Degradation of High Concentrations of a Phosphorothioic Ester by

20 Degradation of High Concentrations of a Phosphorothioic Ester by Hydrolase

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R. HONEYCUTT,L.BALLANTINE, H. LEBARON, D. PAULSON, and V. SEIM— CIBA-GEIGY Corporation, Greensboro, NC 27409 C. GANZ—EN-CAS Laboratories, Winston-Salem, NC 27103 G. MILAD—Biospherics, Inc., Rockville, MD 20852

Greenhouse s o i l was treated at 500, 1000, 2000 and 5000 ppm with Diazinon 4E. Parathion hydrolase was added to the s o i l to determine the efficacy of the enzyme to rapidly degrade diazinon during a s p i l l situation. The h a l f - l i f e of the diazinon in the 500 ppm treatment without enzyme present was 9.4 days while the h a l f - l i f e of diazinon in the 500 ppm treatment with enzyme present was one hour. The half-lives of diazinon in the 1000, 2000 and 5000 ppm treatments with enzyme present were 1.2, 5.6 and 128 hours (5.3 days), respectively. These data indicate that parathion hydrolase can be used effectively to rapidly reduce large concentrations of diazinon in s o i l . At diazinon concentrations above 2000 ppm the enzyme is less effective. Parathion hydrolase is readily soluble in water, is reasonably stable and can be easily handled in the f i e l d . Further research is needed to evaluate the efficacy of parathion hydrolase to decontaminate diazinon under actual s p i l l conditions.

Chemical spills can be d e v a s t a t i n g to the environment as w e l l as to the pocketbook of those r e s p o n s i b l e f o r i t s cleanup. The cost of cleanup of a s i n g l e spill can approach $200,000. I t immediately occurs to one that a much e a s i e r and cheaper way must e x i s t to accomplish the cleanup of chemical spills. At CIBA-GEIGY we have formed a task force to study a l t e r n a t e ways to c l e a n up spills. One of the first p r o j e c t s the task force undertook was to develop a simple inexpensive means to c l e a n up d i a z i n o n spills which may occur on land or in water. 0097-6156/84/0259-0343$06.00/0 © 1984 American Chemical Society

Krueger and Seiber; Treatment and Disposal of Pesticide Wastes ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

344

TREATMENT AND DISPOSAL O F PESTICIDE WASTES

Chakrabarty has e x t e n s i v e l y reviewed the biodégradation o f p e s t i c i d e s (1). Table I shows the r e s u l t s of s e v e r a l s t u d i e s on the enzymatic a c t i v i t y of m i c r o b i a l c e l l - f r e e e x t r a c t s f o r p e s t i c i d e degradation. C l e a r l y , there i s s u b s t a n t i a l evidence to suggest that enzymes might be used i n the development o f biotechnology f o r use i n degradation of p e s t i c i d e s . D i a z i n o n (Figure 1) i s widely used throughout the U n i t e d States and other c o u n t r i e s f o r c o n t r o l of v a r i o u s i n s e c t s such as cutworms, grubs, ants, cockroaches and s i l v e r f i s h . In the past, d i a z i n o n has been degraded using a c i d or sodium hypoc h l o r i t e degradation (2,3). Recently, enzymatic h y d r o l y s i s f o r decontamination of d i a z i n o n s p i l l s has been studied by Munnecke, et^ j a l . (4,5). Using an enzyme, p a r a t h i o n hydrolase, from a mixed c u l t u r e of pseudomonas sp., Munnecke was able to show that p a r a t h i o n hydrolase would degrade the organophosphate parathion 2450 times f a s t e r than 0.1N NaoH a t 40°C ( 4 ) . Table I I shows the comparative h y d r o l y s i s r a t e s o f s e v e r a l organophosphates using parathion hydrolase vs. a chemical h y d r o l y s i s method. P a r a t h i o n hydrolase outperforms chemical h y d r o l y s i s methods f o r most organophosphates that Munnecke looked a t . Subsequent s t u d i e s showed that the enzyme would degrade 1000 ppm d i a z i n o n (25% EC formulation) i n s o i l by 97% i n 24 hours

Most of the studies done by Munnecke were small scale laboratory s t u d i e s . The e f f i c a c y o f parathion hydrolase has not been tested under f i e l d c o n d i t i o n s . I t was the major obj e c t i v e of our study to determine the usefulness o f parathion hydrolase f o r the decontamination of high concentrations of formulated d i a z i n o n i n s o i l under greenhouse c o n d i t i o n s . A secondary, but very important, o b j e c t i v e was to determine i f the enzyme could be handled i n a p r a c t i c a l f a s h i o n as would be done i n the f i e l d and r e t a i n i t s a b i l i t y to degrade d i a z i n o n . METHODS M a t e r i a l s : Parathion hydrolase was obtained from Doug Munnecke (4,5). The s p e c i f i c a c t i v i t y was measured by a method of Munnecke (4,5) and was found to be 0.1 pmole d i a z i n o n hydrolyzed/mg t o t a l protein/minute. Diazinon 4E was obtained from CIBA-GEIGY, Greensboro, NC. D e s c r i p t i o n o f Greenhouse Study - In 1981, s i x r e c t a n g u l a r flats(2 Χ 3' X 3") of Georgia loamy sand s o i l were prepared. The s o i l i n each f l a t was then placed i n a l a r g e Hobart mixer (80 quart bowl) and the appropriate amount of Diazinon 4E f


20.

HONEYCUTT ET AL.

Table I .

Enzymatic A c t i v i t y of M i c r o b i a l C e l l - F r e e E x t r a c t s For P e s t i c i d e Degradation

Pesticide Organaophospate s Acephate Aspon Cyanophos Diazinon Dursban DCVMP DEFP DFP EPN Fenitrothion Fensulfothion Methyl P a r a t h i o n Monocrotophos Paraoxon Parathion Propetamphos Quinalphos Triazophos TEPP Phenylureas Carboxin Chlorbromuron Linuron Metabromuron Monalide Monolinuron Monuron Pyracarbolid

Note:

345

Degradation by Hydrolase

Enzyme C l a s s

Esterase Esterase Esterase Esterase Esterase Lyase Lyase Lyase Esterase Esterase Esterase Esterase Esterase Esterase Esterase Esterase Esterase Esterase Lyase

Acylamidase Acylamidase Acylamidase Acylamidase Acylamidase Acylamidase Acylamidase Acylamidase

Enzyme A c t i v i t y (NMOL o f Substrate Transformed/Min/ mg P r o t e i n )

52 110 58 301,1200,A 600 A A A 12 217 238 600,A 133 13,3600,A 259,3000, 7000,A 50 1410 4350 A

252 11,15 18,20,130 16,18 29,238 15,20 4 35

A = i n s u f f i c i e n t data t o c a l c u l a t e enzyme a c t i v i t y .

Reprinted with permission from Ref. 1. Copyright 1982, CRC P r e s s , Inc.


346

T R E A T M E N T A N D DISPOSAL O F PESTICIDE WASTES

Table I I .

P a r a t h i o n Hydrolase S t u d i e s . Enzymatic v s . Chemical H y d r o l y s i s of Organophosphate I n s e c t i c i d e s

Solution Concentration MM

Pesticide Parathion Triazophos Paraoxon EPN Diazinon Methyl P a r a t h i o n Dursban

R a t i o Enzymatic H y d r o l y s i s Rate/ Chemical Rate 2450 1005 525 11 143 122 40

45 110 50 2 72 150 8

(Λ7.5% a c t i v e i n g r e d i e n t ) was slowly mixed i n t o i t over a p e r i od of 5-10 minutes. At this point, 2 l i t e r s of a s o l u t i o n of parathion hydrolase i n 10 triM trihydroxy-methy1-amino methane b u f f e r (TRIS) - 5 mM cobalt c h l o r i d e , pH 8.5, were sprayed onto the s o i l using a 2-gallon garden hand sprayer. The mixture was then mixed f o r an a d d i t i o n a l 15-30 minutes to achieve homogene i t y , at which time 300 grams of s o i l were taken f o r a n a l y s i s . These are r e f e r r e d to as 0.5 hr. samples. Subsequent s o i l samples were taken at 2, 4, 24 and 48 hours, 7 days, and 3 weeks. Table I I I shows the d e s c r i p t i o n of s o i l f l a t s for the greenhouse study. Between sampling periods a l l f l a t s were kept i n the greenhouse at 24-27°C and 50-70% r e l a t i v e humidity.

Table I I I .

Diazinon Concentrations Flats

Soil Soil Soil Soil Soil Soil

-

All

Flat Flat Flat Flat Flat Flat

A Β C D Ε F

i n Georgia Greenhouse

Enzyme Only Diazinon 500 ppm - No Enzyme Diazinon 500 ppm + Enzyme Diazinon 1000 ppm + Enzyme Diazinon 2000 ppm + Enzyme Diazinon 5000 ppm + Enzyme

f l a t s kept at 24-27°C and 50-70% r e l a t i v e

humidity.


Soil

20.

HONEYCUTT ET AL.


347

A n a l y t i c a l Methods D i a z i n o n concentrations i n s o i l were measured using e x t r a c t i o n - p a r t i t i o n followed by gas chromatography. F i g u r e 2 gives an o u t l i n e of t h i s procedure. F i f t y grams of s o i l was f i r s t e x t r a c t e d with 200 ml acetone. Water (500 ml) was added t o a 140 ml a l i q u o t of the e x t r a c t and the mixture p a r t i t i o n e d with 35 ml hexane. The hexane was analyzed f o r d i a z i n o n using by GLC and a flame photometric d e t e c t o r ( F i g u r e 3). Oxypyrimidine, the major product o f d i a z i n o n degradation, (4 hydroxy-6methyl-2-isopropyl-pyrimidine) was determined i n s o i l by ext r a c t i o n with methanol:water (80:20) followed by a n a l y s i s by HPLC using a reverse phase Whatman p a r t i s i l ODS-3 column and a 50/50 mixture of methanol and 0.01M Na^PO^ i n the i s o c r a t i c mode (Figure 4 ) . Results S o l u b i l i t y of P a r a t h i o n Hydrolase During S o i l A p p l i c a t i o n P a r a t h i o n hydrolase i s s o l u b l e i n water and e a s i l y handled during a p p l i c a t i o n to s o i l . For p r a c t i c a l use on s o i l , i t was found that l a r g e amounts of the enzyme should be d i s s o l v e d i n t o a minimum amount of 10 mM TRIS - 5 mM c o b a l t c h l o r i d e , pH 8.5, and the mixture blended with a Waring blender at low speed f o r 1-2 minutes to achieve a homogenous suspension. At t h i s time more b u f f e r can be added g r a d u a l l y with constant a g i t a t i o n to f i n a l l y achieve the des i r e d d i l u t e s o l u t i o n . T h i s i s not a complicated procedure. Most workers w i l l have ready access to the equipment needed t o c a r r y out the procedure. Some s e t t l i n g of the enzyme w i l l occur over a p e r i o d of time. However, the s o l u t i o n remains homogeneous enough t o pass through common garden spray equipment. T h i s would make the enzyme q u i t e p r a c t i c a l f o r use i n ground a p p l i c a t i o n equipment. S t a b i l i t y of P a r a t h i o n Hydrolase The s t a b i l i t y of parathion hydrolase was determined f o r r e f r i gerator storage and room temperature storage. The enzyme s o l u t i o n i s stable under r e f r i g e r a t i o n ( 4 ) . The enzyme a l s o remains s t a b l e a t room temperature f o r long p e r i o d s ( 4 ) .

American Chemical Society Library 1155 16th St. N. W. Krueger and Seiber; Treatment andD. Disposal of Pesticide Wastes Washington, C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

348

TREATMENT AND DISPOSAL OF PESTICIDE WASTES

CH,

"

CH 3

DIAZINON

un

^CH I CH-C .

'1 H/ ^c-O-P^ 0

Ν

C I 1

2-CH

J 3

0-CH -CH 2

3

C

^ÇH

OXYPYRIMI DINE

CH 3 \ CH ^ 3

F i g u r e 1.

Chemical S t r u c t u r e s

506 SOIL

EXTRACT 2 0 0 ML ACETONE

FILTRATE

FILTER

FILTER

CAKE

ADD WATER

PARTITION WITH HEXANE

HEXANE

F i g u r e 2.

WATER

A n a l y t i c a l Methods to Determine D i a z i n o n i n S o i l


HONEYCUTT ET AL.


COLUMN 6'X2MM GLASS PACKED WITH 10X D C - 2 0 0 ON 8 0 / 1 0 0 GAS CHROfl Q

OVEN TEMPERATURE •

GAS FLOWS:

DETECTOR:

Figure 3 .

170*L

CARRIER ( H E ) -

40 M L / M I Ν .

HYDROGEN

-

50 ML/MIN.

AIR

-

100 M L / M I N .

FLAME PHOTOMETRIC (SULFUR MODE)

GC Conditions f o r A n a l y s i s o f D i a z i n o n

10G SOIL EXTRACT 80:20 METHANOL : WATER CENTRIFUGE

PELLET

SUPERNATANT

HPLC REVERSE PHASE WHATMAN PARTISIL ODS-3 ISOCRATIC MODE ME0H/NAH2P0i|(0.01M) 50/50 Figure 4.

HPLC A n a l y s i s o f Oxypyrimidine i n S o i l


350

TREATMENT AND DISPOSAL OF PESTICIDE WASTES

Degradation lase

of D i a z i n o n i n Greenhouse S o i l by P a r a t h i o n Hydro-

P a r a t h i o n hydrolase degrades d i a z i n o n r a p i d l y at high concent r a t i o n s i n s o i l . Table 4 shows a summary of the r e s u l t s of the attempt to degrade high concentrations of d i a z i n o n with p a r a t h i o n hydrolase. The f i r s t column shows that d i a z i n o n degrades very slowly a t 500 ppm with no enzyme present. The subsequent columns show that p a r a t h i o n hydrolase r a p i d l y and e f f e c t i v e l y degrades high concentrations of d i a z i n o n . At 2000 ppm the h a l f - l i f e of d i a z i n o n , when enzyme i s present, i s about 4-5 hours. At 5000 ppm the enzyme i s not as e f f e c t i v e . This may be due to the l a r g e amount of D i a z i n o n 4E present which could i n h i b i t the enzyme. Table IV.

E f f i c a c y of P a r a t h i o n Hydrolase Decontamination D i a z i n o n 4E Georgia Sandy Loam S o i l

of

1

Average ppm of D i a z i n o n Remaining i n S o i l from Treatments , Time A f t e r 500 A p p l i c a t i o n (No Enzyme) 0.0 Hour 0.5 Hour 2.0 Hours 4.0 Hours 1 Day 2 Days 1 Week 3 Weeks

3

500 618+ 91 398+133 627+ 25 547+ 26 634+ 12 501+ 9 109+ 11

500

3

500 102+13 33+ 8 15+ 2 3+ 0 2+ 0.7

Degradation of High Concentrations of a Phosphorothioic Ester by

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