Environ. Sci. Technol. 2004, 38, 6645-6655
Using 19F NMR Spectroscopy to Determine Trifluralin Binding to Soil M A R K S T R Y N A R , † J E R Z Y D E C , * ,‡ ALAN BENESI,§ A. DANIEL JONES,§ RODERICK A. FRY,§ AND JEAN-MARC BOLLAG‡ National Exposure Research Laboratory, U.S. EPA, Research Triangle Park, North Carolina 27711, Laboratory of Soil Biochemistry, Penn State Institutes of the Environment, 107 Research Building C, University Park, Pennsylvania 16802, and Department of Chemistry, Eberly College of Science, The Pennsylvania State University, University Park, Pennsylvania 16802
Trifluralin is a widely used herbicide for the control of broad leaf weeds in a variety of crops. Its binding to soil may result in significant losses in herbicidal activity and a delayed pollution problem. To investigate the nature of soilbound trifluralin residues, 14C-labeled herbicide was incubated for 7 weeks with four soils under anoxic conditions. As determined by radiocounting, trifluralin binding ranged between 10 and 53% of the initial 14C depending on the soil tested. 19F NMR analyses of the methanol extracts and different fractions of the extracted soil suggested that bound residue formation largely involved reduced metabolites of the herbicide. A 2,6-diamino product of trifluralin reduction with zero-valent iron (Fe-TR), and the standard of a 1,2-diaminotrifluralin derivative (TR6) formed covalent bonds with fulvic acid (FA), as indicated by the 19F NMR spectra taken periodically over a 3-week contact time. At short contact times, TR6 and Fe-TR formed weak physical bonds with FA, as the respective spin-lattice relaxation times (T1) decreased from the range 1300-1831 ms for TR6 or Fe-TR analyzed in the absence of FA to the range 150410 ms for TR6/FA or Fe-TR/FA mixtures. In general, the results indicated that trifluralin immobilization involved a variety of mechanisms (covalent binding, adsorption, sequestration), and with time it became increasingly stable.
Introduction Trifluralin (R,R,R-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) is a dinitroaniline pre-emergence herbicide used for the control of broadleaf weeds in a wide variety of crops including cotton, soybeans, and alfalfa (1, 2). It is among the top five herbicides produced annually in the United States (24 000 t yr-1) with $300 million in annual sales (1) despite being a suspected carcinogen. Previous studies on trifluralin have included its fate and transport in soils (3, 4), the transformation under low redox or anoxic conditions (5-7), the co-metabolism in sewage systems (8), and trifluralin photodecomposition (9-11). To maintain the viability of the herbicide and prevent photodecomposition, it was recom* Corresponding author phone: (814)863-0843; fax: (814)865-7836; e-mail:
[email protected]. † U.S. EPA. ‡ Penn State Institutes of the Environment. § The Pennsylvania State University. 10.1021/es0403110 CCC: $27.50 Published on Web 11/12/2004
2004 American Chemical Society
mended that trifluralin be injected under the soil surface (1, 12). However, subsurface application may not necessarily prevent losses in herbicidal activity, since as demonstrated by Grover et al. (1), in wet soils of high organic matter contents, reduced forms of trifluralin can become incorporated into soil organic matter. The incorporation, in turn, may evolve into a delayed pollution problem in case of future release of bound trifluralin residues under favorable conditions. To date, relatively little has been done to investigate the mechanism of trifluralin binding despite the progress in determining other xenobiotic interactions with soil (13-15). Recent research in this area involved contaminants labeled with both 14C and stable isotopes to facilitate the analyses of xenobiotic/soil organic matter complexes by NMR techniques. In the studies of Strynar et al. (16) using [15N]-2,4,6trinitrotoluene (TNT), NMR evidence suggested that reducing the nitro groups under anoxic conditions led to the formation of very stable covalent bonds with soil and compost humic materials. Among other studies on binding interactions that involved xenobiotics labeled with stable isotopes were those using [15N]-aniline (17), [13C]-2,4-dichlorophenol (18), and [13C]-cyprodinil (19). Presently, 19F emerges as a useful tracer for research in this area. Benefits of using 19F over the use of stable isotopes include lack of background 19F signal in soils, increased sensitivity and spectral range relative to 15N and 13C, and perhaps most important, no need to synthesize 19F-labeled xenobiotics as 19F is 100% abundant in fluorinated compounds and constitutes an elemental tracer. A limitation is that only F-containing xenobiotics may be investigated, though Chien et al. (20) used atrazine derivatized with fluorine for their investigation. Previously, 19F NMR has been used to study interactions between fluorinated atrazine (20, 21) or 4′-fluoro-1′-acetoonaphthone (22) and various humic materials. Other studies used this technique to investigate fipronil degradation pathways (23), characterize photolysis products of 3-trifluoromethyl-4-nitrophenol (24), detect the chlorodifluoroacetic acid ions (25), and evaluate sorption of hexafluorobenzene to soil organic matter (26), sediments, polymers, or activated carbon (27). Mabury and Crosby (11) appear to be the only researchers who investigated trifluralin in depth by 19F NMR, mainly to identify photodegradation products that could react with soil and form bound residues. In this study, 19F NMR was applied to investigate the formation of soil-bound residues of trifluralin under anoxic metabolic conditions. The hypothesis was that, as was the case for TNT (16), the nitro groups of the trifluralin molecule would be reduced to amino groups which would then react with soil organic matter. In an analogous manner, aromatic amines reacted with humates via carbonyl or quinone moieties (28). On the basis of previous reports (1, 3), bound residues of trifluralin were assumed to retain the trifluoromethyl (CF3) group. No study, to date, has suggested mineralization of the CF3 group, at least in the short term (3 yr). There are indications, however, that eventually the CF3 group may be oxidized to the carboxyl group (3). The major goal of this study was to investigate the mechanism of trifluralin binding to soil. Despite the 100% abundance of 19F in the molecule of test compound, the concentration of trifluralin had to be 3 orders of magnitude higher than the recommended application rate to ensure obtaining clear NMR signals. However, elevating pesticide concentrations to study binding mechanisms is an acceptable approach that was applied in several previous investigations (14, 17, 19). VOL. 38, NO. 24, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Chemicals Used for Experiments
Experimental Section Chemicals. Unlabeled and 14C-labeled trifluralins as well as five standards of reduced trifluralin derivatives were donated by Dow AgroSciences (formerly Dow-Elanco) (Indianapolis, IN). To obtain two additional trifluralin reduction products, 1 mg of trifluralin in 20:80 water:methanol was treated with 50 mg of zero-valent iron nanoparticles or zero-valent iron nanoparticles with palladium (both donated by Dr. Thomas Mallouk, The Pennsylvania State University) and centrifuged. The supernatant was passed through a 0.45-µm nylon filter and analyzed by LC-MS and 19F NMR. The chemical names, structures of trifluralin, and its transformation products are listed in Table 1. 6646
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Experimental Setup. Fifty-gram samples of four uncontaminated local soils (designated Pope, Hagerstown, Chagrin, and Carlisle) were thoroughly mixed with 10 mg of trifluralin and 0.23 µCi of uniformly ring-labeled [14C]-trifluralin dissolved in methanol to result in an initial herbicide concentration of 200 mg kg-1 and an initial radioactivity of 4.6 µCi kg-1. The recommended application rate for this herbicide is 0.25-0.5 mg kg-1 (1); however, for analytical purposes, a much higher trifluralin concentration was necessary. Table 2 lists physicochemical properties of the soils. Soils were collected 1 week prior to use and stored at 4 °C. Soil characteristics were determined by The Pennsylvania State University Agricultural Analytical Testing service using
TABLE 2. Physicochemical Properties of Soil OM % sand % silt % clay % texture CEC (mequiv/100 g) N% pH class.
Pope
Hagerstown
Chagrin
Carlisle
3.3 55.9 25.4 18.7 sandy loam 9.8 0.21 5.1 Fluventic Dystrochrept
2.7 29.9 46.2 24.0 loam 9.2 0.15 6.2 Typic Hapludalf
2.8 27.7 38.0 34.2 clay loam 17.0 0.15 7.6 Dystric Fluventic Eutrochrept
7.4 35.9 28.9 35.2 clay loam 19.9 0.31 5.1 Typic Medisaprist
standard soil testing procedures. The samples were distributed into 100-mL serum bottles, amended with an anaerobic microbial consortium from swine manure slurry (2.0 mL) and glucose (3 mL of a 5% glucose solution w/v), flushed with N2 gas to create anoxic conditions, crimp-sealed with a butyl rubber stopper, and incubated in darkness for 7 weeks at 25 °C. Soil Fractionation. On the completion of the incubation, soil samples were extracted by shaking for 24 h with 50 mL of methanol, centrifuged at 10000g for 1 h, and rinsed 3 times by resuspending in 10 mL of methanol and shaking for 1 h. The supernatants were combined, and the total extract was prepared for analysis by radiocounting, thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), mass spectrometry (MS), and 19F NMR spectroscopy. The solids were air-dried, extracted by a 24-h shaking with N2-purged 0.1 M NaOH (75 mL), centrifuged for 1 h at 10000g, and rinsed 5 times with 30 mL of 0.1 M NaOH. The resulting humin fraction was analyzed by radiocounting (after combustion to 14CO2 and trapping in scintillation cocktail). The NaOH extract (combined with the washings) was acidified with 5 M HCl to pH HA > FA (5-35%, 2-15%, and 4-6%, respectively). TLC analysis of the methanol extracts showed three major radioactive peaks, one of which represented unaltered herbicide, as indicated by its Rf value (∼0.9) that matched that for the [14C]-trifluralin standard (Figure 2). The Pope soil extract showed only the trifluralin peak. No trifluralin peak, however, was generated by the Chagrin soil extract that, on the other hand, generated two peaks at Rf 0.0 and 0.5, representing transformation products. Methanol extracts from the remaining soils (Carlisle and Hagerstown) showed three spots at Rf 0.0, 0.5, and 0.9 for transformation products and residual trifluralin, respectively. 19F NMR Analysis of the Methanol Extracts. The 19F liquidstate NMR spectra of the soil methanol extracts are shown in Figure 3. The chemical shift for trifluralin relative to CFCl3 (0.000 ppm) was -61.994 ppm (Figure 4). The Pope soil 19F NMR spectrum showed only one peak (-61.989 ppm) that represented unaltered herbicide (Figure 3A). Resonances for residual trifluralin also appeared in the spectra for Hagerstown soil (-61.995 ppm, Figure 3B) and Carlisle soil (-61.990 ppm, Figure 3D), but the spectrum for the Chagrin soil did not show a trifluralin resonance (Figure 3C). All spectra, except that for Pope soil, showed additional resonances that represented trifluralin metabolites. Several common resonances appeared at -60.868 to -60.874, -62.703 to -62.710, and -60.699 to -60.706 ppm (Figure 3B-D). However, none of these resonances matched the 19F peaks generated by the five standards from Dow AgroSciences (Table 2 and Figure 4). 19F NMR Analysis of Humic and Fulvic Acids. The 19F solid-state NMR spectra of humic acids isolated (after incubation) from the Chagrin and Carlisle soils are presented in Figure 5 along with the spectra of trifluralin and the mixture of trifluralin with a control humic acid (from Chagrin soil). 6648
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FIGURE 2. Radioscanning of thin-layer chromatography (TLC) plates for methanol extracts from different soils. Both the Carlisle and Chagrin samples showed clear fluorine signals in the form of large broad resonances at -60.8 and -61.2 ppm, respectively. The signals were centered between several much smaller signals that might be spinning sidebands resulting from weak interactions between trifluralin and humic acid. A chloroform-extracted FA fraction from the Chagrin soil analyzed by liquid-state 19F NMR showed two broad resonances at -60.598 and -61.135 ppm (Figure 6). 19F NMR Analysis of Fe-TR and Pd-Fe-TR and Their Coupling Products with Fulvic Acid. As tentatively determined by mass spectrometry, the reaction with zero-valent iron resulted in the formation of 2,6-diamino analogue trifluralin (Fe-TR) (m/z 276) with a 19F signal at -62.653 ppm and an unidentified product at -62.612 ppm (data not shown). These signals did not match any of the 19F resonances collected for methanol extracts from the incubated soils, probably because Fe-TR-like products were involved in bound residue formation. The same two products were also formed, although in lesser quantities, in the presence the palladium zero-valent iron; however, additionally a product was formed with a chemical shift at -60.879 ppm that closely matched 19F signals shown in the spectra of methanol extracts from Hagerstown, Chagrin, and Carlisle soils (-60.874, -60.870, and -60.868 ppm, respectively) (Figure 3). As determined by mass spectrometry, this product (Pa-Fe-TR) had the same mass (m/z 276) as the zero-valent iron product (Fe-TR), and was tentatively identified as a 2,6-diamino derivative of trifluralin with an intramolecular hydrogen bond between two of the three nitrogen atoms (see chemical structure in Table 1). When Fe-TR was mixed with FA, a 19F signal at -62.676 ppm gradually disappeared over a 3-week contact time, giving rise to an emergence and a gradual increase in the size of a broad signal at -61.150 (Figure 7A). Similarly, a gradual disappearance of an 19F signal (at -61.450 ppm) and a gradual buildup of a new 19F signal (at -60.215 ppm) was observed
FIGURE 3. 19F NMR liquid-state spectra of methanol extracts from soils incubated for 7 weeks with trifluralin under anoxic conditions: (A) Pope soil, (B) Hagerstown soil, (C) Chagrin soil, and (D) Carlisle soil. for the Dow AgroSciences standard 2,6-diaminotrifluralin (TR6) when the latter was mixed with FA (Figure 7B). In the absence of FA, T1 values for both Fe-TR (1192 ms) and TR6 (1831 ms) were much greater than those for Fe-TR (151.9 ms) or TR6 (248 to 410 ms) mixed with FA (Figure 8). Chloroform extracts from FA complexes generated 19F three signals at -60.166, -60.284, and -61.347 ppm (for Fe-TR/ FA) and seven signals at -60.269 to -60.856 ppm (for TR6/ FA) but did not generate any signals corresponding to free Fe-TR or TR6 (data not shown). The chloroform-extracted FA solutions showed some changes in their 19F NMR features (data not shown) as compared to 19F signals collected before extraction. The changes included reduced sizes of the major TR6/FA peaks (centered around -61.438 and -60.213 ppm) and, in the case of the Fe-TR/FA, the appearance of new small signals (-60.789 and -61.525 ppm) on either side of the main peak (-61.191 ppm).
Discussion The soils under investigation differed significantly in their ability to transform and immobilize trifluralin under anoxic incubations. On the basis of radioscanning of the TLC plates (Figure 2), most of trifluralin incubated with Pope soil (80.9%
of the initial 14C) remained unaltered, and only 9% occurred in the bound form at the completion of the 7-week incubation (Figure 1). In contrast, the Chagrin soil showed no free trifluralin (Figure 2), but it produced considerable amounts of methanol-extractable transformation products (58%) and bound material (40%) (Figure 1). Similarly, as a result of considerable transformation and binding (Figure 1), only small amounts of free trifluralin were extracted from the Carlisle soil (Figure 2). The differences may have been a result of different soil properties (Table 2). Carlisle soil that had the highest organic matter content (7.4%), clay content (35.2%), and cation exchange capacity (19.9 mequiv/100 g) showed the greatest amount of bound residues (53%) despite the presence of some unaltered herbicide (Figure 2). In contrast, Pope soil, which had less organic matter (3.3%), more sand (55.9%), less clay (18.7%), and smaller cation exchange capacity (9.8 mequiv/100 g) than Carlisle soil, was the least effective in trifluralin transformation and binding (Figures 1 and 2). Chagrin soil with its relatively low organic matter content (2.8%) and high pH (7.6) was the most effective in trifluralin transformation, but not as effective in binding as Carlisle soil (probably due to increased microbial activity as compared to other soils). The complete disappearance of VOL. 38, NO. 24, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 4.
19F
NMR liquid-state spectra of trifluralin and five metabolite standards dissolved in methanol-d4.
trifluralin in Chagrin soil might be due to increased microbial activity as a result of an optimal pH (7.6) (29). The 19F liquid-state NMR analysis confirmed TLC plate findings of the presence of only unaltered trifluralin (with chemical shift at -61.989 ppm) in the methanol extract from Pope soil (Figure 3A) and the absence of trifluralin (no corresponding peak in the NMR spectrum) in the Chagrin soil extract (Figure 3C). Trifluralin signals for Hagerstown and Carlisle soils (-61.995 and -61.990 ppm, respectively) (Figure 3B and D) were considerably reduced relative to that for Pope soil (Figure 3A), which is in agreement with TLC radioscans (Figure 2). In NMR spectra (Figure 3), methanol-extractable transformation products with Rf values of 0 and 0.51, 0.56, or 0.61 (as determined by radio-scanning of the TLC; Figure 2) showed many signals representing at least 14 different compounds with chemical shifts ranging from -60.656 to -62.721 ppm. The signals were very narrow and well resolved, suggesting that they represented free trifluralin metabolites rather than trifluralin metabolites complexed with methanolsoluble components of soil organic matter. Golab et al. (3) reported 31 extractable trifluralin metabolites formed in soil under field conditions, 28 of which were identified. The identified products belonged to four categories: (i) products with the dealkylated amino group and/or reduced nitro groups, (ii) heterocyclic benzimidazole products, (iii) azoxy and azo dimeric products, and (iv) miscellaneous products of oxidation/hydroxylation (3). The latter category is probably not represented by any of the 19F signals collected in this study for methanol extracts, because the experiments were carried out under anoxic conditions. The anoxic incubation in the study of Golab et al. (3), created by flooding the soil, resulted in the formation of three major products including compounds with one or two nitro groups reduced to amino 6650
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groups and a heterocyclic benzimidazole (3). In the study of Golab et al. (3), the same compounds were also formed, although in smaller amounts, under oxic growth conditions, and it was believed that some of these compounds were involved in the formation of bound residues. The chemical shifts of metabolite standards (listed as TR2, TR6, TR15, 4-ABT, and 2-NITRO in Table 2) (Figure 4) did not match any of the resonances shown by the methanol extracts. This suggested that, if any of them were formed, they underwent covalent binding and thus could not be extracted. The possibility of covalent binding was supported by solid-state 19F NMR analysis. The NMR spectra were acquired with magic angle spinning (MAS) one-pulse experiments, without cross polarization or proton decoupling. This was to obtain signals for all 19F present in the samples experiments with cross polarization magic angle spinning (CP-MAS) would only show 19F close enough to H to be cross polarized. Humic acid samples from Carlisle and Chagrin soils, analyzed by 19F solid-state NMR, generated large, broad resonances at -60.8 and -60.2 ppm (Figure 5). Prior to NMR analysis, the samples were exhaustively extracted with methanol and subjected to alkaline hydrolysis and 48-h dialysis; therefore, the resonances could be attributed to trifluralin residues covalently associated with organic matter. An additional indication of covalent interactions was the width of NMR signals (Figure 5). In the absence of humic acid, trifluralin standard showed a sharp well-resolved peak at -63.7 ppm (Figure 5 top left). When the standard was just mixed with the control humic acid, trifluralin peak (at -63.4 ppm) remained sharp and well-resolved (Figure 5 top right). In contrast, humic acid samples extracted from the Chagrin and Carlisle soils with incorporated reduced forms of trifluralin showed broad resonances (Figure 5 bottom). Line broadening is in general a strong indication of covalent
FIGURE 5. 19F NMR solid-state spectra of trifluralin, trifluralin mixed with the dialyzed control HA (from Chagrin soil) at a ratio 1:10 (5 mg/50 mg), and the dialyzed HA from Carslisle and Chagrin soils containing bound residues of trifluralin. interactions between xenobiotics and humic materials (30). Due to the heterogeneous nature of humic acid, bound xenobiotic molecules assume a multitude of slightly different structural conformations, showing broad, composite NMR signals, as was the case for bound residues of trifluralin in this study. The chloroform-extracted fulvic acid from the Chagrin soil also generated large, broad resonances (at -60.598 and -61.135 ppm) (Figure 6), which seemed to represent reduced trifluralin products involved in covalent interactions with organic matter. The presence of two major resonances, rather than one, as was the case for humic acid, indicated that the fulvic acid-bound residues of trifluralin existed in two distinctly different chemical environments or underwent binding via two distinctly different reduced metabolites of trifluralin. It should be emphasized that the resolution of liquid-state 19F NMR is superior to solid-state 19F NMR so that broad resonances observed in the solid-state spectra may represent more than one trifluralin product. No free trifluralin products could contribute to the signals in Figures 5 and 6, because all free material was removed during the 48-h dialysis of humic acids or by chloroform extraction of fulvic acid. Chloroform extraction of fulvic acid was used to extract free residues of the herbicide and was unlikely to remove any trifluralin residues that were covalently bound to fulvic acid. In the study of Williams and Feil (32), for instance, chloroform extraction of rumen microbial media incubated with 14C-labeled trifluralin resulted in the recovery of 88-93% of radioactivity that represented only free residues of the herbicide. Free xenobiotics usually generate sharp and well-resolved NMR peaks (19), similar to the signal generated by the trifluralin standard (-63.4 ppm) that was mixed with humic acid shortly before the analysis (Figure 5). A slight
broadening (by paramagnetic substances) of this signal as compared to the trifluralin peak collected in the absence of humic acid and the appearance of the semi-symmetrical sidebands next to this signal suggested weak interactions between the herbicide molecule and humic acid (e.g., van der Waals forces or hydrogen bonding) (30). As already mentioned, at long contact times (weeks to months rather than hours), a given compound may react with different functional groups present in humic acid and generate a large number of overlapping resonances that appear on the NMR spectra as one broad resonance (17, 19). Two smaller broad resonances (at ∼-30 and -95 ppm) that appeared in the humic acid spectra (Figure 5) resembled the sidebands shown by the mixture of trifluralin with the control humic acid, but they probably represented trifluralin metabolites involved in covalent binding rather than weak interactions. This is based on the expectation that extended dialysis and acid-base treatment should have removed all metabolites held by weak interactions leaving only covalently bound metabolites. Experiments involving reactions between reduced trifluralin products (Fe-TR and TR6) and fulvic acid (Figure 7) supported the hypothesis that the broad resonances generated by solid and liquid soil fractions resulted from covalent binding of amino metabolites of trifluralin, probably through nucleophilic addition to quinone moieties of humic or fulvic acids. As already mentioned, Fe-TR was obtained by incubating trifluralin with zero-valent iron nanoparticles. It generated 19F resonances (at -62.653 and -62.612 ppm; data not shown) that did not match any of the 19F resonances generated by the methanol extracts (Figure 3), which suggested that Fe-TR-like products (2,6-diamines) might be involved in bound residue formation. On the other hand, the VOL. 38, NO. 24, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 6.
19F
NMR liquid-state spectrum of the chloroform-extracted Chagrin fulvic acid containing bound residues of trifluralin.
reaction with the palladium zero-valent iron produced a 2,6diamine derivative that according to MS analysis had the same molecular weight as the zero-valent iron derivative but showed a different chemical shift (at -60.879 ppm; data not shown), probably because of an intramolecular hydrogen bond between two of the three nitrogen atoms (see chemical structure in Table 1). The methanol extracts from three soils showed the presence of trifluralin products with 19F resonances (Figure 3) matching closely that of Pd-Fe-TR (-60.879 ppm), which suggested that the intramolecular hydrogen bonding made 2,6-diamine derivatives unreactive and thus not capable of binding. The gradual reduction in the size of Fe-TR and TR6 resonances (at -62.446, -62.635, and -62.676 ppm and at -61.450 ppm, respectively) and the gradual buildup of new broad signals (at -61.150 ppm and at -60.215, -60.414, -60.441, and -61.796 ppm, respectively) during the 3-week incubation of Fe-TR and TR6 with fulvic acid (Figure 7) supported the hypothesis that 2,6-diamine products of trifluralin metabolism are formed and are bound to humic material with time. Aromatic amines have been shown to react quickly but reversibly with soil organic matter via imine bonds and slower but irreversibly via nucleophilic addition of aromatic amines to soil organic matter quinone moieties (28). The unextractable nature of the FA complexes, formation of broad 19F resonances, and growth of these resonances with time are consistent with the formation of covalent bonds. The reduced spin-lattice T1 values for Fe-TR and TR6 that were complexed with fulvic acid as compared with those determined for Fe-TR and TR6 analyzed in the absence of humic acid (Figure 8) are consistent with strong covalent interactions between diamino derivatives of trifluralin and soil organic matter. Measuring spin-lattice (T1) or spin6652
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spin (T2) relaxation times has been used in the past to examine xenobiotic interactions with humic materials. Nanny et al. (31), for instance, used 13C NMR T1 relaxation to investigate noncovalent interactions between acenaphthenone and fulvic acid samples in aqueous solutions. Their approach was based on the assumption that nuclei of xenobiotics associated with humic material through either strong linkages (covalent binding) or weak linkages (hydrogen bonding, sorption, sequestration) should relax faster than those of free xenobiotics. Depending on the concentration of the reaction components, the T1 of acenaphthenone adsorbed to fulvic acid ranged between 24 and 26 s, whereas the T1 of free acenaphthenone was about 30 s. In this study, the T1 measured for Fe-TR or TR6 in the absence of fulvic acid were 1192-1518 and 1831 ms, respectively, and they were reduced to 152 and 248-410 ms, respectively, when fulvic acid was added (Figure 8). From this observation, we can conclude that interactions occurred between FA and reduced metabolites of trifluralin, although interactions may not necessarily be covalent. Dixon et al. (22) used 19F NMR T1 and T2 relaxation measurements to investigate interactions between 4′-fluoro1′-acetonaphthone and Suwannee River fulvic acid (SRFA). Addition of 4.5 mg of SRFA mL-1 reduced T1 relaxation times from 1781 to 555 ms, with observed broadening of resonances. However, Dixon et al. (22) attributed interactions to sorption either by hydrophobic interactions or hydrogen bonding. In this investigation, similar reductions in T1 relaxation measurements are reported (Figure 8). After mixing with fulvic acid, the Fe-TR signal moved from -62.007 ppm (T1 1192 ms) for Fe-TR alone to -61.188 ppm (T1 152 ms) after a 66-h contact with fulvic acid and showed considerable line broadening (Figure 8), demonstrating
FIGURE 7.
19F
NMR liquid-state spectra of (A) Fe-TR metabolite and (B) TR6 metabolite involved in binding to Chagrin fulvic acid.
xenobiotic-humic associations consistent with covalent binding. Line broadening could be due to heterogeneity of environments in which the Fe-TR was immobilized. In the case of TR6, the initial signals at -61.510 ppm remained in the spectrum (at -61.438 ppm) even after the addition of fulvic acid (Figure 8). This peak decreased and considerably broadened with time, as new broad resonances appeared maximizing at -60.213 and -61.793 ppm (Figure 8). It was expected that T1 measurements for these new peaks would be different than those for the remainder of the original peak; however, all relaxation times were in a narrow range of 248 to 337 ms. This demonstrated that, in some cases, it is not possible to distinguish whether changes in T1 arise from either covalent or noncovalent binding interactions. In an attempt to verify the covalent nature of bonds, the Fe-TR and TR6 complexes with fulvic acid (identified based on T1 relaxation measurements) were extracted with chloroform and again analyzed by 19F NMR (data not shown).
Chloroform extracts generated no 19F signals for free Fe-TR or TR6 (at -62.007 and -61.510 ppm as shown in Figure 8, panels A and C, respectively), suggesting the covalent nature of associations between these compounds and fulvic acid. Three chemical shifts at -60.166, -61,284, and -61.347 ppm (for Fe-TR chloroform extract) and seven signals ranging from -60.269 to -60.856 ppm (for TR6 chloroform extract) can probably be attributed to Fe-TR and TR6 associated with chloroform-soluble fulvic acid components. The chloroform extraction of the Fe-TR/FA (Fe-TR metabolite incubated with FA) resulted in a reduction of the peak at -61.188 ppm (shown for the unextracted complex in Figure 8B), and the appearance of two new peaks at -60.789 and -61.525 ppm (data not shown) that probably resulted from changes in the structural arrangement of the chemically bound Fe-TR molecules after the removal of the chloroformsoluble fulvic acid complex. VOL. 38, NO. 24, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 8. 19F NMR liquid-state spectra and the respective spin-lattice relaxation times (T1) of (A) Fe-TR metabolite, (B) Fe-TR with Chagrin FA, (C) TR6 metabolite, and (D) TR6 metabolite with Chagrin FA. As a result of chloroform extraction of the TR6/FA complex, the large sharp peak at -61.438 ppm (shown for the unextracted complex in Figure 8D) decreased somewhat (more in height rather than in size or peak area); and the center of the peak at -60.213 ppm shifted to -60.249 ppm (data not shown). In view of this relatively minor modification, the latter peak probably represented reduced trifluralin molecules that were covalently attached to fulvic acid. The peak at -61.438 ppm probably represented the unaltered TR6 molecules that may have been retained by strong electrostatic attraction between the negative charges of fulvic acid and the positive charges of the protonated amino groups and, therefore, resisted chloroform extraction. In general, broad resonances generated by the fulvic acid complexes appeared in the same regions of the spectra as those collected for the fulvic acid fraction of the incubated soil (Figure 6). These and other NMR data (lack of extractable amino metabolites, broad 19F resonances in FA and HA from trifluralin/soil incubations) collected in this study can be considered a strong confirmation of the hypothesis that trifluralin binding in soil proceeds through the formation of 6654
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amino derivatives that in turn interact with soil organic matter. The differences in the location of 19F signals in the NMR spectra could represent varying numbers and positions of amino groups (3-, 4-, and 5- relative to the CF3 group) in different transformation products. The 19F NMR technique shows great potential for investigating the fate of important fluorinated pollutants in soil environments. If combined with a sufficient number of reference standards and traditional approaches (e.g., using 14C-labeled xenobiotics), it could quickly provide reliable information on both transformation pathways and the mechanisms of binding.
Acknowledgments This research was supported by the Office of Research and Development, U.S. Environmental Protection Agency (Grant R-826646). Although this work was reviewed by the U.S. EPA and approved for publication, it may not necessarily reflect official Agency policy. Partial support was also provided by Penn State Biogeochemical Research Initiative for Education (BRIE) sponsored by NSF (IGERT) Grant DGE-9972759.
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Received for review January 2, 2004. Revised manuscript received September 19, 2004. Accepted September 20, 2004. ES0403110
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