Volatile Metabolites from Dimethyldithiocarbamate Fungicide Residues by JoHN W. HYLINand BYONGHAN CttlN 1

Department of Agricultural Biochemistry, University of Hawaii, Honolulu, Hawaii

The decomposition of dithiocarbamic acids to amines and carbon disulfide in the presence of strong acid is well known and forms the basis for the analytical determination of dithiocarbamates (i). Evolution of gaseous products from dilute solutions of dithiocarbamate fungicides has also been reported (2, 3). Cox (4) and Sisler and Cox (5) showed that carbon disulfide was present in the air above dilute solutions of thiram and n a b a m in contact with cultures of Fusarium roseum.

The

Taken in part f r o m a Ph. D. dissertation in Horticulture by Byong Han Chin.

This work was supported by funds f r om Western Regional R e s e a r c h Project W-45, !'Pesticide R e s i d u e s , " and is published with the approval of the Director of the Hawaii Agricultural Experiment Station as Technical Paper No. 960. Present address:

Mellon Institute, Pittsburgh, Pennsylvania.

322 Bulletin of Environr~ental Contamination & Toxicology, Vol. 3, No. 6, 1968, published by Springer-Verlag New York Inc.

fungicides were presumably decomposed by acids excreted into the medium by the microorganism.

No similar study with higher plants has

been r e p o r t e d , yet the vast majority of dithiocarbamate fungicides are applied to protect higher plants.

During a study of the total fate of z i r a m

r es id u es on Carica papaya (papaya), it appeared conceivable that carbon disulfide and dimethylamine could be f orm ed as volatile metabolites. F u r t h e r m o r e , it seemed likely that if dimethylamine were formed, it could be further metabolized to carbon dioxide.

This paper presents

the r e s u l t s of experiments designed to detect, identify and quantitate these volatile products. Materials and Methods Dithiocarbamates: Radioactive potassium dimethyldithiocarbamate (KDDC) labeled with 35S was purchased f r o m Volk Radiochemical Co., Chicago, Illinois.

Prior to use it was exhaustively extracted with

benzene to r e m ove elemental 35S which was the principal radiochemical impurity resulting f r o m radiodecomposition. Radioactive KDDC labeled with 14C in the methyl groups was synthesized f r o m 14C dimethylamine and carbon disulfide.

Zixam [Zn(DDC)2], either 35S or methyl 14C

labeled, was pr e pa r ed f r o m the r e s pect i ve KDDC by addition of a stoichiometric amount of zinc chloride, dissolved in water, to an aqueous solution of the dithiocarbamate. The precipitated Zn(DDC) 2 was extracted into chloroform and isolated by rem oval of the solvent on a

323

Figure i.

Apparatus used for the collection of volatile decomposition products.

324

rotating evaporator.

Prior to use all d i t h i o c a r b a m a t e s w e r e a s s a y e d f o r

purity and r a d i o p u r i t y by c h r o m a t o g r a p h y (6) and r a d i o c h r o m a t o g r a p h y . W a t e r - s o l u b l e d i t h i o c a r b a m a t e s w e r e d i s s o l v e d in 0. 02M p o t a s s i u m phosphate b u f f e r , pH 7. 4, containing 800 p p m T r i t o n X-100 as a wetting agent, i m m e d i a t e l y p r i o r to application to plants.

Zn (DDC) 2 w a s applied

e i t h e r as a finely d i s p e r s e d s u s p e n s i o n in H20 with 800 p p m T r i t o n X-100, or d i s s o l v e d in d i m e t h y l f o r m a m i d e .

D i m e t h y l f o r m a m i d e solutions did

not p r o d u c e any o b s e r v a b l e phytotoxicity. Plant m a t e r i a l s :

Papaya plants (Solo v a r i e t y , line 5) w e r e grown

f r o m s e e d s g e r m i n a t e d in V e r m i c u l i t e in the g r e e n h o u s e .

Three-week-

old seedlings w e r e t r a n s p l a n t e d to s t e r i l e soil and the plants w e r e u s e d when t h r e e months old. Procedure:

Known a m o u n t s of r a d i o a c t i v e d i m e t h y l d i t h i o c a r b a m a t e s

w e r e applied to the l e a v e s of the plant with a m i c r o p i p e t .

When the

solvents had e v a p o r a t e d the w e l l - w a t e r e d plant w a s p l a c e d in the a p p a r a t u s shown in F i g u r e 1.

It w a s i l l u m i n a t e d by two c i r c u l a r f l u o r e s c e n t

l a m p s giving an a v e r a g e of 250-foot candies a t the leaf s u r f a c e .

Air

w a s d r a w n through the s y s t e m by a s m a l l mechanical v a c u u m pump. I n c o m i n g a i r f i r s t p a s s e d through a t r a p containing acidified 0. 1M p o t a s s i u m p e r m a n g a n a t e to r e m o v e i m p u r i t i e s which o t h e r w i s e p r o duced an e p i n a s t i c r e s p o n s e in the plant within 4 h o u r s . a i r s t r e a m p a s s e d through two t r a p s in s e r i e s .

The effluent

In the c a s e of 35S-

l a b e l e d m a t e r i a l s , the f i r s t of t h e s e t r a p s contained 20~o zinc a c e t a t e

325

to r e t a i n H2S, and the second contained Viles r e a g e n t (7) for CS2.

With

1 4 C - l a b e l e d s u b s t a n c e s , the f i r s t t r a p contained 1. ON HC1, to r e t a i n d i m e t h y l a m i n e , and the second contained 15% KOH to t r a p CO 2.

The

t r a p p i n g solutiuns w e r e changed at suitable i n t e r v a l s . Radioactivity d e t e r m i n a t i o n s :

Hydrogen sulfide w a s r e m o v e d f r o m

the t r a p p i n g solution by addition of 7. 8 m g of c a r r i e r sodium sulfide. The r e s u l t i n g p r e c i p i t a t e was collected on a g l a s s f i b e r disc, washed, d r i e d and weighed.

Aliquots of the c a r b o n disulfide and d i m e t h y l a m i n e

t r a p p i n g solutions w e r e t r a n s f e r r e d to p l a n c h e t s , d r i e d and weighed. The r a d i o a c t i v i t y in t h e s e s a m p l e s w a s d e t e r m i n e d with a N u c l e a r Chicago thin window g a s flow counter with an e f f i c i e n c y of 26% for 14C. R a d i o a c t i v e c a r b o n dioxide w a s p r e c i p i t a t e d a s b a r i u m c a r b o n a t e by addition of 10% b a r i u m chloride to the t r a p p i n g solution.

The p r e c i p i -

tate w a s c o l l e c t e d on a s i n t e r e d g l a s s f i l t e r , w a s h e d with w a t e r and 95% ethanol, and dried.

A portion of the p r e c i p i t a t e w a s ground to a fine

powder, t r a n s f e r r e d to a t a r e d 20 m l liquid scintillation counting vial and d r i e d to constant weight.

The vial w a s filled with C a b - O - S i l thixo-

t r o p i c agent and 20 m l of s c i n t i l l a t o r solution (8).

These samples were

counted in a P a c k a r d T r i - C a r b Liquid Scintillation S p e c t r o m e t e r which had an efficiency of 55~o for 14C.

The r e s u l t s of r a d i o a c t i v i t y d e t e r -

m i n a t i o n s w e r e c o r r e c t e d for background, d e c a y , and seli a b s o r p t i o n .

326

TABLE 1 Radioactive Carbon Disulfide Released by Plants Treated with Radioactive Dimethyldithiocarbamates (Average of 3 Replications)

Days after application

Compound applied 35S_KDDC 35S_Zn(DDC)2 cpm cpm

1

105,600

1,290

2+3

218,800

2,790

4+5+6

270, 800

4,110

7+8+9

36,300

3,980

6, 270

5,230

Total

637, 770

17, 400

co_unts applied

1.65 x 107

10+11+12+13

.......

1.36 x 107

TABLE 2 Radioactive Carbon Dioxide Released by Plants Treated with Radioactive Dimethyldithiocarbamates (Average of 3 Replications) Compound applied Days after applicatio~

14CH3-KDDC .cpm

14CH3-Zn(DDC)2 . .cpm . . . . .

1-3

2,001

547

4-6

2,295

582

7-9

2,351

568

10-12

2,227

543

8,874

2,240

Total Counts applied

2 . 9 x 105

327

2.04 x 106

Results and Discussion The possibility that volatile p r o d u c t s could be f o r m e d f r o m r e s i d u e s of d i t h i o c a r b a m a t e s on higher plants s e e m s to have been overlooked.

Our

data, p r e s e n t e d in T a b l e s 1 and 2, d e m o n s t r a t e that such products axe f o r m e d by higher plants, and that significant amounts of carbon disulfide axe r e l e a s e d f r o m r e s i d u e s of both KDDC and Zn(DDC)2.

The r a t e of

r e l e a s e (Fig. 2) is m u c h g r e a t e r with KDDC shortly after application, but by the ninth day the r a t e s of carbon disulfide evolution a r e e s s e n t i a l l y the s a m e for both compounds.

The much slower evolution of carbon di-

sulfide f r o m Zn(DDC)2 r e s i d u e s is p r e s u m a b l y due to the low-water solubility of this substance when c o m p a r e d with that of the p o t a s s i u m salt.

The d e c r e a s e in carbon disulfide production o b s e r v e d with the

l a t t e r m a y also be due to solubility, since it has been shown that KDDC r e s i d u e s a r e gradually oxidized to t h i r a m on leaf s u r f a c e s (9). The site of d e c o m p o s i t i o n m a y be e i t h e r the leaf s u r f a c e or within the outer l a y e r s of cells.

The plants w e r e in an a t m o s p h e r e at or n e a r

100~o r e l a t i v e humidity, and under these conditions a film of m o i s t u r e on the leaf s u r f a c e could be sufficiently acidic, due to d i s s o l v e d carbon dioxide, to catalyze a r e a c t i o n s i m i l a r to that p r o p o s e d by Zuman and Zahradnik (10) for the acid decomposition of dithiocaxbamates

(CH3) 2 n

_c~S

"s-

+ H+~(CH3)2

+ S ~-C ~ ~

I >..s-

H

(CH3) 2 NH + CS2

l[ m?~ (CH3)2NH2HCO3

a28

5.0

J

|

.

I

/'I

I

I

I

I

.. I

!

I

I

1

.

./

. / 1.0

~" 0.5

o L.

0.1

0.05

0.01

0

2

4

6

8

Days after

F i g u r e 2.

10

12

application

Evolution of volatile products from plants treated with radioactive dimethyldithioca_rbamates. CS2 fromS5S-KDDC 0 ; CS 2 from 35S-Zn(DDC)2 9 ; CO2 from 14CH3-KDDC 9 ; CO 2 from 14CH3-Z-(DDC)20. 329

Hallaway (11) has shown that the half life of d i e t h y l d i t h i o c a r b a m a t e in acid solution r a n g e s f r o m 2 min at pH 4 . 0 to 860 min at pH 6. 6.

At pH

5 . 7 , the a p p r o x i m a t e pH of m o i s t leaf s u r f a c e s , the haK life was 107 rain.

Although these r e s u l t s a r e for the diethyl d e r i v a t i v e , it is r e a s o n -

able to e x p e c t that the dimethyl compound would behave in a s i m i l a r fashion.

The KDDC compounds w e r e applied in pH 7. 4 phosphate buffer

in an attempt to r e d u c e the effects of dissolved CO 2.

The other p r o d u c t

of acidic decomposition, t r i m e t h y l a m m o n i u m b i c a r b o n a t e , would be unstable under these conditions, and p r e s u m a b l y give r i s e to appreciable quantities of t r i m e t h y l a m i n e .

Only t r a c e amounts (0. 013~o of applied

14C-KDDC and 0. 016~o of applied 14C-Zn(DDC)2) of this substance w e r e d e t e c t e d o v e r a 14-day period, indicating that e i t h e r decomposition at the leaf s u r f a c e is a m i n o r phenomenon, or that the l i b e r a t e d t r i m e t h y l amine or t r i m e t h y l a m m o n i u m b i c a r b o n a t e is r a p i d l y and s e l e c t i v e l y a b s o r b e d by the plant t i s s u e s . The a l t e r n a t i v e to s u r f a c e decomposition is degradation of the d i t h i o c a r b a m a t e s in the outer cell l a y e r s of the leaf.

Z i r a m and

t h i r a m , or at l e a s t DDC ions d e r i v e d t h e r e f r o m , a r e known to be a b s o r b e d by intact l e a v e s (9, 12).

D i r e c t decomposition of these c o m -

pounds by acidic c e l l u l a r fluids would follow the r e a c t i o n a l r e a d y d i s cussed.

R e l e a s e d c a r b o n disulfide would diffuse f r o m the cells into

the a t m o s p h e r e while the t r i m e t h y l a m i n e would be oxidatively metabolized.

330

It would a p p e a r that DDC ion is not d e m e t h y l a t e d p r i o r to r e l e a s e of CS 2 since this would give r i s e to m e t h y l i s o t h i o c y a n a t e by r e a r r a n g e m e n t of the m o n o m e t h y l d i t h i o c a r b a m a t e , and this s u b s t a n c e w a s not detected. Neither w a s another potential volatile d e c o m p o s i t i o n product, hydrogen sulfide, d e t e c t e d in these e x p e r i m e n t s . DDC ions a r e conjugated in higher plants to f o r m d e r i v a t i v e s of g l u c o s e and alanine (13, 14).

T h e s e s u b s t a n c e s a r e in e q u i l i b r i u m with

e a c h other and with a s m a l l amount of DDC ion (15).

It is p r o b a b l e that

p a r t or all of the c a r b o n disulfide p r o d u c e d l a t e r than s e v e n days a f t e r application of KDDC is d e r i v e d f r o m d e c o m p o s i t i o n of DDC ions p r e s e n t in this e q u i l i b r i u m . A m o r e c o m p l e t e a n a l y s i s of the m e c h a n i s m s u n d e r l y i n g the r e l e a s e of volatile d e c o m p o s i t i o n p r o d u c t s of d i t h i o c a r b a m a t e fungicide r e s i d u e s on higher plants is not p o s s i b l e at this t i m e .

The s a l i e n t point i l l u s -

t r a t e d by our r e s u l t s is that such volatile p r o d u c t s a r e f o r m e d and constitute one p o s s i b l e r o u t e for the d i s s i p a t i o n of these p e s t i c i d e s .

331

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,

C. E. COX, H. D. SISLER, and R. A. SPURR, Science 114, 643 (1951). H. D. SISLER and C. E. COX, Phytopathology 41, 565 (1961).

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332