Environ. Sci. Technol. 2009, 43, 698–703
Photochemically Induced Changes in Dissolved Organic Matter Identified by Ultrahigh Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry M I C H A E L G O N S I O R , * ,†,§ BARRIE M. PEAKE,† WILLIAM T. COOPER,‡ DAVID PODGORSKI,‡ JULIANA D’ANDRILLI,‡ AND WILLIAM J. COOPER§ Chemistry Department, Otago University, P.O. Box 56, Dunedin, New Zealand, Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida, and Urban Water Research Center, Department of Environmental and Civil Engineering, University of California, Irvine, Irvine, California 92697
Received August 14, 2008. Revised manuscript received November 22, 2008. Accepted November 26, 2008.
Sunlight-induced molecular changes have been observed in two samples of dissolved organic matter (DOM) collected in the Cape Fear River system, North Carolina, USA. The molecular composition of a water sample collected in the Black River (sample B210, salinity 0) and another water sample collected within the Cape Fear River estuary (sample M61, salinity 13.7) were analyzed using an ultrahigh resolution 9.4 Tesla (T) electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer. Additionally, the Ultraviolet/Visible (UV/vis) absorbance as well as the excitation emission matrix (EEM) fluorescence spectra were determined to identify changes in the optical properties associated with photochemical reactions of the chromophoric DOM (CDOM). The molecular formulas for the Cape Fear River Estuary (M61) sample before the irradiation experiments indicated the presence of highly aromatic compounds which were not present in the unirradiated Black River sample (B210). These aromatic compounds, with oxygensubtracted double bond equivalents (DBE-O) values greater than nine, are more photoreactive and readily photodegraded relative to saturated compounds. Compounds with DBE-O values below nine are less photoreactive. The UV/vis absorbance and EEM fluorescence results supported this different photodegradation behavior, suggesting that the photoreactivity of CDOM is highly dependent on the molecular composition of the CDOM.
Introduction Dissolved organic matter (DOM) plays an important role in the global carbon cycle (1). For example, the mass of the marine dissolved organic carbon (DOC) pool has been * Corresponding author phone: 949-273-0399; fax 949-824-3620; e-mail:
[email protected]. † Otago University. § University of California, Irvine. ‡ Florida State University. 698
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estimated to be 680-700 gigatons carbon (2) which is approximately equal to the carbon content of atmospheric carbon dioxide. The optically active (chromophoric) components of DOM (CDOM) influence ocean color (3) and modulate the intensity of the underwater light field, affecting aquatic biogeochemical processes such as those induced by exposure to ultraviolet radiation (4). Furthermore, this CDOM is photoreactive under natural light conditions and undergoes photodegradation (5-7). Many studies have suggested that such photochemically induced processes may well be the most important removal pathway of terrestrially derived CDOM (8-10) and may also lead to the production of smaller molecules which may undergo further microbiological processing, although such a pathway is not without controversy. Some research groups have reported that photodegradation of CDOM leads to products that can be consumed by bacteria (11-13), while others have reached the opposite conclusion (7, 14, 15). Therefore, it is important to determine the molecular composition of CDOM influenced by solar-induced photodegradation in order to better understand the role of photochemical processes in the overall biogeochemical cycling of CDOM. The molecular characterization of photochemically altered CDOM is a major goal in modern photobiogeochemistry and can potentially be used to explain its unique spectral properties. Such characterizations might also assist in extracting fundamental molecular information from optical properties of CDOM provided by bulk techniques such as Ultraviolet/Visible (UV/vis) absorbance and fluorescence. For example, the aromatic content of DOM has been estimated using absorption spectral slope and molar absorptivity (16, 17). In addition, the ratio of fluorescence to absorption intensities has been used to estimate the aromatic content of DOM (18, 19). However, these bulk optical measurements suffer from the major disadvantage that they provide no detailed molecular insights into the changes in molecular composition caused by photoprocesses (20-22), Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) is becoming increasingly important in the effort to explain the apparent changes in optical properties associated with sunlightinduced degradation processes (23, 24). ESI-FT-ICR-MS at high magnetic fields (g9 T) can provide unambiguous molecular formula assignments for masses up to about 500 Da. Molecular formulas greater than 500 Da can be assigned based on exact mass differences using techniques described previously (25). The present study uses the same combination of bulk optical properties and molecular ultrahigh resolution mass spectrometric techniques as those reported in a study of the outwelling of mangrove-derived DOM into an estuary along the northeastern coast of Brazil (24). High molecular weight, highly unsaturated components of mangrove CDOM were shown to be lost as it moved from its source to the coastal shelf. These molecular changes were accompanied by specific losses in emission intensities which could be explained in part by photodegradation of the high molecular weight and highly unsaturated component of the CDOM. However, other biogeochemical processes are associated with DOM outwelling, and it is thus important to isolate the specific photochemical effects that change DOM composition. In the present study, the molecular composition of DOM in water samples collected from a freshwater river and estuary in the Cape Fear River (North Carolina) system was determined before and after 21 h of simulated sunlight. Molecular formulas obtained from ESI-FT-ICR-MS were sorted according to elemental ratios (H/C, O/C), double bond 10.1021/es8022804 CCC: $40.75
2009 American Chemical Society
Published on Web 01/06/2009
TABLE 1. Changes in DOC levels, aCDOM(355), aCDOM(355)/DOC, Spectral Slope, and EEMtotal Fluorescence Intensities after the Exposure to Simulated Sunlight in Samples B210 and M61 sample
DOC (µM C)
aCDOM(355) (m-1)
aCDOM(355)/DOC (L mg-1 m-1)
EEMtotal (peak integral)
spectral slope S
B210 M61
930 ( 21 630 ( 4
32.10 16.32
before irradiance 2.9 2.2
3168592 2025070
0.0150 ( 0.0003 0.0155 ( 0.0002
B210 M61
965 ( 8 651 ( 4
31.98 14.81
after irradiance 2.8 1.9
2557021 1448700
0.0151 ( 0.0003 0.0159 ( 0.0002
equivalencies, and aromatic indices. Formulas that appeared or disappeared from the DOM spectra after irradiation were identified and plotted on Van Krevelen diagrams (H/C vs O/C). Optical properties of the original and irradiated DOM sample were determined by UV/vis absorbance and excitation emission matrix (EEM) fluorescence spectroscopy. This work is novel in that there have been no previous reports that link molecular composition with bulk spectral properties of photoirradiated CDOM collected from natural aquatic systems.
Materials and Methods Sampling and Sample Preparation. Water samples (2 L) were collected from the Black River (sample station B210, salinity 0, N 34° 25.883 W 78° 08.677), and the Cape Fear River Estuary, North Carolina (sample station M61, salinity 13.7, N 34° 11.626 W 77° 57.435) into which the Black River discharges, in acidcleaned (10% v/v HCl) glassware and filtered through Millipore GV 0.22 µm filters. The watershed of the Black River is influenced by forest and some agriculture, and the water is characterized by high levels of CDOM, low turbidity, and low pH. In contrast, the lower Cape Fear River is influenced by industry and urban areas such as Wilmington. The water at sample station M61 is tidal and shows high levels of CDOM, high turbidity, changing salinity, and neutral pH. More details about these two watersheds and sample stations B210 and M61 can be found at the Lower Cape Fear River Program web page maintained by the University of North Carolina, Wilmington (http://www.uncwil.edu/cmsr/aquaticecology/lcfrp/ (last accessed August 2008)). One liter from each sample was irradiated for 21 h using a 1000 W Spectral Energy solar simulator, which mimicked very closely the midsummernoon solar global and spectral irradiation at 34° N (26). The irradiation of all filtered samples was carried out in open beakers at a constant temperature (10 °C) to avoid any limitation of oxygen exchange with the atmosphere. The remaining liter of the same water sample was stored in the dark at 4-6 °C and used as a dark control. Irradiated and nonirradiated samples (20 mL) were collected for the analysis of dissolved organic carbon (DOC), the UV/vis absorbance (27), and excitation emission matrix fluorescence (28) (see Supporting Information). The samples (≈980 mL) were acidified to pH 2 and extracted using Varian Mega Bond Elut PPL solid-phase extraction (SPE) cartridges filled with 1 g of a functionalized styrene-divinylbenzene polymer (PPL) resin. ESI-FT-ICR-MS Analysis. Cape Fear and Black River samples were analyzed using a 9.4 T FT-ICR-MS built inhouse at the National High Magnetic Field Laboratory (NHMFL), Tallahassee, Florida (29, 30). Electrospray ionization (ESI) was used to generate largely intact negative ions at atmospheric pressure (31). Additional information about the ESI-FT-ICR-MS techniques used in this study are given in the Supporting Information. An important method used to interpret the data was to create Van Krevelen diagrams, which are effective for
visualizing large numbers of exact molecular formulas (32). Since major chemical classes typically found in DOM have characteristic H/C and O/C ratios, they cluster within specific regions of the Van Krevelen diagram. For example, highly aromatic compounds show low O/C (e.g., 0.2-0.6) and low H/C ratios (e.g., 0.4-1.1), whereas highly saturated molecules have medium to high O/C ratios (e.g., 0.6-1) and high H/C ratios (e.g., 1.1-2). Useful parameters in the characterization of the unsaturation and aromaticity of molecular formulas arising from this ESI-FT-ICR-MS analysis include the following. Double bond equivalents (DBE): DBE ) 1 + 0.5 × (2C - H + N)
(2)
Double bond equivalents minus oxygen (DBE-O): DBE-O ) (1 + 0.5(2C - H + N)) - O
(3)
Aromaticity index (AI) (adapted from ref (33)): AI ) (1 + C - O - 0.5H) ⁄ (C - O - N)
(4)
Kendrick mass defect (KMD): KMD ) (Nominal Mass - Kendrick Mass)
(5)
In eqs 2, 3, and 4, C, H, N, and O denote the number of carbon, hydrogen, nitrogen, and oxygen atoms, respectively. We believe the DBE-O parameter is a better indicator for the unsaturation of the carbon skeleton compared to the DBE, since most oxygen atoms in DOM molecules are a part of carboxyl group that is counted as one DBE. It should be noted that not all oxygen atoms in DOM exist as a part of a carboxyl group, as alcohols and ethers have also been identified (34). Thus, the DBE-O parameter may overestimate the carbon skeleton unsaturation to some extent. Nevertheless it is an unambiguous indicator that removes the contribution of oxygen to a DBE value.
Results and Discussion Photochemically Induced Changes in the UV/vis Absorbance of DOM. After exposure to 21 h of simulated sunlight, the relative decrease in the absorption coefficients of the Black River sample (B210, 0.3% loss in aCDOM(355)) was less compared to the Cape Fear River Estuary sample (M61, 9.3% loss in aCDOM(355)) indicating that photodegradation had occurred in both samples, but that it was more pronounced in sample M61 (Table 1 and Supporting Information Figure S1). The differences in the relative decrease in aCDOM(355) are potentially influenced by the initial differences in the aCDOM(λ) (Figure S1A) and therefore, a less pronounced selfshading effect in sample M61. However, the decrease in aCDOM(λ) over the entire wavelength range suggested a wavelength-dependent photodegradation with distinct differences between the Black River sample (B210) and the Cape Fear estuary sample (M61) in the UV range (Figure S1B). The present results are in general agreement with those very recently reported for photoirradiation of aquatic humic substance material extracted from the Amazon River Basin VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Absolute changes in the EEM fluorescence intensities arising from 21 h solar irradiation in sample B210 (A) and M61 (B).
FIGURE 2. Mass spectra before and after 21 h solar simulated irradiance of sample M61. (35). In that work the authors suggested that a larger decrease in absorbance in the range 200-550 nm upon irradiation of summer compared to winter samples was attributed to increased aromatic character in the former material, as determined by 13C NMR spectroscopy. The increase in spectral slope S with irradiation of the B210 sample was very small compared to a slightly higher change in S observed for irradiation of the M61 sample (see Table 1). This trend of S may also suggest a different wavelength-dependent photodegradation of these two samples, but given the very small overall changes, these results are not significant. Photochemically Induced Changes in the EEM Fluorescence of DOM. An EEM fluorescence spectrum can be used to distinguish between fluorescent compound groups based on differences in their excitation and emission maxima. Several spectral regions representative of distinct fluorophore groups have been previously observed in the EEM spectra of DOM (21, 36, 37). The total EEM fluorescence intensity (EEMtotal) decreased by 24% for the freshwater B210 sample and 40% for the Cape Fear River estuarine M61 sample after the exposure to 21 h of simulated sunlight (EEM spectra before and after irradiation are given in the Supporting Information data and Figure S2). The comparison of the decrease in fluorescence with the decrease in aCDOM(355) suggests a more pronounced photodegradation of CDOM components containing fluorescent chromophores compared to nonfluorescent chro700
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mophores (see Table 1 and Figure 1). Furthermore, not only was a difference in the overall photodegradation apparent between B210 and M61, but also a change of fluorescent intensity for specific regions of the EEM spectra (Figure 1). The maximal fluorescence decrease occurred at the excitation (ex)/emission (em) couple of ex:260nm/em:480nm in sample B210 and of ex:250nm/em:460nm in sample M61 suggests a change in the molecular composition of the photodegraded fluorescent CDOM (FDOM). Photochemically Induced Changes in Dissolved Organic Carbon (DOC) Levels. The DOC values did not significantly change during irradiation for either of the samples (see Table 1) suggesting that no photomineralization occurred under the present photoirradiation conditions. The use of open containers might have contributed to slight increases in DOC levels arising from laboratory sources, but this does not change the general observation that significant irradiationinduced decreases in DOC levels were not observed and the transformation of photolabile DOM into other organic compounds appeared to be more likely. Photochemically Induced Changes in the Molecular Composition of DOM. Figure 2 shows mass spectra obtained from sample M61 before and after the exposure to 21 h solar simulated sunlight as well as a series of peaks which were influenced by the irradiation. Within the given mass window (529.0-529.3 Da), it was obvious that an entire homologous series disappeared and a different series reappeared as a result of the solar irradiation. Furthermore, within this mass
FIGURE 3. Van Krevelen diagrams illustrating changes in the molecular composition of samples B210 (A and B) and M61 (C and D) after exposure to 21 h simulated sunlight (lines separate areas with aromaticity indices (AI) of AI > 0.5, AI ) 0.3-0.5, and AI < 0.3). window, all assigned molecular formulas that disappeared showed DBE-O values of 9 in contrast to the new molecular formulas apparent after irradiation which all showed DBE-O values of -5. This example illustrates the shift from unsaturated/aromatic compounds (high DEB-O) toward more saturated compounds (low DBE-O). A detailed list of the 100 most abundant compounds, which were photodegraded and photoproduced, is given in the Supporting Information in Tables S1 and S2. Prior to irradiation, the molecular formulas assigned to peaks in the mass spectrum for sample B210 showed marked differences compared to the molecular formulas assigned to sample M61 (see Supporting Information Figure S3). The M61 sample appeared to contain proportionally more highly aromatic compounds with aromaticity indices of AI g 0.5, which is an unambiguous minimum criterion for aromatic molecules (30). The B210 sample exhibited significantly less of these highly aromatic compounds. Fifty-eight percent of all assigned molecular formulas in the estuarine M61 sample were also observed in the freshwater B210 sample. The remaining 41% of the molecular formulas present in the M61 sample but not in the B210 sample exhibited low H/C and O/C ratios characteristic of the highly aromatic region of the Van Krevelen plot (see Figure 3). These differences in the molecular composition of DOM are quite intriguing, but, because molecular composition data are only available for these two sample stations, we cannot draw any conclusive relationship between source and composition. Comparison of Van Krevelen plots (see Supporting Information Figure S3) of the two irradiated samples suggested that solar irradiation did not change the molecular composition of the freshwater B210 sample in the same manner as the estuarine M61 sample. Regardless of the DOM source, these data provide, for the first time, direct evidence for the linkage between the photochemical reactivity and molecular composition of DOM. In sample B210, 525 molecular formulas observed in the mass spectrum disappeared upon irradiation. The majority
of these photodegraded compounds showed H/C and O/C ratios indicative of unsaturated compounds. These H/C and O/C ratios were higher compared to sample M61 and the photodegraded compounds in sample B210 were less aromatic. This observation is in general agreement with a previous study (2), where a substantial fraction of the initial molecular formulas disappeared after a long-term irradiation experiment. Those authors hypothesized that compounds with initially high DBE and low oxygen values (unsaturated and/or aromatic compounds) may be converted to compounds with low DBE and high oxygen content as a result of photodegradation, which in turn would potentially produce compounds with the same molecular formulas already assigned for the nonirradiated sample and therefore appear to be lost. They also suggested the possibility of irradiation producing nonionizable neutral molecules which would not appear in a mass spectrum. The formation of covalent linking between smaller units seemed unlikely, since no evidence of the formation of higher molecular weight compounds was observed (molecules up to about 1500 Da can be reliably measured). Only 188 new compounds were formed in sample B210 after irradiation and showed increased H/C ratios without any obvious shift in the O/C ratios (see Figure 4A and B). The results from photoirradiation of the estuarine M61 sample were strikingly different compared to either the present freshwater sample B210 in this study or previous DOM photoirradiation results (21). These differences appear to be related to the highly aromatic compounds present in the initial (unirradiated) estuarine M61 sample but not in the freshwater B210 sample. The van Krevelen plot determined for the irradiated M61 sample indicated almost a complete loss of the highly aromatic compounds upon irradiation (see Figure 3C and D). The 397 newly formed compounds with high H/C and O/C ratios appeared and supported the hypothesis of a transformation of unsaturated/ aromatic compounds into much more saturated molecules. This trend is very similar to the previous observation of the VOL. 43, NO. 3, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4. Number of molecular formulas associated with the specific DBE-O values before and after sunlight exposure for samples B210 (A) and M61 (B). preferential loss of these highly aromatic compounds (AI g 0.5) as mangrove-derived DOM migrated from source porewaters through an estuary (24). However, that study observed changes in DOM as a result of all biogeochemical processes associated with outwelling, including photochemical, biochemical, and physical (e.g., sorption onto particles, precipitation, hydrolysis), and the changes observed in DOM composition could not be unequivocally linked to photochemical changes. In the present study we have “isolated” photochemistry as the only source of DOM variability and for the first time directly linked source composition to photosusceptibility. This photosusceptibility appears to be a function of the aromaticity of the DOM, a plausible result which we believe has never been validated before in such a direct way. To further evaluate the effects of unsaturation on the observed differences in the photoreactivity of these two DOM samples, the numbers of assigned molecular formulas associated with specific DBE-O values were summed for each sample before and after the irradiation and then plotted on a frequency histogram (Figure 4). There was a clear shift from high DBE-O values toward low values in the estuarine M61 sample induced by the simulated sunlight exposure. This trend was not observed for irradiation of the freshwater B210 sample. However, the total number of molecular formulas for each DBE-O value decreased, indicating a loss of compounds with the same DBE-O values after irradiation. Given the constant DOC content, these data support a previous suggestion (23) that irradiation may generate some nonionizable compounds which are not observable by the ESI-FT-ICR-MS technique. The DBE-O plot for the estuarine M61 DOM sample offers convincing evidence that high levels of unsaturation and/or aromaticity are associated with high photoreactivity as has been previously noted for mangrove (24) and riverine (35) DOM. In the latter study, aromaticity was determined by solid-state 13C NMR, which is a bulk technique when used to assess DOM; it does not have the resolution to identify the specific aromatic compounds which are responsible for photoreactivity. Here we have linked aromatic and oxygen content and identified a trend that suggests the double bond levels of DOM molecules must exceed the number of oxygen atoms by some threshold value to be highly photoreactive. We must therefore conclude that the photoreactivity of DOM is highly dependent on the initial molecular composition. DOM compounds with DBE-O values greater than about 9 are highly photoreactive and readily photodegraded into more highly saturated and oxidized compounds with much lower DBE-O values. Molecular compositional analysis by 702
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FT-ICR-MS is the only technique at present that can identify these additional features of the photoreactive components of CDOM. These data also provide some further insights into theories of Del Vecchio and Blough (10) regarding donoracceptor charge-transfer complexes, in that we have been able to put some formula constraints (double bonds and oxygen content) on the types of molecules that are photoreactive. The present results also indicate that the optical properties of DOM after solar irradiation are dependent on its initial composition. Changes in the absorbance and EEM fluorescence intensities were quite different for the two DOM samples studied, and these differences are also reflected in the changes in the molecular composition. For example, sample M61 showed a maximum fluorescence decrease at 250 nm excitation and 460 nm emission wavelengths: compared to sample B210 at 265 nm excitation and 475 nm emission wavelengths. The absorbance also showed a much higher decrease for the M61 compared to the B210 sample at wavelengths lower than 320 nm. The distinct differences in the watersheds of sample location B210 and M61 may be responsible for the photochemically induced changes in the optical properties, but this suggestion is not conclusive due to the small sampling size. Ultrahigh resolution ESI-FT-ICR-MS techniques were used to identify the photoreactive component of the estuarine DOM that was not present in the freshwater DOM sample. This photoreactive component is characterized by high DBE-O values, an aromaticity index of AI e 0.5 and specific fluorescence properties. Loss of this photoreactive component was also associated with an increase in highly saturated and oxidized products that were still ionizable and therefore detectable by ESI-FT-ICR-MS. The freshwater DOM, in contrast, exhibited little of this photoreactive component. The ESI-FT-ICR-MS results are in agreement with the changes in the optical properties and also indicate that DOM samples from different locations and salinity can exhibit very different sunlight-induced reactions. Future work will include using the same ESI-FT-ICR-MS technique to evaluate the molecular changes induced by sunlight irradiation of a variety of other DOM samples to better identify the sources of these highly aromatic and very photoreactive DOM compounds. We are also characterizing the composition of DOM from a variety of marine sources in an effort to validate our hypothesis that these photochemically induced changes produce a suite of stable molecules that are observable by ESI-FT-ICR-MS and can serve as markers of altered terrestrial organic matter in the ocean.
Acknowledgments Mass spectra were obtained at the National High-Field Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Facility (NSF CHE-99-09502) at the National High Magnetic Field Laboratory in Tallahassee, FL. Financial support was provided by the University of Otago and the Otago University Chemistry Department (PhD scholarships, M. Gonsior) and the US National Oceanic and Atmospheric Administration, Grant NA05OAR4311162. We also thank Lori Tremblay for help in acquiring Fourier Transform Ion Cyclotron Resonance mass spectra. This is contribution 30 from the University of California, Irvine, Urban Water Research Center.
Supporting Information Available Additional figures and text relevant to this work. This material is available free of charge via the Internet at http:// pubs.acs.org.
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