JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH

Part B—Pesticides, Food Contaminants, and Agricultural Wastes

Vol. B38, No. 6, pp. 737–746, 2003

Biodegradation of Chlorsulfuron and Metsulfuron-Methylby Aspergillus niger in Laboratory Conditions

Giovanna Boschin,1,* Alessandra D’Agostina,1 Anna Arnoldi,1

Ester Marotta,3 Elisabetta Zanardini,2 Marco Negri,2

Anna Valle,2 and Claudia Sorlini2

1Dipartimento di Scienze Molecolari Agroalimentari (DISMA), Sezione di

Chimica, and 2Dipartimento di Scienze, Tecnologie Alimentari e Microbiologiche

(DISTAM), Sezione MAAE, Universita degli Studi di Milano, Milano, Italy3Istituto di Scienze e Tecnologie Molecolari, CNR, Padova, Italy

ABSTRACT

Two sulfonylurea herbicides, chlorsulfuron and metsulfuron-methyl, were studied

under laboratory conditions, in order to elucidate the biodegradation pathway operated

by Aspergillus niger, a common soil fungus, which is often involved in the de-

gradation of xenobiotics. HPLC-UV was used to study the kinetic of degradation,

whereas LC-MS was used to identify the metabolites structure. In order to avoid the

chemical degradation induced by a decrease in pH, due to the production of citric acid

by the fungus, the experiments were performed in a buffered neutral medium. No

significant degradation for both compounds was observed in mineral medium with

0.2% sodium acetate. On the contrary, in a rich medium, after 28 days the de-

gradations, chemical degradation excluded, were about 30% for chlorsulfuron and

33% for metsulfuron-methyl. The main microbial metabolites were obtained via

*Correspondence: Giovanna Boschin, Dipartimento di Scienze Molecolari Agroalimentari

(DISMA), Sezione di Chimica, Universita degli Studi di Milano, via Celoria 2, I-20133, Milano,

Italy; E-mail: giovanna.boschin@unimi.it.

737

DOI: 10.1081/PFC-120025557 0360-1234 (Print); 1532-4109 (Online)

Copyright D 2003 by Marcel Dekker, Inc. www.dekker.com

cleavage of the sulfonylurea bridge. In addition the fungus seems to be able to

hydroxylate the aromatic ring of chlorsulfuron. In the case of metsulfuron-methyl the

only detected metabolite was the triazine derivative, while the aromatic portion was

completely degraded. Finally, the demethylation of the methoxy group on the triazine

ring, previously observed with a Pseudomonas fluorescens strain, was not observed

with A. niger.

Key Words: Chlorsulfuron; Metsulfuron-methyl; Sulfonylurea herbicides; Microbial

degradation; Aspergillus niger; LC-MS.

INTRODUCTION

Since the second half of 1970’s sulfonylurea herbicides are used on a wide range

of crops, such as wheat, barley, rice, corn, soybean, oilseed rape, cotton, potato, and

sugar beet, either in pre- or post-emergence treatments. They are very competitive as

alternatives to conventional herbicides since they are characterized by low field rates

(2–100 g/ha), high herbicidal activity, a broad spectrum of action, good crop

selectivity, and low toxicity towards humans and animals (DL50 on rat general-

ly > 5000 mg/Kg).[1 – 3] They interfere with protein synthesis, since they inhibit

acetolactate synthase (ALS), a key enzyme in the biosynthetic pathway of the branched

amino acids (valine, leucine, and isoleucine) present in plant and micro-organisms, but

not in animals.[1,2] Their structure is characterized by an aryl group, a sulfonylurea

bridge and a nitrogen-containing heterocyclic portion. Soil pH is an important

environmental factor in chemical and biological processes: as these herbicides are weak

acids (pKa = 3.3–5.2), they are prevalently present in soil in the dissociated form.

Sulfonylureas present a high mobility in soil and their degradation is faster in acidic

conditions, and in the presence of organic matter.[4 – 12] They do not present any

remarkable volatility and photodegradability, even if some recent studies have reported

photodegradation under lab conditions.[13 – 17]

Chemical hydrolysis and microbial degradation represent the main degradation

processes of pesticides in soil. As the degradation of sulfonylureas results faster and

more effective in non-sterile compared to sterile soil, probably it depends prevalently

on soil microflora.[18 – 22] Although the microbial degradation processes prevalently

occur in aerobic conditions, there are some evidences that they may take place also in

an anoxic and methanogenic environment.[23]

The half-life of these herbicides is relatively short, ranging from a few days to

8 weeks, but under particular climatic and pedological conditions, some of them can

persist for longer times and show phytotoxicity and damages in crop rotation.[24] In

addition some of these herbicides can reduce the microbial biomass and the metabolic

activity in soil, in particular the nitrification process and the nitrogen fixation by

symbiotic microrganisms.[25 – 33]

In a previous work[34] we have studied the chemical and microbial degradation

of chlorsulfuron 1 and metsulfuron-methyl 2, two herbicides that may show high

persistence in soil under specific conditions. Microbial degradation was performed

using a Pseudomonas fluorescens strain, previously selected by us from a sulfonylureas

treated soil.[34]

738 Boschin et al.

The general aim of this work was to study, under laboratory conditions, the in

batch degradation of these two sulfonylureas by Aspergillus niger. A. niger is a

common soil fungus, which is able to degrade several xenobiotics.[35 – 37] Only few

authors have reported studies on sulfonylureas with pure cultures;[18,38,39] most of these

studies were performed in soil or with mixed cultures.[40 – 42] On the contrary we were

interested to investigate the contribution of pure strain of A. niger to the total bio-

degradation of sulfonylureas.

MATERIALS AND METHODS

Materials

Solvents and reagents were used without any purification. Chlorsulfuron (analytical

grade 99.5%) and metsulfuron-methyl (analytical grade 97.4%) were kindly provided

by DuPont-Italy. Product 4 was obtained as previously described.[34] HPLC-grade

methanol was from Baker, and the HPLC water was produced with a Milli-Q Water

Purification System (Millipore). The eluents and the samples for HPLC were filtered

through disposable nylon filters (0.45 mm, Alltech, Italy).

Degradation Tests

The degradation tests were performed under different conditions: 1) mineral con-

ditions in M9 medium adding the herbicide as sole carbon and energy source; 2) co-

metabolic conditions using mineral M9 medium added with 0.2% sodium acetate; and

3) co-metabolic conditions using a rich medium (peptone 0.5%, yeast extract 0.3%,

glucose 1%).

M9 medium composition was: Na2HPO4 (7 g), KH2PO4 (3 g), NaCl (0.5 g), NH4Cl

(1 g), H2O (1000 mL).[43] In all the experiments the liquid media were buffered at

neutral pH (6.5–7) with phosphate buffer and pH was monitored periodically in order

to avoid chemical degradation by acidic hydrolysis. The herbicides were added at the

concentration of 100 mg/L. All the experimental trials were incubated at the temper-

ature of 28–30�C for four weeks. A. niger strain was from the DISTAM culture

collection, University of Milan.

In the degradation cultures (biotic tests), 0.2 mL of A. niger spore suspension (106

spores/mL) were added as inocula to 20 mL of the different media. Controls (abiotic

tests) were run in order to evaluate the chemical degradation.

Biodegradation of Chlorsulfuron and Metsulfuron-Methyl 739

All experiments were performed in triplicate and the data were analysed

statistically using the program COSTAT 2.00 (Cohort Software) and the values were

processed by ANOVA analysis.

HPLC-UV Analyses

Standard volumes of broth-cultures were taken at regular intervals and stored at

�20�C until HPLC analysis. Initially the samples were analysed at 254 nm simply after

filtration on 0.45 mm nylon filters (Alltech, Italy). When degradation was observed, the

samples were extracted with ethyl acetate, the combined extracts were dried with

anhydrous sodium sulfate, and the solvent was evaporated under vacuum at 30�C. The

residues were dissolved in methanol (1 mg/mL) and analysed by HPLC.

HPLC analyses were conducted with a Hewlett-Packard HP-1050 quaternary pump

fitted with a Rheodyne injector (20 mL, loop) and equipped with a HP-1050 Variable

Wavelength Detector (HPLC-VWD). Data were processed with a HP Chemstation

(Agilent Technologies, Palo Alto, CA, USA). The chromatographic separation was

achieved on a Lichrospher1 100 RP-18 column (5 mm, 250 � 4 mm, Merck, Darmstadt,

Germany). The analyses were carried out using a linear gradient: from 20:80 metha-

nol/1% acetic acid in water to 100:0 methanol/1% acetic acid in water, over 50 min,

then 5 min isocratic; the flow rate was 1 mL/min. Chromatograms were recorded at

254 nm, each analysis was repeated three times.

LC-MS Analyses

The LC-MS analyses were performed with a mass spectrometer LC-MSD Trap

Agilent 1100 Series (Agilent Technologies, Palo Alto, CA, USA). LC analyses were

achieved on a Lichrospher1 100 RP-18 column (5 mm, 250 � 4 mm, Merck,

Darmstadt, Germany) with a linear gradient from 0:100 acetonitrile/0.1% acetic acid in

water to 50:50 acetonitrile/0.1% acetic acid in water, over 16 min; the flow rate was

0.2 mL/min. Mass analyses were performed in ESI positive mode, the capillary voltage

was set at 4 kV and the mass range was m/z 50–2000.

RESULTS AND DISCUSSION

The biodegradation tests with A. niger were performed under three different

conditions: in a mineral M9 medium with the sulfonylurea herbicide as sole carbon and

energy source, in a mineral medium added with 0.2% sodium acetate, and in a rich

medium. In the first two cases no significant degradation was observed (data not

shown), probably because even 0.2% sodium acetate, is not rich enough to support a

suitable growth of A. niger for the degradation of the two sulfonylureas.

On the contrary in co-metabolic conditions with a rich medium, after four

weeks the total degradation of chlorsulfuron was 40%, with 30% of biotic degra-

dation, whereas the total degradation of metsulfuron-methyl was 43%, with 33%

of biotic degradation. Figure 1 represents the biodegradation of the two herbicides

during time.

740 Boschin et al.

Recently some authors[38,39] have shown that, under their laboratory conditions, the

microbial degradation observed with pure cultures of A. niger and Streptomyces

griseolus was, as a matter of fact, a chemical degradation induced by a decrease of pH

due to the microbial metabolism, in particular to the formation of citric acid. To avoid

this problem, in our experiments the medium was buffered in order to keep a neutral

pH. Monitoring during all the biodegradation experiments, confirmed that the pH did

not change, excluding any chemical degradation induced by acidification of the

medium. Thus our experiments confirm that A. niger makes a specific contribution in

the biodegradation of these sulfonylureas.

In order to separate the metabolites, the microbial culture broth were extracted

with ethyl acetate and then analysed by HPLC-UV. No additional clean-up or isolation

procedure was required before analyses. A typical HPLC chromatogram of the

degradation products from chlorsulfuron is shown in Figure 2.

The products identification was performed by positive LC-ESI-MS and confirmed

by comparison with authentic standards. Two metabolites were detected. The

metabolite with tR = 6.3 min showed an [M + H]+ ion at m/z 141 and was identified

as 2-amino-4-methoxy-6-methyltriazine (3). Its structure was confirmed by comparison

with the standard obtained by chemical decomposition of chlorsulfuron or metsulfuron-

methyl in DMSO.[34] It derives from the cleavage of the sulfonylurea bridge.

Figure 1. Changes in chlorsulfuon and metsulfuron-methyl concentration during biodegrada-

tive tests.

Biodegradation of Chlorsulfuron and Metsulfuron-Methyl 741

The LC-MS positive ion analysis of the second metabolite with tR = 17.5 min

showed a [M + H]+ ion at m/z 250 and a [M + Na]+ at m/z 272. A tentative structure 4is proposed; its formation requires the hydroxylation of the aromatic ring and the

acetylation of the sulfonamide residue after cleavage of the sulfonylurea bridge.

We have already observed the formation of this compound in the biodegradation of

chlorsulfuron by a P. fluorescens strain, isolated from soil treated with sulfonylurea

herbicides.[34] The hydroxylation of the benzene ring in meta position has been cited as

a minor degradation pathway in soil[44] and represents the first step of the major

degradation pathway in plants.[45] Some authors showed that Streptomyces griseolus

metabolises many sulfonylurea herbicides via hydroxylation of phenyl group by

inducible cytochrome P450 monoxygenases.[40,46 – 48] In addition, hydroxy compounds

were also detected in photodegradation experiments on other sulfonylureas.[13]

In the case of metsulfuron-methyl, only one metabolite with tR = 6.2 min was

detectable: its [M + H]+ ion at m/z 141 confirmed that it was product 3. With this

Figure 2. HPLC-UV chromatogram of chlorsulfuron and its degradation products extracted with

ethyl acetate from broth culture after 28 days.

742 Boschin et al.

herbicide no compound deriving from an oxidative pathway, as in the case of chlor-

sulfuron, was observed.

There are some similarities and differences in the behaviour of the P. fluorescens

strain previously studied[34] and A. niger. The main metabolite was in both cases the 2-

amino-triazine derivative 3, however A. niger is able to produce the oxidative

metabolite 4 only on chlorsulfuron and not on metsulfuron-methyl, whereas P.

fluorescens can oxidise both sulfonylureas. In addition only P. fluorescens is able to

demethylate of triazinic methoxy group to produce compounds 5a and 5b.

Thus it is possible to hypothesise that the cleavage of the sulfonylurea bridge is the

primary step in the degradation of these solfonylureas either in bacteria or fungi.

In order to investigate the fate and stability of the aromatic moiety other ex-

periments were carried out on two possible metabolites (compounds 6 and 7).

Experiments were performed both in M9 with 0.2% sodium acetate and in rich

medium. The results were comparable in both media: compound 6 undergoes mainly

chemical degradation (30–40%), while biodegradation is not significant. On the

contrary A. niger degrades totally compound 7 after 7 days, forming metabolites, which

were not detectable with our method.

In conclusion the reported results showed the contribution of A. niger to the total

biodegradation of these two sulfonylureas in nutrient rich medium; the main

degradative pathways were the sulfonylurea bridge cleavage and the hydroxylation of

benzene ring.

ACKNOWLEDGMENT

The authors are indebted to Dr. Bonacini (DuPont-Italy) for chlorsulfuron and

metsulfuron-methyl samples.

Biodegradation of Chlorsulfuron and Metsulfuron-Methyl 743

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Received May 5, 2003

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