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The fate of dichlorvos in soil
0045-6535/78/1001-0807~02.00/0
Chemosyhere No. 10, pp 807 - 814. ©Pergamon
Press Ltd. 1978. Printed in Great Britain.
THE
FATE
OF
DICHLORVOS
IN
SOIL +
Robert J. Lamoreaux and Leo W. Newland Department of Biology and the Environmental Sciences Progr~n Texas Christian University Ft. Worth, Texas 76129, U.S.A.
Abstract
Experiments were conducted to determine the factors responsible for the loss (adsorption, c~emical hydrolysis, microbial degradation, etc.) of dichlorvos (2,2-dichlorovinyl O,O--dimethyl phosphate) in soil perfusion systems of Houston Black clay. The rate of disappearance from the p~rfusate (hence the rate of dichlorvos degradation in soil) was related directly to the preseance of Bacillus cereus in the perfusion system, the pH of the system, and the extent of dichlorvos adsorption. Gas liquid chromatographic analyses of the perfusates showed that dichlorvos disappearance was rapid when B. cereus was added to a previously sterilized soil perfusion system (50% in 3.9 days). Under ~ter~-~econditions, 50% of the added dichlorvos was recovered after 10 days. When B. cereus was added to a mineral salts medium containing dichlorvos as sole carbon source, 49% of the-~nitial dichlorvos concentration was degraded in 4 days. The organism was not capable of utilizing dichlorvos as a sole phosphorus source. Chemical hydrolysis of d{chlorvos in aqueous, buffered, soil-free systems showed that hydrolysis did not occur in very acid systems (< pH 3.3), but increased with increasing pH values(26% in 4 days at pH 6.9), and wcs rapid at pH 9.3 (> 99% in 2 days). The extent of dichlorvos adsorption was dete~nined by comparing the initial loss of dichlorvos in a sterile, soil-free extract solution with the initial ~css in a sterile soil perfusion system. The rapid initial disappearance of dichlorvos in the presence of sterilized soil was attributed to soil adsorption of the pesticide. After 10 days beth systems contained equal concentrations(50%) of dichlorvos. Non-biological mechanisms accounted for 70% of the total degradation of dichlorvos, while bacterial degradation accounted for 36% in the soil perfusion systems. Introduction
Organic phosphorus insecticides display a wide range of stability towards environmental degradation but are generally regarded as not persistent and are known to be degraded chemically and by a variety of microor~ani~ns I . Dichlorvos or DDVP (2,2-dichlorovinyl-O,O-dimethyl phosphate), synthesized in 1955z and introduced primarily for use against flies, mosquitoes, and other d~nestic pests decomposes "slowly in the presence of water, with 50% hydrolyzed at 20% in approximately 62 days". In the presence of alkali, hydrolysis occurs rapidly forming primarily dimethyl phosphate and dichloroacetaldehyde 3 . In acidic solutions the reaction occurs more slowly 4 Hodgson and Casida 5 studied the metabolism of DDVP in mammalian tissues and reported hydrolysis products of dimethyl phosphate and des~ethyldichlorvos which further hydrolyzed to monoethyl phosphate and eventually to inorganic phosphate. The dichlorovinyl portion of the molecule was
Presented at the Fourth International Congress of Pesticide Chemistry ~978, Zurich, Switzerland.
807
(IUPAC), July 24-28,
808
No. I0
t r a n s f o r m e d to d i c h l o r o a c e t a l d e h y d e w h i c h was further r e d u c e d to d i c h l o r o e t h a n o l and trace quantities of d i c h l o r o a c e t i c acid. Studies of the d e g r a d a t i o n of D D V P by b a c t e r i a and other m i c r o o r g a n i s m s are few. M a t s u m u r a and Boush 6 have shown that the soil fungus Trichoder~a uiride rapidly d e g r a d e d DDVP. The bacterium Pseudomonas melophthora, an o b l i g a t e e x t r a c e l l u l a r symbiote of the apple maggot, Rhagoletis pc~oneZ~a, was shown to r a p i d l y degrade DDVP with only 8.3% of the o r i g i n a l insecticide remaining after incubation for 24 hours at 30°C 7. The m a j o r p a t h w a y of d e g r a d a t i o n a p p e a r e d to be hydrolysis by esterases. By m e a s u r i n g the loss of toxicity of several o r g a n o p h o s p h a t e insecticides tc m o s q u i t o larvae (Culex pipiens), Yasuno et al. 8 r e p o r t e d that DDVP h a d completely lost its i n s e c t i c i d a l a c t i v i t y w i t h i n 16 days. The i n a c t i v i a t i o n was a t t r i b u t e d to and a s s o c i a t e d with the g r o w t h of Bacillus subtilis. A t t e m p t s were m a d e in this i n v e s t i g a t i o n to study the d e g r a d a t i o n activities of Bacillus cereus, a spore forming o r g a n i s m w h i c h occurs w i d e l y in soil. Spore forming b a c t e r i a constitute an i m p o r t a n t p a r t of the soil mieroflora. Five to twenty p e r c e n t of the organisms f o u n d in the A h o r i z o n (top soil) have b e e n reported to be strains of Bacillus, w i t h B. cer~{~, ~. ~atoYe~z6~i and B. subtilis being e n c o u n t e r e d m o s t frequently 9. In the U.S.S.R., soils have b e e n characterized by particular groups of Bacillus species p r e s e n t l 0 . B a c t e r i a l endospores of Bacillus are capable of w i t h s t a n d i n g u n f a v o r a b l e enviror~nental conditions such as lack of water, d e p l e t i o n of available food, and m a r k e d t e m p e r a t u r e deviations. Since Bacillus cereus spores are u b i q u i t o u s in soil, it m a y be feasible to add an i n o c u l a t e d energy source, e.g., b i o l o g i c a l wastes to aid in the d e t o x i f i c a t i o n of soils by a c c e l e r a t i n g the b r e a k d o w n of DDVP. The r e s e a r c h d e s c r i b e d h e r e i n was d e s i g n e d to i n v e s t i g a t e the factors w h i c h determine the fate of d i c h l o r v o s in and to examine the ability of Bacillus cereus to degrade DDVP in soil.
M e t h o d s and M a t e r i a l s
Reagents and Solvents: D i c h l o r v o s (100%) was supplied by City C h e m i c a l Corporation, New York. The solvent, r e a g e n t g r a d e n-hexane, u s e d in the e x t r a c t i o n p r o c e d u r e was p u r c h a s e d from M a t h e s o n Chemical Company and p u r i f i e d by glass d i s t i l l a t i o n . A n h y d r o u s , g r a n u l a r sodium sulfate was activated by h e a t i n g at 300°C o v e r n i g h t and storing at 130°C II. G l a s s w a r e for use in gas liquid c h r o m a t o g r a p h y was cleaned a c c o r d i n g to p r o c e d u r e No. 4 o u t l i n e d in Bevenue et al. 12 w h i c h involved w a s h i n g the glassware in sodium d i c h r o m a t e - c o n c e n t r a t e d sulfuric acid. B a c t e r i a l Isolation and Classification. Pure cultures of Bacillus cereus were o b t a i n e d by streaking d i r e c t l y from a soil water m i x ture agar (Difco) plates. C o l o n i e s with a rough, w h i t i s h appearance and w h i p - l i k e o u t g r o w t h s were s e l e c t e d a f t e r 48 h o u r s i n c u b a t i o n at 26°C and t r a n f e r r e d again to n u t r i e n t agar plates. Each culture was tested and c o m p a r e d m o r p h o l o g i c a l l y and b i o c h e m i c a l l y to a s c e r t a i n its identity. I d e n t i f i c a t i o n was based on the a c c e p t e d c h a r a c t e r i s t i c s in B e r g e y ' s M a n u a l 13. Sterile b a c t e r i o logical p r o c e d u r e s were s t r i n g e n t l y followed in all cases. DDVP as a C and P Source. The i s o l a t e d organism, B. cereus, was tested for its ability to u t i l i z e the pesticide as a sole source of carbon and p h o s p h o r u s by s u b s t i t u t i n g D D V P for glucose or K2HPO4, respectively, in a m i n e r a l salts s o l u t i o n (MSS). C o m b i n a t i o n s of ingredients u s e d in this e x p e r i m e n t are d e s c r i b e d in Table i. The u n a m e n d e d MSS contained the following ingredients: M g S O 4 . 7 H 2 0 , @.ig; C a S O 4 . 2 H 2 0 , 0.1g; NaNO3, 0.1g; NH4CI, 0.2g; F e C I ] . 6 H 2 0 , 0.002g; KNO3, 0.4g; and I liter d i s t i l l e d water. The m e d i u m was a u t o c l a v e d for 15 m i n u t e s at 1.02 atm. (15 psi) and 121°C to achieve sterilization. The final pH was a d j u s t e d to 6.7 u s i n g NaOH or HCI. The amended MSS was p r e p a r e d by adding sufficient amounts of a 10% g l u c o s e solution, p r e v i o u s l y s t e r i l i z e d by filtration, for a final concentration of i%. The K 2 H P O 4 (l.6g/l) was added p r i o r to s t e r i l i z a t i o n for those e x p e r i m e n t s req u i r i n g it. F o l l o w i n g s t e r i l i z a t i o n of the medium, s u f f i c i e n t amounts of p e s t i c i d e were added so that the final c o n c e n t r a t i o n was 1.000g/l. Portions of 300 m l each were p l a c e d in sterile flasks and i n o c u l a t e d w i t h i ml of a 24-hour r e s t i n g cell suspension. Duplicate inoculated flasks were then p l a c e d on a r e c i p r o c a l shaker and incubated at room tempertaure (26°C). After about 2 days, some of the m e d i a became turbid indicative of b a c t e r i c a l growth.
No.
i0
809
Table i.
B a c t e r i a l g r o w t h and D D V P b r e a k d o w n in v a r i o u s aqueous m e d i a
Media
MSS
(No DDVP)
Growth
% D D V P Remaining
x
O
M S S + K 2 H P O 4 (No DDVP)
O
M S S + K2HPO 4 + D D V P
+
51
M S S + K 2 H P O 4 + D C V P + glucose
+
61
M S S + D D V P + glucose
0
94
MSS + DDVP
0
80
DDVP
0
84
X A n a l y z e d by G L C after 4 days of i n c u b a t i o n
Scil P e r f u s i o n To determine the extent of a d s o r p t i o n and the rate of d e g r a d a t i o n of D ~ P in soil, a p e r f u sion was employed. The p e r f u s i o n technique was similar to that u s e d b y Lees and m o d i f i e d b y A r m s t r o n g et aZ. 15 and i n v o l v e d c o n t i n u a l l y perfusing a column of soil w i t h an aqueous s o l u t i o n c o n t a i n i n g 1000mg/l DDVP. The soil had b e e n p r e v i o u s l y s t e r i l i z e d and then an inoculum of B. o e P ~ 8 had been added. The p e r f u s i o n u n i t was e m p l o y e d b e c a u s e it p r o v i d e d an aerobic, enriched soil m e d i u m with r e l a t i v e l y clear p e r f u s a t e for analysis. It also allowed a c o n t r o l l e d c o n s t a n t rate of p e r f u s i o n w i t h o u t contact with p l a s t i c or rubber tubing w h i c h m a y adsorb the insecticide. Samples of the p e r f u s a t e were r e m o v e d p e r i o d i c a l l y through a sampling tube, w i t h o u t d i s c o n t i n u a n c e of perfusion, and a n a l y z e d for DDVP content, pH, and b a c t e r i a l numbers. The p e r f u s i o n u n i t was s t e r i l i z e d s e p a r a t e l y from the soil by a u t o c l a v i n g p r i o r to the a d d i t i o n of the p e s t icide and bacteria. The air u s e d to perfuse the soil was f i l t e r e d through a sterilized cotton plug. Initially, p e r f u s i o n was p e r f o r m e d u s i n g s t e r i l i z e d soil to a s c e r t a i n the rate and e x t e n t of adsorption. F o r the b a c t e r i a l d e g r a d a t i o n investigations, i m l of an a c t i v e l y growing pure b r o t h culture was p i p p e t t e d into a sterile soil p e r f u s i o n u n i t containing Houston Black clay. The initial number of b a c t e r i a i n t r o d u c e d into the p e r f u s i o n unit, and the rate of b a c t e r i a l growth were e s t i m a t e d by the decimal dilution, spread plate technique. A series of 9 m l sterile buffer w a t e r blanks 16 was u s e d to obtain the d e s i r e d ten-fold dilutions. To obtain plates with b e t w e e n 30 and 300 colonies, 0.1 m l q u a n t i t i e s were t r a n s f e r r e d to 100 x 15 n~n sterile p l a s t i c petri dishes c o n t a i n i n g n u t r i e n t agar and were incubated for 48 hours at room temperature. Each d i l u t i o n was p r e p a r e d in triplicate with an average of three countable plates used for the actual counts. Soil Analysis The glass column of the p e r f u s i o n u n i t was p a c k e d with a p p r o x i m a t e l y I00 grams of Houston Black clay collected from e a s t e r n T a r r a n t County, Texas. The p H of the soil was d e t e r m i n e d in a I:i soil to distilled w a t e r suspension w i t h a glass electrode 17, u s i n g a S a r g e n t - W e l c h M o d e l LS p H meter. Soil p H was 7.7 p r i o r to autoclaving, and 8.0 after. Soil o r g a n i c m a t t e r content was 3.26% and was d e t e r m i n e d by w e i g h t a f t e r ignition 17. W a t e r was d r i v e n off by heating at IIC°C overnight; the organic m a t t e r was oxidized by h e a t i n g at 3 5 0 - 4 0 0 ° C for 7-8 hours. Soil S t e r i l i z a t i o n The soil was s t e r i l i z e d p r i o r to its use in the p e r f u s i o n u n i t to d e t e r m i n e the d e g r a d a t i o n c a p a c i t y of the s e l e c t e d b a c t e r i a l species. L i c h t e n s t e i n and Schulz 18 have shown that soil autoclaved at 1.05 kg/cm 2 and 120°C for 4 days (7 hours daily) was not s t e r i l i z e d b u t the number of m i c r o o r g a n i s m s was reduced considerably. In their investigation, the f i r s t week a f t e r autoclaving s h o w e d s u b s t a n t i a l increases in the number of m i c r o o r g a n i s m s . Therefore, the authors conducted tests to d e t e r m i n e the amount of a u t o c l a v i n g n e c e s s a r y to achieve s t e r i l i z a t i o n of the Houston Black clay soil. I n t e r m i t t a n t a u t o c l a v i n g (Tyndallization) at 1.02 arm and 121°C
810
No. I0
for three l-hour p e r i o d s at l-day intervals (for 3 days total) proved s a t i s f a c t o r y for the Houston Black clay. 19 G e t z i n and R o s e f i e l d d e t e r m i n e d that nonviable, h e a t - l a b i l e substances were p r i m a r i l y resp o n s i b l e for the loss of d i c h l o r v o s in soil. They m e a s u r e d the d i f f e r e n c e b e t w e e n the amount of D[YVP d e c o m p o s e d in autoclaved, irradiated, and n o n s t e r i l e soil, and found that the amount of i n s e c t i c i d e d e g r a d e d a f t e r one d a y was 17, 88 and 99%,respectively. Because the soil to be used in this i n v e s t i g a t i o n was s u b j e c t e d to severe h e a t sterilization, it was n e c e s s a r y to determine the extent to w h i c h the a d s o r p t i v e p r o p e r t i e s of the Houston Black clay were altered. For this d e t e r m i n a t i o n , i g r a m each of nonsterile and sterile soil was placed in separate 30 m l centrifuge tubes containing 20 m l d i s t i l l e d w a t e r and I00 mg/l DDVP. Tubes were shaken v i g o r o u s l y for 3 m i n u t e s f o l l o w e d by the low speed centrifugation, extraction, and analysis of the p a r e n t cemp~ind. A 158 em x 4 m m Pyrex c h r e m a t o g r a p h i c column packed with 2% Reoplex-400 (polypropylene glycol adipate) on 80/100 m e s h Gas Chrom Q was employed. However, with continued use, severe column b l e e d i n g o c c u r r e d r e q u i r i n g an alternate column packing. Ten p e r c e n t DEGS (diethylene g l y c o l succinate) on 60/80 m e s h G a s Chrem Q proved s a t i s f a c t o r y as an alternate for the separation and i d e n t i f i c a t i o n of DDVP. Operating conditions (Table 2) were chosen to achieve optimum b a l a n c e b e t w e e n s e n s i t i v i t y and r e s o l u t i o n with a relatively symmetrical peak and a reasonable r e t e n t i o n time. I d e n t i f i c a t i o n was b a s e d on retention time and c o n c e n t r a t i o n was determined by m e a s u r e m e n t of peak heights 20 and cempared to peak heights of k n o w n D D V P concentrations.
T a b l e 2. G a s Liquid c h r o m a t o g r a p h operating parameters. Liquid p h a s e
2% Reoplex-400
10% DEGS
Solid support
Gas Chrom Q
Gas Chrom
M e s h size
80/100
60/80
30 m l / m i n
86 m l / m i n
C a r r i e r gas
(N 2) flow rate
Hydrogen flow rate
60 m l / m i n
55 m l / m i n
O x y g e n flow rate
38 m l / m i n
40 m l / m i n
Air flow rate
200 m l / m i n
170 m l / m i n
Inlet t e m p e r a t u r e
170°C
170°C
Detector temperature
200°C
200°C
Column t e m p e r a t u r e
140°C
150°C
A p p r o x i m a t e retention time
1.5 m i n
3 min
Extraction. The e x t r a c t i o n p r o c e d u r e was similar to the m i c r o e x t r a c t i o n m e t h o d d e s c r i b e d by Halvorson 8~ aZ. 21 and was d e s i g n e d to recover insecticides from small samples (5 ml). DDVP was extracted be adding 5 m l each of sample and hexane to a 30 m l centrifuge tube and m i x i n g v i g o r o u s l y for 3C seconds on a V o r t e x G e n i e mixer. Emulsions were b r o k e n b y centrifugation. The water layer c o n t a i n i n g the i n s e c t i c i d e was e x t r a c t e d twice more, each time cc~bining the extracts and filtering the hexane layer through activated, p r e w e t t e d with distilled n-hexane, anydrous sodium sulfate to remove any e n t r a i n e d water. The combined extracts were c o n c e n t r a t e d utilizing a 250 m l c a p a c i t y K u d e r n a - D a n i s h concentrator fitted with a 3-ball Snyder column. Special cleanup procedures w e r e not n e c e s s a r y p r i o r to analysis by GLC. The e f f i c i e n c y of the e x t r a c t i o n p r o c e d u r e was d e t e r m i n e d to be 97-99%.
Results and D i s c u s s i o n
D i c h l o r v o s D i s s i p a t i o n in Soil Perfusion. D i c h l o r v o s c o n c e n t r a t i o n d e c r e a s e d r a p i d l y d u r i n g the I0 d a y i n c u b a t i o n period for the sterile and the n o n s t e r i l e soil p e r f u s i o n systems. The data in F i g u r e s 1 and 2 show losses of
No.
i0
811
71 and 50% under n o n s t e r i l e and sterile conditions, r e s p e c t i v e l y . T h e d a t a in F i g u r e i i n d i c a t e that i n s e c t i c i d e d i s a p p e a r a n c e was e n h a n c e d by the g r o w t h a c t i v i t i e s of ~icil~us cereus in the soil p e r f u s i o n system. H a l f - l i f e v a l u e s of D D V P (t½), were 3.9 and i0 days for n o n s t e r i l e and sterile systems, r e s p e c t i v e l y . However, the d e c r e a s e in c o n c e n t r a t i o n of d i c h l o r v o s in the n o n s t e r i l e system was largely due to m e c h a n i s m s o t h e r than those a s s o c i a t e d w i t h b a c t e r i a l g r o w t h since as m u c h as 50% was lost from the sterile p e r f u s i o n system.
pH
2
~
pH
5
5
90
7
1oo\
--
70
-~
60
5
~
OOVP
6
2
9O
"~
8O
.E
60
•-
40
'~ ~c
30
~.
20
50
40 30 cm
20 10
__1
3
10
l l l l t l l l l l
i
0 1 2 3 4 5 6 7 8 9 1 0
0
Time, days Figure
5
70
~
50 =
•
100
80
~
~
i. D i c h l o r v o s d e g r a d a t i o n soil p e r f u s i o n system.
U s e of D D V P as a C a r b o n
I
i
1 Z
i
i
3 4
i
i
,
5
6
7
~
8
,
i
9
10
__
Time, days in n o n s t e r i l e
and Phosphorus
Figure
2. D i c h l o r v o s in sterile p e r f u s i o n system.
soil
Source.
E v i d e n c e s u p p o r t i n g b a c t e r i a l d e g r a d a t i o n is seen in Table i. BacilZas cereus u t i l i z e d D D V P as a sole carbon source. A f t e r 4 days of incubation, 49% of the a d d e d DDVP was b r o k e n down, i.e., 51% was remaining. The o r g a n i s m was also capable of using D D V P s u p p l i e d as an a d d i t i o n a l carbon so'irce a l t h o u g h to a lesser e x t e n t than w h e n supplied as the sole source of carbon. These r e s u l t s s u g g e s t that the p h e n o m e n a of enzyme induction, the s y n t h e s i s of an enzyme o n l y w h e n a s u b s t r a t e is p r e s e n t 22, m a y have b e e n involved. By such a m e c h a n i s m D D V P could induce the s y n t h e s i s of an e s t e r a s e w h i c h u t i l i z e s the p e s t i c i d e as a substrate. Bacillus cereus has b e e n r e p o r t e d as an e s t e r a s e - a c t i v e m i c r o o r g a n i s m 23. The organism m a y m a n u f a c t u r e the enzyme r e q u i r e d f o r m e t a b o l i s m of D D V P supplied as a sole c a r b o n source, b u t w h e n an a d d i t i o n a l carbon source such as g l u c o s e is i n c l u d e d in the medium, the o r g a n i s m shows a p r e f e r e n c e for the a l t e r n a t e carbon source and p r o d u c e d little or no d i c h l o r v o s - u t i l i z i n g e s t e r a s e . F u r t h e r e x p e r i m e n t a t i o n w o u l d be r e q u i r e d to confirm these suggestions. D i c h l o r v o s was not u t i l i z e d by B. cereus w h e n supplied as the sole source of p h o s p h o r u s since no g r o w t h o c c u r r e d in those media. The b r e a k d o w n of D D V P in those m e d i a w h e r e no g r o w t h o c , ~ r r e d m u s t be a c c o u n t e d for by chemical hydrolysis. Effect
of pH on D D V P Loss.
B e c a u s e D D V P h y d r o l y s i s is c a t a l y z e d by the p r e s e n c e of OH-, an e x p e r i m e n t was c o n d u c t e d to examine the e f f e c t of pH on D D V P h y d r o l y s i s in aqueous s o i l - f r e e systams. Buffer s o l u t i o n s w e r e p r e p a r e d a c c o r d i n g to G o r t n e r 24 by v a r y i n g the amounts of sodium b o r a t e and h y d r o c h l o r i c acid or sodium h y d r o x i d e and r a n g e d from p H 2.0 to 9.3. R e s i d u a l D D V P was q u a n t i f i e d by G L C at 24 hour i n t e r v a l s for 96 hours (Table 3).
812
No. i0
Table 3. P e r c e n t D D V P r e m a i n i n g after i n c u b a t i o n in buffered, solutions.
soil-free aqueous
Hours of Incubation
pH
24
48
72
96
2.0
99
I00
i00
93 100
3.3
i00
100
i00
6.2
92
91
85
81
6.9
89
91
83
74
7.8
84
69
57
49
8.2
82
47
31
25
8.7
36
12
5
9.3
i0
< i
0
< 0
The data in Table 3 clearly show that dichlorvos is quite stable under acidic conditions. At pH v a l u e s less than or equal to 3.3, D D V P remained e s s e n t i a l l y u n c h a n g e d t h r o u g h o u t the 96 h o u r i n c u b a t i o n period. S u s c e p t i b i l i t y to hydrolysis increases as neutral pH was approached. At p H 6.9, 74% of the a d d e d insecticide r e m a i n e d after 96 hours. Hydrolysis was e x c e e d i n g l y rapid w i t h i n c r e a s i n g basicity. A t pH 9.3, D D V P could not be d e t e c t e d a f t e r 72 hours. Since the buffer solutions r e m a i n e d free of c o n t a n i n a t i n g m i c r o o r g a n i s m s , this rapid hydrolytic b r e a k d o w n m u s t be a t t r i b u t e d to pH. In the n o n - s t e r i l e soil p e r f u s i o n system (Figure i), pH increases from 6.2 to 7.4 in the f i r s t four days of i n c u b a t i o n and r e m a i n e d at 7.4 for the r e m a i n d e r of the incubation period. This increase in p H o c c u r r e d during and after the logarithmic and s t a t i o n a r y b a c t e r i a l growth phases, indicating that the change in pH is associated with the g r o w t h of B. c e r e u s . Approximately 25% of the initial p e s t i c i d e c o n c e n t r a t i o n was d e g r a d e d during these g r o w t h phases. Only 29% of the a d d e d insecticide could be r e c o v e r e d u p o n t e r m i n a t i o n of incubation. This rapid diss i p a t i o n of DDVP was a p p a r e n t l y due to both b a c t e r i a l d e g r a d a t i o n and the associated increase iE pH. However, in the sterile soil p e r f u s i o n system (Figure 2) p H v a l u e s tended toward an acid c o n d i t i o n i n d i c a t i n g that p H alone does not account for the d i s s i p a t i o n of DLVP from sterile soil and i m p l i c a t i n g s u r f a c e - c a t a l y z e d adsorption. A d s o r p t i o n of DDVP to Soil. To determine the extent of chemical d e g r a d a t i o n DDVP loss was m o n i t o r e d in sterilized soil extract. The soil e x t r a c t was p r e p a r e d according to A l l e n 25 and filtered through W h a t m a n No. 42 filter paper to remove soil particles. Figure 3 shows the r e s u l t s of this experiment. A compari son of the d a t a in F i g u r e s 2 and 3 indicate that a d s o r p t i o n a c c o u n t e d for the r a p i d initial disa p p e a r a n c e of the p e s t i c i d e since the curve r e p r e s e n t i n g DDVP loss in the sterile soil p e r f u s i o n system was non-linear. It should be p o i n t e d out that a d s o r p t i o n does not n e c e s s a r i l y m e a n degradation, o n l y a removal from the aqueous phase. At the end of the incubation period the perc6nt of p e s t i c i d e r e m a i n i n g in the solution was the same for both systems. The t½ v a l u e s for both systems was i0 days. C h e m i c a l and B a c t e r i o l o g i c a l Degradation. The e x p e r i m e n t a l data suggest that the d i s s i p a t i o n of D D V P from soil is the result of both c h e m i c a l and b a c t e r i o l o g i c a l m e c h a n i s m s . C h e m i c a l d e g r a d a t i o n is a p p a r a r e n t l y due to b a s e - c a t a lyzed hydrolysis. The relative i n v o l v e m e n t of these two d e g r a d a t i v e m e c h a n i s m s (chemical and bacteriological) was c a l c u l a t e d a c c o r d i n g to Walker and Stojanovic 26. The results are p r e s e n t e d in T a b l e 4.
No. l0
813
if
pH
--I ~
5
7 6 5
100 2
90 80
¢)
70 =
00
.~
50
N
40
~
30
~N
2O 10 I
I
I
I
]
I
I
I
L
0 1 2 3 4 5 0 7 8 9 1 0 Time, days Figure
Table
3. D i c h l o r v o s d e g r a d a t i o n in s t e r i l i z e d soil solution.
4. R e l a t i v e types of D D V P d e g r a d a t i o n of i n c u b a t i o n
Total D e g r a d a t i o n ,
Non-sterile Soil P e r f u s i o n
Black
clay f o l l o w i n g
% Total D e g r a d a t i o n
%
Sterile Soil P e r f u s i o n
71
+ % chemical
in Houston
Chemical + Mechanisms
50
Due
10 days
to
Bacteriological m e c h a n i e m s ++
70
30
degradation
++ % bacteriological
= % d e g r a d a t i o n under s t e r i l e c o n d i t i o n s % total d e g r a d a t i o n (non-sterile) d e g r a d a t i o n = i00 - % chemical d e g r a d a t i o n
The r e s u l t s in Table 4 indicate that the m a j o r m e c h a n i s m D D V P frc~ soil was chemical degradation.
responsible
x I00
for the d i s a p p e a r a n c e
of
CONCLUSIONS
I. The rapid d i s a p p e a r a n c e of d i c h l o r v o s observed in H o u s t o n B l a c k clay is the r e s u l t of a d s o r p tion to soil particles, and c h e m i c a l and b a c t e r i a l degradation. 2. Soil adsorption, and chemical d e g r a d a t i o n a c c o u n t for 70% of the rapid d i s a p p e a r a n c e of D D V P from the soil. 3. A d i c h l o r v o s - u t i l i z i n g esterase, i n d u c e d by S° oereTls, is h y p o t h e s i z e d to have b e e n r e s p o n sible f o r the loss of D D V P from a m i n e r a l salts s o l u t i o n c o n t a i n i n g the p e s t i c i d e as a sole carbon source. This enzyme system m a y also be i m p l i c a t e d in the d i s s i p a t i o n of D D V P in soil since ft. c ~ r ~ s is r e s p o n s i b l e for about 30% of the total d e g r a d a t i o n of the p e s t i c i d e in the soil p e r f u s i o n system. 4. The r a t e of chemical h y d r o l y s i s is a function of pH, i n c r e a s i n g at h i g h e r p H values.
814
No. i0
ACKNOWLEDGEMENT
The authors express appreciation to the Texas Christian University Research Foundation for support of this research.
REFERENCES
I. ~. 7. 4. ~. C. 7. ~. <,. 10.
R.J. Lamoreaux and L.W. Newland, Biol. Conserv., ii, 59 (1977). A.M. Mattson, J.T. Spillane, and G.W. Pearce, J. Agr. Food Chem., 3, 319 (1955). Shell Chemical Company, PMS-G-913/69, 6, (1969). N.N. Melnikov, Chemistry of Pesticides, Springer-Verlag, New York, (1971). E. Hodgson and J.E. Casida, J_2_.A@r. Food Chem., i0, 207 (1962). F. Matsumura and G.M. Boush, J. Econ. Entemol., 61, 610, (1968). G.M. Boush and F. Matsumura, J. Econ. Entomol., 60, 918, (1967). M. Yasu~o, S. Hirakoso, M. Sasa, and M. Uchida, Japan Jour. Exp. Med., 35, 545, (1965). M. Alexander, Introduction to Soil Microbiology, John Wiley and Sons, Inc., New York, (1961) E.N. Nishustin and V.A. Nisroeva, The Ecology of Soil Bacteria, University of Toronto Press, (1968). 1~. D.F. Goerlitz and E. Brown, Methods for Analysis o f Organic Substances in Water, Washington, U.S. Government Printing Office, (1972). i~I. T. Bevenue, T.W. Kelley, and J.W. Hylin, J. Chromatog., 54, 17, (1971). I~. R.S. Breed, E.G.D. Murray, and N.R. Smith, Ber~ey's Manual of Determinative Bacteriology, 7th. ed., The Williams and Wilkins Co., Baltimore, (1957). l.l.H. Lees, J_t.Agr. Sci., 37, 27, (1947). 15. D.E. Armstrong, G. Chester s, and R.F. Harris, Soil Sci. Soc. Amer. Proc., 31, 61 (1967). i~). American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 13th. ed., Washington, D.C., (1971). 1"I. M.L. Jackson, Soil Chamical Analysis, Prentice Hall, Inc., Englewood Cliffs, N.J., (1958). I;3. E.P. Lichtenstein, and K.R. Schulz, J. Econ. Entomol., 53, 192 (1960). 19. L.W. Getzin and I. Rosefield, J__u.Agr. Food Chem., 16, 958 (1968). 2). U.S. Department of Health, Education and Welfare, Pesticide Analytical Manual, (1975). 21. H. Halvorson, M. Ishaque, J. Solomon, and O.W. Grussendorf, J__t.Microbiol., 17, 585, (1971). 22. T.D. Brock, Biology o f Microorganisms, Prentice-Hall, Inc., Englewood Cliffs, N.J., (1970). 23. S. Aarsonson, Experimental Microbial Ecology, Academic Press, New York,(1970). 21. R.A. Gortner, Outlines o f Biochemistry , John Wiley and Sons, Inc., New York, (1950). 25. O.N. Allen, Experiments i__n_nSoil Bacteriology, Burgess Publishing, Minneapolis, (1949). 26. W.W. Walker, and B.J. Stojanovic, J__u.Environ. Quality, ~, 229, (1973).
(Received in The Netherlands 28 August 1978)
Chemosyhere No. 10, pp 807 - 814. ©Pergamon
Press Ltd. 1978. Printed in Great Britain.
THE
FATE
OF
DICHLORVOS
IN
SOIL +
Robert J. Lamoreaux and Leo W. Newland Department of Biology and the Environmental Sciences Progr~n Texas Christian University Ft. Worth, Texas 76129, U.S.A.
Abstract
Experiments were conducted to determine the factors responsible for the loss (adsorption, c~emical hydrolysis, microbial degradation, etc.) of dichlorvos (2,2-dichlorovinyl O,O--dimethyl phosphate) in soil perfusion systems of Houston Black clay. The rate of disappearance from the p~rfusate (hence the rate of dichlorvos degradation in soil) was related directly to the preseance of Bacillus cereus in the perfusion system, the pH of the system, and the extent of dichlorvos adsorption. Gas liquid chromatographic analyses of the perfusates showed that dichlorvos disappearance was rapid when B. cereus was added to a previously sterilized soil perfusion system (50% in 3.9 days). Under ~ter~-~econditions, 50% of the added dichlorvos was recovered after 10 days. When B. cereus was added to a mineral salts medium containing dichlorvos as sole carbon source, 49% of the-~nitial dichlorvos concentration was degraded in 4 days. The organism was not capable of utilizing dichlorvos as a sole phosphorus source. Chemical hydrolysis of d{chlorvos in aqueous, buffered, soil-free systems showed that hydrolysis did not occur in very acid systems (< pH 3.3), but increased with increasing pH values(26% in 4 days at pH 6.9), and wcs rapid at pH 9.3 (> 99% in 2 days). The extent of dichlorvos adsorption was dete~nined by comparing the initial loss of dichlorvos in a sterile, soil-free extract solution with the initial ~css in a sterile soil perfusion system. The rapid initial disappearance of dichlorvos in the presence of sterilized soil was attributed to soil adsorption of the pesticide. After 10 days beth systems contained equal concentrations(50%) of dichlorvos. Non-biological mechanisms accounted for 70% of the total degradation of dichlorvos, while bacterial degradation accounted for 36% in the soil perfusion systems. Introduction
Organic phosphorus insecticides display a wide range of stability towards environmental degradation but are generally regarded as not persistent and are known to be degraded chemically and by a variety of microor~ani~ns I . Dichlorvos or DDVP (2,2-dichlorovinyl-O,O-dimethyl phosphate), synthesized in 1955z and introduced primarily for use against flies, mosquitoes, and other d~nestic pests decomposes "slowly in the presence of water, with 50% hydrolyzed at 20% in approximately 62 days". In the presence of alkali, hydrolysis occurs rapidly forming primarily dimethyl phosphate and dichloroacetaldehyde 3 . In acidic solutions the reaction occurs more slowly 4 Hodgson and Casida 5 studied the metabolism of DDVP in mammalian tissues and reported hydrolysis products of dimethyl phosphate and des~ethyldichlorvos which further hydrolyzed to monoethyl phosphate and eventually to inorganic phosphate. The dichlorovinyl portion of the molecule was
Presented at the Fourth International Congress of Pesticide Chemistry ~978, Zurich, Switzerland.
807
(IUPAC), July 24-28,
808
No. I0
t r a n s f o r m e d to d i c h l o r o a c e t a l d e h y d e w h i c h was further r e d u c e d to d i c h l o r o e t h a n o l and trace quantities of d i c h l o r o a c e t i c acid. Studies of the d e g r a d a t i o n of D D V P by b a c t e r i a and other m i c r o o r g a n i s m s are few. M a t s u m u r a and Boush 6 have shown that the soil fungus Trichoder~a uiride rapidly d e g r a d e d DDVP. The bacterium Pseudomonas melophthora, an o b l i g a t e e x t r a c e l l u l a r symbiote of the apple maggot, Rhagoletis pc~oneZ~a, was shown to r a p i d l y degrade DDVP with only 8.3% of the o r i g i n a l insecticide remaining after incubation for 24 hours at 30°C 7. The m a j o r p a t h w a y of d e g r a d a t i o n a p p e a r e d to be hydrolysis by esterases. By m e a s u r i n g the loss of toxicity of several o r g a n o p h o s p h a t e insecticides tc m o s q u i t o larvae (Culex pipiens), Yasuno et al. 8 r e p o r t e d that DDVP h a d completely lost its i n s e c t i c i d a l a c t i v i t y w i t h i n 16 days. The i n a c t i v i a t i o n was a t t r i b u t e d to and a s s o c i a t e d with the g r o w t h of Bacillus subtilis. A t t e m p t s were m a d e in this i n v e s t i g a t i o n to study the d e g r a d a t i o n activities of Bacillus cereus, a spore forming o r g a n i s m w h i c h occurs w i d e l y in soil. Spore forming b a c t e r i a constitute an i m p o r t a n t p a r t of the soil mieroflora. Five to twenty p e r c e n t of the organisms f o u n d in the A h o r i z o n (top soil) have b e e n reported to be strains of Bacillus, w i t h B. cer~{~, ~. ~atoYe~z6~i and B. subtilis being e n c o u n t e r e d m o s t frequently 9. In the U.S.S.R., soils have b e e n characterized by particular groups of Bacillus species p r e s e n t l 0 . B a c t e r i a l endospores of Bacillus are capable of w i t h s t a n d i n g u n f a v o r a b l e enviror~nental conditions such as lack of water, d e p l e t i o n of available food, and m a r k e d t e m p e r a t u r e deviations. Since Bacillus cereus spores are u b i q u i t o u s in soil, it m a y be feasible to add an i n o c u l a t e d energy source, e.g., b i o l o g i c a l wastes to aid in the d e t o x i f i c a t i o n of soils by a c c e l e r a t i n g the b r e a k d o w n of DDVP. The r e s e a r c h d e s c r i b e d h e r e i n was d e s i g n e d to i n v e s t i g a t e the factors w h i c h determine the fate of d i c h l o r v o s in and to examine the ability of Bacillus cereus to degrade DDVP in soil.
M e t h o d s and M a t e r i a l s
Reagents and Solvents: D i c h l o r v o s (100%) was supplied by City C h e m i c a l Corporation, New York. The solvent, r e a g e n t g r a d e n-hexane, u s e d in the e x t r a c t i o n p r o c e d u r e was p u r c h a s e d from M a t h e s o n Chemical Company and p u r i f i e d by glass d i s t i l l a t i o n . A n h y d r o u s , g r a n u l a r sodium sulfate was activated by h e a t i n g at 300°C o v e r n i g h t and storing at 130°C II. G l a s s w a r e for use in gas liquid c h r o m a t o g r a p h y was cleaned a c c o r d i n g to p r o c e d u r e No. 4 o u t l i n e d in Bevenue et al. 12 w h i c h involved w a s h i n g the glassware in sodium d i c h r o m a t e - c o n c e n t r a t e d sulfuric acid. B a c t e r i a l Isolation and Classification. Pure cultures of Bacillus cereus were o b t a i n e d by streaking d i r e c t l y from a soil water m i x ture agar (Difco) plates. C o l o n i e s with a rough, w h i t i s h appearance and w h i p - l i k e o u t g r o w t h s were s e l e c t e d a f t e r 48 h o u r s i n c u b a t i o n at 26°C and t r a n f e r r e d again to n u t r i e n t agar plates. Each culture was tested and c o m p a r e d m o r p h o l o g i c a l l y and b i o c h e m i c a l l y to a s c e r t a i n its identity. I d e n t i f i c a t i o n was based on the a c c e p t e d c h a r a c t e r i s t i c s in B e r g e y ' s M a n u a l 13. Sterile b a c t e r i o logical p r o c e d u r e s were s t r i n g e n t l y followed in all cases. DDVP as a C and P Source. The i s o l a t e d organism, B. cereus, was tested for its ability to u t i l i z e the pesticide as a sole source of carbon and p h o s p h o r u s by s u b s t i t u t i n g D D V P for glucose or K2HPO4, respectively, in a m i n e r a l salts s o l u t i o n (MSS). C o m b i n a t i o n s of ingredients u s e d in this e x p e r i m e n t are d e s c r i b e d in Table i. The u n a m e n d e d MSS contained the following ingredients: M g S O 4 . 7 H 2 0 , @.ig; C a S O 4 . 2 H 2 0 , 0.1g; NaNO3, 0.1g; NH4CI, 0.2g; F e C I ] . 6 H 2 0 , 0.002g; KNO3, 0.4g; and I liter d i s t i l l e d water. The m e d i u m was a u t o c l a v e d for 15 m i n u t e s at 1.02 atm. (15 psi) and 121°C to achieve sterilization. The final pH was a d j u s t e d to 6.7 u s i n g NaOH or HCI. The amended MSS was p r e p a r e d by adding sufficient amounts of a 10% g l u c o s e solution, p r e v i o u s l y s t e r i l i z e d by filtration, for a final concentration of i%. The K 2 H P O 4 (l.6g/l) was added p r i o r to s t e r i l i z a t i o n for those e x p e r i m e n t s req u i r i n g it. F o l l o w i n g s t e r i l i z a t i o n of the medium, s u f f i c i e n t amounts of p e s t i c i d e were added so that the final c o n c e n t r a t i o n was 1.000g/l. Portions of 300 m l each were p l a c e d in sterile flasks and i n o c u l a t e d w i t h i ml of a 24-hour r e s t i n g cell suspension. Duplicate inoculated flasks were then p l a c e d on a r e c i p r o c a l shaker and incubated at room tempertaure (26°C). After about 2 days, some of the m e d i a became turbid indicative of b a c t e r i c a l growth.
No.
i0
809
Table i.
B a c t e r i a l g r o w t h and D D V P b r e a k d o w n in v a r i o u s aqueous m e d i a
Media
MSS
(No DDVP)
Growth
% D D V P Remaining
x
O
M S S + K 2 H P O 4 (No DDVP)
O
M S S + K2HPO 4 + D D V P
+
51
M S S + K 2 H P O 4 + D C V P + glucose
+
61
M S S + D D V P + glucose
0
94
MSS + DDVP
0
80
DDVP
0
84
X A n a l y z e d by G L C after 4 days of i n c u b a t i o n
Scil P e r f u s i o n To determine the extent of a d s o r p t i o n and the rate of d e g r a d a t i o n of D ~ P in soil, a p e r f u sion was employed. The p e r f u s i o n technique was similar to that u s e d b y Lees and m o d i f i e d b y A r m s t r o n g et aZ. 15 and i n v o l v e d c o n t i n u a l l y perfusing a column of soil w i t h an aqueous s o l u t i o n c o n t a i n i n g 1000mg/l DDVP. The soil had b e e n p r e v i o u s l y s t e r i l i z e d and then an inoculum of B. o e P ~ 8 had been added. The p e r f u s i o n u n i t was e m p l o y e d b e c a u s e it p r o v i d e d an aerobic, enriched soil m e d i u m with r e l a t i v e l y clear p e r f u s a t e for analysis. It also allowed a c o n t r o l l e d c o n s t a n t rate of p e r f u s i o n w i t h o u t contact with p l a s t i c or rubber tubing w h i c h m a y adsorb the insecticide. Samples of the p e r f u s a t e were r e m o v e d p e r i o d i c a l l y through a sampling tube, w i t h o u t d i s c o n t i n u a n c e of perfusion, and a n a l y z e d for DDVP content, pH, and b a c t e r i a l numbers. The p e r f u s i o n u n i t was s t e r i l i z e d s e p a r a t e l y from the soil by a u t o c l a v i n g p r i o r to the a d d i t i o n of the p e s t icide and bacteria. The air u s e d to perfuse the soil was f i l t e r e d through a sterilized cotton plug. Initially, p e r f u s i o n was p e r f o r m e d u s i n g s t e r i l i z e d soil to a s c e r t a i n the rate and e x t e n t of adsorption. F o r the b a c t e r i a l d e g r a d a t i o n investigations, i m l of an a c t i v e l y growing pure b r o t h culture was p i p p e t t e d into a sterile soil p e r f u s i o n u n i t containing Houston Black clay. The initial number of b a c t e r i a i n t r o d u c e d into the p e r f u s i o n unit, and the rate of b a c t e r i a l growth were e s t i m a t e d by the decimal dilution, spread plate technique. A series of 9 m l sterile buffer w a t e r blanks 16 was u s e d to obtain the d e s i r e d ten-fold dilutions. To obtain plates with b e t w e e n 30 and 300 colonies, 0.1 m l q u a n t i t i e s were t r a n s f e r r e d to 100 x 15 n~n sterile p l a s t i c petri dishes c o n t a i n i n g n u t r i e n t agar and were incubated for 48 hours at room temperature. Each d i l u t i o n was p r e p a r e d in triplicate with an average of three countable plates used for the actual counts. Soil Analysis The glass column of the p e r f u s i o n u n i t was p a c k e d with a p p r o x i m a t e l y I00 grams of Houston Black clay collected from e a s t e r n T a r r a n t County, Texas. The p H of the soil was d e t e r m i n e d in a I:i soil to distilled w a t e r suspension w i t h a glass electrode 17, u s i n g a S a r g e n t - W e l c h M o d e l LS p H meter. Soil p H was 7.7 p r i o r to autoclaving, and 8.0 after. Soil o r g a n i c m a t t e r content was 3.26% and was d e t e r m i n e d by w e i g h t a f t e r ignition 17. W a t e r was d r i v e n off by heating at IIC°C overnight; the organic m a t t e r was oxidized by h e a t i n g at 3 5 0 - 4 0 0 ° C for 7-8 hours. Soil S t e r i l i z a t i o n The soil was s t e r i l i z e d p r i o r to its use in the p e r f u s i o n u n i t to d e t e r m i n e the d e g r a d a t i o n c a p a c i t y of the s e l e c t e d b a c t e r i a l species. L i c h t e n s t e i n and Schulz 18 have shown that soil autoclaved at 1.05 kg/cm 2 and 120°C for 4 days (7 hours daily) was not s t e r i l i z e d b u t the number of m i c r o o r g a n i s m s was reduced considerably. In their investigation, the f i r s t week a f t e r autoclaving s h o w e d s u b s t a n t i a l increases in the number of m i c r o o r g a n i s m s . Therefore, the authors conducted tests to d e t e r m i n e the amount of a u t o c l a v i n g n e c e s s a r y to achieve s t e r i l i z a t i o n of the Houston Black clay soil. I n t e r m i t t a n t a u t o c l a v i n g (Tyndallization) at 1.02 arm and 121°C
810
No. I0
for three l-hour p e r i o d s at l-day intervals (for 3 days total) proved s a t i s f a c t o r y for the Houston Black clay. 19 G e t z i n and R o s e f i e l d d e t e r m i n e d that nonviable, h e a t - l a b i l e substances were p r i m a r i l y resp o n s i b l e for the loss of d i c h l o r v o s in soil. They m e a s u r e d the d i f f e r e n c e b e t w e e n the amount of D[YVP d e c o m p o s e d in autoclaved, irradiated, and n o n s t e r i l e soil, and found that the amount of i n s e c t i c i d e d e g r a d e d a f t e r one d a y was 17, 88 and 99%,respectively. Because the soil to be used in this i n v e s t i g a t i o n was s u b j e c t e d to severe h e a t sterilization, it was n e c e s s a r y to determine the extent to w h i c h the a d s o r p t i v e p r o p e r t i e s of the Houston Black clay were altered. For this d e t e r m i n a t i o n , i g r a m each of nonsterile and sterile soil was placed in separate 30 m l centrifuge tubes containing 20 m l d i s t i l l e d w a t e r and I00 mg/l DDVP. Tubes were shaken v i g o r o u s l y for 3 m i n u t e s f o l l o w e d by the low speed centrifugation, extraction, and analysis of the p a r e n t cemp~ind. A 158 em x 4 m m Pyrex c h r e m a t o g r a p h i c column packed with 2% Reoplex-400 (polypropylene glycol adipate) on 80/100 m e s h Gas Chrom Q was employed. However, with continued use, severe column b l e e d i n g o c c u r r e d r e q u i r i n g an alternate column packing. Ten p e r c e n t DEGS (diethylene g l y c o l succinate) on 60/80 m e s h G a s Chrem Q proved s a t i s f a c t o r y as an alternate for the separation and i d e n t i f i c a t i o n of DDVP. Operating conditions (Table 2) were chosen to achieve optimum b a l a n c e b e t w e e n s e n s i t i v i t y and r e s o l u t i o n with a relatively symmetrical peak and a reasonable r e t e n t i o n time. I d e n t i f i c a t i o n was b a s e d on retention time and c o n c e n t r a t i o n was determined by m e a s u r e m e n t of peak heights 20 and cempared to peak heights of k n o w n D D V P concentrations.
T a b l e 2. G a s Liquid c h r o m a t o g r a p h operating parameters. Liquid p h a s e
2% Reoplex-400
10% DEGS
Solid support
Gas Chrom Q
Gas Chrom
M e s h size
80/100
60/80
30 m l / m i n
86 m l / m i n
C a r r i e r gas
(N 2) flow rate
Hydrogen flow rate
60 m l / m i n
55 m l / m i n
O x y g e n flow rate
38 m l / m i n
40 m l / m i n
Air flow rate
200 m l / m i n
170 m l / m i n
Inlet t e m p e r a t u r e
170°C
170°C
Detector temperature
200°C
200°C
Column t e m p e r a t u r e
140°C
150°C
A p p r o x i m a t e retention time
1.5 m i n
3 min
Extraction. The e x t r a c t i o n p r o c e d u r e was similar to the m i c r o e x t r a c t i o n m e t h o d d e s c r i b e d by Halvorson 8~ aZ. 21 and was d e s i g n e d to recover insecticides from small samples (5 ml). DDVP was extracted be adding 5 m l each of sample and hexane to a 30 m l centrifuge tube and m i x i n g v i g o r o u s l y for 3C seconds on a V o r t e x G e n i e mixer. Emulsions were b r o k e n b y centrifugation. The water layer c o n t a i n i n g the i n s e c t i c i d e was e x t r a c t e d twice more, each time cc~bining the extracts and filtering the hexane layer through activated, p r e w e t t e d with distilled n-hexane, anydrous sodium sulfate to remove any e n t r a i n e d water. The combined extracts were c o n c e n t r a t e d utilizing a 250 m l c a p a c i t y K u d e r n a - D a n i s h concentrator fitted with a 3-ball Snyder column. Special cleanup procedures w e r e not n e c e s s a r y p r i o r to analysis by GLC. The e f f i c i e n c y of the e x t r a c t i o n p r o c e d u r e was d e t e r m i n e d to be 97-99%.
Results and D i s c u s s i o n
D i c h l o r v o s D i s s i p a t i o n in Soil Perfusion. D i c h l o r v o s c o n c e n t r a t i o n d e c r e a s e d r a p i d l y d u r i n g the I0 d a y i n c u b a t i o n period for the sterile and the n o n s t e r i l e soil p e r f u s i o n systems. The data in F i g u r e s 1 and 2 show losses of
No.
i0
811
71 and 50% under n o n s t e r i l e and sterile conditions, r e s p e c t i v e l y . T h e d a t a in F i g u r e i i n d i c a t e that i n s e c t i c i d e d i s a p p e a r a n c e was e n h a n c e d by the g r o w t h a c t i v i t i e s of ~icil~us cereus in the soil p e r f u s i o n system. H a l f - l i f e v a l u e s of D D V P (t½), were 3.9 and i0 days for n o n s t e r i l e and sterile systems, r e s p e c t i v e l y . However, the d e c r e a s e in c o n c e n t r a t i o n of d i c h l o r v o s in the n o n s t e r i l e system was largely due to m e c h a n i s m s o t h e r than those a s s o c i a t e d w i t h b a c t e r i a l g r o w t h since as m u c h as 50% was lost from the sterile p e r f u s i o n system.
pH
2
~
pH
5
5
90
7
1oo\
--
70
-~
60
5
~
OOVP
6
2
9O
"~
8O
.E
60
•-
40
'~ ~c
30
~.
20
50
40 30 cm
20 10
__1
3
10
l l l l t l l l l l
i
0 1 2 3 4 5 6 7 8 9 1 0
0
Time, days Figure
5
70
~
50 =
•
100
80
~
~
i. D i c h l o r v o s d e g r a d a t i o n soil p e r f u s i o n system.
U s e of D D V P as a C a r b o n
I
i
1 Z
i
i
3 4
i
i
,
5
6
7
~
8
,
i
9
10
__
Time, days in n o n s t e r i l e
and Phosphorus
Figure
2. D i c h l o r v o s in sterile p e r f u s i o n system.
soil
Source.
E v i d e n c e s u p p o r t i n g b a c t e r i a l d e g r a d a t i o n is seen in Table i. BacilZas cereus u t i l i z e d D D V P as a sole carbon source. A f t e r 4 days of incubation, 49% of the a d d e d DDVP was b r o k e n down, i.e., 51% was remaining. The o r g a n i s m was also capable of using D D V P s u p p l i e d as an a d d i t i o n a l carbon so'irce a l t h o u g h to a lesser e x t e n t than w h e n supplied as the sole source of carbon. These r e s u l t s s u g g e s t that the p h e n o m e n a of enzyme induction, the s y n t h e s i s of an enzyme o n l y w h e n a s u b s t r a t e is p r e s e n t 22, m a y have b e e n involved. By such a m e c h a n i s m D D V P could induce the s y n t h e s i s of an e s t e r a s e w h i c h u t i l i z e s the p e s t i c i d e as a substrate. Bacillus cereus has b e e n r e p o r t e d as an e s t e r a s e - a c t i v e m i c r o o r g a n i s m 23. The organism m a y m a n u f a c t u r e the enzyme r e q u i r e d f o r m e t a b o l i s m of D D V P supplied as a sole c a r b o n source, b u t w h e n an a d d i t i o n a l carbon source such as g l u c o s e is i n c l u d e d in the medium, the o r g a n i s m shows a p r e f e r e n c e for the a l t e r n a t e carbon source and p r o d u c e d little or no d i c h l o r v o s - u t i l i z i n g e s t e r a s e . F u r t h e r e x p e r i m e n t a t i o n w o u l d be r e q u i r e d to confirm these suggestions. D i c h l o r v o s was not u t i l i z e d by B. cereus w h e n supplied as the sole source of p h o s p h o r u s since no g r o w t h o c c u r r e d in those media. The b r e a k d o w n of D D V P in those m e d i a w h e r e no g r o w t h o c , ~ r r e d m u s t be a c c o u n t e d for by chemical hydrolysis. Effect
of pH on D D V P Loss.
B e c a u s e D D V P h y d r o l y s i s is c a t a l y z e d by the p r e s e n c e of OH-, an e x p e r i m e n t was c o n d u c t e d to examine the e f f e c t of pH on D D V P h y d r o l y s i s in aqueous s o i l - f r e e systams. Buffer s o l u t i o n s w e r e p r e p a r e d a c c o r d i n g to G o r t n e r 24 by v a r y i n g the amounts of sodium b o r a t e and h y d r o c h l o r i c acid or sodium h y d r o x i d e and r a n g e d from p H 2.0 to 9.3. R e s i d u a l D D V P was q u a n t i f i e d by G L C at 24 hour i n t e r v a l s for 96 hours (Table 3).
812
No. i0
Table 3. P e r c e n t D D V P r e m a i n i n g after i n c u b a t i o n in buffered, solutions.
soil-free aqueous
Hours of Incubation
pH
24
48
72
96
2.0
99
I00
i00
93 100
3.3
i00
100
i00
6.2
92
91
85
81
6.9
89
91
83
74
7.8
84
69
57
49
8.2
82
47
31
25
8.7
36
12
5
9.3
i0
< i
0
< 0
The data in Table 3 clearly show that dichlorvos is quite stable under acidic conditions. At pH v a l u e s less than or equal to 3.3, D D V P remained e s s e n t i a l l y u n c h a n g e d t h r o u g h o u t the 96 h o u r i n c u b a t i o n period. S u s c e p t i b i l i t y to hydrolysis increases as neutral pH was approached. At p H 6.9, 74% of the a d d e d insecticide r e m a i n e d after 96 hours. Hydrolysis was e x c e e d i n g l y rapid w i t h i n c r e a s i n g basicity. A t pH 9.3, D D V P could not be d e t e c t e d a f t e r 72 hours. Since the buffer solutions r e m a i n e d free of c o n t a n i n a t i n g m i c r o o r g a n i s m s , this rapid hydrolytic b r e a k d o w n m u s t be a t t r i b u t e d to pH. In the n o n - s t e r i l e soil p e r f u s i o n system (Figure i), pH increases from 6.2 to 7.4 in the f i r s t four days of i n c u b a t i o n and r e m a i n e d at 7.4 for the r e m a i n d e r of the incubation period. This increase in p H o c c u r r e d during and after the logarithmic and s t a t i o n a r y b a c t e r i a l growth phases, indicating that the change in pH is associated with the g r o w t h of B. c e r e u s . Approximately 25% of the initial p e s t i c i d e c o n c e n t r a t i o n was d e g r a d e d during these g r o w t h phases. Only 29% of the a d d e d insecticide could be r e c o v e r e d u p o n t e r m i n a t i o n of incubation. This rapid diss i p a t i o n of DDVP was a p p a r e n t l y due to both b a c t e r i a l d e g r a d a t i o n and the associated increase iE pH. However, in the sterile soil p e r f u s i o n system (Figure 2) p H v a l u e s tended toward an acid c o n d i t i o n i n d i c a t i n g that p H alone does not account for the d i s s i p a t i o n of DLVP from sterile soil and i m p l i c a t i n g s u r f a c e - c a t a l y z e d adsorption. A d s o r p t i o n of DDVP to Soil. To determine the extent of chemical d e g r a d a t i o n DDVP loss was m o n i t o r e d in sterilized soil extract. The soil e x t r a c t was p r e p a r e d according to A l l e n 25 and filtered through W h a t m a n No. 42 filter paper to remove soil particles. Figure 3 shows the r e s u l t s of this experiment. A compari son of the d a t a in F i g u r e s 2 and 3 indicate that a d s o r p t i o n a c c o u n t e d for the r a p i d initial disa p p e a r a n c e of the p e s t i c i d e since the curve r e p r e s e n t i n g DDVP loss in the sterile soil p e r f u s i o n system was non-linear. It should be p o i n t e d out that a d s o r p t i o n does not n e c e s s a r i l y m e a n degradation, o n l y a removal from the aqueous phase. At the end of the incubation period the perc6nt of p e s t i c i d e r e m a i n i n g in the solution was the same for both systems. The t½ v a l u e s for both systems was i0 days. C h e m i c a l and B a c t e r i o l o g i c a l Degradation. The e x p e r i m e n t a l data suggest that the d i s s i p a t i o n of D D V P from soil is the result of both c h e m i c a l and b a c t e r i o l o g i c a l m e c h a n i s m s . C h e m i c a l d e g r a d a t i o n is a p p a r a r e n t l y due to b a s e - c a t a lyzed hydrolysis. The relative i n v o l v e m e n t of these two d e g r a d a t i v e m e c h a n i s m s (chemical and bacteriological) was c a l c u l a t e d a c c o r d i n g to Walker and Stojanovic 26. The results are p r e s e n t e d in T a b l e 4.
No. l0
813
if
pH
--I ~
5
7 6 5
100 2
90 80
¢)
70 =
00
.~
50
N
40
~
30
~N
2O 10 I
I
I
I
]
I
I
I
L
0 1 2 3 4 5 0 7 8 9 1 0 Time, days Figure
Table
3. D i c h l o r v o s d e g r a d a t i o n in s t e r i l i z e d soil solution.
4. R e l a t i v e types of D D V P d e g r a d a t i o n of i n c u b a t i o n
Total D e g r a d a t i o n ,
Non-sterile Soil P e r f u s i o n
Black
clay f o l l o w i n g
% Total D e g r a d a t i o n
%
Sterile Soil P e r f u s i o n
71
+ % chemical
in Houston
Chemical + Mechanisms
50
Due
10 days
to
Bacteriological m e c h a n i e m s ++
70
30
degradation
++ % bacteriological
= % d e g r a d a t i o n under s t e r i l e c o n d i t i o n s % total d e g r a d a t i o n (non-sterile) d e g r a d a t i o n = i00 - % chemical d e g r a d a t i o n
The r e s u l t s in Table 4 indicate that the m a j o r m e c h a n i s m D D V P frc~ soil was chemical degradation.
responsible
x I00
for the d i s a p p e a r a n c e
of
CONCLUSIONS
I. The rapid d i s a p p e a r a n c e of d i c h l o r v o s observed in H o u s t o n B l a c k clay is the r e s u l t of a d s o r p tion to soil particles, and c h e m i c a l and b a c t e r i a l degradation. 2. Soil adsorption, and chemical d e g r a d a t i o n a c c o u n t for 70% of the rapid d i s a p p e a r a n c e of D D V P from the soil. 3. A d i c h l o r v o s - u t i l i z i n g esterase, i n d u c e d by S° oereTls, is h y p o t h e s i z e d to have b e e n r e s p o n sible f o r the loss of D D V P from a m i n e r a l salts s o l u t i o n c o n t a i n i n g the p e s t i c i d e as a sole carbon source. This enzyme system m a y also be i m p l i c a t e d in the d i s s i p a t i o n of D D V P in soil since ft. c ~ r ~ s is r e s p o n s i b l e for about 30% of the total d e g r a d a t i o n of the p e s t i c i d e in the soil p e r f u s i o n system. 4. The r a t e of chemical h y d r o l y s i s is a function of pH, i n c r e a s i n g at h i g h e r p H values.
814
No. i0
ACKNOWLEDGEMENT
The authors express appreciation to the Texas Christian University Research Foundation for support of this research.
REFERENCES
I. ~. 7. 4. ~. C. 7. ~. <,. 10.
R.J. Lamoreaux and L.W. Newland, Biol. Conserv., ii, 59 (1977). A.M. Mattson, J.T. Spillane, and G.W. Pearce, J. Agr. Food Chem., 3, 319 (1955). Shell Chemical Company, PMS-G-913/69, 6, (1969). N.N. Melnikov, Chemistry of Pesticides, Springer-Verlag, New York, (1971). E. Hodgson and J.E. Casida, J_2_.A@r. Food Chem., i0, 207 (1962). F. Matsumura and G.M. Boush, J. Econ. Entemol., 61, 610, (1968). G.M. Boush and F. Matsumura, J. Econ. Entomol., 60, 918, (1967). M. Yasu~o, S. Hirakoso, M. Sasa, and M. Uchida, Japan Jour. Exp. Med., 35, 545, (1965). M. Alexander, Introduction to Soil Microbiology, John Wiley and Sons, Inc., New York, (1961) E.N. Nishustin and V.A. Nisroeva, The Ecology of Soil Bacteria, University of Toronto Press, (1968). 1~. D.F. Goerlitz and E. Brown, Methods for Analysis o f Organic Substances in Water, Washington, U.S. Government Printing Office, (1972). i~I. T. Bevenue, T.W. Kelley, and J.W. Hylin, J. Chromatog., 54, 17, (1971). I~. R.S. Breed, E.G.D. Murray, and N.R. Smith, Ber~ey's Manual of Determinative Bacteriology, 7th. ed., The Williams and Wilkins Co., Baltimore, (1957). l.l.H. Lees, J_t.Agr. Sci., 37, 27, (1947). 15. D.E. Armstrong, G. Chester s, and R.F. Harris, Soil Sci. Soc. Amer. Proc., 31, 61 (1967). i~). American Public Health Association, Standard Methods for the Examination of Water and Wastewater, 13th. ed., Washington, D.C., (1971). 1"I. M.L. Jackson, Soil Chamical Analysis, Prentice Hall, Inc., Englewood Cliffs, N.J., (1958). I;3. E.P. Lichtenstein, and K.R. Schulz, J. Econ. Entomol., 53, 192 (1960). 19. L.W. Getzin and I. Rosefield, J__u.Agr. Food Chem., 16, 958 (1968). 2). U.S. Department of Health, Education and Welfare, Pesticide Analytical Manual, (1975). 21. H. Halvorson, M. Ishaque, J. Solomon, and O.W. Grussendorf, J__t.Microbiol., 17, 585, (1971). 22. T.D. Brock, Biology o f Microorganisms, Prentice-Hall, Inc., Englewood Cliffs, N.J., (1970). 23. S. Aarsonson, Experimental Microbial Ecology, Academic Press, New York,(1970). 21. R.A. Gortner, Outlines o f Biochemistry , John Wiley and Sons, Inc., New York, (1950). 25. O.N. Allen, Experiments i__n_nSoil Bacteriology, Burgess Publishing, Minneapolis, (1949). 26. W.W. Walker, and B.J. Stojanovic, J__u.Environ. Quality, ~, 229, (1973).
(Received in The Netherlands 28 August 1978)