are K OC values measured under different experimental conditions; p values are for pair-wise statistical comparison of each set of data with the reference ambient, open shutter data. Last column shows open shutter, ambient KOC values adjusted for baseline decay; p values are for comparison between these data and the low O2, closed shutter data.
for triphenylene. The triphenylene plots showed slight curvature toward the abscissa but were analyzed in the same way as the others for the sake of consistency (r2 generally exceeded 0.9). Because of this, the KOC reported here for triphenylene probably represents an underestimate of the actual value. In addition to the fluorescence quenching experiments, experiments were conducted to monitor the baseline fluorescence intensity of each PAH over time. These were conducted at various PAH and humic concentrations using procedures and solution conditions identical to the fluorescence quenching experiments, except that the concentration of the humic acid, if present, was held constant.
Results and Discussion
FIGURE 2. Stern-Volmer plots for three replicate experiments for pyrene under ambient conditions with the shutter open. Data have been corrected for background fluorescence, the inner filter effect, and dilution. under low O2 conditions, the fluorometer was placed inside a glove bag flushed with N2. During experiments a slight positive pressure of about 1 psi was maintained within the bag. Chromatographic analyses performed with grab samples of bag gas indicated that the oxygen concentration was less than 1%. Sample solutions were purged with N2 for 30 min before they were placed in the bag, reducing dissolved oxygen concentrations to about 1.5 mg/L measured using a dissolved oxygen probe. Each experiment was begun 15-20 min after the bag was closed, allowing time for the sample to approach equilibrium with the gas phase in the bag, so that the O2 concentration in the sample was expected to be between 0.4 and 1.5 mg/L. Because the humic stock solution was not de-aerated, the dissolved O2 concentration may have increased during the course of the low O2 experiments, but in the worst case situation could not have exceeded 3 mg/L. The resulting Stern-Volmer plots (based on eq 4, with F replaced by Fcorr) were quite linear for phenanthrene, anthracene, and pyrene. An example is shown in Figure 2. Linearity of the Stern-Volmer plots has been cited as support for the assumptions of static quenching and complete quenching of bound PAH. However, the range of data is often too small for apparent linearity to be conclusive. In this work, the maximum values of F0/Fcorr were 1.42 for anthracene, 1.61 for phenanthrene, 1.86 for pyrene, and 2.75
The results of the fluorescence quenching experiments involving the four PAHs, ambient conditions vs low oxygen conditions, and open vs closed shutter are given in Table 2. The ambient, closed shutter conditions (column B) are typical of the procedures that have been used in most recent studies. The results agree well with data given in the literature. Restricting the review to data for soil humic material, several comparisons can be made with studies involving phenanthrene, anthracene, and pyrene. Magee et al. (8) reported KOC ) 4.38 × 104 mL/g of C for phenanthrene and watersoluble soil organic matter. Gauthier et al. (6) determined KOC ) 5.0 × 104 mL/g of C for phenanthrene and a soil humic acid. For several soil humic acids, KOC for anthracene ranged from 3.7 to 8.5 × 104 mL/g of C based on fluorescence quenching measurements and from 1.1 to 6.1 × 104 mL/g of C by reverse phase separation (6). Values of 8.8 to 32 × 104 mL/g of C were found for pyrene and several soil humic acids (7). Herbert et al. (13) reported KOC ) 17 × 104 mL/g of C for pyrene and a soil humic acid. The closed shutter, low oxygen experiments should be the least influenced by experimental artifacts associated with both UV radiation and oxygen. Based on the results in column D of Table 2, phenanthrene and anthracene have similar KOC values, triphenylene has the highest KOC, and pyrene has an intermediate value. The relative KOC values reflect the trends in KOW values of these compounds more so than their solubilities (see Table 1). Comparing columns A, B, C, and D in Table 2, it is clear that the results for anthracene and pyrene were more affected by UV radiation and oxygen exposure than the results for phenanthrene and triphenylene. In fact, no significant difference was observed in the KOC values measured for triphenylene; the p values for pair-wise comparison of the KOC data for which exposure to UV radiation, dissolved oxygen, or both was minimized (columns B, C, and D) with the reference ambient, open shutter data (column A) all exceeded
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fluorescence decay when the shutter was opened than phenanthrene and triphenylene under these conditions as well, under both ambient and low O2 conditions.
TABLE 3. Long-Term Baseline Decay Rates of PAH Fluorescence Intensitya ambient low O2 conditions R conditions R λex/λem (nm) (%/5 min) (%/5 min) p value phenanthrene anthracene pyrene triphenylene
288/364 252/380 334/390 250/361
0.99 (0.11) 4.08 (0.47) 1.55 (0.20) 0.81 (0.23)
1.00 (0.28) 2.90 (0.63) 1.33 (0.12) 1.01 (0.01)
0.93 0.0012 0.11 0.33
a Samples were exposed (open shutter) at the excitation and emission wavelengths given for 40 min total. Long-term baseline decay is based on data after 15 min (20 min for anthracene) and is expressed as % decay/5 min. Standard deviation is given in parentheses. Concentration of humic acid in these samples ranged from 0 to 5.35 mg of C/L.
0.8. For phenanthrene, the observed influence of UV radiation and oxygen was small, but not entirely insignificant. When only exposure to UV radiation was minimized, apparent KOC decreased from 4.39 × 104 mL/g of C to 3.79 × 104 mL/g of C, but the difference was not significant (p ) 0.50). Similarly, when only exposure to dissolved oxygen was minimized, no significant difference resulted. However, when exposure to both UV radiation and dissolved oxygen was minimized, the decrease in apparent KOC was statistically significant (p ) 0.027). The results for anthracene showed dramatic effects of both UV radiation and oxygen, even when only one was minimized. Similar effects, but smaller in magnitude, were observed for pyrene. In this case, minimization of UV radiation exposure alone did not yield a significantly different KOC value (p ) 0.27), although minimization of oxygen alone did (p ) 0.011). An apparent KOC value can be inflated relative to the actual value by any experimental artifact that acts to decrease the fluorescence intensity of a sample, since any unaccounted for loss of intensity during an experiment is attributed to quenching by humic material. That UV radiation and oxygen exposure resulted in a decrease in fluorescence intensity over time (and therefore artificially increased apparent KOC), especially for anthracene and pyrene, was confirmed by observations of the baseline fluorescence intensity of PAH solutions. Figure 1 shows examples of the decays in baseline fluorescence intensity that were observed for the four PAHs used in this study. The data shown here are for measurements made under ambient, open shutter conditions with no humic acid added. Some decay was observed for all four PAHs although it was more dramatic for anthracene and pyrene than for phenanthrene and triphenylene. When humic acid was present (up to 5.35 mg of C/L), the same long-term baseline decay was observed. The rapid initial decrease in fluorescence, characteristic of anthracene in particular, was muted by the presence of humic acid, perhaps in part due to the decreased magnitude of the fluorescence intensity and/ or UV filtering by the humic acid. For a particular PAH, the long-term baseline decay rate, expressed as a percentage of fluorescence intensity, was independent of both PAH and humic concentration, within experimental error. Table 3 summarizes the results of 43 open shutter experiments that were conducted under ambient conditions and low O2 conditions using various concentrations of PAH and humic acid at pH 4 and [NaNO3] ) 0.001 M. Comparing these two sets of data, O2 clearly had an influence on the baseline fluorescence decay rate of anthracene (p ) 0.0012) and, although it is somewhat less conclusive, pyrene (p ) 0.11). On the other hand, no significant effect of minimizing O2 exposure alone was observed for phenanthrene and triphenylene. For closed shutter experiments, baseline decay rates were difficult to quantify since data were only collected for 1 min at a time. However, qualitatively it was observed that anthracene and pyrene were more susceptible to baseline
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Several preliminary experiments were conducted to determine if experimental characteristics other than light and/ or oxygen may have influenced the observed baseline fluorescence decay rates. It should be noted that in tests using rhodamine dye, the fluorescence intensity remained steady, indicating that instrument drift did not occur over the time scale of these experiments. The choice of electrolyte appeared to have no impact on the observed baseline decay based on observations of pyrene fluorescence in the presence of 0.001 M NaNO3, NaClO4, or Na2CO3. The fluorescence decay of pyrene was also the same whether methanol, propanol, or acetone was used as the co-solvent. That the observed fluorescence decays cannot be attributed to sorption to the quartz cell was supported by several independent observations. When a cell was allowed to equilibrate with a PAH solution for 1 h and then refilled with a fresh PAH solution, the observed fluorescence decay was the same as in a cell that had not been pre-equilibrated with the PAH. When a cell containing a PAH solution was monitored continuously for 1 h and then spiked with additional PAH, once again the fluorescence decay mirrored that of the original solution. Furthermore, if sorption were the source of the baseline fluorescence decay, the magnitude of the effect would be expected to be similar for the more hydrophobic compounds triphenylene and pyrene and less important for anthracene, which is not the case. Other investigators using anthracene, phenanthrene, and pyrene have indicated that negligible sorption occurred in their studies (10, 14), although Herbert et al. (13) reported significant sorption of pyrene to glass vials. Sorption can be significant for more hydrophobic compounds such as perylene (10, 14). Temperature effects are usually not analytically important in these types of measurements (37). On the other hand, Ellis (26) stated that fluorescence intensity usually decreases with increasing temperature, at the rate of about 1 unit of intensity/°C. The temperature of a sample usually increased by a few degrees during the course of an open shutter experiment and in some cases up to 8 °C. The effect of an 8 deg temperature change on the fluorescence intensity of phenanthrene solutions was tested. The difference in fluorescence intensity never exceeded 6 intensity units. It is possible that temperature variations contributed in a small way to the baseline fluorescence decays observed in this study. However, even for phenanthrene and triphenylene the decays in baseline fluorescence intensity that were observed under ambient conditions exceeded 20-30 intensity units. Temperature effects alone cannot adequately account for these decays. While the results of this study are not sufficient to prove detailed mechanisms, dynamic quenching by O2 and photooxidation reactions appear to be among the likely sources of the observed decay in baseline fluorescence intensity. Dynamic quenching by O2 is a diffusion-limited process. It would be expected to reach steady-state quickly when a sample is continuously exposed to UV radiation. While it may contribute to some initial decay, dynamic quenching by O2 would not appear to account adequately for the long-term baseline decay in the open shutter experiments. Danielsen et al. (15) suggested that there might be an influence of humic material at high concentrations on dynamic quenching by O2, but the concentrations of humic acid used in these studies were much lower. The relative behavior of anthracene, pyrene, and phenanthrene observed in this study is consistent with other studies of photoreactions involving PAHs. It is well-known that some PAHs are more susceptible to photooxidation than others
(23, 26-28, 34, 36). Direct comparisons are difficult since most studies have been of PAHs in organic solvents or on particle surfaces. For example, Sanders et al. compared the effect of irradiation on phenanthrene, anthracene, and pyrene dissolved in dichloromethane (36). All three compounds photodegraded: anthracene was the fastest and phenanthrene the slowest. Other investigators have reported the same relative susceptibility of these three compounds to photodegradation when they were adsorbed on alumina particles and on silica (34) and in a study of the use of solar irradiation to treat organic-contaminated soils (35). Anthracene is the most photoreactive compound of the four studied in this work, had the highest baseline decay rate, and was the most sensitive to changes in the extent of sample exposure to UV radiation and dissolved oxygen. Regardless of the origin of baseline fluorescence intensity decay, an attempt can be made to account for it in the SternVolmer analysis if the decay can be quantitatively characterized through a sufficiently simple constant. In this case, it was observed that the long-term baseline decay rate for a particular compound appeared to be constant on a percentage basis and independent of PAH and humic concentrations in the ranges used. Therefore, we were able to introduce a correction factor into the ambient open shutter data. Using the corresponding decay rate R from Table 3, for each point on the Stern-Volmer plot, F0 was incrementally multiplied by a correction factor (100 - R)% to reflect the baseline decay that occurred since the last data point was taken (i.e., 5 min). By using this correction factor for each subsequent point on the Stern-Volmer plot, that portion of the decrease in fluorescence intensity that was not due to the addition of humic acid was prevented from artificially inflating the apparent extent of binding of the PAH. The Fcorr values themselves were not altered, resulting in F0/Fcorr ratios that were smaller than before the baseline correction. Then, KOC,adj was determined from the slope of the corresponding SternVolmer plot, just as the more directly determined KOC values were. The adjusted Stern-Volmer plots were also linear, and the resulting KOC, adj values are shown in column E of Table 2. In this column, the p values represent a comparison between KOC,adj data and the low O2, closed shutter KOC data in column D. This empirical correction resulted in close agreement with the column D data for phenanthrene and anthracene. The correction did not work as well for triphenylene, but still yielded a value that was within experimental error of the low O2, closed shutter KOC. Although the adjusted value for pyrene does not match closely with the low O2, closed shutter value, it is a great improvement over the unadjusted value in column A. This type of a correction should be applied with care, and only when the baseline fluorescence intensity decay rate can be characterized using a simple fractional rate that is constant over the range of conditions being used. A more generally applicable recommendation is to simply avoid using potentially troublesome PAHs, such as anthracene, since even short-term exposure to UV radiation and oxygen may be sufficient to cause substantial decay. It is evident that exposure to dissolved oxygen and UV radiation can lead to baseline fluorescence decay over time for some compounds, potentially resulting in significant overestimation of KOC. Furthermore, more photochemically active compounds, such as anthracene and pyrene, are more sensitive to these experimental artifacts. In all cases observed here, the influence on the determination of KOC was less than an order of magnitude. Nevertheless, these artifacts can be large enough to complicate interpretation of data comparing different PAHs or different solution conditions when internal consistency in the data is particularly important.
Acknowledgments We gratefully acknowledge the anonymous reviewers for their valuable comments on the manuscript.
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Received for review March 14, 1996. Revised manuscript received September 19, 1996. Accepted September 24, 1996.X ES960237O X
Abstract published in Advance ACS Abstracts, December 1, 1996.
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