Photoproduction of One-Electron Reducing Intermediates by

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Photoproduction of one-electron reducing intermediates by chromophoric dissolved organic matter (CDOM): Relation to O2- and H2O2 photoproduction and CDOM photooxidation Yi Zhang, and Neil V. Blough Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b02919 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on October 2, 2016

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Environmental Science & Technology

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Photoproduction of one-electron reducing intermediates by

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chromophoric dissolved organic matter (CDOM): Relation to O2- and

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H2O2 photoproduction and CDOM photooxidation

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Yi Zhang and Neil V. Blough* Department of Chemistry and Biochemistry, University of Maryland College Park, MD 20740

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*Corresponding Author Email: (N.V.B) [email protected]

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Abstract A molecular probe, 3-amino-2,2,5,5,-tetramethy-1-pyrrolydinyloxy (3ap), was employed

10

to determine the formation rates of one-electron reducing intermediates generated

11

photochemically from both untreated and borohydride-reduced Suwanee River fulvic and humic

12

acids (SRFA and SRHA, respectively). This stable nitroxyl radical reacts rapidly with reducing

13

radicals and other one-electron reductants to produce a relatively stable product, the

14

hydroxylamine, which can be derivatized with fluorescamine, separated by HPLC and quantified

15

fluorimetrically. We provide evidence that O2 and 3ap compete for the same pool(s) of photo-

16

produced reducing intermediates, and that under appropriate experimental conditions, the initial

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rate of hydroxylamine formation (RH) can provide an estimate of the initial rate of superoxide

18

(O2-) formation. However, comparison of the initial rates of H2O2 formation (RH2O2) to that of

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RH show far larger ratios of RH/RH2O2 (~6-13) than be accounted for by simple O2- dismutation

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(RH/RH2O2 = 2), implying a significant oxidative sink of O2- (~67-85 %). Because of their high

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reactivity with O2- and their likely importance in the photochemistry of CDOM, we suggest that

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co-produced phenoxy radicals could represent a viable oxidative sink. Because O2-/phenoxy

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ACS Paragon Plus Environment


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radical reactions can lead to more highly oxidized products, O2- could be playing a far more

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significant role in the photooxidation of CDOM than has been previously recognized.

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Introduction Over the past 30 years, chromophoric dissolved organic matter (CDOM) has been shown

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－

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to generate a variety of reactive intermediates upon irradiation with UV and near-visible light.1

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17

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oxygen species (ROS),18 including superoxide (O2-),19 hydrogen peroxide (H2O2),20 singlet

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dioxygen (1O2),21, 22 peroxy radicals (RO2),23 and organic peroxides (ROOH).1,24 Among the ROS

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generated photochemically, H2O2 has been one of the most intensively investigated due to its

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significant environmental importance and relative ease of measurement. This ROS can affect

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trace metal speciation and impact the photochemical and thermal degradation of natural and

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anthropogenic organic compounds in aquatic systems.1,2,25,26 In addition, as a relatively stable

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ROS, detection of the photo-produced H2O2 can serve as an informative measure of electrons

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transferred from the organic matter pool to dioxygen, providing a lower bound to estimates of the

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net photochemical oxidation.1,27

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In aerobic waters, many of these species can react with dioxygen to produce various reactive

Past work has shown that H2O2 arises primarily through the dismutation of superoxide

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(O2-), the one-electron reduction product of dioxygen.28- 30 Recently, Zhang et al.31 provided

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evidence that O2 - is formed via reaction of O2 with reduced electron acceptors generated by

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intramolecular electron transfer from excited singlet-state electron donors to ground-state

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electron acceptors within the CDOM.31 However, past studies have suggested that not all of this

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photo-produced O2- undergoes dismutation to form H2O2. Earlier work by Petasne and Zika,29

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who employed the enzyme, superoxide dismutase (SOD), to catalyze O2-dismutation, indicated

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that 24-41% of the O2- flux did not lead to H2O2 formation, suggesting that a significant portion

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of the O2- is being consumed through reactions other than dismutation. Waite and coworkers32

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later argued that the contribution of these reactions to the loss of O2- are even larger, ~1.7-fold

3



49

greater than that previous proposed by Petasne and Zika.29 More recently, Powers et al.33 and

50

Powers and Miller27 found that estimated rates of O2- formation were significantly larger than

51

those of H2O2, with the ratio of rates averaging ~4 but ranging up to 10, far larger than the ratio

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of 2 expected for dismutation, or the ratio of 1 expected through a further one-electron reduction

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of O2-. These results suggest that a substantial portion of the photo-produced O2- is being

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consumed by as yet unknown oxidative reactions.

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Measurements of the photochemical formation rates of O2-reducing intermediates and the

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stoichiometric relationship of these rates to those of O2- and H2O2 formation would allow a far

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better understanding of the magnitude of O2 - production and the degree to which other reaction(s)

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of O2– are competing with dismutation. Here, we employed the molecular probe, 3-amino-

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2,2,5,5,-tetramethy-1-pyrrolydinyloxy (3ap)34,35 to determine the formation rates of one-electron

60

reducing intermediates generated photochemically by CDOM. Having similar reactivity to

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dioxygen, 35-37 this stable nitroxide radical reacts rapidly with reducing radicals and one-electron

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reductants to produce a relatively stable product, the hydroxylamine. Following derivatization of

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the hydroxylamine with fluorescamine, the fluorescent derivative can be separated by HPLC and

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quantified fluorometrically (Fig.S1).38-40 As shown here, this probe can provide estimates of the

65

photochemical formation rates of O2-reactive, one-electron reductants and thus indirectly of O2-

66

formation rates. Consistent with past results,27,29,32 our data suggest far higher production rates

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for O2- than H2O2, implying the presence of a significant oxidative sink of O2-.

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Materials and Methods

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Chemicals. Boric acid, sodium chloride, sodium phosphate, sodium dihydrogen phosphate,

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fluorescamine and 3-carbamoyl-2,2,5,5,-tetramethy-1-pyrrolydinyloxy (3cp) were purchased

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from Sigma-Aldrich, whereas 3-amino-2,2,5,5,-tetramethy-1-pyrrolydinyloxy (3ap) and sodium

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dithionite were purchased from Acros. Acetic acid, phosphoric acid and HPLC grade methanol

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were obtained from Fisher. Suwannee River fulvic acid (SRFA) and Suwannee River humic acid

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(SRHA) were obtained from the International Humic Substance Society. Pure water for all

76

experiments was obtained from a Millipore Milli-Q system.

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Experiment Apparatus. Hewlett-Packard 8425A spectrophotometer was employed to acquire

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UV/VIS absorption spectra. Absorption spectra over the range of 190 to 820 nm for 5 and 10

79

mg/L samples were recorded using 1 cm cuvette referenced to the solvent. The HPLC consisted

80

of a Dionex Model P580 pump, 4 µm C-18 reversed-phase column (Waters) and a 50 µL

81

injection loop. All separations are performed at room temperature. The fluorescence detector

82

used was a Hitachi model L7480 set at 390 nm (excitation) and 490 nm (emission). The mobile

83

phase consisted of 55% methanol and 45% acetate buffer (50 mM, pH= 4.0).

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Electron paramagnetic resonance (EPR) spectra were recorded on a

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BrukerBioSpinGmBH instrument. Standard instrument settings were: microwave frequency,

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9.42 GHz; power, 2.00 mW; modulation amplitude, 1.0 G; modulation frequency 100 KHz; time

87

constant, 10.24 ms. Samples were drawn into 50 µL capillaries, which were then sealed with

88

Critoseal@ and placed inside 3 mm diameter quartz EPR tubes.

89 90

Borohydride reduction of samples were performed employing a 25-fold mass excess of borohydride.31,41 H2O2 measurements were performed using a chemiluminescence-based flow

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91

injection method as previously described.31 The chemiluminescence reagent, acridinium ester 10-

92

methyl-9(p-formylphenyl)-acridinium carboxlate trifluoromethanesulfonate (AE) was provided

93

by Waterville Analytical Company. The reaction buffer was 0.1 M Na2CO3, pH 11.2. Irradiated

94

samples were introduced via injection with a glass syringe, while AE, buffer and carrier water

95

were pumped into the reaction vessel by peristaltic pump. Mixing of AE and irradiated samples

96

in reaction buffer yielded chemiluminescence signals, which were converted to H2O2

97

concentration by a daily constructed calibration curve. At least three measurements were

98

acquired for each sample, with the uncertainty provided by the standard deviation of these

99

replicate measurements. Hydrogen peroxide concentrations were observed to increase linearly

100

over 15 min, and thus this time interval was employed for the measurement of initial rates.

101

Determination of photoreductants. SRFA or SRHA at concentrations of either 5 or 10 mg/L

102

were prepared in 50 mM borate buffer (pH =8.0), with differing amounts of 3ap added to these

103

solutions. For the polychromatic irradiations, a 300 W xenon arc lamp combined with a 325 nm

104

long-pass cutoff filter was employed to approximate the solar spectrum. A 20 cm water jacket

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was placed in front of the light source to remove infrared irradiation. Monochromatic irradiations

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were performed using 1000 W Hg-Xe lamp and a Spectral Energy GM 252 monochromator set

107

to a 20 nm band pass. Samples were held in 1 cm pathlength quartz cuvettes (3 mL volume) that

108

were sealed with a Teflon cap septum through which gases were introduced via a small Teflon

109

line and vented with a needle. Differing dioxygen concentrations, ranging from 0 to 1250 µM,

110

were achieved by mixing of N2, O2 or air with a rotameter, with the solutions purged with the

111

appropriate gas mixture for 20 min prior to irradiation; the headspace was then purged slowly

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throughout the irradiation. As describe in a previous study,31 sodium dithionite was employed

113

to check for possible O2 leaks within this system.

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Following sample irradiation, 200 µL of the solution was withdrawn with a gas-tight

115

syringe through the sealed septum cap of the cuvette, and immediately derivatized via addition to

116

50 µL of a 5 mM stock fluorescamine solution within a Teflon container. Following vigorous

117

mixing and a one-minute incubation in the dark, the derivatized hydroxylamine was then

118

separated by HPLC and detected fluorometrically. The stock fluorescamine solution was

119

prepared daily in acetonitrile and stored in the dark at room temperature.

120

Because 3ap is highly hygroscopic, concentrations of 3ap were determined by EPR using

121

3cp as a primary standard. The concentration of the stock 3cp solution was determined from the

122

weighed mass of 3cp, with lower concentrations obtained by serial dilution of this stock.

123

Concentrations of 3ap were then determined by EPR from a standard curve of the double integral

124

of the EPR signal versus concentration of the 3cp standard. The molar absorptivity of 3ap at 316

125

nm (12.5 ±0.2 M-1cm-1) was determined from the absorption at 316 nm and 3ap concentration,

126

with the molar absorptivity then employed to acquire 3ap concentrations in all later experiments.

127

(Fig. S2) All solutions were prepared in pH=8, 50 mM borate buffer.

128

Concentrations of the hydroxylamine were determined from calibration curves

129

constructed through anoxic titrations of 3ap employing dithionite as reductant (Fig. S3).40 The

130

reliability of this calibration was checked through the use of two types of titrations that employed

131

either dithionite or 3ap as the limiting reagent(titrant); both provided equivalent results (Fig. S3

132

Top vs Bottom). The limiting reagent, either 3ap or dithionite, was added to a solution within

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Teflon-capped, sealed cuvette that had been purged with N2 for 20 min. A second, sealed cuvette

134

that had also been purged with N2 for 20 min contained the titrant. Nitrogen was purged through

135

the headspace of both cuvettes during the titrations to maintain anoxic conditions. Employing a

136

gas-tight syringe, titrant was then transferred anaerobically to the cuvette containing the limiting

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137

reagent. Following each titrant addition, 200 µL of sample was withdrawn, derivatized, separated

138

by HPLC and detected fluorometrically as described above. Stock dithionite concentrations were

139

determined from the molar absorptivity at 316 nm (8000 M-1 cm-1).42,43

140

Determination of the initial rate of hydroxylamine formation, RH. The initial hydroxylamine

141

formation rate (RH, nM/s) was calculated using equation 1, R =

T − T (1) 15 × 60

142

where T0 is the derivatized product yield from samples of the humic substance (HS) spiked with

143

[3ap] before irradiation (blank), and T15 is the yield at the end of a 15 min irradiation. The

144

concentration of the hydroxylamine increased linearly over the first 15 minutes of irradiation,

145

and thus initial rates were calculated over this time interval.

146 147

148

Apparent quantum yields for polychromatic irradiations, ɸ, were calculated using the following expression,

ɸ=

(2)

149

where Rx is the initial rate of formation of the measured species, either the hydroxylamine or

150

H2O2 (corresponding to RH or RH2O2, respectively); REX is the rate of light excitation

151

R = a(λ) × I(λ)dλ(3)

152

where a(λ) is the Naperian absorption coefficient of the sample (cm-1), and (λ) is the irradiance

153

at wavelength λ (photons cm-2 s-1nm-1) as measured with an Ocean Optics spectroradiometer at

154

the front face of the cuvette, with REX then converted to mole photons L-1s-1.

155

For the monochromatic irradiations, ϕ (λ) were acquired using the following equation: 8


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"●#

ϕ = = $(%)●(&'(()) )

156

(4)

157

where L is the path length of the cell (cm-1), A is the absorbance, and (1 − 10&* ) is the fraction

158

of light absorbed by the sample solution.

159

Results and Discussion

160

Hydroxylamine formation rates (RH) under anoxic conditions. Irradiation of SRFA under

161

anoxic conditions in the presence of 3ap resulted in the formation of the hydroxylamine, with the

162

initial rate of hydroxylamine formation, RH, increasing with increasing 3ap concentration (Fig.

163

S4, Fig 1). The hydroxylamine was not observed in the absence of either 3ap or HS. Further, no

164

hydroxylamine formation was observed following irradiation with the polychromatic light source

165

of 600 µM 3ap in the presence of 100 mM phenol, precluding the possibility of hydrogen atom

166

abstraction by the excited state nitroxide from good hydrogen atom donors such as phenols.44

167

Further, direct reduction of 3ap by electron transfer from excited singlet or triplet states is also

168

highly unlikely, because the quenching of these states by nitroxides proceeds almost exclusively

169

through other pathways,45-49 in a fashion similar to dioxygen (see also below). RH increased

170

approximately in proportion to the SRFA and SRHA concentration (Figs. 1, 2), indicating that

171

the humic substances were the source of the photo-produced reductant(s) of 3ap.

172

Above ~200 to 300 µM 3ap, RH exhibited evidence of saturation (Fig.1), suggesting

173

complete trapping of the reducing intermediate(s). This result is consistent with a competition

174

between reaction of 3ap with the reducing species and their loss either through recombination or

175

other pathways.

176

Dependence of RH on [O2]. RH decreased with increasing [O2] for both SRFA and SRHA,

177

consistent with 3ap and dioxygen competing for the same reducing intermediates37(Figs.1 ,2),

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Control experiments indicated that the hydroxylamine was stable to air oxidation over the

179

timescale of the experiments (see Fig. S5). Also, the substantial decrease in RH with increasing

180

[O2] cannot be attributed to the re-oxidation of the hydroxylamine to the nitroxide by reaction

181

with co-produced O2-; rate constants for the reaction of O2- with the hydroxylamine (~103 M-1s-

182

1 50

183

of O2- at the pH of our measurements (~6x104 M-1s-1).51 Reaction of O2- with nitroxides leads to

184

formation of the corresponding oxoammonium cations,52, 53 which can be recycled to the

185

nitroxide via rapid reduction by O2-, and thus act as dismutases. However, rate constants for the

186

oxidation of pyrrolidinyl nitroxides by O2- is exceedingly small, < 1x103 M-1s-1,52,53 so this

187

reaction as well as the dismutase activity are also very small at pH 8 making it highly unlikely

188

that hydroxylamine concentrations are affected by this set of reactions as well.52,53 Finally,

189

because nitroxides, like O2, are also effective physical quenchers of excited triplet states,

190

particularly those of ketones (~109 M-1s-1),48,49 formation of singlet dioxygen (1O2) will be largely

191

suppressed at high [3ap]; thus, the variations in RH with [3ap] and [O2] (see below) cannot be

192

explained as due primarily to the re-oxidation of the hydroxylamine by 1O2. Additional evidence

193

that hydroxylamine concentrations are not largely affected by air oxidation or by O2- and 1O2

194

reactions is provided below. Instead, this behavior can be understood within the following

195

reaction scheme,

), are over an order of magnitude smaller than the rate constant for the uncatalyzed dismutation

kO2 [O2]

Rf

*I

k3ap

O2-

(5)

hydroxylamine

(6)

NR

(7)

[3ap]

kd 196

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197 198

where *I represents a photo-produced reducing intermediate and Rf, the rate of its formation, with

199

the dependence of RH on [3ap] and [O2] provided by the following expression: +, = -

200

201

./,0 2 .1 0

=

+3 /3560

89. +9;2 =;2 >?

7

+/3560@

9356

AB

= C+B (8)

A nonlinear least squares fitting routine was employed to fit the data in Fig. 1 to Equation (9. +9;2 /;2 0)

202

8, with the parameters, A = Rf, and B=

203

kd/k3ap, thus kd = B0×k3p, whereas at [O2] = 50 µM, E FG =

9356

, provided in Table 1. At [O2] = 0 µM, B0= E

HIJ

(KLM &KM )

.

E

204

Values of E FG for untreated SRFA and SRHA are roughly 10, indicating that dioxygen

205

reacts with the reducing intermediate(s) much faster than 3ap (Table 1). Fitting the dependence

206

of RH on [3ap] in the presence of 250 µM dioxygen provides far less reliable values, owing to the

207

substantial quenching of RH at this higher dioxygen concentration. This is also true for the

208

borohydride reduced samples, where little evidence of curvature was observed with increasing

209

[3ap] at 50 and 250 µM O2 (see Fig. 1, Table 1)

210

HIJ

Thus, to examine the effect of [O2] on RH in better detail, [3ap] was kept fixed while [O2]

211

was varied (Fig. 2). At [3ap] = 600 µM, RH decreased rapidly with increasing [O2] (Fig. 2),

212

providing further evidence of a direct competition between O2 and 3ap for these reductants.

213

However, significant values of RH (2-4 nM/s) remained even under [O2]>250µM (Fig. 2),

214

suggesting a second, much smaller reductant pool that is less reactive with O2 than with 3ap.

215

These two pools are more readily apparent when the data are plotted in the following

216

rearranged form of Eqn.8,

11



RO 9Q + 9R /; 0 1 9Q 9R = 1 + × = 1+ + /; 0 (9) /3560 +P 9STU 9STU /3560 9STU /3560 EFG (Fig. EHIJ /STU0

217

where the slope represents

218

much larger than those for [O2] > 250 µM (Table 2), indicating minimally two pools of reducing

219

intermediates. The 9R /k3ap values obtained from the slope when [O2] < 250 µM for SRFA and

220

SRHA are very similar in magnitude to the values obtained from the fits to the data in Fig. 1 (~

221

10), providing further evidence that the largest pool of reductant reacts with dioxygen at a rate

222

constant that is approximately an order of magnitude larger than 3ap (Table 2). Importantly, the

223

fact that a very similar rate constant ratio was observed for kO2/k3ap under conditions in which

224

3ap was kept fixed at a high concentration and the [O2] varied (Figs. 2, 3), and under conditions

225

in which [O2] concentration was kept fixed at a low concentration and the [3ap] concentration

226

varied (Table 1, Fig. 1) provides further strong evidence that hydroxylamine concentrations were

227

largely unaffected by air oxidation or by reactions with O2- or 1O2. At [O2] > 250 µM, 9R /k3ap is

228

less than 1 for the untreated SRFA and SRHA, indicating that 3ap reacts faster than O2 with this

229

much smaller pool.

230

Effect of borohydride reduction on RH and RH2O2. In all cases, borohydride reduction led to an

231

approximately 20 to 30% increase in RH (Fig. 1; Table 3). This small increase in rate combined

232

with the significant loss of absorption due to borohydride reduction produced a significant

233

increase in apparent monochromatic quantum yields (Table3, 4). As observed previously,31 the

234

initial rates of H2O2 production, RH2O2, also slightly increased or stayed approximately constant

235

following borohydride reduction (Table 3), leading to enhanced apparent quantum yields for

236

H2O2 production as well 31(Table 3). Although not of the same magnitude, both processes show

237

similar behavior, arguing that they originate from common intermediate. As previously noted,31

3). The slopes acquired for [O2] < 250 µM are clearly

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238

the observation that borohydride reduction does not largely affect RH2O2 and only slightly

239

enhances RH, further argues that neither (aromatic) ketones/aldehydes nor charge transfer

240

transitions play a major role in the photochemical production of the reductant(s) or H2O2.

241

Within the uncertainties of the data, the dependence of RH on [3ap] and [O2] were similar

242

for the untreated and reduced samples (Figs. 1-3, Table 2), suggesting that borohydride reduction

243

does not change the lifetime(s) of the reducing intermediates dramatically. We showed

244

previously that the dependence of RH2O2 on [O2] was also largely unaffected by borohydride

245

reduction,31 providing additional evidence for a common source of these products. Still further

246

evidence for a common source is provided by the approximately proportional decrease in RH and

247

RH2O2 with increasing wavelength for both untreated and borohydride-reduced samples (Table 3).

248

Comparison between production rates and quantum yields for hydroxylamine and H2O2

249

formation. Despite the strong evidence for a common source, substantially higher values were

250

observed for RH than RH2O2 under comparable conditions of reaction, namely RH measured under

251

anoxic conditions with [3ap]= 600 µM and RH2O2 measured under aerated conditions with [O2]=

252

250 µM (Tables 3,4). Under these conditions, nearly quantitative reaction with the reducing

253

intermediate(s) is achieved by both O2 (see Fig. 3 in ref. 31) and 3ap (Fig. 1).Thus, given that

254

every reducing equivalent trapped by 3ap is also trapped by O2 to form O2- (Figs. 1-3), with the

255

O2- then simply dismutating to H2O2, a ratio of two for RH/RH2O2 would be expected. Instead,

256

ratios of RH/RH2O2 of approximately six were observed under monochromatic irradiation (Table

257

3), with even higher ratios observed under polychromatic irradiation (Table 4, see below).

258 259

These results thus suggest that a significant fraction of O2 -is lost through secondary reaction pathways. These pathways must be oxidative, because reductive pathways would

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260

produce H2O2. The fraction of O2- lost through oxidative pathways (PO2-) can be calculated from

261

the following expression (see SI for derivation), +,

262

+,2;2

=

2+;− (1W15X) 2

/+;− (1W15X) -1−Y;− (WBZ.51Z[\) 20 2

2

=6

(10)

263

where the value of six is estimated from the values observed in the monochromatic irradiations

264

(Table 3). Employing this value, we calculate that ~67% of the O2- proceeds through oxidative

265

pathways. This value is higher than that reported by Petasne and Zika (24-41%),29 but is

266

consistent with that reported by Garg et al. (lower limit,~70%) 32

267

Although a number of explanations are possible, it remains unclear why the RH/RH2O2

268

ratios (and the corresponding quantum yield ratios) acquired under polychromatic irradiations

269

are even higher (~13 for the untreated samples; Table 4) than those obtained under the

270

monochromatic irradiations (Table 3). One possible explanation is that RH is enhanced relative

271

to RH2O2 at longer wavelengths in the near UV and blue portion of the visible where Rex is higher,

272

although prior preliminary work that examined longer wavelengths appears inconsistent with this

273

explanation. 54 A second possible explanation is that the use of polychromatic light activates

274

additional pathways for O2- removal; however, significant further work would be required to test

275

this possibility. A third possibility is that 3ap is reacting with additional pools of photo-

276

reductants that do not react with O2, although this seems highly improbable on the basis of the

277

3ap/O2 competition experiments (Figs. 1-3). Regardless of this difference, the results from both

278

the monochromatic and polychromatic irradiations provide evidence that significantly higher

279

yields of O2- are produced than can be accounted for by H2O2 yields, and thus that a substantial

280

fraction of photo-produced O2- is lost through oxidative pathways (from 67-85% for RH/RH2O2

281

ranging from ~6-13; Eqn.10).

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282

Possible Origin of the Oxidative Sink. Substantial evidence has accumulated that humic

283

substances and terrestrially-sourced CDOM contain phenols 1, 55,56 Further, these species have

284

been strongly implicated as important electron donors in excited state electron transfer reactions

285

within CDOM.1 Thus, the co-production of phenoxy radicals and O2- would be anticipated

286

through the following set of reactions: H+

287

DA + hν

D*A or DA*

D ● A - ● + O2

288

D ● A- ●

D ● A + O2-●

(11)

(12)

289

where D represents a phenol electron donor and A an electron acceptor, perhaps quinones within

290

CDOM,18,31 while D● represents phenoxy radicals. Phenoxy radicals are known to react very

291

rapidly with O2-, with rate constants (~109 M-1S-1),57-59 far exceeding the rate constants for the

292

uncatalyzed dismutation of O2- at neutral to alkaline pH. The reaction of phenoxy radicals with

293

O2- is thought to produce an intermediate hydroperoxide, which can then react further either to

294

form oxidized products or to regenerate the phenol and O2.57,58

295

296

297 oxidized products (13) 298

Phenoxy + HO2/O2 -

Hydroperoxide Phenol + O2

299

15


(14)


300

While capable of lowering the yield of H2O2, Eqn.14 represents a recombination reaction

301

that produces no net chemistry. By contrast, Eqn.13 represents an additional O2--dependent

302

oxidation reaction leading potentially to ring-opened products.57,58 The branching ratio between

303

recombination (Eqn.14) and further oxidation (Eqn.13) can vary widely (0.11-0.94) depending

304

on the structure of the phenol.58 Depending on the phenol composition of CDOM, O2- could thus

305

be playing a far more significant role in the photooxidation of CDOM than has been previously

306

recognized. Because, in general, O2- reacts rapidly with radicals,60,61 other (donor) radicals

307

besides phenoxy could also be involved.

308

This work has shown that the molecular probe, 3ap, can provide estimates of the

309

photochemical formation rates of one-electron reductants that also undergo reaction with O2

310

(Figs. 1-3). Thus we believe this approach can provide reliable independent estimates of the

311

rates of O2- formation. Rates obtained in this fashion are far larger than the rates of H2O2

312

formation anticipated from simple O2- dismutation, implying a substantial oxidative sink of O2- in

313

accord with a number of other recent studies. 29,32,33 Because of their high reactivity with O2- and

314

their likely importance in the photochemistry of CDOM, we believe that phenoxy radicals

315

represent a viable sink, although other photo-generated radicals might also be involved. If

316

Eqn.14 dominates, then the primary result will simply be a lowering of the H2O2 yield. However,

317

if Eqn.13 dominates, then additional oxidation will take place following a single charge

318

separation event (Eqn. 11). In this case, O2- could play a critical role in the photoxidation of HS

319

and CDOM. In the future, we hope to compare directly the initial rates of O2- formation to those

320

of hydroxylamine formation to test this idea further.

321

Acknowledgements

16


Page 16 of 29

Page 17 of 29


322 323 324

This work was supported by grants from the NSF (OCE 1032223) and (OCE 0648414) to N.V. Blough and R. Del Vecchio. We thank Professor M. Al-Shiekhly for access to the EPR spectrometer.

325

Supporting Information Available.

326 327 328 329 330 331

Supporting information includes: 1) scheme illustrating hydroxylamine formation; 2) determination of 3ap molar absorptivity; 3) construction of hydroxylamine calibration curve; 4) examples of HPLC chromatograms of hydroxylamine formation; 5) tests of the stability of the hydroxylamine under aerobic conditions; 6) derivation of Equation 10. This material is available free of charge via the Internet at http://pubs.acs.org.

332

References

333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363

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Page 20 of 29

Page 21 of 29


502 503 504

21



Page 22 of 29

505 506

Tables

507

Table 1: Parameters obtained from fits of the data in Fig. 1 (see text).a

508

509

Parameter A (nM/s)

Parameter B

O2=0

(µM)

kO2/k3ap

O2=0

O2=50

6.8±0.7

O2=50 7.9±2.3

109±35

515±267

8.1

5 mg/L SRHA

7.8±0.9

9.4±3.4

252±64

868±480

12.3

10 mg/L SRFA

16.2±0.7

12.9±1.9

40±8

417±129

7.5

10 mg/L SRHA

20.4±1.7

13.7±5

108±35

469±306

7.2

10 mg/L Reduced SRFA

28.3±3.2

-

119±35

-

-

10 mg/L Reduced SRHA

32.1±4.0

-

119±61

-

-

510

5 mg/L SRFA

511

512

513

514

515 516

a

517

Fig. 1.

Uncertainties in these values were obtained from the standard errors of the fits to the data in

518

22


Page 23 of 29


519

Table2: Slopes and

520

expression of

521

]H_` /Sfg0

]^G

a

b

]^G

]H_`

values obtained from linear least-square fits of the data in Fig. 3 to the

= 1 +

]c d]^G ×/eG 0 ]H_`

× /Sfg0 = 1 + ]

]c

H_` /Sfg0

+ ]

]^G

H_` /Sfg0

/O 0. Slope represents

, where [3ap]=600 µM.a

522 523 524

a

Rf represents the initial rate of formation of the reducing intermediates; RH, the initial rate of

525

hydroxylamine formation; kd, the rate constant for recombination; k3ap, the rate constant for 3-ap

526

reaction with reducing intermediates, kO2, the rate constant for dioxygen reaction with the

527

reducing intermediates. Uncertainties are estimated from the standard error of the slopes.

528 529 530

23



Page 24 of 29

531

Table 3: Comparison of the RH with RH2O2 under monochromatic irradiation employing

532

equivalent irradiance; RH was measured under anoxic conditions in the presence of 600 µM 3ap,

533

whereas RH2O2 was measured under aerobic conditions, [O2]=250 µM.a

302 nm SRFA H2O2 nM/s 2.2±0.5 (Φ×10-4) ( 8.3±0.5) Hydroxylamine (nM/s) 14.4±2.7 ratio

6.6±2.8

313 nm

365 nm

SRHA

RESRFA

RESRHA

SRFA

SRHA

RESRFA

RESRHA

SRFA

SRHA

RESRFA RESRHA

2.0±0.2 (5.9±0.3)

1.8±0.8 (9.8±0.6)

2.1±0.2 (6.4±0.5)

1.7±0.3 (8.7±0.9)

1.4±0.5 (6.6±0.1)

1.3±0.4 (11±0.4)

1.8±0.5 (7.2±0.1)

0.4±0.2 (6.08±0.2)

10.9±2.2

18.3±3.2

12.4±2.5

10.2±2.0

7.8±2.2

14.5±3.4

8.2±2.2

2.1 ±1.1

1.9±0.5

3.7±0.8 5.0±1.0

5.5±2.2

10.2±3.3

5.8±2.6

6.0±2.1

5.6+2.3

11.1±3.4

4.7±2.3

5.0±1.2

6.4±0.6

9.6±0.8 11.1±.3

0.3±0.3 0.4±0.2 0.5±0.3 (3.6±0.2) (6.2±1.0) (3.9±0.5)

534 535 536

a

RH and RH2O2 represent the initial rate of hydroxylamine and H2O2 formation, respectively,

537

whereas RH/RH2O2 represents the rate ratio. Uncertainties in rates are based on the standard

538

deviation acquired from at least three independent measurements. Uncertainties in the ratios are

539

propagated from the uncertainties in the hydroxylamine and H2O2 formation rates. Uncertainties

540

in the quantum yields are propagated from the uncertainties in both the rate and irradiance

541

measurements.

24


Page 25 of 29


542

Table 4: Apparent polychromatic quantum yields and quantum yield ratios of hydroxylamine

543

and H2O2 formation for both untreated and borohydride-reduced SRFA and SRHA.a

polychromatic

RH

Quantum yield ×10-8 M/s SRFA 1.6 ± 0.7 SRHA 1.8 ± 0.7 ReSRFA 2.5 ± 1.1 ReSRHA 2.6 ± 0.9

REX

Quantum yields

×10-9 M/s

φH ×10-3 φH2O2 ×10-4 6.8 ± 1.3 5.1 ± 0.2 4.4 ± 1.2 3.8 ± 0.3 25 ± 5.8 9.8 ± 0.3 13 ± 4.1 6.2 ± 0.5

2.4E-09 4.0E-09 1.0E-09 2.0E-09

Φ ratio φH /φH2O2 13±1.3 13±1.2 25±5.8 21±4.1

544 545

a

546

deviation acquired from at least three independent measurements, with the uncertainties

547

propagated for the derived values.

Rex stands for the rate of light excitation. Uncertainties in RH are based on the standard

548

549

25



550

Figure 1: Dependence of the initial rate of hydroxylamine formation, RH (nM/s), on [3ap] for 5

551

and 10 mg/L SRFA, SRHA and their corresponding borohydride-reduced samples (Black line:

552

O2 =0 µM, Red Line O2 = 50 µM, Blue Line O2 = 250µM); lines are the non-l fits to Eqn. 8.

553

Error bars represent the standard deviation of at least three independent measurements under

554

each condition of [3ap] and [O2].

555

556 557

26


Page 26 of 29

Page 27 of 29


558

Figure 2: Dependence of RH on [O2] in the presence of 600 µM 3ap for untreated and

559

borohydride-reduced SRFA and SRHA.

560

27



561

Figure 3: Dependence of the ratio Rf/RH on [O2] for untreated and borohydride-reduced SRFA

562

and SRHA in the presence of 600 µM 3ap. Here, Rf represents the initial rate of hydroxylamine

563

formation in the presence of 600 µM 3ap, but absence of O2, whereas RH represents the variation

564

in the initial rate of hydroxylamine formation with [O2].

565 566

567

28


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568


Table of Content

569 570

Photograph was taken by the paper author, Yi Zhang

29


Photoproduction of One-Electron Reducing Intermediates by

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