Correspondence: Partha P. Choudhury, Directorate of Weed Science Research, Maharajpur, Madhya Pradesh, India. Tel.: +91 9179457045; fax: +91 761 2353921; e-mail: email@example.com
Chlorimuron-ethyl, ethyl-2-[[[[(4-methoxy-6-chloro-pyrimidin-2-yl)amino]carbonyl]amino] sulfonyl]benzoate, is used as a pre- and postemergence herbicide for the control of important broadleaved weeds in soybean and maize. Due to its phytotoxicity to rotation crops, concerns regarding chlorimuron contamination of soil and water have been raised. Although it is degraded in the agricultural environment primarily via pH- and temperature-dependent chemical hydrolysis, microbial transformation also has an important role. Fungi such as Fusarium and Alternaria are unable to survive in artificial media containing chlorimuron-ethyl at 25 mg L−1. However, Aspergillus niger survived in minimal broth containing chlorimuron at 2 mg mL−1. Aspergillus niger degraded the herbicide to harvest energy through two major routes of degradation. One route involves the cleavage of the sulfonylurea bridge, resulting in the formation of two major metabolites, namely ethyl-2-aminosulfonylbenzoate (I) and 4-methoxy-6-chloro-2-amino-pyrimidine (II). The other route is the cleavage of sulfonylamide linkage, which generates the metabolite N-(4-methoxy-6-chloropyrimidin-2-yl) urea (III). Two other metabolites, saccharin (IV) and N-methyl saccharin (V), formed from metabolite II, were also identified. A metabolic pathway for the degradation of chlorimuron-ethyl by A. niger has been proposed.
Chlorimuron-ethyl, ethyl-2-[[[[(4-methoxy-6-chloro-pyrimidin-2-yl)amino]carbonyl]amino] sulfonyl]benzoate, is used as a postemergence herbicide for the control of important broadleaved weeds in soybean (Worthing & Hance, 1991). It is also used as part of combination formulations for rice (Singh et al., 2008; Saha & Rao, 2009). Chlorimuron-ethyl exerts carry-over effects on succeeding crops such as sugar beet, corn and cotton. It reduced the yield of sugar beet planted 1 year after its application (Renner & Powell, 1991). Chlorimuron-residue haremed corn (Curran et al., 1991), and also harmed sunflower, watermelon, cucumber and mustard when observed 16 weeks after application (Johnson & Talbert, 1993). Although its persistence is moderate in soil [half-life (T1/2) 30 days], like many other sulfonylurea herbicides, its persistence increases with increasing pH. The T1/2 of chlorimuron under acidic conditions (pH 5) is 17–25 days, whereas at higher pH this may increase to 70 days. The half-life of chlorimuron in a silt-loam soil was 7 days at pH 6.3 and 18 days at pH 7.8 (Brown, 1990). By using a root bioassay technique, Schroeder (1994) determined the half-life of chlorimuron in soils of different pH-ranges as 12–50 days. Bedmar et al. (2006) observed a wide range of half-life for chlorimuron in soil from 30 days at pH 5.9 to 69 days at pH 6.8.
Chlorimuron-ethyl degrades in the agricultural environment primarily via pH- and temperature-dependent chemical hydrolysis (Beyer et al., 1988; Brown, 1990; Hay, 1990), as observed for many sulfonylurea herbicides, such as sulfometuron-methyl (Harvey et al., 1985), chlorsulfuron (Sabadie, 1990), metsulfuron-methyl (Sabadie, 1991), rimsulfuron (Schneiders et al., 1993), nicosulfuron (Sabadie, 2002) and flazasulfuron (Bertrand et al., 2003). The phototransformation of chlorimuron by sunlight also takes place on the soil surface (Choudhury & Dureja, 1996a) and in water (Venkatesh et al., 1993; Choudhury & Dureja, 1996b). Within the surface soil chlorimuron is also considered to serve as a source of carbon, nitrogen and sulfur for microorganisms. There are reports on the utilization of sulfonylurea herbicides by microorganisms. The metabolic pathways for the degradation of chlorsulfuron and metsulfuron-methyl by Streptomyces griseolus (Joshi et al., 1985; Reiser & Steiglitz, 1990), and trisulfuron by S. griseolus in artificial media (Dietrich et al., 1995) have been established. At low pH the degradation of trisulfuron-methyl takes place by chemical hydrolysis, whereas in neutral to alkaline soil, microorganisms play the dominant role in its degradation (Peeples et al., 1991), and the major degradation route is cleavage of the sulfonylurea bridge (Vega et al., 2000). Streptomyces griseolus can also de-esterify and O-dealkylate the chlorimuron-ethyl molecule (Reiser & Steiglitz, 1990). A bacterium, Pseudomonas sp., isolated from chlorimuron-ethyl-contaminated soil degrades the herbicide by cleaving the sulfonylurea bridge (Ma et al., 2009), and a yeast strain, Sporobolomyces sp., was isolated as a chlorimuron-degrading organism (Xiaoli et al., 2009). The transformation of chlorimuron-ethyl by microorganisms, more specifically by fungi, has not yet been studied in detail. In the context of the ecological risk of long-term chlorimuron-ethyl application, it is necessary to understand the interaction between the herbicide and microorganisms. The present investigation was undertaken to isolate chlorimuron-ethyl-degrading fungi from agricultural soil and its degradation pathway.
Materials and methods
A laboratory sample of chlorimuron-ethyl (95% purity) was supplied by DuPont Far East Inc., and was purified further by repeated crystallization from benzene and hexane until a constant melting point of 185 °C was achieved. Laboratory-grade reagents and solvents were procured locally. All solvents were dried and distilled before use. HPLC-grade solvent and reagents were used during chromatographic and spectroscopic analysis. Deionized water was obtained from the Milli-Q SP Reagent water system (Millipore).
Soil samples were collected from different fields of the Directorate of Weed Science Research (DWSR) farm with a previous history of chlorimuron-ethyl application.
Isolation and identification of chlorimuron-ethyl-degrading fungi
The collected soil was enriched with chlorimuron-ethyl (5 mg in 100 g of soil) and incubated for 1 week at 30 °C. For selection of fungi as a suitable chlorimuron-degrading agent, were used serial dilution following agar plating of incubated soil. Fungi that appeared on potato-dextrose agar (PDA) plates (200 g potato, 20 g dextrose, 20 g agar and 1000 mL water) after 5 days of incubation were further plated to obtain pure cultures. The fungi screened from chlorimuron-enriched soil were again incubated for 7 days in minimal PDA broth (10 g potato, 20 g dextrose and 1000 mL of water) containing different levels of chlorimuron-ethyl (25, 50, 100 and 200 mg per 100 mL broth). The most efficient fungus was screened out on the basis of its growth and was further inoculated on PDA plates. After 2 days of incubation, colony morphology of the isolate was examined. On the basis of colony morphology and microscopy of spore structures the fungus was characterized.
Degradation of chlorimuron-ethyl by A. niger
For degradation studies, 25 mg chlorimuron-ethyl was added to 100 mL of sterile dextrose-minimal broth (100 g potato, 10 g dextrose and 1000 mL water) in 250-mL flasks. The chlorimuron was allowed to dissolve overnight on a shaker before use. Twenty such flasks were incubated with isolated A. niger in the dark at 25 °C for 27 days in a BOD incubator. Three flasks with minimal broth and chlorimuron, and without incubation with A. niger were kept in the dark as controls.
Degraded products were extracted by partitioning in chloroform from the broth sampled after 3, 9, 16, and 27 days of incubation. Solvent was then evaporated under low pressure in a rotary vacuum evaporator to obtain a crude mixture of products. Products were purified by preparative thin-layer chromatography and characterized by spectroscopic techniques.
Liquid chromatography-mass spectroscopy
An API 4000 Qtrap mass spectrometer linked to an Agilent 1200 series HPLC machine was used to characterize metabolites. Chromatographic separation was carried out on an Atlantis dC18 (Waters India Ltd.) column (150 mm × 2.1 mm, i.d. 5 μm). The mobile phase comprised A = methanol/water with 5 mM ammonium formate (20 : 80) and B = methanol/water with 5 mM ammonium formate (90 : 20); the gradient programme was: 0–1 min 98% A, 1–8 min 98–5% A, 8–12 min 5% A, 12–13 min 5–98% A and 13–20 min 98% A phases. The column oven temperature was set at 35 °C with a flow rate of 0.4 mL min−1. An aliquot of 10 μL was injected through an auto sampler. Mass spectrometric analysis was performed with electrospray ionization (ESI) in positive (5500 eV) modes for each sample. The nebulizer gas and heater gases were adjusted at 30 and 55 p.s.i., respectively. The ion source temperature was set at 500 °C. A hybrid triple quadrupole linear ion trap mass spectrometer (QqQLIT) was used by integrating an EMS-triggered IDA-enhanced production (EPI), resulting in enhanced sensitivity at trace level. IDA-EPI experiments were automatically triggered to obtain product ion mass spectra of these peaks. In the IDA experiment, the parameters included rolling collision energy with scan speed of 4000 amus−1, and dynamic trap fill time as a dependent scan.
Preparation of major metabolites
Acid hydrolysis of chlorimuron-ethyl
Chlorimuron-ethyl (50 mg) was dissolved in distilled water (100 mL). The pH of the solution was adjusted to 2.5 by the addition of concentrated sulfuric acid (2 mL). The solution was stirred magnetically for 48 h at 42 °C and then kept for 4 days at room temperature. Products formed were separated by preparative thin-layer chromatography, purified by crystallization from benzene and characterized by spectroscopic methods. The compounds were 4-methoxy-6-chloro-2-amino pyrimidine (III) [IR (cm−1): 3460, 3323, 802; 1H-NMR (CDCl3) δ: 6.2 (s, 1H, aromatic), 5.3 (s, 2H, NH2), 3.85 (s, 3H, OCH3); mass spectrum: 159 (M+, 27.7%, 129 (M+ - 30), 94 (M+-66,12.6%) and ethyl-2-(aminosulfonyl)benzoate (IV) [IR (cm−1): 3382, 3278, 2367, 1723; 1H-NMR (CDCl3) δ: 8.15 (d, 1H, aromatic, J = 7 Hz), 7.85 (d, 1H, aromatic, J = 5 Hz), 7.65 (t, 2H, aromatic, J = 5 Hz), 5.84 (s, 2H, NH2), 4.46 (q, 2H, OCH2CH3, J = 5 Hz), 1.46 (t, 3H, OCH2CH3, J = 7 Hz); mass spectrum: 229 (M+, 8.5%), 212 (10.6%), 184 (100%) and 121].
Results and discussion
Isolation and characterization of chlorimuron-ethyl-degrading fungus
Fungi isolated from rice rhizosphere soil were allowed to grow in minimal media with chlorimuron-ethyl as the carbon and nitrogen source. Only one fungus survived and grew in medium with chlorimuron as high as 200 mg L−1 (Fig. 1). The mycelia of the isolated fungus were nonseptate with a foot-cell, and conidiophores ended in a terminal enlarged ellipsoidal spherical swelling. This spherical vesicle bearded phialides that covered its entire surface and therefore the head of the conidia was mop-like. They were highly branched; multinucleate mycelia bore a large number of conidiophores, which arose individually as hyphae. Chains of conidia arose on the sterigma, giving the appearance of strings of beads. This fungus was characterized as A. niger.
Degradation of chlorimuron-ethyl by A. niger
Aspergillus niger, isolated from soil samples of the DWSR, survived in chlorimuron-ethyl-enriched media (200 mg in 100 mL of broth). At this high concentration, other isolated fungi, namely Alternaria alternate, Aspergillus sp. and Fusarium oxysporium, were unable to survive. Aspergillus niger degraded chlorimuron-ethyl by releasing extracellular enzymes, which acted upon it, converting into simpler forms that enabled the microorganism to derive energy from the herbicide for growth and maintenance. The degraded products were characterized structurally by the mass spectra found from LC-MS/MS and the structures were further confirmed based on the spectra of synthesized molecules and previously reported degraded compounds of chlorimuron-ethyl. There was no major degradation of chlorimuron-ethyl during incubation without A. niger under similar conditions (pH 7.0, 28 °C). Metabolites isolated from this biodegradation by A. niger were ethyl-2-aminosulphonyl benzoate (I, Fig. 2), 4-methoxy-6-chloro-2-amino-pyrimidine (II, Fig. 3), N-(4-methoxy-6-chloropyrimidin-2-yl)urea (III, Fig. 4), o-benzoic sulf-N-methylimide (IV, Fig. 5) and o-benzoic sulfimide (V, Fig. 6).
On the basis of the structures of the metabolites, a pathway of degradation is proposed (Fig. 7). The initial degradation of the compound is suggested to take place via cleavage of the sulfonylurea bridge. The presence of two metabolites, ethyl-2-aminosulphonyl benzoate (I) and 4-methoxy-6-chloro-2-amino-pyrimidine (II), supported this suggestion. This is basically a decarboxylation reaction of the sulfonylurea bridge, and a decarboxylase-type enzyme is catalyses the reaction. However, the presence of the metabolite N-(4-methoxy-6-chloropyrimidin-2-yl) urea (III) suggests a different mode of degradation. Formation of this metabolite is possible through cleavage of the sulfonyl amide linkage. This reaction involves hydrolysis at the sulfonyl amide bond, and a hydrolase-type enzyme was probably utilized by A. niger to catalyse the reaction. The presence of three metabolites, i.e. I, II and III, suggests the simultaneous occurrence of both mechanisms. The other degradation products were formed from these three basic metabolites. In the metabolite o-benzoic sulf-N-methylimide (IV), a methyl group is attached with an imide-nitrogen atom. The source of this methyl group is either the –CH2CH3 of carboxylic ester or the methyl of the methoxy group attached to a pyrimidine ring. Therefore, a dealkylation process, either O-dealkylation or C-dealkylation, is involved in generating the methyl group. The N-dealkylation of metabolite IV led to the formation of o-benzoic sulfimide (V), commonly known as saccharin.
Chlorimuron-ethyl appears to have the ability to inhibit the growth of some fungi present in soil, as it shows a deleterious effect on Fusarium and Alternaria. But its biodegradation, both in soil and in media, by Aspergillus indicates that the appropriate consortium of fungi can remove chlorimuron-ethyl from soil and water. Further investigation to isolate other chlorimuron-ethyl-degrading fungi is needed.
We are grateful to the Director, Directorate of Weed Science Research (ICAR), Jabalpur, MP, India, for providing the research facilities to complete the PG dissertation work of S.S.