Low Molecular Weight Components in an Aquatic Humic SubstanceAs Characterized by Membrane Dialysis and Orbitrap MassSpectrometryChristina K. Remucal,, Rose M. Cory, Michael Sander, and Kristopher McNeill*,

Institute of Biogeochemistry and Pollutant Dynamics (IBP), ETH Zurich, 8092 Zurich, SwitzerlandEnvironmental Sciences & Engineering, University of North Carolina, Chapel Hill, North Carolina, United States

*S Supporting Information

ABSTRACT: Suwannee River fulvic acid (SRFA) wasdialyzed through a 100500 molecular weight cuto dialysismembrane, and the dialysate and retentate were analyzed byUVvisible absorption and high-resolution Orbitrap massspectrometry (MS). A signicant fraction (36% based ondissolved organic carbon) of SRFA passed through the dialysismembrane. The fraction of SRFA in the dialysate had adierent UVvisible absorption spectrum and was enriched inlow molecular weight molecules with a more aliphaticcomposition relative to the initial SRFA solution. Comparisonof the SRFA spectra collected by Orbitrap MS and Fouriertransform ion cyclotron resonance MS (FT-ICR MS)demonstrated that the mass accuracy of the Orbitrap MS issucient for determination of unique molecular formulas of compounds with masses

(FT-ICR MS), with

isotopologue was detected with 100 in the mass spectra are shown, corresponding to 4550% of identied peaks in this experiment due to dilute samples.

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Known concentrations (01 M) of benzoic acid, esculetin,and umbelliferone were added to SRFA (nal concentration =50 mg/L) and analyzed by Orbitrap MS as described above.The magnitudes of the m/z values of the spiked compoundswere normalized to the magnitudes of 812 nearby peaks andplotted versus the added compound concentration to estimatethe maximum concentration of each compound in SRFA.

RESULTS AND DISCUSSIONDialysis Separation of LMW Components from SRFA.

A SRFA solution was dialyzed through a 100500 MWCOdialysis membrane against Nanopure water for 7 days. Over thecourse of the dialysis, the non-purgeable organic carbon(NPOC) decreased in the retentate and increased in thedialysate (Figure 1a). Using the measured NPOC values andthe known solution volumes, it was possible to close the carbonmass balance (99.7%). Of the initial mass of 3.50 0.09 mg of

carbon added to the dialysis tube, 2.24 0.05 and 1.25 0.02mg of carbon were recovered in the retentate and dialysatesolutions, respectively.In a control experiment, a solution containing riboavin

(MW = 376.36 g/mol) and Rose Bengal (MW = 973.67 g/mol;smallest cross-sectional dimension = 14 )39 was dialyzedthrough a separate dialysis membrane against Nanopure water.The concentration of riboavin in the dialysate increased to 3.0M (268 = 31 600 cm

1 M1)40 after 7 days of dialysis,corresponding to 91% of the expected concentration atpartition equilibrium (Supporting Information, Figure S2).Conversely, Rose Bengal was conserved in the retentate andwas not detected in the dialysate, as expected based on its MW,which is larger than the 500 Da MWCO of the dialysismembrane. It is possible that molecules in SRFA withmolecular masses >500 Da diused through the membraneprovided that they had suciently small cross-sectional areas

Figure 2. (a) Orbitrap ESI negative mass spectrum of SRFA in the 100600 m/z range and SRFA m/z relative magnitude distributions of identiedions measured by (b) Orbitrap and (c) FT-ICR MS. The relative magnitude cutos of 10.2 and 8.2 for Orbitrap and FT-ICR, respectively,correspond to 65% of the identied peaks and are indicated by dashed lines. The relative magnitude cuto corresponds to an absolute magnitudecuto of 100 for the Orbitrap spectra.

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(e.g., long aliphatic molecules). However, the results from thecontrol experiment strongly support that the molecules in thedialysate of SRFA were low in molecular weight (250 nm. The opposite isobserved in the 200250 nm wavelength range, with thedialysate showing higher relative absorption compared to theretentate. After correcting for solution volumes, summing thespectra of the retentate and dialysate produces a spectrum thatis identical to the starting SRFA solution (SupportingInformation, Figure S3), demonstrating that the light-absorbingcomponents of SRFA were conserved.The NPOC-normalized spectra of the dialysate has a much

weaker specic absorbance than the retentate (SupportingInformation, Figure S3), suggesting that the dialysate isdepleted in UVvis light absorbing moieties relative to theretentate. For example, the molar absorptivity at 280 nm [L(mol OC)1 cm1] increased from 420 to 480 in the retentateand only reached 194 in the dialysate after 7 days, in agreementwith previous observations that lower molecular weightfractions of fulvic acids have lower molar absorptivities.18

Additionally, the specic UV absorbance at 254 nm [SUVA254;L (mg C)1 m1]47 of the retentate increased from 4.68 to 5.20

after dialysis, suggesting an increase in aromaticity. Thedialysate had a lower SUVA254 value of 2.36, further indicatingthat this fraction was depleted in aromatic components relativeto the initial SRFA solution.The UVvis absorbance data clearly indicate that the SRFA

components that passed through the dialysis membranegenerally had lower absorbance at all wavelengths andparticularly weakened absorbance at longer wavelengthscompared with the initial SRFA solution. The absorbanceindices all point to the same conclusion that this LMW materialwas depleted in aromatic and other unsaturated chromophores.This conclusion is reinforced by high-resolution MS analysis ofthe solutions, as discussed below.High-Resolution Mass Spectra of SRFA. Prior to the

analysis of the retentate and dialysate of SRFA, the massspectrum of nondialyzed SRFA was collected on an Orbitrapinstrument to validate the analysis method. The mass spectrumof SRFA showed a pattern of intense clusters of ions at oddm/z values and less intense groups at even m/z values (Figure2a). This pattern is attributable to ions containing only C, H,and O, which are the dominant atoms in SRFA.22,23,26 The lessintense clusters of ions observed at even m/z values are due tothe presence of heteroatoms (e.g., N and P) and 13C1

12Cn1isotopologues from singly charged ions.21 The SRFA massspectrum has a repeating pattern with a period of 14.0156 massunits, corresponding to the addition of CH2 groups.

22,23,25 Thesame features have previously been reported for DOM spectracollected by FT-ICR MS.2123,25,26 The Orbitrap-detected massspectrum of SRFA showed the highest intensities at m/z values50 000 in theOrbitrap MS (data not shown). However, a conservativenominal mass cuto of 600 Da was used for molecular formulaidentication. The mass accuracy of the Orbitrap MS issuciently high to derive unique chemical formulas for the C-,H-, and O-containing peaks detected in the negative ESI SRFAspectrum in this mass range.The chemical formulas of ions identied by Orbitrap analysis

of the SRFA prior to dialysis and of the retentate and dialysateafter 7 d of dialysis are presented in the form of van Krevelendiagrams (i.e., the ratio of H:C plotted versus the ratio of O:C;Figure 1ce). The mass spectra of all three solutions showedclusters of intense ions at odd m/z values and less intense ionsat even m/z values (Supporting Information, Figure S4),consistent with the spectrum presented in Figure 2a. Note thatthe mass spectra of the SRFA prior to dialysis and the retentatewere collected from dilute solutions to match the intensity ofions in the mass spectrum of the dialysate and that all formulasidentied in the dialysate were also identied in the initialSRFA solution (formulas not presented in Figure 1c werebelow the magnitude cuto). The spectrum obtained fromanalyzing a concentrated SRFA solution is discussed in detailbelow.The van Krevelen plots clearly show that the chemical

formulas of the compounds in the dialysate diered from thosein the retentate (Figure 1ce), conrming that a distinctfraction of the SRFA starting material was separated by dialysis.While the retentate showed intense ions in regions of the vanKrevelen diagram where aromatic and lignin compounds are

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found (H:C < 1), the dialysate showed ions with higher H:Cand lower O:C ratios, indicating a more aliphatic composition.The changes in the van Krevelen diagrams must be carefully

interpreted since the abundance of an ion in a single massspectrum is not quantitative. The magnitudes of ions producedby ESI depend on the ionization eciency of their functionalgroups (e.g., carboxylic acids, alcohols, amines)29 and also maydepend on the eciency of the detector to detect the ions; theydo not necessarily correlate with the abundance of thosecompounds in a mixture such as SRFA.33 Nevertheless, it ispossible to compare relative magnitudes of the same ion beforeand after the dialysis to reveal trends.48 Changes in the relativemagnitude (M) of ions were calculated by comparing theirmagnitudes in the retentate (t7), normalized to the sum ofmagnitudes of all common ion masses in the retentate, to theirnormalized magnitudes in the dilute SRFA solution (t0) beforeit was dialyzed:

=

M M

M M

M Mlog / log

/

/m z

m zt t

/t

allt

/t

allt7 0

7 7

0 0(1)

According to eq 1, ions in the retentate after dialysis withnegative and positive log Mt7/Mt0 values represent compoundswith decreasing and increasing abundance, respectively. Dialysisresulted in an increasing relative abundance of compounds witharomatic/lignin signatures in the retentate (Figure 1f).Numerous ions with more saturated formulas (i.e., high H:Cratios) that were detected in the dilute SRFA solution prior todialysis were not detected in the retentate with sucientresolution of the 13C1

12Cn1 isotopologue (Figure 1f), but werefound in the dialysate (Figure 1e). The dialysate thereforecontained compounds that were less aromatic and moresaturated than compounds in the retentate, consistent with theresults of the UVvis absorbance analysis. Our results are alsoin agreement with previous observations that the amount ofaromatic moieties in aquatic humic substances increases withincreasing molecular weight.18

The relative mass distribution conrms that the retentatesolution had fewer compounds in the 300500 mass rangeafter dialysis and was slightly enriched in the higher massesrelative to the starting material (Supporting Information, FigureS5). While some of the low molecular weight components inthe mass spectra of the dialysate may have resulted fromfragmentation of compounds with larger masses, as discussedbelow, the predominance of low molecular weight ions in thedialysate (400Da. In fact, many of these masses (57%) were detected by theOrbitrap MS but were excluded from analysis because the13C1

12Cn1 isotopologue was not detected with sucientcondence. The formulas only identied by FT-ICR MSoverlap with those identied in both data sets, with slightlyhigher O:C in some cases (Figure 3a), in agreement withprevious observations that HMW fractions of SRFA havehigher O content.25 Note that general shape of the vanKrevelen diagram from the FT-ICR MS data set for 600800m/z is the same as the 290600 m/z range (SupportingInformation, Figure S7), suggesting that trends in the classcomposition of SRFA in the 290600 m/z range may beapplicable to higher masses as well. The compounds that wereunique to the Orbitrap data set generally have masses

(Supporting Information, Figure S6) and are found in the samesection of the van Krevelen diagram as condensed aromaticsand black carbon.2,23,32,5759

It is important to note that the previous FT-ICR MS studiesof SRFA generally did not consider masses below 200Da.22,23,26 An advantage of the Orbitrap MS is the assessmentof ions in this low mass range. The molecular formulasidentied in the 100200 m/z range occupied a larger space inthe van Krevelen diagram than the ions in the 200290 m/zrange (Figure 3b,c). A similar wide pattern in the van Krevelendiagram was previously reported for SRFA ions in the 150344m/z range obtained by SEC-TOF-MS.48

This study demonstrates that Orbitrap and FT-ICR MSproduce complementary information about the chemicalcomposition of SRFA. For the 290600 mass range, theagreement between the two methods shows that Orbitrap MScan be used to determine the molecular composition ofcomponents in complex mixtures, such as SRFA. Additionally,the high resolving power of the Orbitrap MS in the 100290mass range and the high magnitudes of these ions in thedetected mass spectra demonstrate that the Orbitrap MS iswell-suited for analyzing the LMW fraction of SRFA and likelyother aquatic humic substances with low N and S contents.Compound Identication and Quantication. The

complexity of DOM and the presence of multiple compoundsat the same nominal mass limit the application of MSMS forstructural elucidation,2 although insights can be gained bytechniques such as single ion isolation using on-resonanceCID.26 Eorts were made to identify some of the additionalprominent compounds observed in the 100200 mass range,such as those containing only hydrogen and carbon (Figure3b). Even with these low masses and only two possibleelements, H and C could be combined in numerous potentialstructures, illustrating the diculty with compound identi-cation from mass alone. For example, the neutral mass116.0623, corresponding to the formula C9H8, matches 41possible structures in the ChemSpider database,60 of whichindene may be considered the most likely based on its presencein environmental systems61 and its ability to ionize to form arelatively stable negative ion.62 By contrast, the mass 120.0935(C9H12) matches >250 possible structures, with no obviousnaturally occurring compound presenting itself as the clearassignment.Nonetheless, it is possible to tentatively identify some

compounds by comparing the unique molecular formulasobserved in SRFA with lists of compounds previously identiedin lignin, DOM, plants, and black carbon (SupportingInformation, Figure S8 and Tables S1S3). Lignin, forexample, is thought to be a major constituent of SRFA.22,63

Although this identication approach does not provide directevidence for the presence of these compounds, thecombination of accurate mass determination with knowledgeof the origin of DOM (i.e., derived from plants) does enablethe identication of likely structures. While only one likelyisomer was found for some masses (Supporting Information,Table S1), multiple potential isomers could be identied forothers (Supporting Information, Table S2), and it is possiblethat the masses assigned to a unique structure here couldcorrespond to other isomers not included in this screening.Many masses corresponded to those previously reported tobelong to class types such as lignin or carboxyl-rich alicylicmolecules (CRAM),64,65 and no structures were identied(Supporting Information, Table S3). The tentatively identied

Figure 3. van Krevelen diagram for (a) compounds identied bynegative-ion ESI showing compounds unique to FT-ICR and OrbitrapMS for 290600 m/z, as well as compounds identied by bothmethods. van Krevelen diagrams for compounds identied bynegative-ion ESI by Orbitrap MS for (b) 100200 and (c) 200290 m/z with magnitudes >100.

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compounds span most of the area covered by the van Krevelendiagram of SRFA, with many of the uniquely identiedcompounds found in the 100200 mass range (SupportingInformation, Figure S8).Three of the tentatively identied compounds were added to

SRFA in increasing amounts according to the standard additionmethod. The resulting increase in the relative magnitude ofeach mass peak was then used to estimate the maximumconcentration of the compound that could have been originallypresent in SRFA (Supporting Information, Figure S9 and TableS4), assuming that no isomeric compounds contributed to theoriginal MS peak intensity. Benzoic acid, esculetin, andumbelliferone were selected because they have low masses,are known plant-derived compounds,66,67 and their masseswere detected in SRFA with high intensities (SupportingInformation, Figure S8 and Table S1). The calculatedconcentrations represent the maximum possible concentrationof the standard compound and do not account for otherisomers. For example, in the case of umbelliferone, it is possiblethat other hydroxycoumarins are present in SRFA and wouldionize with comparable eciency. Benzoic acid had a lowresponse factor, and as a result, the error on the maximumbenzoic acid concentration is high. Neglecting isomers at thesame exact mass, the selected compounds could comprise ahigh percentage (ca. 1%) of SRFA by mass (SupportingInformation, Table S4). The linear response of the OrbitrapMS with increasing model concentration demonstrates that thismethod could be used for further quantication of targetcompounds in NOM.Environmental and Technical Implications. The view of

DOM has evolved from it being composed of high molecularweight polymeric structures to supramolecular associations ofrelatively small organic molecules.7,8,50 Fulvic acids areconsidered smaller than humic acid14,15 and the averagereported molecular weight of SRFA ranges from 800 to 1500Da.14,15,1719 Our results show that 36% of the carbon mass ofSRFA passed through a 100500 MWCO dialysis membrane,demonstrating that a signicant fraction of SRFA falls withinthe low (i.e.,

(9) Amon, R. M. W.; Benner, R. Bacterial utilization of different sizeclasses of dissolved organic matter. Limnol. Oceanogr. 1996, 41 (1),4151.(10) Meyer, J. L.; Edwards, R. T.; Risley, R. Bacterial growth ondissolved organic carbon from a blackwater river. Microbial Ecol. 1987,13 (1), 1329.(11) Benner, R.; Hedges, J. I. A test of the accuracy of freshwaterDOC measurements by high-temperature catalytic oxidation and UV-promoted persulfate oxidation. Mar. Chem. 1993, 41 (13), 161165.(12) Hedges, J. I.; Cowie, G. L.; Richey, J. E.; Quay, P. D.; Benner,R.; Strom, M.; Forsberg, B. R. Origins and processing of organicmatter in the Amazon River as indicated by carbohydrates and aminoacids. Limnol. Oceanogr. 1994, 39 (4), 743761.(13) Aiken, G. R.; McKnight, D. M.; Thorn, K.; Thurman, E.Isolation of hydrophilic organic acids from water using nonionicmacroporous resins. Org. Geochem. 1992, 18 (4), 567573.(14) Thurman, E. M.; Wershaw, R.; Malcolm, R.; Pinckney, D.Molecular size of aquatic humic substances. Org. Geochem. 1982, 4 (1),2735.(15) Beckett, R.; Jue, Z.; Giddings, J. C. Determination of molecularweight distributions of fulvic and humic acids using flow field-flowfractionation. Environ. Sci. Technol. 1987, 21 (3), 289295.(16) Leenheer, J.; Rostad, C.; Gates, P.; Furlong, E.; Ferrer, I.Molecular resolution and fragmentation of fulvic acid by electrosprayionization/multistage tandem mass spectrometry. Anal. Chem. 2001,73 (7), 14611471.(17) Aiken, G. R.; Malcolm, R. L. Molecular weight of aquatic fulvicacids by vapor pressure osmometry. Geochim. Cosmochim. Acta 1987,51 (8), 21772184.(18) Chin, Y. P.; Aiken, G.; OLoughlin, E. Molecular weight,polydispersity, and spectroscopic properties of aquatic humicsubstances. Environ. Sci. Technol. 1994, 28 (11), 18531858.(19) Reid, P. M.; Wilkinson, A. E.; Tipping, E.; Jones, M. N.Determination of molecular weights of humic substances by analytical(UV scanning) ultracentrifugation. Geochim. Cosmochim. Acta 1990, 54(1), 131138.(20) Brown, T.; Rice, J. Effect of experimental parameters on the ESIFT-ICR mass spectrum of fulvic acid. Anal. Chem. 2000, 72 (2), 384390.(21) Stenson, A.; Landing, W.; Marshall, A.; Cooper, W. Ionizationand fragmentation of humic substances in electrospray ionizationFourier transform-ion cyclotron resonance mass spectrometry. Anal.Chem. 2002, 74 (17), 43974409.(22) Stenson, A.; Marshall, A.; Cooper, W. Exact masses andchemical formulas of individual Suwannee River fulvic acids fromultrahigh resolution electrospray ionization Fourier transform ioncyclotron resonance mass spectra. Anal. Chem. 2003, 75 (6), 12751284.(23) Hertkorn, N.; Frommberger, M.; Witt, M.; Koch, B. P.; Schmitt-Kopplin, P.; Perdue, E. M. Natural organic matter and the eventhorizon of mass spectrometry. Anal. Chem. 2008, 80 (23), 89088919.(24) Kujawinski, E. B.; Del Vecchio, R.; Blough, N. V.; Klein, G. C.;Marshall, A. G. Probing molecular-level transformations of dissolvedorganic matter: Insights on photochemical degradation and protozoanmodification of DOM from electrospray ionization Fourier transformion cyclotron resonance mass spectrometry. Mar. Chem. 2004, 92 (14), 2337.(25) Reemtsma, T.; These, A.; Springer, A.; Linscheid, M.Differences in the molecular composition of fulvic acid size fractionsdetected by size-exclusion chromatographyon line Fourier transformion cyclotron resonance (FT-ICR) mass spectrometry. Water Res.2008, 42 (12), 6372.(26) Witt, M.; Fuchser, J.; Koch, B. P. Fragmentation studies of fulvicacids using collision induced dissociation Fourier transform ioncyclotron resonance mass spectrometry. Anal. Chem. 2009, 81 (7),26882694.(27) Cory, R. M.; McNeill, K.; Cotner, J. P.; Amado, A.; Purcell, J.M.; Marshall, A. G. Singlet oxygen in the coupled photochemical and

biochemical oxidation of dissolved organic matter. Environ. Sci.Technol. 2010, 44 (10), 36833689.(28) Kujawinski, E.; Hatcher, P.; Freitas, M. High-resolution Fouriertransform ion cyclotron resonance mass spectrometry of humic andfulvic acids: Improvements and comparisons. Anal. Chem. 2002, 74(2), 413419.(29) Kujawinski, E.; Freitas, M.; Zang, X.; Hatcher, P.; Green-Church, K.; Jones, R. The application of electrospray ionization massspectrometry (ESI MS) to the structural characterization of naturalorganic matter. Org. Geochem. 2002, 33 (3), 171180.(30) Krauss, M.; Singer, H.; Hollender, J. LChigh resolution MS inenvironmental analysis: From target screening to the identification ofunknowns. Anal. Bioanal. Chem. 2010, 397 (3), 943951.(31) Cortes-Francisco, N.; Flores, C.; Moyano, E.; Caixach, J.Accurate mass measurements and ultrahigh-resolution: Evaluation ofdifferent mass spectrometers for daily routine analysis of smallmolecules in negative electrospray ionization mode. Anal. Bioanal.Chem. 2011, 112.(32) Kim, S.; Kramer, R.; Hatcher, P. Graphical method for analysisof ultrahigh-resolution broadband mass spectra of natural organicmatter, the van Krevelen diagram. Anal. Chem. 2003, 75 (20), 53365344.(33) Reemtsma, T. Determination of molecular formulas of naturalorganic matter molecules by (ultra-) high-resolution mass spectrom-etry. J. Chromatogr. A 2009, 1216 (18), 36873701.(34) Kim, S.; Rodgers, R. P.; Marshall, A. G. Truly exact mass:Elemental composition can be determined uniquely from molecularmass measurement at

(48) These, A.; Reemtsma, T. Structure-dependent reactivity of lowmolecular weight fulvic acid molecules during ozonation. Environ. Sci.Technol. 2005, 39 (21), 83828387.(49) Chin, W. C.; Orellana, M. V.; Verdugo, P. Spontaneousassembly of marine dissolved organic matter into polymer gels. Nature1998, 391 (6667), 568572.(50) Conte, P.; Piccolo, A. Conformational arrangement of dissolvedhumic substances. Influence of solution composition on association ofhumic molecules. Environ. Sci. Technol. 1999, 33 (10), 16821690.(51) Kerner, M.; Hohenberg, H.; Ertl, S.; Reckermann, M.; Spitzy, A.Self-organization of dissolved organic matter to micelle-like micro-particles in river water. Nature 2003, 422 (6928), 150154.(52) Reemtsma, T.; These, A. On-line coupling of size exclusionchromatography with electrospray ionization-tandem mass spectrom-etry for the analysis of aquatic fulvic and humic acids. Anal. Chem.2003, 75 (6), 15001507.(53) Hunt, S. M.; Sheil, M. M.; Belov, M.; Derrick, P. J. Probing theeffects of cone potential in the electrospray ion source: Consequencesfor the determination of molecular weight distributions of syntheticpolymers. Anal. Chem. 1998, 70 (9), 18121822.(54) Gabelica, V.; Pauw, E. D. Internal energy and fragmentation ofions produced in electrospray sources. Mass Spectrom. Rev. 2005, 24(4), 566587.(55) Cole, R. B. Some tenets pertaining to electrospray ionizationmass spectrometry. J. Mass Spectrom. 2000, 35 (7), 763772.(56) These, A.; Reemtsma, T. Limitations of electrospray ionizationof fulvic and humic acids as visible from size exclusion chromatographywith organic carbon and mass spectrometric detection. Anal. Chem.2003, 75 (22), 62756281.(57) Kramer, R. W.; Kujawinski, E. B.; Hatcher, P. G. Identificationof black carbon derived structures in a volcanic ash soil humic acid byFourier transform ion cyclotron resonance mass spectrometry. Environ.Sci. Technol. 2004, 38 (12), 33873395.(58) Kujawinski, E. B.; Behn, M. D. Automated analysis ofelectrospray ionization Fourier transform ion cyclotron resonancemass spectra of natural organic matter. Anal. Chem. 2006, 78 (13),43634373.(59) Sleighter, R. L.; Hatcher, P. G. Molecular characterization ofdissolved organic matter (DOM) along a river to ocean transect of thelower Chesapeake Bay by ultrahigh resolution electrospray ionizationFourier transform ion cyclotron resonance mass spectrometry. Mar.Chem 2008, 110 (34), 140152.(60) ChemSpider; http://www.chemspider.com/.(61) Wammer, K. H.; Peters, C. A. Polycylic aromatic hydrocarbonbiodegradation rates: A structure-based study. Environ. Sci. Technol.2005, 39, 25712578.(62) Bordwell, F. G.; Drucker, G. E. Acidities of indene and phenyl-,diphenyl-, and triphenylindenes. J. Org. Chem. 1980, 45 (16), 33253328.(63) Haiber, S.; Herzog, H.; Burba, P.; Gosciniak, B.; Lambert, J.Two-dimensional NMR studies of size fractionated Suwannee Riverfulvic and humic acid reference. Environ. Sci. Technol. 2001, 35 (21),42894294.(64) Hertkorn, N.; Benner, R.; Frommberger, M.; Schmitt-Kopplin,P.; Witt, M.; Kaiser, K.; Kettrup, A.; Hedges, J. Characterization of amajor refractory component of marine dissolved organic matter.Geochim. Cosmochim. Acta 2006, 70 (12), 29903010.(65) Lam, B.; Baer, A.; Alaee, M.; Lefebvre, B.; Moser, A.; Williams,A.; Simpson, A. J. Major structural components in freshwater dissolvedorganic matter. Environ. Sci. Technol. 2007, 41 (24), 82408247.(66) Egan, D.; OKennedy, R.; Moran, E.; Cox, D.; Prosser, E.;Thornes, R. D. The pharmacology, metabolism, analysis, andapplications of coumarin and coumarin-related compounds. DrugMetab. Rev. 1990, 22 (5), 503529.(67) Hollman, P. C. H. Evidence for health benefits of plant phenols:Local or systemic effects? J. Sci. Food Ag. 2001, 81 (9), 842852.(68) Kim, S.; Kaplan, L. A.; Hatcher, P. G. Biodegradable dissolvedorganic matter in a temperate and a tropical stream determined from

ultra-high resolution mass spectrometry. Limnol. Oceanogr. 2006, 51(2), 10541063.

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