Direct Evidence of a Light-Dependent Sink of Superoxide within Chromophoric Dissolved Organic Matter.

Superoxide (O2• -) is produced photochemically in natural waters by chromophoric dissolved organic matter (CDOM) via the reaction of molecular oxygen with photoproduced one-electron reductants (OERs) within CDOM. In the absence of other sinks (metals or organic radicals), O2• - is believed to undergo primarily dismutation to produce hydrogen peroxide (H2O2). However, past studies have implicated the presence of an additional light-dependent sink of O2• - that does not lead to H2O2 production. Here, we provide direct evidence of this sink through O2• - injection experiments. During irradiations, spikes of O2• - are consumed to a greater extent (∼85-30% loss) and are lost much faster (up to ∼0.09 s-1) than spikes introduced post-irradiation (∼50-0% loss and ∼0.03 s-1 rate constant). The magnitude of the loss during irradiation and the rate constant are wavelength-dependent. Analysis of the H2O2 concentration post-spike indicates that this light-dependent sink does not produce H2O2 at low spike concentrations. This work further demonstrates that simply assuming that the O2• - production is twice the H2O2 production is not accurate, as previously believed.

Photoproduction Rates of One-Electron Reductants by Chromophoric Dissolved Organic Matter via Fluorescence Spectroscopy: Comparison with Superoxide and Hydrogen Peroxide Rates.

One-electron reductants (OER) photoproduced by chromophoric dissolved organic matter (CDOM) have been shown to be likely precursors for the formation of superoxide and subsequently hydrogen peroxide. An improved method that employs a nitroxide radical probe (3AP) has been developed and utilized to determine the photoproduction rates of OER from a diverse set of CDOM samples. 3AP reacts with OER to produce the hydroxylamine, which is then derivatized with fluorescamine and quantified spectrofluorometrically. Although less sensitive than traditional methods for measuring RO2•-, measuring RH provides a simpler and faster method of estimating RO2•- and is amenable to continuous measurement via flow injection analysis. Production rates of OER (RH), superoxide (RO2•-), and hydrogen peroxide (RH2O2) have a similar wavelength dependence, indicating a common origin. If all the OER react with molecular oxygen to produce superoxide, then the simplest mechanism predicts that RH/RH2O2 and RO2•-/RH2O2 should be equal to 2. However, our measurements reveal RH/RH2O2 values as high as 16 (5.7-16), consistent with prior results, and RO2•-/RH2O2 values as high as 8 (5.4-8.2). These results indicate that a substantial fraction of superoxide (65-88%) is not undergoing dismutation. A reasonable oxidative sink for superoxide is reaction with photoproduced phenoxy radicals within CDOM.

Time-Resolved Fluorescence Spectra of Untreated and Sodium Borohydride-Reduced Chromophoric Dissolved Organic Matter.

Time-resolved fluorescence spectra of chromophoric dissolved organic matter (CDOM) from different sources were acquired using UV (280 and 375 nm) and visible light (440 and 640 nm) excitation to probe the structural basis of the emission properties of CDOM. Emission decays were faster at the blue and red edges, particularly at the red edge, relative to those acquired from 480 to 550 nm. Based on the lifetime distribution and multiexponential analysis of the emission decays recorded at different time resolution, current findings demonstrate that the components recovered based on a superposition model have no defined physical meaning. A substantial increase in steady-state fluorescence intensity and only small changes (<30%) of amplitude-weighted average lifetime caused by sodium borohydride reduction suggest that intramolecular fluorescence quenching occurs mainly through formation of ground state charge-transfer interactions. Short-lived species (lifetime < 100 ps) dominate the emission decays over wavelengths from 400 to 800 nm, particularly under excitation at long wavelengths (440 and 640 nm). Compared to locally excited (LE) states, the contribution of charge-transfer excited (ECT) states and other short-lived species to the steady-state emission is small because of their very rapid nonradiative relaxation. This study suggests that a careful choice of observation wavelength is needed to distinguish LE states from ECT states.

Contribution of ketone/aldehyde-containing compounds to the composition and optical properties of Suwannee River fulvic acid revealed by ultrahigh resolution mass spectrometry and deuterium labeling.

A prior method of mass labeling ketone-/aldehyde-containing species in natural dissolved organic matter (DOM) is further developed and applied. This application involved the treatment of Suwannee River fulvic acid (SRFA) with increasing concentrations of sodium borodeuteride (NaBD4), followed by detection of reduced species via negative mode electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI FTICR MS). The extent of reduction, as determined by ESI FTICR MS, resulting from increasing concentrations of NaBD4 correlated well with changes in the absorption and emission spectra of the corresponding untreated and borodeuteride-reduced samples, providing evidence that ketone/aldehyde functional groups contribute substantially to the bulk optical properties of SRFA. Furthermore, the differences in the reactivity and abundance of ketone-/aldehyde-containing species for various regions in Van Krevelen plots were revealed, thus showing how this mass labeling method can be used to provide more detailed structural information about components within complex DOM samples than that provided by the determination and analysis of molecular formulae alone. Graphical abstract.

Combined Effects of pH and Borohydride Reduction on Optical Properties of Humic Substances (HS): A Comparison of Optical Models.

The combined effects of pH and borohydride reduction on the optical properties of a series of humic substances and a lignin model were examined to probe the molecular moieties and interactions that give rise to the observed optical properties of these materials. Increasing the pH from 2 to 12 produced significantly enhanced absorption across the spectra of all samples, with distinct spectral responses observed over pH ranges attributable to the deprotonation of carboxylic acids and phenols. Borohydride reduction substantially attenuated the broadband absorption enhancements with pH, clearly indicating that the loss of absorption due to ketone/aldehyde reduction is coupled with the pH-dependent increase in absorption due to deprotonation of carboxylic acids and phenols. These results cannot be easily explained by a superposition of the spectra of independently absorbing chromophores (superposition model) but are readily interpretable within a charge transfer (CT) model. Changes of fluorescence emission with pH for both untreated and borohydride reduced samples suggest that a pH-dependent structural reorganization of the HS may also be influencing the fluorescence emission. Independent of optical model, these results demonstrate that chemical tests targeted to specific moieties can identify distinct structural differences among HS sources as well as provide insight into the molecular moieties and interactions that produce the observed optical and photochemical properties.

Contribution of Quinones and Ketones/Aldehydes to the Optical Properties of Humic Substances (HS) and Chromophoric Dissolved Organic Matter (CDOM).

The molecular basis of the optical properties of chromophoric dissolved organic matter (CDOM) and humic substances (HS) remains poorly understood and yet to be investigated adequately. This study evaluates the relative contributions of two broad classes of carbonyl-containing compounds, ketones/aldehydes versus quinones, to the absorption and emission properties of a representative suite of HS as well as a lignin sample. Selective reduction of quinones to hydroquinones by addition of small molar excesses of dithionite to these samples under anoxic conditions produced small or negligible changes in their optical properties; however, when measurable, these changes were largely reversible upon exposure to air, consistent with the reoxidation of hydroquinones to quinones. With one exception, estimates of quinone content based on dithionite consumption by the HS under anoxic conditions were in good agreement with past electrochemical measurements. In contrast, reduction of ketones/aldehydes to alcohols employing excess sodium borohydride produced pronounced and largely, but not completely, irreversible changes in the optical properties. The results demonstrate that (aromatic) ketones/aldehydes, as opposed to quinones, play a far more prominent role in the optical absorption and emission properties of these HS, consistent with these moieties acting as the primary acceptors in charge-transfer transitions within these samples. As a method, anoxic dithionite titrations may further allow additional insight into the content and impact of quinones/hydroquinones on the optical properties of HS and CDOM.

Photoproduction of One-Electron Reducing Intermediates by Chromophoric Dissolved Organic Matter (CDOM): Relation to O2- and H2O2 Photoproduction and CDOM Photooxidation.

A molecular probe, 3-amino-2,2,5,5,-tetramethy-1-pyrrolydinyloxy (3ap), was employed to determine the formation rates of one-electron reducing intermediates generated photochemically from both untreated and borohydride-reduced Suwanee River fulvic and humic acids (SRFA and SRHA, respectively). This stable nitroxyl radical reacts rapidly with reducing radicals and other one-electron reductants to produce a relatively stable product, the hydroxylamine, which can be derivatized with fluorescamine, separated by HPLC and quantified fluorimetrically. We provide evidence that O2 and 3ap compete for the same pool(s) of photoproduced reducing intermediates, and that under appropriate experimental conditions, the initial rate of hydroxylamine formation (RH) can provide an estimate of the initial rate of superoxide (O2-) formation. However, comparison of the initial rates of H2O2 formation (RH2O2) to that of RH show far larger ratios of RH/RH2O2 (∼6-13) than be accounted for by simple O2- dismutation (RH/RH2O2 = 2), implying a significant oxidative sink of O2- (∼67-85%). Because of their high reactivity with O2- and their likely importance in the photochemistry of CDOM, we suggest that coproduced phenoxy radicals could represent a viable oxidative sink. Because O2-/phenoxy radical reactions can lead to more highly oxidized products, O2- could be playing a far more significant role in the photooxidation of CDOM than has been previously recognized.

Are Extracted Materials Truly Representative of Original Samples? Impact of C18 Extraction on CDOM Optical and Chemical Properties.

Some properties of dissolved organic matter (DOM) and chromophoric dissolved organic matter (CDOM) can be easily measured directly on whole waters, while others require sample concentration and removal of natural salts. To increase CDOM content and eliminate salts, solid phase extraction (SPE) is often employed. Biases following extraction and elution are inevitable, thus raising the question of how truly representative the extracted material is of the original. In this context, we investigated the wavelength dependence of extraction efficiency for C18 cartridges with respect to CDOM optical properties using samples obtained from the Middle Atlantic Bight (MAB) and the Equatorial Atlantic Ocean (EAO). Further, we compared the optical changes of C18 extracts and the corresponding whole water following chemical reduction with sodium borohydride (NaBH4). C18 cartridges preferentially extracted long-wavelength absorbing/emitting material for samples impacted by riverine input. Extraction efficiency overall decreased with offshore distance away from riverine input. Spectral slopes of C18-OM samples were also almost always lower than those of their corresponding CDOM samples supporting the preferential extraction of higher molecular weight absorbing material. The wavelength dependence of the optical properties (absorption, fluorescence emission, and quantum yield) of the original water samples and their corresponding extracted material were very similar. C18 extracts and corresponding water samples further exhibited comparable optical changes following NaBH4 reduction, thus suggesting a similarity in nature (structure) of the optically active extracted material, independent of geographical locale. Altogether, these data suggested a strong similarity between C18 extracts and corresponding whole waters, thus indicating that extracts are representative of the CDOM content of original waters.

Enhanced photoproduction of hydrogen peroxide by humic substances in the presence of phenol electron donors.

Addition of a series of phenol electron donors to solutions of humic substances (HS) enhanced substantially the initial rates of hydrogen peroxide (H2O2) photoproduction (RH2O2), with enhancement factors (EF) ranging from a low of ∼3 for 2,4,6-trimethylphenol (TMP) to a high of ∼15 for 3,4-dimethoxyphenol (DMOP). The substantial inhibition of the enhanced RH2O2 following borohydride reduction of the HS, as well as the dependence of RH2O2 on phenol and dioxygen concentrations are consistent with a mechanism in which the phenols react with the triplet excited states of (aromatic) ketones within the HS to form initially a phenoxy and ketyl radical. The ketyl radical then reacts rapidly with dioxygen to regenerate the ketone and form superoxide (O2-), which subsequently dismutates to H2O2. However, as was previously noted for the photosensitized loss of TMP, the incomplete inhibition of the enhanced RH2O2 following borohydride reduction suggests that there may remain another pool of oxidizing triplets. The results demonstrate that H2O2 can be generated through an additional pathway in the presence of sufficiently high concentrations of appropriate electron donors through reaction with the excited triplet states of aromatic ketones and possibly of other species such as quinones. However, in some cases, the much lower ratio of H2O2 produced to phenol consumed suggests that secondary reactions could alter this ratio significantly.

The importance of charge-transfer interactions in determining chromophoric dissolved organic matter (CDOM) optical and photochemical properties.

Absorption of sunlight by chromophoric dissolved natural organic matter (CDOM) is environmentally significant because it controls photic zone depth and causes photochemistry that affects elemental cycling and contaminant fate. Both the optics (absorbance and fluorescence) and photochemistry of CDOM display unusual properties that cannot easily be ascribed to a superposition of individual chromophores. These include (i) broad, unstructured absorbance that decreases monotonically well into the visible and near IR, (ii) fluorescence emission spectra that all fall into a single envelope regardless of the excitation wavelength, and (iii) photobleaching and photochemical quantum yields that decrease monotonically with increasing wavelength. In contrast to a simple superposition model, these phenomena and others can be reasonably well explained by a physical model in which charge-transfer interactions between electron donating and accepting chromophores within the CDOM control the optical and photophysical properties. This review summarizes current understanding of the processes underlying CDOM photophysics and photochemistry as well as their physical basis.

Selective mass labeling for linking the optical properties of chromophoric dissolved organic matter to structure and composition via ultrahigh resolution electrospray ionization mass spectrometry.

The mass spectra acquired by ESI FT-ICR MS of untreated, borohydride-reduced, and borodeuteride-reduced samples of Suwannee River fulvic acid (SRFA) and a C18 extract from the upper Delaware Bay were compared to one another. Treatment of these samples with sodium borodeuteride was shown to produce unique mass labels for species which contain one or two ketone/aldehyde moieties. Approximately 30% of all identified peaks in the two samples were shown to comprise ketone/aldehyde-containing species. The molecular formulas of the majority of these species had O/C and H/C molar ratios typically attributed to lignin-derived compounds and/or carboxylic rich alicyclic molecules (CRAM). However, the significant loss of UV-vis absorption following reduction supports a lignin-based origin for the optical (and photochemical) properties of these samples. The mass-labeling method described and tested herein shows great promise as a means to further characterize the structure and composition of complex natural samples, especially in terms of identifying specific subsets of chemical species that contribute significantly to the optical and photochemical properties of such samples.

Investigating the mechanism of hydrogen peroxide photoproduction by humic substances.

The mechanism(s) by which hydrogen peroxide (H(2)O(2)) is photoproduced by humic substances and chromophoric dissolved organic matter was probed by examining the dependence of the initial H(2)O(2) photoproduction rate (R(H(2)O(2))) and apparent H(2)O(2) quantum yields on dioxygen concentration for both untreated and borohydride-reduced samples. Although borohydride reduction substantially reduced light absorption, the R(H(2)O(2)) values were largely unaffected. Apparent monochromatic and polychromatic quantum yields thus increased following reduction. The results indicate that light absorption by charge-transfer states or by (aromatic) ketone/aldehydes does not lead to significant H(2)O(2) photoproduction. High concentrations of triplet quenchers relative to that of dioxygen produced only small decreases (sorbic acid) or small increases (Cl(-) and Br(-)) in R(H(2)O(2)), indicating that neither (1)O(2) nor excited triplet states of quinones contribute significantly to H(2)O(2) photoproduction. The dependence of R(H(2)O(2)) on O(2) concentration provides evidence that the intermediate(s) reacting with O(2) to produce superoxide are relatively long-lived (approximately tens of microseconds or more). Evidence of the photochemical formation of O(2)-reducing intermediates under anaerobic conditions was also obtained; these reducing intermediates appeared to be relatively stable in the absence of O(2). Our data suggest that these O(2)-reducing intermediates are generated by intramolecular electron transfer from short-lived excited states of electron donors to ground-state acceptors.

Investigating the mechanism of phenol photooxidation by humic substances.

To probe the mechanism of the photosensitized loss of phenols by humic substances (HS), the dependence of the initial rate of 2,4,6-trimethylphenol (TMP) loss (R(TMP)) on dioxygen concentration was examined both for a variety of untreated as well as borohydride-reduced HS and C(18) extracts from the Delaware Bay and Mid-Atlantic Bight. R(TMP) was inversely proportional to dioxygen concentration at [O(2)] > 50 µM, a dependence consistent with reaction with triplet excited states, but not with (1)O(2) or RO(2). Modeling the dependence of R(TMP) on [O(2)] provided rate constants for TMP reaction, O(2) quenching, and lifetimes compatible with a triplet intermediate. Borohydride reduction significantly reduced TMP loss, supporting the role of aromatic ketone triplets in this process. However, for most samples, the incomplete loss of sensitization following borohydride reduction, as well as the inverse dependence of R(TMP) on [O(2)] for these samples, suggests that there remains another class of oxidizing triplet sensitizer, perhaps quinones.

Optical properties of humic substances and CDOM: effects of borohydride reduction.

Treatment of Suwanee River humic (SRHA) and fulvic (SRFA) acids, a commercial lignin (LAC), and a series of solid phase extracts (C18) from the Middle Atlantic Bight (MAB extracts) with sodium borohydride (NaBH(4)), a selective reductant of carbonyl-containing compounds including quinones and aromatic ketones, produces a preferential loss of visible absorption (> or = 50% for SRFA) and substantially enhanced, blue-shifted fluorescence emission (2- to 3-fold increase). Comparison of the results with those obtained from a series of model quinones and hydroquinones demonstrates that these spectral changes cannot be assigned directly to the absorption and emission of visible light by quinones/hydroquinones. Instead, these results are consistent with a charge transfer model in which the visible absorption is due primarily to charge transfer transitions arising among hydroxy- (methoxy-) aromatic donors and carbonyl-containing acceptors. Unlike most of the model hydroquinones, the changes in optical properties of the natural samples following NaBH(4) reduction were largely irreversible in the presence of air and following addition of a Cu(2+) catalyst, providing tentative evidence that aromatic ketones (or other similar carbonyl-containing structures) may play a more important role than quinones in the optical properties of these materials.

Prefluorescent nitroxide probe for the highly sensitive determination of peroxyl and other radical oxidants.

Fluorescamine derivatized 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (I) is shown to undergo an irreversible reaction with peroxyl radicals and other radical oxidants to generate a more highly fluorescent diamagnetic product (II) and thus can be used as a highly sensitive and versatile probe to determine oxidant production optically, either by monitoring the changes in fluorescence intensity, by HPLC analysis with fluorescence detection, or by a combination of both approaches. By changing the [O2]/[I] ratio, we show that peroxyl radicals can be detected and quantified preferentially in the presence of other radical oxidants. Detection of photochemically produced peroxyl radicals is achieved by employing 3-amino-2,2,5,5,-tetramethyl-1-pyrrolidinyloxy (3-ap) alone, followed by derivatization with fluorescamine. With employment of HPLC analysis, the detection limit of II at a S/N of 2 is approximately 3 nM for a 125 microL injection. Preliminary applications include the detection of peroxyl radicals generated thermally in soybean phosphatidylcholine liposomes and produced photochemically in tap water.

Optical properties of humic substances and CDOM: relation to structure.

The spectral dependencies of absorption and fluorescence emission (emission maxima (lamdamax), quantum yields (phi), and mean lifetimes (taum)) were acquired for a commercial lignin, Suwannee River humic (SRHA) and fulvic (SRFA) acids, and a series solid phase extracts (C18) from the Middle Atlantic Bight (MAB extracts). These parameters were compared with the relative average size and total lignin phenol content (TLP). TLP was strongly correlated with absorption at 280 and 355 nm for the MAB extracts, SRHA, and SRFA. The spectral dependence of lamdamax, phi), and taum was very similar for all samples, suggesting a common photophysical and thus structural basis. A strong decrease of phi and taum with increasing average size indicates that intramolecular interactions must be important. When combined with previous work, the results lead us to conclude that the optical properties commonly associated with terrestrial humic substances and chromophoric dissolved organic matter arise primarily from an ensemble of partially oxidized lignins derived from vascular plant sources. Theyfurther provide additional support for an electronic interaction model in which intramolecular energy transfer, excited-state electron transfer, as well as charge transfer likely play important roles in producing the observed optical and photochemical properties of these materials.

Fluorescence anisotropy as a means to determine extracellular polysaccharide hydrolase activity in environmental samples.

Current approaches to measure the activities of microbial extracellular enzymes in aquatic environments are hampered by slow throughput or by differences between the structure of simple substrate proxies and macromolecules. Here we show that measurements of fluorescence anisotropy can be used to determine the hydrolysis rate of two fluorescently labeled polysaccharides, laminarin and xylan, in environmental samples. A simple analysis shows that the anisotropy of these fluorescently labeled polysaccharides can be approximated using a modification of the Perrin equation.

Hydrogen peroxide and dioxygen activation by dinuclear copper complexes in aqueous solution: hydroxyl radical production initiated by internal electron transfer.

Dinuclear Cu(II) complexes, CuII2Nn (n = 4 or 5), were recently found to specifically cleave DNA in the presence of a reducing thiol and O2 or in the presence of H2O2 alone. However, CuII2N3 and a closely related mononuclear Cu(II) complex exhibited no selective reaction under either condition. Spectroscopic studies indicate an intermediate is generated from CuII2Nn (n = 4 or 5) and mononuclear Cu(II) solutions in the presence of H2O2 or from CuI2Nn (n = 4 or 5) in the presence of O2. This intermediate decays to generate OH radicals and ligand degradation products at room temperature. The lack of reactivity of the intermediate with a series of added electron donors suggests the intermediate discharges through a rate-limiting intramolecular electron transfer from the ligand to the metal peroxo center to produce an OH radical and a ligand-based radical. These results imply that DNA cleavage does not result from direct reaction with a metal-peroxo intermediate but instead arises from reaction with either OH radicals or ligand-based radicals.