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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Microbial conversion of fungicide vinclozolin a
a
L.A. Golovleva , Z.I. Finkelstein , A.V. b
a
Polyakova , B.P. Baskunov & M.Yu. Nefedova
a
a
Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Pushchino, Moscov Region, 142292 b
Rostov State University, R/D, Rostov, 344711 Version of record first published: 21 Nov 2008.
To cite this article: L.A. Golovleva , Z.I. Finkelstein , A.V. Polyakova , B.P. Baskunov & M.Yu. Nefedova (1991): Microbial conversion of fungicide vinclozolin, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 26:3, 293-307 To link to this article: http://dx.doi.org/10.1080/03601239109372736
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/ terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
Downloaded by [McGill University Library] at 20:48 23 April 2013
The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
J. ENVIRON. SCI. HEALTH, B26(3), 293-307 (1991)
Downloaded by [McGill University Library] at 20:48 23 April 2013
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
Key words: vinclozolin, soil microorganisms, bioconversion, environmental safety
L.A. Golovleva*, Z.I. F i n k e l s t e i n * , A.V. Polyakova**, B.P. Baskunov* and M.Yu. Nefedova* * I n s t i t u t e of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Pushchino, Moscov Region, 142292
** Rostov State University, Rostov, 344711 R/D
ABSTRACT
An ecological safety study of using vinclozolin in
field
and
laboratory
experiments
showed
that
the
effect of the preparation led to a decrease in the abundance of actinomycetes and mycelial fungi and an enhancement of nitrification. The residual amounts of vinclozolin in soil after 12 months were 6-12% of the dose introduced. The
persistent chlorinated deriva293
Copyright© 1991 by Marcel Dekker, Inc.
294
GOLOVLEVA ET AL.
tives of the toxicant were found. Microbial strains pertaining to the genera Pseudomonas and Bacillus were isolated that utilized vinclozolin as the sole source
Downloaded by [McGill University Library] at 20:48 23 April 2013
of carbon and energy.
INTRODUCTION
I n v e s t i g a t i o n of t h e behaviour of p e s t i c i d e s i n the environment, t h e i r e f f e c t s on t h e major biochemical processes and s o i l microflora, s t u d i e s of t h e i r metabolisms and pathways of conversion are of extreme importance for assessing t h e ecological s a f e t y of an extensively used preparation. The fungicide vinclozolin
(3-(3.5-dichlorophenyl)-S-methyl-S-vinyl-l,3-oxa-
zolidine-2,4-dione) i s a dicarboxyimide fungicide. I t i s used t o control the diseases of grapevine, strawberry, vegetable crops and ornamental
cultures etc.
Vinclozolin has been reported t o y i e l d t o photochemical conversion and chemical hydrolysis (Scwack, Bourgeois 1989, Szeto et al. 1989b). The metabolism of t h e preparation i n wine and pea leaves h a s been gated
( P i r i s i et al. 1986,
investi-
Szeto e t al. 1989a) . I n
s o i l v i n c l o z o l i n i s known t o be depleted r a t h e r rapidl y ; 50% of t h e p r e p a r a t i o n being degraded within 23 days. In a c i d i c s o i l s i t s conversion i s slower (Walker et al. 1986). At a repeated treatment of s o i l t h e r a t e
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
295
of depletion increases. A vinclozolin conversion product 3,5-dichloroaniline was found (Walker 1987). The aim of this work was to investigate the effect of vinclozolin on the major groups of soil microorganisms in various climatic zones, to study the poten-
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t i a l i t i e s of soil microflora to convert the fungicide, to elucidate the pathways of i t s degradation by active microbial strains.
MATERIALS AND METHODS
Vinclozolin was investigated experiments regions
and under
of Russia.
field
in laboratory model
conditions
in various
Under laboratory conditions the
amounts of fungicide introduced into soil were 5, 25, 125 and 625 mg/kg. In field experiments
the prepara-
tion was distributed surface-wise over 10 m2
plots,
the doses exceeding those commonly used 10- and 100fold (7.5 and 75 kg/ha). The samples were taken in 5 (14) days and 1, 2, 3, months after the treatment. The effect
of vinclozolin
on
soil
microorganisms
was
studied by the e a r l i e r described methods (Golovleva et al.
1984). Active
microorganisms were identified by
the
common techniques
(Buchanan
and Gibbons 1974,
Zvyagintsev et al. 1980). The a b i l i t y of microorganisms t o degrade vinclozol i n was revealed using enrichment cultures with the
296
GOLOVLEVA ET AL.
soils under study. The enrichment cultures were reinoculated and the active strains were investigated on a mineral 0.8;
medium containing
(g/1): NaCl, 0.5;
MgSO^,
(NH)2HPO4, 1.5; KH2PO4, 0.7. The amount of vin-
clozolin introduced was 100-200 mg/1; the additional
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carbon sources - glucose, sucrose, glycerol, calcium lactate - 2 g/1. Cultivation conditions were aerobic and microaerophilic. Vinclozolin was extracted from the culture broth three times with diethyl ether after i t s acidification to pH 2.O. Then the extract was concentrated by evaporating the solvent. Vinclozolin
and i t s conversion
products were qualitatively determined
by thin-layer
chromatography (TLC) on Silufol-254 plates using the mixture
of solvents benzene:ethyl
hexane:acetone
acetate 10:1 ( I ) ,
5:2 (II) and benzene:diethyl
ether:
ethyl acetate 6:4:2 ( I I I ) . The substances were visualized by UV light absorption as well as using solution of AgNO , the Ehrlich reagent or the mixture of iron trichloride and potassium hexacyanoferrate ( I I I ) . The quantitative assays were done by the methods of gas chromatography (GC) and high-performance liquid chromatography (HPLC) . GC was performed a t a Pye Unicam 304 Chromatograph (Philips) with an electron capture detector on a 1.5 m x 0.2 mm column f i l l e d with 1.5% OV-101 on Supelcoport (80-100 mesh). The tempera-
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
297
ture of the columns, injector and detector was 170, 200 and 200°C, respectively. HPLC was carried out a t a LKB Instruments Ine Bromma Chromatograph. A Spherisorb ODS 2 reversed-phase column (250 χ 4 mm) was used; the size of particles was 5 μιη; the mobile phase consisted
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of 80% methanol and 20% solution of KH PO
(0.005 M) .
The compounds were recorded a t a wavelength of 280 nm. The products of vinclozolin conversion were ident i f i e d by physico-chemical
methods. The mass spectra
were recorded a t an LKB 2091 gas chromatograph-mass spectrometer using a column packed with Chromosorb W/HP with 2% OV-101 as well as a t a Finnigan Matt 8430 mass spectrometer. IR Fourier spectra were recorded a t a Perkin Elmer 1710 spectrophotometer in CCI and KBr.
Results and Discussion
As shown by the laboratory studies, the most sens i t i v e t o vinclozolin were mycelial fungi and actinomycetes whose abundance decreased with the increase in the concentration of the fungicide. In three months the
abundance of these microbial groups
was s t i l l
lower than in the control (Fig. 1) . The preparation was shown to have no prolonged and irreversible effect on
the t o t a l
abundance of heterotrophic bacteria,
cellulose-degrading organisms, the respiratory a c t i v i -
GOLOVLEVA ET AL.
298
5 days
90 days nxlO
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ΠχΙΟ 0.4-
0.4 -
0.3 -
0.3-
0.2-
0.2-
0.1-
0.1-
5
125
I 0
625
mg
0.3-
nxlO' 0.3 -
0.2-
0.2-
0.1-
0.1-
ηχ10~
5 125 v i n c1 ./ k g
625 s o i l
Β
0
5
125
0
625
mg
days 15 ^
mg N-NO days 15-
10-
10-
mg
N-NO
5 125 v i n e l ,
/ k g
625 s o i l
5-
5-
5
125
625
0 mg
5 125 v i n e l ,
/ k g
625 s o l l
FIGURE 1
Effect of different doses of vinclozolin on the abundance of actinomycetes (A), fungi (B) and nitrification activity in soil (C)
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
299
ty of the soil, as well as on the microorganisms involved in denitrification and nitrogen fixation processes; however, vinclozolin activated the microorganisms
responsible
for
nitrification
(Fig. 1).
The
major results of the laboratory assays were confirmed
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by the field experiments. For the actinomycetes and mycelial
fungi
the
strong
inhibiting effect
was
observed immediately upon the treatment of soil with the preparation and was preserved until the end of the experiment. The toxic effect of vinclozolin was
espe-
cially pronounced after 3 months if the preparation was used at a concentration which exceeded that of the working dose 100-fold. From the enrichment cultures with soils we isolated 36 microbial strains which converted vinclozolin. Four of
these, as the most active, were used for
further studies. The microorganisms were identified as Bacillus cereus 625/1, Bacillus brevis 625/2, Pseudomonas fluorescens 10/3 and 8/28. I t was shown that the strains Bacillus cereus 625/1 and Pseudomonas fluorescens 8/28
utilize vinclozolin as the sole source of
carbon and energy by 87-90% under aerobic conditions and by 85% under microaerophilic conditions. Aadditional carbon sources
did not contribute to the more
complete conversion of the preparation by these cultures. Addition of glucose slightly increased degrada-
300
GOLOVLEVA ET AL.
tion of vinclozolin by the culture P. fluorescens
10/3
from 58 to 63% under aerobic conditions and by Bacillus brevis 625/2 from 52 to 65% under microaerophilxc conditions. Based on these data, we investigated the conver-
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sion of vinclozolin used as the sole source of carbon. When an acetone solution of vinclozolin was introduced into a mineral medium, the medium became turbid due to the weak solubity of the preparation in water; as
i t was
converted
by
microorganisms the optical
density of the medium decreased.
Therefore,
i t was
difficult to trace the culture growth nephelometrically. At the same time, due to the small concentration of the carbon source the biomass increment determined by the weight method was too small (3-5 mg in 4 days). However, the amount of vinclozolin
in the culture
broth decreased and i t s conversion products accumulated
(Table 1) . Upon separation of the culture broth
extracts by TLC
these compounds were isolated as pre-
parations to determine their structures. Table 2 gives the Chromatographie characteristics of the vinclozolin conversion products. All the compounds were visualized by AgNO solution, i.e., are chlorine-containing metabolites.
Compound 3 was
also revealed
on
Silufol
plates by the Ehrlich reagent and the mixture of iron (III) chloride and potassium hexacyanoferrate.
MICROBIAL CONVERSION
301
OF FUNGICIDE VINCLOZOLIN
TABLE 1
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Content of vinclozolin and products of i t s conversion by Pseudomonas fluorescens 8-28 after 10 days of cultivation (determined by HPLC)
Content,, % Variant
vinclozolin
aerobic conditions 10.2 microaerophilic conditions 31.5 88.6 control
compound 1
compound 2
compound 3
other compounds
38.4
42.7
3.5
5.2
24.7 6.2
27.1 4.2
8.2 0
8.5 0.9
TABLE 2 Chromatographie characteristics of vinclozolin and i t s metabolites. Compound
HPLC t
TLC ret
I
vinclozolin compound 1 compound 2 compound 3 compound 4 compound 5
II
III
6.71
0.85
0.56
0.84
5.83
0.20
0.12
0.75
5.58
0.35
0.25
0.70
4.99
0.65
0.31
0.92
3.48
0.13
0.19
0.42
0.50
0.38
0.70
-
302
GOLOVLEVA ET AL.
Identification
of
the compounds
chemical methods revealed spectrometric
their
characteristics
by
physico-
structure.
The mass
of the compounds are
given in Table 3. Compounds 1 and 2 were also investigated by IR
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spectroscopy. Analysis of the element composition of molecular ion from compound 1 and i t s low intensity may point to the opening of the oxazolidine ring as a r e s u l t of i t s hydration. The
difference Fourier
against vinclozolin spectra
3540
1
cm"
in CCL
IR spectra of compound 1 contained the absorbance
(carboxylic
hydroxyl),
3440 cm"
(-NH of the secondary amide), 1752 cm"1 (amide-l-N-COO-urethane structure), 1722 cm"1 (carboxylic CO), 1590 cm"1 (-C=C-), 1528"1 (amide 11), 1415, 1235, 1107 cm"1 (characteristic
absorbance
bands
of the carboxylic
group). Compound 1 was treated with diazomethane, as a result of which R
of the methylated compound changed
in solvent mixture I I from 0.12 t o 0.70. Thus, compound 1 can be presented as the product of opening the five-membered
vinclozolin
ring
by
the C-N
bond:
2[(3,5-dichlorophenyl)carbamoyl]oxy]2-methyl]-3-butenoic
acid.
In storage,
this
compound
i s partially
converted t o vinclozolin. The
spectrum of compound 2 featured an intensive
fragment with m/z 71 that can be formed as a r e s u l t of
o
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TABLE 3 Mass spectrometric
data of vinclozolin and i t s metabolites
Compound
Ion
m/z measured calculated
Vinclosolin
285
284.9949
284.9960
303
303.0065 186.9591
C
187
303. 0054 186.9578
161
160. 9778
160.9800
C 6 H 5 NC1 2
99
9 9 . 0436
99.0446
C
5H7°2
259
259. 0170
259.0167
C
11H11NO2C12
71
7 1 . 0499
71.0497
C
4H7°
C
6H5NC12
161
-
-
Composition
Major intensive peaks in mass spectrum m/z (%) M 285(55),287(37),241(13),243(8),212(55),213(55), 214(53),215(37),198(50),200(13),187(67), 189(42),178(42),180(13),124(50)
12H11N°4C12 C 7 H 3 NOC1 2
Μ+303(3),305(2),285(4),287(2),187(72),189(46), 161(100),163(63),124(37),126(22),99(32),71(56)
S < M PO
en tH O S3 O •η
O O M
Μ+259(12),261(8),217(12),219(8),191(12),161(48), 163(32),71(100),72(45),43(45) Μ+161(100),163(65),126(13),128(4),99(22),101(8),
2! O f O Ν O f
90(21),63(20) 203
348
-
347.9422
-
347.9391
C H NOCÍ 8 7 2
Μ+203(18),205(12),161(100),165(66),167(10),
C
Μ 348(11),350(14),352(7),187(21),189(14),161(100),
Η Ν 0C1 13 8 2 4
63(15) , 4 3 ( 8 0 )
163(67),165(10),124(12),126(14),128(5),99(21), 101(7) ,90(21) οι Ο Οι
304
G0L0VLEVA ET AL.
the carbon bond break in the carboxylic group of the opened oxazolidine ring which supports the element composition of the above fragment. I t s IR spectrum contains the following absorbance bands:
3615 cm"1,
t e r t i a r y hydroxyl; 3402 cm"1, NH; 1675 cm"1, amide I
Downloaded by [McGill University Library] at 20:48 23 April 2013
of the secondary amide; 1527 cm" , amide I I of the secondary amide; 1585 cm"1, the aromatic ring; 3104 cm"1, vinyl =CH . Thus, product 2 also i s a compound with the oxazolidine ring opened a t another C-N bond (in contrast to compound 1) and the C=0 group cleaved off: 3,5-dichloro-2-hydroxy-2-methylbut-3enanilide. The mass spectrum of compound 3 i s similar t o that of
3,5-dichloroaniline which was confirmed
by the
coincidence of the Chromatographie characteristics and the character of revealing this compound and that of the standard. The mass spectrum of compound 4 was observed t o be intensively broken off molecular ion of the 43 a.m.u. fragment producing the stable ion of dichloroaniline, which i s characteristic of i t s N-aceto derivative. This compound was synthesized (Gale et al. 1985). The Chromatographie mobility of the compound investigated and the one obtained was the same in the three systems: in I , 0.13; in I I , 0.19; in I I I , 0.42. Comparing the relative intensities of peaks of the chlorine isotopic ions in molecular ion of compound 5
305
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
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H
CH3
fj-N-C-O-C-COOH Cr 0 CH=CH2
C-C-OH η I
0 CH=CH2
FIGURE 2
Products of vinclozolin conversion by Pseudomonas fluorescens 8/28
and based on i t s element composition (Table 3), one can assert that this compound i s formed from two molecules of dichloroacetanilide or a molecule of dichloroaniline and a molecule of dichloroacetanilide. The scheme of vinclozolin conversion by P. fluorescens 8/28, based on these data, i s presented in Fig. 2. I t i s known that vinclozolin, especially in alkaline solutions, i s unstable and i s hydrolyzed forming
306
G0LOVLEVA ET AL.
products with the opened oxazolidine
ring - metabo-
l i t e s 1 and 2 (Szeto et al. 1989). Table 1 shows that the major products of vinclozolin conversion in the culture broth
are compounds 1 and 2; however, the
amounts of these products point to their more inten-
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sive formation under the action of p. fluorescens.
The
same compounds were also found when other strains (P. fluorescens
10/3, Bac. cereus 625/1 and Bac. brevis
625/2 were grown in the medium with vinclozolin as a sole source of carbon. Compounds 1 and 2 were found in the leaves of beans and pea if during their growth the nutrient solution contained vinclozolin; and compound 2, in wine (Szeto et al. 1989a, Pirisi et al. 1986). The works on vinclozolin-degradation
in soil investigated
only the depletion of the preparation, the metabolite revealed
being 3,5-dichloroaniline.
the behaviour of vinclozolin
Investigation of
in soil and in model
experiments showed that the residual amounts of vinclozolin after 12 months were 6-17% depending on the i n i t i a l dose. Compounds 1, 2 and 5 were ed. Dichloroaniline
also detect-
and chlorinated acetanilide were
revealed in earlier times. Dichloroaniline
i s not an
ecologically safe metabolite. I t i s preserved in soil for a long time, i s able to be sorbed by soil components. Besides i t i s established to be acetylated in
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
307
soil as well as to form condensed compound 5 which was found in soil after a year. Thus, i t was shown that the treatment of soil with vinclozolin slightly decreases the stability of microbiocenoses due to i t s effect on the abundance of fungi, actinomycetes and on the nitrification activity. Downloaded by [McGill University Library] at 20:48 23 April 2013
Vinclozolin
was
not totally degraded in experiments
with enrichment and pure microbial cultures. I t was
metabolized forming chlorinated products
which were
stable in the environment. Therefore, the preparation
can not be attributed to ecologically safe fungicides. Its use in agriculture should be strictly controlled. References
Buchanan R.E., Gibbons N.C. Bergey's Manual of Determinative Bacetriology, 8th ed., The Williams & Wilkis Co., Baltimore (1974). Gale G.T., Hofberg A.M., J . Assoc. Anal. Chem. 68 (3) 570-572 (1985). P i r i s i F.M., Meloni M., Cabras P., Bionducci M.R., Serra Α., P e s t i c . S c i . 17:109-118 (1986). Schwack V., Bourgeois B. Z. Lebensmrnters Forsch., 188:346-347 (1989). Szeto S.Y., Burlinson N.E., Rahe J.E., Oloffs P.C., J. Agric. Food Chem. 37:529-534 (1989a). Szeto S.Y., Burlinson N.E., R e t t i g S.J., T r o t t e r J . , J . Agric. Food Chem. 37:1103-1108 (1989b). Thin-Layer Chromatography, ed. by Stahl E. Moscow, Mir, 476-498 (1965) ( t r a n s l a t e d from German). Walker Α., Brown P.A., Entwistle A.R., P e s t i c . S c i . 17:183-193 (1986). Walker A. P e s t i c . Sci. 21:233-240 (1986). Received: March 5, 1991
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Microbial conversion of fungicide vinclozolin a
a
L.A. Golovleva , Z.I. Finkelstein , A.V. b
a
Polyakova , B.P. Baskunov & M.Yu. Nefedova
a
a
Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Pushchino, Moscov Region, 142292 b
Rostov State University, R/D, Rostov, 344711 Version of record first published: 21 Nov 2008.
To cite this article: L.A. Golovleva , Z.I. Finkelstein , A.V. Polyakova , B.P. Baskunov & M.Yu. Nefedova (1991): Microbial conversion of fungicide vinclozolin, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 26:3, 293-307 To link to this article: http://dx.doi.org/10.1080/03601239109372736
PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/ terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
Downloaded by [McGill University Library] at 20:48 23 April 2013
The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.
J. ENVIRON. SCI. HEALTH, B26(3), 293-307 (1991)
Downloaded by [McGill University Library] at 20:48 23 April 2013
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
Key words: vinclozolin, soil microorganisms, bioconversion, environmental safety
L.A. Golovleva*, Z.I. F i n k e l s t e i n * , A.V. Polyakova**, B.P. Baskunov* and M.Yu. Nefedova* * I n s t i t u t e of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Pushchino, Moscov Region, 142292
** Rostov State University, Rostov, 344711 R/D
ABSTRACT
An ecological safety study of using vinclozolin in
field
and
laboratory
experiments
showed
that
the
effect of the preparation led to a decrease in the abundance of actinomycetes and mycelial fungi and an enhancement of nitrification. The residual amounts of vinclozolin in soil after 12 months were 6-12% of the dose introduced. The
persistent chlorinated deriva293
Copyright© 1991 by Marcel Dekker, Inc.
294
GOLOVLEVA ET AL.
tives of the toxicant were found. Microbial strains pertaining to the genera Pseudomonas and Bacillus were isolated that utilized vinclozolin as the sole source
Downloaded by [McGill University Library] at 20:48 23 April 2013
of carbon and energy.
INTRODUCTION
I n v e s t i g a t i o n of t h e behaviour of p e s t i c i d e s i n the environment, t h e i r e f f e c t s on t h e major biochemical processes and s o i l microflora, s t u d i e s of t h e i r metabolisms and pathways of conversion are of extreme importance for assessing t h e ecological s a f e t y of an extensively used preparation. The fungicide vinclozolin
(3-(3.5-dichlorophenyl)-S-methyl-S-vinyl-l,3-oxa-
zolidine-2,4-dione) i s a dicarboxyimide fungicide. I t i s used t o control the diseases of grapevine, strawberry, vegetable crops and ornamental
cultures etc.
Vinclozolin has been reported t o y i e l d t o photochemical conversion and chemical hydrolysis (Scwack, Bourgeois 1989, Szeto et al. 1989b). The metabolism of t h e preparation i n wine and pea leaves h a s been gated
( P i r i s i et al. 1986,
investi-
Szeto e t al. 1989a) . I n
s o i l v i n c l o z o l i n i s known t o be depleted r a t h e r rapidl y ; 50% of t h e p r e p a r a t i o n being degraded within 23 days. In a c i d i c s o i l s i t s conversion i s slower (Walker et al. 1986). At a repeated treatment of s o i l t h e r a t e
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
295
of depletion increases. A vinclozolin conversion product 3,5-dichloroaniline was found (Walker 1987). The aim of this work was to investigate the effect of vinclozolin on the major groups of soil microorganisms in various climatic zones, to study the poten-
Downloaded by [McGill University Library] at 20:48 23 April 2013
t i a l i t i e s of soil microflora to convert the fungicide, to elucidate the pathways of i t s degradation by active microbial strains.
MATERIALS AND METHODS
Vinclozolin was investigated experiments regions
and under
of Russia.
field
in laboratory model
conditions
in various
Under laboratory conditions the
amounts of fungicide introduced into soil were 5, 25, 125 and 625 mg/kg. In field experiments
the prepara-
tion was distributed surface-wise over 10 m2
plots,
the doses exceeding those commonly used 10- and 100fold (7.5 and 75 kg/ha). The samples were taken in 5 (14) days and 1, 2, 3, months after the treatment. The effect
of vinclozolin
on
soil
microorganisms
was
studied by the e a r l i e r described methods (Golovleva et al.
1984). Active
microorganisms were identified by
the
common techniques
(Buchanan
and Gibbons 1974,
Zvyagintsev et al. 1980). The a b i l i t y of microorganisms t o degrade vinclozol i n was revealed using enrichment cultures with the
296
GOLOVLEVA ET AL.
soils under study. The enrichment cultures were reinoculated and the active strains were investigated on a mineral 0.8;
medium containing
(g/1): NaCl, 0.5;
MgSO^,
(NH)2HPO4, 1.5; KH2PO4, 0.7. The amount of vin-
clozolin introduced was 100-200 mg/1; the additional
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carbon sources - glucose, sucrose, glycerol, calcium lactate - 2 g/1. Cultivation conditions were aerobic and microaerophilic. Vinclozolin was extracted from the culture broth three times with diethyl ether after i t s acidification to pH 2.O. Then the extract was concentrated by evaporating the solvent. Vinclozolin
and i t s conversion
products were qualitatively determined
by thin-layer
chromatography (TLC) on Silufol-254 plates using the mixture
of solvents benzene:ethyl
hexane:acetone
acetate 10:1 ( I ) ,
5:2 (II) and benzene:diethyl
ether:
ethyl acetate 6:4:2 ( I I I ) . The substances were visualized by UV light absorption as well as using solution of AgNO , the Ehrlich reagent or the mixture of iron trichloride and potassium hexacyanoferrate ( I I I ) . The quantitative assays were done by the methods of gas chromatography (GC) and high-performance liquid chromatography (HPLC) . GC was performed a t a Pye Unicam 304 Chromatograph (Philips) with an electron capture detector on a 1.5 m x 0.2 mm column f i l l e d with 1.5% OV-101 on Supelcoport (80-100 mesh). The tempera-
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
297
ture of the columns, injector and detector was 170, 200 and 200°C, respectively. HPLC was carried out a t a LKB Instruments Ine Bromma Chromatograph. A Spherisorb ODS 2 reversed-phase column (250 χ 4 mm) was used; the size of particles was 5 μιη; the mobile phase consisted
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of 80% methanol and 20% solution of KH PO
(0.005 M) .
The compounds were recorded a t a wavelength of 280 nm. The products of vinclozolin conversion were ident i f i e d by physico-chemical
methods. The mass spectra
were recorded a t an LKB 2091 gas chromatograph-mass spectrometer using a column packed with Chromosorb W/HP with 2% OV-101 as well as a t a Finnigan Matt 8430 mass spectrometer. IR Fourier spectra were recorded a t a Perkin Elmer 1710 spectrophotometer in CCI and KBr.
Results and Discussion
As shown by the laboratory studies, the most sens i t i v e t o vinclozolin were mycelial fungi and actinomycetes whose abundance decreased with the increase in the concentration of the fungicide. In three months the
abundance of these microbial groups
was s t i l l
lower than in the control (Fig. 1) . The preparation was shown to have no prolonged and irreversible effect on
the t o t a l
abundance of heterotrophic bacteria,
cellulose-degrading organisms, the respiratory a c t i v i -
GOLOVLEVA ET AL.
298
5 days
90 days nxlO
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ΠχΙΟ 0.4-
0.4 -
0.3 -
0.3-
0.2-
0.2-
0.1-
0.1-
5
125
I 0
625
mg
0.3-
nxlO' 0.3 -
0.2-
0.2-
0.1-
0.1-
ηχ10~
5 125 v i n c1 ./ k g
625 s o i l
Β
0
5
125
0
625
mg
days 15 ^
mg N-NO days 15-
10-
10-
mg
N-NO
5 125 v i n e l ,
/ k g
625 s o i l
5-
5-
5
125
625
0 mg
5 125 v i n e l ,
/ k g
625 s o l l
FIGURE 1
Effect of different doses of vinclozolin on the abundance of actinomycetes (A), fungi (B) and nitrification activity in soil (C)
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
299
ty of the soil, as well as on the microorganisms involved in denitrification and nitrogen fixation processes; however, vinclozolin activated the microorganisms
responsible
for
nitrification
(Fig. 1).
The
major results of the laboratory assays were confirmed
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by the field experiments. For the actinomycetes and mycelial
fungi
the
strong
inhibiting effect
was
observed immediately upon the treatment of soil with the preparation and was preserved until the end of the experiment. The toxic effect of vinclozolin was
espe-
cially pronounced after 3 months if the preparation was used at a concentration which exceeded that of the working dose 100-fold. From the enrichment cultures with soils we isolated 36 microbial strains which converted vinclozolin. Four of
these, as the most active, were used for
further studies. The microorganisms were identified as Bacillus cereus 625/1, Bacillus brevis 625/2, Pseudomonas fluorescens 10/3 and 8/28. I t was shown that the strains Bacillus cereus 625/1 and Pseudomonas fluorescens 8/28
utilize vinclozolin as the sole source of
carbon and energy by 87-90% under aerobic conditions and by 85% under microaerophilic conditions. Aadditional carbon sources
did not contribute to the more
complete conversion of the preparation by these cultures. Addition of glucose slightly increased degrada-
300
GOLOVLEVA ET AL.
tion of vinclozolin by the culture P. fluorescens
10/3
from 58 to 63% under aerobic conditions and by Bacillus brevis 625/2 from 52 to 65% under microaerophilxc conditions. Based on these data, we investigated the conver-
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sion of vinclozolin used as the sole source of carbon. When an acetone solution of vinclozolin was introduced into a mineral medium, the medium became turbid due to the weak solubity of the preparation in water; as
i t was
converted
by
microorganisms the optical
density of the medium decreased.
Therefore,
i t was
difficult to trace the culture growth nephelometrically. At the same time, due to the small concentration of the carbon source the biomass increment determined by the weight method was too small (3-5 mg in 4 days). However, the amount of vinclozolin
in the culture
broth decreased and i t s conversion products accumulated
(Table 1) . Upon separation of the culture broth
extracts by TLC
these compounds were isolated as pre-
parations to determine their structures. Table 2 gives the Chromatographie characteristics of the vinclozolin conversion products. All the compounds were visualized by AgNO solution, i.e., are chlorine-containing metabolites.
Compound 3 was
also revealed
on
Silufol
plates by the Ehrlich reagent and the mixture of iron (III) chloride and potassium hexacyanoferrate.
MICROBIAL CONVERSION
301
OF FUNGICIDE VINCLOZOLIN
TABLE 1
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Content of vinclozolin and products of i t s conversion by Pseudomonas fluorescens 8-28 after 10 days of cultivation (determined by HPLC)
Content,, % Variant
vinclozolin
aerobic conditions 10.2 microaerophilic conditions 31.5 88.6 control
compound 1
compound 2
compound 3
other compounds
38.4
42.7
3.5
5.2
24.7 6.2
27.1 4.2
8.2 0
8.5 0.9
TABLE 2 Chromatographie characteristics of vinclozolin and i t s metabolites. Compound
HPLC t
TLC ret
I
vinclozolin compound 1 compound 2 compound 3 compound 4 compound 5
II
III
6.71
0.85
0.56
0.84
5.83
0.20
0.12
0.75
5.58
0.35
0.25
0.70
4.99
0.65
0.31
0.92
3.48
0.13
0.19
0.42
0.50
0.38
0.70
-
302
GOLOVLEVA ET AL.
Identification
of
the compounds
chemical methods revealed spectrometric
their
characteristics
by
physico-
structure.
The mass
of the compounds are
given in Table 3. Compounds 1 and 2 were also investigated by IR
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spectroscopy. Analysis of the element composition of molecular ion from compound 1 and i t s low intensity may point to the opening of the oxazolidine ring as a r e s u l t of i t s hydration. The
difference Fourier
against vinclozolin spectra
3540
1
cm"
in CCL
IR spectra of compound 1 contained the absorbance
(carboxylic
hydroxyl),
3440 cm"
(-NH of the secondary amide), 1752 cm"1 (amide-l-N-COO-urethane structure), 1722 cm"1 (carboxylic CO), 1590 cm"1 (-C=C-), 1528"1 (amide 11), 1415, 1235, 1107 cm"1 (characteristic
absorbance
bands
of the carboxylic
group). Compound 1 was treated with diazomethane, as a result of which R
of the methylated compound changed
in solvent mixture I I from 0.12 t o 0.70. Thus, compound 1 can be presented as the product of opening the five-membered
vinclozolin
ring
by
the C-N
bond:
2[(3,5-dichlorophenyl)carbamoyl]oxy]2-methyl]-3-butenoic
acid.
In storage,
this
compound
i s partially
converted t o vinclozolin. The
spectrum of compound 2 featured an intensive
fragment with m/z 71 that can be formed as a r e s u l t of
o
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TABLE 3 Mass spectrometric
data of vinclozolin and i t s metabolites
Compound
Ion
m/z measured calculated
Vinclosolin
285
284.9949
284.9960
303
303.0065 186.9591
C
187
303. 0054 186.9578
161
160. 9778
160.9800
C 6 H 5 NC1 2
99
9 9 . 0436
99.0446
C
5H7°2
259
259. 0170
259.0167
C
11H11NO2C12
71
7 1 . 0499
71.0497
C
4H7°
C
6H5NC12
161
-
-
Composition
Major intensive peaks in mass spectrum m/z (%) M 285(55),287(37),241(13),243(8),212(55),213(55), 214(53),215(37),198(50),200(13),187(67), 189(42),178(42),180(13),124(50)
12H11N°4C12 C 7 H 3 NOC1 2
Μ+303(3),305(2),285(4),287(2),187(72),189(46), 161(100),163(63),124(37),126(22),99(32),71(56)
S < M PO
en tH O S3 O •η
O O M
Μ+259(12),261(8),217(12),219(8),191(12),161(48), 163(32),71(100),72(45),43(45) Μ+161(100),163(65),126(13),128(4),99(22),101(8),
2! O f O Ν O f
90(21),63(20) 203
348
-
347.9422
-
347.9391
C H NOCÍ 8 7 2
Μ+203(18),205(12),161(100),165(66),167(10),
C
Μ 348(11),350(14),352(7),187(21),189(14),161(100),
Η Ν 0C1 13 8 2 4
63(15) , 4 3 ( 8 0 )
163(67),165(10),124(12),126(14),128(5),99(21), 101(7) ,90(21) οι Ο Οι
304
G0L0VLEVA ET AL.
the carbon bond break in the carboxylic group of the opened oxazolidine ring which supports the element composition of the above fragment. I t s IR spectrum contains the following absorbance bands:
3615 cm"1,
t e r t i a r y hydroxyl; 3402 cm"1, NH; 1675 cm"1, amide I
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of the secondary amide; 1527 cm" , amide I I of the secondary amide; 1585 cm"1, the aromatic ring; 3104 cm"1, vinyl =CH . Thus, product 2 also i s a compound with the oxazolidine ring opened a t another C-N bond (in contrast to compound 1) and the C=0 group cleaved off: 3,5-dichloro-2-hydroxy-2-methylbut-3enanilide. The mass spectrum of compound 3 i s similar t o that of
3,5-dichloroaniline which was confirmed
by the
coincidence of the Chromatographie characteristics and the character of revealing this compound and that of the standard. The mass spectrum of compound 4 was observed t o be intensively broken off molecular ion of the 43 a.m.u. fragment producing the stable ion of dichloroaniline, which i s characteristic of i t s N-aceto derivative. This compound was synthesized (Gale et al. 1985). The Chromatographie mobility of the compound investigated and the one obtained was the same in the three systems: in I , 0.13; in I I , 0.19; in I I I , 0.42. Comparing the relative intensities of peaks of the chlorine isotopic ions in molecular ion of compound 5
305
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
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H
CH3
fj-N-C-O-C-COOH Cr 0 CH=CH2
C-C-OH η I
0 CH=CH2
FIGURE 2
Products of vinclozolin conversion by Pseudomonas fluorescens 8/28
and based on i t s element composition (Table 3), one can assert that this compound i s formed from two molecules of dichloroacetanilide or a molecule of dichloroaniline and a molecule of dichloroacetanilide. The scheme of vinclozolin conversion by P. fluorescens 8/28, based on these data, i s presented in Fig. 2. I t i s known that vinclozolin, especially in alkaline solutions, i s unstable and i s hydrolyzed forming
306
G0LOVLEVA ET AL.
products with the opened oxazolidine
ring - metabo-
l i t e s 1 and 2 (Szeto et al. 1989). Table 1 shows that the major products of vinclozolin conversion in the culture broth
are compounds 1 and 2; however, the
amounts of these products point to their more inten-
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sive formation under the action of p. fluorescens.
The
same compounds were also found when other strains (P. fluorescens
10/3, Bac. cereus 625/1 and Bac. brevis
625/2 were grown in the medium with vinclozolin as a sole source of carbon. Compounds 1 and 2 were found in the leaves of beans and pea if during their growth the nutrient solution contained vinclozolin; and compound 2, in wine (Szeto et al. 1989a, Pirisi et al. 1986). The works on vinclozolin-degradation
in soil investigated
only the depletion of the preparation, the metabolite revealed
being 3,5-dichloroaniline.
the behaviour of vinclozolin
Investigation of
in soil and in model
experiments showed that the residual amounts of vinclozolin after 12 months were 6-17% depending on the i n i t i a l dose. Compounds 1, 2 and 5 were ed. Dichloroaniline
also detect-
and chlorinated acetanilide were
revealed in earlier times. Dichloroaniline
i s not an
ecologically safe metabolite. I t i s preserved in soil for a long time, i s able to be sorbed by soil components. Besides i t i s established to be acetylated in
MICROBIAL CONVERSION OF FUNGICIDE VINCLOZOLIN
307
soil as well as to form condensed compound 5 which was found in soil after a year. Thus, i t was shown that the treatment of soil with vinclozolin slightly decreases the stability of microbiocenoses due to i t s effect on the abundance of fungi, actinomycetes and on the nitrification activity. Downloaded by [McGill University Library] at 20:48 23 April 2013
Vinclozolin
was
not totally degraded in experiments
with enrichment and pure microbial cultures. I t was
metabolized forming chlorinated products
which were
stable in the environment. Therefore, the preparation
can not be attributed to ecologically safe fungicides. Its use in agriculture should be strictly controlled. References
Buchanan R.E., Gibbons N.C. Bergey's Manual of Determinative Bacetriology, 8th ed., The Williams & Wilkis Co., Baltimore (1974). Gale G.T., Hofberg A.M., J . Assoc. Anal. Chem. 68 (3) 570-572 (1985). P i r i s i F.M., Meloni M., Cabras P., Bionducci M.R., Serra Α., P e s t i c . S c i . 17:109-118 (1986). Schwack V., Bourgeois B. Z. Lebensmrnters Forsch., 188:346-347 (1989). Szeto S.Y., Burlinson N.E., Rahe J.E., Oloffs P.C., J. Agric. Food Chem. 37:529-534 (1989a). Szeto S.Y., Burlinson N.E., R e t t i g S.J., T r o t t e r J . , J . Agric. Food Chem. 37:1103-1108 (1989b). Thin-Layer Chromatography, ed. by Stahl E. Moscow, Mir, 476-498 (1965) ( t r a n s l a t e d from German). Walker Α., Brown P.A., Entwistle A.R., P e s t i c . S c i . 17:183-193 (1986). Walker A. P e s t i c . Sci. 21:233-240 (1986). Received: March 5, 1991
