Environ. Sci. Technol. 1997, 31, 2991-2997
Use of a Silylation Procedure and 13C-NMR Spectroscopy To Characterize Bound and Sequestered Residues of Cyprodinil in Soil J E R Z Y D E C , † K O N R A D H A I D E R , †,‡ A N D R E A S S C H A¨ F F E R , § , | ERROL FERNANDES,† AND J E A N - M A R C B O L L A G * ,† Laboratory of Soil Biochemistry, Center for Bioremediation and Detoxification, The Pennsylvania State University, University Park, Pennsylvania 16802, and Novartis Crop Protection AG, Basel, Switzerland
Soil-bound residues of the fungicide cyprodinil (4cyclopropyl-6-methyl-2-phenylaminopyrimidine), which was labeled with 13C and 14C either in the phenyl ring or the pyrimidyl ring, were analyzed by a silylation procedure and 13C-NMR spectroscopy. After a 6-month incubation of soil with 3 or 500 ppm cyprodinil, bound residues amounted to about 50% and 18% of the initial radioactivity, respectively. The isolated humic acid fraction and the NaOH-extracted soil (the humin fraction) were suspended in chloroform and silylated by overnight shaking with trimethylchlorosilane. Analysis of the silylated extracts by 13C-NMR revealed that the formation of soil-bound residues in the 500 ppm samples involved: (1) sequestration of the unaltered or slightly altered fungicide in the humin fraction and (2) cleavage of the cyprodinil molecule between the aromatic rings followed by covalent binding of the separated moieties to humic acid. The sequestered fungicide (phenyl label) generated triplet NMR signals at 121.0, 125.0, 128.7, and 135.8 ppm, which closely coincided with the resonances shown by the silylated standard of cyprodinil. TLC analysis indicated that, in the 3-ppm samples, cleavage products were also subject to sequestration in humin. As determined by sizeexclusion chromatography, the molecular size of the humic acid fraction with the incorporated cleavage products was in the range of 2 × 103 Da.
Introduction The impact of xenobiotics on the biosphere may be considerably modified by the immobilization phenomena occurring in soil. For that reason, the nature of soil-bound pollutants has become the subject matter of many studies (1, 2). By definition, bound residues represent the portion of the pollutant that cannot be extracted from soil by methods that do not alter the chemical character of the immobilized compounds (3). Xenobiotics retained through adsorption are considered reversibly bound because they can be desorbed through extraction with organic solvents. According to recent * Corresponding author: phone: (814) 863-0843; fax: (814) 8657836; e-mail:
[email protected]. † The Pennsylvania State University. ‡ Present address: Kastanienallee 4, 82041 Deisenhofen, Germany. § Novartis Crop Protection AG. | Present address: RWTH Aachen, Biology V, Environmental Analytical Chemistry, 52074 Aachen, Germany.
S0013-936X(97)00228-9 CCC: $14.00
1997 American Chemical Society
observations, however, the desorption rates are subject to reduction with the time that chemicals remain in soil (4). Apparently, during prolonged residence or aging in soil, xenobiotic molecules undergo an entrapment (sequestration) in structural voids and hydrophobic interiors of micelle-like humic aggregates (6-8) or in the micropores of organic matter and clay minerals (4, 5, 9). As a result of these phenomena, chemicals exhibit declining availability to extraction or biodegradation (9). In addition to sorption and sequestration, the immobilization mechanisms involve covalent binding of xenobiotics to soil organic matter. The chemicals that are bound through stable covalent linkages are considered to be an integral part of humus, thus representing little or no threat to the environment (2). The evaluation of soil-bound residues using 13C- or 15Nenriched xenobiotics is a novel approach that was applied in recent studies with aniline, anilazine, 2,4-dichlorophenol, and cyprodinil (11, 13-17). The method consisted of NaOH extraction of soil, dialysis and freeze-drying of the humic acid fraction, and 13C-NMR analysis of humic acid redissolved in 1% NaOD. In addition, solid state (CPMAS) 13C NMR was applied for analysis of soil extracted with organic solvents. Bound residues were characterized based on changes in the chemical shifts of the labeled atoms. The analysis was possible due to the increased intensities of the NMR signals as compared to the background signals resulting from the natural abundance of 13C or 15N in humic acid. In the study of Haider et al. (10, 11), soil-bound residues of the fungicide anilazine were analyzed by a silylation procedure and NMR spectroscopy. According to Drozd (12), the silylation reaction involves the substitution of a silyl moiety for the hydrogen atom in various functional groups (-OH, dNH, -NH2, -SH, -COOH) without further alteration of the derivatized molecule. Haider et al. (10, 11) demonstrated that upon silylation significant amounts of soil humus together with bound residues of 14C- and 13C-labeled anilazine became soluble in organic solvents. Apparently, the replacement of the active hydrogens in the soil matrix with silyl groups caused a disintegration of humic aggregates or micelles into smaller fragments that normally were held together by the hydrogen bonds and other non-covalent interactions. The disintegrative effect of silylation on humic substances brought about a remarkable improvement in the resolution of 13C-NMR spectra as compared to that achieved for the NaOH extracts. Moreover, silylation proved to be capable of releasing significant amounts of organic substances from humin, the fraction that cannot be released by alkali extraction. Since the silylated humic materials were soluble in organic solvents, they could be analyzed by size-exclusion, thin-layer, and highperformance chromatography to address questions regarding whether, upon silylation, the xenobiotic residues were released in the bound or in the free form, whether binding mechanisms differed in specific soil fractions, and whether any changes occurred in the mode of binding when xenobiotics were aged in soil. In the present investigation, the silylation procedure combined with 13C-NMR spectroscopy and various chromatographic techniques was applied to evaluate soil-bound residues of cyprodinil (4-cyclopropyl-6-methyl-2-phenylaminopyrimidine), a new fungicide manufactured by Novartis Crop Protection AG, Basel, Switzerland. The objectives of the study were to confirm the results of previous research on the incorporation of cyprodinil residues into humic acid (17) and to evaluate the immobilization mechanism in the humin fraction.
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Materials and Methods Chemicals. The silylating reagent, trimethylchlorosilane, was purchased from Aldrich Chemical Company, Inc. (Milwaukee, WI). Unlabeled, 14C-labeled, and 13C-labeled cyprodinil were provided by Novartis Crop Protection AG, Basel, Switzerland. The labeled carbons of cyprodinil were located either in the phenyl ring (uniformly labeled) or in the pyrimidyl ring at the C-2 position. The specific radioactivity of both the U-phenyland the 2-pyrimidyl-labeled cyprodinil was 1.85 MBq/mg. The enrichment of cyprodinil with carbon 13C amounted to 98% of the total carbon at the specified position. Incubation of Cyprodinil in Soil. The incubation experiments were carried out using a Hagerstown soil (Typic Hapludalf). The physicochemical properties of this soil and the storage conditions prior to incubation with cyprodinil were described previously (17). The initial concentrations of the fungicide in soil were 0 (control) and 3 or 500 ppm. Soil samples (200 g dry weight; sieved through a 2-mm screen) were distributed into 1-L Erlenmeyer flasks, inserted into airflow systems, and incubated for 197 days with 3 ppm and 169 days with 500 ppm, respectively, as described by Dec et al. (17). The 500 ppm incubations were carried out to prepare samples for 13C-NMR analysis. As shown in previous studies (17), the concentration of 3 ppm that represented the recommended field rate (1-3 ppm) was too low to obtain discernible signals on NMR spectra. The contribution of the 13C-labeled compound to the initial concentration of 500 ppm was 498 ppm; the remainder was contributed by 14C-labeled cyprodinil. The 3 ppm samples were prepared using only the 14C-labeled fungicide. The initial radioactivity per incubation flask for the 3 ppm and 500 ppm samples was 1.1 and 0.74 MBq, respectively. Fractionation and Silylation of Soil-Bound Residues. After incubation, soil samples (10 g dry weight) were extracted four times with methanol (the fourth extract contained less than 0.1% of the radioactivity found in the first), centrifuged, and air-dried overnight. One set of the methanol-extracted samples (designated as whole soil) was saved for silylation. Other samples were extracted for 24 h by shaking with 50 mL of 0.5 M NaOH under nitrogen. The NaOH extract was separated by centrifugation, and the soil pellet was washed five times with 0.1 M NaOH. The NaOH-extracted soil (designated as humin) was dried and saved for silylation. The combined NaOH solution (extract plus washings) was acidified to pH < 1 with 5 M HCl, stored overnight at 4 °C for complete precipitation of humic acid, and centrifuged. The humic acid precipitate was redissolved in NaOH solution and centrifuged at 12000g to remove insoluble fine solids. The supernatant was dialyzed for 48 h against frequently changed deionized water using a membrane with 6000-8000 Da cutoff (Spectrum, Houston, TX) and freeze-dried, yielding on average 55 mg of humic acid. After freeze-drying, the purified humic acid was considered ready for silylation; however, samples of the whole soil and humin first had to be completely dehydrated because traces of moisture inhibit the silylation reaction. For that purpose, after the preliminary air-drying, pellets of the soil containing humin were crushed to a fine mesh and freeze-dried. Samples of the whole soil were kept under nitrogen and first shaken with 0.5 M NaOH for 2 h. The suspension of soil in the NaOH extract was sonicated under nitrogen for 5 min, frozen and freeze-dried, then crushed into small particles, and freezedried again overnight. The dehydrated samples of humic acid, whole soil, and humin were placed in 500-mL round-bottom flasks, resuspended in 80 mL of freshly distilled dry chloroform, and treated with 3-7 mL of freshly distilled trimethylchlorosilane and three to six pieces of granular NaOH (to neutralize HCl generated during silylation). The reaction flask was plugged with an inverted form tube (Fisher Scientific, Pittsburgh, PA) packed with a drying agent; the inner joint of the tube was
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sealed with a Teflon sleeve. After a 3-h shaking at room temperature, another 3-7-mL portion of trimethylchlorosilane and three to six pieces of granular NaOH were added, and shaking was continued overnight. The reaction mixture was then centrifuged, and the chloroform supernatant was analyzed for radioactivity. The solids were transferred into a Soxhlet thimble and extracted exhaustively with freshly distilled dry acetone. The acetone extract was analyzed by radiocounting, combined with the chloroform fraction, and evaporated to dryness under vacuum (4 × 10-3 Torr) at
