Environ. Sci. Technol. 2009, 43, 718–723
Quantifying Interactions between Singlet Oxygen and Aquatic Fulvic Acids R O S E M . C O R Y , †,‡,§ J A M E S B . C O T N E R , * ,† A N D K R I S T O P H E R M C N E I L L * ,‡ Department of Ecology, Evolution and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, Minnesota, and Department of Chemistry, University of Minnesota, 207 Pleasant St SE, Minneapolis, Minnesota
Received July 8, 2008. Revised manuscript received October 31, 2008. Accepted November 7, 2008.
Singlet oxygen (1O2) is a reactive oxygen species produced by dissolved organic matter (DOM) in sunlit waters. While the production of 1O2 by DOM has been studied, little is known on interactions between 1O2 and DOM. The central objective of this work was to quantify the rate constants of reaction and quenching of 1O2 with Suwannee River and Pony Lake fulvic acids, the terrestrial and microbial end-member reference aquatic humic substances of the International Humic Substance Society. Fulvic acids were reacted with 1O2 generated through visible light irradiation of Rose Bengal. Uptake of 1O2 by the fulvic acids was followed through changes in dissolved oxygen concentrations via membrane inlet mass spectrometry (MIMS). Results from multiple diagnostic tests for 1O2processes in solution suggested that 64-70% of the observed uptake of oxygen by the fulvic acid solutions was due to reaction with 1O2; the remaining O2 uptake was likely due to non1 O2 processes initiated by the excited-state sensitizer. The rate constants of reaction (krxn) and physical quenching (kphys) with 1O2 were determined to be 2.6 × 105 M-C-1 s-1 and 2.7 × 105 M-C1- s-1 (krxn) and 1.5 × 105 M-C1- s-1 and 1.3 × 106 M-C1s-1 (kphys) for Suwannee River and Pony Lake fulvic acids, respectively. Results from this study demonstrated that 1O2 reacts with microbially and terrestrially derived DOM at rate constants comparable to phenols, naphthols, or aromatic amines, on a per carbon basis.
Introduction Singlet oxygen (1O2, 1∆g) is a reactive oxygen species formed by the interaction of sunlight and dissolved organic matter (DOM) in natural waters (1). Dissolved organic matter is a heterogeneous mixture of the organic compounds excreted by microorganisms and the breakdown products from decomposing plant and soil organic material (2). Singlet oxygen is known to react with biomolecules such as proteins, DNA, lipids, and lignin (3), which likely comprise a large fraction of DOM. It was recently demonstrated that within the hydrophobic interior of isolated fractions of DOM, * Address correspondence to either author. E-mail: cotne002@ umn.edu (J.B.C.);
[email protected] (K.M.). † Department of Ecology, Evolution and Behavior. ‡ Department of Chemistry. § Current address: Department of Environmental Science & Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. 718
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concentrations of 1O2 (ca. 10-12 M) were 2-3 orders of magnitude higher than bulk 1O2 measurements (ca. 10-14 M) (4). Given the reactivity of 1O2 and potentially high concentrations of 1O2 in the hydrophobic fraction of DOM, interactions between 1O2 and DOM may be more important in the photooxidation of DOM than has been previously recognized. Singlet oxygen is formed when dissolved oxygen (O2) interacts with photochemically produced triplet states of chromophoric DOM (1). Once formed in natural waters, 1O2 is readily quenched by water. In the presence of 1O2-labile substrates that may exist in DOM, reaction and physical quenching of 1O2 by DOM could serve as additional sinks for 1 O2. Thus, DOM may act as both the sensitizer and substrate for 1O2 in natural waters. Our objectives were to investigate interactions between 1 O2 and the fulvic acid fraction of DOM. Fulvic acids are the dominant hydrophobic and light-absorbing fraction of DOM isolated from aquatic systems (2), and thus are likely “hotspots” for 1O2 reactions. Fulvic acids are derived from two classes of precursor organic material, i.e., decomposed plant material and soils of terrestrial (allochthonous) origin and organic material produced by algae and bacteria (autochthonous). Chemical characteristics of aquatic fulvic acids fall on a continuum between the International Humic Substance Society (IHSS) reference fulvic acid from the Suwannee River in Georgia, representing a terrestrial endmember, and fulvic acid isolated from Pony Lake in Antarctica, representing an autochthonous end-member (5, 6). Suwannee River fulvic acid has high aromatic carbon content and is low in nitrogen, reflective of its higher plant precursor material such as lignin. In contrast, Pony Lake fulvic acid contains roughly 10 times the nitrogen of Suwannee River fulvic acid and is significantly less aromatic; consistent with its derivation from microbial organic matter. Variation in fulvic acid reactivity in many important biogeochemical processes has repeatedly been linked to differences in fulvic acid chemical character attributable to the relative amount of the two major classes of precursor organic matter (7, 8). Thus, because Suwannee River and Pony Lake fulvic acids encompass the variation in chemical character of aquatic fulvic acids, we investigated interactions between these fulvic acids and 1O2 in order to address whether 1 O2 can react with the range of naturally occurring organic matter present in all aquatic systems. As the target structures for 1O2 within DOM are not known, this “end-member” approach also served to address whether lignin-rich terrestrial DOM or protein-rich microbial DOM would be more reactive with 1O2. We quantified oxygen uptake by fulvic acids by measuring the change in dissolved oxygen (O2) when fulvic acid solutions were irradiated in the presence of 1O2-sensitizing dyes (e.g., Rose Bengal). When 1O2-sensitizing dyes are used, oxygen uptake by the substrate can occur by two pathways: reaction with 1O2, and non-1O2 reactions due to processes mediated by the excited-state sensitizer (9). Evidence for the non-1O2 reaction pathway was recently presented in a study on the degradation of lignin model compounds using 1O2-sensitizing dyes (9). Because our central objective was to quantify the rate at which 1O2 reacts with different fulvic acid samples, we employed diagnostic tests to quantify the oxygen uptake due to excited-state sensitizer (non-1O2 reactions). Accounting for the non-1O2 pathway, we measured rate constants for reactions between 1O2 and each fulvic acid as well as rate constants for physical quenching of 1O2 by each fulvic acid. 10.1021/es801847g CCC: $40.75
2009 American Chemical Society
Published on Web 12/18/2008
Experimental Section Chemicals and DOM Isolates. Furfuryl alcohol, Rose Bengal, Methylene Blue, β-carotene, and sodium azide were purchased from Sigma-Aldrich. Deuterium oxide (D2O, 99%) was obtained from Cambridge Isotoptes Laboratories. The two reference end-member fulvic acids, Suwannee River and Pony Lake (5, 10, 11), were obtained from the International Humic Substance Society (http://www.ihss.gatech.edu).Chemical characteristics of the fulvic acids and details on solution preparation are presented in the Supporting Information. Fulvic acids were characterized by dissolved organic carbon (DOC), UV-vis absorbance, and fluorescence before and after reaction with1O2 as described previously (12) and as shown in the Supporting Information (SI) Section S3. 1O2 quantum yield values (Φ∆) were measured for each fulvic acid solution by FFA degradation (13), through comparison to perinaphthenone as a quantum yield standard (Φ ) 0.98 (14)). Reaction with 1O2. Reaction between 1O2 and the fulvic acid fraction of DOM was quantified as the uptake of 1O2 by fulvic acid isolates under conditions selective for 1O2 (15). To selectively generate 1O2, fulvic acid solutions were irradiated in the presence of 40 µM Rose Bengal, a 1O2 photosensitizer. The visible light originated from a metal halide bulb, the output of which was passed through an Edmond Optics longpass filter having a cutoff position of 550 ( 6 nm to remove light of wavelengths shorter than 550 nm. Reactions involving 1 O2 and fulvic acid not leading to the uptake of oxygen by fulvic acid would not be quantified by our approach. Loss of dissolved oxygen (O2) in the solution during irradiation was quantified by a membrane inlet mass spectrometer (MIMS) which provided high precision measurements of dissolved gases in water (16). Sample (20 mL) was continuously pumped from the reaction flask to the MIMS via a peristaltic pump fitted with Viton tubing at a flow rate of 1 mL min-1. Consumption of dissolved gases by the MIMS as the sample was continuously recycled was quantified at a linear loss rate of 2 ( 2 nM s-1; sample measurements were corrected for this. Concentrations of dissolved N2, O2, and Ar were recorded every two seconds by the MIMS during the experiment and dissolved O2 was measured as the ratio of O2/Ar in the sample relative to airequilibrated Nanopure water at the same temperature (25 °C) and pressure (atmospheric (16)). The sample was open to the atmosphere, continuously stirred, and maintained at a temperature of 25 °C. The rate of 1O2 formation from the Rose Bengal sensitizer, kf, was quantified by measuring the loss of dissolved O2 in a solution of furfuryl alcohol (FFA), a well-characterized substrate for 1O2 (13). FFA concentrations ranged from 0-150 µM in H2O or D2O. FFA degradation was also used to quantify 1 O2 steady state concentrations ([1O2]ss). A range of [1O2]ss was achieved by varying the Rose Bengal concentration from 0.5 to 40 µM. Rates of oxygen uptake and [1O2]ss measured as a function of Rose Bengal concentration were corrected for light screening using the absorbance of Rose Bengal at 550 nm (17). Control Experiments. Control experiments were conducted with fulvic acid solutions to quantify how much of the observed uptake of dissolved oxygen by each substrate was due to reaction with 1O2. Solutions of FFA were analyzed alongside the fulvic acid solutions in order to compare the effects of the following control experiments between a wellknown 1O2 substrate and the fulvic acids. First, loss of O2 was quantified in a solution of fulvic acid or FFA and Rose Bengal without visible light (dark control). Fulvic acids or FFA were also irradiated with visible light in the absence of Rose Bengal. Second, the kinetic solvent isotope effect (KSIE) effect was investigated by changing from H2O to D2O solvent. The KSIE is commonly used to verify 1O2 processes in aqueous
solutions because the solvent-dependent 1O2 deactivation rate constant is 16 times slower for D2O than it is for H2O (18). The measurable effect of the KSIE should be an increased O2 uptake by any 1O2 substrate dissolved in D2O relative to H2O. Third, to investigate the role of excited-state Rose Bengal in the observed uptake of oxygen by the DOM isolates, another 1O2 sensitizer, Methylene Blue, was investigated under identical experimental conditions. Owing to differences in light absorption and excited-state energies, these sensitizers differ in their 1O2 formation rate (kf). Variation in the fulvic acid oxygen uptake in the presence of each sensitizer should vary proportionally to the kf of the sensitizer if all the observed loss of O2 was due to reaction with 1O2. Finally, several different 1O2 quenchers were used to aid in the quantification of 1O2 processes in the observed uptake of oxygen by the fulvic acids or FFA solutions. Quenchers included 1 mM sodium azide (quenching rate constant of 9 × 108 M-1 s-1 (13)), and nanomolar concentrations of β-carotene. β-carotene, a hydrophobic 1O2 quencher insoluble in water, has been shown to quench 1O2 in aqueous DOM solutions at nanomolar levels, likely due to association with the DOM (4). Laser Flash Photolysis. Laser flash photolysis (LFP) was employed to detect the change in the phosphorescent decay of 1O2 in the presence of each SRFA and PLFA (0-8 mM C) to quantify the total interaction (ktot, quenching and/or reaction) between the fulvic acid and 1O2 (9). Samples containing SRFA or PLFA and 1O2 sensitizer (40 µM Rose Bengal) in D2O were excited by 4 ns pulses at 532 nm (Nd/ YAG, Continuum Minilite II). Details of the LFP experiment and data analysis employed for this study have been described previously (9).
Results Effects of Rose Bengal and Visible Light on Oxygen Uptake by Fulvic Acids. Singlet oxygen is formed at rate (kf), which is proportional to the light intensity, as well as the light absorption coefficient and quantum yield of the sensitizer (see scheme in the SI). Quenching of 1O2 by the solvent (H2O; ksolv) or by the substrate (fulvic acid; kphys) does not result in a net loss of O2. Only reaction with 1O2 and the substrate, described by the rate constant krxn, results in net loss of O2. There was no detectable loss of dissolved O2 for a solution of substrate only (SRFA exposed to >550 nm visible light), nor was there loss of O2 for a solution of SRFA with Rose Bengal in the dark (see SI for data). There was no detectable change in O2 for a solution of Rose Bengal only, i.e., no substrate for 1O2. Loss of O2 occurred only in the presence of the 1O2 sensitizer (Rose Bengal plus visible light) and a 1O2 substrate (SRFA). These results suggest that the observed loss rate of oxygen, - (dO2)/(dt)(rxn), was due to 1O2 uptake by SRFA, described by eq 1, where S represents the substrate (e.g., SRFA): -
kfkrxn[S] dO2 (rxn) ) dt ksolv + (krxn + kphys)[S]
(1)
When a solution of SRFA with Rose Bengal was exposed to visible light, detectable loss of O2 occurred for the duration of the irradiation. When the light was switched off, the O2 concentration in solution was replenished until the equilibrium O2 concentration was reached (see SI). The replenishment rate of O2 in solution is controlled by mass transfer of O2 across the O2 air-water interface, which is a function of the O2 concentration gradient: dO2 D A (replenishment) ) · {[O2]t - [O2]i} dt l V
(2)
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FIGURE 1. Effect of D2O (∆) vs H2O (b) on logarithmic decay for O2 for 50 µM FFA (left) and 4.5 mM-C SRFA (right). Sensitizer ) 40 µM Rose Bengal + visible light. D is the diffusivity of O2 in water, V is the volume of solution, A is the surface area of the air-water interface, and l is the thickness of the aqueous boundary layer (19). Net loss of O2 (-(dO2)/(dt)(total)), assuming loss of O2 is due to reaction of fulvic acids with 1O2, is described by eq 3: dO2 kfkrxn[S] D A (total) ) - · {[O2]t - [O2]i} + dt l V ksolv + (krxn + kphys)[S] (3) The observed rate constant for oxygen replenishment due to mass transfer, 2 × 105 s-1, was at least an order of magnitude lower than the observed rate constant for loss of oxygen due to reaction with 1O2, at the lowest concentration of organic matter. Therefore, at early reaction times, when the oxygen concentration gradient term (eq 2) is small relative to the oxygen loss term (eq 1), the rate of oxygen loss simplifies to -
kfkrxn[S] dO2 (rxn) ) dt ksolv
(4)
assuming (kphys + krxn)[S] , ksolv. The initial first-order kinetic regime for oxygen loss is visualized by plotting ln([O2]t/[O2]i) vs time. The logarithmic decay was linear over the initial irradiation time when the O2 loss was within 5% (H2O) or 10% (D2O) of the initial O2 concentration. Thus, 1O2 uptake by all substrates (fulvic acids and FFA) was calculated as initial rates of oxygen loss as follows: -
dO2 (rxn) ) kobs[O2i]i dt
(5)
where kobs is the slope of ln([O2]t/[O2]i) vs time. Kinetic Solvent Isotope Effect (KSIE). The kinetic solvent isotope effect (KSIE), which can be tested by switching solvents from H2O to D2O, is commonly used to verify 1O2 processes in aqueous solution. For FFA, the kobs (and thus the initial rate of O2 loss) was six times greater in D2O vs H2O (Figure 1). For the 50 µM FFA solution (Figure 1), the expected enhancement due to the KSIE, calculated as described in the SI, was a factor of 12. The initial rate of O2 uptake by SRFA in D2O was 8.6 times greater than in H2O (Table 1; Figure 1). Assuming the difference between the calculated and measured enhancement factors was due to non-1O2 oxygen uptake mediated by the excited-state sensitizer (which would not be affected by solvent), then the 1O2 reaction with the fulvic acid accounts for 63% of the total measured rate of oxygen uptake. This leaves an estimated 36% contribution from the excited-state sensitizer (non-1O2) pathway in the measured rate of oxygen uptake. Effect of Different 1O2 Sensitizers. To investigate the role of excited-state sensitizer in the observed uptake of oxygen by the DOM isolates, two sensitizers for 1O2, Rose Bengal 720
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TABLE 1. Effects of Solvent and Quencher on O2 Uptake by SRFAa test condition
krel, experimental
krel, calculated
99% D2OR 1 mM N3-γ 40 µM Methylene Blueδ 55 nM β-carotene
8.6 0.50 1.9 0.25
13.6 0.22 1.9 0.053ζ
a krel refers to the rate of O2 uptake under the experimental condition relative to the same concentration of SRFA: in H2OR, without N3- γ, 40 µM Rose Bengalδ, or without β-carotene. ζ
and Methylene Blue, were investigated. The rate of formation of 1O2, kf, was calculated for each sensitizer by measuring the initial rate of O2 loss for a range of FFA concentrations in H2O. Plotting the initial rate of O2 loss vs FFA for each sensitizer yielded a straight line with a slope equal to eq 4, assuming (kphys + krxn)[S] , ksolv (Figure 2). Given that the krxn is known for FFA (8.3 × 10-7 M-1 s-1 in H2O (20), the kf for each sensitizer was calculated to be 9 µM s-1 and 17 µM s-1 for Rose Bengal and Methylene Blue, respectively. Thus, the initial rate of O2 uptake should have been greater by a factor of 1.9 for a fulvic acid solution with Methylene Blue compared to Rose Bengal. Upon changing sensitizers from Rose Bengal to Methylene Blue, the initial rate of O2 uptake was 1.9-fold greater for SRFA and 2.0-fold greater for PLFA (Table 1 and SRFA shown for example in Figure 2). These results suggest that either the rate of oxygen uptake was due only to 1O2 or that both sensitizers are equally efficient at instigating the non-1O2 pathway for oxygen uptake. Effect of 1O2 Quenchers. Addition of 1 mM azide slowed the rate of O2 loss in the presence of FFA or SRFA. The expected effect of azide was a 4.6 fold decrease in O2 uptake (see SI). For 150 µM FFA in H2O with 40 µM Rose Bengal, there was a factor of 2.0 decrease in the initial rate of O2 loss (data not shown). Similarly, the effect of 1 mM azide on SRFA (4.7 mM-C in H2O) resulted in a 1.7-fold decrease in O2 uptake (Table 1). Thus, FFA and SRFA both showed roughly a 2-fold decrease in O2 uptake upon addition of azide, less than the expected 4.6 fold decrease. For the SRFA, again assuming the difference in the calculated and experimental rate of oxygen uptake was due to the role of the non-1O2 pathway (not affected by azide), then reaction between 1O2 and SRFA accounted for 70% of the rate of oxygen uptake. The addition of a hydrophobic 1O2-quencher, β-carotene (55 nM), to the SRFA solution (7.3 mM-C) in H2O containing Rose Bengal as the sensitizer significantly slowed the uptake of O2 by a factor of 0.25 (Table 1). At the concentration of β-carotene used, the effect of β-carotene in the bulk aqueous phase (unassociated with DOM) on the quenching of oxygen uptake is calculated to be negligible (less than 1%), given the quenching rate constant of β-carotene (1010 M-1 s-1; (4, 18)).
FIGURE 2. Effect of varying sensitizer on kf (left) and O2 uptake by SRFA in H2O (right). Left:. Initial rate of O2 loss vs [FFA] with 40 µM Rose Bengal) and 40 µM Methylene Blue (∆). Rose Bengal: y ) 3.1 × 10-3x r2 ) 0.96; Methylene Blue: y ) 5.8 × 10-3x, r2 ) 0.95. Right: Logarithmic decay of O2 in 2.5 mM-C SRFA vs time. Rose Bengal: y ) 7.70 × 10-5x - 1.2 × 10-4, r2 ) 0.97; Methylene Blue: y ) 1.45 × 10-4x - 9.9 × 10-5, r2 ) 0.99.
FIGURE 3. Determination of ktot and krxn for SRFA in D2O (O), SRFA in H2O (b), and PLFA in D2O (∆) Left: ktot, kobs/kobs(0) vs [FA], in D2O; error bars smaller than points. SRFA: y ) 0.028x+1.0, r2 ) 0.99; PLFA: y ) 0.18x + 1.0, r2 ) 0.97; Right: Initial rate of O2 uptake normalized to [FA] vs [1O2]ss; Values were corrected for light-screening. Error bars shows standard deviation. For SRFA, krxn experiments were conducted in D2O and H2O; PLFA, krxn was investigated in D2O only. The point for SRFA in H2O was higher than some D2O points because the highest concentration of Rose Bengal (40 µM) was used for the H2O experiments. SRFA: y ) 2.6 × 105x + 6.9 × 10-6, r2 ) 0.99; PLFA: y ) 2.7 × 105x + 1.8 × 10-5, r2 ) 0.99. We hypothesize that the observed enhanced quenching by β-carotene of 1O2 in the presence of DOM is due to association of β-carotene with the DOM (4). This association leads to an enhanced concentration of β-carotene inside the DOM phase relative to the bulk aqueous phase. For example, we estimated the H2O: DOM volume ratio to be ∼11 × 103, based on the concentration of SRFA used for this experiment, which yields an expected concentration of β-carotene in the DOM phase of ∼600 µM. It follows that the oxygen uptake due to reaction with 1O2 upon addition of β-carotene should be 0.053 times the rate of uptake without β-carotene, assuming all the β-carotene is associated with the DOM. Although the calculated effect of β-carotene (associated with DOM) was stronger than the observed quenching effect (Table 1), the association between β-carotene with the DOM and the predicted enhanced concentration rationalizes the fact that a small added concentration of β-carotene can lead to significant quenching of 1O2 in the presence of DOM. Quantifying ktot and krxn and for Fulvic Acids. Figure 3 shows that as the fulvic acid concentration increased, the decay constant for the 1O2 transients in solution increased as measured by laser flash photolysis (LFP). In other words, the lifetime of 1O2 in solution decreased in proportion to the fulvic acid concentration due to both quenching and reaction. The rate constant for the combination of these processes, ktot, was obtained from the plot of the observed decay constant (kobs) versus the fulvic acid concentration (Figure 3, Table 2). PLFA was found to have a greater ktot than SRFA (Figure 3, Table 2). To quantify the rate constant for reaction of 1O2 with each fulvic acid, the initial rate of oxygen uptake was investigated
TABLE 2. 1O2 Reaction and Quenching Rate Constants for the Fulvic Acids rate constant (M-C-1 s-1)
SRFA
ktot krxn kphys
4.1 × 10 2.6 × 105 1.5 × 105
PLFA 5
1.6 × 106 2.7 × 105 1.3 × 106
as a function of [1O2]ss in both H2O and D2O. The total measured rate of oxygen uptake can be expressed as the contribution from reaction of 1O2 with the fulvic acid as well as the non-1O2 pathway due to the excited-state sensitizer (eq 6). Plotting the rate of oxygen uptake normalized to [FA] vs [1O2]ss, the slope is equal to krxn and the intercept corresponds to k′, the non-1O2 oxygen uptake rate constant (eq 7; Figure 3). initial rate ) krxn[1O2]ss[FA] + k[FA]
(6)
initial rate ) krxn[1O2]ss + k [FA]
(7)
Figure 3 verifies that the rate of oxygen uptake varied linearly as a function of [1O2]ss for each fulvic acid, and that each fulvic acid had a nonzero intercept, corresponding to the non-1O2 pathway for oxygen uptake. Based on the magnitude of the intercepts, the non-1O2 pathway accounted for 3-30% of the total rate of oxygen uptake for SRFA and PLFA, respectively. SRFA and PLFA rate constants for reaction with 1O2 were of the same order of magnitude, with PLFA having a greater VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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krxn (Table 2; Figure 3). The rate constant for physical quenching, kphys, for each fulvic acid was calculated as the difference between ktot and krxn because ktot ) kphys + krxn (Table 2). The quenching rate constant for PLFA was an order of magnitude greater than that measured for SRFA (Table 2). Effects of 1O2 on DOC, Absorbance and Fluorescence. Under the experimental conditions (1 µM Rose Bengal, kf ) 1.5 µM s-1), there were no detectable changes in DOC concentration, absorbance spectra, or emission intensities for SRFA and PLFA (see SI for data). Differences in reacted SRFA emission intensities were ( 10% of control intensities across all measured excitation and emission wavelengths. These differences were determined to be insignificant based on instrument reproducibility. No shifts in excitation or emission peak positions were observed for either fulvic acid.
Discussion All experiments aimed at verifying 1O2 processes in solution, including the KSIE, varying the 1O2 sensitizer, addition of 1O2 quenchers, and dark controls, suggested that 64-70% of the observed uptake of oxygen by the fulvic acids was due to reaction between 1O2 and the fulvic acid. There was no detectable uptake of oxygen by the fulvic acids without the presence of the 1O2 sensitizer. The effect of the KSIE on the rate of oxygen uptake by FFA and the fulvic acids was a 6.0-8.6 fold enhancement in oxygen uptake in D2O compared to H2O, less than the theoretical enhancement factor of 12-16 (Figure 1; Table 1). The difference in the rate of oxygen uptake by SRFA and PLFA in methylene blue compared to Rose Bengal differed in direct proportion to the difference between the 1O2 rates of formation of these sensitizers (Figure 2; Table 1). Addition of 1O2 quenchers, azide and β-carotene, slowed the rate of oxygen uptake by SRFA (Table 1). While our results converged around 64-70% of the observed O2 uptake as attributable to 1O2, overall we observed a range of 3% (best case) to 36% (worst case) of the O2 uptake which may have been due to non-1O2 processes. This range is consistent with studies demonstrating that Rose Bengal can photochemically generate non-1O2 oxidants such as superoxide, or take up oxygen, with efficiencies as high as 0.2 (21, 22). The non-1O2 photooxidation reactions initiated by Rose Bengal have been shown to depend strongly on solution conditions (21, 22). For example, superoxide production by Rose Bengal bound to macromolecules in aqueous solutions was concluded to be a very inefficient process (21). Under different conditions (Rose Bengal concentration, pH, alternative sources of organic matter), the proportion of oxygen uptake due to non-1O2 mediated reactions initiated by Rose Bengal may vary from the range reported here. The terrestrial and microbial end-member fulvic acids investigated here, SRFA and PLFA, respectively, sensitize, react with, and quench 1O2. The 1O2 quantum yield (Φ∆) of each fulvic acid was less than 1% (shown in SI), similar to the 1O2 Φ∆ values reported previously at 355 nm (23) and 366 nm (24). The Φ∆ values reported here for SRFA and PLFA are also in the same range as those reported previously for various natural waters and natural organic matter samples (23, 25). This is the first study to report reaction rate constants of fulvic acids with 1O2. The fulvic acids reacted with 1O2 with rate constants similar to those of a range of aromatic substrates, including phenols, naphthols, and aromatic amines on a per carbon basis (18). However, because the fulvic acid rate constants are likely averages representing reaction rate constants of a range substrates, comparison to rate constants of model 1O2 substrates does not necessarily pinpoint these compounds as the predominant fulvic acid substrates involved in the reaction with 1O2. Indeed, it has been concluded that 1O2 did not control DOM-sensitized photodegradation of a range of phenols (26). The ktot 722
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constants for the fulvic acids (Table 2) are of the same magnitude as those measured previously for plant and soilderived humic substances: 3.1 × 105 M-C1- s-1 to 5.3 × 105 M-C1- s-1 (27). No significant changes were observed in DOC concentration or in fulvic acid spectral properties after reaction with 1 O2. Lack of detectable concentration and spectral effects may be due to the small extent of the 1O2 uptake allowed to occur. These experiments aimed at identifying changes in fulvic acid quantity and quality were investigated at a lower concentration of Rose Bengal than was used to quantify the interactions between 1O2 and the fulvic acids, due to interferences at high Rose Bengal concentration. Even at high Rose Bengal concentrations, the observed oxygen uptake corresponded to a maximum 5% increase in the oxygen content of SRFA. While the 1O2-labile substrates in SRFA and PLFA may not be those that give rise to dominant spectral features (thus not contributing to changes in spectral properties upon reaction), it is possible that even at the low concentration of Rose Bengal used for these experiments, the Rose Bengal itself may have masked clear detection of shifts in fulvic acid spectral properties. Despite significant differences in the chemical characteristics of the two end-members, it is striking that these two fulvic acids had similar reaction rate constants with 1O2. Because the chemical properties of fulvic acids isolated from diverse environments fall on a continuum between these end-members, we hypothesize that in sunlit surface waters where 1O2 is continuously produced by DOM, reaction of 1O2 with the fulvic acid fraction of DOM is a ubiquitous process. We propose that the fulvic acid fraction of DOM is a sink for the 1O2 produced photochemically by DOM. The extent to which 1O2 may oxidize aqueous contaminants (28) or viruses (29, 30) may be limited by the quality and quantity of 1O2labile substrates of the DOM itself. The fact that 1O2-mediated-oxygen uptake by the fulvic acid did not result in detectable loss of DOC strongly suggests that this reaction leads to increased oxygen content of the DOM, but not to mineralization of the DOM to CO or CO2. Previous work has shown that for all photochemical reactions involving DOM, the ratio of DOC loss to oxygen consumption was g1 (31). Our findings suggest that oxidation of DOM by 1 O2 should lead to a DOC loss to O2 consumption ratio less than 1; thus the 1O2 pathway likely lowers the total observed ratio of DOC loss to oxygen consumption. Our findings demonstrate that reaction with 1O2 is a mechanism by which oxygen is incorporated into DOM, contributing to the increased O/C ratios of DOM compared to biomass precursor material. Given the higher oxygen content of SRFA relative to PLFA (43 vs 31%,), our results tentatively suggests that SRFA has “experienced” more 1O2-mediated oxidation. Because the oxygen content of DOM has been negatively correlated with DOM uptake by microorganisms (7), 1O2 oxidation of DOM could impact the turnover of DOM by aquatic microorganisms.
Acknowledgments We thank three anonymous reviewers for helpful comments. This work was supported by the National Science Foundation under grant number OCE-0527196.
Supporting Information Available Kinetic model for the formation and loss of 1O2. Chemical characteristics of end member fulvic acids. Experimental solution preparation and analysis. Photochemical uptake of 1 O2 as quantified by membrane inlet mass spectrometry (MIMS): controls. Calculation of enhancement and quenching factors on rates of 1O2 uptake. Effect of 1O2 DOC concentration, absorbance, and emission spectra of SRFA.
This material is available free of charge via the Internet at http://pubs.acs.org.
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