Research Major Structural Components in Freshwater Dissolved Organic Matter BUUAN LAM,† ANDREW BAER,† MEHRAN ALAEE,‡ BRENT LEFEBVRE,§ ARVIN MOSER,§ ANTONY WILLIAMS,§ AND A N D R É J . S I M P S O N * ,† Department of Chemistry, University of Toronto Scarborough, Toronto, Ontario, Canada, M1C 1A4, National Water Research Institute, Environment Canada, 867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario, Canada, L7R 4A6, and Advanced Chemistry Development Inc., 110 Yonge Street, 14th floor, Toronto, Ontario, Canada, M5C 1T4
Received June 2, 2007. Revised manuscript received October 7, 2007. Accepted October 9, 2007.
Dissolved organic matter (DOM) contains a complex array of chemical components that are intimately linked to many environmental processes, including the global carbon cycle, and the fate and transport of chemical pollutants. Despite its importance, fundamental aspects, such as the structural components in DOM remain elusive, due in part to the molecular complexity of the material. Here, we utilize multidimensional nuclear magnetic resonance spectroscopy to demonstrate the major structural components in Lake Ontario DOM. These include carboxyl-rich alicyclic molecules (CRAM), heteropolysaccharides, and aromatic compounds, which are consistent with components recently identified in marine dissolved organic matter (1). In addition, long-range proton-carbon correlations are obtained for DOM, which support the existence of material derived from linear terpenoids (MDLT). It is tentatively suggested that the bulk of freshwater dissolved organic matter is aliphatic in nature, with CRAM derived from cyclic terpenoids, and MDLT derived from linear terpenoids. This is in agreement with previous reports which indicate terpenoids as major precursors of DOM (2). At this time it is not clear in Lake Ontario whether these precursors are of terrestrial or aquatic origin or whether transformations proceed via biological and/ or photochemical processes.
Introduction Dissolved organic matter (DOM) is a complex, heterogeneous mixture found ubiquitously in nature. It comprises a major mobile fraction of organic carbon on Earth and is an intimate link between the terrestrial and aquatic environment (3, 4). Terrestrial and freshwater DOM experiences an annual flux of approximately 0.4 × 1015 g C/year via riverine discharge (5) to the marine environment. It is believed that DOM plays a significant role in the enhanced solubility (6) of chemical * Corresponding author phone: 1-416-287-7547; fax: 1-416-2877279; e-mail:
[email protected]. † University of Toronto Scarborough. ‡ Environment Canada. § Advanced Chemistry Development Inc. 8240
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contaminants and may potentially be a shuttle for the longrange transport of chemicals globally. Thus, the cycling of DOM from freshwater to marine sources is not only important in the global carbon cycle, but is a significant mediator in the fate and transport of pollutants in the environment. Despite this importance, there is still much to be revealed regarding the structural components that make up this complex environmental mixture and how these compounds vary between freshwater and marine environments. Here, dissolved organic matter from Lake Ontario, Canada is studied in detail using multidimensional nuclear magnetic resonance (NMR) spectroscopy. Lake Ontario covers just over 19000 km2, contains over 1600 cubic kilometers of freshwater (7), and is part of the Great Lakes, which represent the world’s largest freshwater lakes system. Recently a pivotal paper by Hertkorn et al. (1) utilized a range of modern 1-D and 2-D NMR approaches in combination with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to identify carboxyl-rich alicyclic molecules (CRAM) in oceanic DOM. This pioneering paper has been essential in providing key assignments making further NMR based studies possible. Here, we build upon the work of Hertkorn et al. (1) who have reported on major structural and refractory components of marine DOM, extending these initial findings to show that marine and freshwater DOM share many structural similarities. Two dimensional solution-state NMR spectroscopy (1, 8–15) and three-dimensional NMR (16, 17) are becoming widely employed and very powerful techniques to study structures and interactions in environmental chemistry. Long-range proton-carbon correlations have been especially useful for identifying structures in terrestrial derived materials (9, 14, 18). However, collecting such data is extremely challenging given the relatively low sensitivity of the experiments and the fast relaxation in DOM. Combining recent improved long-range NMR experiments with relaxation optimized delays (19) permits weak long-range correlations toberecordedforaquaticDOM.Thelong-rangeproton-carbon correlations help confirm previous assignments of CRAM and support the presence of an aliphatic material derived from linear terpenoids in freshwater DOM.
Materials and Methods Sample Preparation. Freshwater was pumped from a depth of 50 cm and at a distance of 20 m from the Lake Ontario shoreline (Darlington Provincial Park, Ontario, Canada). Lake Ontario DOM (LO-DOM) was isolated as described by Simpson et al. (20). Briefly, water from Lake Ontario was prefiltered through 0.22 µm poly(vinylidene difluoride) (PVDF) filters. DOM was isolated on diethylaminoethylcellulose resin, recovered using 0.1 M NaOH, ion-exchanged, (note: pH was adjusted to ∼6 after ion-exchanging), and freeze-dried. Excess salts were removed from the sample by extensive dialysis against double-distilled water using 100 Dalton molecular weight cutoff cellulose ester tubing. The sample was once again freeze-dried to obtain a powder. NMR Analysis. Sample (100 mg) was resuspended in 1 mL of deuterium oxide (D2O) and titrated to pH 13.1 using NaOD (40% by weight) to ensure complete solubility. Samples were analyzed using a Bruker Avance 500 MHz NMR spectrometer equipped with a 1H-BB-13C 5 mm, triple resonance broadband inverse probe. Average 1H relaxations were estimated at ∼380 ms (T2) and ∼630 ms (T1) for the LO-DOM. 1-D solution state 1H NMR experiments were performed with 512 scans, a recycle delay of 3 s, 32768 time domain points, and an acquisition time of 1.6 s. Solvent 10.1021/es0713072 CCC: $37.00
2007 American Chemical Society
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suppression was achieved by presaturation utilizing relaxation gradients and echoes (21). Spectra were apodized through multiplication with an exponential decay corresponding to 1 Hz line broadening, and a zero filling factor of 2. Diffusion-edited experiments were performed using a bipolar pulse longitudinal encode-decode sequence (22). Scans (1024) were collected using a 1.25 ms, 53.5 gauss/cm, sine-shaped gradient pulse, a diffusion time of 50 ms, 8192 time domain points, 410 ms acquisition time, and a sample temperature of 298 K. Spectra were apodized through multiplication with an exponential decay corresponding to 10 Hz line broadening and zero filling factor of 2. 13 C data were acquired using a 5 mm broadband probe using WALTZ-16 decoupling. Scans (37888) were collected using inverse gated decoupling, an 8 s recycle delay, and an acquisition time of 186 ms. Multiplicity editing was carried out using distortionless enhanced polarization transfer and a quaternary only sequence (23). All 13C spectra were processed with an exponential function corresponding to a 35 Hz line broadening. Heteronuclear multiple quantum coherence (HMQC) spectra were collected in phase-sensitive mode using echo/ antiecho gradient selection. 1024 scans were collected for each of the 256 increments in the F1 dimension. 1024 data points were collected in F2, a 1J 1H-13C value of 145 Hz, a relaxation delay of 1 s was employed, and an acquisition time of 77 ms. The F2 dimension was multiplied by an exponential function corresponding to a 15 Hz line broadening, whereas the F1 dimension was processed using a sinesquared function with a π/2 phase shift and a zero-filling factor of 2. Heteronuclear multiple bond correlation (HMBC) were carried out in phase-sensitive mode using echo/antiecho gradient selection (19) and a relaxation optimized delay of 25 ms for the evolution of long-range couplings. 2048 scans were collected for each of the 128 increments in the F1 dimension. 2048 data points were collected in F2, a relaxation delay of 1 s, and acquisition time of 154 ms were used. The F2 dimension was multiplied by an exponential function corresponding to a 15 Hz line broadening, whereas the F1 dimension was processed using a sine-squared function with a π/2 phase shift and a zero-filling factor of 2. Spectral predictions were carried out using Advanced Chemistry Development’s ACD/SpecManager and ACD/2D NMR Predictor using Neural Network Prediction algorithms (version 10.02). Parameters used for prediction including spectral resolution, and base frequency were chosen to match those of the real data sets as closely as possible. For comparison of predicted versus real shifts, narrow line widths of 2 Hz 1H and 5 Hz 13C were used to accurately evaluate the predicted shift data. For HMQC, only one bond 1H-13C couplings are considered, whereas in HMBC, only 2 and 3 bond couplings are considered, and in rare cases, 4 bond couplings may also be included as a further investigative tool. In Figure 4A the spectral line widths have been increased to 10 Hz (1H) and 20 Hz (13C). This is for visualization purposes only such that the crosspeaks are enlarged and easier to see.
Results and Discussion Characterization of Lake Ontario DOM using 1D and 2D NMR data. Figure 1 shows the 1H NMR data of freshwater DOM from Lake Ontario (LO-DOM). Major structural components present include aliphatics (I), carboxyl-rich alicyclic molecules (CRAM) (II), carbohydrates (III), and aromatics (IV). Further discussion is provided later in this paper. Signals from larger macromolecular and/or aggregated species can be further emphasized by the use of diffusion editing, which retains only signals from macromolecular and/or aggregated species (24). The diffusion edited spectrum (Figure 1B) compared to that of the conventional 1H NMR spectrum
FIGURE 1. 1H NMR spectra showing (A) freshwater DOM from Lake Ontario (LO-DOM), and (B) the diffusion edited spectrum for LO-DOM. Resonances from I, aliphatics; II, CRAM; III, carbohydrates; and IV, aromatics. (Figure 1A) shows a generally similar profile, indicating that the structures present as stable aggregates and/or macromolecular species have a generally similar composition when compared to the sample as a whole. At this time, it is not possible to distinguish whether the species are macromolecular in nature or simply aggregated/associated due to the high concentration of the sample, furthermore it is not possible to provide any insight into how much of the sample may be present in the form of aggregates/macromolecules. Future studies based on diffusion ordered spectroscopy are planned to address this aspect of DOM (16). Figure 2 depicts the 13C spectrum (A) of the LO-DOM sample, and the corresponding, scaled, multiplicity edited spectra (B). The LO-DOM contains strong resonances from CRAM (see ref 1 and later) and has a lower CH/CH2 ratio 1.6) in comparison to oceanic DOM (ratio 2.3) isolated by ultrafiltration (1). This higher relative abundance of CH2 vs CH may be partially explained by the presence of materials derived from linear terpenoids (MDLT). This component and others present are discussed later in this paper. Due to the large degree of overlap, extracting detailed structural information from the 1D NMR alone is difficult. 2D NMR experiments provide increased spectral dispersion as well as additional connectivity information, which permits further characterization of the chemical functionalities present in DOM. Figure 2C shows the HMQC NMR spectrum for the LO-DOM sample. The HMQC experiment detects one bond 1H-13C connectivities in an organic structure (13). The HMQC NMR spectrum identifies a range of chemical constituents present, including anomeric units in carbohydrates (1), functionalized and/or conjugated olefins (also see “MDLT section” later for further considerations) (2), aromatics (3), N-acetyl and/or O-acetyl, S-CH3 (4), aliphatics (5), carboxyl-rich alicyclic molecules (6) (CRAM) ref (1) (see later for discussion), methyl esters (7), methylene (CH2) from carbohydrates (8), and methine (CH) from carbohydrates (9). Note, assignments offered here are also consistent with total correlation spectroscopy (TOCSY), nuclear overhauser effect spectroscopy, and edited heteronuclear 2D correlations (data not shown), as well as literature assignments (1). It is interesting to note that the methoxy group from lignins, often the most intense signal in soil organic matter (13), is not present in LO-DOM, indicating that terrestrial inputs are quickly transformed in Lake Ontario. The methyl ester region (region 7, Figure 2C) should not be confused with the methoxy from lignin which is not present in the LO-DOM sample. It is important to add that elemental analysis (C:N ratio 20:1) and TOCSY NMR data (not shown) were also collected for the LO-DOM sample. TOCSY and HMQC (HMQC assignment VOL. 41, NO. 24, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 2. 13C NMR spectra of LO-DOM. (A) 13C NMR spectrum (all carbons), PMCLT ) predominantly material derived from cyclic and linear terpenoids, SCA ) region will contain a small contribution from acetate (see HMQC Figure 2C and Hertkorn et al. (ref 1) for more details). (B) Edited spectrum showing nonprotonated (NP) (blue); methine (CH, black); methylene (CH2, red), and methyl (CH3, green) carbon. Inset indicates the percentage of each type of carbon present. It is important to note that the spectra contain considerable overlap and assignments refer to only the predominant species expected in each region. (C) Heteronuclear multiple quantum coherence (HMQC) spectrum for the LO-DOM (left) and zoom region of the HMQC spectrum with contours reduced by a factor of 5 for clarity (right). Specific assignments include: 1 ) anomeric carbon from carbohydrates, 2 ) conjugated unsaturated aliphatics, 3 ) aromatics, 4 ) N-acetyl and/or O-acetyl and S-CH3, 5 ) aliphatics, 6 ) CRAM, 7 ) methyl esters, 8 ) methylene from carbohydrates, and 9 ) methine from carbohydrates, 10 ) region where specific methines from intact cyclic terpenoids would be expected. NCDB ) nonconjugated double bonds. not discussed explicitly, due to its low abundance) contain very weak contributions from peptides/proteins (10, 12). Protein has been considered as a potential DOM constituent but is only present as a minor/trace component in this sample and cannot account for the major resonances assigned to terpenoids in this manuscript. HMBC provides long-range 1H-13C connectivities (generally up to 3 bonds) and provides critical information as to how H-C units are structurally organized (13). Information gained from HMBC spectra (Figure 3A-C), is discussed below in relation to specific structural components. Identification of Major Components in LO-DOM. Several structural components have been isolated and shown to comprise marine DOM, including polymeric carbohydrate moieties (1, 25–27), long chain aliphatic compounds (1, 27, 28), acetyl (1, 27, 28), aromatics (1, 27), peptide/protein (1, 11), and recently, carboxyl-rich alicyclic molecules (CRAM) (1). In comparison, the 1H NMR spectra obtained for LODOM in this study has a general overall profile that is similar to many of the DOM spectra presented in the literature from 8242
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FIGURE 3. 2D HMBC spectra of LO-DOM. (A) Expansion including the full CRAM (I) and MDLT (II) regions. (B) Expansion of (A) showing the 5–115 ppm (carbon) region, (C) expansion of (A) showing the carboxyl region. Notations La, Lb, Lc, Ld are used for identification of crosspeaks in the HMBC, see text for discussion. both marine and freshwater sources (1, 29). The LO-DOM sample appears remarkably similar to a DOM sample from the Pacific Ocean described by Hertkorn et al. (1). In this sample, Hertkorn et al. (1) described the three major components as carbohydrates, aliphatics (including peptidederived aliphatics), and CRAM (including some weaker contributions from peptides). Using the same means of integration (see ref 1), quantification of the 1H NMR resonances for LO-DOM resulted in values of ∼17% for the
carbohydrate region, ∼12% for the aliphatic region, and ∼62% for the CRAM region. These three regions combined comprised the majority (∼91%) of the proton signals from LODOM, similar to the quantities reported by Hertkorn et al. (1) for marine DOM. This is further supported by the carbon data and quantitative estimates. Using the integration procedure outlined in ref 1) it can be determined that over 75% of the carbon is likely terpenoid derived, including CRAM and a fraction derived from linear terpenoids (see later). Additionally, up to 17% appears to be from carbohydrate derived material, and up to 8% from aromatics (some of which may be associated with the terpenoid derived materials). Carbohydrates. Carbohydrate components have been shown to represent up to 50% of high-molecular weight (HMW) surface marine DOM but comprise a much smaller proportion of deeper ocean waters (1, 30). Of these however, only a small fraction are simple carbohydrate structures, which contribute only a small percentage to the total composition of marine DOM (27). The majority of carbohydrates identified in the literature appear to comprise complex polymeric structures referred to as heteropolysaccharides (HPS) (25, 30) or acyl polysaccharides (APS) which contain carbohydrates and acetate to varying degrees (31). These polymeric carbohydrates have been shown to be major constituents of ultrafiltered DOM and a rapidly cycling component of marine surface waters (1, 31). Similarly, the freshwater LO-DOM contains a contribution from carbohydrates which are not removed during diffusion editing (Figure 1B) and may potentially be associated with acetyl groups (Figure 2C, region 4). However, 1H and 13C NMR (note especially the weak anomeric signal in the carbon data, Figure 2A) for the LO-DOM indicate that the quantity of carbohydrates in the LO-DOM sample (up to 17%), to be less than that observed in the deep (27%) or surface (44.6%) ocean (1). However, close examination of the HMQC spectrum (Figure 2C) indicates the contour shapes to be roughly similar indicating that while the relative quantities of materials may be less in the LO-DOM, both lake and ocean DOM contain complex carbohydrates that may be generally similar. Unfortunately, in the case of carbohydrates, the 2D HMBC data does not provide additional information as to the structures present in freshwater DOM, mainly due to the majority of carbohydrate signals being below the detection limit of the HMBC. Further work is needed to confirm the similarity of the carbohydrates in freshwater and marine DOM, both in terms of origin and structure. Carboxyl-Rich Alicyclic Molecules (CRAM). Spectral characteristics in the proton chemical shift region from 1.7–3.3 ppm, closely resembling those depicted in Figure 1A and 1B, have been shown to be prevalent in a vast majority of the 1H NMR spectra in the literature for both marine and freshwater DOM (1, 20, 28–31). However, the components that comprise this region were not adequately defined until a recent study by Hertkorn et al. (1), who showed this region to largely contain CRAM. Hertkorn et al. (1) describe CRAM as a major refractory component of marine DOM which is likely derived from sterols and hopanoids (both categories of terpenoids) and is consistent with carboxylated alicyclic structures with carboxyl to aliphatic carbon ratios of approximately 1:2 to 1:7. Although not conclusively shown to be present in freshwater DOM, as Hertkorn et al. (1) points out, the presence of CRAM in freshwater is likely due to the global distribution of biomolecules and the similarity in biogeochemical processes that occur within the environment. It is not surprising therefore, that evidence for CRAM-like structures in the LO-DOM sample are seen in both the 1D and 2D NMR spectra. It is also interesting to note that signals characteristic of intact “parent” cyclic terpenoids (mainly diterpenoids and higher) are not observed in the HMQC
spectrum indicating that DOM constituents are highly transformed and functionalized (Figure 2C, region 10). Longrange correlations from the HMBC data (Figure 3A, region I) not only substantiate the presence of CRAM in LO-DOM, but also provide strong evidence to corroborate the structure of CRAM proposed by Hertkorn et al. (1). As previously noted, HMBC identifies long-range 1H-13C correlations. HMBC correlations (Figure 3A, region I) show carboxylic moieties directly coupled to alicyclic rings in the LO-DOM sample. This is consistent with the CRAM structures found by Hertkorn et al. (1) in marine DOM. In fact, it would appear that the CRAM present in LO-DOM contain structures with multiple fused, nonaromatic rings, with a high ratio of substituted carboxyl groups most consistent with Isomer I proposed by Hertkorn et al. (1). The HMBC data also shows that the protons attributed to CRAM mainly correlate with carboxylic moieties (cf. above) but not with other functionalities [i.e., methyl ((13C) < 25 ppm) and hydroxyl-substituted carbon ((13C): 60–90 ppm)]; hence, the lack of respective 1H-13C HMBC cross peaks. These findings were supported by computation of HMBC spectra for a range of carboxylated terpenoids. However, only those computed spectra derived from alicyclic, nonaromatic rings with a high ratio of substituted carboxyl groups produced a similar HMBC profile. As an example, Figure 4A shows the predicted HMBC spectrum for Isomer II (see ref 1) for discussion of the isomers) which based on HSQC data alone could be a feasible CRAM component (1). However, the correlation between the LO-DOM HMBC data (Figure 3A) and the predicted HMBC data is poor. On the other hand, the predicted HMBC spectrum for a molecule closely related to isomer I (1) shows a much stronger correlation to the observed HMBC data for LO-DOM. Note that using high resolution mass spectrometry, Hertkorn et al. (1) found over 600 ions with molecular compositions suggestive of CRAM in their marine DOM sample; therefore, it is impossible to provide an exact “structure” of CRAM, as numerous, structurally different components are likely contributing to the signals in this region. The structure shown as isomer I in Figure 4A is shown as an example only, but does contain two key features that are likely characteristic of all CRAM structures; a cyclic terpenoid backbone and a high degree of carboxylation. Further details, for example, additional substitutions, cross-linkages, molecular size, etc., cannot be determined from the NMR data at hand. Material Derived from Linear Terpenoids (MDLT). Terpenoids constitute the largest family of natural products known with over 30000 identified compounds (32). Terpenoids are classified by the number of five carbon isoprene units in their structures. Both linear and cyclic terpenoids are prolific in nature, with many larger cyclic terpenoids being synthesized from their linear counterparts (33). Carotenoids represent just one class of linear terpenoids and are known to be prevalent in the aquatic environment (34–36). Over 650 carotenoid species have currently been identified in aquatic organisms, with a net annual production estimated at over 100 million tons from photosynthetic organisms alone (37). The fate of linear terpenoids is not well understood (34), especially in freshwater environments, in which they are thought to be preferentially preserved (36) or produce various transformation products (38). Considering this, it is feasible that material derived from linear terpenoids could be present in DOM. Most linear terpenoids contain a high degree of unsaturation with many containing extensive conjugated double bond systems (35, 39). Interestingly, characteristic resonances VOL. 41, NO. 24, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. (A) Predicted HMBC for isomer II and one closely related to isomer I; both isomers from ref 1. From HMBC data isomer I is most representative of CRAM in LO-DOM (see text for details). (B) Schematic outlining potential reactions of a methylated double bond in DOM and conjugated linear terpenoids (see text for details). Shaded regions (a-d) represent those substituents which give rise to the corresponding aliphatic cross peaks in the HMBC (Figure 3A, region II, La-d), see text for discussion. PAP ) photo and auto-oxidation product (44). from conjugated double bonds are visible in the HMQC spectrum of LO-DOM (Figure 2C, region 2) which suggests the presence of conjugated linear terpenoids in the LO-DOM sample (40). Predictions (not shown) indicate that the relatively “tight grouping” in this region fits very well with conjugated double bond materials. Note these signals fall out of the typical chemical shift region for common aromatic natural organic matter (NOM) constituents (41). It is im8244
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portant to stress that some chemical shifts from fivemembered heterocycles (for example in certain substituted furans, pyrroles, etc.) can also resonate in this region (also in many cases overlapping into region 1). Predictions (not shown) indicate, in some cases, resonances from other positions in the five membered heterocyclic rings are not well represented in the DOM HMQC data. However, due to spectral overlap, the diversity of five-membered heterocycles,
which could potentially be DOM constituents, and that N-aromatics have been identified in coastal DOM ( (42), (43)), the presence of five membered heterocyclic rings cannot be ruled out, and future research will be required to fully assess the contributions of these compounds. The HMBC data further supports the presence of DOM constituents derived from linear terpenoids (Figure 3A, region II). First, it is clear from Figure 3C that the carboxyl groups from region II, do not have the same distribution of carbon chemical shifts as those from CRAM (region I). Indeed, extensive predictions indicate that if region II were derived from partially formed CRAM, the carboxyl groups would be expected on the same carbon plane and would also fall over a very wide 1H region spanning 1.5–3 ppm. This region is marked with a gray shaded box in Figure 3C. The same argument holds true for the CH, CH2 region for CRAM (Figure 3B, region Ca) which only partially overlaps with region II. This has also been confirmed with extensive spectral predictions (both HMQC and HMBC, data not shown) which support the presence of CRAM but not the presence of partially carboxylated cyclic terpenoids at any abundance. Consider, for example, that unaltered terpenoids give rise to peaks in the region labeled 10 (Figure 2C) that are not observed. These observations indicate that region II is unlikely to result from CRAM or partially formed CRAM. Second, region II cannot be predominantly derived from simple long chain aliphatic molecules (for example cellular lipids, etc.). Any molecule containing a significant proportion of unsubstituted (not in close proximity to a functionality) methylene would contain a dominating carbon signal at ∼29 ppm in the carbon spectrum. From the CH2-only carbon spectrum (Figure 2B) and conventional carbon spectrum (Figure 2A) this is not the case. Furthermore, in simple straight chain structures (for example, many lipids) a COOH is most often connected to an adjacent carbon which is protonated (methylene in the case of a terminal COOH, methine in the case of a mid chain COOH (these protons resonate from ∼2.0–2.5 ppm). In such cases, the long-range correlation would not occur as observed in region II (Figure 3C) but in the region shaded with a red box. These considerations, in combination with extensive chemical shift predictions, indicate that region II cannot arise from long chains that contain COOH groups substituted on nonquaternary carbons. Third, the chemical shifts for region II (Figure 3A and 3B) are somewhat unusual. The spectra demonstrate that region La (mainly CH3, see Figure 2B) is correlated to a COOH Ld. This indicates that COOH and CH3 are connected over distance of 3 bonds or less (the distance observable in an HMBC experiment). Furthermore, other oxygenated functional groups (region Lc) are also in close proximity. Region Lc is especially interesting as it resonates at an unusually high chemical shift, 70–95 ppm, and the apex of this resonance (∼86 ppm, carbon) corresponds mainly to nonprotonated carbons (see Figure 2B). Note correlations to double bonds are not observed due to lack of crosspeaks in the region highlighted with a box in Figure 3A. Thus the question arises how can a CH3, be in close proximity to a carboxyl group and an oxygenated quaternary carbon, yet still resonant at ∼1.3 ppm (1H)? Extensive chemical shift prediction (HMQC and HMBC, not shown) indicate the general structures that best fits the spectral information are oxidation products of methylated straight chain polyolefins (which are characteristic of linear terpenoids). It is important to point out to the reader that the following structures discussed are only tentative suggestions. While the authors are reasonably certain that material derived from linear terpenoids (MDLT) is present in the LO-DOM, the exact chemical form of MDLT cannot be assigned with any certainty. Figure 4B (structure 1) is phytoene, a common
precursor molecule from which many linear terpenoids are biosynthesized (37). However, the structure itself-is not likely to be present in any abundance in LO-DOM due to the absence of nonconjugated double bonds (NCDB) in the HMQC data (Figure 2C). The molecule is shown only as an example containing methylated double bonds, and at this point of the discussion the reader should only consider the fate of the double bond highlighted in orange in structure 2. Such a unit can undergo photo and auto-oxidation to form structures 3, 4, and 5 in Figure 4B (44). Such structures could feasibly undergo further oxidation to also form products 6 and 7 (tentative structures). When considered together, spectral predictions (not shown) indicate that these structures could give rise to structures that correspond to the general regions observed in the HMBC spectrum of LO-DOM (see Figure 3A). However, Region Lc in the LO-DOM HMBC spectrum resonates at ∼86 ppm, which is unusually high. The highest chemical shift assigned to region Lc from structures 3–7 is at 85 ppm (most occur in the range of 72–80 ppm). However, the most common linear terpenoid structures in the aquatic environment are fully conjugated systems (35). A common example is zeaxanthin (Figure 4B, structure 8). In fully conjugated systems the orange highlighted region (Figure 4B, structure 10) may react as described above, but the adjacent double bonds (structure 10 red) may also undergo various reactions. Note there are no peaks in the HMQC spectrum for NCDB (Figure 2C) indicating the double bonds are not left intact in the DOM. Figure 4B (structure 10) shows a simple example where the double bond is hydrated (45), and thus additional OH groups substituted close to products 3–7 (discussed above). With the proximity of these additional oxygens the chemical shifts in region Lc (Figure 4B, structures 3–7) increase to ∼80–90 ppm region, which is consistent with those observed in the LO-DOM HMBC data. Finally, it is important to consider the terminal groups of zeaxanthin which are common in many linear terpenoids. Few studies have followed the fate of linear terpenoids in the environment (38, 46) but one key study has identified loliolide (and its isomers) as the major degradation products (46). Interestingly if the double bond is excluded, the resulting cyclic structure fits fairly well with the HMBC data. This is because this particular cyclic structure contains a carboxyl, in close proximity to another nonprotonated carbon in the 80–90 ppm range (region Lc) and CH3 groups. In summary, both the conjugated chains and the terminal end groups of linear terpenoids could give rise to the structures observed in the HMBC data. At present, the exact structures formed cannot be determined and the complex patterns of overlapping crosspeaks suggest that the structures themselves display a great range of diversity. At this point it is not possible to provide absolute quantification of the CRAM vs MDLT in the LO-DOM sample. However, using methods outlined for CRAM quantification (1) it seems very likely that CRAM is in much greater abundance than MDLT. However, MDLT cannot be a minor component as it was detected easily by the relatively insensitive HMBC procedure. Future research will be needed to refine both the exact composition and structures of CRAM and MDLT after which more definite quantification of LODOM should be possible. Further Considerations. It has been demonstrated that CRAM is potentially the largest contributor to freshwater DOM in the Great Lakes system. This is consistent with findings reported for Pacific Ocean DOM (1). While CRAM appears to be derived from cyclic terpenoids, another fraction MDLT is identified in freshwater DOM and appears to be derived from linear terpenoids. In addition, smaller amounts of heteropolysaccharides and aromatics also contribute to the LO-DOM. At present, little detail can be obtained from VOL. 41, NO. 24, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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the aromatic compounds in LO-DOM, mainly due to their relatively low abundance in the sample. The fact that aquatic DOM from Lake Ontario is mainly derived from terpenoids is consistent with earlier reports that suggests the majority of dissolved organic matter from landfill sites, surface water and groundwater is also of terpenoid origin (2). Terpenoids comprise the most abundant family of natural compounds originating from both the terrestrial and aquatic environments. Given the presence of structurally similar precursor components in both freshwater and marine environments, it is difficult to ascertain whether the constituents of DOM in freshwater are of terrestrial or aquatic origin. However, in light of this, it is known that certain terpenoid structures are specific to certain species (35, 37). Thus it will be interesting to observe whether the signatures of specific “tracer” terpenoids are preserved in DOM over time, potentially providing a rich source of information as to the origins and dynamics of dissolved carbon on a global scale. Finally, it is important to point out that the multidimensional NMR approaches employed here and in other works (1), are not just helping to unravel the key structural components present in a major global carbon pool, but these approaches are also permitting more detailed assignments of complex NMR data sets. This is critical, as once assignments can be made, the full arsenal of modern nuclear magnetic resonance techniques can be better employed to understand key processes, such as aggregation, flocculation, and contaminant interactions. The understanding of these processes has been hampered by a lack of understanding of the principal structural components present in major carbon pools such as dissolved organic matter.
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Acknowledgments We thank the Canadian Foundation for Climate and Atmospheric Sciences (CFCAS) (GR-520), the Natural Science and Engineering Research Council (NSERC) (Discovery Grant, A.J.S), the International Polar Year (IPY), and Ontario Government (Early Researcher Award, A.J.S) for funding this research.
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