Terpenoids as Major Precursors of Dissolved Organic Matter in

Apr 17, 2003 - Significant amounts of lignin precursors, commonly postulated to be the major source of DOM, were found only in trace quantities by the...
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Research Terpenoids as Major Precursors of Dissolved Organic Matter in Landfill Leachates, Surface Water, and Groundwater J E R R Y A . L E E N H E E R , * ,† MARK A. NANNY,‡ AND CAMERON MCINTYRE§ U.S. Geological Survey, Building 95, Denver Federal Center, Denver, Colorado 80225, School of Civil Engineering and Environmental Science, The University of Oklahoma, 202 West Boyd, Norman, Oklahoma 73019, and Department of Chemistry, Division of Environmental and Life Sciences, Macquarie University, Sydney, Australia 2109

13C

NMR analyses of hydrophobic dissolved organic matter (DOM) fractions isolated from a landfill leachate contaminated groundwater near Norman, OK; the Colorado River aqueduct near Los Angeles, CA; Anaheim Lake, an infiltration basin for the Santa Ana River in Orange County, CA; and groundwater from the Tomago Sand Beds, near Sydney, Australia, found branched methyl groups and quaternary aliphatic carbon structures that are indicative of terpenoid hydrocarbon precursors. Significant amounts of lignin precursors, commonly postulated to be the major source of DOM, were found only in trace quantities by thermochemolysis/gas chromatography/mass spectrometry of the Norman Landfill and Tomago Sand Bed hydrophobic DOM fractions. Electrospray/tandem mass spectrometry of the Tomago Sand Bed hydrophobic acid DOM found an ion series differing by 14 daltons, which is indicative of aliphatic and aryl-aliphatic polycarboxylic acids. The product obtained from ozonation of the resin acid, abietic acid, gave a similar ion series. Terpenoid precursors of DOM are postulated to be derived from resin acid paper sizing agents in the Norman Landfill, algal and bacterial terpenoids in the Colorado River and Anaheim Lake, and terrestrial plant terpenoids in the Tomago Sand Beds.

Introduction Dissolved organic matter (DOM) is a complex mixture of humic and nonhumic substances (1). Aquatic humic substances, consisting of humic and fulvic acid, are operationally defined by isolation of hydrophobic DOM acids on resin sorbents followed by precipitation of humic acid at pH 1 (2). Humic substances are generally considered to be derived from lignins, tannins, carbohydrates, and proteins. This is based upon extensive studies of fulvic acid extracted from blackwaters such as the Suwannee River (1, 2) that have large inputs of lignins and tannins. Degradative procedures, such * Corresponding author phone: (303)236-3977; fax: (303)236 3934; e-mail: [email protected]. † U.S. Geological Survey. ‡ The University of Oklahoma. § Macquarie University. 10.1021/es0264089 CCC: $25.00 Published on Web 04/17/2003

 2003 American Chemical Society

as chemical oxidation (3), pyrolysis (4), and thermochemolysis (5), applied to both unfractionated DOM and aquatic fulvic and humic acids, always found fragment compounds derived from lignins, tannins, carbohydrates, proteins, and amino sugars. However, a few recent studies of aquatic fulvic acid and NOM found waters that could be characterized as “low humic” (6, 7) and “nonhumic” (8). This nonhumic fulvic acid was proposed to be derived from the biodegradation of terpenoids based upon infrared and 13C NMR spectral characterization of the isolated fulvic acid (8). Terpenoids have been found to be incorporated into soil humus (9), but they have not been reported as a major precursor of DOM. Terpenoids are formed by plants, phytoplankton, and bacteria from isoprene synthesized from mevalonic acid (10). Major plant terpenoids consist of terpene hydrocarbons such as pinene, resin acids such as abietic acid, and essential oils such as menthol. Algae produce the terpenoids geosmin and 2-methylisoborneol, which cause taste and odor problems in water. Bacteria produce large terpenoids (hopanepolyols) that degrade to hopanoids, which are used as geochemical markers in sediment (11). Terpenoids are classified into hemiterpenoids (5-carbon compounds), monoterpenoids (10-carbon compounds), sesquiterpenoids (15-carbon compounds), diterpenoids (20carbon compounds), triterpenoids (30-carbon compounds) that include sterols, tetraterpenoids (40-carbon compounds) that include carotenoids, and polymeric rubber that contains 3000 to 6000 isoprene units (10). Terpenoids may also be combined with other compound classes such as porphyrins (phytol side chain in chlorophyll), phloroglucinol derivatives such as humulone with isoprene side chains, and alkaloids that incorporated nitrogen into steroid structures. Terpenoids occur in both open chain and cyclic structures (aliphatic alicyclic and aromatic) in almost every possible structural arrangement that gives them great mixture complexity when analyzed. Open chain terpenoids, alkene, and aromatic structures are much more readily degraded by bacteria (12) than are alicyclic ring structures (13). Studies of pinene (a monoterpenoid with an alicyclic ring) degradation by bacteria (13) and atmospheric oxidation (14) found a series of aliphatic mono- and dicarboxylic acid products formed by oxidation of alkene and methylene hydrocarbon groups with aliphatic quaternary carbons and branched methyl groups being the most resistant to oxidation. It is also these aliphatic quaternary carbons and branched methyl groups that distinguish terpenoids from other major possible precursors such as plant lignins, tannins, carbohydrates, proteins, and waxes such as cutin and suberin. Therefore, major indicators of terpenoid precursors in nonhumic DOM should be quaternary aliphatic carbons, branched methyl groups, and ring structures that are refractory to biodegradation. The objectives of this report are to (1) report the discovery of chemical and spectral indicators of terpenoid precursors in landfill leachate from Norman, OK and (2) assay DOM from diverse environments (Colorado River and Anaheim lake surface waters, and infiltrated Anaheim Lake and Tomago Sand Bed groundwaters) for indicators of DOM terpenoid precursors. Various biogeochemical processes are proposed that transform parent terpenoids into DOM constituents.

Methods Samples. Norman Landfill. The Norman Landfill Research Site is a closed municipal landfill located south of Norman, VOL. 37, NO. 11, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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OK. The landfill accepted municipal solid waste from the early 1920s until 1985 when the landfill was closed and capped (15). The groundwater and surface water hydrology of the Norman Landfill Research Site is described by Christenson et al. (16) and organic and inorganic contaminants are leaching into the groundwater from the landfill waste to form a leachate plume (17). A site map, well sampling locations, and dissolved organic carbon (DOC) concentrations in the leachate plume are given in Cozzarelli et al. (17). Twenty liters of water from wells 35B and 40B downgradient from the landfill were sampled during April, 1996. Two leachate contaminated groundwater samples were taken at different wells to assess if there was significant variability in organic solute composition within the leachate plume. After the wells were purged, groundwater samples were collected using a peristaltic pump. The samples were field filtered using 25 µm porosity (Balston DH) and 0.3 µm porosity (Balston AAH) glass fiber cartridge filters in series (18). The filtered samples were collected in precleaned 20 L high-density polyethylene cubitainers, and the samples were shipped on ice in coolers to Denver for analyses that were completed within 2 weeks after receipt of the samples. Colorado River Water. Colorado River water (1000 L) was sampled in February 1998 at the input to the water treatment plant of the Metropolitan Water District of Southern California in La Verne, CA. This large sample was part of an AWWARF funded study on drinking water treatment and characterization of the polar fraction of NOM with respect to disinfection byproduct formation (19). The sample was filtered (1 µm), sodium-softened, and concentrated to 20 L by reverse osmosis at the water treatment plant before shipment in a cubitainer to the USGS laboratory. Anaheim Lake. Anaheim Lake is one of several spreading basins located near Anaheim, CA used by the Orange County Water District to recharge the Santa Ana River into the Orange County groundwater basin. A map of the site and a description of recharge and groundwater flow are given by Gamlin et al. (20). Anaheim Lake (123 L) was sampled February 6, 2001, Well DP-alk1-15 (187 L) was sampled February 6, 2001, Well AMD 9/1 (229 L) was sampled March 7, 2001, and Well AM 44 (189 L) was sampled May 22, 2001 using similar methods to those described previously for the Norman Landfill. Groundwater that had infiltrated 15 feet under Anaheim Lake was sampled from Well DP-alk1-15 and 200 feet under Anaheim Lake was sampled from well AMD 9/1. Well AM 44 is located about 600 ft west (downgradient) of Anaheim Lake and is screened at a depth of 165 ft. Differences in well sampling dates for the Anaheim Lake samples was the time required for the same parcel of infiltrated water to travel to each well to observe DOM attenuation and changes during groundwater infiltration. Tomago Sand Beds. The Tomago Sand Beds (TSB) are located 200 km north of Sydney, Australia and are part of an extensive aquifer system that is used by the local water corporation to supplement water supplies to the nearby city of Newcastle. The sand beds have been mined for minerals for at least 30 years and a description of the site can be found in Prosser and Roseby (21). Overlying vegetation at the site is dominated by species rich in terpenoid resins (21). One hundred and fifty liters of groundwater was mechanically pumped from a control piezometer (3502/2) located at UTM 56H 372940 1371020 on March 18, 2002. The piezometer sampling depth and the water table depth were 4.3 m and 0.5 m, respectively, below ground level. The shallow depth of the water table has resulted in the dominance of humus podzols in areas such as the study site with iron rich podzols restricted to areas of higher relief (21). The sample was pressure filtered with high purity nitrogen through a 0.45 µM cellulose acetate membrane (MFS) into 50-L HDPE 2324

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drums. The filtered sample was acidified to pH 2 with concentrated hydrochloric acid and transported to the laboratory and processed immediately. DOM Fractionation and Isolation. DOM fractionations differ from DOC fractionations in that the entire organic element (C,H,O,N, S, P) mass is measured in DOM fractionations as opposed to the organic carbon mass in DOC fractionations. The preparative DOM fractionation procedure of Leenheer and Noyes (18) was used for the Norman Landfill samples with an additional column of borate-saturated anionexchange resin used to adsorb the hydrophilic neutral (HPI-N) fraction (22). An improved DOM fractionation procedure that also recovered the colloid fraction (23) was used for the Anaheim Lake samples. Aquatic fulvic acids are operationally defined to occur in the hydrophobic acid (HPOA) fraction isolated by the XAD resins; however, the hydrophobic neutral (HPO-N) and the hydrophobic acid fractions from the Colorado River water were coisolated from the XAD resin and this fraction was called the hydrophobic NOM fraction (19). The groundwater sample obtained from the Tomago Sand Beds was subject to a DOC fractionation procedure detailed elsewhere (24). The hydrophobic acid (TSB-FA) fraction was isolated and analyzed without further fractionation. This fraction has been determined to be 98% fulvic acid (24). 13 C NMR Spectral Analysis. Solid-State Cross Polarization Magic Angle Spinning (CPMAS) 13C NMR spectra were obtained on 20-200 mg of NOM samples. Freeze-dried samples were packed in zirconia rotors. CPMAS-13C NMR spectra were obtained on a 200 MHz (4.7 T field strength) Chemagnetics CMX spectrometer with a 7.5 mm diameter probe. The following parameters were used: spinning rate of 5000 Hz, contact times of 1 ms (Colorado River water and Norman Landfill fulvic acid) and 5 ms (Anaheim Lake fulvic acids and TSB-FA), pulse delay of 1 s, a pulse width of 4.5 µs for the 90° pulse, and line broadening of 100 Hz. The long 5 ms contact time was used in the Anaheim Lake and TSBFA samples to better detect quaternary-substituted carbon atoms (25). The dipolar dephased spectra of the HPO-N fraction used a 1 ms contact time and a coupling time of 30 µs. Stable Carbon Isotopes. Stable carbon isotopes were determined by established methods (26). TMAH Thermochemolysis. Tetramethylammonium hydroxide (TMAH) thermochemolysis was done with an accurately weighed 0.2 mg sample of the HPO-A fraction from the Norman Landfill well 35B according to the established protocol for humic substances isolated from landfill leachate (27). Pyrolysis-GC/MS. A 1.4 mg sample of the HPO-A fraction from Norman Landfill well 35B, containing 1.4 µg of poly(4-tert-butylstyrene) (Polyscience Inc.) as an internal standard, was held at 30 °C for 2 min before being heated to a final temperature of 600 °C at 50 °C/min (Pyran System TCMS, Ruska Laboratories). Pyrolysis products were captured in a cryotrap held at -60 °C. The trap was then heated at a rate of 5 °C/min to a final temperature of 300 °C, at which it was held isothermally for 21 min. Pyrolysis products were analyzed by gas chromatography/mass spectrometry using a DB-5 MS capillary column (30 m × 0.32 mm i.d., J&W Scientific) and an INCOS 50 XL (Finnigan MAT) quadrupole mass spectrometer. Peaks were identified based on their mass spectra. Green River Shale (in-house standard) and Suwannee River fulvic acid (International Humic Substances Society) were used as pyrolysis reference standards. Electrospray Ionization Mass Spectrometry (ESI-MS). Details of the instrumentation and conditions used can be found elsewhere (28). Other details relevant to this study are as follows. The groundwater sample was analyzed using 75%

TABLE 1. Dissolved Organic Matter (DOM) Fraction Concentrations and Percentages of Isolated DOM in Norman Landfill Leachate Samples Well 35B

Well 40B

fraction

concn, mg DOM/L

percent of DOM

concn, mg DOM/L

percent of DOM

HPO-N HPO-A HPI-A1 HPI-A2 HPI-N HPI-B

18.3 63.3 10.4 2.3 1.9 9.9

17.2 59.7 9.8 2.2 1.8 9.3

35.1 119.3 21.2 4.0 9.6 19.1

16.9 57.2 10.2 1.9 4.6 9.2

methanol/water. Abietic acid (tech., 70%) was purchased from the Aldrich Chemical Co. and used as received. Abietic acid was ozonized according to the method of Bailey (29) and the product obtained by rotary evaporation of the final solution and drying in a vacuum oven overnight at 60 °C. The groundwater sample was dissolved at a concentration of 1 mg/mL. Abietic acid and its ozonylsis product were prepared at a concentration of 200 µM. All MS/MS spectra were acquired for 10 min with a scan time of 2 s using a 100 µL injection of the sample solution.

Results and Discussion Discovery of Tepenoid Indicators in DOM from the Norman Landfill. DOM fraction concentrations for the two landfill leachate contaminated groundwater samples are presented in Table 1. The complete DOM fractionation is presented because terpenoid indicators were found in multiple fractions. The DOM compositions of the two landfill leachate samples are similar, but DOM concentrations are different indicating varying degrees of landfill leachate dilution with native groundwater. The CPMAS-13C NMR spectra of DOM fractions from the landfill leachate (well 40B) are shown in Figure 1, and the normal and dipolar dephased CPMAS-13C NMR spectra of the HPO-N fraction of well 35B are shown in Figure 2. Elemental analyses had destroyed the HPO-N fraction from well 40B, and dipolar-dephased spectra were acquired after elemental analyses. Sample mass limitations are also the reason 13C NMR spectra are presented for only one sample. The CPMAS-13C NMR spectral profiles shown in Figure 1 are generally typical of aquatic DOM fractions (30) except for some minor features that are indicative of specific contaminants, biogeochemical processes and precursor structures. The most unusual feature is that there is no anomeric carbon indicative of carbohydrates near 105 ppm in any of the fractions. As the anomeric carbon results from cyclization of an aldehyde or ketone group with an alcohol in a sugar, it is possible that the highly reducing conditions of the landfill (17) resulted in biochemical reduction of aldehydes and ketones in sugars to alcohols seen as the broad peak from 60 to 90 ppM. However, this reduction process must be specific to carbohydrates as aliphatic ketones and/ or aldehydes are seen as a broad peak from 200 to 220 ppm in the HPO-N and HPO-A fractions. The presence of pentaerythritol in the HPI-N fraction of the landfill leachate was confirmed by running a standard of this compound as shown in Figure 1. Pentaerythritol is used as a cross-linking agent in paints and resin acids. A major use of resin acids, which are terpenoid byproducts of the pulp mill industry, is as paper sizing agents. Detection of pentaerythritol is an indicator of resin acids that are released during degradation of paper in the landfill. The HPO-N fraction was selected for the dipolar diphased CPMAS-13C NMR study (Figure 2) that specifically detects methyl and quaternary carbons because it is the richest in

FIGURE 1. CPMAS-13C NMR spectra of DOM fractions isolated from landfill leachate sampled from well 40B plus Pentaerythritol standard (PE).

FIGURE 2. CPMAS-13C NMR spectrum (top) and dipolar dephased spectrum (bottom) of the hydrophobic neutral fraction isolated from landfill leachate sampled from well 35B. aliphatic hydrocarbon components characteristic of terpenoids. In the aliphatic hydrocarbon region from 0 to 60 ppm, four peaks at 20, 30, 37, and 46 ppm were detected in the normal spectrum. The dipolar-dephased spectrum indicated that the 20 ppm peak was methyl, and a portion of the 37 and 46 ppm peaks was attributed to quaternary carbons. This pattern is indicative of highly branched hydrocarbon structures in resin acids as contrasted to linear hydrocarbons whose methylene peak occur near 30 ppm. The stable carbon isotope results for DOM fractions from well 35B are as follows: δ13C ) -25.02‰ for the HPO-N fraction, -27.77‰ for the HPO-A fraction, and -23.14‰ for the HPI-A1 fraction. Again, these results are typical of aquatic fulvic acid (31). δ13C values for possible DOM precursors are as follows: monocotyledon plants such as grasses ) -12 to -19‰, dicotylendon plants ) -23 to -32‰, and petroleum VOL. 37, NO. 11, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. TMAH thermochemolysis GC/MS total ion current profile of the hydrophobic neutral fraction isolated from Norman Landfill leachate (Well 35B). L ) lignin-derived compounds, PG ) polyethylene and polypropylene glycol-derived compounds, N ) nucleotides, A ) carboxylic and dicarboxylic acid-derived compounds, numbers are peak designations. / ) n-eicosane standard. hydrocarbons ) -28 to -32‰ (28). Within the dicot plants, carbohydrates are isotopically heaviest (δ13C ) -17 to -19‰), followed by proteins (δ13C ) -18 to -22‰), lipids (δ13C ) -24 to -28‰), and lignins and tannins (δ13C ) -28 to -32‰). The HPI-A1 fraction is heavier than the two hydrophobic fractions which may indicate carbohydrate precursors for the HPI-A1 fraction, but its greater carboxyl group content (as indicated by 13C NMR spectral data in Figure 1) would result in a heavier isotopic composition as the carboxyl group is about 18‰ heavier than the hydrocarbon portion of the carboxylic acids (32). Sources such as cellulose in paper and grass clippings appeared to be ruled out by the spectral and isotopic results, although cellulose in paper might be an important source of semivolatile DOM, such as acetate and ethanol that were not recovered in this study. The δ13C values for the HPO-N and HPO-A fractions fall within the lipid range which encompasses the resin acids. TMAH Thermochemolysis-GC/MS. Thermochemolysis with TMAH concurrently hydrolyzes and methylates ester and aromatic ether bonds, producing gas chromatographic peaks for numerous lignin derived methylated monomer units (5, 33-36). Figure 3 contains the total ion current profile (TIC) of TMAH thermochemolysis products for the Norman Landfill leachate HPO-A fraction. Unlike typical TMAH thermochemolysis results from terrestrially derived humic and fulvic acids (37) that display numerous and prominent lignin-derived products, only four thermochemolysis products from the Norman Landfill leachate HPO-A fraction come from lignin. Moreover, none of these products originates from syringyl monomers. Three of the products (L2, L3, and L4) are guiacyl monomers, and L1 is a cinnamyl monomer. The low concentration of lignin-based products (less than 0.1% of the total sample mass, assuming that all lignin is detected and that the response factor of lignin TMAH products is roughly similar to eicosane) indicates that lignin is not a major contributor of nonvolatile organic carbon in the Norman Landfill leachate HPO-A fraction. Cellulose derived TMAH thermochemolysis products were not detected in the Norman Landfill leachate HPO-A fraction. None of the TMAH thermochemolysis products of pure cellulose (∼20 micron, Aldrich) (data not shown) matched any of the Norman Landfill leachate hydrophobic acid fraction thermochemolysis products, either in retention time or mass spectra. 2326

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The absence of branched aliphatic TMAH products, which are expected based upon 13C NMR results, suggests that such branched aliphatic components are linked primarily by carbon-carbon bonds within fulvic acid molecules and therefore are not amenable to TMAH thermochemolysis. Under nitrate-reducing conditions, acyclic, mono-, and bicyclic alcohol monoterpenes are microbially reduced, often with concurrent carbon skeleton rearrangement, to monoterpenes (38-40). Such reactions suggest that a microbially initiated route for fulvic acid formation from terpenes is feasible under anaerobic conditions. Pyrolysis-GC/MS. Figure 4 displays the TIC of pyrolysis products for the HPO-A fraction from well 35B. Identified pyrolysis products are as follows: BTEX, alkylated monoaromatics, naphthalene and C1 to C3 methylated naphthalenes, and phenanthrene and C1 to C4 methylated phenanthrenes. All of these are found in gasoline and light oils (41). These hydrocarbons are not contaminants that are extractable by organic solvents but are partitioned into HPO-A structures and are released during pyrolysis (unpublished research). Despite their prominence, the total mass of all the detected aromatic compounds comprise approximately 0.55% of the total sample mass based upon the internal standard. Also present is a large, late-eluting hump (∼15% of the total sample mass) representative of a complex mixture of unidentified compounds. The lack of significant amounts of other pyrolysis products (excepting the hump) is in contrast to pyrolysis-GC/MS studies of aquatic (freshwater and marine) and terrestrial humic acids that all contained a wide range of biologically related materials characteristic of proteins, carbohydrates, and peptidoglycans as well as variable amounts of phenol and methylphenol (42). This indicates that the Norman Landfill leachate hydrophobic acid fraction may originate from different precursors or by different mechanisms than do aquatic and terrestrial hydrophobic acids, i.e., humic and fulvic acids. The lack of lignin pyrolysis products reinforces the fact that lignin contributes very little to the Norman Landfill leachate HPO-A fraction. This is consistent with the finding (43) that lignin degradation under anaerobic conditions in landfills is extremely slow relative to cellulose degradation. Release of plant resin acids from paper-sizing agents such as rosin tall oils during cellulose degradation is a hypothetical

FIGURE 4. Pyrolysis GC/MS total ion current profile of the hydrophobic acid fraction isolated from Norman Landfill leachate (Well 35B), B ) benzene, T ) toluene, EB ) ethylbenzene, m,o,p-X ) m-, o-, and p- xylene, B1-B4 ) alkylbenzenes, N ) naphthalenes and alkylnaphthalenes, C ) cresol, P ) phenanthrenes and alkyl-phenanthrenes, numbers are peak designations. / ) poly(4-tert butylsytrene) standard. process that could explain the origin and nature of the landfill leachate HPO-A fraction. These resin acids are highly branched terpenoids that are consistent with the spectral, isotopic, and pyrolysis characterizations of the leachate hydrophobic DOM fractions. Terpenoid Indicators in DOM from the Colorado River. The normal and dipolar dephased CPMAS-13C NMR spectra of the hydrophobic NOM fraction of the Colorado River Water is presented in Figure 5. The normal spectrum (Figure 5B) is very similar to the spectrum of hydrophobic acid fraction (Figure 5A) from the Norman Landfill that indicates terpenoid derived fulvic acid. This NOM fraction also gave low yields of lignin degradation products and low yields of chlorinated disinfection byproducts typically associated with tannin and lignin precursors (19). It is likely that fulvic acids derived from tannins and lignins in the Colorado River basin are removed by a combination of adsorption on iron-rich soils and photolysis during their long residence time in the river, reservoirs, and canals during the transport of this water to Los Angeles. Fulvic acids derived from terpenoids, especially the aliphatic alicyclic ring structures, should not adsorb the UV radiation causing photolytic oxidation, as do the more aromatic fulvic acids derived from tannins and lignins. Carboxyl groups distributed across aliphatic ring structures in terpenoid-derived fulvic acid are less likely to form strong metal complexes with iron that causes precipitation and adsorption on iron-rich soils. Carboxyl groups in fulvic acid derived from tannins and lignins are clustered so that they form polydentate chelate structures that strongly bind to various metal ions (44). Conventional treatment of Colorado River Water with alum and ferric chloride only removed 14% of the DOC by coagulation/flocculation, whereas waters containing high percentages of fulvic acid

FIGURE 5. CPMAS-13C NMR spectrum (A) of hydrophobic acid fraction isolated from Norman landfill leachate sampled from well 40B. CPMAS-13C NMR spectrum (B) and dipolar dephased spectrum (C) of the hydrophobic NOM fraction isolated from Colorado River Water. derived from tannins and lignins typically give 50% or greater DOC removal by conventional treatment (19). Terpenoid Indicators in DOM from Anaheim Lake and Infiltrated Groundwater. The CPMAS-13C NMR spectra of the HPO-A (fulvic acid) fractions of Anaheim Lake and infiltrated groundwater samples are presented in Figure 6. Groundwater infiltration results in a progressive decrease in the aromatic carbon fraction (110-160 ppm) of these fulvic acids. The difference 13C NMR spectrum (spectrum not VOL. 37, NO. 11, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. CPMAS-13C NMR spectra (top four spectra) and dipolar dephased spectrum (bottom) of the hydrophobic acid fraction isolated from Anaheim Lake and infiltrated groundwater samples. shown) between Anaheim Lake and well AMD 9/1 also shows a phenol band (140-155 ppm) indicative of tannins and lignins precursors that are removed during infiltration. In the aliphatic carbon region (0-90 ppm), the peak at 40-50 becomes more intense and well defined with SAT treatment, a valley deepens near 30 ppm, and the peak at 77 ppm shifts to 85 ppm. The dipolar-dephased spectra (Well AM 44) of Figure 6 indicates branched methyl groups near 20 ppm, quaternary carbons near 50 ppm, and quaternary C-O bonds near 85 ppm. These spectral changes are consistent with the bacterial degradation of the alicyclic ring of a terpenoid such as camphor shown in the following reaction sequence (12).

Ring opening in the above reaction sequence occurs by insertion of oxygen to form a lactone (steps 3 and 5) followed by hydrolysis of the ring ester to a carboxylate (steps 4 and 6). Ring methylenes are destroyed in this reaction sequence, but branched methyl groups (NMR peak at 20 ppm) and the quaternary carbon (NMR peak near 50 ppm) to which the two methyl groups are attached survive the biodegradation. A quaternary C-O linkage (shown in bold with a NMR peak near 85 ppm) is created; in addition many terpenoids already have quaternary C-O linkages in the parent structures (10) that may be selectively preserved during biodegradation. Additional diagenetic processes probably cause the loss or condensation of olefinic bonds in the reaction products. Likewise, acyclic, alcohol monoterpines undergo microbial degradation reactions forming unsaturated lactones with

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FIGURE 7. Normal CPMAS-13C NMR spectrum (A) and dipolar dephased spectrum (B) of fulvic acid isolated from the Tomago Sand Beds (TSB-FA). Normal CPMAS-13C NMR spectra of abietic acid (C) and the ozonation product of abietic acid (D).

quaternary C-O linkages (shown in bold with an NMR peak near 85 ppm in the dipolar-dephased spectrum of Figure 6). For example, linalool is degraded by a soil pseudomonad to form, among several products, an unsaturated lactone (45). This is believed to occur through the epoxidation of the 7,8 olefinic bond (step 1) (46), which upon further oxidation, produces the linanol oxide (step 2) and the unsaturated lactone (step 3). The dipolar dephased spectrum of Figure 6 is also similar to the dipolar dephased spectra of Figures 1, 2, and 5 which is an indication of the terpenoid component in fulvic acids in landfill leachate (Figure 1), surface water (Figure 2), and infiltrated groundwater (Figure 5). The dipolar dephased spectrum of the Suwannee River fulvic acid (derived from lignin and tannin) is very different with most of the quaternary carbon occurring in the aromatic and CdO regions of the spectrum (47). Terpenoid Indicators in DOM from Groundwater, Tomago Sand Beds, Australia. The CPMAS-13C NMR spectrum of the TSB-FA, the dipolar dephased spectrum, and the spectra of abietic acid and its ozone oxidation product are presented in Figure 7. The dipolar-dephased spectrum indicates methyl and quaternary aliphatic carbon indicative of terpenoid precursor structures that are similar to samples discussed previously, but the aromatic carbon content (100-160 ppm) in the CPMAS-13C NMR spectrum is much greater than the previous samples discussed in this study. The aromatic carbon may be caused by residual aromatic structures from tannins and lignins that are not degraded or retained by adsorption on the iron and aluminum sesquioxides in the spodic horizon during groundwater infiltration; however, aromatic carbon structures associated with phenols indicative of tannin and lignins (145-160, 110-120 ppm) are not prominent in the spectrum. Also, aliphatic substituted succinic acid structures derived from aromatic ring cleavage biodegradation reactions in lignin (47) give a major peak near 35 ppm, but the major aliphatic carbon peak of the TSB-FA spectrum is near 45 ppm which is indicative of alicyclic ring structures and quaternary carbon as shown by the CPMAS-13C NMR spectra of abietic acid and its ozone oxidation product.

FIGURE 8. Electrospray ionization mass spectra of fulvic acid isolated from the Tomago Sand Beds (TSB-FA) (A) and the ozonation product of abietic acid (B). Product ion spectra of m/z 311 for each sample are shown as insets. An alternative hypothesis to account for the aromatic structures in TSB-FA is that certain bacteria oxidatively degrade alicyclic rings by aromatizing the ring, followed by insertion of phenols, oxidation to orthoquinones, and ring cleavage to muconic acid (13). Abietic acid is also known to convert to dehydroabietic acid (one aromatic ring) and retene (three aromatic rings) in abiotic oxidative reactions in pine tar (48). The groundwater of the Tomago Sand Beds is acid (pH 4.90) as opposed to the near neutral pH values of the previous samples of this study. This acid environment is likely to result in a different microbiology with different biodegradation pathways as contrasted to neutral pH environments. Electrospray Ionization Mass Spectrometry. ESI-MS provides detailed molecular level mass data for the polycarboxylic acids found in the fulvic acid while minimizing fragmentation (24). The ESI-MS mass spectra and tandem mass spectra for TSB-FA and the ozonation product of abietic acid are shown in Figure 8. The ESI-MS mass spectrum for TSB-FA (Figure 8a) is typical of that observed for aquatic substances and shows a distribution of ions at odd masses reflecting the complex mix of organic polycarboxylic acids (24, 49, 50). Although the distribution of ions is thought to represent only the polycarboxylic acids that ionize most efficiently and is likely to include molecular ions, fragments, adducts and multiply charged ions, certain structural characteristics can be revealed (24, 50, 51, 52). The ESI-MS mass spectrum for TSB-FA contains a series of peaks separated by 2 Da and submaxima separated by 14 Da. Stenson et al. (53) recently showed for Suwannee River fulvic acid using highresolution mass spectrometry that these were attributable to compounds differing in structure by double bounds or ring structures (2 Da) and CH2 groups (14 Da). High-resolution data for older samples of TSB-FA (data not shown) gave similar results. This suggests, and is consistent with, samples from this site containing a mixture of aliphatic and arylaliphatic carboxylic acid compounds derived from terpenoid precursors (24).

To further substantiate the data, the terpenoid resin acid, abietic acid, was subject to oxidation (ozonation), and its product was subjected to ESI-MS analysis. Abietic acid was subjected to ozonolysis as this reaction can be used to cleave olefinic bonds to carboxylic acids (29). The reaction scheme for abietic acid is shown below.

The abietic acid was of 70% purity, and the supplier indicated that impurites were likely to be other resin acids such pimaric and palaustric acid. This was confirmed by GC/MS of the methyl ester derivative, which revealed minor amounts of at least seven other resin acids. The mixture of resin acids thus, once subjected to ozonolysis, produces a mixture of oxidized resin acids that may simulate terpenoids that have been degraded in nature. Ring opening reactions occur during the degradation of olefinic compounds such as resin acids (13, 14). A mixture of several different products, with the major product shown above, was confirmed by GC/ MS of the methyl ester derivatives. The ESI-MS mass spectrum for the abietic ozonation product (Figure 8b) showed a distribution of ions separated by 2 Da and submaxima separated by 14 Da that was comparable to that of TSB-FA. The spectrum has fewer ions present as it is a simpler mixture of compounds; however, it is the range over which they occur and the spacing of the ions that further supports the hypothesis that degraded terpenoids have a contribution to the TSB-FA sample. VOL. 37, NO. 11, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Product ion tandem mass spectrometric experiments were conducted for selected masses in both samples to confirm similarities. Product ion spectra for m/z 311 are shown as insets in Figure 8. This mass was selected as it gave good signal-to-noise ratios for both samples and did not correspond to a major abieitc acid oxidation product, such that it may represent several compounds with this nominal mass. The product ion spectra are characteristic of aquatic humic substances showing the major ions separated by 44 and 18 Da due to CO2 and H2O losses from carboxylic acids (24). The occurrence of lower mass ions in clusters separated by 14 Da, such as those at m/z 59 and 73 (corresponding to acetic and propanoic acid fragment ions), are an indication of fragmentation of aliphatic structures. The similarity of the product ion spectra for both samples strongly suggests that similar aliphatic structures are present. The ESI-MS mass spectra for terrestrial HS samples can reveal the presence of lignin degradation products (28). ESIMS (spectra not shown), TMAH thermochemolysis, and 13C NMR analysis revealed that lignin derived compounds have only a minor contribution (