Journal of Environmental Science and Health, Part B
ISSN: 0360-1234 (Print) 1532-4109 (Online) Journal homepage: http://www.tandfonline.com/loi/lesb20
Microbial degradation of propoxur in turfgrass soil L.‐T. Ou , P. Nkedi‐Kizza , J.L. Cisar & G.H. Snyder To cite this article: L.‐T. Ou , P. Nkedi‐Kizza , J.L. Cisar & G.H. Snyder (1992) Microbial degradation of propoxur in turfgrass soil, Journal of Environmental Science and Health, Part B, 27:5, 545-564 To link to this article: http://dx.doi.org/10.1080/03601239209372800
Published online: 14 Nov 2008.
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Date: 07 November 2015, At: 12:16
J. ENVIRON. SCI. HEALTH, B27(5), 545-564 (1992)
Downloaded by [University of Florida] at 12:16 07 November 2015
MICROBIAL DEGRADATION OF PROPOXUR IN TURFGRASS SOIL Key Words: Propoxur, baygon, i n s e c t i c i d e , t u r f g r a s s , s o i l , biodegradation, m i n e r a l i z a t i o n , metabolites, microorganisms L.-T. Ou1, P. Nkedi-Kizza1, J.L. Cisar 2 and G.H. Snyder3 1Soil 2
Science Department, University of Gainesville, Florida, 32611, U.S.A.
Florida,
Ft. Lauderdale Research and Education Center, University of Florida, Ft. Lauderdale, Florida 33314
3Everglades Research and Education Center, University of Florida, Belle Glade, Florida 33430
ABSTRACT This
study
degradation r a t e s
was conducted in turfgrass
t o determine t h e soil
over a 12-month
period a f t e r a s i n g l e f i e l d a p p l i c a t i o n of propoxur and to
isolate
microorganisms
from t h e s o i l
capable of
degrading t h e i n s e c t i c i d e . Soil samples were c o l l e c t e d from a t u r f g r a s s experimental s i t e near Fort Lauderdale, FL one week before t h e f i e l d a p p l i c a t i o n of propoxur, and over a 12-month period a f t e r t h e f i e l d
application.
Mineralization r a t e s i n surface (0 - 15 cm depth) and subsurface (15 - 30 cm depth) s o i l samples
545 Copyright© 1992 by Marcel Dekker, Inc.
collected
546
.
OU ET AL.
before t h e f i e l d a p p l i c a t i o n were low. M i n e r a l i z a t i o n i n surface
and subsurface
samples c o l l e c t e d
1, 6 and 8
months a f t e r t h e f i e l d a p p l i c a t i o n was much higher t h a n f o r corresponding samples c o l l e c t e d before
Downloaded by [University of Florida] at 12:16 07 November 2015
a p p l i c a t i o n . Mineralization collected
t h ef i e l d
i n t h e subsurface
samples
12 months a f t e r t h e f i e l d a p p l i c a t i o n had
r e v e r t e d back t o t h e s i m i l a r r a t e f o r t h e corresponding sample c o l l e c t e d
before f i e l d application.
Half-life
values (t 1/2 ) f o r propoxur showed s i m i l a r trends t o t h e r e s u l t s of mineralization.
After a s i n g l e a p p l i c a t i o n of
propoxur, degradation i n t u r f g r a s s s o i l was enhanced. Such enhancement l a s t e d
l e s s than 12 months f o r t h e
subsurface, but more than 12 months f o r the surface. A s t r a i n of Arthrobacter sp. capable of degrading propoxur was
i s o l a t e d from t h e s o i l . INTRODUCTION The
i n s e c t i c i d e propoxur [2-(1-methylethoxy)phenyl
methylcarbamate] i s used f o r s t r u c t u r a l pest
control
including t u r f , domestic p e t s , ornamental, and mosquito c o n t r o l . Although t h i s chemical has been'used since 1959 (Thomson, 1985), l i t t l e i s known regarding i t s f a t e i n s o i l s , including those used for t u r f g r a s s . As a c l a s s of carbamate pesticide, i t i s anticipated that i t s primary route of degradation in soils is by hydrolysis (Kazano et al.,
.1972;
Ou
et a l . , 1982; Venkateswarlu
and
Sethunathan, 1978). The hydrolysis product for propoxur would be 2-isopropoxyphenol (2-IPP).
DEGRADATION OF PROPOXUR
547
A number of carbamate p e s t i c i d e s have been r e p o r t e d t o have enhanced d e g r a d a t i o n i n s o i l s a f t e r one o r more f i e l d a p p l i c a t i o n s (Racke and Coats, 1990). These i n c l u d e t h e h e r b i c i d e s EPTC ( s - e t h y l dipropylthiocarbamate) (Bean
Downloaded by [University of Florida] at 12:16 07 November 2015
e t a l . , 1988a; Obrigawitch e t a l . , 1982) and b u t y l a t e ( s e t h y l d i i s o b u t y l t h i o c a r b a m a t e ) (Bean e t a l . , 1988a), and the insecticides carbofuran (2,3-dihydro-2,2-dimethyl-7benzofuranyl-N-methylcarbamate)
(Felsot et a l . ,
1981;
Suett, 1986) and carbaryl (1-naphthyl methylcarbamate) (Rajagopal et a l . , 1986). However, whether previous exposure to propoxur for turfgrass soils or other soils can cause subsequent enhanced degradation of propoxur needs to be demonstrated. The frequent irrigation and fertilization practices used for turfgrass are conducive to
high microbial activity.
Therefore, propoxur in
turfgrass may be degraded rapidly and enhanced may occur after 'priming'. Bean et al. (1988b) reported that the duration for enhanced degradation of EPTC in Crete s i l t y clay loam from Nebraska after a single field application of the herbicide was about 30 months. Ou (1991) found that the duration for enhanced degradation of the thiophosphorus nematicide fenamiphos in Ellzey fine sand from Florida was between 3 and 4 years after a single field application to an experimental site cropped to potatoes. The objectives of this study were to determine degradation
rates,
mineralization
rates
and parent
chemical disappearance rates, in turfgrass soil collected
548
OU ET AL.
before and during a 12-months period a f t e r a s i n g l e f i e l d application to a turfgrass site, and to isolate and characterize microorganisms capable of degrading propoxur from turfgrass soil.
Downloaded by [University of Florida] at 12:16 07 November 2015
MATERIALS AND METHODS
Soils Soil
samples
(Margate
fine
sand,
a siliceous,
hyperthermic Mollic Psammaqvent, 0 - 1 5 and 15 - 30 cm depth) were collected from a turfgrass experimental s i t e near
Fort
Lauderdale,
FL. Although
this
site
was
established in 1969, prior t o this study i t had never been treated with propoxur or with any other carbamate pesticides
such as carbofuran, carbaryl, EPTC,
etc.
Propoxur was applied t o the s i t e in late February, 1989, at a rate of 12.2 kg/ha. Nitrogen (N) and potassium (K) f e r t i l i z e r s in the forms of (NH4)2SO4 and KC1 were applied monthly and bimonthly, respectively, a t the rate of 100 kg
of
nutrient/ha/application.
Soil
samples
were
collected 1 week prior t o the field application, and 1, 6, 8 and 12 months after the field
application of
propoxur. Soil samples were stored in the dark a t 4 °C, and were used within 60 days. Key soil properties are shown in Table 1. The two soil horizons from which soil samples were collected were visually different with the top
horizon being black due t o i t s high organic matter
content and the subsurface horizon being a leached white sand. In addition, s o i l samples ( 0 - 1 5 and 15 - 30 cm
DEGRADATION OF PROPOXUR
549
depth) from a nearby n o n t u r f g r a s s s i t e t h a t had never been exposed t o any p e s t i c i d e s ( i n c l u d i n g propoxur) o r f e r t i l i z e r s were also included in the study. Chemicals
Downloaded by [University of Florida] at 12:16 07 November 2015
Uniformly
ring-labeled
l4
C-propoxur
(specific
activity 1.395 GBq and radiopurity >99%) and analytical grade
propoxur were provided
(Kansas
City,
poxyphenol
MO) .
by Mobay Chemical Co.
Metabolite
standards
2-isopro-
(2-IPP) and catechol were purchased
from
Aldrich Chemical Co. (Milwaukee, WI). Uniformly ringlabeled uC-2-IPP was prepared by alkaline hydrolysis of 14
C-propoxur. A 15 mL glass vial containing 185 kBq ofI4C-
propoxur and 2.5 mL of 0.1 mol/L NaOH was placed in a waterbath
a t 60
°C
overnight.
The
solution was
subsequently neutralized with 2.5 mL of 0.1 mol/L HCl. Degradation Studies For determination of mineralization rates of l4Cpropoxur in turfgrass s o i l , 100 g of soil were placed in a 250 mL Erlenmeyer flask which contained 0.01 g of propoxur and 18.5 kBq of 14c-propoxur. After mixing, each flask was weighed and then closed tightly with a rubber stopper under which a stainless steel vial was hung from a stainless steel wire. Each vial contained 0.5 mL of 8 mol/L KOH for trapping 14CO2. At predetermined intervals, vials
in the flasks
containing fresh KOH.
were replaced with w
new vials
c-activity in the KOH traps was
determined by liquid s c i n t i l l a t i o n counting. At the same
550
OU ET AL.
time, weights of t h e f l a s k s were checked, and s t e r i l e water was added t o compensate f o r any intervening water l o s s . Upon completion of t h e incubation period (42 t o 70 days), 10 g of s o i l were removed from each f l a s k and
Downloaded by [University of Florida] at 12:16 07 November 2015
placed i n a 50 mL g l a s s c u l t u r e tube with t e f l o n - l i n e d cap for solvent extraction. For determination of metabolites in soil, 200 g of soil were placed in a 1 L glass bottle which contained 2 mg of analytical grade propoxur. Each bottle was weighed and then closed tightly with a screw cap. The bottles were incubated in the dark at 25 °C. At predetermined intervals, 10 g of each treated soil were removed and placed in a 50 mL glass culture tube as described above. Soil samples (10 g) in culture tubes were extracted with 30 mL of an equal volume of organic solvent mixture, benzene, acetone and methanol, in a reciprocal shaker for 2 hours. The extracts were vacuum-filtered through Whatman no. 42 f i l t e r paper and the extracted soils were washed twice with 5 mL of the organic mixture, followed by vacuum f i l t r a t i o n . The extracts were concentrated under a gentle stream of nitrogen gas to 1 mL. Propoxur and i t s metabolites in those concentrated extracts were determined by a high performance liquid Chromatographie (HPLC) method using a Spectra Physics HPLC system (San Jose, CA) equipped with an UV detector. The wavelength of the detector was set a t 220 nm. A Nova-Pak reversed-phase column (Haters, Milford, MA) was used with mobile phase
DEGRADATION OF PROPOXUR
551
of 50% methanol and 50% water a t a c o n s t a n t flow r a t e of 1 mL/minute. R e t e n t i o n times f o r propoxur, 2-IPP and c a t e c h o l were 3.9, 5.4 and 2.1 minutes, r e s p e c t i v e l y . 14
Downloaded by [University of Florida] at 12:16 07 November 2015
if
14
C-activity remaining i n t h e e x t r a c t e d s o i l samples,
C-fenamiphos was used, was combusted
t o14CO2 i n a
sample o x i d i z e r d e s c r i b e d p r e v i o u s l y by Ou e t a l . (1982) . I s o l a t i o n of Propoxur-Deqradincr Microorganisms B a t c h - c u l t u r e enrichment t e c h n i q u e s were employed t o i s o l a t e microorganisms capable of degrading propoxur from t h e t u r f g r a s s s o i l . S o i l samples ( 0 - 1 5 and 15 - 30 cm depth) collected
6 months after the field
application
were treated i n i t i a l l y with analytical grade propoxur a t a rate of 10 Mg/g. At 14-day intervals, propoxur was applied at increasingly higher rates each time, and a f t e r 3 successive applications the final application rate was 50 ftg/g. Ten g of soil from each sample were transferred to a s t e r i l e 250 mL Erlenmeyer flask containing 100 ml· of basal mineral medium and 200 ßq/tiL of analytical grade propoxur. All flasks were incubated in a rotary shaker a t 25 °C. After a series of l-to-10 dilutions a t 7-day intervals, turbidity was developed in the propoxur-basal mineral medium. Pure bacterial cultures were isolated by streaking
a small amount of the culture medium t o a
propoxur-basal mineral agar plate. Bacterial
isolates
were characterized by conventional morphological and biochemical
methods.
The basal
mineral
medium was
composed of K2HPO4, 4.8 g; KH2PO4, 1.2 g; NH4NO3, 1.0 g;
552
OU ET AL.
MgSO4.4H2O, 0.25 g; Ca(NO3)2.4H2O, 0.04 g; Fe2(SO4)2, 0.001 g; and deionized H2O, 1 L. 14
C-propoxur and l4C-2-IPP were used t o determine t h e
capacity of the bacterial isolates t o mineralize the two
Downloaded by [University of Florida] at 12:16 07 November 2015
chemicals.
14
C-propoxur (3.7 kBq) and nonlabeled propoxur
(200 /ig/mL), or I4C-2-IPP (2.5 kBg) and nonlabeled 2-IPP (200 /ig/mL), were added to a sterile 250 mL Erlenmeyer flask which contained 50 mL of basal mineral medium and ' 1 mL of 2-day old bacterial culture. A 0.5 mL of the culture
solution
determination
of
was
immediately
initial
scintillation counting.
14
C-activity
removed by
for
liquid
Each flask was then
closed
tightly with a rubber stopper, under which was suspended a stainless steel vial containing 0.5 mL of 8 mol/L KOH as described above. The flasks were incubated in a rotary shaker a t 25 °C. At adequate intervals the vials were replaced with new vials containing fresh KOH. 14C-activity in the traps was quantified by liquid scintillation counting.
Similar
conditions
were
employed for
determination of metabolite formation in the culture fluid. In this case, only nonlabeled compounds were used and hence KOH traps were not employed. The HPLC method described above was used to determine metabolites in the culture fluid. RESULTS AND DISCUSSION Although
the soil
samples
collected
from t h e
experimental s i t e before and a f t e r f i e l d application of
DEGRADATION OF PROPOXUR propoxur
possessed
553
similar
chemical,
physical
and
biological properties (Table l ) , a single application of propoxur stimulated
degradative activity of propoxur-
degrading microorganisms. Prior to the field application
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of propoxur, propoxur was mineralized slowly in both horizons (Fig. 1), with only 10.8 and 4.7% of the applied 14
C-propoxur
respectively,
in
surface
and
subsurface
being mineralized
during
70
samples, days
of
incubation. More propoxur was mineralized in the surface samples than in the subsurface sample. Mineralization of propoxur in soil samples collected 1 month after the field
application was much higher than for samples
collected before the field application. During the f i r s t 3 days of incubation,
10.6 and 11.2% of applied
I4
C-
propoxur in surface and subsurface samples, respectively, were mineralized. Unlike the samples collected before the field
application,
mineralization
horizon samples were now
rates
of
the two
similar. Mineralization
of
propoxur in soil samples collected 6 and 8 months after the field application was
even more rapid
than for
samples collected 1 month after field the application. In contrast to the samples collected before the field application, for soil samples collected 6 and 8 months after
the field
application, more propoxur was
now
mineralized by the subsurface samples than that by the surface
samples. Twelve months following the field
application, the degradation pattern reverted back to
554
OU ET AL.
Table 1 Key s o i l properties of Ssoil samples collected from the turfgrass
Downloaded by [University of Florida] at 12:16 07 November 2015
experimental s i t e near Fort Lauderdale, FL
Soil depth (cm)
pH
Water content (g/kg)
Organic C (g/kg)
Size Distribution Sand
Silt
Bacteria Fungi (cfu/kgxio9) (cfu/kgxicf)
Clay
1 week before field application 0 - 1 5 6.7
88
8.1
97 '
15 - 30 6.2
44
0.1
99
1
2
8.76
21.22
ISSN: 0360-1234 (Print) 1532-4109 (Online) Journal homepage: http://www.tandfonline.com/loi/lesb20
Microbial degradation of propoxur in turfgrass soil L.‐T. Ou , P. Nkedi‐Kizza , J.L. Cisar & G.H. Snyder To cite this article: L.‐T. Ou , P. Nkedi‐Kizza , J.L. Cisar & G.H. Snyder (1992) Microbial degradation of propoxur in turfgrass soil, Journal of Environmental Science and Health, Part B, 27:5, 545-564 To link to this article: http://dx.doi.org/10.1080/03601239209372800
Published online: 14 Nov 2008.
Submit your article to this journal
Article views: 13
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=lesb20 Download by: [University of Florida]
Date: 07 November 2015, At: 12:16
J. ENVIRON. SCI. HEALTH, B27(5), 545-564 (1992)
Downloaded by [University of Florida] at 12:16 07 November 2015
MICROBIAL DEGRADATION OF PROPOXUR IN TURFGRASS SOIL Key Words: Propoxur, baygon, i n s e c t i c i d e , t u r f g r a s s , s o i l , biodegradation, m i n e r a l i z a t i o n , metabolites, microorganisms L.-T. Ou1, P. Nkedi-Kizza1, J.L. Cisar 2 and G.H. Snyder3 1Soil 2
Science Department, University of Gainesville, Florida, 32611, U.S.A.
Florida,
Ft. Lauderdale Research and Education Center, University of Florida, Ft. Lauderdale, Florida 33314
3Everglades Research and Education Center, University of Florida, Belle Glade, Florida 33430
ABSTRACT This
study
degradation r a t e s
was conducted in turfgrass
t o determine t h e soil
over a 12-month
period a f t e r a s i n g l e f i e l d a p p l i c a t i o n of propoxur and to
isolate
microorganisms
from t h e s o i l
capable of
degrading t h e i n s e c t i c i d e . Soil samples were c o l l e c t e d from a t u r f g r a s s experimental s i t e near Fort Lauderdale, FL one week before t h e f i e l d a p p l i c a t i o n of propoxur, and over a 12-month period a f t e r t h e f i e l d
application.
Mineralization r a t e s i n surface (0 - 15 cm depth) and subsurface (15 - 30 cm depth) s o i l samples
545 Copyright© 1992 by Marcel Dekker, Inc.
collected
546
.
OU ET AL.
before t h e f i e l d a p p l i c a t i o n were low. M i n e r a l i z a t i o n i n surface
and subsurface
samples c o l l e c t e d
1, 6 and 8
months a f t e r t h e f i e l d a p p l i c a t i o n was much higher t h a n f o r corresponding samples c o l l e c t e d before
Downloaded by [University of Florida] at 12:16 07 November 2015
a p p l i c a t i o n . Mineralization collected
t h ef i e l d
i n t h e subsurface
samples
12 months a f t e r t h e f i e l d a p p l i c a t i o n had
r e v e r t e d back t o t h e s i m i l a r r a t e f o r t h e corresponding sample c o l l e c t e d
before f i e l d application.
Half-life
values (t 1/2 ) f o r propoxur showed s i m i l a r trends t o t h e r e s u l t s of mineralization.
After a s i n g l e a p p l i c a t i o n of
propoxur, degradation i n t u r f g r a s s s o i l was enhanced. Such enhancement l a s t e d
l e s s than 12 months f o r t h e
subsurface, but more than 12 months f o r the surface. A s t r a i n of Arthrobacter sp. capable of degrading propoxur was
i s o l a t e d from t h e s o i l . INTRODUCTION The
i n s e c t i c i d e propoxur [2-(1-methylethoxy)phenyl
methylcarbamate] i s used f o r s t r u c t u r a l pest
control
including t u r f , domestic p e t s , ornamental, and mosquito c o n t r o l . Although t h i s chemical has been'used since 1959 (Thomson, 1985), l i t t l e i s known regarding i t s f a t e i n s o i l s , including those used for t u r f g r a s s . As a c l a s s of carbamate pesticide, i t i s anticipated that i t s primary route of degradation in soils is by hydrolysis (Kazano et al.,
.1972;
Ou
et a l . , 1982; Venkateswarlu
and
Sethunathan, 1978). The hydrolysis product for propoxur would be 2-isopropoxyphenol (2-IPP).
DEGRADATION OF PROPOXUR
547
A number of carbamate p e s t i c i d e s have been r e p o r t e d t o have enhanced d e g r a d a t i o n i n s o i l s a f t e r one o r more f i e l d a p p l i c a t i o n s (Racke and Coats, 1990). These i n c l u d e t h e h e r b i c i d e s EPTC ( s - e t h y l dipropylthiocarbamate) (Bean
Downloaded by [University of Florida] at 12:16 07 November 2015
e t a l . , 1988a; Obrigawitch e t a l . , 1982) and b u t y l a t e ( s e t h y l d i i s o b u t y l t h i o c a r b a m a t e ) (Bean e t a l . , 1988a), and the insecticides carbofuran (2,3-dihydro-2,2-dimethyl-7benzofuranyl-N-methylcarbamate)
(Felsot et a l . ,
1981;
Suett, 1986) and carbaryl (1-naphthyl methylcarbamate) (Rajagopal et a l . , 1986). However, whether previous exposure to propoxur for turfgrass soils or other soils can cause subsequent enhanced degradation of propoxur needs to be demonstrated. The frequent irrigation and fertilization practices used for turfgrass are conducive to
high microbial activity.
Therefore, propoxur in
turfgrass may be degraded rapidly and enhanced may occur after 'priming'. Bean et al. (1988b) reported that the duration for enhanced degradation of EPTC in Crete s i l t y clay loam from Nebraska after a single field application of the herbicide was about 30 months. Ou (1991) found that the duration for enhanced degradation of the thiophosphorus nematicide fenamiphos in Ellzey fine sand from Florida was between 3 and 4 years after a single field application to an experimental site cropped to potatoes. The objectives of this study were to determine degradation
rates,
mineralization
rates
and parent
chemical disappearance rates, in turfgrass soil collected
548
OU ET AL.
before and during a 12-months period a f t e r a s i n g l e f i e l d application to a turfgrass site, and to isolate and characterize microorganisms capable of degrading propoxur from turfgrass soil.
Downloaded by [University of Florida] at 12:16 07 November 2015
MATERIALS AND METHODS
Soils Soil
samples
(Margate
fine
sand,
a siliceous,
hyperthermic Mollic Psammaqvent, 0 - 1 5 and 15 - 30 cm depth) were collected from a turfgrass experimental s i t e near
Fort
Lauderdale,
FL. Although
this
site
was
established in 1969, prior t o this study i t had never been treated with propoxur or with any other carbamate pesticides
such as carbofuran, carbaryl, EPTC,
etc.
Propoxur was applied t o the s i t e in late February, 1989, at a rate of 12.2 kg/ha. Nitrogen (N) and potassium (K) f e r t i l i z e r s in the forms of (NH4)2SO4 and KC1 were applied monthly and bimonthly, respectively, a t the rate of 100 kg
of
nutrient/ha/application.
Soil
samples
were
collected 1 week prior t o the field application, and 1, 6, 8 and 12 months after the field
application of
propoxur. Soil samples were stored in the dark a t 4 °C, and were used within 60 days. Key soil properties are shown in Table 1. The two soil horizons from which soil samples were collected were visually different with the top
horizon being black due t o i t s high organic matter
content and the subsurface horizon being a leached white sand. In addition, s o i l samples ( 0 - 1 5 and 15 - 30 cm
DEGRADATION OF PROPOXUR
549
depth) from a nearby n o n t u r f g r a s s s i t e t h a t had never been exposed t o any p e s t i c i d e s ( i n c l u d i n g propoxur) o r f e r t i l i z e r s were also included in the study. Chemicals
Downloaded by [University of Florida] at 12:16 07 November 2015
Uniformly
ring-labeled
l4
C-propoxur
(specific
activity 1.395 GBq and radiopurity >99%) and analytical grade
propoxur were provided
(Kansas
City,
poxyphenol
MO) .
by Mobay Chemical Co.
Metabolite
standards
2-isopro-
(2-IPP) and catechol were purchased
from
Aldrich Chemical Co. (Milwaukee, WI). Uniformly ringlabeled uC-2-IPP was prepared by alkaline hydrolysis of 14
C-propoxur. A 15 mL glass vial containing 185 kBq ofI4C-
propoxur and 2.5 mL of 0.1 mol/L NaOH was placed in a waterbath
a t 60
°C
overnight.
The
solution was
subsequently neutralized with 2.5 mL of 0.1 mol/L HCl. Degradation Studies For determination of mineralization rates of l4Cpropoxur in turfgrass s o i l , 100 g of soil were placed in a 250 mL Erlenmeyer flask which contained 0.01 g of propoxur and 18.5 kBq of 14c-propoxur. After mixing, each flask was weighed and then closed tightly with a rubber stopper under which a stainless steel vial was hung from a stainless steel wire. Each vial contained 0.5 mL of 8 mol/L KOH for trapping 14CO2. At predetermined intervals, vials
in the flasks
containing fresh KOH.
were replaced with w
new vials
c-activity in the KOH traps was
determined by liquid s c i n t i l l a t i o n counting. At the same
550
OU ET AL.
time, weights of t h e f l a s k s were checked, and s t e r i l e water was added t o compensate f o r any intervening water l o s s . Upon completion of t h e incubation period (42 t o 70 days), 10 g of s o i l were removed from each f l a s k and
Downloaded by [University of Florida] at 12:16 07 November 2015
placed i n a 50 mL g l a s s c u l t u r e tube with t e f l o n - l i n e d cap for solvent extraction. For determination of metabolites in soil, 200 g of soil were placed in a 1 L glass bottle which contained 2 mg of analytical grade propoxur. Each bottle was weighed and then closed tightly with a screw cap. The bottles were incubated in the dark at 25 °C. At predetermined intervals, 10 g of each treated soil were removed and placed in a 50 mL glass culture tube as described above. Soil samples (10 g) in culture tubes were extracted with 30 mL of an equal volume of organic solvent mixture, benzene, acetone and methanol, in a reciprocal shaker for 2 hours. The extracts were vacuum-filtered through Whatman no. 42 f i l t e r paper and the extracted soils were washed twice with 5 mL of the organic mixture, followed by vacuum f i l t r a t i o n . The extracts were concentrated under a gentle stream of nitrogen gas to 1 mL. Propoxur and i t s metabolites in those concentrated extracts were determined by a high performance liquid Chromatographie (HPLC) method using a Spectra Physics HPLC system (San Jose, CA) equipped with an UV detector. The wavelength of the detector was set a t 220 nm. A Nova-Pak reversed-phase column (Haters, Milford, MA) was used with mobile phase
DEGRADATION OF PROPOXUR
551
of 50% methanol and 50% water a t a c o n s t a n t flow r a t e of 1 mL/minute. R e t e n t i o n times f o r propoxur, 2-IPP and c a t e c h o l were 3.9, 5.4 and 2.1 minutes, r e s p e c t i v e l y . 14
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if
14
C-activity remaining i n t h e e x t r a c t e d s o i l samples,
C-fenamiphos was used, was combusted
t o14CO2 i n a
sample o x i d i z e r d e s c r i b e d p r e v i o u s l y by Ou e t a l . (1982) . I s o l a t i o n of Propoxur-Deqradincr Microorganisms B a t c h - c u l t u r e enrichment t e c h n i q u e s were employed t o i s o l a t e microorganisms capable of degrading propoxur from t h e t u r f g r a s s s o i l . S o i l samples ( 0 - 1 5 and 15 - 30 cm depth) collected
6 months after the field
application
were treated i n i t i a l l y with analytical grade propoxur a t a rate of 10 Mg/g. At 14-day intervals, propoxur was applied at increasingly higher rates each time, and a f t e r 3 successive applications the final application rate was 50 ftg/g. Ten g of soil from each sample were transferred to a s t e r i l e 250 mL Erlenmeyer flask containing 100 ml· of basal mineral medium and 200 ßq/tiL of analytical grade propoxur. All flasks were incubated in a rotary shaker a t 25 °C. After a series of l-to-10 dilutions a t 7-day intervals, turbidity was developed in the propoxur-basal mineral medium. Pure bacterial cultures were isolated by streaking
a small amount of the culture medium t o a
propoxur-basal mineral agar plate. Bacterial
isolates
were characterized by conventional morphological and biochemical
methods.
The basal
mineral
medium was
composed of K2HPO4, 4.8 g; KH2PO4, 1.2 g; NH4NO3, 1.0 g;
552
OU ET AL.
MgSO4.4H2O, 0.25 g; Ca(NO3)2.4H2O, 0.04 g; Fe2(SO4)2, 0.001 g; and deionized H2O, 1 L. 14
C-propoxur and l4C-2-IPP were used t o determine t h e
capacity of the bacterial isolates t o mineralize the two
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chemicals.
14
C-propoxur (3.7 kBq) and nonlabeled propoxur
(200 /ig/mL), or I4C-2-IPP (2.5 kBg) and nonlabeled 2-IPP (200 /ig/mL), were added to a sterile 250 mL Erlenmeyer flask which contained 50 mL of basal mineral medium and ' 1 mL of 2-day old bacterial culture. A 0.5 mL of the culture
solution
determination
of
was
immediately
initial
scintillation counting.
14
C-activity
removed by
for
liquid
Each flask was then
closed
tightly with a rubber stopper, under which was suspended a stainless steel vial containing 0.5 mL of 8 mol/L KOH as described above. The flasks were incubated in a rotary shaker a t 25 °C. At adequate intervals the vials were replaced with new vials containing fresh KOH. 14C-activity in the traps was quantified by liquid scintillation counting.
Similar
conditions
were
employed for
determination of metabolite formation in the culture fluid. In this case, only nonlabeled compounds were used and hence KOH traps were not employed. The HPLC method described above was used to determine metabolites in the culture fluid. RESULTS AND DISCUSSION Although
the soil
samples
collected
from t h e
experimental s i t e before and a f t e r f i e l d application of
DEGRADATION OF PROPOXUR propoxur
possessed
553
similar
chemical,
physical
and
biological properties (Table l ) , a single application of propoxur stimulated
degradative activity of propoxur-
degrading microorganisms. Prior to the field application
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of propoxur, propoxur was mineralized slowly in both horizons (Fig. 1), with only 10.8 and 4.7% of the applied 14
C-propoxur
respectively,
in
surface
and
subsurface
being mineralized
during
70
samples, days
of
incubation. More propoxur was mineralized in the surface samples than in the subsurface sample. Mineralization of propoxur in soil samples collected 1 month after the field
application was much higher than for samples
collected before the field application. During the f i r s t 3 days of incubation,
10.6 and 11.2% of applied
I4
C-
propoxur in surface and subsurface samples, respectively, were mineralized. Unlike the samples collected before the field
application,
mineralization
horizon samples were now
rates
of
the two
similar. Mineralization
of
propoxur in soil samples collected 6 and 8 months after the field application was
even more rapid
than for
samples collected 1 month after field the application. In contrast to the samples collected before the field application, for soil samples collected 6 and 8 months after
the field
application, more propoxur was
now
mineralized by the subsurface samples than that by the surface
samples. Twelve months following the field
application, the degradation pattern reverted back to
554
OU ET AL.
Table 1 Key s o i l properties of Ssoil samples collected from the turfgrass
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experimental s i t e near Fort Lauderdale, FL
Soil depth (cm)
pH
Water content (g/kg)
Organic C (g/kg)
Size Distribution Sand
Silt
Bacteria Fungi (cfu/kgxio9) (cfu/kgxicf)
Clay
1 week before field application 0 - 1 5 6.7
88
8.1
97 '
15 - 30 6.2
44
0.1
99
1
2
8.76
21.22
