Investigation of Humic Substance Photosensitized Reactions via

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Investigation of Humic Substance Photosensitized Reactions via Carbon and Hydrogen Isotope Fractionation Ning Zhang,† Janine Schindelka,‡ Hartmut Herrmann,‡ Christian George,§ Mònica Rosell,∥ Sara Herrero-Martín,† Petr Klán,⊥ and Hans H. Richnow*,† †

Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research-UFZ, Permoserstrasse 15, 04318 Leipzig, Germany ‡ Department of Chemistry, Leibniz Institute for Tropospheric Research (TROPOS), Permoserstrasse 15, 04318 Leipzig, Germany § Université de Lyon, Lyon, F-69626, France; Université Lyon 1, Lyon, F-69626, France; CNRS, UMR5256, IRCELYON, Institut de Recherches sur la Catalyse et l’Environnement de Lyon, Villeurbanne, F-69626, France ∥ Grup de Mineralogia Aplicada i Medi Ambient, Departament de Cristal·lografia, Mineralogia i Dipòsits Minerals, Facultat de Geologia, Universitat de Barcelona, C/Martí i Franquès s/n, 08028 Barcelona, Spain ⊥ Department of Chemistry and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic S Supporting Information *

ABSTRACT: Humic substances (HS) acting as photosensitizers can generate a variety of reactive species, such as OH radicals and excited triplet states (3HS*), promoting the degradation of organic compounds. Here, we apply compound-specific stable isotope analysis (CSIA) to characterize photosensitized mechanisms employing fuel oxygenates, such as methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE), as probes. In oxygenated aqueous media, Λ (Δδ2H/Δδ13C) values of 23 ± 3 and 21 ± 3 for ETBE obtained by photosensitization by Pahokee Peat Humic Acid (PPHA) and Suwannee River Fulvic Acid (SRFA), respectively, were in the range typical for H-abstraction by OH radicals generated by photolysis of H2O2 (Λ = 24 ± 2). However, 3HS* may become a predominant reactive species upon the quenching of OH radicals (Λ = 14 ± 1), and this process can also play a key role in the degradation of ETBE by PPHA photosensitization in deoxygenated media (Λ = 11 ± 1). This is in agreement with a model photosensitization by rose bengal (RB2−) in deoxygenated aqueous solutions resulting in one-electron oxidation of ETBE (Λ = 14 ± 1). Our results demonstrate that the use of CSIA could open new avenues for the assessment of photosensitization pathways.

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INTRODUCTION The light-induced transformation of organic compounds is an important degradation mechanism naturally occurring in surface waters and atmospheric media. Two different mechanisms have been proposed for photomediated reactions: direct substrate photolysis and indirect photolysis promoted by naturally present photosensitizers.1,2 Examples of photosensitizers include various aromatic compounds and, more generally, dissolved organic matter (DOM), which is ubiquitous in diverse aquatic environments and is a major source of photochemically generated oxidants.3,4 Humic substances (HS), the major fraction of DOM, can strongly absorb light and, upon excitation, lead to the formation of a variety of reactive species (e.g., 3HS*, OH•, 1O2, O2•−, and eaq−), which can play an important role in photodegradation pathways.5,6 It has been shown that OH radicals and excited triplet states (3HS*) produced from HS are of prime importance to the fate of organic matter in sunlit aquifers.7−9 The photochemical properties of dissolved HS have been studied for decades, and dozens of research papers have endeavored to elucidate the © XXXX American Chemical Society

underlying mechanisms of the photosensitization processes by measuring the different types of reactive species produced by natural-water HS and HS isolates,10 by employing probe molecules,11 by studying probe degradation upon the addition of quenchers,12 and by applying photochemical models.13,14 Nevertheless, the identification and interpretation of the complex photosensitized mechanisms initiated by a diverse range of reactive intermediates still face many challenges. Compound-specific stable isotope analysis (CSIA) offers a new avenue for characterizing reaction mechanisms that can be reflected by the magnitude of the kinetic isotope effect (KIE) depending on the rate-limiting step of a given reaction mechanism.15−17 The isotope fractionation of elements reflects the bond changes involved in this rate-limiting step. Using the isotope fractionation data for only one element can lead to a Received: June 8, 2014 Revised: November 20, 2014 Accepted: November 26, 2014

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dx.doi.org/10.1021/es502791f | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

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dimensional isotope slopes (Λ), and apparent kinetic isotope effects (AKIEs) were compared to those of reference reactions in order to investigate the underlying reaction mechanisms. Of the various reactive species formed upon photosensitization, the OH radical may be the likely candidate contributing to the degradation of fuel oxygenates through hydrogen atom abstraction (H-abstraction).30,40 Although the mechanism still remains unclear, there is evidence of several pathways for the production of OH radicals by HS irradiation, including an oxygen-dependent pathway through the disproportionation of O2•−/HO2•6 and an anoxic pathway through the oxidation of water or OH− by the excited triplet state of HS.42−44 Therefore, a reference reaction based on photolysis of H2O2 was conducted to generate OH radicals to compare the isotope fractionation in HS model systems. An additional reactive species, the excited triplet state of HS (3HS*), is formed from the singlet excited state of various HS chromophores via intersystem crossing.45 The triplet states can promote electron exchange with organic compounds or produce singlet oxygen (1O2) via energy transfer to ground-state molecular oxygen.8,46 Rose bengal (RB2−, carries two negative charges at pH 7) was thus employed as a model photosensitizer to generate the reactive triplet state of RB2− (3RB2−) and subsequently 1O247 in order to compare the fuel oxygenate degradation mechanisms to those when HS acted as a sensitizer. The use of CSIA for the investigation of photosensitized reaction pathways is an innovative approach which has not been proved before. The proposed approach may open new perspectives for distinguishing HS photosensitized degradation pathways involving a variety of photogenerated reactive species.

biased interpretation of the experimental data in the characterization of the degradation pathway.18 Hence, the combination of multiple elements (mainly δ13C vs δ2H, but also δ13C vs δ37Cl or δ15N) is more suitable for assessing the underlying reaction mechanism as masking effects typically occurring in one-dimensional CSIA are almost canceled.19,20 For instance, a wide range of chemical and biological degradation mechanisms of fuel oxygenates have been characterized by a dual (δ13C vs δ2H) isotope approach.21−23 However, only a few studies have investigated isotope fractionation during the light-induced transformations of organic compounds.24,25 We built a conceptual model system to explore how photosensitizers initiate the reaction with organic chemicals. Pahokee Peat Humic Acid (PPHA) and Suwannee River Fulvic Acid (SRFA) were selected as representative photosensitizers. Although some properties of HS vary by source, the general properties of HS samples are similar regardless of their origins.26 HS have the absorbance over a wide wavelength range (200−800 nm), which is particularly strong in the UV region because of the presence of aromatic chromophores.27 The carboxyl group and phenolic group contents were estimated by potentiometric titration, for example, giving 2.05 and 2.91 mequiv g−1 of phenolic groups for PPHA and SRFA, respectively.28 Fuel oxygenates, such as methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME), may serve as model organic compounds because of their particular properties. First, as they are transparent to UV and visible light, they do not undergo direct photolysis, and thus the reaction can only be induced by reactive species formed by irradiation of photosensitizers, which simplifies the model design. Second, the presence of the tert-butyl-oxygen bond offers a broad spectrum of diverse reactions, such as oxidation reactions initiated by free radicals29,30 and SN1-type31 and SN2-type hydrolysis.23 This makes them suitable model compounds for investigating complex photosensitized mechanisms. Third, they are especially suitable for a two-dimensional CSIA approach, which has been widely used to distinguish fuel oxygenate degradation reaction pathways,18,21−23 providing very useful information for comparing the results and interpreting the relevant mechanisms. Moreover, fuel oxygenates are still of considerable concern for health officials and water utilities because, despite being banned in some countries, they are still in use in several countries in Asia, Europe, and the Middle East.32 Fuel oxygenates can enter surface water systems by direct release from industrial facilities, atmospheric deposition,33 and stormwater runoff.34 Additionally, these compounds are present in the atmosphere as a result of evaporative emissions and incomplete combustion.35−38 Wallington et al.39 reported that reaction with photochemically produced OH radicals is the main determining factor affecting the atmospheric fate of these compounds; the half-life of atmospheric MTBE can be as short as 3 days in a regional airshed.36 Guillard et al.40 reported average MTBE concentrations of 4 ppbv in the atmosphere and 0.8 mg L−1 in atmospheric droplets at 293 K. Fuel oxygenates in atmospheric water droplets (rain, clouds, fog) may then react with various reactive species generated from the irradiation of atmospheric humic matter or humic-like substances.41 In our study, photosensitized degradation reactions employing fuel oxygenates as probes in solutions containing either PPHA or SRFA were investigated by CSIA. Carbon and hydrogen isotope enrichment factors (εC and εH), two-

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EXPERIMENTAL SECTION Chemicals. All chemicals were of analytical grade and used without further purification. MTBE (anhydrous, 99.8%), ETBE (99%), TAME (97%), rose bengal (4,5,6,7-tetrachloro2′,4′,5′,7′-tetraiodofluorescein, dye content 95%), and 2,4,6trimethylphenol (99%) were purchased from Sigma-Aldrich Chemie GmbH (Munich, Germany). Hydrogen peroxide (30%, w/w) and 2-propanol (anhydrous, 99.5%) were supplied by Merck (Darmstadt, Germany). Deuterium oxide (99.8%) was purchased from ARMAR (Europa) GmbH (Leipzig, Germany). Pahokee Peat Humic Acid (PPHA; 1R103H-2) and Suwannee River Fulvic Acid (SRFA; 1S101F) were obtained from the International Humic Substances Society (IHSS). Photoirradiation of H2O2. A schematic diagram of the photoreaction system is shown in Figure S1 of the Supporting Information. The reactor used for photochemical reactions consists of a 400 mL Pyrex cylindrical flask with a quartz window. A 150-W xenon lamp (Type L2175, Hamamatsu, Japan; Figure S2) was used as a light source to achieve a quartzfiltered irradiation (> 250 nm) of the reaction. The reactor was filled with 400 mL of 5 mM fuel oxygenates (MTBE, ETBE, or TAME) in a phosphate buffer solution (pH 7.0, 50 mM). The reactor was almost completely filled with the solution to minimize the headspace and volatility losses. Excess hydrogen peroxide (initial molar ratio to fuel oxygenates = 30:1)48 was added for the reference reaction of UV/H2O2 to form OH radicals. The solution was stirred with a magnetic stirrer. For sampling, the reaction mixture was transferred into gastight vials at given times via a syringe through the rubber septum of the reactor. The vials were immediately sealed with Teflonlined septum caps and stored at −20 °C prior to analysis to avoid evaporation effects.49 B



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Figure 1. Degradation of MTBE (a), ETBE (b), and TAME (c) by irradiation of H2O2 (diamond). Two types of control experiments in the absence of H2O2 (circle) or in the dark in the presence of H2O2 (square). Rayleigh plots of carbon (d) and hydrogen (e) isotope fractionation for MTBE (square), ETBE (circle), and TAME (triangle) under irradiation of H2O2. Two-dimensional (2D) plots (f) of hydrogen versus carbon for MTBE (square), ETBE (circle), and TAME (triangle) under irradiation of H2O2.

HS Photosensitized Reactions. For the HS photosensitized degradation of fuel oxygenates, PPHA was dissolved in a phosphate buffer solution (pH 7.0, 50 mM) by adding 0.1 M sodium hydroxide dropwise to achieve complete dissolution at a final concentration of 0.4 g L−1. The pH was adjusted to 7.0 by the dropwise addition of 0.1 M sulfuric acid. SRFA can be dissolved directly in a phosphate buffer solution (pH 7.0) at a concentration of 0.4 g L−1. The corresponding amount of pure MTBE or ETBE was added to a PPHA/SRFA solution to obtain a concentration of 5 mM. The pH of the experimental solutions was monitored with a pH meter (Mettler Delta 320, UK). Maintaining the pH value at 7.0 is critical for ensuring the occurrence of the corresponding degradation pathway; otherwise, the mechanism would be different because of the

Photoirradiation of Rose Bengal. For the reactions in the presence or absence of singlet oxygen, rose bengal (0.4 g) was dissolved into 400 mL of the pH 7.0 buffered solution, and the solution was bubbled with oxygen or argon for 1 h prior to the addition of pure ETBE at a final concentration of 5 mM. A balloon filled with oxygen or argon (in the case of deoxygenated solutions) was applied to maintain a constant gas pressure (Figure S1). For the reaction in heavy water (D2O), a procedure similar to that in the presence of oxygen was performed by preparing a buffer solution (pH 7.0) in D2O instead of in H2O. The samples were transferred into gastight vials at regular times and treated as described for the photoirradiation of H2O2 experiments. C



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Table 1. Comparison of the Carbon and Hydrogen Isotopic Enrichment Factors (ε), Apparent Kinetic Isotope Effects (AKIEs), Dual-Isotope Plots (Λ), and Half-Lives (t1/2) for Photoreactions of Fuel Oxygenates photoirradiation H2O2

rose bengal/O2 rose bengal/O2-free rose bengal/O2/D2O SRFA/O2 PPHA/O2 PPHA/O2/280 nm cutoff filter PPHA/O2/2-propanol PPHA/O2/2-propanol/TMP PPHA/O2-free PPHA/O2-free/TMP a

fuel oxygenate MTBE ETBE TAME ETBE ETBE ETBE MTBE ETBE MTBE ETBE ETBE ETBE ETBE ETBE ETBE

εC ± 95%CI [‰] −1.6 −1.0 −1.5 −2.2 −1.7 −1.7 −2.4 −1.8 −2.1 −1.7 −1.4 −1.9 −2.0 −1.9 −1.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

εH ± 95%CI [‰]

0.3 0.1 0.4 0.3 0.1 0.1 0.6 0.3 0.2 0.3 0.1 0.3 0.2 0.2 0.1

−34 −25 −32 −24 −27 −25 −47 −41 −32 −42 −36 −29 −24 −24 −26

AKIE values were calculated according to the mechanism suggested.

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

7 3 8 1 2 2 10 5 4 6 3 5 2 4 3

AKIECa 1.0086 1.0060 1.0091 1.0134 1.0103 1.0103 1.0121 1.0109 1.0106 1.0103 1.0085 1.0115 1.0121 1.0115 1.0097

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0020 0.0006 0.0024 0.0019 0.0006 0.0006 0.0031 0.0018 0.0010 0.0018 0.0006 0.0018 0.0012 0.0012 0.0006

AKIEHa 1.689 1.539 1.812 1.038 1.044 1.044 2.294 2.347 1.623 2.427 2.016 1.047 1.039 1.039 1.042

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.245 0.103 0.386 0.001 0.003 0.003 0.692 0.398 0.127 0.520 0.172 0.009 0.003 0.007 0.005

Λ ± 95%CI

t1/2 (h)

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5 3 3 17 17 17 38 19 33 18 485 36 94 38 112

20 24 21 10 14 13 19 21 15 23 23 14 11 11 14

6 2 3 2 1 1 4 3 2 3 1 1 1 1 2

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H+ produced during the reaction. The final solution (400 mL) was mixed well with a magnetic stirrer, and samples were transferred into gastight vials at regular times as described above. The concentration of oxygen in the experimental solutions was monitored using a Clark electrode (Biolytik, Bochum, Germany). A dark control (in which the conditions were identical to the treatment experiments except that the lamp was switched off) was conducted for the irradiation of both the H2O2 and HS systems. A second control used aqueous solutions of fuel oxygenates subjected to irradiation in the absence of H2O2 or HS. For the OH radical-quenching experiment, 2-propanol (initial molar ratio to ETBE = 100:1) was added to the mixed solution of PPHA and ETBE before irradiation, as described above. For oxygen-free experiments, the PPHA solution was bubbled with argon for 1 h prior to the addition of pure ETBE at a final concentration of 5 mM. The well-known 3HS* quencher, 2,4,6-trimethylphenol (TMP),3 was employed (4 mM) to investigate the role of 3HS* in the degradation of ETBE in oxygenated media when OH radicals were quenched, or in deoxygenated media. In addition, the effect of a filter with a 280 nm cutoff wavelength on the HS photosensitized reactions was investigated. The filter was applied to the degradation of ETBE by photosensitized PPHA in oxygenated media. The experimental procedure was the same as previously described. The reaction rate, isotope fractionation, and reaction products were measured for comparison with the reaction in the absence of a filter. Analytical Methods. The concentration of fuel oxygenates was determined using a gas chromatograph equipped with a flame ionization detector (GC-FID), and the degradation products were analyzed by gas chromatography−mass spectrometry (GC-MS). The carbon and hydrogen stable isotope composition of the fuel oxygenates was determined using a gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) system described in our previous work.50 A Zebron ZB1 column (60 m length × 0.32 mm ID, 1 μm film thickness; Phenomenex, Aschaffenburg, Germany) was used for separation. Each sample was analyzed via headspace injection in triplicate. A detailed description of the analytical methods is provided in the Supporting Information.

RESULTS AND DISCUSSION Reaction of OH Radicals Formed by Photolysis of H2O2. Because fuel oxygenates do not absorb light, the degradation of fuel oxygenates can only be initiated by OH radicals formed by the direct photolysis of H2O2 (Figure 1a−c). Since the amount of generated OH radicals was not determined, we use half-life (t1/2) as an indication of the reaction rate (Table 1). The concentrations remained constant in both control experiments: that in which irradiation was performed in the absence of H2O2 and that in which the solution was kept in the dark in the presence of H2O2 (Figure 1a−c). Upon irradiation, the change in the concentration and enrichment of heavy isotopes in the reactant were used to calculate the isotopic enrichment factors for both carbon and hydrogen (εC, εH) using the Rayleigh equation (see the equations in the Supporting Information). The degradation experiments using MTBE, ETBE, and TAME were evaluated with a degradation of no more than 97%, yielding an εC of −1.6 ± 0.3‰, −1.0 ± 0.1‰, and −1.5 ± 0.4‰ (Figure 1d) and an εH of −34 ± 7‰, −25 ± 3‰, and −32 ± 8‰ (Figure 1e), respectively. The correlation of hydrogen and carbon isotope fractionation (ΔC = δt13C − δ013C, ΔH = δt2H − δ02H) yielded Λ values of 20 ± 6, 24 ± 2, and 21 ± 3 for MTBE, ETBE, and TAME, respectively (Figure 1f). These Λ values approximately corresponded to the ratio of the isotopic enrichment factors εH/εC and were in agreement with the previous values reported for C−H bond cleavage.21 Tert-butyl formate (TBF), tert-butyl alcohol (TBA), and acetone (AC) were the major reaction products detected under the photoirradiation of solutions containing H2O2 (Figure S3), suggesting that OH radicals initiated the degradation of MTBE. The methoxy group hydrogens of MTBE were most prone to be abstracted by OH radicals.51 The newly formed alkyl radicals could be immediately trapped by molecular oxygen to produce peroxyl radicals (Scheme 1a). In the aqueous phase, peroxyl radicals could undergo different reactions to produce compounds with the carbonyl and hydroxyl groups as well as unstable alkoxy radicals which could react further.52,53 ETBE and TAME in the presence of OH radicals provided analogous products to those of MTBE degradation: acetone and tert-butyl alcohol (TBA) and acetone and tert-amyl alcohol (TAA). tert-Butyl acetate was formed probably by a subsequent reaction of ETBE peroxyl D



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Scheme 1. Pathways for Degradation of ETBE under Various Reaction Conditions

Figure 2. (a) Remaining concentration (Ct/C0%) of ETBE and (b) two-dimensional (2D) plots of hydrogen versus carbon under irradiation of rose bengal in the presence of O2 (square), in the absence of O2 (circle), and in the presence of O2 in D2O (triangle).

Photosensitization by Rose Bengal: Reactive Triplet State and 1O2 Formation. Upon irradiation, rose bengal

radical intermediate (Scheme 1a) but was not detected in the case of TAME degradation (tert-amyl formate; Figure S3). E



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(RB2−) forms the triplet state (3RB2−) which can transfer energy to ground-state molecular oxygen to form 1O2.54 A possible mechanism for the degradation of ETBE by an initial one-electron oxidation involving 3RB2− or an oxidized form RB•− (produced by 3RB2− disproportionation55) to compete the reaction with 1O2 was evaluated. Irradiation of RB2− was performed in the presence or absence of O2, thus facilitating or preventing the production of 1O2, respectively. The same reaction rate with a half-life (t1/2) of 17 h was observed (Figure 2a). Furthermore, irradiation of RB2− was conducted in D2O instead of H2O to determine the contribution of 1O2 to the degradation of ETBE. The lifetime of 1O2 is approximately 13 times longer in D2O than in H2O;56 thus an enhanced reaction rate would be expected. The same reaction rate (t1/2 = 17 h) was again observed (Figure 2a). Hence, 1O2 did not react with ETBE under the experimental conditions, which is in agreement with the fact that the second-order rate constant for quenching of 1O2 by tertiary alkyl ethers (

Investigation of Humic Substance Photosensitized Reactions via

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