Metabolites of Alachlor in Water: Identification by Mass Spectrometry

Nov 26, 1997 - Reductive Dechlorination of Chloroacetanilide Herbicide (Alachlor) Using Zero-Valent Iron Nanoparticles. Jay M. Thompson , Bret J. Chis...
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Environ. Sci. Technol. 1997, 31, 3637-3646

Metabolites of Alachlor in Water: Identification by Mass Spectrometry and Chemical Synthesis S . M A N G I A P A N , * ,† E . B E N F E N A T I , † P. GRASSO,† M. TERRENI,‡ M. PREGNOLATO,‡ G. PAGANI,‡ AND D. BARCELO ´ § Istituto di Ricerche Farmacologiche “Mario Negri”, Milan, Italy, Dipartimento di Chimica Farmaceutica, Universita` degli Studi, Pavia, Italy, and Centro de Investigacion y Desarrollo-Centro Superior de Investigaciones Cientificas (CID-CSIC), Barcelona, Spain

This biodegradation study of alachlor in water was designed to assess the presence and the identity of metabolites. Alachlor was incubated in river water for 28 days. The pesticide and its transformation products were extracted by solid phase extraction and analyzed by gas chromatography/ mass spectrometry. Selected samples were also analyzed by liquid chromatography/mass spectrometry. The disappearance of the pesticide was not significative, but several compounds were identified as alachlor metabolites. Nine compounds were confirmed by comparison with synthetic standards. One metabolite has never been reported. For seven molecules, formulae were presumed on the basis of spectrum interpretation and literature data.

Introduction Alachlor, 2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide, is a pre-emergence herbicide of the chloroacetanilide family, widely used on different crops since 1969. In Italy, its use has grown since the ban on atrazine (1). Although the persistence of alachlor in the environment is limited, its complete mineralization to CO2, H2O, and NH3 has never been reported (2), and in fact, a lot of transformation products (TP) are generated by degradation. Several studies of the environmental biotic and abiotic degradation have been reported, and different compounds have been found, depending on the experimental conditions. The range of alachlor metabolites produced during metabolic studies in vitro or in vivo in plants and animals is likewise very wide. The large series of alachlor TP, detected in various studies, is presented in reviews by Chesters et al. (3) and Sharp (4). Moreover, the number of alachlor metabolites is continually rising, and recent studies report new compounds (5, 6). The picture of alachlor TP is therefore complex, and we only know the environmental and toxicological properties of a few compounds (7-9). The U.S. Environmental Protection Agency classifies alachlor as a probable carcinogen for humans (group B2) (10) and most of its TP are structurally similar (aniline derivates). The Commission of the European Community * Corresponding author address: Istituto di Ricerche Farmacologiche Mario Negri, via Eritrea 62, 20157 Milano, Italy. Fax: +39 2 39001916. E-mail: [email protected]. † Istituto di Ricerche Farmacologiche Mario Negri. ‡ Dipartimento di Chimica Farmaceutica. § Centro de Investigacion y Desarrollo.

S0013-936X(97)00380-5 CCC: $14.00

 1997 American Chemical Society

has classified alachlor among the “high-priority pesticides”, including those products used in amounts over 50 tonnes per annum and with some potential to leach (11). As alachlor degradation products are generally of lower molecular weight and more oxidized than the parent compound, they may be consequently more water soluble, more mobile, and have a greater potential to leach (5). Therefore, concern about alachlor degradation is mainly focused on the possibility of detecting its TP in surface and ground water, which they can contaminate by run-off or leaching, or where they can originate from the pesticide. Indeed, in some cases TP can be as least as toxic as the parent compound (12). A series of TP of alachlor has been found in rivers and wells and in treated soils (5, 13, 14). The maximum permitted concentration, according to a EC directive for pesticides in drinking water, i.e., 0.1 µg/L, also extends to “related products” (15). Therefore, it is fundamental to identify the TP of the most widely used pesticides in order to optimize analytical methods and plan monitoring programs. Because most environmental studies on alachlor are about mineralization in soil or degradation by isolated microorganisms, we undertook a laboratory study of alachlor biotic degradation in an aquatic system in order to detect more metabolites or confirm already detected ones.

Experimental Section Degradation Studies. We made several tests with different inocula and media over 2 years. A detailed description of all biodegradation experiments and the results about degradation kinetics have been previously reported (16). In short, in the metabolism tests, alachlor (Alltech, Deerfield, IL; purity 98.5%) was incubated at a concentration of 1 mg/L in dark bottles filled with 2-5 L of river water, stirred at 20 °C for 28 days. Dark bottles were used to avoid photodegradation, since the study was focused on biodegradation in water. The water samples were collected from the Olona river (Italy) in May 1993 and May and September 1994. In order to follow the disappearance of alachlor every 7 days, a 10 mL subsample was extracted with a C18 cartridge and analyzed in GC with a NPD detector (16). At the end of the biodegradation experiment, the remaining water was filtered (0.45 µm) and extracted on a C18 cartridge and subsequently on a Carbopack-B column to ensure better recovery of polar compounds. Three samples with relative blanks were obtained from the incubations and analyzed in the metabolism study. Sample Extraction. C18 cartridges employed to extract weekly subsamples (1 mL cartridge) and the final whole samples (3 mL cartridge) were from J. T. Baker (Phillipsburg, NJ). The phase was washed with ethyl acetate and then activated with methanol. After passage of the sample, the column was eluted with ethyl acetate. The final whole water sample was extracted according to Di Corcia and Marchetti (17). Briefly, a glass column containing 400 mg of Carbopack-B phase (Supelco, Bellefonte, PA) was employed. The phase was washed with 10 mL of a mixture of CH2Cl2:CH3OH (4:1) followed by 5 mL of CH3OH and then activated with 20 mL of 10 mg/mL ascorbic acid solution in HCl 0.01 M. The water sample passed through the column at a flux rate of 20 mL/min. Compounds adsorbed on the phase were eluted by 10 mL of a mixture of CH2Cl2: CH3OH (4:1) for neutral fraction recovery and by the same mixture acidified with trifluoroacetic acid (0.2% v/v) for the acidic fraction. Finally, the extracts were concentrated under a nitrogen stream to 0.05-0.5 mL. All solvents (Carlo Erba, Milan, Italy) were for the analysis of pesticide residues.

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SCHEME 1. Synthesis Scheme of Standards

Before injection on the GC, samples from Carbopack-B extracts were derivatized with diazomethane or silylating agent (BSTFA). Diazomethane was prepared in the laboratory by distillation from Diazald ethereal solution (Aldrich-Chemie, Steinheim, Germany) added dropwise to a potassium hydroxide ethanol mixture. BSTFA was from Fluka Chemie (Buchs, Switzerland). We used these two derivatization reagents to make possible, or simply to improve, the detectability in GC analysis of any TP-containing functional groups with active hydrogens such as COOH, OH, NH, and SH, typical of some alachlor TP. Diazomethane is used to convert carboxylic acids into methyl esters, while BSTFA substitutes a trimethylsilyl group for the acidic hydrogen of OH, NH, and SH groups. Derivatization was done by adding to subsamples, dried under N2, 0.5 mL of the ethereal solution of diazomethane or 30 mL of BSTFA. The reactions with diazomethane and BSTFA lasted 10 min and were maintained at room temperature and 60 °C, respectively. Instrumental Analysis. The extracts were analyzed in GC/ MS on two different instruments: a Varian 3400 gas chromatograph coupled with a Varian Saturn II ion trap mass spectrometer and a HP 5890 series II gas chromatograph equipped with a HP MSD 5971 series mass spectrometer. For the first instrument, a CP-Sil 8 CB Chrompack (Middleburg, The Netherlands) fused silica capillary column (25 m long; 0.25 mm i.d.; 0.25 µm film thickness) was used. The carrier gas (He) head pressure was 30 kPa. Inlet and transfer line temperatures were set at 240 and 280 °C. The oven program was raised from 80 °C (1 min) to 300 °C (4 min) at a rate of 10 °C/min. On the HP instrument, the following fused silica capillary columns were used: the same CP-Sil 8 CB as on the ion trap

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instrument; a SE 52 (25 m long; 0.25 mm i.d.; 0.25 µm film thickness) and an Easy 1701 (25 m long; 0.25 mm i.d.; 0.25 µm film thickness), the latter both from Analytical Technology (Cernusco s/N, Milan, Italy). The inlet and transfer line were held at 260 and 280 °C, respectively. The carrier gas (He) head pressure was 70 kPa. For the CP-Sil 8 CB and SE 52 columns, the oven program went from 80 °C (1 min) to 300 °C (5 min) at a rate of 6 °C/min; for the Easy 1701, operating temperatures were raised from 60 to 275 °C at a rate of 6 °C/min. The ion trap mass spectrometer was employed in electron ionization (EI) and in positive chemical ionization (PCI) modes, with CH4 as ionizing gas, to obtain more information on molecular weight. On both instruments, the extracts were injected (2 µL) with a splitless system. Selected extracts were also analyzed in LC/MS with a highflow pneumatically assisted electrospray interface (LC-ESPMS), using a VG Platform ESP from Fisons Instruments (Manchester, U.K.). The analyses were conducted in the positive ionization mode at isocratic condition (60:40 CH3CN) for 35 min on a C18 column (2.1 mm i.d., 30 cm, packed with 10 µm particles) from Waters-Millipore (Milford, MA). The operational ESP parameters were flow, 0.3 mL/min; source temperature, 150 °C; extraction voltage, 20 V. Reference Standards and Their Synthesis. We used several standards to verify the identity of the presumed alachlor metabolites detected in our samples. Two standards were bought: 2,6-diethylaniline (DEA) (I) from Alltech (Deerfield, IL) and chloroacetic acid from AldrichChemie (Steinheim, Germany). Another compound, N-(2,6diethylphenyl)-N-(methoxymethyl)acetamide (MW, 235), was synthetized at CID-CSIC, Barcelona. The other 12 reference

SCHEME 2. Synthesis Schemes of Standards

compounds were synthetized in our laboratories. The synthesis schemes are shown in Schemes 1 and 2. Identities of the new synthetized products were established by MS and also by 1H NMR for some of them. The 300 MHz 1H NMR spectra were recorded on a Bruker ACE-300 spectrometer, in deuterochloroform using deuterated tetramethylsilane as internal standard; chemical shifts are in d (parts per million) and coupling constants (J) in hertz. DISMS spectra were obtained on a VG TS 250 instrument by EI at 70 eV. The TLC analyses were run on silica gel 60 F254 Merck and flash column chromatography on silica gel 60 (60-200 µm, Merck). The formulae and the mass spectra of the synthetized compounds and of the two commercial standards, analyzed by HP gas chromatography mass spectrometry, are reported in Figures 1 and 2. N-(2,6-Diethylphenyl)methyleneamine (II), 2,6-Diethylformanilide (XII), and 2-Chloro-N-(2,6-diethylphenyl)acetamide (XVI). These compounds were synthetized as reported in ref 5, and analytical data are in agreement with those reported. (XII) 1H NMR (CDC13) d: 1.20, (t, CH3CH2Ar, 6H), 2.65 (m, CH3CH2Ar, 4H), 6.85 (bs, NH, 1H), 7.10-7.35 (m, Ar, 3H), 8.05 (d, J ) 11.5 Hz, CHO, 0.55 from 1H), 8.45 (s, CHO, 0.45 from 1H). 2-Acetoxy-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide (III). To a solution of II (6 mmol) in dry cyclohexane (20 mL) 2-acetoxyacetyl chloride (0.645 mL), dry methanol (1.5 mL), and triethylamine (1.5 mL) were added. The reaction mixture was stirred for 1 h at room temperature, then water (20 mL) was added, and organic products were extracted with ethyl acetate (4 × 50 mL). The combined organic phases were dried (anhydrous sodium sulfate) and evaporated under reduced pressure. The pure product was used in the next reaction without further purification. Analytical data are in agreement with those reported (6). 2-Acetoxy-N-(2,6-diethylphenyl)acetamide (IV), 2-Acetoxy-N-(2,6-diethylphenyl)-N-(methyl)acetamide (V), 2-Hydroxy-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide (VI), 2-Hydroxy-N-(2,6-diethylphenyl)acetamide (VII), 2-Hydroxy-N-(2,6-diethylphenyl)-N-(methyl)acetamide (VIII), and 2,6-diethylacetanilide (XIII). These compounds were synthetized as reported (6) and analytical data are in agreement with those reported. (VII) 1H NMR (CDC13) d: 1.18, (t, CH3CH2Ar, 6H), 2.55 (q, CH3CH2Ar, 4H), 3.70 (bs, OH, 1H), 4.15 (s, COCH2OH, 2H), 7.12-7.28 (m, Ar, 3H), 7.95 (bs, NH, 1H). (VIII) 1H NMR (CDC13) d: 1.24, (t, CH3CH2Ar, 6H), 2.51 (q, CH3CH2Ar, 4H), 3.24 (s, CH3N, 3H), 3.38 (t, J ) 5 Hz, OH, 1H), 3.58 (d, J ) 5 Hz, COCH2OH, 2H), 7.14-7.38 (m, Ar, 3H).

2-Oxo-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide (IX), 2-Oxo-N-(2,6-diethylphenyl)acetamide (X), and 2-Oxo-N-(2,6-diethylphenyl)-N-(methyl)acetamide (XI). Neat DMSO (4 mmol) was added dropwise, at -60 °C under N2, to a stirred solution of oxalylchloride (1.44 mmol) in dry CH2Cl2 (6 mL). After 5 min, the yellow solution was forced by N2 pressure through a double-tipped needle, into a solution of the appropriate alcohol (VI-VIII) (0.48 mmol) in dry CH2Cl2 (10 mL), at -60 °C with stirring. After 15 min, 500 mL of Et3N was injected into the solution that was warmed to room temperature and poured into 20 mL of H2O. Organic products were extracted with CH2Cl2 and washed once with 2 M HCl and with NaHCO3, with re-extraction of each aqueous layer with CH2Cl2. The collected organic layers were dried (anhydrous sodium sulphate) and evaporated under reduced pressure to give the pure products (IX-XI) (see GC/MS analysis). 2-Methylthio-N-(2,6-diethylphenyl)acetamide (XIV). 2-(Methylthio)acetylchloride (4.3 mmol) was added to a solution of 2,6-diethylaniline (3.3 mmol) and NaHCO3 (10 mmol) in CH2Cl2 (50 mL), and the mixture was stirred at reflux until reaction was completed. Water (20 mL) was added, and organic products were extracted with CH2Cl2. The combined organic phases were dried (anhydrous sodium sulfate) and evaporated under reduced pressure. The pure product (see GC/MS analysis) was used without further purification. N-Methyl-2,6-diethylformanilide (XV). Methyl iodide (1.2 mmol) was added to a stirred mixture of 2,6-diethylformanilide (XII) (0.5 mmol), 18-crown-6 (0.05 mmol) and 50% KOH/NaOH (5 mL) in toluene (5 mL) at room temperature. After 15 min, the organic layer was washed once with H2O, dried (anhydrous sodium sulfate), evaporated under reduced pressure, and the residue was purified by flash chromatography (n-hexane/ethyl acetate (6:4)) to give the pure product (see GC/MS analysis).

Results and Discussion The disappearance of alachlor was not significative, taking into account the analytical variability (10%), in any incubation performed to study alachlor metabolism, during the 4 weeks of the experiments (16). However, GC/MS analysis of the final extracts, representing the incubated samples concentrated from 4000 to 40 000 times, found about 20 alachlor degradation products. Several chromatographic peaks not present in the blanks and, in some cases, characterized by typical ions of alachlor fragmentation appeared in the chromatograms. The areas of these peaks were generally less than 1% of the alachlor peak. These results

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FIGURE 1. GC-MS spectra and formulae of synthetic standards used to confirm the metabolites. (a) Commercial standard. (b) Standard synthetized at CID-CSIC, Barcelona, Spain. On the upper left-hand side of the spectra, the Roman numeral is the synthesis scheme number (see Schemes 1 and 2), and the Arabic number is the one assigned to identify the compound (see Table 2).

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FIGURE 2. GC-MS spectra and formulae of synthetic standards not found as metabolites in our samples. The Roman numeral, on the upper left-hand side of the spectra, is the synthesis scheme number (see Schemes 1 and 2). agreed with findings of other authors operating in similar conditions (18). First, we tried to assign a structural formula to the molecules detected on the basis of literature data, hypothetical fragmentation and comparison with corresponding PCI spectra. This enabled us to verify the molecular weight. The mass spectra libraries NIST 92 and Wiley 5 helped us identify DEA and chloroacetic acid. Because different alachlor degradation pathways, caused by physical, chemical or biological agents, can give rise to the same final TP, we compared our metabolites with those reported in the literature, resulting from different degradation processes. Then, we synthetized the presumed metabolites. The synthetic compounds and the commercial standards were analyzed in GC/MS in the same conditions as the water samples. Retention time and mass spectrum of standards and presumed metabolites were compared. The synthetic standards VI, VII, and VIII, characterized by OH groups, were also derivatized with BSTFA and mass spectrum mean ions are reported in Table 1. Derivatization of the extracts with diazomethane and BSTFA did not give good results; a lot of new peaks, difficult to resolve, appeared in the chromatogram, and we could not identify new TP with reactive groups. Confirmed Metabolites. We identified 9 metabolites, for which Table 2 reports the following parameters: number assigned to identify the compound, molecular weight, mass

spectrum mean ions and relative abundance, chemical name, extraction solid phases used, samples in which it was found, reference number of the standard used to confirm, and literature references. Formulae and spectra of the standards used to confirm these compounds are shown in Figure 1. The metabolites identified have a molecular weight lower than alachlor, from which they originate mainly by hydrolytic and oxidation reactions operated by microorganisms. The two compounds 6 and 7 were already present as impurities in the alachlor standard used in the incubation experiments (standard purity 98.5%). We analyzed this standard in GC/MS (in triplicate) and compared the levels of each impurity with the alachlor peak, measuring the area ratios. In the same way, we measured the TP/alachlor ratio in the samples. The ratios of 6 and 7 to alachlor were, respectively, 0.0066 and 0.000 28 in the standard and 0.045 and 0.000 27, as average, in the samples. Therefore, we could establish for sure that 7 was formed during incubation, but nothing can be said about the other product. Metabolites 6 and 7 derive from loss of the methoxymethyl group and chlorine, respectively. To our knowledge, 5 has not been previously reported; we only found it in the C18 extract of the May 94 sample. All other metabolites in Table 2 have been reported. This compound might derive from metabolite 6 by loss of the chlorine atom and subsequent oxidations to the hydroxylic (the correspondent metabolite was not found) and then the carbonylic group.

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161

177 205 225

235

249

251

3

4 5 6

7

8

9

219 (30)

217 (15)

235 (33)

177 (100) 205 (43) 227 (4)

161 (52)

94 (10) 149 (40)

206 (8)

204 (4)

178 (74)

162 (31) 190 (2) 225 (13)

146 (100)

52 (31) 134 (100)

221 251

207

MW

188 (98)

188 (50)

161 (100)

160 (38) 176 (100) 210 (1)

130 (27)

50 (100) 120 (14)

b

45 (100)

45 (100)

146 (97)

148 (65) 158 (19) 176 (100)

118 (74)

49 (46) 119 (22)

May 94 Sept. 94 May 93 May 94 Sept. 94 May 93 May 94 Sept. 94 C18 Carbopack-B/n

2-hydroxy-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide

Numbers of the synthesis scheme; comm ) commercial standard.

C18 Carbopack-B/n

2-oxo-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide

C18

N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide

Carbopack-B/n

C18 C18 C18 Carbopack-B/n

2,6-diethylformanilide 2-oxo-N-(2,6-diethylphenyl)acetamide 2-chloro-N-(2,6-diethylphenyl)acetamide

C18

N-(2,6-diethylphenyl)methyleneamine

samples

264 (77) 336 (6) 278 (69) 308 (26)

May 94 May 93 May 94 Sept. 94 May 94 Sept. 94 May 94 May 94 May 93 May 94 Sept. 94 May 93

extraction phasea

279 (3) 351 (10) 293 (4) 323 (1)

C18 C18 Carbopack-B/n,a

+1 TMSb (75%)c +2 TMS (25%) +1 TMS +1 TMS

VI

IX

3, 4, 5, 6, 18, 22

18

5, 18, 22

3, 4, 18, 19, 20, 21

5

5

3, 4 3, 4, 5, 19, 20

ref

73 (36) 73 (100) 190 (100) 45 (100)

standard synthetized at CID-CSIC, Barcelona

XII X XVI

II

comm I comm

standardsb

176 (100) 248 (73) 262 (13) 188 (81)

mean ions and relative abundance of silylated standards

chloroacetic acid 2,6-diethylaniline (DEA)

name

TMS ) Trimethylsilyl group. c Percentage of compound that reacted.

mean ions and relative abundance

b

Carbopack B/n,a: n ) neutral fraction, a ) acidic fraction.

94 149

1 2

a

MW

no.

TABLE 2. Confirmed Metabolites

Numbers of the synthesis scheme.

VIII VI

2-hydroxy-N-(2,6-diethylphenyl)-N-(methyl)acetamide 2-hydroxy-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide

a

VII

standardsa

2-hydroxy-N-(2,6-diethylphenyl)acetamide

name of standards not silylated

TABLE 1. Standards Derivatized with BSTFA

TABLE 3. Presumed Transformation Products no. MW

mean ions and relative abundance

10 147 147 (50)

77 (20)

7-ethylindoline

extraction phase

samples

ref

May 93 5 May 94 11 175 (60) 160 (100) 146 (61) 134 (46) alachlor metabolite May 94 12 191 191 (32) 160 (100) 144 (20) 132 (37) alachlor metabolite May 93 May 94 Sep. 94 13 219 (100) 189 (36) 188 (27) 174 (55) alachlor metabolite C18 May 94 Sept. 94 14 221 221 (63) 189 (38) 160 (100) 146 (69) alachlor metabolite Carbopack-B/n,a May 93 Sept. 94 15 223 (49) 208 (43) 174 (42) 146 (100) 1-chloroacetyl-2,3-dihydro-7-ethylindole C18 May 94 3, 4, 19 Sept. 94 16 235 (43) 189 (20) 160 (100) 146 (43) alachlor metabolite Carbopack-B/n Sept. 94 17 285 285 (12) 253 (48) 204 (88) 176 (100) 2′-(1-hydroxyethyl)-6′-ethyl-2-chloroC18 May 93 21, 24 N-(methoxymethyl)acetanilide Carbopack-B/n Sept. 94 18 301 (7) 252 (15) 224 (19) 176 100) alachlor metabolite C18 May 94 19 313 (22) 267 (25) 234 (35) 188 (100) alachlor metabolite C18 May 93 Carbopack-B/n a

132 (100) 117 (39)

presumed compound

C18 Carbopack-B/n C18 C18 Carbopack-B/n

MW is reported when it was possible to confirm the molecule by PCI.

In two alachlor degradation studies, 2 and 6 were produced by the soil fungus Chaetomium globosum (19) and by Chironomid larvae (20), and in both these experiments, the metabolites’ identities were confirmed by authentic standards. Compound 6 was also identified during an incubation of alachlor with male rat liver microsomes (21). DEA (metabolite 2) originates from loss of both functional groups on N of alachlor. A mutagenic potential is documented for this compound, probably linked to its oxidation to nitrosobenzene (7, 8). In a recent study, groundwater samples were collected beneath a Massachusetts cornfield and analyzed in GC/MS for alachlor metabolites; metabolites 2, 3, 7, and 9 were detected and confirmed by standards (5). Metabolite 3 might originate from metabolite 4 through a reduction followed by dehydration. Metabolite 9 appeared silylated after reaction with BSTFA, giving ions at m/z 323, 308, 188, and 45, in agreement with the standard behavior (see Table 1). Suba and Pearson (18), in several studies of alachlor degradation, identified many degradation products, some of which correspond to our confirmed metabolites. Compounds 7, 8, and 9 were found as major photoproducts of the sensitized photolysis (in presence of acetone) of alachlor, and compound 9 together with 6 was found in soil photolysis studies. The same authors (18) also found 8 and 9 in an aerobic soil metabolism study and 7 in an anaerobic aquatic metabolism study. Compounds 9 and 7 were identified as alachlor photodegradation products by Somich et al. (22) and 9 by Chiron et al. (6) and by Pen ˜ uela and Barcelo´ (23). Metabolites 8 and 9 originate from the progressive oxidation of the carbon bound to chlorine in the alachlor molecule. The Microtox test showed that hydroxyalachlor (metabolite 9) had toxicities higher than or not significantly different from the parent compound (9). A very small peak (metabolite 4) with a spectrum and retention time corresponding to the standard XII (2′,6′diethylformanilide) was seen by us after derivatization of the C18 extract with BSTFA. The BSTFA did not react with 4, but probably reacted with a coeluting polar component, changing its retention time. This compound was found in Massachusetts groundwater samples (5). It might originate from metabolite 5 through oxidation followed by loss of CO2. The formation of TP, which can reach groundwater and can be in some cases at least as toxic as the parent compound, represents a potential threat to the environment and human health.

Presumed TP. Spectra of the remaining presumed metabolites were compared with literature data, when available. Table 3 shows the same parameters as Table 2 for every presumed metabolite. Spectra and hypotheses about the formulae of some of these compounds are presented in Figure 3. For metabolites 12, 14, and 17, the molecular weights of 191, 221, and 285 were confirmed by PCI spectrum. 7-Ethylindoline (MW, 147) was tentatively identified by Potter and Carpenter (5), on the basis of the mass spectrum. We could not confirm the identity of this metabolite because attempts to synthetize it gave two peaks both with a parent ion at m/z 147 and with very similar MS spectra. From spectral analysis, we conclude that metabolites 11, 12, and 13 do not present chlorine and the methoxymethyl group; the latter, when present, gives a strong ion at m/z 45 (in metabolite 12 there is a loss of 45, but the ion at m/z 45 is absent). The metabolite 11 may have a MW of 175, and the losses of 15 and 29 may correspond to CH3 and CHO groups. On the basis of these considerations, a hypothetical structural formula is presented in Figure 3. A metabolite with the same formula, formed in flooded soil, is reported by Chesters et al. (3). By comparison with authentic standards, compound 12 of MW 191 did not correspond to either standard XIII or XV (Figure 2). The first, 2,6-diethylacetanilide, was found in Massachusetts’ groundwater samples by Potter and Carpenter (5) and in sensitized photolysis and aerobic soil metabolism experiments by Suba and Pearson (18). A hypothetical formula of 12 is reported in Figure 3. Metabolite 13 does not correspond to standard XI (Figure 2b). A hypothetical formula is reported in Figure 3. Metabolite 14, with MW 221, differed from the standard VIII, which was found by some authors (6, 22, 23). It may therefore correspond to 2′,6′-diethyl-N-(methoxymethyl)formanilide (Figure 3). Observing the spectrum, parent ion M+ (221) could lose CH3O• to give the ion at m/z 190 and CH3OH to give the ion at m/z 189. The combined losses of CH3OH and CHO• can give the ion at m/z 160, which is typical of alachlor’s fragmentation pattern. Further evidence is that the metabolite did not react with BSTFA. We have not found this presumed metabolite in the literature. 1-Chloroacetyl-2,3-dihydro-7-ethylindole (MW, 223) was observed as a major component in the alachlor degradation study with C. globosum, and its structural formula was assigned on the basis of MS and IR spectra but was not confirmed by comparison with an authentic standard (19).

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FIGURE 3. GC-MS spectra and presumed formulae of some transformation products not confirmed by comparison with standards. The Arabic numeral, on the upper left-hand side of the spectra, is the number assigned to identify the compound (see Table 3).

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TABLE 4. TPs Detected by LC-MS other no. MW (M + H)+ (M + Na)+ ions 6a

225

226

248

7a 8a 20 21

235 249 233 281

236 250 234 282

258 272 256 304

a

name

extraction phase

ref

243 2-chloro-N-(2,6-diethylphenyl)acetamide

Carbopack-B/a 3, 4, 18, 19, 20, 21 162 N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide Carbopack-B/n 5, 18, 22 2-oxo-N-(2,6-diethylphenyl)-N-(methoxymethyl)acetamide Carbopack-B/n 18 202 8-ethyl-N-methoxymethyl-4-methyl-2-oxytetrahydroquinoline Carbopack-B/a 18, 22 2-methylthio-N-(methoxymethyl)-N-(2,6-diethylphenyl)acetamide Carbopack-B/n 4, 18

Already seen and confirmed by GC-MS (see Table 2).

TABLE 5. Standards Synthetized but Not Found in the Incubated Samples mean ions

MW 191 191 207 219 221 237 a

191 191 207 219 221 237

176 174 192 204 205 176

148 162 176 190 190 160

134 147 160 162 175 148

name

standardsa

ref

2′,6′-diethylacetanilide 2′,6′-diethyl-N-methylformanilide 2-hydroxy-N-(2,6-diethylphenyl)acetamide 2-oxo-N-(2,6-diethylphenyl)-N-(methyl)acetamide 2-hydroxy-N-(2,6-diethylphenyl)-N-(methyl)acetamide 2-methylthio-N-(2,6-diethylphenyl)acetamide

XIII XV VII XI VIII XIV

3, 5, 6, 18, 22 3 6, 22

Numbers of the synthesis scheme.

The spectrum of this compound is consistent with that of compound 15, found among our samples. Feng et al. identified 2′-(1-hydroxyethyl)-6′-ethyl-2-chloroN-(methoxymethyl)acetanilide (MW, 285) as the most abundant metabolite produced during incubation of alachlor with male rat liver microsomes. The metabolite was confirmed by identification of its acid hydrolysis product, 2-ethylaniline (21). The PCI-MS spectrum described in this work is very similar to the PCI-MS spectrum of metabolite 17 for the presence of a protonated molecular ion at m/z 286, a base ion at m/z 254 (MH+-CH3OH), and ions at m/z 268 (MH+-H2O) and m/z 218 (MH+-CH3OH-HCl). The EI spectrum of 17 shows a fragmentation pattern very similar to that of the PCI spectrum: presence of the molecular ion and loss of CH3OH and of H2O from the molecular ion to give ions at m/z 253 and 267. There is also the loss of CH3OCH2• from the molecular ion to give an ion at m/z 240 and the ion itself CH3OCH2+ at m/z 45. Finally, the ion at m/z 204 corresponds to the ion at m/z 188 in the alachlor EI spectrum. In fact, both are produced from the correspoding molecular ions by loss of ClCH2• and CH3OH with H abstraction from the ortho ethyl on the ring followed by cyclization (20). A probable isomer of this compound appeared in the chromatogram of an extract analyzed by GC/MS. It was characterized by a slightly longer retention time and an almost identical MS spectrum. An isomer of 2′-(1-hydroxyethyl)6′-ethyl-2-chloro-N-(methoxymethyl)acetanilide was identified by Feng and Patanella (21), too. They suggested that the two isomers originate because the rotation about the aromatic nitrogen bond is much more restricted in the presence of an adjacent benzylic hydroxyl group. A compound tentatively identified as 2′-(1-hydroxyethyl)6′-ethyl-2-chloro-N-(methoxymethyl)acetanilide was found by Bonfanti et al. (24) in an in vitro experiment, incubating alachlor with rat hepatocytes. Metabolites 16, 18, and 19 present typical ions of the alachlor spectrum (m/z at 132, 146, 160, 176, 188, etc.). Since chromatogram peaks were very low, probably MS spectra present interfering ions. We have not formulated any hypothesis about their structures. HPLC-MS Analysis. Neutral and acidic fractions of Carbopack-B extracts of the May and September 1994 samples were also analyzed in LC-ESP-MS. This indicated 5 metabolites, three (6, 7, and 8) already seen and confirmed by

GC/MS; two others (MW, 233 and 281) were assumed on the basis of their spectrum and comparison with literature (4, 18, 22). All the metabolites detected are reported in Table 4. The metabolite with MW 233 was already found by Chiron et al. (6) and confirmed as a photodegradation product. The molecular ion plus hydrogen (M + H)+ and molecular ion plus sodium (M + Na)+ can generally be seen in the LCESP-MS spectrum. In one case (metabolite 6), the molecular ion plus ammonium was present. The voltage used (20 V) does not generally enable us to see other compound fragments but this voltage improves sensitivity, and we can therefore detect more TP, even if the spectrum is less informative. However, for metabolite 7, the ion at m/z 162, representing the loss of OCH3 and COCH3 groups, and the ion at m/z 202 (M-OCH3) for metabolite 20 are present. By LC-ESP-MS, we did not find [(methoxymethyl)(2,6-diethylphenyl)amino]oxoacetic acid and 2-[(methoxymethyl)(2,6-diethylphenyl)amino]-2-oxoethanesulfonic acid, the two major metabolites identified in previous studies of alachlor in soil (18) and water (25), deriving from its microbial degradation. Table 5 summarizes the standards identified by other authors as corresponding to alachlor metabolites (see the ref column in Table 5), that we synthetized and analyzed but did not find in our samples. Figure 2 shows spectra and formulae of these compounds. Because compounds VII and VIII present an OH group, we searched for the corresponding trimethylsilyl ethers in derivatized extracts, but found none.

Acknowledgments We thank Dr. A. Messeguer, CID-CSIC, Barcelona, for his help with metabolite synthesis. This work was supported by the Commission of the European Community (EC Project EV5VCT92-0061).

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(5) Potter, T. L.; Carpenter, T. L. Environ. Sci. Technol. 1995, 29, 1557-1563. (6) Chiron, S; Abian, J.; Ferrer, M.; Sanchez-Baeza, F.; Messeguer, A.; Barcelo´ D. Environ. Toxicol. Chem. 1995, 14, 1287-1298. (7) Brown, M. A.; Kimmel, E. C.; Casida, J. E. Life Sci. 1988, 43, 2087-2094. (8) Lyons, C. D.; Katz, S. E.; Bartha, R. Bull. Environ. Contam. Toxicol. 1985, 35, 696-703. (9) Kross, B. C.; Vergara, A.; Raue, L. E. J. Toxicol. Environ. Health. 1992, 37, 149-159. (10) Regulating Pesticides in Food. National Academy Press. Washington, 1987. (11) Fielding, M. (Ed.) Pesticides in ground and drinking water; Commission of the European Communities: E. Guyot SA, Brussels (Water Pollution Research Report 27), 1992. (12) Galassi, S.; Provini, A.; Halfon, E. Intern. J. Environ. Anal. Chem. 1996, 65, 331-344. (13) Kolpin, D. W.; Thurman, E. M.; Goolsby, D. A. Environ. Sci. Technol. 1996, 30, 335-340. (14) Thurman, E. M.; Goolsby, D. A.; Aga, D. S.; Pomes, M. L.; Meyer, M. T. Environ. Sci. Technol. 1996, 30, 569-574. (15) CEE Directive no. 80/778. (16) Galassi, S.; Provini, A.; Mangiapan, S.; Benfenati, E. Chemosphere 1996, 32, 229-237. (17) Di Corcia, A.; Marchetti, M. Environ. Sci. Technol. 1992, 26, 6674.

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(18) Suba, L. A.; Pearson, D. A. The environmental studies of alachlor; Monsanto Final Report MSL-0860, Monsanto Agricultural Company, 1979. (19) Tiedje, J. M.; Hagedorn, M. L. J. Agric. Food Chem. 1975, 23, 77-81. (20) Wei, L. Y.; Vossbrinck, C. R. J. Agric. Food Chem. 1992, 40, 16951699. (21) Feng, P. C. C.; Patanella, J. E. Pestic. Biochem. Physiol. 1989, 33, 16-25. (22) Somich, C. J.; Kearney, P. C.; Muldoon, M. T.; Elsasser, S. J. Agric. Food Chem. 1988, 36, 1322-1326. (23) Pen ˜ uela, G. A.; Barcelo´, D. J. Chromatogr. A 1996, 754, 187195. (24) Bonfanti, M.; Taverna, P.; Chiappetta, L.; Villa P.; D’Incalci, M.; Bagnati, R.; Fanelli, R. Toxicology 1992, 72, 207-219. (25) Hoobler, M. A.; Letendre L. J. The aquatic metabolism studies of alachlor; Monsanto Final Report MSL-5307, Monsanto Agricultural Company, 1986.

Received for review April 29, 1997. Revised manuscript received October 10, 1997.X ES970380X X

Abstract published in Advance ACS Abstracts, October 15, 1997.