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Limnol. Ocennogr., 42(6), 1997, 1454-1462 0 1997, by the American Society of Limnology and Oceanography, Inc. Photooxidation of triglycerides and fatty acids in seawater: Implication toward the formation of marine humic substances Robert J. Kieber, Linda H. Hydra, and Pamela J. Seaton Department of Chemistry and Marine Science Program, University Wilmington, North Carolina 28403-3297 of North Carolina at Wilmington, Abstract Unsaturated fatty acids rapidly photodecomposed in filtered seawater to aliphatic aldehydes and an o-oxocarboxylic acid (CHO-(CH,),,-COOH) upon a 5-h exposure to ambient sunlight. The photoreactivity of fatty acids increased as their degree of unsaturation increased. Palmitic acid displayed no photodegradation while monounsaturated oleic acid photodecomposed to nonanal and 9-oxononanoic, respectively. Photooxidation of the polyunsaturated linolcic acid produced significantly higher concentrations, primarily hexanal, and a wider variety of aldehydes. The rate of photodegradation was more than 10 times greater for the polqtnsaturated fatty acids than for the monounsaturated fatty acids. Photolysis of the triglyceride, trilinolein, gave En aldehyde profile similar to its component fatty acid, indicating each undergoes a similar mechanism of photodegradation. Twenty percent of the initial triglyceride was photodegraded during the 6-h irradiation. The rapid photoreactivity of unsaturated fatty acids may explain their low in situ levels relative to saturated fatty acids, the latter of which are found in the dissolved phase at relatively higher levels. Fluorescent hydrophobic dissolved organic matter, operationally defined as humic substances, increased after irradiation of trilinolein-treated seawater over a 14-d period. The concentraticn of humic products was enhanced by postphotolysis addition of ammonia. Dissolved ammonia concentrations declined as the dissolved organic nitrogen of the extracted humics increased, indicating the added ammonia was becoming incorporated into the organic structure. Fatty acids are chemically and biologically labile compounds that comprise -3% of the total dissolved organic matter (DOM) in seawater (Kattner et al. 1983b). They, along with triglycerides, are involved in many biogeochemically significant processes occurring within the sea. Of particular importance is their potential role as precursors to several other important types of DOM, including aldehydes and humic substances. Several mechanisms have been proposed for the autooxidation of fatty acids to aldehydes in seawater. It has been suggested that oleic acid (A9 C,,,,) is autooxidized to octanal, nonanal, and decanal while palmitoleic acid (A9 C,,,,) is autooxidized to hexanal, heptanal, and octanal (Schauenstein et al. 1977; Gschwend 1979; Gschwend et al. 1980, 1982). o-Oxocarboxylic acids have been detected in marine aerosols and the dominance of the C, o-oxoacid (CHO-(CH,),COOH) was proposed to be due to photooxidation of A9 unsaturated precursors such as oleic (A9 C,,,,), palmitoleic (A9 C,,,,), linoleic (89,12 C,,:,), and linolenic (A9,12,15 C,,:,) fatty acids (Kawamura and Gagosian 1987). Although mechanisms for the photooxidation of fatty acids have been proposed, they have never been verified experimentally in seawater. This paper describes photoproduction of aldehydes and o-oxocarboxylic acids from fatty acids in seawater, under ambient sunlight conditions, as well as in situ aldehyde concentration in four coastal marine waters. Understanding the photolability of fatty acids and triglycerides in seawater is important for reasons beyond their role Acknowledgments Financial assistance was provided by the Center for Marine Science Research (No. 142) at UNCW. We also thank Andrea Li for assistance in the nitrogen analyses. in the production of aliphatic aldehydes and o-oxocarboxylic acids. Fatty ac-.d and triglyceride photooxidation is significant because 0:’ the suggested role this process plays in the formation of marine humic substances. As early as 1972, researchers prcposed that marine humic acids form in situ from degradation products of marine plankton, rather than being transported from continents (Nissenbaum and Kaplan 1972). There are two prevailing views concerning the formation of marine humic substances. The polymerization view considers that humic substances are macromolecules formed through abiotic or enzyme-mediated polymerization of simple organic molecules, including amino acids, fatty acids, sugars, and phenols. However, it is very unlikely that simple organic molecules will aggregate and polymerize to humic substances, given the micro- or nanomolar concentrations at which they exist in ocean waters, or in large, dilute, freshwater lakes, such as the Antarctic lakes, where extracted humic substances were found to have significant similarities to marine humics (M&night et al. 199 1, 1994). A second proposed pathway for humic substance formation is through continued oxid ation, condensation, and transformation of existing biopolymers. Polyunsaturated lipids released into the water column by plankton have been invoked as a potential precursor for humic substance formation. Harvey and Boran (1982a,b) and Harvey et al. (1983, 1984) autooxidized trilinolein in a seawater matrix under both light and dark conditions and found that the XAD-extracted product had infrared, ‘H-NMR, and 13C-NMR spectra that were very similar to those of marine humic substances. The 613Cvalue of their synthetic product was also similar to the S13Cvalues of natural marine fulvic acids (Harvey and Boran 1985). The authors proposed a mechanism that involves free radical autooxidative cross-linking of unsaturated fatty acid chains of 1454 Photooxidation of triglycerides the triglyceride, producing humic substances with both aliphatic and aromatic regions containing hydroxyl, ether, peroxide, and carbonyl functional groups. This mechanism provides an insightful starting point to our understanding of the formation of marine humic substances. However, it does not address the role of the photodegradation products of triglycerides, such as aldehydes, in the humification process. These photooxidation products are potentially more reactive than their precursors and may therefore be significant source material in the formation of hydrophobic DOM such as humics. Marine humic substances are composed of between 3 and 5% nitrogen, although the chemical form of N in marine humics is poorly known. Synthetic humic substances produced from trilinolein-treated seawater, however, contained only 0.86% N (Harvey et al. 1984; Schnitzer 1985). The possible photooxidation of triglycerides and fatty acids has important ramifications with respect to the inclusion of N into humics. In addition to being a direct source material for humification, aldehyde photoproducts are much more reactive toward ammonia or amino-containing organic compounds, relative to the parent unoxidized molecules. The question regarding the incorporation of N into marine humic substances is particularly important because of the suggested role humics have in the stimulation of primary productivity (Moran and Hodson 1990, 1994; Carlsson and Graneli 1993; Graneli and Moreira 1990; Moran et al. 1991). The primary goals of this research are to describe, both qualitatively and quantitatively, the degree of fatty acid and triglyceride photodegradation in seawater under ambient sunlight and to explore possible mechanisms for the incorporation of N into humic substances resulting from the reactive photodegradation products. Materials and methods Reagents and standards-All chemicals were obtained from Fisher Scientific or Aldrich Chemical and were reagent grade or HPLC grade unless stated otherwise. Aldehydes were purchased from Aldrich Chemical. Fatty acids and triglycerides were purchased from Sigma Chemical. 9-0x0nonanoic acid was synthesized through oxidative cleavage (KMnOJNaIO,) of oleic acid. The oxo-acid was converted to its dinitrophenylhydrazone (DNPH) derivative, which was purified through column chromatography for use as a standard (Seaton and Kieber unpubl.) Stock fatty acid (lo-15 mM) and triglyceride (-3 mM) standards were prepared in acetonitrile. and chloroform, respectively. All fatty acid and triglyceride stock solutions were stored at 4°C where they were stable for several months. 2,4-DNPH was recrystallized twice from a 70 : 30 (vol : vol) solution of acetonitrile and water and a third time from 100% acetonitrile. Crystals were dried under vacuum and stored in the dark in airtight 30-ml Teflon vials. The derivatizing reagent was prepared by dissolving 20 mg of recrystallized 2,4-DNPH in a 15-ml solution containing concentrated HCl, water, and acetonitrile (2:5: 1) (vol: vol : vol). Carbonyl contamination was removed daily with a 2-ml extraction using redistilled (inhouse) carbon tetrachloride (Ccl,) added to the reagent, shaken on a wrist action shaker for 5 min, and centrifuged and fatty acids 1455 at 2,000 rpm for 5 min. The organic layer was removed and the reagent solution reextracted twice more as described above. Stock solutions of aldehydes (-0.1 mM) were prepared in acetonitrile and kept in the dark at 4°C where they were stable for several months. Immediately prior to HPLC analysis serial dilutions of the stock were made in deionized water (Milli-R06 Plus/Milli-Q Plus, Millipore) and derivatized with 2,4-DNPH. HPLC instrumentation-The HPLC mobile-phase gradient was generated by an E-Lab model 2020 gradient programmer connected to data acquisition software (OMS Tech) installed in an IBM-compatible PC and to an Eldex model B-100-S pump (Eldex Laboratories) containing an inert solenoid valve placed on its low pressure side. Derivatized, concentrated, and eluted (from C,, cartridges) samples were injected into a Valco six-port injector (Valco Instruments) containing a 2,000+~,1 sample loop. Derivatized hydrazones were separated on an 8-mm i.d. X 20-cm C,, reversed-phase Radial-pak cartridge housed in a Radial compression module (Millipore), detected by an Isco V4 variable wavelength absorbance detector (Isco) at 370 nm, and processed by the ELab system. A two-solvent gradient elution at 2 ml min- I was utilized with solvent A as 10% acetonitrile in water, and solvent B 100% acetonitrile: isocratic at 40% B for 10 min, 40% B to 75% B in 7 min, 75% B to 100% B in 11 min, isocratic at 100% B for 8 min, then isocratic at 40% B for 9 min. Aldehydes analysis -Aldehydes were determined by the method of Kieber and Mopper (1990). Filtered samples (50250 ml) were derivatized with 2,4-DNPH reagent for 1 h in the dark at ambient temperatures. Derivatized hydrazones were extracted and concentrated using C,, cartridges that were precleaned and preconditioned by rinsing with 2 X 5 ml of acetonitrile followed by 2 X 5 ml of filtered Gulf Stream seawater. To avoid contamination from plastic tubing, derivatized samples were placed in 50-ml glass-syringe reservoirs (Fisher) that were connected in line to C,, cartridges. The derivatized aliquots were pulled through C,, cartridges via a dialysis pump (Cole-Parmer) at a flow rate of 3.5 ml min-l. After the sample was loaded onto the cartridge, salts were rinsed off with deionized water and the remaining liquid blown out with air. Hydrazones were eluted with 2 ml of acetonitrile into 7-ml Teflon vials, and 3 ml of deionized water was added to mimic the HPLC mobile phase. Diluted derivatized extracts were analyzed by HPLC, Photochemical studies-Fatty acids and trilinolein from stock standards were added to 0.2~pm filter-sterilized Gulf Stream seawater (1 FM and 0.33 ~.LM concentrations, respectively) prior to placement in six sterile 500-ml quartz, round-bottomed flasks with ground glass quartz stoppers. The quartz flasks containing the fatty acid- or triglyceridetreated seawater were placed directly on cork rings in direct sunlight for a minimum of 5 h on a grassy area near the chemistry building on the UNC-Wilmington campus. Dark samples were analogous in every way to the light samples except they were wrapped in aluminum foil to restrict light exposure. 1456 Kieber et al. To determine the degree of thermal degradation of fatty acids during the photolysis studies, various fatty acids were added to 0.2~pm filter-sterilized Gulf Stream seawater, placed in 500-ml of round-bottomed quartz flasks wrapped in aluminum foil, and submerged under isothermal conditions at 25 or 40°C for 5 h. The latter temperature was the highest attained during the unthermostated photolysis studies. Samples were analyzed for aldehyde concentrations initially and after the 5-h exposure at the two different temperatures. No statistical difference was found between the 25 and 40°C samples (P < 0.01, n = 28), indicating undetectable thermal degradation of fatty acids at 40°C during the 5-h exposure. Photolysis of trilinolienPostphotolysis time studies of trilinolein were also preformed, and the rate of humic substance formation in two separate experiments was determined. In the first experiment, trilinolein (0.33 ~.LM)in filtersterilized Gulf Stream seawater was irradiated (as described above) and the water from three quartz flasks was combined in a sterile 2-liter Erlenmeyer flask. Aliquots were taken over a 14-d period for analysis of UV absorbance at 250 nm and fluorescence, as described below. In the second experiment, trilinolein was photolyzed as above, but after combining the water from the three quartz flasks, ammonia was added to final concentration of 1 ~.LM.Aliquots were again taken over a 14-d period for absorbance and fluorescence. Ammonia concentrations and dissolved organic nitrogen (DON) levels in the C,, cartridge-extracted DOM in this latter experiment were also measured, as described below. Hydrophobic DOM extraction-Fluorescent, hydrophobic DOM, operationally defined here as humic substances, was extracted using C,, cartridges by the method of Amador et al. (1990). This technique was chosen over conventional methods using XAD resins because C,, is more efficient at removing the chromophoric humic substances from seawater. Amador et al. (1990) reported that C,, extraction of humic substances was between 22 and 84% more effective relative to XAD-2. Kuo et al. (1993) also recovered 83% of an aquatic fulvic acid using C,, solid-phase extraction. In addition, Amador et al. (1990) found C,, extraction was better able to retain the UV-visible and fluorescence characteristics of the isolated humic material relative to XAD. Because these were the characteristics of most interest in this study, C,, extraction was the most logical choice of an extraction procedure. A second advantage of using C,, extraction is that methanol is the eluant rather than 0.1 M NaOH. Therefore any degradation due to high pH is avoided. Note that some of the unreacted triglycerides and fatty acids will also be retained by the C,,. However, their fluorescence and absorbance are negligible relative to the isolated humic material. C,, cartridges were preconditioned by washing with 2 X 5 ml methanol followed by 2 X 5 ml humic-free Gulf Stream water. Humic-free Gulf Stream water was obtained by passing filtered Gulf Stream water through C,, cartridges, preconditioned by washing with 2 X 5 ml methanol, followed by 2 X 5 ml of deionized water. Samples for humic extraction were filtered, acidified to pH 2.2-2.5 with 0.3 M HCl, and loaded onto C,, cartridges. The cartridges were washed with 2 X 5 n-l of deionized water to remove residual salts and humic su3stances were eluted with 4 ml of methanol, which was brought to a final volume of 5 ml with methanol. The relative c,ancentration of humics in the extract was determined by fluorescence intensity of the extract in a manner similar to Amador et al. (1990). Fluorescence was chosen because it is a highly sensitive spectrophotometric technique capable of detecting extremely small quantities of humic substances prcduced. It is also highly selective because two wavelengths are employed, allowing us to distinguish between other nonpolar entities that might be extracted by the C,, packing. The sample was diluted 4/10 in methanol and fluorescence rr easurements (Turner model 112) made against a methanol blank at 360 nm excitation and 490 nm emission, respectively. Fluorescence is reported as quinine sulfate units (QSU) where 1 QSU is the fluorescence of a 1.3 nM solution of quinine sull’ate in 0.1 N H,SO,. A 100 QSU solution in 0.1 N H,SO, v/as set to read -73 fluorescence units against a 0.1 N H,SO, blank and readings converted to change in (A) QSU liter- I relative to initial fluorescence values taken immediately after photolysis. Relative humic concentrations were also detemined by UV absorbance relative to a methanol blank at Z!50 nm and converted to a change in (A) absorbance liter- l relative to initial absorbance values taken immediately after photolysis. Nitrogen anczlyses-Total dissolved nitrogen was analyzed using a persulfate oxidation method in which nitrogen compounds are oxidized to nitrate by potassium peroxodisulfate under alkaline conditions (Valderrama 1981). The resulting nitrate is quantified by Technicon Autoanalyzer. Ammonia (NH,) concentrations were determined by the method of Parsons et al. (198 9) in which ammonia reacts with hypochlorite under slightly alkaline conditions to form monochloramine, which, in the presence of phenol, nitroprusside ions, and an excess of hypcchlorite, gives indophenol blue that is measured at 630 nm. Results Photooxidation of fatty acids-Unsaturated fatty acids added to sterile seawater and irradiated for 5 h under ambient sunlight conditions photooxidized to produce a variety of aliphatic aldehydes (Figs. 1, 2; Table 1). There were undetectable changes in the concentration of aldehydes produced in dark controls suggesting little or no degradation of the parent fatty acd (Table 1). Photodegradation increased as the degree of tinsaturation of the precursor fatty acid increased. Photodegradation of palmitic acid was negligible (Fig. 1B) while oleic acid produced nonanal and 9-oxononanoic acid (Fig:;. lC, 2). Diunsaturated linoleic acid degraded to a greater extent relative to oleic acid and yielded higher concentrations and a greater variety of aldehydes (Fig. 1E). Hexanal and 9-oxononanoic acid were the principal aldehydes produced, along with smaller amounts of butanal, pentanal, and sevel-al unidentified carbonyl compounds. Triunsaturated linolenic acid also photooxidized primarily to hexanal and 9-oxononanoic acid as well as several unidentified carbonyl compounds (Fig. 1F). The rate of Jdehyde production, based on the primary Photooxidation of triglycerides and fatty acids 1457 STANDARD -.. PALMTIC LINOLENIC$ 7 Time (min) Fig. 1. HPLC chromatograms of derivatized aldehydes after photolysis in the presence of 1 FM fatty acid-treated filter-sterilized Gulf Stream seawater. (A) Aldehydes standard (45.5-364 nM range). (B) Palmitic (C,,,,) acid. (C) Palmitoleic (A9 C,,,.) acid. (D) Oleic (A9 C,,,,) acid. (E) Linoleic (A9,12 C,,,,) acid. (F) Linolenic (A9,12,15 C,,,.) acid. Cl-Cl0 = straight-chain aliphatic aldehydes, MG = methyl glyoxal, GLY = glyoxal, 9-0X = 9-oxononanoic acid. aldehyde produced and normalized to light intensity, is presented in Table 1. The rate was over 10 times greater for the diunsaturated fatty acid and triglyceride relative to the monounsaturated species.The percent conversion of the initial fatty acid or triglyceride after photolysis is also presented in Table 1. Twenty percent of the original triglyceride was photodegraded during the 6-h irradiation with lesser amounts of degradation for the individual fatty acids. These should be viewed as minimum conversion and production rates because several of the peaks in the chromatograms presented in Fig. 1, especially for the diunsaturated species, were not identifiable by our analytical scheme. The above photooxidation of fatty acids to aldehyde results are consistent with earlier reports and proposed mechanisms (Jalliffier-Merlon et al. 1991; Gschwend 1979; Gschwend et al. 1980,1982; Kattner et al. 1983b; Kawamura and Gagosian 1987). Photolysis of unsaturated fatty acids results in oxidative cleavage of the double bond, producing aliphatic aldehydes and an o-oxocarboxylic acid. Oleic acid (A9 C&, for example, produced nonanal and 9-oxononanoic acid (1, 2, and 3, respectively, Fig. 2). 9-Oxononanoic acid is also produced upon photolysis of palmitoleic (A9 C,,,,), linoleic (A9,12 C&, and linolenic (A9,12,15 C,,,,) acids, all of which have their first double bond at C,-,, (Fig. 1). The photochemical lability of unsaturated fatty acids reported above may explain in part why in situ concentrations of dissolved unsaturated fatty acids are low in natural waters relative to their saturated counterparts (Scribe et al. 1991; Hama 1991; Kattner et al. 1983a,b). Comparison of fatty acid profiles of particulate and dissolved fractions from a spring phytoplankton bloom in the North Sea (Kattner et al. 1983a,b) showed that unsaturated fatty acids were significantly less prevalent than saturated fatty acids in the dissolved fraction than in the particulate fraction. The dissolved fraction contained -63% saturated, 32% monounsaturated, and 6% polyunsaturated acids, whereas coexisting particles contained -5 1% saturated, 37% monounsaturated, and 12% polyunsaturated acids. Photooxidation of trilinolein--The triglyceride trilinolein containing three linoleic acid chains were also investigated for its photochemical reactivity (Fig. 3). The primary photodegradation product was hexanal (66 &I). This concentration was similar in magnitude to the hexanal (42 nM) produced during photolysis of linoleic acid (Fig. 1). Photolysis of trilinolein also produced relatively smaller amounts of butanal, pentanal, and 9-oxononanoic acid, which is the same suite of degradation products found upon irradiation of linoleic acid. This similarity suggeststhat a similar mechanism is responsible for the photodegradation of the triglyceride and its component fatty acid. Natural aldehydes-Natural aldehyde concentrations (Fig. 4) were determined at four coastal North Carolina sites (Fig. 5). Straight-chain, aliphatic aldehydes from butanal through decanal and methyl glyoxal were detected at sites 1, 2, and 1458 Kieber et al. A 0 10 30 Time (min) 0 2o 30 10 Time (rniii 1 Fig. 2. HPLC chromatogramsfrom photolysis of 1 PM oleic (A9 C,,:,). (A) Prior to photolysis. (B) After 5 h photolysis. Fig. 3. HPLC chromatograms from photolysis of 0.33 FM trilinolein-treated filter-sterilized Gulf Stream seawater. (A) Prior to photolysis. (B) After 5 h photolysis. 3 at relatively low concentrations (<2 nM). The more UVabsorbent waters of the Alligator River contained higher concentrations (2-10 nM) of the same aldehydes found at the other sites. Their presence in the water column is suggested to be due, in part, to the photooxidation of various fatty acid precursors described above. The relatively low concentrations reported here might result from the biological and chemical lability of aliphatic aldehydes (Hydro 1996). Formation qf humic substances from trilinolein photolys&-The photooxidative lability of unsaturated fatty acids and triglycerides reported above may play an important role in the production of marine humic substances. The generation of reactive photolysis products, such as 9-oxononanoic acid, is significant because the reactivity of the aldehyde functionality WI th ammonia or organic amines provides an avenue for incorporation of nitrogen into the humic struc- Table 1. Rate of aldehyde production, normalized to light intensity, from photolysis cBa variety of fatty acids and triglycerides. Percent conversion is the difference between initial and final fatty acid concentrations where the final fatty acid concentration was estimated based on the concentration of the primary aldehyde photoproduct generatedduring 5 h of irradiation. ND = not detectable. Fatty acid Trilinolein Linoleic Oleic Palmitoleic Initial aldehyde (nM) 4 1 ND 0.4 Aldehyde (nM) Darkt=5h Light t = 5 11 3 66 2 42 2.5 ND 0.6 2.6 Rate X 100 [nM(W-h)-I cm21 240 110 6 6 Percent conversion 20 4 0.3 0.3 Photooxidation of triglycerides 1459 and fatty acids Site 1 (Myrtle Grove) J s 8 E 8 8 E $ 2: ss~s~~p Aldehyde Aldehyde Site 3 (Chowan River) 8 s 8 s s Aldehyde 8 8 susssegg $ Aldehyde Fig. 4. Distribution of aldehydes in coastal North Carolina seawater.Error bars represent + 1 SD based on y1= 3. Absorbance values at 300 nm were 0.06-o. 16 for sites 1, 2, and 3. Absorbance value at 300 nm for site 4 was 1.7. Note scale change for site 4. ture. In order to test this hypothesis, two separate, postphotolysis studies with trilinolein were performed and the rate of humic formation monitored over time. In the first experiment, photolyzed trilinolein-treated seawater was the only reactant. In the second experiment, after photolysis of the trilinolein-treated seawater, ammonia was added to a final concentration of 1 FM. Aliquots, taken from each experiment, were analyzed for aldehydes, UV absorbance, and fluorescence over a 14-d period. In the trilinolein-only-treated sample the concentration of humic substances, as measured by change in fluorescence intensities of C,, extract, decreased after irradiation through 799/v 78w 77W 76w 74% TE4 36’N - .Z 5 J 75ow - 36ON Atlantic Ocean 35’N - 73W - 35’N Longitude Fig. 5. Map of North Carolina sampling locations. W = Wilmington; E = Edenton; C = Columbia; WB = Wrightsville Beach. the first day relative to fluorescence values taken immediately after photolysis (Fig. 6A). These early fluorescence data most likely represent photobleaching of fluorescence from ambient humic-type material in the seawater used as the matrix for the photolysis study. This would be consistent with photobleaching already known to occur during irradiation of a variety of oceanic samples by ambient sunlight (Kouassi 1986; Kieber et al. 1990); however, it is unclear why the process continues after irradiation ceases. After the postphotolysis minimum, the concentration of humic substances gradually increased, reaching approximately starting levels after 2 weeks. It is unclear from these data whether new humic-type material was formed from the spiked trilinolein, or if there is a reversal of the photobleaching of the ambient DOM. The key feature to these photolysis studies, and the primary focus of this research, is the dramatic impact of postphotolysis addition of ammonia tp the photolyzed, trilinolein-treated seawater. This is particularly important, given the photolability of the triglyceride reported earlier and the reactivity of the photofragments toward ammonia or organic amines. When ammonia was added after irradiation, the magnitude of the increase in fluorescence and humic substances concentration was greatly enhanced (Fig. 6B). In this latter experiment the final concentration of humic substances dramatically exceeded initial levels and therefore cannot be due simply to reversible photobleaching of ambient DOM present in the water. Another significant feature of ammonia addition is the lack of a postphotolysis fluorescence minimum. This implies that addition of ammonia enhances the rate of the humification process. Similar conclusions can be drawn when humic substance concentrations are monitored by change in UV absorbance rather than fluorescence (Fig. 6). In the trilinolein-treated sample there was an initial decrease in absorbance followed by a gradual increase over 1460 Kieber et al. :: ;c-0-q 8 \/C-O-CH I 4 II II I 5 IO 15 I ]A Time (d) hv, 02 P -O-C& 8-0-CH f .Z 0.06 L 0.03 =E {g u)cv a4 d --4-i, 1 B 6 0 -0.03 -0.06 0 5 I 15 IO it-0-CH -4 8 I I HflC/C3\--b 6 Fig. 6. Concentration of humic substances as measured by fluorescence change (A QSU quinine sulfate units liter-l) and absorbance change over time of C,, cartridge extract in methanol after photolysis of 0.33 p,M trilinolein-treated Gulf Stream seawater. (A) Trilinolein only. (B) Tiilinolein with postphotolysis addition of ammonia (3 FM). time. As with fluorescence, the magnitude of the increase was enhanced by addition of ammonia, and no postphotolysis minimum was observed. During the postphotolysis addition of ammonia experiment, aqueous ammonia concentrations and DON concentrations of C,,-extracted humic substances were also monitored over the 14-d period (Fig. 7). Aqueous ammonia concentrations declined over time, and DON in the extracted humic substances increased. The loss in dissolved ammonia T T fc-o-c& 2O Time (d) 0.8 NH3 I 0.3 go=6 so.4 v go.2 02, OH, 03, Hz0 many steps I OH 0 7 Fig. 8. Proposed mechanisms for the incorporation of nitrogen into marine hunic substances from photolyzed trilinolein. Structures 6 and 7 are representative of a family of related structures containing covalently bound nitrogen that might be formed after photolysis. and concurrent increase in humic-bound nitrogen suggest that ammonia is incorporated into the humic structure as it is enhancing the rate of humic substances formation. 0 0 0 10 5 Discussion 15 Time (d) Fig. 7. Aqueous ammonia (A) and dissolved organic nitrogen (B) concentrations as a function of time. Error bars represent k 1 SD based on y1 = 3. The aldehydes generated during the photolysis of trilinolein may play an important role in humification of marine triglycerides. Aldol-type condensations between the photogenerated aldehyde and carbonyls on one of the chains of the partially oxidized triglyceride would result in increased Photooxidation of triglycerides crosslinking in the humic substances. Additionally, the reactivity of aldehydes toward ammonia and organic amines may explain the observation that addition of ammonia to photolyzed trilinolien in seawater resulted in increased humification and incorporation of nitrogen into the humic structure. Aldehydes react readily with ammonia and amines to form Schiff bases. Schiff bases have been proposed as intermediates in sedimentary and aqueous humic substance formation (Francois 1990; Thorn and Mikita 1992). One possible mechanism for the synthesis of marine humic substances from photolyzed triglycerides is proposed in Fig. 8 and is very similar to the earlier mechanism proposed by Harvey et al. (1983, 1985), except that it includes a significant addition, namely, the incorporation of nitrogen into the humic structure. Photooxidation of trilinolein (4) first produces hexanal and the aldehyde (5). This initial photooxidation step was verified experimentally during our photolysis studies. Reaction of the aldehyde (5) with ammonia would produce a Schiff base (6). Free radical-initiated crosslinking of the fatty acid strands and subsequent oxidation, cyclization, dehydration, and ester hydrolysis, as proposed by Harvey et al. (1983, 1985) would give a product incorporating covalently bound nitrogen, such as 7. This mechanism is also consistent with the experimentally observed fluorescence and absorbance increases in extracted humic substances after addition of ammonia to photolyzed trilinolien in seawater. Structures, such as 6 and 7 in Fig. 8, are proposed as possible intermediates in the humification process and are meant to represent a family of related structures that contain covalently bound nitrogen. Although this mechanism suggests one possible pathway for the incorporation of ammonia into humic substances, other organic amines, such as amino acids, amino sugars, and nucleic bases (purines and pyrimidines), could also be incorporated into the humic structure through similar processes. Our findings contribute a significant addition to our understanding of how nitrogen might be incorporated into marine humic substances. 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Received: 1 July 1996 Accepted: 20 December 1996