Chemical and Biological Transformation of the Fungicide Vinclozolin

Article abstract of DOI:10.1021/jf980275m

Two mixed bacterial cultures were isolated from a French soil adapted to the dicarboximide fungicide vinclozolin. The vinclozolin was transformed by the mixed bacterial cultures according to two degradation pathways: (a) the formation of 2-[[(3,5-dichlorophenyl)carbamoyl]oxy]-2-methyl-3-butenoic acid and then 3,5-dichloroaniline or (b) the formation of 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide and then 3,5-dichloroaniline. The structure of 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid was unambiguously established by ¹H and ¹³C 2-D NMR analysis. A bacterial strain isolated from soil that degrades this compound was identified as a strain of Corynebacterium sp. Attempts to obtain pure vinclozolin-degrading strains via 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide were unsuccessful.

Full text of DOI:10.1021/jf980275m

J. Agric. Food Chem. 1998, 46, 3817−3822
3817
Ch em ica l a n d Biologica l Tr a n sfor m a tion of th e F u n gicid e
Vin clozolin
Christine Mercadier, Danielle Vega,* and J ean Bastide
Centre de Phytopharmacie URA CNRS 461, Universit e´ de Perpignan, 52 Avenue de Villeneuve,
6
6860 Perpignan Cedex, France
Two mixed bacterial cultures were isolated from a French soil adapted to the dicarboximide fungicide
vinclozolin. The vinclozolin was transformed by the mixed bacterial cultures according to two
degradation pathways: (a) the formation of 2-[[(3,5-dichlorophenyl)carbamoyl]oxy]-2-methyl-3-
butenoic acid and then 3,5-dichloroaniline or (b) the formation of 3′,5′-dichloro-2-hydroxy-2-
methylbut-3-enanilide and then 3,5-dichloroaniline. The structure of 2-[[(3,5-dichlorophenyl)-
1
13
carbamoyl]oxy]-2-methyl-3-butenoic acid was unambiguously established by H and C 2-D NMR
analysis. A bacterial strain isolated from soil that degrades this compound was identified as a
strain of Corynebacterium sp. Attempts to obtain pure vinclozolin-degrading strains via 3′,5′-dichloro-
2
-hydroxy-2-methylbut-3-enanilide were unsuccessful.
Keyw or d s: Vinclozolin; fungicide-degrading bacteria; chemical transformation
INTRODUCTION
drolysis rate at the same pH observed by Clark (1983),
Melkebeke et al. (1986), Szeto et al. (1989), and Villedieu
et al. (1994).
The experiments described in this paper were de-
signed to investigate both the chemical hydrolysis of
vinclozolin and the microbial degradation of the fungi-
cide by mixed and pure bacterial cultures isolated from
vinclozolin-degrading soil.
Vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-
oxazolidine-2,4-dione] is marketed by BASF under the
trade name Ronilan. This cyclic imide fungicide is used
to control diseases of grapevine, fruit trees, and veg-
etable crops caused by Botritys spp., Sclerotinia spp.,
and Monilinia spp.
A loss of efficacy of vinclozolin after several treat-
ments has been demonstrated in studies performed in
several countries over the past 10 years. For example,
such findings have been observed in France by Martin
et al. (1991), in the United Kingdom by Walker et al.
MATERIALS AND METHODS
Vin clozolin a n d Its Degr a d a tion P r od u cts. Vinclozolin
(1) [3-(3,5-dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-di-
one] in the form of commercial wettable powder formulation
(
1986), Walker (1987), and Mitchell and Cain (1996),
-
1
(
Ronilan 500 g kg of active ingredient) was purchased from
and in New Zealand by Slade et al. (1992). This
phenomenom was related by these authors to the
enhanced microbial degradation of the fungicide. Fur-
thermore, with the exception of very acidic soils (pH <5),
the degradation rate of vinclozolin was found to increase
according to the number of applications of the pesticide
BASF (Limburgerhof, Germany). The active ingredient was
extracted from Ronilan according to the method of Villedieu
et al. (1994). 2-[[(3,5-Dichlorophenyl)carbamoyl]oxy]-2-methyl-
3
3
-butenoic acid (2) and 3′,5′-dichloro-2-hydroxy-2-methylbut-
-enanilide (4) were synthesized according to the method of
Clark (1983). Analytical grade 3,5-dichloroaniline (5) was
purchased from Aldrich Chemical (Saint Quentin Fallavier,
France). Structures of vinclozolin and its metabolites are
shown in Figure 1.
(Walker, 1987)
Melkebeke et al. (1986), Szeto et al. (1989), and
Villedieu et al. (1994) have observed that the rate of
chemical transformation of vinclozolin in liquid medium
was related to the pH of the medium and that the
fungicide was more stable in acidic buffer. Transforma-
tion rates of vinclozolin reported by these authors were
similar, but the transformation patterns were different.
Szeto et al. (1989) reported the breakdown of the amide
bond, whereas Clark (1983), Melkebeke et al. (1986),
Pirisi et al. (1986), and Villedieu et al. (1994) suggested
the breakdown of ester function.
Head et al. (1988), Golovleva et al. (1991), and Cain
and Mitchell (1996) described the isolation of vinclozo-
lin-degrading strains of bacteria from soils exhibiting
enhanced degradation of the fungicide. Five bacterial
strains capable of degrading vinclozolin were isolated.
However, these results need to be viewed with caution
because, in liquid medium without the bacterial strain,
the transformation rates of vinclozolin reported in these
studies were not in agreement with the chemical hy-
NMR Tech n iqu es. The NMR spectra of compounds were
1
3
measured on a J EOL EX400 spectrometer at 100 MHz for
C
1
1
13
and 400 MHz for H. The long-range H- C correlation was
measured with a COLOC procedure (Kessler et al., 1984).
An a lytica l Meth od s. Concentrations of vinclozolin and its
degradation products were determined by HPLC. HPLC
analyses were performed on a system with a Beckman pump
and a Shimadzu SPD 2A UV detector (235 nm wavelength).
The operating parameters were as follows: column, Ultrabase
UB 235-5 µm C8; mobile phase, acetonitrile/water/trifluoro-
acetic acid (65:35:0.1) (v/v/v), delivered at a flow rate of 1 mL
-1
min . All compounds studied were quantified using external
standards.
Tr a n sfor m a tion of Vin clozolin or Bu ten oic Acid in
Aqu eou s Bu ffer p H 4.5. Portions (0.5 mL) of vinclozolin or
-1
butenoic acid solution at 1 mg mL
in methanol were
thoroughly mixed with 50 mL of aqueous buffer solution, pH
.5. This buffer was prepared with a mixture of sodium
4
acetate (0.01 M) and acetic acid (0.01 M) and was filter-
sterilized with a 0.22 µm filter. These flasks were incubated
S0021-8561(98)00275-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/29/1998

3818 J. Agric. Food Chem., Vol. 46, No. 9, 1998
Mercadier et al.
F igu r e 1. Structures of vinclozolin (1) and its metabolites: 2-[[(3,5-dichlorophenyl)carbamoyl]oxy]-2-methyl-3-butenoic acid (2);
carbamic acid (3); 3′,5′-dichloro-2-hydroxy-2-methylbut-3-enanilide (4); 3,5-dichloroaniline (5).
at 35 °C in an oven. Duplicate samples were periodically
removed from each flask and analyzed by HPLC.
Growth was monitored by measuring the optical density at
550 nm.
Soil Ad a p ta tion . Soil sample (Saint Nazaire) was collected
from a depth of 0-15 cm in the south of France. The soil
properties have been previously described (Vega et al., 1992).
This soil (pH 6.4) had no previous history of dicarboximide
fungicide treatment. In the laboratory, 1 kg of this soil was
treated with an aqueous suspension of Ronilan to give a final
Vin clozolin Degr a d a t ion b y Tw o Mixed Ba ct er ia l
Cu ltu r es. All of the mixed cultures obtained by scraping the
surface of the plates were subcultured in 30 mL of MM, pH
-
1
6.5, supplemented with vinclozolin (20 µmol L ) in Erlenm-
eyer flasks. After incubation at 30 °C in a rotary shaker at
200 rpm, 1 mL of these cultures was diluted in MM (10 mL)
-
1
-1
concentration of 10 mg kg [active ingredient (ai)] of dry soil
with 20% soil moisture. The soil was then incubated at 28
supplemented with vinclozolin (20 µmol L ) and incubated
under the same conditions as previously described. A control
experiment with noninoculated mineral medium was also
carried out. Samples (0.5 mL) of both bacterial cultures and
noninoculated medium were periodically removed, diluted with
acetonitrile (0.5 mL), and centrifuged briefly to remove solid
matter before injection. The concentration of vinclozolin and
its metabolites was monitored by HPLC.
°
C. When 50% of vinclozolin was degraded, an identical
fungicide treatment was repeated up to six times.
Extr a ction . After each treatment to adaptation, soil
samples (20 g) were removed and extracted by shaking for1 h
with 20 mL of acetonitrile/acetic acid (99:1). After decantation,
the supernatant was centrifuged for 2 min, to remove the solid
matter, and analyzed by HPLC.
Cu ltu r e Med ia . Cultures were carried out in a mineral
medium (MM) containing 50 mL of the Winogradsky stock
salts solution (a) and 1 mL of trace elements stock solution
Vin clozolin a n d Com p ou n d 2 Degr a d a tion by a n
Isola ted P u r e Ba cter ia Str a in . Microorganisms from a
single colony were harvested from MM agar plates and
inoculated in 30 mL of MM, pH 6.5, supplemented with
(b) adjusted to 1 L with distilled water. The mineral medium
-1
vinclozolin or compound 2 (10 µmol L ). Incubations, the
was adjusted to pH 6.5 and sterilized by autoclaving for 20
min at 121 °C. When required, this mineral medium was
control experiments, and HPLC analyses were carried out as
previously described.
-
1
solidified by the addition of agar (15 g L ).
a) The composition of the Winogradsky stock salts solution
was as follows: K HPO , 5 g; MgSO , 2.5 g; NaCl, 2.5 g; Fe
SO , 50 mg in 1 L of distilled water.
b) The composition of the trace elements stock solution was
(
RESULTS AND DISCUSSION
2
4
4
2
-
(
4 3
)
Str u ctu r e of Com p ou n d Obta in ed by Ba sic Hy-
d r olysis of Vin clozolin . A compound X was obtained
by synthesis according to the experimental method of
Clark (1983). This compound had the same physico-
(
as follows: K
Co(NO ‚6H
mg; ZnSO ‚7H
2
MoO
O, 50 mg; CdSO
4
, 50 mg; NaBO
‚8H
2
‚4H
O, 50 mg; CuSO
‚5H O, 50 mg in 1 L of
2
O, 50 mg; FeCl
3
, 3 mg;
3
)
2
2
4
2
4
‚5H
2
O, 50
4
2
O, 50 mg; MnSO
4
2
1
distilled water. These two solutions, (a) and (b), were sterilized
at 121 °C for 20 min before storage.
chemical characteristics (mp, mass spectrum, H NMR)
as those reported by Clark (1983), with the formula
proposed by Clark being the carbamic acid 3. It is
known that the chemical stability of this type of
compound is generally low, such that spontaneous
decarboxylation usually occurs. However, we observed
that the compound X was stable and easily isolated.
A second structure of a vinclozolin transformation
product, compound 2, was described by Szeto et al.
(1989) and Golovleva et al. (1991), although the method
of synthesis and the physicochemical characteristics
were not reported.
Acetone solutions of vinclozolin or butenoic acid (5 g L-1)
were filter-sterilized (FG 0.22 µm pore size filter, Millipore,
Saint Quentin Yvelines, France) and added to cooled and
sterile MM to obtain a final concentration of 10-70 µmol L
To isolate colonies, nutrient agar (Difco, Detroit, MI) was
prepared.
-
1
.
Isola tion of Vin clozolin -Degr a d in g Micr oor ga n ism s
(
Mixed Cu ltu r es). A vinclozolin-degrading soil (2.5 g) was
added to MM (pH 6.5, 50 mL) supplemented with vinclozolin
-
1
(
70 µmol L ) in an Erlenmeyer flask. This soil suspension
was incubated at 30 °C in a rotary shaker at 200 rpm.
Samples of the culture (0.5 mL) were removed at regular
intervals, diluted with acetonitrile (0.5 mL), and centrifuged
briefly to remove solid matter before injection into the chro-
matographic column for analysis. When 50% of vinclozolin
was degraded, 5 mL of these soil suspensions was subcultured
in 45 mL of fresh MM with vinclozolin as the sole source of
carbon and nitrogen. After ∼10 h of incubation, 100 µL of this
The H and 13C NMR spectra of compound X were
measured, and the assignment of the different protons
and carbons was established (Figure 2). Long-range
1
1
13
H- C correlation from CH3 and C7 and N-H and C2
(C6) of the compound were in accordance with the
structure of 2. The chemical shifts of the two acidic
hydrogens were 10.2 and 13.05 ppm, corresponding to
N-H and CO2H functions, respectively, and thus con-
firming the structure of compound X to be the same as
that of 2.
-1
suspension was spreaded on plates of MM plus agar (15 g L
)
or on plates of nutrient broth, both supplemented with
-
1
vinclozolin (50 mg L ). The plates were incubated at 30 °C.
After several days of incubation, all of the cells on the plate
surface were scraped and dispersed in 5 mL of mineral
medium. These mixed cultures will be used as inocula.
Isolates were purified by restreaking single colonies onto
plates of MM plus agar supplemented with vinclozolin (50 mg
Hyd r olysis of Vin clozolin a n d Com p ou n d 2 a t
p H 4.5. The chemical transformation of both vinclozolin
and compound 2 was studied in a pH 4.5 buffer under
the same conditions as those described by Szeto et al.
-
1
L
).

Chemical and Biological Transformation of Vinclozolin
J. Agric. Food Chem., Vol. 46, No. 9, 1998 3819
F igu r e 2. 1H and 13C NMR chemical shifts of compound X,
which are the same as for compound 2.
F igu r e 3. Vinclozolin transformation in aqueous buffer at pH
4
.5. Mean values and standard deviations are given for
vinclozolin (9), compound 2 (b), compound 4 (4), and 3,5-
dichloroaniline 5 (2).
F igu r e 5. Vinclozolin degradation in nonadapted soil (a) and
in adapted soil (b): vinclozolin (9), compound 2 (b), compound
4
(4), and 3,5-dichloroaniline 5 (2).
5
5
a), whereas 4 and 5 were formed in treated soil (Figure
b). The quantities of 5 and 4 found at the beginning
of the curve in Figure 5b correspond to residues of the
degradation products that had accumulated during the
soil adaptation.
En r ich m en t a n d Isola tion of Vin clozolin -De-
gr a d in g Micr oor ga n ism s. Mixed enrichment cul-
tures of microorganisms that were able to degrade
vinclozolin and showed enhanced degradation of the
fungicide were obtained from the Saint Nazaire soil.
After plating on mineral salt medium plus agar or on
nutrient medium plus agar, two different mixed bacte-
rial cultures, named MA and MB, respectively, were
obtained.
Three bacterial strains were isolated from MA, but
only one of these isolates was able to grow on and
degrade vinclozolin in liquid mineral medium with the
fungicide as the sole source of carbon and nitrogen. This
strain was grown aerobically, forming a yellow, smooth,
circular colony. It was Gram-positive, motile, and rod-
shaped and was tentatively identified as Corynebacte-
rium sp. by the Institut Pasteur (Lyon, France).
All attempts to isolate vinclozolin-degrading pure
strains from MB failed.
F igu r e 4. Compound 2 transformation in aqueous buffer at
pH 4.5. Data are the average of duplicate measurements for
vinclozolin (9), compound 2 (b), compound 4 (4), and 3,5-
dichloroaniline 5 (2).
(1989). Vinclozolin was transformed into 2, while at the
same time, 4 was also rapidly formed (Figure 3). Under
the same incubation conditions, compound 2 was rapidly
transformed into vinclozolin and 4 (Figure 4). Com-
pound 2 showed the same reactivity as the compound
obtained by Szeto et al. (1989). Our findings suggest
that the transformation pathway of vinclozolin proposed
by Clark (1983), Pirisi et al. (1986), and Villedieu et al.
(1994) was not supported in this instance: the carbamic
acid 3 was never isolated in these experiments, and the
structure of the isolated intermediate was 2.
Soil Ad a p ta tion . The transformation rate of vin-
clozolin in soil from Saint Nazaire increased after
repeated applications of the fungicide, such that the
half-life of vinclozolin was 22 days after the first
treatment and 2.15 h after the sixth treatment. The
compounds formed in untreated and treated soils were
different: 2 and 5 were formed in untreated soil (Figure
Vin clozolin Degr a d a tion by th e Mixed Ba cter ia l
Cu ltu r es, MA a n d MB. In mineral medium, pH 6.5,
with vinclozolin as the sole source of carbon and
nitrogen, the mixed bacterial culture MA degraded the
fungicide into 2 and 5 (Figure 6). The transformation
rate of vinclozolin was greater in the inoculated medium

3820 J. Agric. Food Chem., Vol. 46, No. 9, 1998
Mercadier et al.
F igu r e 7. Vinclozolin degradation in pH 6.5 mineral medium
at 30 °C inoculated with Corynebacterium sp. (full line) and
without strain (broken line): vinclozolin (9), compound 2 (b),
compound 4 (4), and 3,5-dichloroaniline 5 (2).
F igu r e 8. Compound 2 degradation in pH 6.5 mineral
medium at 30 °C inoculated with Corynebacterium sp. (full
line) and without strain (broken line): compound 2 (b), 3,5-
dichloroaniline 5 (2), and unidentified compound A (O).
Corynebacterium sp. culture was 3 times higher with
the OD increasing from 0.018 to 0.06.
F igu r e 6. Vinclozolin degradation by mixed cultures MA and
MB in pH 6.5 mineral medium with incubation at 30 °C in a
rotary shaker at 200 rpm.
Com p ou n d 2 Degr a d a tion by Cor yn eba cter iu m
sp . Compound 2 was chemically stable in the mineral
medium (Figure 8). In the same medium inoculated
with Corynebacterium sp., 2 was very rapidly degraded.
Two metabolites were formed: 5 and an unidentified
compound (compound A). A small quantity of this
compound was isolated, but the data obtained by NMR
and MS did not permit the unambiguous determination
of its chemical structure. However, we noticed that the
5 ring was present in this molecule. We postulated that
the molecular absorption coefficients of compounds A
and 5 were similar. This allowed an evaluation of the
concentration of compound A by measuring and com-
paring the UV absorption of this compound to the UV
absorption of a solution of 5 at known concentrations.
These two results (Figures 7 and 8) were consistent
with the hypothesis that the slight increase in the
degradation rate of vinclozolin in the liquid Corynebac-
terium sp. culture might be related to the changing
chemical equilibrium between vinclozolin and 2.
(
)
half-life ) 15 h) than in the uninoculated one (half-life
40 h). After incubation, the final pH of the culture
remained unchanged.
Under the same culture conditions, the mixed bacte-
rial culture MB degraded vinclozolin more rapidly than
MA, as a measured half-life of 6 h was found in the
presence of MB. Moreover, the degradation pathway
was different, with 4 and 5 being formed (Figure 6). A
small amount of 2 corresponding to the chemical hy-
drolysis was detected.
Vin clozolin Degr a d a t ion b y Cor yn eba cter iu m
sp . When the Corynebacterium sp. strain was inocu-
lated in mineral medium supplemented with vinclozolin,
the transformation rate of vinclozolin was only slightly
increased (Figure 7). Nevertheless, the products formed
were different from those obtained by chemical hydroly-
sis; the breakdown of vinclozolin led to the formation
of 2, which rapidly disappeared. 5 was formed in large
amounts.
CONCLUSION
The mechanism of the vinclozolin transformation
seems more complex than the one of other dicarboximide
fungicides, such as iprodione (Athiel et al., 1995; Mer-
cadier et al., 1997). Indeed, two different bonds of
In mineral medium supplemented with vinclozolin,
a slight increase in the bacterial population was ob-
served. After 48 h of incubation, the turbidity of the

Chemical and Biological Transformation of Vinclozolin
J. Agric. Food Chem., Vol. 46, No. 9, 1998 3821
F igu r e 9. Suggested pathways for chemical and biological vinclozolin transformation.
the 2,4-oxazolidinedione cycle of vinclozolin may be
broken. A scheme of the transformation pathway of
vinclozolin is proposed in Figure 9.
the chemical instability of vinclozolin and to the diver-
sity of the isolated microorganisms.
In this study, we reported the isolation of mixed and
pure cultures involved in the degradation of vinclozolin
in liquid medium. Are these microorganisms respon-
sible for the enhanced degradation of vinclozolin in
adapted soils? The addition of degrading microorgan-
isms to soils can increase pesticide transformation rates
(Barles et al., 1979; Edgehill and Finn, 1983; Milhomme
et al., 1989; Cork and Krueger, 1991). The transforma-
tion rate of vinclozolin was not increased by the addition
of Corynebacterium sp. to untreated Saint Nazaire soil
(data not shown), although 3,5-dichloroaniline (5) was
the only metabolite detected in the soil. On the other
hand, the transformation of vinclozolin in untreated soil
led to the formation of both 2 and 5 (results not shown).
This result suggests that this strain is not responsible
for the enhanced degradation of vinclozolin in adapted
soils, although it was able to degrade compound 2 into
5. No similar experiment was carried out with the
mixed culture MB. Nevertheless, in liquid medium
inoculated with MB, vinclozolin was converted into 4
and 5. We noticed that this transformation pathway
was similar to that observed in the Saint Nazaire
adapted soil. These findings suggest that microorgan-
isms of the mixed culture MB might be involved in the
vinclozolin degradation in soils and might also be
responsible for the enhanced degradation of the fungi-
cide in the Saint Nazaire adapted soil.
We were able to show that vinclozolin was converted
by chemical hydrolysis into an open form, the butenoic
acid derivative 2, and that the fungicide was in chemical
equilibrium with this intermediate. Vinclozolin was
also transformed into a carbamic acid derivative, 3. This
unstable intermediate 3 was then converted into an
enanilide, 4. These two chemical transformation path-
ways were observed by monitoring biological degrada-
tion. However, there was evidence of a biological
transformation due to the appearance of the 3,5-dichlo-
roaniline, this being compound 5. Indeed, the interme-
diate compounds 2 and 4 might then be metabolized into
5
.
We succeeded in isolating two mixed bacterial cul-
tures, MA and MB, involved in the vinclozolin degrada-
tion, with two degradation patterns observed. In media
inoculated with MA, vinclozolin was converted into 2,
which was then degraded into 5. In the presence of MB,
the degradation of vinclozolin produced 4, which was
then converted into 5.
A pure bacterial strain was isolated from the mixed
culture MA and tentatively identified as Corynebacte-
rium sp. All attempts to show that this isolate degraded
vinclozolin failed. However, we demonstrated that
Corynebacterium sp. was able to degrade 2 into 5 and
into an unidentified compound A. Several authors have
described the degradation of vinclozolin by pure isolated
strains. Golovleva et al. (1991), for example, showed
that the isolates were involved in the degradation of
vinclozolin and that the compounds 2 and 4 were
produced. However, the incubation time (10 days) and
the metabolites formed were more in agreement with a
chemical transformation. Cain and Mitchell (1996)
detected only one metabolite, this being 3,5-dichloroa-
niline (5), with no other intermediate reported. Head
at al. (1988) demonstrated that vinclozolin was first
degraded to an unidentified metabolite by a mixed
bacterial culture, with this metabolite then leading to
the formation of 3,5-dichloroaniline (5). The diversity
of results reported in the literature may be related to
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