Marine Pollution Bulletin 79 (2014) 54–60
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Photoproduction of hydrogen peroxide in aqueous solution from model compounds for chromophoric dissolved organic matter (CDOM) Catherine D. Clark, Warren de Bruyn ⇑, Joshua G. Jones School of Earth and Environmental Sciences, Schmid College of Science and Technology, Chapman University, Orange, CA 92866, United States
a r t i c l e
i n f o
Keywords: Hydrogen peroxide Humic Fulvic CDOM Quinone
a b s t r a c t To explore whether quinone moieties are important in chromophoric dissolved organic matter (CDOM) photochemistry in natural waters, hydrogen peroxide (H2O2) production and associated optical property changes were measured in aqueous solutions irradiated with a Xenon lamp for CDOM model compounds (dihydroquinone, benzoquinone, anthraquinone, napthoquinone, ubiquinone, humic acid HA, fulvic acid FA). All compounds produced H2O2 with concentrations ranging from 15 to 500 lM. Production rates were higher for HA vs. FA (1.32 vs. 0.176 mM h1); values ranged from 6.99 to 0.137 mM h1 for quinones. Apparent quantum yields (Happ; measure of photochemical production efficiency) were higher for HA vs. FA (0.113 vs. 0.016) and ranged from 0.0018 to 0.083 for quinones. Dihydroquinone, the reduced form of benzoquinone, had a higher production rate and efficiency than its oxidized form. Post-irradiation, quinone compounds had absorption spectra similar to HA and FA and 3D-excitation– emission matrix fluorescence spectra (EEMs) with fluorescent peaks in regions associated with CDOM. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Hydrogen peroxide (H2O2) is an important intermediate in aquatic redox processes that affects water quality (Cooper et al., 1989). It has been shown to reduce fecal indicator bacteria (FIB) levels in wastewater treatment (Davies-Colley et al., 1997) and implicated as a possible cause of observed diel variability in FIB levels in surf-zone waters at a recreational bathing beach (Boehm et al., 2002). Concentrations from 10 to 300 nM have been measured in surface waters in near to off-shore marine environments (see review Clark et al., 2008a and references therein; Garg et al., 2011). Photochemical production from chromophoric dissolved organic material (CDOM) is considered the primary source in natural waters. This is based on observations of diel cycles in H2O2 concentrations with afternoon maxima and early morning minima, and higher measured H2O2 concentrations and production rates in marine waters with higher CDOM levels (Cooper et al., 1988; Fujiwara et al., 1993; Moore et al., 1993; Scully et al., 1996; Herut et al., 1998; Obernosterer et al., 2001; O’Sullivan et al., 2005). The initial step in the photochemical production of H2O2 from CDOM is the absorption of sunlight by CDOM to create an excited state that reacts with oxygen to generate superoxide O 2 , which dismutates to form H2O2 (Zhang et al., 2012). There are a number of possible pathways by which O 2 could form from photoexcited ⁄ CDOM reacting with oxygen. Prior studies have shown minor ⇑ Corresponding author. Tel.: +1 714 628 7353. E-mail address: [email protected] (W. de Bruyn). 0025-326X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2014.01.001
contributions from the photoionization of ⁄CDOM to give a hydrated electron which then reacts with O2 (Cooper et al., 1989) and from the direct reduction of excited-state singlet O2 by ⁄CDOM (Dalrymple et al., 2010). Two recent studies have provided evidence for a primary production pathway via (1) the production of reducing radical intermediates, from an intramolecular excited state electron transfer process in ⁄CDOM, which then react with O2 (Zhang et al., 2012) and (2) direct reduction of ground-state ⁄ triplet oxygen to O 2 by redox active chromophores in CDOM (Garg et al., 2011). The identity of the chromophores in CDOM which are responsible for the absorption of light and subsequent production of H2O2 is not clear, but a number of lines of evidence suggest that quinones could play a key role. Fluorescence spectroscopy studies have shown the ubiquitous presence of oxidized and reduced quinones in CDOM, with approximately 50% of the observed optical properties of CDOM samples attributed to quinone type species (Cory and McKnight, 2005). A quinone photochemistry study carried out in air-equilibrated aqueous solution using optical properties and radical trapping techniques has shown the production of hydroxylating intermediates, singlet oxygen and superoxide (Garg et al., 2007), the same reactive intermediates produced from photoexcited CDOM (Cooper et al., 1989; Scully et al., 1996). This study also directly measured hydrogen peroxide production from disodium anthraquinone-2-6-disulfonate (AQDS) in water (Garg et al., 2007). However, to the best of our knowledge this is the only study that has directly shown that hydrogen peroxide is produced photochemically from quinones.
C.D. Clark et al. / Marine Pollution Bulletin 79 (2014) 54–60
To expand on this work and test the feasibility of hydrogen peroxide in natural waters being produced from quinone moieties in CDOM, we measured the photoproduction of H2O2 from a series of model quinone compounds and a humic and fulvic acid in aqueous solution. We also determined optical parameters commonly used to characterize DOM in natural water samples, specifically spectral slope and shifts in the peak maxima for excitation and emission in fluorescence spectra. We chose model compounds with a range of sizes and degrees of conjugation and included an oxidation–reduction pair to assess the effects of quinone redox state on H2O2 production. We compare hydrogen peroxide production rates and optical property changes for the model compounds to recent measurements we have made in our laboratory on salt marsh and coastal seawater samples. 2. Methods and materials 2.1. Solutions Solutions of 1,4-benzoquinone, 2,5-dihydroxyquinone, 1,2naphthoquinone, anthraquinone, and ubiquinone 50 (Sigma–Aldrich) were prepared at 0.13 mM, except for naphthoquinone and ubiquinone which were prepared with a concentration 10 lower because of reduced solubility. Water used in preparing solutions was de-ionized by passing distilled water through a Nanopure Infinity Ultrapure Water System (Model D8961, Barnstead). Post irradiation all solutions were diluted with nanopure water to 130 nM for hydrogen peroxide measurements. Standard Suwannee River humic and fulvic acids were obtained from the International Humic Substances Society (IHSS). Humic and fulvic acid solutions were prepared at concentrations of 0.1 mg mL1 to give similar absorbances as the quinone model compound solutions and similarly diluted for hydrogen peroxide analysis. 2.2. Optical measurements Absorbance spectra were obtained from 200 to 700 nm with a UV/VIS spectrophotometer (Agilent Technologies, Model 8453) in 1 mm quartz cells. Absorbance spectra were measured at time intervals of 0, 5, 10, 15, 20, 30 and 60 min during irradiations. Emission spectra were obtained in a 1 1 cm quartz fluorometer cell with a scanning fluorometer (Quantamaster, PTI). Three dimensional Excitation–Emission Matrix fluorescence spectra (EEMs) were run on all solutions pre and post irradiation. EEMs were obtained by ranging excitation wavelengths from 260 to 430 nm and emission wavelengths from 270 to 650 nm in 5 nm increments. Details are given in Clark et al. (2008a,b). A water EEM was generated daily to subtract out the water Raman peak, and spectra were corrected for instrumental response. Intensities are reported in photons s1. Spectral slopes (S) were calculated by fitting data to Eq. (1) with a first-order linear regression for the 300–400 nm region for comparison to our previous work on natural water samples (Clark et al., 2008a,b, 2009, 2010).
S ¼ ½ðln Abs=A0 Þ=ðk k0 Þ
ð1Þ
Here, Abs is the absorbance at wavelength k and A0 is the absorbance at reference wavelength k0 (Green and Blough, 1994; Moran et al., 2000). 2.3. Irradiations 4 mL of solution was irradiated in 5–10 min increments up to 60 min in a 1 1 cm quartz cell with a 200 W ozone-free Xenon mercury arc lamp (Oriel Instruments, Model 6292; lamp specifications http://www.newport.com/Technical-Note-Arc-Lamp-Spec-
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tral-Irradiance-Data/409171/1033/content.aspx). A 1 1 cm quartz cell with deionized water was placed in front of the sample cell as an IR filter. A cooling fan (Radio Shack) was placed at 90° to the side of the sample cell. The temperature inside the cell was monitored with a digital thermometer as a function of irradiation time. With the IR filter and cooling fan, temperature increased by 1.5 °C within 5 min of irradiation, and remained constant throughout the remainder of the irradiation time. A lamp photon flux of 1.1 1021 photons s1 m2 was measured by nitrite/benzoic acid/ hydroxybenzoic acid chemical actinometry (Jankowski et al., 1999).
2.4. Hydrogen peroxide analyses Concentrations were measured with an enzyme-mediated fluorescence peroxidase technique (Zika and Saltzman, 1982) used for most previous surface seawater measurements. A filter field fluorometer (Turner Designs Model 10-AU-0) with a near-UV DOM/ Ammonium lamp and 25 mL sample cuvette was used, with excitation centered at 354 nm and emission at 496 nm. Aqueous solutions for the hydrogen peroxide analysis included a phosphate buffer (0.5 M; JT Baker), hydrogen peroxide (1 105 M; Aldrich), scopoletin (5 105 M; Sigma–Aldrich) and horseradish peroxidase (HRP; 1 104 M; Sigma–Aldrich). Details of the method have been previously published (Clark et al., 2009). 2–4 replicate measurements on a single sample were averaged, with an average r < 18%. Error bars are reported as ±r.
3. Results and discussion 3.1. Quinone structure and photochemistry Structures of the model quinone compounds are shown in Fig. 1. Molecular structures range in size and complexity from benzoquinone (BQ) and dihydroquinone (DHQ) (1 ring) through the larger napthaquinone (NQ) (2 ring) and anthraquinone (AQ) (3 ring). Ubiquinone (UQ) has a substituted aromatic ring bound to an aliphatic tail with 6–10 CH2 repeating units. Molecular structures for fulvic (FA) and humic acids (HA) have not been definitively characterized but complex macromolecular structures with multiple rings and substituents such as quinone moieties and carboxyl groups have being proposed (Leenheer and Croue, 2003). DHQ is the reduced form of BQ. Most quinone photochemistry studies have been carried out in organic solvents. When irradiated, quinones can undergo a range of reactions including cycloaddition reactions like dimerization, abstraction of hydrogen from substrates to form hydroquinones and electron transfer (Patai and Bruce, 1974). Irradiated quinones in aqueous solution generally produce semi-quinone intermediates and hydroquinones and hydroxyl-substituted quinones as photoproducts (Patai and Bruce, 1974; Ononye and Bolton, 1986; Alegria et al., 1997 and 1999; Gan et al., 2008). A number of photoxidation mechanisms have been proposed. These include hydrogen atom abstraction from water (Ononye and Bolton, 1986), water-quinone exciplex formation and intramolecular charge transfer (Gan et al., 2008), electron abstraction from water to form quinone anions (Roy and Aditya, 1983; Parker et al., 1992), and photooxidation of water to produce reactive oxygen species like superoxide and hydroxyl radicals (Alegria et al., 1997, 1999; Garg et al., 2007) and hydrogen peroxide (Garg et al., 2007). A recent study showed the production of hydroxylating intermediates, singlet oxygen and superoxide reactive intermediates (Garg et al., 2007). Possible production pathways for these species include quenching of the quinone excited state by ground-state triplet O2 to produce excited
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NQ DHQ
AQ
BQ
UQ
HA
Fig. 1. Structures of model compounds: dihydroquinone (DHQ), benzoquinone (BQ), napthaquinone (NQ), anthraquinone (AQ), ubiquinone (UQ) and a proposed structure for humic acid (HA; Stevenson, 1994).
singlet oxygen or direct reduction of oxygen to produce the superoxide ion O 2. 3.2. Absorbance CDOM has an absorbance spectrum that decreases exponentially from the UV through the visible region (200–600 nm). Del Vecchio and Blough (2004) proposed that the absorption spectrum arises from intramolecular charge-transfer interactions between hydroxyl-aromatic donors and quinoid acceptors formed by the partial oxidation of lignin species. Quinones are characterized by intense absorption bands in the 250–300 nm range and weaker overlapping bands in the 300–400 nm range due to P–P⁄ transitions associated with the benzene and quinone moieties (Cory and McKnight, 2005). A change in redox state shifts the UV–VIS spectra, with reduction increasing the intensity of the P–P⁄ transition especially in the visible range (Cory and McKnight, 2005). Absorbance spectra pre-irradiation are given in Fig. 2a. FA and HA showed the same exponential decrease from 200 to 600 nm (Fig. 2), characteristic of CDOM (Del Vecchio and Blough, 2004), with HA having higher absorption in the 200–400 nm range. Similar featureless spectra were obtained for a wide range of humic and fulvic acids in a prior study, with the humic acids having higher absorbances than the fulvic acids at the same concentration (Mignone et al., 2012). In comparison, the quinone compounds pre-irradiation had more structural features in the 200–300 nm range with several distinct peaks presumably associated with P–P⁄ transitions, with the exception of UQ and AQ which were similar to FA. The most striking is that for BQ and DHQ, the oxidation reduction pair. One can also clearly see the shift in absorbance maximum to longer wavelengths in the reduced form. In Table 1 we report pre-irradiation absorbances at 300 nm. This is a wavelength frequently given in studies of CDOM optical properties in natural waters (see for example Clark et al., 2009 and 2010). Values range from 0.75 for BQ to 4.7 for HA; however, it should be noted that DHQ has an absorbance maximum at 300 nm, unlike all the other species studied here. For comparison purposes, absorbances of 0.03–0.04 at 300 nm were obtained in our laboratory for coastal water samples (Clark et al., 2009). Absorbance spectra post-60 min irradiation are shown in Fig. 2b. After irradiation, all 5 quinone compounds had spectra like that of FA, with no distinct peaks and decreasing absorbance with increasing wavelength, similar to those observed for CDOM in natural water samples (Del Vecchio and Blough, 2004 and references therein). BQ, DQ and NQ have lost their distinct P–P⁄ structural
Fig. 2. Absorbance from 200 to 600 nm for dihydroquinone (DHQ), benzoquinone (BQ), napthaquinone (NQ), anthraquinone (AQ), ubiquinone (UQ), fulvic acid (FA) and humic acid (HA) (a) prior to irradiation and (b) after 60 min of irradiation.
features during irradiation. We report absorbance changes preand post-irradiation in the visible region at 490 nm (Table 2) since reduction has been shown to increase absorbance in this region for
C.D. Clark et al. / Marine Pollution Bulletin 79 (2014) 54–60 Table 1 Absorbance pre-irradiation at 300 nm, initial peroxide photoproduction rates (mM h1), and apparent quantum yields (Happ) of hydrogen peroxide produced after irradiation for model compounds. Compound
a (300)
Rate (mM h1)
Happ
Dihydroquinone (DHQ) Benzoquinone (BQ) Napthaquinone (NQ) Anthraquinone (AQ) Ubiquinone (UQ) Fulvic acid (FA) Humic acid (HA)
0.211 0.0033 0.0033 0.0061 0.0364 0.0113 0.205
6.99 1.39 0.137a 0.588 0.372a 0.176 1.324
0.083 0.135 0.0018 0.057 0.025 0.016 0.113
a Concentrations were 0.13 mM for DHQ, BQ and AQ but 10 lower at 0.013 mM for NQ and UQ due to reduced solubility; HA and FA were prepared as 0.1 mg L1.
quinone compounds (Cory and McKnight, 2005). Decreases are observed for NQ, UQ, and HA, whereas AQ, DHQ and FA show increases in absorbance. BQ shows decreasing absorbance at 490 nm after irradiation, possibly due to a net decrease in the reduced form of this compound with irradiation. Another absorbance based method used to characterize CDOM in natural waters is to calculate the spectral slope (S). S increases as CDOM is photobleached in oxidative environments and decreases with aging in sub-oxic soils and sediments due to increasing aromaticity and humification (Stabenau et al., 2004; Tzortziou et al., 2007). Spectral slopes (S) for natural water samples in prior studies in our laboratory range from 0.016 to 0.021 for surf zone water (Clark et al., 2009) to 0.010–0.012 for salt marsh waters (Clark et al., 2008b). Table 2 shows S values calculated pre- and post-irradiation for all model compounds however, because of the strong P–P⁄ structural features prior to irradiation for some of these species, the initial spectral slope is not very meaningful. Post-irradiation, S range from 0.004 to 0.034 consistent with previous natural water sample results (Clark et al., 2008b and 2009). S values scale approximately with the number of rings in the structure for the 4 quinones in their oxidized state. During irradiation, S for FA stays the same while S for HA increases from 0.019 to 0.034, consistent with photobleaching.
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3.3. Photoproduction Hydrogen peroxide concentrations for the irradiated model compounds and fulvic and humic acids in aqueous solution are shown in Fig. 3 as a function of irradiation time. For all compounds, there was an initial rapid increase in H2O2 concentrations over the first 10 min of irradiation to around 15 lM for the larger quinones (UQ, NQ) and FA and 150 lM for HA, AQ and BQ. DHQ the reduced form of benzoquinone produced higher concentrations of 500 lM. In general, a slower increase was then observed from 10 to 60 min irradiation time or in some cases a steady state was reached. DHQ concentrations decreased with continued irradiation time, possibly due to loss processes like the interconversion of redox states. Final hydrogen peroxide concentrations after 60 min irradiation ranged from 15 to 500 lM. Initial photochemical production rates were calculated from the concentration of hydrogen peroxide produced in the first 5 min (Rate = [H2O2]at 5 min/0.0833 h (5 min in units of h); Table 1). Production rates varied over more than an order of magnitude, from 0.14 for NQ to 7 mM h1 for DHQ. Production rates for NQ, UQ and FA averaged 0.23 ± 0.13 mM h1, whereas rates were higher at an average of 1.10 ± 0.44 mM h1 for HA, AQ and BQ. A recent study using triplet quenchers to probe the mechanism of hydrogen peroxide production from irradiated humic and fulvic acids concluded that excited triplet states of quinones do not
Table 2 Optical properties pre- and post-irradiation at 0 and 60 min for model compounds: absorbance at 490 nm, spectral slope (300–400 nm), steady-state fluorescence intensity (Flu, 103 photons s1; ex = 350 nm, em = 450 nm) and locations of excitation/emission peak maxima from EEMs. Compound
a (490)
S
Flu
Ex/em
Ex/em
Dihydroquinone 0 min 60 min
0.0136 0.0225
0.017 0.015
80.9 48.6
260/442 260/472
330/440 330/452
Benzoquinone 0 min 60 min
0.0103 0.0048
0.019 0.004
3.66 27.1
290/332 260/484
330/462
Napthaquinone 0 min 60 min
0.0013 0.0002
0.017 0.010
2.63 21.1
Anthraquinone 0 min 60 min
0.0016 0.0038
0.019 0.017
17.7 6.37
260/456 279/484
325/443 325/453
Ubiquinone 0 min 60 min
0.0143 0.0129
0.013 0.009
3.00 2.13
270/474 260/510
390/482 355/473
Fulvic acid 0 min 60 min
0.0004 0.0061
0.021 0.021
130 77.7
260/456 260/486
325/437 330/454
Humic acid 0 min 60 min
0.0192 0.0138
0.019 0.034
145 249
330/462 320/442
376/508 367/502
Fig. 3. Concentrations of hydrogen peroxide (lM) produced from irradiated solutions as a function of irradiation time (min) for (a) anthraquinone j, dihydroxyquinone d, benzoquinone Nand humic acid and (b) napthaquinone h, ubiquinone s, and fulvic acid D. Lines shown are for ease of viewing.
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contribute significantly to hydrogen peroxide production (Zhang et al., 2012). Garg et al. (2011) fitted their kinetic data for the production of hydrogen peroxide from fulvic acid with a model based on a chromophore (Q) which they attributed to a photoexcited triplet state quinone-like redox moiety (or another type of electron acceptor) which forms semi-quinone like radicals (Q) through intramolecular charge transfer that then reduces oxygen to O 2, which subsequently forms H2O2. In our study, DHQ (the reduced form of BQ) had the highest production rate at 6.99 mM h1, suggesting that the reduced state may be better at hydrogen peroxide production. This would be consistent with a semi-quinone intermediate in the mechanism if the H atom on the hydroquinone – OH substituent is readily abstracted by water to leave –O, which reacts readily with oxygen to produce O 2. H2O2 production in aqueous solution has previously been measured for humic and fulvic acids and one quinone compound. A direct quantitative intercomparison among studies is difficult due to the use of different irradiation systems with different wavelength characteristics and photon fluxes. Garg et al. (2011) measured 60 nM H2O2 for a 1.0 mg L1 Suwanee River fulvic acid solution irradiated for 60 min by a 150 W Xenon lamp equipped with an AM1 filter to simulate the solar spectrum at the earth’s service. A study of a quinone compound (disodium 9,10-anthraquinone1,5-dilsulfonate (AQDS)) using the same irradiation setup measured higher concentrations of 1200 nM H2O2 after 60 min of irradiation of a 3 lM solution (Garg et al., 2007); rates scaled almost linearly with AQDS concentration in the low lM range.
Zhang et al. (2012) report production rates of 1.4 nM s1 and 1.2 nM s1 (0.005 mM h1) for 10 mg L1 Suwanee River humic and fulvic acid solutions irradiated with a 300 W Xenon lamp with a 320 nm long bandpass cut off filter, with similar rates of 1.1 and 0.8 nM s1 obtained for C-18 extracts of fresh and coastal waters. In a prior study of surf zone water samples in our laboratory (Clark et al. 2009), we measured 1500 nM H2O2 after 60 min of irradiation with an unfiltered 300 W Xenon lamp, giving a production rate of 2.5 nM s1 (or 0.009 mM h1). Ambient steady-state concentrations in sunlight-exposed marine waters are an order of magnitude lower, typically ranging from 10 to 300 nM, with net production rates range from 10 nM h1 to 60 nM h1, 2 orders of magnitude lower than the production rate obtained in laboratory irradiations with a lamp (Clark et al., 2008a, 2009). These differences between in situ and laboratory studies are likely due to differences in irradiation output between sunlight and the arc lamp light source. One way to compare photoproduction efficiencies for different compounds is to calculate the quantum yield (H), a ratio of the moles of a particular photoproduct of interest to the moles of incident photons absorbed. A quantum yield of 1 for hydrogen peroxide would indicate that one molecule of hydrogen peroxide is produced for each photon absorbed by the quinone compound. To calculate a true H, it is necessary to know the concentration and extinction coefficient of the chromophore to calculate the amount of light absorbed by the reactant molecule. Since this information is not available for material like CDOM, which does not have a well-defined structure and associated molecular mass,
Fig. 4. Contour plots of 3D excitation-emission matrix (EEM) fluorescence spectra for humic acid, fulvic acid, ubiquinone and anthraquinone pre-irradiation. Excitation and emission wavelengths in nm are shown on the y and x axes respectively.
C.D. Clark et al. / Marine Pollution Bulletin 79 (2014) 54–60
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Fig. 5. Contour plots of 3D excitation-emission matrix (EEM) fluorescence spectra for dihydroquinone and napthaquinone post-irradiation. Excitation and emission wavelengths in nm are shown on the y and x axes respectively.
researchers typically calculate an apparent quantum yield (Happ) for natural water samples (Moore et al., 1993). Happ is calculated by dividing the amount of photoproduct by the absorbance of the solution and the number of incident photons measured by actinometry. Values of Happ calculated by this method (using the absorbance at 300 nm) averaged 0.06 ± 0.05, ranging from a low of 0.0018 for NQ to a high of 0.135 for BQ, with values of 0.113 and 0.016 for HA and FA respectively (Table 1). Note that the value for DHQ, the fully reduced form of BQ, has a lower Happ value than might be expected from its high production rate due to the unusually high absorbance from the intense peak in its spectrum at 300 nm. For comparison purposes, Happ for H2O2 from coastal seawater samples in a study in our laboratory averaged 0.09 ± 0.04 (Clark et al., 2009), within the same range obtained here for the model compounds. Increasing aromaticity appeared to decrease the efficiency of production of H2O2 from the quinone compounds. For example, Happ for BQ is 0.135, 0.057 for AQ and 0.025 for UQ.
3.4. Fluorescence Decreases in fluorescence intensity may also be observed when CDOM is irradiated. Fluorescence intensities at an excitation wavelength of 350 nm and an emission wavelength of 490 nm are reported in Table 2 for the model compounds pre- and post-irradiation. As expected, since reduction increases fluorescence intensity in the P–P⁄ transition region (Cory and McKnight, 2005), DHQ, the reduced form of BQ, has an intensity 20 times higher than that for BQ pre-irradiation. After irradiation for 60 min, fluorescence intensity decreased by 30–60% for DHQ, AQ, UQ and FA, suggesting oxidation may be occurring. In contrast, increases were observed for BQ, NQ and HA, possibly due to reduction. In a prior photochemical study, the excited triplet state of BQ was shown to abstract a hydrogen atom from water to produce the reduced semi-quinone form (Ononye et al., 1986). In addition to intensity changes in fluorescence, shifts in peak emission maxima and minima may also be observed with irradiation. Fig. 4 shows examples of contour plots of 3D excitation-emission-matrix fluorescence spectra (EEMs) for FA, HA, AQ and UQ pre-irradiation showing one peak for HA and two for each of the other compounds. A prior study showed comparable EEMs for fulvic acids from lake waters (Cory and McKnight, 2005). Fig. 5 shows EEMs for DHQ and NQ post-irradiation, showing two and one major peak respectively. The excitation and emission wavelengths associated with the maximum fluorescence intensity of each peak for each compound pre- and post-irradiation are given in Table 2. In general, the emission peak maxima are red-shifted by 10–30 nm to longer wavelengths for most of the compounds after irradi-
Fig. 6. Fluorescence excitation and emission peak maxima (in nm) for all compounds (a) pre- and (b) post- irradiation for 60 min. Circles correspond to regions characteristic of marine and terrestrial humic type peaks in CDOM studies (Coble, 1996). Upper group (peaks M and C): excitation wavelengths 320–380 nm; emission maxima 440–500 nm. Lower group (peak A): excitation wavelengths 260– 290 nm; emission maxima 450–500 nm.
ation, whereas the excitation peak maxima remain the same or are blue-shifted to lower wavelengths by 10–30 nm. A prior study of humic and fulvic acids correlated red shifts in the emission wavelength (excitation wavelength 340 nm) to increasing molecular size and maturation degree (aromaticity, oxidation of functional
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groups like quinones) of the dissolved humic substances (Mignone et al., 2012). A study of dissolved organic matter and fulvic acids from diverse aquatic environments showed that a combination of reduced and oxidized quinone-type fluorophores accounted for 50% of fluorescence based on a comparison of EEMs to model quinone spectra (Cory and McKnight, 2005). A spectrum for a fulvic acid derived from forest material in a prior study was comparable to that for FA observed in this work, and was attributed by the authors to the presence of a quinone–hydroquinone group as the principal type of fluorophore in the fulvic acid (Ariese et al., 2004). Fig. 6 shows the excitation and emission peak maxima pre- and post-irradiation for 60 min. After irradiation, fluorescent peaks were observed in regions typically associated with humic-type material in CDOM natural water samples, specifically peaks A, C and M using the Coble designation (1996). In natural water studies, peak A is typically attributed to humic material, peak C to terrestrially derived humic material and peak M to marine-derived humic material (Coble, 1996). Peak M has been associated with biological activity in seawater but also observed in wetlands and agricultural environments (Stedmon and Markager, 2005; Clark et al., 2008b). Peaks A and C are observed in terrestrial, coastal and oceanic waters (Stedmon and Markager, 2005; Clark et al., 2008b). In this study, Peak A was associated with excitation wavelengths of 260–270 nm and emission wavelengths around 480–490 nm; this was observed for FA, DHQ, BQ, AQ and UQ. Peak C is associated with excitation wavelengths of 370 nm and emission wavelengths around 500 nm, and was only observed for HA. Peak M is associated with excitation wavelengths of 320–330 nm and emission wavelengths 440–460 nm, and was observed for FA, DHQ, BQ, NQ, and AQ. For most compounds, these peaks were present both pre- and post-irradiation. However, for BQ, peaks A and M only appeared after irradiation; BQ only had one peak pre-irradiation in a different region of the fluorescence spectrum at an excitation of 290 nm and emission of 332 nm. These results are consistent with the spectra obtained for BQ in a prior study at pH 10 and pH 4 respectively (Ariese et al., 2004), where higher pH favors a shift of the redox equilibrium towards the quinone form. 4. Summary Photochemical production of hydrogen peroxide at the micromolar level was measured for 5 quinone compounds at rates and concentrations comparable to humic and fulvic acids used as models for CDOM. Absorbance and fluorescence optical data for the quinone compounds post-irradiation were comparable to spectra for the humic and fulvic acids and prior results for CDOM. These results support the hypothesis that quinone moieties contribute to the chemistry and optical properties of CDOM in natural water systems. Acknowledgement The authors thank the Office of Naval Research for funding this work (ONR Grant #N000160110609). References Alegria, A.E., Ferrer, A., Sepulveda, E., 1997. Photochemistry of water-soluble quinines. Production of a water-derived spin adduct. Photochem. Photobiol. 66, 436–442. Alegria, A.E., Ferrer, A., Santiago, G., Sepulveda, E., Flores, W., 1999. Photochemistry of water-soluble quinones. Production of the hydroxyl radical, singlet oxygen and the superoxide ion. J. Photochem. Photobiol. A: Chem. 127, 57–65.
Ariese, F., van Assema, S., Gooijer, C., Bruccoleri, A.G., Langford, C.H., 2004. Comparison of Laurentian fulvic acid luminescence with that of the hydroquinone/quinone model system: evidence from low T fluorescence studies and EPR spectroscopy. Aquat. Sci. 66, 86–94. Clark, C.D., De Bruyn, W., Jakubowski, S., 2008a. Hydrogen peroxide production in marine bathing waters: implications for fecal indicator bacteria mortality. Marine Poll. Bull. 56, 397–401. Clark, C.D., Litz, L.P., Grant, S.B., 2008b. Saltmarshes as a source of chromophoric dissolved organic matter to Southern California coastal waters. Limnol. Oceanogr. 53, 1923–1933. Clark, C.D., De Bruyn, W.J., Jones, J.G., 2009. Photochemical production of hydrogen peroxide in size-fractionated Southern California coastal waters. Chemosphere 76, 141–146. Clark, C.D., De Bruyn, W.J., Hirsch, C.M., Jakubowski, S.D., 2010. Hydrogen peroxide measurements in recreational marine bathing waters in Southern California, USA. Water Res. 44, 2203–2210. Coble, P.G., 1996. Characterization of marine and terrestrial dissolved organic matter in seawater using excitation-emission matrix spectroscopy. Mar. Chem. 51, 325–346. Cooper, W.J., Zika, R.G., Petasne, R.G., Fischer, A.M., 1989. Sunlight-induced photochemistry of humic substances in natural waters: major reactive species. In: Suffet, I.H., MacCarthy, P. (Eds.), Aquatic Humic Substances: Influence on Fate and Treatment of Pollutants. American Chemical Society, Washington, DC. Cory, R.M., Mcknight, D.M., 2005. Fluorescence spectroscopy reveals ubiquitous presence of oxidized and reduced quinones in dissolved organic matter. Environ. Sci. Technol. 39, 8142–8149. Del Vecchio, R., Blough, N.V., 2004. On the origin of the optical properties of humic substances. Environ. Sci. Technol. 38, 3885–3891. Garg, S., Rose, A.L., Waite, T.D., 2007. Production of reactive oxygen species on photolysis of dilute aqueous quinone solutions. Photochem. Photobiol. 83, 904– 913. Garg, S., Rose, A.L., Waite, T.D., 2011. Photochemical production of superoxide and hydrogen peroxide from natural organic matter. Geochim. Cosmochim. Acta 75, 4310–4320. Green, S.A., Blough, N.V., 1994. Optical absoprtion and fluorescence properties of dissolved organic matter in natural waters. Limnol. Oceanogr. 39, 1903–1916. Leenheer, J.A., Croue, J.-P., 2003. Characterizing aquatic dissolved organic matter. Environ. Sci. Technol. 37, 18A–26A. http://dx.doi.org/10.1021/es032333c. Mignone, R.A., Martin, M.V., Moran Vieyra, F.E., Palazzi, V.I., Lopez de Mishima, B., Martire, D.O., Borsarelli, C.D., 2012. Modulation of optical properties of dissolved humic substances by their molecular complexity. Photochem. Photobiol. 88, 792–800. Moore, C.A., Farmer, C.T., Zika, R.G., 1993. Influence of the Orinoco River on hydrogen peroxide distribution and production in the Eastern Caribbean. J. Geophys. Res. 98, 2289–2298. Moran, M.A., Sheldon, W.M.J., Zepp, R.G., 2000. Carbon loss and optical property changes during long-term photochemical and biological degradation of estuarine dissolved organic matter. Limnol. Oceanogr. 43, 1254–1264. Ononye, A.I., Bolton, J.R., 1986. Mechanism of the photochemistry of pbenzoquinone in aqueous solutions. 2. Optical flash photolysis studies. J. Phys. Chem. 90, 6270–6274. Parker, A.W., Hester, R.E., Phillips, D., Umpathy, S., 1992. Time-resolved resonance raman spectroscopic investigations of the photochemistry of ubiquinone. J. Chem. Soc. Faraday Trans. 88, 2649–2653. Patai, S., Bruce, J.M., 1974. The Chemistry of the Quinonoid Compounds Chapter 9: Photochemistry of Quinones. Wiley and Sons, New York, pp 466–530. Roy, A., Aditya, S., 1983. Primary process in the photochemistry of aqueous anthraquinone-2-sulphonate by flash photolysis. J. Photochem. 22, 361–367. Scully, N.M., McQueen, D.J., Lean, D.R.S., Cooper, W.J., 1996. Hydrogen peroxide formation: the interaction of ultraviolet radiation and dissolved organic carbon in lake waters along a 43–75N gradient. Limnol. Oceanogr. 41, 540–548. Stabenau, E.R., Zepp, R.G., Bartels, E., Zika, R.G., 2004. Role of the seagrass Thalssia testudinum as a source of chromophoric dissolved organic matter in coastal South Florida. Mar. Ecol. Prog. Ser. 282, 59–72. Stedmon, C.A., Markager, S., 2005. Resolving the varaibility in disolved organic matter fluorescence in a temperate estuary and its catchment using PARAFAC analysis. Limnol. Oceaongr. 50, 686–697. Stevenson, F.J., 1994. Humus Chemistry: Genesis, Composition, Reactions. John Wiley and Sons, New York. Tzortziou, M., Osburn, C.L., Neale, P.J., 2007. Photobleaching of dissolved organic material from a tidal marsh-estuarine system of the Chesapeake Bay. Photochem. Photobiol. 83, 782–792. http://dx.doi.org/10.1562/2006-09-28RA-1048. Zhang, Y., Del Vecchio, R., Blough, N., 2012. Investigating the mechanism of hydrogen peroxide photoproduction by humic substances. Environ. Sci. Technol. 46, 11836–11843. Zika, R.G., Saltzman, E.S., 1982. Interaction of ozone and hydrogen peroxide in water: implications for the analysis of H2O2 in air. Geophys. Res. Lett. 9, 231– 234.
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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Photoproduction of hydrogen peroxide in aqueous solution from model compounds for chromophoric dissolved organic matter (CDOM) Catherine D. Clark, Warren de Bruyn ⇑, Joshua G. Jones School of Earth and Environmental Sciences, Schmid College of Science and Technology, Chapman University, Orange, CA 92866, United States
a r t i c l e
i n f o
Keywords: Hydrogen peroxide Humic Fulvic CDOM Quinone
a b s t r a c t To explore whether quinone moieties are important in chromophoric dissolved organic matter (CDOM) photochemistry in natural waters, hydrogen peroxide (H2O2) production and associated optical property changes were measured in aqueous solutions irradiated with a Xenon lamp for CDOM model compounds (dihydroquinone, benzoquinone, anthraquinone, napthoquinone, ubiquinone, humic acid HA, fulvic acid FA). All compounds produced H2O2 with concentrations ranging from 15 to 500 lM. Production rates were higher for HA vs. FA (1.32 vs. 0.176 mM h1); values ranged from 6.99 to 0.137 mM h1 for quinones. Apparent quantum yields (Happ; measure of photochemical production efficiency) were higher for HA vs. FA (0.113 vs. 0.016) and ranged from 0.0018 to 0.083 for quinones. Dihydroquinone, the reduced form of benzoquinone, had a higher production rate and efficiency than its oxidized form. Post-irradiation, quinone compounds had absorption spectra similar to HA and FA and 3D-excitation– emission matrix fluorescence spectra (EEMs) with fluorescent peaks in regions associated with CDOM. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Hydrogen peroxide (H2O2) is an important intermediate in aquatic redox processes that affects water quality (Cooper et al., 1989). It has been shown to reduce fecal indicator bacteria (FIB) levels in wastewater treatment (Davies-Colley et al., 1997) and implicated as a possible cause of observed diel variability in FIB levels in surf-zone waters at a recreational bathing beach (Boehm et al., 2002). Concentrations from 10 to 300 nM have been measured in surface waters in near to off-shore marine environments (see review Clark et al., 2008a and references therein; Garg et al., 2011). Photochemical production from chromophoric dissolved organic material (CDOM) is considered the primary source in natural waters. This is based on observations of diel cycles in H2O2 concentrations with afternoon maxima and early morning minima, and higher measured H2O2 concentrations and production rates in marine waters with higher CDOM levels (Cooper et al., 1988; Fujiwara et al., 1993; Moore et al., 1993; Scully et al., 1996; Herut et al., 1998; Obernosterer et al., 2001; O’Sullivan et al., 2005). The initial step in the photochemical production of H2O2 from CDOM is the absorption of sunlight by CDOM to create an excited state that reacts with oxygen to generate superoxide O 2 , which dismutates to form H2O2 (Zhang et al., 2012). There are a number of possible pathways by which O 2 could form from photoexcited ⁄ CDOM reacting with oxygen. Prior studies have shown minor ⇑ Corresponding author. Tel.: +1 714 628 7353. E-mail address: [email protected] (W. de Bruyn). 0025-326X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.marpolbul.2014.01.001
contributions from the photoionization of ⁄CDOM to give a hydrated electron which then reacts with O2 (Cooper et al., 1989) and from the direct reduction of excited-state singlet O2 by ⁄CDOM (Dalrymple et al., 2010). Two recent studies have provided evidence for a primary production pathway via (1) the production of reducing radical intermediates, from an intramolecular excited state electron transfer process in ⁄CDOM, which then react with O2 (Zhang et al., 2012) and (2) direct reduction of ground-state ⁄ triplet oxygen to O 2 by redox active chromophores in CDOM (Garg et al., 2011). The identity of the chromophores in CDOM which are responsible for the absorption of light and subsequent production of H2O2 is not clear, but a number of lines of evidence suggest that quinones could play a key role. Fluorescence spectroscopy studies have shown the ubiquitous presence of oxidized and reduced quinones in CDOM, with approximately 50% of the observed optical properties of CDOM samples attributed to quinone type species (Cory and McKnight, 2005). A quinone photochemistry study carried out in air-equilibrated aqueous solution using optical properties and radical trapping techniques has shown the production of hydroxylating intermediates, singlet oxygen and superoxide (Garg et al., 2007), the same reactive intermediates produced from photoexcited CDOM (Cooper et al., 1989; Scully et al., 1996). This study also directly measured hydrogen peroxide production from disodium anthraquinone-2-6-disulfonate (AQDS) in water (Garg et al., 2007). However, to the best of our knowledge this is the only study that has directly shown that hydrogen peroxide is produced photochemically from quinones.
C.D. Clark et al. / Marine Pollution Bulletin 79 (2014) 54–60
To expand on this work and test the feasibility of hydrogen peroxide in natural waters being produced from quinone moieties in CDOM, we measured the photoproduction of H2O2 from a series of model quinone compounds and a humic and fulvic acid in aqueous solution. We also determined optical parameters commonly used to characterize DOM in natural water samples, specifically spectral slope and shifts in the peak maxima for excitation and emission in fluorescence spectra. We chose model compounds with a range of sizes and degrees of conjugation and included an oxidation–reduction pair to assess the effects of quinone redox state on H2O2 production. We compare hydrogen peroxide production rates and optical property changes for the model compounds to recent measurements we have made in our laboratory on salt marsh and coastal seawater samples. 2. Methods and materials 2.1. Solutions Solutions of 1,4-benzoquinone, 2,5-dihydroxyquinone, 1,2naphthoquinone, anthraquinone, and ubiquinone 50 (Sigma–Aldrich) were prepared at 0.13 mM, except for naphthoquinone and ubiquinone which were prepared with a concentration 10 lower because of reduced solubility. Water used in preparing solutions was de-ionized by passing distilled water through a Nanopure Infinity Ultrapure Water System (Model D8961, Barnstead). Post irradiation all solutions were diluted with nanopure water to 130 nM for hydrogen peroxide measurements. Standard Suwannee River humic and fulvic acids were obtained from the International Humic Substances Society (IHSS). Humic and fulvic acid solutions were prepared at concentrations of 0.1 mg mL1 to give similar absorbances as the quinone model compound solutions and similarly diluted for hydrogen peroxide analysis. 2.2. Optical measurements Absorbance spectra were obtained from 200 to 700 nm with a UV/VIS spectrophotometer (Agilent Technologies, Model 8453) in 1 mm quartz cells. Absorbance spectra were measured at time intervals of 0, 5, 10, 15, 20, 30 and 60 min during irradiations. Emission spectra were obtained in a 1 1 cm quartz fluorometer cell with a scanning fluorometer (Quantamaster, PTI). Three dimensional Excitation–Emission Matrix fluorescence spectra (EEMs) were run on all solutions pre and post irradiation. EEMs were obtained by ranging excitation wavelengths from 260 to 430 nm and emission wavelengths from 270 to 650 nm in 5 nm increments. Details are given in Clark et al. (2008a,b). A water EEM was generated daily to subtract out the water Raman peak, and spectra were corrected for instrumental response. Intensities are reported in photons s1. Spectral slopes (S) were calculated by fitting data to Eq. (1) with a first-order linear regression for the 300–400 nm region for comparison to our previous work on natural water samples (Clark et al., 2008a,b, 2009, 2010).
S ¼ ½ðln Abs=A0 Þ=ðk k0 Þ
ð1Þ
Here, Abs is the absorbance at wavelength k and A0 is the absorbance at reference wavelength k0 (Green and Blough, 1994; Moran et al., 2000). 2.3. Irradiations 4 mL of solution was irradiated in 5–10 min increments up to 60 min in a 1 1 cm quartz cell with a 200 W ozone-free Xenon mercury arc lamp (Oriel Instruments, Model 6292; lamp specifications http://www.newport.com/Technical-Note-Arc-Lamp-Spec-
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tral-Irradiance-Data/409171/1033/content.aspx). A 1 1 cm quartz cell with deionized water was placed in front of the sample cell as an IR filter. A cooling fan (Radio Shack) was placed at 90° to the side of the sample cell. The temperature inside the cell was monitored with a digital thermometer as a function of irradiation time. With the IR filter and cooling fan, temperature increased by 1.5 °C within 5 min of irradiation, and remained constant throughout the remainder of the irradiation time. A lamp photon flux of 1.1 1021 photons s1 m2 was measured by nitrite/benzoic acid/ hydroxybenzoic acid chemical actinometry (Jankowski et al., 1999).
2.4. Hydrogen peroxide analyses Concentrations were measured with an enzyme-mediated fluorescence peroxidase technique (Zika and Saltzman, 1982) used for most previous surface seawater measurements. A filter field fluorometer (Turner Designs Model 10-AU-0) with a near-UV DOM/ Ammonium lamp and 25 mL sample cuvette was used, with excitation centered at 354 nm and emission at 496 nm. Aqueous solutions for the hydrogen peroxide analysis included a phosphate buffer (0.5 M; JT Baker), hydrogen peroxide (1 105 M; Aldrich), scopoletin (5 105 M; Sigma–Aldrich) and horseradish peroxidase (HRP; 1 104 M; Sigma–Aldrich). Details of the method have been previously published (Clark et al., 2009). 2–4 replicate measurements on a single sample were averaged, with an average r < 18%. Error bars are reported as ±r.
3. Results and discussion 3.1. Quinone structure and photochemistry Structures of the model quinone compounds are shown in Fig. 1. Molecular structures range in size and complexity from benzoquinone (BQ) and dihydroquinone (DHQ) (1 ring) through the larger napthaquinone (NQ) (2 ring) and anthraquinone (AQ) (3 ring). Ubiquinone (UQ) has a substituted aromatic ring bound to an aliphatic tail with 6–10 CH2 repeating units. Molecular structures for fulvic (FA) and humic acids (HA) have not been definitively characterized but complex macromolecular structures with multiple rings and substituents such as quinone moieties and carboxyl groups have being proposed (Leenheer and Croue, 2003). DHQ is the reduced form of BQ. Most quinone photochemistry studies have been carried out in organic solvents. When irradiated, quinones can undergo a range of reactions including cycloaddition reactions like dimerization, abstraction of hydrogen from substrates to form hydroquinones and electron transfer (Patai and Bruce, 1974). Irradiated quinones in aqueous solution generally produce semi-quinone intermediates and hydroquinones and hydroxyl-substituted quinones as photoproducts (Patai and Bruce, 1974; Ononye and Bolton, 1986; Alegria et al., 1997 and 1999; Gan et al., 2008). A number of photoxidation mechanisms have been proposed. These include hydrogen atom abstraction from water (Ononye and Bolton, 1986), water-quinone exciplex formation and intramolecular charge transfer (Gan et al., 2008), electron abstraction from water to form quinone anions (Roy and Aditya, 1983; Parker et al., 1992), and photooxidation of water to produce reactive oxygen species like superoxide and hydroxyl radicals (Alegria et al., 1997, 1999; Garg et al., 2007) and hydrogen peroxide (Garg et al., 2007). A recent study showed the production of hydroxylating intermediates, singlet oxygen and superoxide reactive intermediates (Garg et al., 2007). Possible production pathways for these species include quenching of the quinone excited state by ground-state triplet O2 to produce excited
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C.D. Clark et al. / Marine Pollution Bulletin 79 (2014) 54–60
NQ DHQ
AQ
BQ
UQ
HA
Fig. 1. Structures of model compounds: dihydroquinone (DHQ), benzoquinone (BQ), napthaquinone (NQ), anthraquinone (AQ), ubiquinone (UQ) and a proposed structure for humic acid (HA; Stevenson, 1994).
singlet oxygen or direct reduction of oxygen to produce the superoxide ion O 2. 3.2. Absorbance CDOM has an absorbance spectrum that decreases exponentially from the UV through the visible region (200–600 nm). Del Vecchio and Blough (2004) proposed that the absorption spectrum arises from intramolecular charge-transfer interactions between hydroxyl-aromatic donors and quinoid acceptors formed by the partial oxidation of lignin species. Quinones are characterized by intense absorption bands in the 250–300 nm range and weaker overlapping bands in the 300–400 nm range due to P–P⁄ transitions associated with the benzene and quinone moieties (Cory and McKnight, 2005). A change in redox state shifts the UV–VIS spectra, with reduction increasing the intensity of the P–P⁄ transition especially in the visible range (Cory and McKnight, 2005). Absorbance spectra pre-irradiation are given in Fig. 2a. FA and HA showed the same exponential decrease from 200 to 600 nm (Fig. 2), characteristic of CDOM (Del Vecchio and Blough, 2004), with HA having higher absorption in the 200–400 nm range. Similar featureless spectra were obtained for a wide range of humic and fulvic acids in a prior study, with the humic acids having higher absorbances than the fulvic acids at the same concentration (Mignone et al., 2012). In comparison, the quinone compounds pre-irradiation had more structural features in the 200–300 nm range with several distinct peaks presumably associated with P–P⁄ transitions, with the exception of UQ and AQ which were similar to FA. The most striking is that for BQ and DHQ, the oxidation reduction pair. One can also clearly see the shift in absorbance maximum to longer wavelengths in the reduced form. In Table 1 we report pre-irradiation absorbances at 300 nm. This is a wavelength frequently given in studies of CDOM optical properties in natural waters (see for example Clark et al., 2009 and 2010). Values range from 0.75 for BQ to 4.7 for HA; however, it should be noted that DHQ has an absorbance maximum at 300 nm, unlike all the other species studied here. For comparison purposes, absorbances of 0.03–0.04 at 300 nm were obtained in our laboratory for coastal water samples (Clark et al., 2009). Absorbance spectra post-60 min irradiation are shown in Fig. 2b. After irradiation, all 5 quinone compounds had spectra like that of FA, with no distinct peaks and decreasing absorbance with increasing wavelength, similar to those observed for CDOM in natural water samples (Del Vecchio and Blough, 2004 and references therein). BQ, DQ and NQ have lost their distinct P–P⁄ structural
Fig. 2. Absorbance from 200 to 600 nm for dihydroquinone (DHQ), benzoquinone (BQ), napthaquinone (NQ), anthraquinone (AQ), ubiquinone (UQ), fulvic acid (FA) and humic acid (HA) (a) prior to irradiation and (b) after 60 min of irradiation.
features during irradiation. We report absorbance changes preand post-irradiation in the visible region at 490 nm (Table 2) since reduction has been shown to increase absorbance in this region for
C.D. Clark et al. / Marine Pollution Bulletin 79 (2014) 54–60 Table 1 Absorbance pre-irradiation at 300 nm, initial peroxide photoproduction rates (mM h1), and apparent quantum yields (Happ) of hydrogen peroxide produced after irradiation for model compounds. Compound
a (300)
Rate (mM h1)
Happ
Dihydroquinone (DHQ) Benzoquinone (BQ) Napthaquinone (NQ) Anthraquinone (AQ) Ubiquinone (UQ) Fulvic acid (FA) Humic acid (HA)
0.211 0.0033 0.0033 0.0061 0.0364 0.0113 0.205
6.99 1.39 0.137a 0.588 0.372a 0.176 1.324
0.083 0.135 0.0018 0.057 0.025 0.016 0.113
a Concentrations were 0.13 mM for DHQ, BQ and AQ but 10 lower at 0.013 mM for NQ and UQ due to reduced solubility; HA and FA were prepared as 0.1 mg L1.
quinone compounds (Cory and McKnight, 2005). Decreases are observed for NQ, UQ, and HA, whereas AQ, DHQ and FA show increases in absorbance. BQ shows decreasing absorbance at 490 nm after irradiation, possibly due to a net decrease in the reduced form of this compound with irradiation. Another absorbance based method used to characterize CDOM in natural waters is to calculate the spectral slope (S). S increases as CDOM is photobleached in oxidative environments and decreases with aging in sub-oxic soils and sediments due to increasing aromaticity and humification (Stabenau et al., 2004; Tzortziou et al., 2007). Spectral slopes (S) for natural water samples in prior studies in our laboratory range from 0.016 to 0.021 for surf zone water (Clark et al., 2009) to 0.010–0.012 for salt marsh waters (Clark et al., 2008b). Table 2 shows S values calculated pre- and post-irradiation for all model compounds however, because of the strong P–P⁄ structural features prior to irradiation for some of these species, the initial spectral slope is not very meaningful. Post-irradiation, S range from 0.004 to 0.034 consistent with previous natural water sample results (Clark et al., 2008b and 2009). S values scale approximately with the number of rings in the structure for the 4 quinones in their oxidized state. During irradiation, S for FA stays the same while S for HA increases from 0.019 to 0.034, consistent with photobleaching.
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3.3. Photoproduction Hydrogen peroxide concentrations for the irradiated model compounds and fulvic and humic acids in aqueous solution are shown in Fig. 3 as a function of irradiation time. For all compounds, there was an initial rapid increase in H2O2 concentrations over the first 10 min of irradiation to around 15 lM for the larger quinones (UQ, NQ) and FA and 150 lM for HA, AQ and BQ. DHQ the reduced form of benzoquinone produced higher concentrations of 500 lM. In general, a slower increase was then observed from 10 to 60 min irradiation time or in some cases a steady state was reached. DHQ concentrations decreased with continued irradiation time, possibly due to loss processes like the interconversion of redox states. Final hydrogen peroxide concentrations after 60 min irradiation ranged from 15 to 500 lM. Initial photochemical production rates were calculated from the concentration of hydrogen peroxide produced in the first 5 min (Rate = [H2O2]at 5 min/0.0833 h (5 min in units of h); Table 1). Production rates varied over more than an order of magnitude, from 0.14 for NQ to 7 mM h1 for DHQ. Production rates for NQ, UQ and FA averaged 0.23 ± 0.13 mM h1, whereas rates were higher at an average of 1.10 ± 0.44 mM h1 for HA, AQ and BQ. A recent study using triplet quenchers to probe the mechanism of hydrogen peroxide production from irradiated humic and fulvic acids concluded that excited triplet states of quinones do not
Table 2 Optical properties pre- and post-irradiation at 0 and 60 min for model compounds: absorbance at 490 nm, spectral slope (300–400 nm), steady-state fluorescence intensity (Flu, 103 photons s1; ex = 350 nm, em = 450 nm) and locations of excitation/emission peak maxima from EEMs. Compound
a (490)
S
Flu
Ex/em
Ex/em
Dihydroquinone 0 min 60 min
0.0136 0.0225
0.017 0.015
80.9 48.6
260/442 260/472
330/440 330/452
Benzoquinone 0 min 60 min
0.0103 0.0048
0.019 0.004
3.66 27.1
290/332 260/484
330/462
Napthaquinone 0 min 60 min
0.0013 0.0002
0.017 0.010
2.63 21.1
Anthraquinone 0 min 60 min
0.0016 0.0038
0.019 0.017
17.7 6.37
260/456 279/484
325/443 325/453
Ubiquinone 0 min 60 min
0.0143 0.0129
0.013 0.009
3.00 2.13
270/474 260/510
390/482 355/473
Fulvic acid 0 min 60 min
0.0004 0.0061
0.021 0.021
130 77.7
260/456 260/486
325/437 330/454
Humic acid 0 min 60 min
0.0192 0.0138
0.019 0.034
145 249
330/462 320/442
376/508 367/502
Fig. 3. Concentrations of hydrogen peroxide (lM) produced from irradiated solutions as a function of irradiation time (min) for (a) anthraquinone j, dihydroxyquinone d, benzoquinone Nand humic acid and (b) napthaquinone h, ubiquinone s, and fulvic acid D. Lines shown are for ease of viewing.
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contribute significantly to hydrogen peroxide production (Zhang et al., 2012). Garg et al. (2011) fitted their kinetic data for the production of hydrogen peroxide from fulvic acid with a model based on a chromophore (Q) which they attributed to a photoexcited triplet state quinone-like redox moiety (or another type of electron acceptor) which forms semi-quinone like radicals (Q) through intramolecular charge transfer that then reduces oxygen to O 2, which subsequently forms H2O2. In our study, DHQ (the reduced form of BQ) had the highest production rate at 6.99 mM h1, suggesting that the reduced state may be better at hydrogen peroxide production. This would be consistent with a semi-quinone intermediate in the mechanism if the H atom on the hydroquinone – OH substituent is readily abstracted by water to leave –O, which reacts readily with oxygen to produce O 2. H2O2 production in aqueous solution has previously been measured for humic and fulvic acids and one quinone compound. A direct quantitative intercomparison among studies is difficult due to the use of different irradiation systems with different wavelength characteristics and photon fluxes. Garg et al. (2011) measured 60 nM H2O2 for a 1.0 mg L1 Suwanee River fulvic acid solution irradiated for 60 min by a 150 W Xenon lamp equipped with an AM1 filter to simulate the solar spectrum at the earth’s service. A study of a quinone compound (disodium 9,10-anthraquinone1,5-dilsulfonate (AQDS)) using the same irradiation setup measured higher concentrations of 1200 nM H2O2 after 60 min of irradiation of a 3 lM solution (Garg et al., 2007); rates scaled almost linearly with AQDS concentration in the low lM range.
Zhang et al. (2012) report production rates of 1.4 nM s1 and 1.2 nM s1 (0.005 mM h1) for 10 mg L1 Suwanee River humic and fulvic acid solutions irradiated with a 300 W Xenon lamp with a 320 nm long bandpass cut off filter, with similar rates of 1.1 and 0.8 nM s1 obtained for C-18 extracts of fresh and coastal waters. In a prior study of surf zone water samples in our laboratory (Clark et al. 2009), we measured 1500 nM H2O2 after 60 min of irradiation with an unfiltered 300 W Xenon lamp, giving a production rate of 2.5 nM s1 (or 0.009 mM h1). Ambient steady-state concentrations in sunlight-exposed marine waters are an order of magnitude lower, typically ranging from 10 to 300 nM, with net production rates range from 10 nM h1 to 60 nM h1, 2 orders of magnitude lower than the production rate obtained in laboratory irradiations with a lamp (Clark et al., 2008a, 2009). These differences between in situ and laboratory studies are likely due to differences in irradiation output between sunlight and the arc lamp light source. One way to compare photoproduction efficiencies for different compounds is to calculate the quantum yield (H), a ratio of the moles of a particular photoproduct of interest to the moles of incident photons absorbed. A quantum yield of 1 for hydrogen peroxide would indicate that one molecule of hydrogen peroxide is produced for each photon absorbed by the quinone compound. To calculate a true H, it is necessary to know the concentration and extinction coefficient of the chromophore to calculate the amount of light absorbed by the reactant molecule. Since this information is not available for material like CDOM, which does not have a well-defined structure and associated molecular mass,
Fig. 4. Contour plots of 3D excitation-emission matrix (EEM) fluorescence spectra for humic acid, fulvic acid, ubiquinone and anthraquinone pre-irradiation. Excitation and emission wavelengths in nm are shown on the y and x axes respectively.
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Fig. 5. Contour plots of 3D excitation-emission matrix (EEM) fluorescence spectra for dihydroquinone and napthaquinone post-irradiation. Excitation and emission wavelengths in nm are shown on the y and x axes respectively.
researchers typically calculate an apparent quantum yield (Happ) for natural water samples (Moore et al., 1993). Happ is calculated by dividing the amount of photoproduct by the absorbance of the solution and the number of incident photons measured by actinometry. Values of Happ calculated by this method (using the absorbance at 300 nm) averaged 0.06 ± 0.05, ranging from a low of 0.0018 for NQ to a high of 0.135 for BQ, with values of 0.113 and 0.016 for HA and FA respectively (Table 1). Note that the value for DHQ, the fully reduced form of BQ, has a lower Happ value than might be expected from its high production rate due to the unusually high absorbance from the intense peak in its spectrum at 300 nm. For comparison purposes, Happ for H2O2 from coastal seawater samples in a study in our laboratory averaged 0.09 ± 0.04 (Clark et al., 2009), within the same range obtained here for the model compounds. Increasing aromaticity appeared to decrease the efficiency of production of H2O2 from the quinone compounds. For example, Happ for BQ is 0.135, 0.057 for AQ and 0.025 for UQ.
3.4. Fluorescence Decreases in fluorescence intensity may also be observed when CDOM is irradiated. Fluorescence intensities at an excitation wavelength of 350 nm and an emission wavelength of 490 nm are reported in Table 2 for the model compounds pre- and post-irradiation. As expected, since reduction increases fluorescence intensity in the P–P⁄ transition region (Cory and McKnight, 2005), DHQ, the reduced form of BQ, has an intensity 20 times higher than that for BQ pre-irradiation. After irradiation for 60 min, fluorescence intensity decreased by 30–60% for DHQ, AQ, UQ and FA, suggesting oxidation may be occurring. In contrast, increases were observed for BQ, NQ and HA, possibly due to reduction. In a prior photochemical study, the excited triplet state of BQ was shown to abstract a hydrogen atom from water to produce the reduced semi-quinone form (Ononye et al., 1986). In addition to intensity changes in fluorescence, shifts in peak emission maxima and minima may also be observed with irradiation. Fig. 4 shows examples of contour plots of 3D excitation-emission-matrix fluorescence spectra (EEMs) for FA, HA, AQ and UQ pre-irradiation showing one peak for HA and two for each of the other compounds. A prior study showed comparable EEMs for fulvic acids from lake waters (Cory and McKnight, 2005). Fig. 5 shows EEMs for DHQ and NQ post-irradiation, showing two and one major peak respectively. The excitation and emission wavelengths associated with the maximum fluorescence intensity of each peak for each compound pre- and post-irradiation are given in Table 2. In general, the emission peak maxima are red-shifted by 10–30 nm to longer wavelengths for most of the compounds after irradi-
Fig. 6. Fluorescence excitation and emission peak maxima (in nm) for all compounds (a) pre- and (b) post- irradiation for 60 min. Circles correspond to regions characteristic of marine and terrestrial humic type peaks in CDOM studies (Coble, 1996). Upper group (peaks M and C): excitation wavelengths 320–380 nm; emission maxima 440–500 nm. Lower group (peak A): excitation wavelengths 260– 290 nm; emission maxima 450–500 nm.
ation, whereas the excitation peak maxima remain the same or are blue-shifted to lower wavelengths by 10–30 nm. A prior study of humic and fulvic acids correlated red shifts in the emission wavelength (excitation wavelength 340 nm) to increasing molecular size and maturation degree (aromaticity, oxidation of functional
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groups like quinones) of the dissolved humic substances (Mignone et al., 2012). A study of dissolved organic matter and fulvic acids from diverse aquatic environments showed that a combination of reduced and oxidized quinone-type fluorophores accounted for 50% of fluorescence based on a comparison of EEMs to model quinone spectra (Cory and McKnight, 2005). A spectrum for a fulvic acid derived from forest material in a prior study was comparable to that for FA observed in this work, and was attributed by the authors to the presence of a quinone–hydroquinone group as the principal type of fluorophore in the fulvic acid (Ariese et al., 2004). Fig. 6 shows the excitation and emission peak maxima pre- and post-irradiation for 60 min. After irradiation, fluorescent peaks were observed in regions typically associated with humic-type material in CDOM natural water samples, specifically peaks A, C and M using the Coble designation (1996). In natural water studies, peak A is typically attributed to humic material, peak C to terrestrially derived humic material and peak M to marine-derived humic material (Coble, 1996). Peak M has been associated with biological activity in seawater but also observed in wetlands and agricultural environments (Stedmon and Markager, 2005; Clark et al., 2008b). Peaks A and C are observed in terrestrial, coastal and oceanic waters (Stedmon and Markager, 2005; Clark et al., 2008b). In this study, Peak A was associated with excitation wavelengths of 260–270 nm and emission wavelengths around 480–490 nm; this was observed for FA, DHQ, BQ, AQ and UQ. Peak C is associated with excitation wavelengths of 370 nm and emission wavelengths around 500 nm, and was only observed for HA. Peak M is associated with excitation wavelengths of 320–330 nm and emission wavelengths 440–460 nm, and was observed for FA, DHQ, BQ, NQ, and AQ. For most compounds, these peaks were present both pre- and post-irradiation. However, for BQ, peaks A and M only appeared after irradiation; BQ only had one peak pre-irradiation in a different region of the fluorescence spectrum at an excitation of 290 nm and emission of 332 nm. These results are consistent with the spectra obtained for BQ in a prior study at pH 10 and pH 4 respectively (Ariese et al., 2004), where higher pH favors a shift of the redox equilibrium towards the quinone form. 4. Summary Photochemical production of hydrogen peroxide at the micromolar level was measured for 5 quinone compounds at rates and concentrations comparable to humic and fulvic acids used as models for CDOM. Absorbance and fluorescence optical data for the quinone compounds post-irradiation were comparable to spectra for the humic and fulvic acids and prior results for CDOM. These results support the hypothesis that quinone moieties contribute to the chemistry and optical properties of CDOM in natural water systems. Acknowledgement The authors thank the Office of Naval Research for funding this work (ONR Grant #N000160110609). References Alegria, A.E., Ferrer, A., Sepulveda, E., 1997. Photochemistry of water-soluble quinines. Production of a water-derived spin adduct. Photochem. Photobiol. 66, 436–442. Alegria, A.E., Ferrer, A., Santiago, G., Sepulveda, E., Flores, W., 1999. Photochemistry of water-soluble quinones. Production of the hydroxyl radical, singlet oxygen and the superoxide ion. J. Photochem. Photobiol. A: Chem. 127, 57–65.
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