Degradation of Carbofuran by Soil Microorganisms by I. H.
WILLIAMS,H.
S.
PEPIN,and
M. J. BRowN
Research Station, Agriculture Canada Vancouver, B. C. V6T 1X2
Carbofuran (2,2 dibydro-2, 2-dimethyl-7-benzofuranyl methylcarbamate) is a useful soil insecticide effective against a broad range of soil inhabiting pests (APPLE et al. 1969; BERRY 1971; TAPPFN 1966). Normally it has ~--re-'ffsonably long half-]ire of up to 50 weeks in neutral or acid soils (GETZIN 1973). In alkaline soils hydrolysis occurs at the carbamate linkage causing rapid degradation, probably to the phenol. Recently when this insecticide was used for the control of phylloxera in a vineyard planted in gravelly loam of pH 6.0, it proved less effective tbaD was anticipated. Analysis of the soil for carbofuran also showed a lower concentration than would have been expected from the rate of application used. On fortifying an untreated sample of this particular soil with a known amount of carbofuran and analysing at intervals over a period of several weeks recovery was found to decrease with time. A test for possible irreversible adsorption by the clay content of the soil proved negative and this led to the logical alternative of possible microbial degradation. Some evidence of this had been reported previously (GETZIN 1973). This paper describes experiments which confirm this hypothesis and identify the more active organisms involved.
FXPERIMENTAL Measurement of microbial degradation: The effect of microbial degradation was measured by comparing the rate of release of I~CO2 from sterile and non-sterile samples of the same soil treated with 1~C~carbonyl labelled carbofuran. The r a d i o l a b e l l e d material was supplied by F.M.C., Middleport, N.Y. Label purity of greater than 99.7% was determined by TLC. Activity was 3..54 mCi/m mol. A standard solution containing 1 ~Ci/ml in acetone was prepared from this material. U n t r e a t e d vineyard
244
Bulletin of Environmental Contamination &Toxieology, Vol. 15, No. 2 9 1976 by Springer-Verlag New York Inc.
soil containing 12% moisture was screened through a 2 mm mesh sieve and 50 g aliouots of this were transferred to two 8 oz. screw-cap jars. One was a u t o c l a v e d for two 1 hr. periods on successive days. An eouivalent of 2 ppm of 14C-labelled carbofuran was added to each sample from the standard solution. Jars were tumbled over night in a Fisher-Kendall mixer. The biometric apparatus used in the experiment consisted of two 300 mm x 20 mm ID c h r o m a t o g r a p h i c columns equipped with stopcocks and ~ 24/40 female joints at the top. Fitted to these were adapters joined to two absorption tubes connected in series. The latter consisted of 20 ml scintillation vials containing 6 ml of 0.5N sodium hydroxide solution. The columns were sterilized and the fortified soil samples transferred to them under aseptic conditions. Air supplied by an a q u a r i u m pump was m o i s t u r i z e d and freed of atmospheric C02 by passage through a scrubber containing 0.5N sodium hydroxide. This air was allowed to percolate slowly upward through the columns, its flow being controlled by a by-pass valve on the scrubber and by the individual column stopcocks. Samples containing any evolved 14CO2 were collected at weekly intervals over a period of eight weeks. After removing the absorption tubes i0 ml of phase combining solution (PCS A m e r s h a m / Searle) was added to each and the absorbed 14C counted in a scintillation spectrometer (Picker Nuclear Liquimat Model 650). After eight weeks the soil was removed from the columns and analysed for total I~C by ignitinr 1 g samples in an induction furnace, collecting the Ir evolved and m e a s u r i n g it by scintillation counting. All counting was corrected for background and quenching. Isolation of soil microorganisms: Organisms used in the study were isolated from samples of furadan-treated soil from Oliver, B.C. One gram of each sample was placed in i00 ml of sterile water in a 250 ml erlenmeyer flask and shaken 30 minutes on a rotary shaker. A dilution series, giving a final dilution of i:i,000,000 was set up, 1 ml of the final dilution being pipetted and swirled on the surface of each of i0 water agar (WA) plates. Some isolations were made directly from soil crumbs placed on potato dextrose agar (PDA) plates. All WA plates and half the PDA plates were incubated at room temperature. The rest of the PDA plates were incubated at 3~C. All developing colonies were transferred to PDA for purification. Stock cultures were stored on oatmeal agar slants. Evaluation of individual m i c r o o r g a n i s m s in degra~in~ carbofuran: Pure cultures of eight different mlcro-
245
o r g a n i s m s w e r e studied. E x a c t l y 0.1 ml of s t a n d a r d 1 ~ C - c a r b o f u r a n s o l u t i o n was a d d e d to each of a series of s t e r i l i z e d W a r b u r g flasks and the s o l v e n t e v a p o rated under aseptic conditions. To each was a d d e d 5 ml of one of the c u l t u r e s to be studied. A slow s t r e a m of m o i s t u r i z e d , C O 2 - f r e e air g e n e r a t e d as p r e v i o u s l y d e s c r i b e d was p a s s e d over each c u l t u r e and any r e l e a s e d 14CO2 was a b s o r b e d in 0.5N s o d i u m h y d r o x i d e as in the p r e v i o u s e x p e r i m e n t and c o u n t e d in the same manner. The a c t i v i t y of each c u l t u r e was a s s e s s e d on the b a s i s of the a m o u n t of CO2 r e l e a s e d d u r i n g a three day p e r i o d as r e l a t e d to the d r i e d w e i g h t of the p a r t i c u l a r o r g a n i s m involved. E f f e c t of t e m p e r a t u r e on the d e g r a d a t i o n of soil~ p p l i e d c a r b o f u r a n a n d its 3 - h y d r o x y m e t a b o l i t e : S a m p l e s of two types of soil from the area b e i n g i n v e s t i g a t e d w e r e f o r t i f i e d at the 1 p p m level w i t h c a r b o furan and 3 - h y d r o x y c a r b o f u r a n . After thorough mixing of the s a m p l e s 20 g a l i q u o t s w e r e r e m o v e d and a n a l y s e d . The r e m a i n d e r of one set was m a i n t a i n e d at 20~ while the o t h e r set was i m m e d i a t e l y t r a n s f e r r e d to the cold r o o m and m a i n t a i n e d at -16-C. At p e r i o d s of 4, 8, and 12 w e e k s f u r t h e r a l i g u o t s w e r e r e m o v e d from b o t h sets and a n a l y s e d .
P2SULTS AND DISCUSSION In Fig. 1 r e l a t i v e rates of r e l e a s e of I~CO2 from s t e r i l e and n o n - s t e r i l e soil are p l o t t e d . F r o m this it is e v i d e n t that under the c o n d i t i o n s of the e x p e r i m e n t the rate of d e g r a d a t i o n of c a r b o f u r a n d u r i n g the s e c o n d w e e k was forty times g r e a t e r in the n o n - s t e r i l e soil t h a n in the s t e r i l e soil. A n a l y s i s for total 14C in the soil a f t e r e i g h t w e e k s i n c u b a t i o n s h o w e d that the a m o u n t of c a r b o f u r a n r e m a i n i n g in the nons t e r i l e soil had d e c r e a s e d to less than 60% of the amount originally present. In the s t e r i l e soil no m e a s u r a b l e loss had o c c u r r e d . Fig. 2 i l l u s t r a t e s the r e l a t i v e a c t i v i t y of v a r i o u s m i c r o o r g a n i s m s in d e g r a d i n g c a r b o f u r a n . H e r e it is seen that some of the a c t i n o m y c e t e s are p a r t i c u l a r l y active. An a t t e m p t is b e i n g m a d e to h a v e t h e s e i d e n t i f i e d t h o u g h this is p r o v i n g d i f f i c u l t .
246
z z~e
~oo
~.~ STIE RILs
SOIL (SCALE
O i l F~:RENCE
X~lOI
W~EKS
F i g u r e 1. Comparison of rates of degradation of s t e r i l e and n o n - s t e r i l e soil f o r t i f i e d with ~ C labelled carbofuran.
20 ,8 1
iiiiiiiiiii iiiiiiiiiii .iiiiiiiiiiii . . . . . iiiiiiiiiii iiiii?i iiiiiiiiiiiiiiiiiiiiiii ii~iiiiiii
1
iiiiiiiiiiil iiiiiiiiiii
Figure
2.
microorganisms
~, 0
iiiiiiiiiiii iiiiiiiiiii
~
~iiiii::i:i~ iiiiiiiiiii iiii!iiiiiii iiiiiiiiiil
~zo
i
Relative in
i
!!!iii!!i. . . . . . iiiiii!!!ii
effectiveness
degrading
of a number
carbofuran
over
a
of three
day period. (a) a c t i n o m y c e t e , (b) penicillium, (c) (d) (e) (f) a c t i n o m y c e t e s , (g) p e n i c i l l i u m , (h) unidentified.
247
~O
O0
12
Storage period (weeks)
I
*
91.9
8].5
34.3
2
-
2
7.8
ND
24.4
1
1
ND
58.3
** none d e t e c t e d
ND
ND
ND
ND
50.5
2
Westbank
90.7
82.8
ND**
3-OH C a r b o f u r a n
at 20~
63.4
63.3
72.5
57.1
76.8
75.0
81.5
82.8
76.6
86.0
53.5
82.7
67.6
83.0
91.9
90.7
3-OH C a r b o f u r a n
at -16~
present)
Storage
conc.
Carbofuran
(% of o r i g i n a l
Carbofuran
Storage
Recovery
1
2 - Oliver
1
Soil No.*
E f f e c t of T e m p e r a t u r e on the D e g r a d a t i o n of C a r b o f u r a n and 3 - H y d r o x y c a r b o f u r a n in Two O k a n a g a n Soils
TABLE
Table I shows the effect of temperature on the rate of degradation of carbofuran and 3 - h y d r o x y c a r b o f u r a n in soil. It is obvious from this that serious losses can occur between the time of sampling the soil and its eventual analysis if it is not kept at sub zero temperatures during this period. Of particular interest is the very rapid disappearance of 3-hydroxycarbofuran. W h e t h e r the same m i c r o o r g a n i s m s responsible for the degradation of carbofuran are also responsible for the disappearance of this m e t a b o l i t e has yet to be determined. F r o m this study it is evident that in a soil containing high levels of actinomycetes rapid degradation of carbofuran may be expected. This should be borne in mind not only in storing samples but also in assessing the efficacy of this insecticide in controlling soil insects. What in some cases may appear to be possible build-up of resistance by the insect for which control is desired may in fact be a lowering of the insecticidal concentration to an ineffective level by microbial degradation. Further studies will be carried out to determine w h e t h e r other carbamates are degrade4 in similar manner by the same organisms.
REFERENCES APPLE,
J.W., WALGENBACH, E.T., J. Econ. Entomol. 62,
and KNEE, W.J.: 1033 (1969).
BERRY,
R.E.:
64,
J. Econ.
Entomol.
GETZIN,
L.W.:
Environ.
Entomol.
[,
TAPPEN,
W.B.:
J. Econ.
Entomol.
59,
249
1325 461
(1971). (1973).
1161
(1966).
WILLIAMS,H.
S.
PEPIN,and
M. J. BRowN
Research Station, Agriculture Canada Vancouver, B. C. V6T 1X2
Carbofuran (2,2 dibydro-2, 2-dimethyl-7-benzofuranyl methylcarbamate) is a useful soil insecticide effective against a broad range of soil inhabiting pests (APPLE et al. 1969; BERRY 1971; TAPPFN 1966). Normally it has ~--re-'ffsonably long half-]ire of up to 50 weeks in neutral or acid soils (GETZIN 1973). In alkaline soils hydrolysis occurs at the carbamate linkage causing rapid degradation, probably to the phenol. Recently when this insecticide was used for the control of phylloxera in a vineyard planted in gravelly loam of pH 6.0, it proved less effective tbaD was anticipated. Analysis of the soil for carbofuran also showed a lower concentration than would have been expected from the rate of application used. On fortifying an untreated sample of this particular soil with a known amount of carbofuran and analysing at intervals over a period of several weeks recovery was found to decrease with time. A test for possible irreversible adsorption by the clay content of the soil proved negative and this led to the logical alternative of possible microbial degradation. Some evidence of this had been reported previously (GETZIN 1973). This paper describes experiments which confirm this hypothesis and identify the more active organisms involved.
FXPERIMENTAL Measurement of microbial degradation: The effect of microbial degradation was measured by comparing the rate of release of I~CO2 from sterile and non-sterile samples of the same soil treated with 1~C~carbonyl labelled carbofuran. The r a d i o l a b e l l e d material was supplied by F.M.C., Middleport, N.Y. Label purity of greater than 99.7% was determined by TLC. Activity was 3..54 mCi/m mol. A standard solution containing 1 ~Ci/ml in acetone was prepared from this material. U n t r e a t e d vineyard
244
Bulletin of Environmental Contamination &Toxieology, Vol. 15, No. 2 9 1976 by Springer-Verlag New York Inc.
soil containing 12% moisture was screened through a 2 mm mesh sieve and 50 g aliouots of this were transferred to two 8 oz. screw-cap jars. One was a u t o c l a v e d for two 1 hr. periods on successive days. An eouivalent of 2 ppm of 14C-labelled carbofuran was added to each sample from the standard solution. Jars were tumbled over night in a Fisher-Kendall mixer. The biometric apparatus used in the experiment consisted of two 300 mm x 20 mm ID c h r o m a t o g r a p h i c columns equipped with stopcocks and ~ 24/40 female joints at the top. Fitted to these were adapters joined to two absorption tubes connected in series. The latter consisted of 20 ml scintillation vials containing 6 ml of 0.5N sodium hydroxide solution. The columns were sterilized and the fortified soil samples transferred to them under aseptic conditions. Air supplied by an a q u a r i u m pump was m o i s t u r i z e d and freed of atmospheric C02 by passage through a scrubber containing 0.5N sodium hydroxide. This air was allowed to percolate slowly upward through the columns, its flow being controlled by a by-pass valve on the scrubber and by the individual column stopcocks. Samples containing any evolved 14CO2 were collected at weekly intervals over a period of eight weeks. After removing the absorption tubes i0 ml of phase combining solution (PCS A m e r s h a m / Searle) was added to each and the absorbed 14C counted in a scintillation spectrometer (Picker Nuclear Liquimat Model 650). After eight weeks the soil was removed from the columns and analysed for total I~C by ignitinr 1 g samples in an induction furnace, collecting the Ir evolved and m e a s u r i n g it by scintillation counting. All counting was corrected for background and quenching. Isolation of soil microorganisms: Organisms used in the study were isolated from samples of furadan-treated soil from Oliver, B.C. One gram of each sample was placed in i00 ml of sterile water in a 250 ml erlenmeyer flask and shaken 30 minutes on a rotary shaker. A dilution series, giving a final dilution of i:i,000,000 was set up, 1 ml of the final dilution being pipetted and swirled on the surface of each of i0 water agar (WA) plates. Some isolations were made directly from soil crumbs placed on potato dextrose agar (PDA) plates. All WA plates and half the PDA plates were incubated at room temperature. The rest of the PDA plates were incubated at 3~C. All developing colonies were transferred to PDA for purification. Stock cultures were stored on oatmeal agar slants. Evaluation of individual m i c r o o r g a n i s m s in degra~in~ carbofuran: Pure cultures of eight different mlcro-
245
o r g a n i s m s w e r e studied. E x a c t l y 0.1 ml of s t a n d a r d 1 ~ C - c a r b o f u r a n s o l u t i o n was a d d e d to each of a series of s t e r i l i z e d W a r b u r g flasks and the s o l v e n t e v a p o rated under aseptic conditions. To each was a d d e d 5 ml of one of the c u l t u r e s to be studied. A slow s t r e a m of m o i s t u r i z e d , C O 2 - f r e e air g e n e r a t e d as p r e v i o u s l y d e s c r i b e d was p a s s e d over each c u l t u r e and any r e l e a s e d 14CO2 was a b s o r b e d in 0.5N s o d i u m h y d r o x i d e as in the p r e v i o u s e x p e r i m e n t and c o u n t e d in the same manner. The a c t i v i t y of each c u l t u r e was a s s e s s e d on the b a s i s of the a m o u n t of CO2 r e l e a s e d d u r i n g a three day p e r i o d as r e l a t e d to the d r i e d w e i g h t of the p a r t i c u l a r o r g a n i s m involved. E f f e c t of t e m p e r a t u r e on the d e g r a d a t i o n of soil~ p p l i e d c a r b o f u r a n a n d its 3 - h y d r o x y m e t a b o l i t e : S a m p l e s of two types of soil from the area b e i n g i n v e s t i g a t e d w e r e f o r t i f i e d at the 1 p p m level w i t h c a r b o furan and 3 - h y d r o x y c a r b o f u r a n . After thorough mixing of the s a m p l e s 20 g a l i q u o t s w e r e r e m o v e d and a n a l y s e d . The r e m a i n d e r of one set was m a i n t a i n e d at 20~ while the o t h e r set was i m m e d i a t e l y t r a n s f e r r e d to the cold r o o m and m a i n t a i n e d at -16-C. At p e r i o d s of 4, 8, and 12 w e e k s f u r t h e r a l i g u o t s w e r e r e m o v e d from b o t h sets and a n a l y s e d .
P2SULTS AND DISCUSSION In Fig. 1 r e l a t i v e rates of r e l e a s e of I~CO2 from s t e r i l e and n o n - s t e r i l e soil are p l o t t e d . F r o m this it is e v i d e n t that under the c o n d i t i o n s of the e x p e r i m e n t the rate of d e g r a d a t i o n of c a r b o f u r a n d u r i n g the s e c o n d w e e k was forty times g r e a t e r in the n o n - s t e r i l e soil t h a n in the s t e r i l e soil. A n a l y s i s for total 14C in the soil a f t e r e i g h t w e e k s i n c u b a t i o n s h o w e d that the a m o u n t of c a r b o f u r a n r e m a i n i n g in the nons t e r i l e soil had d e c r e a s e d to less than 60% of the amount originally present. In the s t e r i l e soil no m e a s u r a b l e loss had o c c u r r e d . Fig. 2 i l l u s t r a t e s the r e l a t i v e a c t i v i t y of v a r i o u s m i c r o o r g a n i s m s in d e g r a d i n g c a r b o f u r a n . H e r e it is seen that some of the a c t i n o m y c e t e s are p a r t i c u l a r l y active. An a t t e m p t is b e i n g m a d e to h a v e t h e s e i d e n t i f i e d t h o u g h this is p r o v i n g d i f f i c u l t .
246
z z~e
~oo
~.~ STIE RILs
SOIL (SCALE
O i l F~:RENCE
X~lOI
W~EKS
F i g u r e 1. Comparison of rates of degradation of s t e r i l e and n o n - s t e r i l e soil f o r t i f i e d with ~ C labelled carbofuran.
20 ,8 1
iiiiiiiiiii iiiiiiiiiii .iiiiiiiiiiii . . . . . iiiiiiiiiii iiiii?i iiiiiiiiiiiiiiiiiiiiiii ii~iiiiiii
1
iiiiiiiiiiil iiiiiiiiiii
Figure
2.
microorganisms
~, 0
iiiiiiiiiiii iiiiiiiiiii
~
~iiiii::i:i~ iiiiiiiiiii iiii!iiiiiii iiiiiiiiiil
~zo
i
Relative in
i
!!!iii!!i. . . . . . iiiiii!!!ii
effectiveness
degrading
of a number
carbofuran
over
a
of three
day period. (a) a c t i n o m y c e t e , (b) penicillium, (c) (d) (e) (f) a c t i n o m y c e t e s , (g) p e n i c i l l i u m , (h) unidentified.
247
~O
O0
12
Storage period (weeks)
I
*
91.9
8].5
34.3
2
-
2
7.8
ND
24.4
1
1
ND
58.3
** none d e t e c t e d
ND
ND
ND
ND
50.5
2
Westbank
90.7
82.8
ND**
3-OH C a r b o f u r a n
at 20~
63.4
63.3
72.5
57.1
76.8
75.0
81.5
82.8
76.6
86.0
53.5
82.7
67.6
83.0
91.9
90.7
3-OH C a r b o f u r a n
at -16~
present)
Storage
conc.
Carbofuran
(% of o r i g i n a l
Carbofuran
Storage
Recovery
1
2 - Oliver
1
Soil No.*
E f f e c t of T e m p e r a t u r e on the D e g r a d a t i o n of C a r b o f u r a n and 3 - H y d r o x y c a r b o f u r a n in Two O k a n a g a n Soils
TABLE
Table I shows the effect of temperature on the rate of degradation of carbofuran and 3 - h y d r o x y c a r b o f u r a n in soil. It is obvious from this that serious losses can occur between the time of sampling the soil and its eventual analysis if it is not kept at sub zero temperatures during this period. Of particular interest is the very rapid disappearance of 3-hydroxycarbofuran. W h e t h e r the same m i c r o o r g a n i s m s responsible for the degradation of carbofuran are also responsible for the disappearance of this m e t a b o l i t e has yet to be determined. F r o m this study it is evident that in a soil containing high levels of actinomycetes rapid degradation of carbofuran may be expected. This should be borne in mind not only in storing samples but also in assessing the efficacy of this insecticide in controlling soil insects. What in some cases may appear to be possible build-up of resistance by the insect for which control is desired may in fact be a lowering of the insecticidal concentration to an ineffective level by microbial degradation. Further studies will be carried out to determine w h e t h e r other carbamates are degrade4 in similar manner by the same organisms.
REFERENCES APPLE,
J.W., WALGENBACH, E.T., J. Econ. Entomol. 62,
and KNEE, W.J.: 1033 (1969).
BERRY,
R.E.:
64,
J. Econ.
Entomol.
GETZIN,
L.W.:
Environ.
Entomol.
[,
TAPPEN,
W.B.:
J. Econ.
Entomol.
59,
249
1325 461
(1971). (1973).
1161
(1966).
