Environ. Sci. Technol. 1997, 31, 1128-1135
Analysis of Soil-Bound Residues of 13C-Labeled Fungicide Cyprodinil by NMR Spectroscopy J E R Z Y D E C , † K O N R A D H A I D E R , †,‡ ALAN BENESI,§ V. RANGASWAMY,† A N D R E A S S C H A¨ F F E R , | UDO PLU ¨ CKEN,| 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, Chemistry Department, The Pennsylvania State University, University Park, Pennsylvania 16802, and Ciba Crop Protection, CIBA (Ciba-Geigy Limited), Basel, Switzerland
13C-NMR spectroscopy was applied to the evaluation of soil-
bound residues of the fungicide cyprodinil (4-cyclopropyl6-methyl-2-phenylaminopyrimidine). A mixture of the 13Cand 14C-labeled fungicide was used to obtain structural information as well as information on the quantitative distribution in the various fractions. Bound residues were accumulated by a 6-month incubation of the labeled compound with a clay loamy soil. Depending on the concentration of [13C]cyprodinil (500, 250, 80, and 3 mg/kg), binding ranged from 18% to 54% of the initial radioactivity. After methanol extraction of soil (10 g dry weight) treated with 500 mg/kg (5.0 mg) of the fungicide, the amount of unextracted bound material was equivalent to 0.9 mg of 13C-labeled cyprodinil. Upon fractionation, 0.21 mg of the bound fungicide was found in the dialyzed humic acid, 0.13 mg in fulvic acid (after extraction with CH2Cl2), and 0.24 mg in humin. The methylene chloride extract from fulvic acid mainly contained unchanged cyprodinil (0.21 mg) that was apparently sequestered in soil by physical forces. The humic acid fraction was dissolved in a 1% solution of NaOD and examined by 13C-NMR. The NMR spectrum of the material from the control sample exhibited all the characteristic features of a typical humic acid. When the control humic acid was spiked with cyprodinil labeled uniformly with 13C at the phenyl ring, four additional signals at 121.9, 124.4, 131.8, and 143.4 ppm could be distinguished in the NMR spectrum. However, when humic acid originated from the soil that was incubated with the phenyllabeled fungicide, only two strong NMR signals, at 122.5 and 131.8 ppm, and two less significant signals around 142 and 162 ppm were observed. The difference in the signal pattern indicated cleavage of the cyprodinil molecule between the aromatic rings and independent binding of the phenyl and pyrimidyl moieties to humic acid.
Introduction Significant amounts of pesticides and other xenobiotics may be present in soil in the form of bound residues that are not extractable by methods that do not alter their chemical nature * Corresponding author phone: (814) 863-0843; fax: (814) 8657836; e-mail:
[email protected]. † Laboratory of Soil Biochemistry. ‡ Present address: Kastanienallee 4, 82041 Deisenhofen, Germany. § Chemistry Department. | Ciba Crop Protection.
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(1). Organic chemicals can be bound to soil through various mechanisms involving either physical sorption or chemical reaction. From an environmental point of view, the immediate effects of binding appear to be beneficial, since the immobilized xenobiotics exhibit diminished bioavailability and toxicity; their transport to groundwater is also greatly restricted (2, 3). However, the formation of bound residues may prevent xenobiotics from being mineralized or transformed to less hazardous derivatives, and it has been argued that soil-bound chemicals may be released at a later time, causing damage to the environment (4). The pioneering research on bound residues was carried out using 14C-labeled xenobiotics. Studies by Katan et al. (5), Helling and Krivonak (6), Fu ¨ hr and Mittelstaedt (7), and Khan and Hamilton (8) demonstrated that bound radioactivity was located in all three major fractions of soil organic matter (humic acid, fulvic acid, and humin). The amount of bound material increased over time, and the rate of binding varied with the chemical structure of the investigated compound and the type of soil. Microbial transformation was usually a prerequisite to binding. Recently, it was suggested that the time factor had much to do with the phenomenon of an entrapment, termed sequestration (3). It appears that the longer a chemical resides or is aged in soil, the more strongly it becomes sequestered. Unlike bound xenobiotics, those that are sequestered can be recovered from soil by exhaustive extraction with organic solvents, but their bioavailability and toxicity gradually diminish as a result of aging. The labeling of xenobiotics with 14C facilitates the quantification of bound residues, but it cannot provide much structural information unless combined with other analytical techniques. Using isothermal heating, for instance, Helling and Krivonak (6) demonstrated that aniline derivatives of 14C-labeled butralin formed covalent bonds with phenolic constituents of soil organic matter. Another application of this technique indicated that immobilization of [14C]prometryn was due to entrapment of the unchanged herbicide in the molecular net of humus (8). In studies involving 14Clabeled atrazine, buturon, cyprodinil, and other pesticides, up to 60% of bound radioactivity could be released from soil in the form of both parent compounds and metabolic products by extraction with supercritical methanol or microwave sonication (9-11). Recently, several studies have demonstrated that further progress in the evaluation of the nature of binding can be achieved by the application of 13C- or 15N-labeled xenobiotics in combination with 13C- or 15N-NMR spectroscopy (12-15). Identification of bound residues relied on the increased intensities of the NMR signals and changes in the chemical shifts of the labeled atoms. In the present study, NMR was applied to the evaluation of soil-bound residues of cyprodinil (4-cyclopropyl-6-methyl-2-phenylaminopyrimidine), a new broad-spectrum fungicide synthesized by Ciba-Geigy Limited, Basel, Switzerland. Contamination of soil with cyprodinil may result from spray drift, rain wash, or runoff. In a study using the fungicide labeled with 14C in either the pyrimidyl or the phenyl moiety, only 10% of the initial radioactivity was recovered from soil after 1 year of incubation; 25% was mineralized to 14CO2, and 65% became bound to soil and could not be extracted with water or organic solvents (16). According to Calderbank (2), the extent of pesticide binding in soil varies from a few percent to 90% depending on the compound structure and soil type. In the present study, 13C- and 14C-labeled cyprodinil was first incubated with soil to accumulate the bound material; subsequently, humic and fulvic acids together with the bound residues of [13C]cyprodinil were isolated from the soil and
S0013-936X(96)00631-1 CCC: $14.00
1997 American Chemical Society
FIGURE 1. Chemical structure of cyprodinil with the assigned carbon numbers. examined by methods.
13C-NMR
spectroscopy or other analytical
Materials and Methods Soil. The experiments were carried out with a Hagerstown soil (Typic Hapludalf, clay loam; sand 27.6%; silt 43.0%; clay 29.4%; pH 5.7; organic carbon 1.6%; nitrogen 0.1%; cation exchange capacity 11.1 mequiv/100 g). The soil was stored in a greenhouse and overgrown with turfgrass. Shortly before the experiment, the soil was sieved through a 2-mm screen, adjusted to 40% of maximum water holding capacity (WHC) (22.3 g of water/100 g of dry soil) and stored at room temperature for 10 days before use. Chemicals. Unlabeled cyprodinil (water solubility 12 mg/L at pH 6.8; log Kow 4.5; Henry’s law constant 2.7 × 10-6 at pH 6.8), and 14C-labeled and 13C-labeled cyprodinil were provided by Ciba-Geigy Ltd., Basel, Switzerland. The chemical structure of cyprodinil with assigned carbon numbers is presented in Figure 1. The labeled carbons 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-phenyl- and 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 positions. Ciba-Geigy Ltd. also provided the unlabeled metabolites of cyprodinil; their chemical structures are shown in Table 1. Incubation of Cyprodinil in Soil. Soil samples (200 g dry weight) were distributed into 1-L Erlenmeyer flasks and thoroughly mixed with 13C- and 14C-labeled cyprodinil dissolved in 0.6 mL of acetone. The control samples were amended with acetone and mixed, but no cyprodinil was added. The initial concentrations of the fungicide in the treated samples were 3, 80, 250, and 500 mg/kg. The contributions of the 13C-labeled compound to these initial concentrations were 0, 77, 247, and 498 mg/kg, respectively. The remainder was contributed by 14C-labeled cyprodinil. The initial radioactivity per incubation flask was 1.1 MBq for the initial concentrations of 3, 80, and 250 mg/kg and 0.74 MBq for the initial concentration of 500 mg/kg. After readjustment of the soil moisture to 40% WHC, the incubation flasks were wrapped in aluminum foil to provide darkness and inserted into an air-flow system operating under slight vacuum. The incubation temperature was 25 °C. Two traps with 4 N NaOH to remove CO2 from the incoming air and one trap with water for moistening the soil were placed on the inlet of the incubation flask. The outlet was followed by one 1 N H2SO4 trap and one ethylene glycol trap to absorb organic volatiles, one empty trap, and three 0.5 N NaOH traps for adsorption of both the respiratory CO2 and the 14CO2 resulting from mineralization of the 14C-labeled cyprodinil. The incubations for each label and concentration were carried out in duplicate for 197 days (3, 80, and 250 mg/kg) or 169 days (500 mg/kg). Trapping solutions were analyzed periodically by radiocounting and titration with HCl according to Stotzky (17). Soil samples (1 g dry weight) taken after the specific incubation times were extracted six times by 1-min vortexing with 3 mL of methanol, centrifuged, and analyzed for extractable and bound radioactivity.
Fractionation of Soil-Bound Residues. Soil samples (10 g dry weight) from the 169-day incubations with 500 mg/kg of [U-phenyl-13C]cyprodinil or [2-pyrimidyl-13C]cyprodinil were extracted four times by shaking for 1 h with 30-mL portions of methanol. After air-drying overnight, the soil was extracted for 24 h by shaking with 50 mL of 0.5 N NaOH under nitrogen. The NaOH extract was separated by centrifugation, and the soil was washed three times with 0.1 N NaOH. The combined solution (extract plus washings) was acidified to pH < 1 with 5 N HCl, stored overnight at 4 °C for complete precipitation of humic acid, and subsequently centrifuged. The supernatant with unprecipitated fulvic acid was extracted three times with methylene chloride, and the extract was analyzed by TLC, radioscanning, and 13C-NMR. The humic acid precipitate was washed three times with acidified water (pH < 1; the washings were combined with fulvic acid for the methylene chloride extractions described above), 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 a 6000-8000 Da cutoff. The dialyzed solution of humic acid was freeze-dried (yielding 54-61 mg of humic acid), dissolved in 1 mL of 1% NaOD, and analyzed by 13C-NMR. For comparison, mixtures of humic acid from the control soil with 13C-labeled cyprodinil were also analyzed by 13C NMR in 1% NaOD. The distribution of the labeled compound among the various fractions was determined by radiocounting. Radiocounting. Aliquots of methanol extracts from soil, NaOH solutions with the adsorbed 14CO2, aqueous solutions of humic and fulvic acids, CH2Cl2 extracts from the unprecipitated fulvic acid, humic acid pellets redissolved in NaOH solution, and other liquid samples were analyzed with Ecoscint scintillation cocktail (National Diagnostics, Atlanta, GA) on a Beta Trac 6895 liquid scintillation counter (Elk Grove, IL). Soil samples before and after extraction (0.3 g) were combusted in a Harvey Biological Oxidizer OX 600 (Hillsdale, NJ) using Harvey Carbon 14 Cocktail for absorption and counting of 14CO2. Radioscanning of TLC plates was carried out on a System 200 imaging scanner (Bioscan, Washington, DC). 13C-NMR Analysis. The 13C-NMR spectra were obtained on four spectrometers, all of which operate in the quadrature mode at 294 ( 1 K. Humic acid fractions (with or without 13C-labeled cyprodinil) dissolved in 1% NaOD were analyzed on either a Bruker AM 500 or a Bruker AMX2-500 spectrometer. Regular 5-mm (o.d.) NMR tubes were used for these samples, and the resonance frequency for 13C in these instruments was 125.76 MHz. Inverse-gated WALTZ decoupling was used, with approximately 150 000 scans for each sample. The relaxation delay was held constant at 0.2 s for all samples dissolved in 1% NaOD. The subtraction spectra (humic acid with labeled cyprodinil minus humic acid control) were obtained with Bruker software. The 13C NMR spectra of the 13C-labeled cyprodinil, unlabeled cyprodinil, and unlabeled metabolites of cyprodinil (in deuterated chloroform) were obtained with continuous WALTZ decoupling on either a Bruker AM 300 or a Bruker WM-360 resonating at 75.47 and 90.56 MHz, respectively, for 13C. Regular 5-mm (o.d.) NMR tubes were used for samples run on these two instruments. Depending on the chemical structure and abundance of the 13C carbon, between 100 and 6100 scans using 45-90° pulses were acquired for each sample, with a relaxation delay of 4 s and an acquisition time of 0.75 s.
Results Formation and Fractionation of Soil-Bound Residues of Cyprodinil. The extent of bound residue formation depended on the initial concentration of cyprodinil in the tested soil (Figure 2). In soil treated with 3 mg/kg of U-phenyl-14C-labeled
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TABLE 1. Chemical Shifts (ppm) of 13C in Molecules of Cyprodinil and Its Metabolites Dissolved in Deuterated Chloroform carbon number no.
chemical structure
1
NH
N
1′
2′
3′
4′
5′
6′
2
140.1
118.5
128.8
121.7
128.8
118.5
159.8
133.1
115.6
121.1
151.0
121.1
115.6
159.9
141.4
105.9
156.2
108.9
129.7
111.0
159.6
141.3
104.1
160.1
107.7
129.4
110.2
159.6
139.3
119.0
128.9
122.7
128.9
119.0
158.0
140.5
118.6
128.4
121.4
128.4
118.6
160.2
139.6
112.3
128.8
122.3
128.8
112.3
159.4
140.0
118.6
128.8
121.8
128.8
118.6
159.7
N CH3 NH
2
N N
HO
CH3 NH
3
N N
OH
CH3 NH
N
4 N CH3
OCH3 NH
N
5 N OCH3
CH3 NH
6
N N COOH
NH
N
7 N CH2OH NH
N
8 N CH2OCH3 H2N
9
N
162.9
N CH3 HO
N
10
160.4 N CH3
fungicide, 54% binding was determined after 197 days of incubation. At higher concentrations of U-phenyl-13C- and 14C-labeled cyprodinil, bound residue formation was reduced to 31.0% for 80 mg/kg, 19.7% for 250 mg/kg, and 18.8% (after 169 days) for 500 mg/kg. Despite this reduction in the percentage of binding, there were increases in the absolute amounts of bound compound; after incubations with 3, 80, 250, and 500 mg/kg, the concentrations of bound material were equivalent to 1.6, 24.8, 49.3, and 94.0 mg/kg of the 13Cand/or 14C-labeled cyprodinil, respectively. The disappearance of cyprodinil was mostly due to binding to soil. Only 6.3% of the applied radioactivity was accounted
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for by mineralization of the compound to 14CO2 during the incubation with 3 mg/kg; in samples treated with 80, 250, and 500 mg/kg, evolved 14CO2 accounted for 3.4%, 2.2%, and 2.1% of the radioactivity, respectively. Time courses for the disappearance and binding of the various concentrations of 2-pyrimidyl-13C- and 14C-labeled fungicide (data not shown) were very similar to those determined for the phenyl-labeled compound. However, the percentages of the evolved 14CO2 from the phenyl ring were approximately 3-fold higher than those from the pyrimidyl ring (2.7%, 1.1%, 0.8%, and 0.5%, respectively), suggesting that the latter was more resistant to mineralization.
FIGURE 2. Binding of U-phenyl-13C- and/or 14C-labeled cyprodinil in soil during incubation with various concentrations of the fungicide (3-500 mg/kg). Increased concentrations of cyprodinil did not affect soil respiration as determined by CO2 trapping and HCl titration. The weekly output of 12CO2, both in controls and in samples treated with U-phenyl-13C- and 14C-labeled cyprodinil, fluctuated between 20 and 40 mg throughout the whole incubation period; outputs for the 2-pyrimidyl label were very similar. The total amounts of CO2 evolved from soil samples during 197 days of incubation with 3, 80, and 250 mg/kg were close to that determined for the control (651 mg). The reduced value of total CO2 evolution (538 mg) from soil incubated with 500 mg/kg of cyprodinil was due to a shorter incubation time. No cyprodinil was lost through volatilization as determined by radiocounting of the 1 N H2SO4 and ethylene glycol traps. The fractionation patterns for the phenyl and pyrimidyl label were very similar. During the repeated methanol extraction of soil (10 g dry weight) incubated with 500 mg/kg of the phenyl-labeled fungicide, 82.6% of the initial radioactivity was removed with less than 0.1% in the fourth methanol extract. The extracted soil retained 18.1% of the initial radioactivity, which was equivalent to 0.9 mg of the labeled compound. The subsequent extraction of soil with 0.5 N NaOH released soluble humic material together with almost two-thirds of the bound residues of cyprodinil (13.2%); the remaining radioactivity (4.9%) was detected in the insoluble humin fraction. After acidification of the NaOH extract and exhaustive washing of the precipitated humic acid with acidified water, 5.7% of bound radioactivity was found in the precipitated humic acid (redissolved in 0.5 N NaOH) and 7.0% in the fulvic acid solution combined with the washings. Dialysis of the humic acid solution resulted in the reduction of bound radioactivity to 4.3% which was equivalent to 0.21 mg of the 13C-labeled cyprodinil. After extraction of fulvic acid with methylene chloride, 4.2% of
radioactivity was found in the organic phase, and 2.7% remained in the aqueous phase, apparently bound to fulvic acid. TLC analysis and radioscanning of the TLC plates indicated that most of the radioactivity (80% or 0.16 mg) contained in the methylene chloride extract represented unchanged fungicide (data not shown), which was confirmed by 13C-NMR. No radioactivity could be extracted with methylene chloride from humic acid in solution. When the humic acid was redissolved and reprecipitated, the aqueous supernatant contained some radioactivity, but this could not be extracted with organic solvents. 13C-NMR Studies. Figure 3 presents the 13C-NMR spectra for unlabeled cyprodinil, U-phenyl-13C-labeled cyprodinil, and 2-pyrimidyl-13C-labeled cyprodinil in deuterated chloroform. The triplets at 76.6-77.3 ppm on all three spectra are due to the deuterated solvent. The unlabeled fungicide generated 11 NMR signals. The peaks at 10.2, 16.8, and 23.8 ppm correspond to the side-chain cyclopropyl-CH2 carbons (CP-CH2), cyclopropyl-CH carbon (CP-CH), and methyl carbon at the C-6 position (C6-CH3), respectively (see Figure 1 for the assignment of carbon numbers). The signals at 110.0, 159.8, 166.5, and 172.5 ppm were assigned to carbons in the pyrimidyl ring (C-5, C-2, C-6, and C-4, respectively). The signals at 118.5, 121.7, 128.8, and 140.1 ppm represent the phenyl ring carbons (C-2′ and C-6′, C-4′, C-3′ and C-5′, and C-1′, respectively).
Four triplets were generated by U-phenyl-13C-labeled cyprodinil (Figure 3). The triplet structure of the signals resulted from the spin-spin coupling of neighboring 13Cenriched carbons in the phenyl ring. The peaks at 117-119, 120-122, 128-130, and 139-141 ppm represent the labeled carbons C-2′ and C-6′, C-4′, C-3′ and C-5′, and C-1′, respectively. The 2-pyrimidyl-13C-labeled cyprodinil gener-
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FIGURE 3. 13C-NMR spectra for the solution of unlabeled cyprodinil, U-phenyl-13C-labeled cyprodinil, and 2-pyrimidyl-13C-labeled cyprodinil in deuterated chloroform. ated only one signal at 159.8 ppm due to the labeled carbon C-2. Carbons C-1′ through C-6′ and C-2 of the unlabeled metabolites of cyprodinil dissolved in deuterated chloroform generated signals at ppm values ranging between 104.1 and 162.9 ppm, depending on the chemical structure of the compound examined (Table 1). The signals representing carbons C-4 through C-6, the carbon of the methoxy group attached to the methyl carbon, and the carbon of the methoxy group substituted at the C-3′ position in the phenyl ring ranged between 10.2 and 193.6 ppm (data not shown). The 13C-NMR spectrum of humic acid (in 1% NaOD) isolated from a control sample of soil that was incubated without cyprodinil is presented in Figure 4. Four characteristic regions can be distinguished on this spectrum, reflecting the presence of aliphatic hydrocarbons (15-50 ppm), C-O/C-N compounds (50-110 ppm), aromatic resonances including those of phenols or amines (110-160 ppm), and compounds with carboxyl or carbonyl functionalities (160-200 ppm). Figure 5 shows the 13C-NMR spectra of U-phenyl- and 2-pyrimidyl-13C-labeled cyprodinil that, shortly before the NMR analysis, were dissolved in the 1% NaOD solution of the control humic acid. The amount of the labeled compound (0.21 mg) was equal to that determined for the residues of 13C- and 14C-labeled cyprodinil bound to the dialyzed humic acid. The pattern of NMR signals generated by the U-phenyl13C-labeled cyprodinil was simlar to the pattern of triplets obtained for the compound dissolved in deuterated chloroform (Figure 3). Due to diminished resolution of the NMR spectrum in the humic acid solution, the triplet structure could not be resolved, and the peaks were slightly shifted downfield (121.9, 124.4, 131.8, and 143.4 ppm for carbons C-2′ and C-6′, C-4′, C-3′ and C-5′, and C-1′, respectively). The resonance for carbon C-4′ (124.4 ppm) appeared only as a shoulder of the resonance for carbons C-2′ and C-6′ (121.9
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ppm). The 2-pyrimidyl-13C-labeled cyprodinil dissolved in the 1% NaOD solution of control humic acid generated a singular peak at 162.5 ppm, representing the labeled carbon C-2. Like signals for the phenyl label, the peak was slightly shifted relative to the corresponding peak generated by the compound dissolved in deuterated chloroform (159.8 ppm). The 13C-NMR spectrum for humic acid isolated from the soil after incubation with 500 mg/kg of the U-phenyl-13Clabeled fungicide is presented in Figure 6A. The labeled component of the sample generated two strong signals, at 122.5 and 131.8 ppm. Both signals were clearly exposed when the background of the control humic acid in Figure 4 was subtracted from the spectrum of humic acid containing the 13 C-labeled material (Figure 6B). In addition, two much weaker signals appeared at 141.7 and 161.7 ppm. It should be noted that the signals at 121.9, 124.4, 131.8, and 143.4 ppm appearing on the spectrum for the mixture of the U-phenyl13 C-labeled fungicide and humic acid (Figure 5) are shifted or even missing in the subtraction spectrum. This change in the original pattern of four NMR signals suggests a significant alteration in the chemical environment of the 13C-labeled carbons in the phenyl moiety of cyprodinil. The most prominent peak at 131.8 ppm coincides with the strongest peak in the aromatic region of humic acid (18). The possibility that this peak originated from unchanged residual cyprodinil must be rejected because of the disappearance or reduction of the original signals seen in Figure 5. Figure 7A shows the spectrum for humic acid isolated from the soil incubated with 500 mg/kg of 2-pyrimidyl-13Clabeled cyprodinil. This spectrum does not differ essentially from that of the humic acid control (Figure 4) except for the presence of a peak at 161.8 ppm originating from labeled C-2 of the bound fungicide. Subtraction of the humic acid control background from this spectrum removed all peaks related to humic material, leaving only the peak at 161.8 ppm (Figure 7B). The peak for bound cyprodinil residues (161.8 ppm) was slightly shifted upfield relative to the peak of free cyprodinil (162.5 ppm), when the latter was dissolved together with humic acid in NaOD (Figure 5).
Discussion Preliminary studies revealed that the natural abundance of 13C in humus would interfere with 13C-NMR measurements unless high concentrations of cyprodinil were applied. Accordingly, despite the dispersion of bound cyprodinil residues among fractions of soil matrix, the accumulation of bound 13C-labeled material in humic acid (0.21 mg) resulting from incubations with 500 mg/kg of cyprodinil was sufficient to obtain discernible signals in the NMR spectra (Figures 5-7). As previously stated, the significant change in the pattern of NMR signals from 13C-phenyl-ring-labeled cyprodinil residues bound to humic acid upon incubation (Figure 6) suggests a major alteration of the 13C-labeled carbons of this fungicide. This change may indicate cleavage of the cyprodinil molecule between the two aromatic rings and separate binding of the phenyl and pyrimidyl moieties to humic acid. Carbons C-2′ and C-6′, C-4′, C-3′ and C-5′, and C-1′ of the free compound are represented in Figure 5 by the signals at 121.9, 124.4, 131.8, and 143.4 ppm, respectively. In Figure 6, the same carbons appear to be represented by two sharp peaks at 122.5 and 131.8 ppm and two weak signals at 141.7 and 161.7 ppm. The possibility of physical entrapment of the intact fungicide molecule in the molecular net of humus must therefore be eliminated, since the spectrum of the entrapped compound would not differ from that of the dissolved one presented in Figure 5. Binding of the intact fungicide involving any site on the phenyl ring would result in significant changes in the chemical shifts for carbons C-2′ through C-6′, but carbon C-1′ connected with the pyrimidyl ring via the -NH group would still be expected to give a strong signal at about 140 ppm. If hydroxylation of the NH- bridge
FIGURE 4.
13
C-NMR spectrum for the 1% NaOD solution of humic acid isolated from a control sample of soil incubated without cyprodinil.
FIGURE 6. (A) 13C-NMR spectrum for the 1% NaOD solution of humic acid extracted from soil after incubation with 500 mg/kg of U-phenyl13C- and 14C-labeled cyprodinil, and (B) 13C-NMR spectrum resulting from the subtraction of the control humic acid spectrum from spectrum A.
FIGURE 5. 13C-NMR spectra for the 1% NaOD solutions of U-phenyl13C-labeled cyprodinil and 2-pyrimidyl-13C-labeled cyprodinil mixed with humic acid isolated from the control soil sample. between the two moieties took place, it would have resulted in an upfield shift of the C-1′ peak, with the chemical shift values of the other phenyl carbons remaining unchanged. The strong resonances observed in Figure 6, panels A and B, are in the aromatic range of resonances from 115 to 147 ppm, with protonated aromatic carbons from 115 to 130 ppm and those substituted by alkyl carbons from 130 to 147 ppm. The most prominent peak at 131.8 ppm is assigned to aromatic carbons substituted by alkyl or carboxyl groups (19) and
strongly suggests incorporation of the phenyl residue from cyprodinil into the aromatic compartment of the humic matrix. The signal at 141.7 ppm cannot be considered an artifact resulting from incomplete subtraction of the spectrum because a similar subtraction for the pyrimidyl label (Figure 7B) resulted in clean removal of all signals from the aromatic region except that for the labeled pyrimidyl carbon at the C-2 position. Based on chemical shift values, the signal at 141.7 ppm can be attributed to hydroxylation of the phenyl moiety. The weak signal at 161.7 ppm most likely arises from ionization of hydroxyl functionalities in the alkaline NaOD solution used for dissolving the humic acid for NMR analysis. Such hydroxylations possibly lead to catechol structures with resonances between 128 and 145 ppm. A rapid binding of catechol and catechol derivatives during soil incubation was reported by Martin et al. (20). The binding occurred even in sterile soil, probably through catalysis by heavy metal oxides.
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FIGURE 7. (A) 13C-NMR spectrum for the 1% NaOD solution of humic acid extracted from soil after incubation with 500 mg/kg of 2-pyrimidyl-13C- and 14C-labeled cyprodinil, and (B) 13C-NMR spectrum resulting from the subtraction of the control humic acid spectrum from spectrum A. Furthermore, Ko¨gel-Knabner et al. (21) observed a decrease of phenolic and methoxyl carbon signals from lignins and a strong increase in signal intensity at about 130 ppm after humification. The separation of the two parts of the cyprodinil molecule was confirmed by plant uptake studies of bound residues from soil previously incubated with 14C-labeled fungicide (16). Due to the cleavage, the uptake of bound 14C from the pyrimidyl-labeled residue was three times higher than that for the phenyl-labeled molecule. Furthermore, prominent metabolites isolated from soil upon incubation with cyprodinil also demonstrated cleavage of the molecule into two parts (11). The C-2 carbon of the free fungicide mixed with control humic acid is represented in Figure 5 by the signal at 162.5 ppm. In Figure 7, the same carbon is represented by the peak at 161.8 ppm. The small difference appears to be the result of the cleavage between the two aromatic rings and the subsequent binding of the pyrimidyl moiety to humic acid. During the measurement of chemical shifts in deuterated chloroform (Table 1), the C-2 carbon of the intact cyprodinil molecule resonated at 159.8 ppm, whereas the corresponding carbon in the amino or hydroxyl derivatives of pyrimidine (see Table 1) has resonances at 162.9 and 160.4 ppm, when dissolved in deuterated chloroform. This slight downfield shift of the carbon in question can be observed upon alkylation or esterification of the hydroxyl group in compound 10 of Table 1. Obviously, the chemical nature of the bound compound cannot be determined based on a slight shift of one NMR signal. To obtain conclusive structural information, all four aromatic carbons of the pyrimidyl moiety need to be enriched with the 13C isotope. With only one carbon labeled, interpretation of the data must be limited to enumeration of the possible pathways, such as incorporation of the pyrimidyl moiety into polysaccharide structures or alkylation of different aromatic sites. Furthermore, shift values of N-heterocyclic compounds vary considerably depending upon the nature of the solvent. Upon fractionation of bound residues, about 60% of the radioactivity could be extracted from fulvic acid with methylene chloride, which was determined to mainly consist of
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the unchanged fungicide. The pattern of four triplet signals, collected during 13C-NMR analysis of the compound after its removal from the TLC plate, was identical to that presented in Figure 3 for U-phenyl-13C-labeled cyprodinil. The unchanged fungicide was apparently sequestered in the soil and could not be recovered by a 4-fold extraction with methanol. The retention of the unaltered cyprodinil in soil may be explained by the process that is often referred to as aging. During aging, many organic compounds become sequestered in inaccessible microsites within the soil matrix (3). They are no longer as easily extractable from soil by organic solvents as absorbed compounds are, but they are not as resistant to extraction as are actually bound chemicals. Sequestration can be considered a process of slow sorption that may require weeks or months to reach equilibrium (3, 22, 23). Huang et al. (23) attributed this sorption to intraparticle diffusion of chemicals to the internal surfaces of meso(20-500 Å) and micro- (
