Quantification of Phenolic Antioxidant Moieties in Dissolved Organic

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Article pubs.acs.org/est

Quantification of Phenolic Antioxidant Moieties in Dissolved Organic Matter by Flow-Injection Analysis with Electrochemical Detection Nicolas Walpen, Martin H. Schroth, and Michael Sander* Institute of Biogeochemistry and Pollutant Dynamics (IBP), Department of Environmental Systems Science, ETH Zurich, Zurich, Switzerland 8092 S Supporting Information *

ABSTRACT: Phenolic moieties in dissolved organic matter (DOM) play important roles as antioxidants in oxidation processes in natural and engineered systems. This work presents an automated and highly sensitive flow injection analysis (FIA) system coupled to both spectrophotometric and electrochemical detection to quantify electron-donating phenolic moieties in DOM by determining the number of electrons that these moieties transfer to an added chemical oxidant, the radical cation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS •+ ). The FIA system was successfully validated using Trolox as a redox standard. Highest method sensitivity was attained when combining the FIA with chronoamperometric detection, resulting in limits of quantification of picomolar amounts of Trolox and nanogram amounts of DOM (corresponding to solutions with 18.2 MΩ cm) from a Barnstead NANOpure Diamond system. All solutions used in electrochemical measurements contained 0.1 M KCl. The FIA reagent and carrier solutions (see details below) were pH-buffered with 0.001 M acetate (pH 5) and 0.1 M phosphate (pH 7), respectively. Characterization of Organic Matter Samples. UV− visible light-absorbance spectra of all samples were collected from 200 to 800 nm on a Varian Cary 100 Bio. The nonpurgable organic carbon (NPOC) was measured on a Shimadzu total organic carbon (TOC-L) analyzer calibrated with a TOC standard (Sigma-Aldrich, Switzerland). Titrated phenol contents for the IHSS HS/NOM samples were obtained from the literature.29,30,44 MEO. Mediated electrochemical oxidation of SRHA was conducted in a glassy carbon cylinder (volume, 9 mL; Sigradur G, HTW, Germany) that served as the WE.45 The cylinder was filled with pH 7 buffer (0.1 M phosphate and 0.1 M KCl) and was polarized to Eh = +0.7 V. A platinum wire was used as a counter electrode and separated from the WE compartment by a porous glass frit. An Ag/AgCl reference electrode was used (ALS, Japan). All potentials are referenced versus the standard hydrogen electrode (SHE). The EDC values were quantified by integrating the oxidative current peaks according to

oxidant, because it has a high water solubility and a standard reduction potential (i.e., Eh0 (ABTS•+/ABTS) = 0.70 V) sufficiently high to oxidize phenols. In these assays, ABTS•+ is obtained through either chemical, enzymatic, or electrochemical one-electron oxidation of ABTS.37−39 The reduction of ABTS•+ to ABTS by an analyte can be quantified either electrochemically37,38 or spectrophotometrically39 as ABTS•+ has a strong absorbance in the red (λmax = 728 nm), whereas ABTS is colorless.40 We recently used the ABTS•+/ABTS redox couple in mediated electrochemical oxidation (MEO), a constantpotential amperometric technique, to quantify phenolic moieties in DOM.8 In these measurements, the addition of DOM samples to electrochemical cells resulted in the reduction of preoxidized ABTS•+ to ABTS, which subsequently was reoxidized at the working electrode (WE) of the cell to ABTS•+. Integration of the resulting oxidative current peaks directly provided the electron-donating capacity (EDC) of the DOM sample (i.e., the amount of electrons transferred per amount of DOM analyzed). The EDC of several humic substances correlated strongly with their titrated phenol contents, supporting the hypothesis that mainly phenolic groups donated electrons to ABTS•+. While the EDC values of as little as a few micrograms of DOM could be quantified by MEO,6 the sensitivity of the method was insufficient to analyze dilute DOM samples (i.e., below approximately 10 mgC L−1). Furthermore, MEO relies on manual sample addition to the electrochemical cells with little opportunity for automation. Finally, the MEO setup has limited portability and hence cannot easily be used for measurements outside the laboratory. These limitations of MEO may, in principle, be overcome by implementing the reduction-based approach on a flow-injection analysis (FIA) platform. Such platforms have been successfully used to quantify antioxidants in other disciplines, including food chemistry.37−39,41−43 Compact, robust, and field-deployable FIA systems can be designed that allow for automated sample analysis. The objective of this work was to develop and validate a FIA system for the accurate and precise quantification of antioxidant phenolic moieties in dilute DOM samples. The system used ABTS•+ as chemical oxidant and spectrophotometric and electrochemical flow cells to detect the extents of ABTS•+ reduction. The system response was validated using Trolox, a redox standard with a known EDC, and using a series of model DOM isolates with published phenol contents and EDC values that were previously quantified by MEO. Finally, the FIA system was used to quantify the EDC values of dilute DOM samples collected from three ombrotrophic bogs and, for one of the peat samples, to monitor changes in the phenol contents of the peat DOM during incubation with a phenol oxidase.

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MATERIALS AND METHODS

Chemicals. ABTS (≥98%), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox, 97%), potassium chloride (≥99%), acetic acid (≥99.8%), and potassium phosphate dibasic (≥99%) were obtained from Sigma-Aldrich (Switzerland). Potassium dihydrogen phosphate was obtained from Merck (Switzerland). Laccase from Trametes versicolor was from Fluka (activity, 22.4 U mg−1) and used as received. The laccase stock solution (0.745 mg mL−1) was prepared in pH 4.75 acetate buffer (1 mM).

EDC =

1 mSRHA

∫ FI dt

(1)

where mSRHA is the mass of analyzed SRHA, I (A) is the baseline-corrected current, and F (= 96 485 C mol−1) is the Faraday constant. 6424

DOI: 10.1021/acs.est.6b01120 Environ. Sci. Technol. 2016, 50, 6423−6432

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Environmental Science & Technology

Figure 1. Scheme of the flow-injection analysis (FIA) system for quantification of the electron-donating capacity (EDC) of dissolved organic matter (DOM) samples. The system contains two separate lines for the reagent (i.e., the oxidant ABTS•+) solution (green syringe) and the carrier solution (blue syringe). The DOM samples are injected into the carrier-solution stream using an injector valve with a 100 μL sample loop. A third syringe (red syringe) is used to load the sample loop. Up to 10 samples can be automatically injected using a 11-port/10-position selector valve. Following mixing of the reagent and carrier streams in a mixing tee, the solution is passed through a 10-m long knitted open-tubular reaction coil. Reduction of the oxidant ABTS•+ to ABTS by electron-donating moieties in DOM (bottom left panel) are detected either via current responses in an electrochemical flow cell (bottom, middle panel) operated in chronoamperometry mode or via absorbance loss at a wavelength of λ = 728 nm in an optical flow cell (bottom, right panel). The absorbance loss reflects the reductive decolorization of ABTS•+ (absorbance in the red) to ABTS (colorless).

Enzymatic Oxidation Experiments. One DOM sample from LM bog was thawed and split into six subsamples of 8 mL each. The pH was adjusted to 4.75 by addition of concentrated acetate buffer (0.1 M acetate; final acetate concentration, 1 mM). This pH falls within the pH range of highest laccase activity46 and was close to the pH of the original sample (pH 4.0). Two subsamples were kept as negative controls (no addition of laccase). Laccase was added to four of the aliquots (final activity of 0.5 U mL−1; initial ratio of enzyme activity to NPOC (excluding the acetate buffer) of 13.4 U mgC−1 (with NPOC = 37.28 mgC L−1)). Two of these aliquots also received ABTS (final concentration, 7.5 μM). All samples were incubated on a horizontal shaker (300 rpm, 25 ± 1 °C) in amber vials for 3 days. Aliquots of 150 μL were repeatedly withdrawn from the subsamples and mixed with phosphate buffer (final concentration 0.1 M phosphate, pH 7, 0.1 M KCl) to inactivate the laccase.46 The quenched aliquots were stored at 4 °C until analysis on the FIA system (Eh = 0.71 V; pH 7).

reduction potential of Eh = +0.82 V. The counter and reference electrodes were the same as used in MEO of SRHA (see above). The bulk electrolysis was terminated when 60% of the added ABTS was oxidized (i.e., CABTS•+/(CABTS + CABTS•+) = 0.6). The total volumetric flow rate (qV = qreagent + qcarrier) was set to 90 μL min−1 unless indicated differently. A volumetric mixing ratio of qcarrier/qreagent = 5 was used in all analyses to minimize sample broadening that results from mixing the carrier and the reagent solutions. All samples were adjusted to have the same pH and electrical conductivity as the carrier solution prior to injection. Sample Selection and Injection. The carrier solution was delivered through an injector valve connected to a 10-position selector valve (both Cetoni, Germany) used for automated sample injection. The injection loop (volume, 100 μL) was filled with sample solutions using a third syringe pump (Cetoni, Germany). The samples were introduced to the carrier stream by redirecting the carrier stream through the injection loop. Mixing and Reaction. The carrier and reagent solutions were combined in a mixing tee (Vici, Switzerland). The mixed solution had a pH of 7 due to the higher buffering capacity of the carrier than the reagent solution. Following the mixing tee, the solution passed through a PTFE knitted open tubular reaction coil (length, 10 m; Biotech, Sweden) that served to increase the reaction time in the system prior to detection. Detection. Two complementary detection methods were used to quantify the extents of ABTS•+ reduction: absorbance measurements in a spectroscopic flow cell and chronoamperometry in an electrochemical flow cell. Spectrophotometric detection was conducted in a Z-flow cell with a 1 cm path length (FIAlab Instruments, Seattle, WA), an HL-2000 light source, and an STS-Vis detector (both Ocean Optics, Dunedin, FL). We detected the reductive decolorization of ABTS•+ to the colorless ABTS. The absorbance was monitored at 728 nm,

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FLOW-INJECTION ANALYSIS SYSTEM General Setup. The FIA system setup is shown schematically in Figure 1 and contains four units in sequential alignment: solution delivery, sample selection and injection, mixing and reaction, and detection. Solution Delivery. Reagent and carrier solutions were continuously delivered through the system by computercontrolled syringe pumps (Cetoni, Germany). The carrier solution was buffered to pH 7 (0.1 M phosphate and 0.1 M KCl). The reagent solution contained the oxidant ABTS•+ (0.75 mM) and was buffered to pH 4 (1 mM acetate; 0.1 M KCl), because the stability of the ABTS•+ in solution was found to decrease with increasing pH (Supporting Information, Figure S2). The reagent solution was obtained by bulk electrolysis of the respective ABTS solution in a 25 mL glassy carbon WE cylinder (Sigradur G, HTW, Germany) at a 6425


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Figure 2. (a) Change in the solution absorbance at 728 nm in the spectrophotometric detector resulting from triplicate injections of Trolox standards with amounts ranging from 6 to 1 nmol. Inset: Linear increase in the amounts of ABTS•+ reduced, Δn ABTS•+, with increasing amounts of injected Trolox, nTrolox. (b) Linear increase in the heights, h, of the negative absorbance peaks (shown in panel a) with increasing amounts of injected Trolox, nTrolox, between 6 and 1 nmol. (c) Oxidative current responses in chronoamperometric detection resulting from triplicate injections of Trolox standards with amounts ranging from 4.6 to 0.02 nmol. (d) Linear increase in the heights h of the oxidative current peaks (shown in panel c) with increasing amounts of injected Trolox, nTrolox, between 4.6 and 0.02 nmol. LOQ and σ correspond to the limits of quantification and the measurement noises, respectively.

the absorbance maximum of ABTS•+ (Supporting Information, Figure S3a). The EDC value of Trolox, a redox standard, was obtained by integrating the negative absorbance peak using OriginPro (version 9.1, OriginLab, Northhampton, MA) according to EDCTrolox =

1

qV

n Trolox l

∫

A(728 nm) dt ε(728 nm)

analyzer (CH Instruments, Austin, TX). All reduction potentials (Eh) applied to the WE are reported against the standard hydrogen electrode. The chronoamperometric responses were analyzed using OriginPro. The EDCj value (mmole‑ gC−1) of an organic matter sample j was obtained from the heights of its oxidative current peak(s), following calibration of the detector with Trolox standards:

(2)

EDCj =

where nTrolox (molTrolox) is the injected amount of Trolox, qV (mL min−1) is the total volumetric flow rate, ε(728 nm) (= 14 000 M−1 cm−1) is the molar absorption coefficient of ABTS•+ (Supporting Information, Figure S3b), l (cm) is the path length in the Z-flow cell, and A(728 nm) is the measured absorbance. The electrochemical cross-flow cell consisted of a glassy carbon WE, a steel tube counter electrode, and a Ag/AgCl reference electrode (all ALS, Japan) and was run in chronoamperometry mode using a 630D electrochemical

hj

1

mj a Trolox

EDCTrolox

(3)

where hj (nA) is the height of the oxidative current peak for sample j, mj (gC,j) is the injected amount of carbon in j determined by NPOC measurements (see above), aTrolox (nA mmolTrolox−1) is the slope of the linear calibration curve of oxidative current peak heights of Trolox standards versus the respective injected Trolox amounts, and EDCTrolox (= 2 mmole‑ mmolTrolox−1) is the electron donating capacity of Trolox. 6426


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current only changes if the ABTS•+/ABTS ratio changes). The noise in the baseline current was σ = 0.39 nA (Supporting Information, Figure S4d). Injections of standard solutions with decreasing amounts of Trolox from 4.6 to 0.02 nmol resulted in decreasing heights of the oxidative current responses (Figure 2c). The highest Trolox amount of 4.6 nmol was chosen such that the ABTS•+/ABTS ratio after reaction with Trolox did not decrease below 0.33 molABTS•+ molABTS−1 and, therefore, that the solution Eh decreased by at most ΔEh = 0.04 V from its initial value (i.e., between 0.71 and 0.72 V; see the Supporting Information, Figure S5 for details). The heights of the oxidative current peaks linearly increased with increasing injected amounts of Trolox, nTrolox (Figure 2d). The linear response range covered more than 2 orders of magnitude in nTrolox. We note that the measured current responses resulted from the re-equilibration of the ABTS•+-ABTS redox couple only in the solution volume that was in direct contact with the WE and not the entire solution volume in the detector. We independently determined that for the electrochemical flow cell used in this work, approximately 15% of the ABTS formed during reaction of Trolox with ABTS•+ was reoxidized to ABTS•+ while passing through the cell (Supporting Information, Figure S6). For this reason, a series of Trolox standards was analyzed in all subsequent DOM analyses to calibrate the electrochemical detection (eq 3). Determination of the Limits of Quantification. The limits of quantification (LOQ) of the FIA method for both the spectrophotometric and electrochemical detections were determined based on the heights of negative absorbance and oxidative current peaks of Trolox standards, using a statistical approach:49−51

RESULTS AND DISCUSSION Spectrophotometric Detection and Validation of the FIA System. The baseline absorbance A in the spectrophotometric cell at 728 nm was relatively constant over several hours at values of A = 1.07 (Figure 2a). Yet, the baseline absorbance showed systematic oscillations that resulted in a relatively large measurement noise of σ = 0.011 (see baseline in Figure 2a labeled “0 nmol” and Figure S4a in the Supporting Information). These oscillations possibly reflected variations in the volumetric mixing ratios of the reagent and carrier solutions in the mixing tee, which resulted in periodic changes in the absolute ABTS•+ concentration entering the spectrophotometric cell. No attempts were made to eliminate these oscillations because they were absent in the more sensitive electrochemical detection and, therefore, did not interfere with the electrochemical quantification of EDC values (see below). We validated the FIA system based on absorbance loss resulting from injections of six different Trolox standard solutions. Trolox was chosen because it is rapidly and completely oxidized to its quinone structure by ABTS•+ in a two-electron transfer reaction.8,47,48 The spectrophotometric detection was used for validation, because, in contrast to the electrochemical detection, it allows quantifying the total amount of ABTS•+ molecules that were converted to ABTS (eq 2). Triplicate injections of decreasing amounts of Trolox from 6 to 1 nmol resulted in decreasing heights of the negative absorbance peaks (Figure 2a). Integration of the peaks yielded an EDC value of 2.06 ± 0.03 mole‑ molTrolox−1 (inset in Figure 2a), which was in very good agreement with the expected EDC value of 2 mole‑ molTrolox−1.8 These values correspond to a Trolox recovery of 103 ± 2%, thereby validating the FIA system. Chronoamperometric Detection. Electron transfer from electron-donating molecules in the injected samples to ABTS•+ lowered the ratio of ABTS•+/ABTS and, as a consequence, the Eh of the solution volume that contained the injected sample. The decrease of the solution Eh relative to the constant Eh applied to the WE in the electrochemical detector resulted in current peak responses relative to the baseline current measured in the absence of injected samples. To maximize sensitivity, the WE was polarized to the open-circuit potential (OCP) that was measured for the ABTS•+/ABTS ratio in the mixed reagent and carrier solutions delivered to the electrochemical cell at the onset of each FIA analysis. We note that the OCP values were between +0.71 V and +0.72 V for all experiments, demonstrating high reproducibility in the electrochemical oxidation of ABTS used to generate the ABTS•+ reagent. As expected, the baseline currents increased with increasing difference between the Eh applied to the WE and the OCP (Supporting Information, Figure S4b,c). Polarizing the WE to the OCP values resulted in very low baseline currents of Ibaseline < 10 nA in the subsequent chronoamperometric detection. While the current baselines typically showed small drifts (1 bar) in the system. Volumetric flow rates below 90 μL min−1 were omitted because they resulted in broader current peaks and increased the intervals between injections, thereby minimizing sample throughput. For each 6427


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Figure 3. (a) Oxidative current responses to injections of different amounts of Suwannee River Humic Acid (SRHA) into the flow injection analysis (FIA) system at total volumetric flow rates of 270 μL min−1 (left; reaction time tReaction= 8.5 min)) and 90 μL min−1 (right; tReaction= 25 min). (b) Electron donating capacity (EDC) values for SRHA obtained by FIA analysis at different flow rates (from 90 to 270 μL min−1) and by mediated electrochemical oxidation (MEO). The EDC values are given in absolute values (left ordinate) and normalized to the EDC values obtained by MEO for integration over 60 min. Inset: Overlaid baseline-corrected oxidative current responses in MEO to triplicate additions of 10 μg of SRHA. (c) Oxidative current responses in the FIA system to injections of Trolox calibration standards (first five peaks) and SRHA (triplicate injections each) at amounts between 600 and 5 ng of SRHA. (d) Linear correlation between the heights of the oxidative current peaks (determined from data in panel c) and the amount of injected SRHA, mSRHA. LOQ and σ correspond to the limits of quantification and the measurement noise, respectively.

tested flow rate, four Trolox calibration standards with amounts between 2 and 0.1 nmol were injected prior to SRHA. The heights of the oxidative current peaks obtained from injections of SRHA solutions decreased linearly with decreasing injected SRHA masses (from 400 to 300 and 200 ng) at all tested flow rates (shown for the highest and lowest tested flow rates of 270 and 90 μL min−1 in Figure 3a). The broadening of the current peaks from 270 to 90 μL min−1 was due to increased time for longitudinal dispersion of ABTS and ABTS•+ during advective transport through the FIA system. While sharper current peaks are preferred because of their higher signal-to-noise ratios, the increase in EDC values of SRHA with decreasing flow rates (Figure 3b) demonstrated that electron transfer from electron donating phenolic moieties in SRHA to ABTS•+ was incomplete at the higher tested flow rates. Conversely, the EDC value of SRHA obtained at the lowest flow rate was in good quantitative agreement with the EDC value of SRHA obtained by MEO (i.e., EDCFIA ≈ 0.96 EDCMEO), which has until now been the state-of-the art approach to determine EDC values of DOM. For comparison,

Figure 3b also shows the baseline-corrected oxidative current responses in MEO to triplicate injections of 10 μg of SRHA (inset) and the corresponding cumulative numbers of electrons transferred obtained by integrating of the current peaks up to 60 min after SRHA injections. The good agreement of EDC values obtained by FIA and MEO confirmed accurate quantification of the EDC values of DOM by the FIA system. Oxidation at a slightly higher Eh in the FIA than MEO systems (i.e., + 0.71 to +0.72 V vs +0.70 V, respectively) likely contributed to the slightly faster reaction in FIA system (reaction time, 25 min at 90 μL min−1) than in the MEO cell. The quantification of EDC values fundamentally differed in the FIA and MEO systems in that ABTS•+ reduction is detected after it has occurred in the reaction coil whereas it is directly monitored over time in MEO. This difference results in very different shapes of the current responses in FIA and MEO. The current peaks in FIA were relatively narrow (peak width of Δt < 10 min) and symmetrical (Figure 3a,c). These peak shapes resulted from small longitudinal dispersion during advective transport of the samples through the FIA system. 6428


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Figure 4. (a) Electron donating capacities (EDC) of selected humic and fulvic acid (HA and FA) and natural organic matter (NOM) isolates used as models for dissolved organic matter (DOM), as determined by flow injection analysis (EDCFIA; Eh= 0.71 V, pH 7) and mediated electrochemical oxidation (EDCMEO; Eh= 0.73 V, pH 7). The EDCMEO data was taken from a previous publication.8 The brown and the blue symbols represent DOM from terrestrial and aquatic systems, respectively. Error bars represent standard deviations of triplicate measurements. (b) EDC values of model DOM isolates and DOM collected from three ombrotrophic bogs versus their specific UV absorbance values at 254 nm (SUVA254). The peat DOM samples were collected from the peat pore water at 125 cm depth (dark green symbols) and from open water pools within the bog at 40 (NR) or 120 cm (LK, LM) depth (light green symbols). Error bars represent standard deviations of triplicate measurements. (c) Comparison of EDC values from tested DOM samples after normalization to SUVA254. The error bars for HS/NOM isolates (blue and brown bars) represent standard deviations of triplicate measurements. For peat DOM sample data, the error bars represent the standard deviations among the EDC averages shown in panel b (either n = 3 or 5 average EDC values, as specified on the data bars). (d) Changes in the EDC values of a selected peatland DOM (peat pore water from LM) incubated at pH 4.75 without laccase (red symbols; control), with laccase (blue symbols) and with laccase and ABTS as electron transfer mediator (green symbols). Shown also are selected oxidative current peak responses for samples from each of the incubations for four selected incubation times. All EDC values were calibrated with five injections of Trolox standards. Error bars represent ranges of duplicate incubations.

Conversely, the current responses in MEO were much wider (width of Δt = 60 min) and asymmetric, a fast initial increase in the current to maximum values was followed by prolonged, approximately exponential decays in the currents over time (Figure 3b). The prolonged current decay challenges accurate integration of small peaks that result from the analysis of small analyte amounts. We estimated the LOQ of the FIA system for DOM by triplicate injections of SRHA at amounts between 600 and 5 ngSRHA (Figure 3c). Each of the triplicate injections resulted in nearly identical current responses, highlighting the high reproducibility of the FIA analysis. Note that the first five

current peaks resulted from Tolox injections and served to calibrate the system (from 2 to 0.092 nmol of Trolox). The LOQ of SRHA was approximately 19 ngSRHA (calculated with σ = 0.39 nA, and RF = 0.206 A gSRHA−1 in eq 4 (Figure 3d)), which corresponds to an approximate NPOC concentration of 0.4 mgC L−1 (using the measured carbon content of SRHA of 0.53 gC gSRHA−1 and an injection volume of 100 μL). These low DOC values imply that the FIA system is sufficiently sensitive to determine the EDC values of dilute DOM samples collected from natural systems. Such dilute DOM solutions cannot be accurately analyzed using MEO. In fact, the addition of 600 ng 6429


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ABTS by laccase (i.e., ABTS is a known substrate for laccase56 and is oxidized by the copper atoms in the active site of the enzyme57,58). The final decrease in EDC values in the presence of ABTS and laccase (i.e., 38%) was, however, only slightly larger than in the systems containing laccase but no ABTS, suggesting that most phenolic moieties in the DOM were directly oxidizable in the active site of the enzyme. The considerable EDC values that remained after laccase treatment likely resulted from the significantly lower solution pH during incubation (pH 4.75) than in the FIA system for EDC quantification (pH 7.0). Because increasing pH favors phenol oxidation both thermodynamically and kinetically,59 the DOM likely contained a subpool of phenolic moieties that were oxidizable by ABTS•+ at pH 7 but not oxidizable by laccase or ABTS•+ at pH 4.75. We note that the FIA analysis could not be conducted at pH 4.75 because laccase from the incubation samples would have oxidized ABTS at this pH and hence interfered with the EDC analysis. Conversely, laccase is inactive at pH 7 and hence did not interfere with the EDC quantification at this pH.

of SRHA into the MEO cell did not result in quantifiable current responses (Supporting Information, Figure S7). FIA Analysis of a Diverse Set of DOM Samples. Figure 4a shows good agreement in the EDC values of 13 HS and NOM isolates quantified by FIA (y-axis) and MEO (x-axis).8 This result demonstrates that the FIA method can be used to quantify EDC values of DOM from very different sources. The EDC values of the HS/NOM determined by FIA also showed a strong linear correlation (R2 = 0.94) with their titrated phenol contents29,30,44 (Supporting Information, Figure S8a), strongly supporting that phenolic moieties in DOM donated electrons to ABTS•+.8 The EDC values of the model DOM quantified by FIA are replotted in Figure 4b versus their specific UV absorbance values at 254 nm (SUVA254). This parameter is linearly correlated to the reported aromaticity values of these DOM (R2 = 0.97, Figure S8b). Combining this information with the linear correlation of EDC values to the phenol contents, Figure 4b,c suggests that electron-donating phenolic moieties in DOM per unit aromaticity (i.e., SUVA254) decrease from aquatic to terrestrial materials. This trend was previously reported and explained by a depletion of phenolic moieties in the older, more oxidatively processed terrestrial DOM than in the younger, less processed aquatic DOM.8,52 DOM samples collected from the pore waters and openwater pools of three ombrotrophic bogs had comparatively high EDC values between 5.34 mmole‑ gC−1 and 7.14 mmole‑ gC−1 (green points, Figure 4b). When normalized to SUVA254 values (from 3.45 to 4.02 L mgC−1 m−1; see Table S2 for DOC and absorbance values used in the calculation of SUVA254 values), the peat EDC values were higher than those of the aquatic and terrestrial HS/NOM isolates (Figure 4c). The concentrations of iron and sulfide in the peat samples were too small for these species to have had major contributions to the measured EDC values (Table S2, Supporting Information). The finding of comparatively high EDC values for peat DOM is consistent with the overall high contents of phenols in peatlands and their low extents of oxidative transformation due to anoxic conditions that prevail in the permanently water-saturated pores of these systems.53−55 Interestingly, the lower SUVA254normalized EDC values of peat-pool than peat-pore water possibly reflected more extensive oxidation of DOM in the oxic peat pools as compared with the anoxic peat-pore water. The analysis of peat DOM demonstrates that the FIA system allows quantifying EDC values of DOM from natural sources. Furthermore, the data suggests that EDC measurements by FIA can be used to monitor the extents of oxidative processing of DOM in natural systems. Enzymatic Oxidation of Phenols in Peatland DOM. To demonstrate that the FIA system can be used to monitor the oxidation of phenolic moieties in DOM, we incubated one of the peat pore-water samples from LM with laccase from Trametes versicolor and monitored the resulting change in EDC values over 3 days. As expected, the EDC values of control DOM samples (i.e., no laccase added) stayed approximately constant during the incubation (Figure 4d; red squares). Conversely, the EDC values of the DOM incubated with laccase decreased by nearly one-third over 2 days and thereafter remained approximately constant (blue lines). Addition of minute amounts of ABTS to samples containing laccase facilitated the initial decrease in the EDC values of the DOM (green lines), consistent with mediated oxidation of the phenolic moieties by ABTS•+ formed via the oxidation of

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IMPLICATIONS This work presents a highly sensitive FIA system with chronoamperometric detection for the automated quantification of electron donating phenolic moieties in DOM. The very low LOQ of this method (i.e., around 20 ngDOM) allows quantifying EDC values for natural samples with NPOC concentrations below 1 mgC L−1. As such, this work largely advances the analytical capabilities to characterize the redox properties of DOM. The superior sensitivity of the FIA method compared to any previously published method is in large part due to using an electrochemical flow cell detector with low background current noise. The FIA system allows characterizing the redox properties of DOM in dilute samples using only very small solution volumes. As such, this technique opens new possibilities to monitor changes in the phenol contents of DOM in oxidation processes at high temporal and spatial resolution. We anticipate that future work will employ the FIA system to monitor changes in the EDC values of DOM during its enzymatic, photochemical, or chemical oxidation. Direct analytical access to the dynamics in DOM phenol pools will contribute to a more holistic understanding of their role in oxidation processes in both natural and engineered systems.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b01120. Additional information and data on peat sampling locations and peat DOM properties, the FIA system, the FIA system, the redox properties of the ABTS•+/ ABTS couple, and the EDC values of model HS and NOM isolates (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +41 (0)44 632-8314; fax: +41 (0)44 633-1122; e-mail: [email protected]. Notes

The authors declare no competing financial interest. 6430


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ACKNOWLEDGMENTS The project was funded by the Swiss National Science Foundation (Project 200020_159692). We thank ETH Zurich for funding the FIA system via the Scientific Equipment Program. We further thank L. Klüpfel, K.H. Jacobson, P. Nauer (all ETH Zurich), M. Lau (IGB Berlin), D. Cervenka, and R. Sander for support during DOM sample collection, L. Klüpfel for helpful discussions, and Ashley Brown (Eawag) for the quantification of total iron in the peat DOM samples.

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