25 Degradation of the Insecticide Pirimicarb in SoilCharacterization of "Bound" Residues I. R. H I L L
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ICI Plant Protection Division, Jealott's Hill Research Station, Bracknell, Berkshire, England
The degradation of pirimicarb (2-dimethylamino-5,6dimethylpyrimidin-4-yl dimethylcarbamate), a fast acting selec tive aphicide, has been studied in several s t e r i l e and non -sterile soils maintained in controlled laboratory conditions. Each soil type was treated with 1 Kg ai/ha of the insecticide radiolabeled at the pyrimidine ring-2-carbon at the carbamate carbonyl-carbon, and incubated 'aerobically' (at 40% of moisture holding capacity) and after flooding the soil with water to a depth of 2.5 cm. Pirimicarb and any degradation products were extracted directly from the moist or wet soils (air drying can result in 'loss' of radioactivity) by refluxing in acetone:water (5:1) (selected after studies of the efficiency of a range of solvents). Chromatographic analyses of the soil extracts i n d i cated that pirimicarb degradation proceeded via cleavage of the carbamate moiety from the ring, N-dealkylation of the 2-dimethyl= amino group and pyrimidine ring cleavage. Degradation was most rapid in alkaline s o i l s . After extraction the soils were freeze-dried, ground to a powder in a hammer m i l l and thoroughly mixed by rotation. As this procedure 'homogenized' the soil sample, making subsequent analyses more accurate, and did not result in any loss of radio activity, all measurements of unextractable radioactivity were made by dry combustion. Radioactivity remaining after exhaustive extraction was considered to be 'bound'. In 'aerobically' incubated s o i l s considerable amounts of the ring-2- C, but relatively little (less than 10% of that applied) of the carbamate- C, radiolabel were present as 'bound' residues. Within an incubation period of 1 year, approximately 20-60% of the applied ring- C became 'bound' in a range of 9 soil types of pH 5.5-8.1 and organic matter content 1.7-10.4%. In one alkaline soil (Gore, pH 7.8), in which the insecticide was very rapidly degraded (50% in 1-2 weeks, 90% in 3-5 weeks), over half of the applied ring- C was 'bound' within 5 weeks incu bation. In s t e r i l e soils (autoclaved and γ-irradiated) both the rate of pirimicarb degradation and the accumulation of 'bound' residues were considerably less than in their non-sterile 14
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equivalents. An a c i d soil (Broadricks, pH 5.5) and an a l k a l i n e soil (Gore) were a l s o incubated under flooded c o n d i t i o n s . In B r o a d r i c k s , the amount o f ' b i n d i n g was very s i m i l a r t o t h a t observed under aerobic c o n d i t i o n s . However, in Gore 20-30%of both the r i n g - and carbamate- radiocarbons became 'bound during an i n c u b a t i o n p e r i o d of 20 weeks. In most s o i l s the amount o f 'bound r a d i o a c t i v i t y increased p r o g r e s s i v e l y throughout the i n c u b a t i o n p e r i o d . I n Broadricks t r e a t e d w i t h r i n g - 2 - l ^ C p i r i m i c a r b and incubated a e r o b i c a l l y f o r 2 years, 70% o f the a p p l i e d r a d i o a c t i v i t y was 'bound . In Gore, whether incubated ' a e r o b i c a l l y ' o r in flooded soil, the amount o f 'bound' r a d i o a c t i v i t y reached a maximum w i t h i n 5-10 weeks, but t h e r e a f t e r d e c l i n e d s t e a d i l y f o r the remainder o f the i n c u b a t i o n p e r i o d . There was no simple r e l a t i o n s h i p between t h e amount o r r a t e o f formation o f 'bound' residues and e i t h e r the r a t e o f p i r i m i c a r b degradation o r the soil pH and organic matter content. Attempts were made t o e x t r a c t and c h a r a c t e r i z e t h e 'bound' p e s t i c i d e residues in the f o l l o w i n g e x t r a c t e d , f r e e z e - d r i e d s o i l s (composite samples from s o i l s incubated f o r a minimum o f 5 weeks): a. Gore ' a e r o b i c a l l y ' incubated w i t h r i n g - 2 - l ^ C p i r i m i c a r b (GAR) ; b. Broadricks ' a e r o b i c a l l y ' incubated w i t h r i n g - 2 - C p i r i m i c a r b (BAR); c. Gore incubated in flooded s t a t e w i t h r i n g - 2- C p i r i m i c a r b (GFR); and d. Gore incubated in flooded s t a t e w i t h carbamate-^C p i r i m i c a r b (GFC). E x t r a c t i o n s were c a r r i e d out w i t h acetone:water (5:1), a c i d , a l k a l i and s a l t s o l u t i o n s by shaking, r e f l u x i n g and u l t r a s o n i c a t i o n . Forallbut the u l t r a s o n i c a t e d treatments e x t r a c t i o n s were c a r r i e d out in 'enclosed' v e s s e l s through which a stream o f a i r was passed. The e f f l u e n t a i r was bubbled through tubes cont a i n i n g , in s u c c e s s i o n , methoxyethanol and ethanolamine o r O.5 M sodium hydroxide, t o t r a p products evolved i n t o the a i r stream during e x t r a c t i o n . S o i l s GAR and BAR contained approximately 40% and 35%, r e s p e c t i v e l y , o f the r a d i o l a b e l e d carbon o r i g i n a l l y a p p l i e d t o the soil as p i r i m i c a r b . V i r t u a l l y all o f t h i s 'bound' r a d i o a c t i v i t y was e x t r a c t e d w i t h O.1 M NaOH and w i t h O.1 M N a P 0 , 60-80% w i t h O.5 M H S 0 and o n l y 10-33% w i t h 1.0 M HC1. No more than 1% o f the r a d i o a c t i v i t y was evolved during a c i d e x t r a c t i o n and no more than 3% during a l k a l i n e o r s a l t e x t r a c t i o n . A f t e r a c i d i f i c a t i o n (to pH 1.0) o f the NaOH e x t r a c t s o f GAR and BAR, almost all o f the r a d i o a c t i v i t y was present in the a c i d a l k a l i s o l u b l e f u l v i c a c i d component. Any 'bound' p y r i m i d i n - 4 - y l carbamate may w e l l be hydrolyzed t o a hydroxypyrimidine during NaOH e x t r a c t i o n which, together w i t h any 'bound' hydroxyp y r i m i d i n e s , would f r a c t i o n a t e w i t h the f u l v i c a c i d s . Refluxing of GAR and BAR with acetone:water e x t r a c t e d 63% and 78%, 1
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r e s p e c t i v e l y , o f the bound r a d i o a c t i v i t y . C l e a r l y the freeze d r y i n g and/or g r i n d i n g had a l t e r e d the s t a t e o f these r e s i d u e s , f o r p r e v i o u s l y these s o i l s had been e x h a u s t i v e l y e x t r a c t e d in the same s o l v e n t . From chromatographic analyses o f these acetone:water e x t r a c t s , 8% and 18% o f the 'bound r a d i o a c t i v i t y in GAR and BAR r e s p e c t i v e l y was i d e n t i f i e d as p i r i m i c a r b , 5% and l e s s than 1% as i t s 2-methylformamido and 2-methylamino d e r i v a t i v e s , and 8% and 38% as hydroxypyrimidines formed by decarbamoylation o f the parent and i t s 2-methylamino degradation product. Thus in both GAR and BAR approximately 5% o f the o r i g i n a l l y a p p l i e d r a d i o a c t i v i t y was present in the 'bound' residues as i d e n t i f i e d p y r i m i d i n - 4 - y l carbamates. This compares approximately w i t h the t o t a l 'bound' r a d i o a c t i v i t y in s o i l s incubated 'aerobically' with carbamate-^^C-pirimicarb. Subsequently, Gore soil t r e a t e d w i t h r i n g - C - p i r i m i c a r b has been used t o i n v e s t i g a t e the e f f e c t o f s e q u e n t i a l acetone :water e x t r a c t i o n s , w i t h combinations o f a i r - d r y i n g , f r e e z e - d r y i n g and g r i n d i n g the soil, on the e x t r a c t a b i l i t y o f p i r i m i c a r b and i t s degradation products. Both methods o f d r y i n g the soil enabled f u r t h e r amounts o f r a d i o a c t i v i t y t o be e x t r a c t e d by repeated acetonerwater r e f l u x . Grinding the soil caused a f u r t h e r but small increase in e x t r a c t a b i l i t y . A f t e r 15 weeks i n c u b a t i o n , 54% of the r a d i o a c t i v i t y present was e x t r a c t e d from the moist soil. Subsequent s e q u e n t i a l d r y i n g and r e f l u x i n g (3 times) e x t r a c t e d an a d d i t i o n a l 15% o f the r a d i o a c t i v i t y . Although d r y i n g may have r e l e a s e d C - m a t e r i a l bound t o soil s u r f a c e s , it is a l s o a p o s s i b i l i t y t h a t the p e s t i c i d e and/or i t s degradation products can accumulate i n s i d e m i c r o b i a l c e l l s . These c e l l s might not all be ruptured without an e x t r a c t i o n from the d r i e d s t a t e . Studies are in progress t o examine t h i s p o s s i b i l i t y . S o i l s GFR and GFC contained approximately 14% and 21%, r e s p e c t i v e l y , o f r a d i o a c t i v i t y derived from the r i n g - ^ c and carbamate-^C r a d i o l a b e l e d p i r i m i c a r b o r i g i n a l l y a p p l i e d . E x t r a c t i o n o f GFC w i t h 1.0 M HC1 (shaken a t room temperature) r e s u l t e d in a l o s s o f 75% o f the bound r a d i o a c t i v i t y from the e x t r a c t i o n v e s s e l , and i t s recovery in the ethanolamine 'trapping' s o l u t i o n . I t was, however, observed t h a t 98% o f the r a d i o a c t i v i t y was a l s o evolved from soil t r e a t e d w i t h carbamate-**C-pirimicarb and ext r a c t e d w i t h 1.0 M HC1 a f t e r o n l y 4 hours i n c u b a t i o n . As 1.0 M CH COONa (adjusted t o pH 5 w i t h g l a c i a l CH3COOH) d i d n o t cause any such ' l o s s ' o f r a d i o a c t i v i t y , soil GFC was a l s o shaken w i t h the acetate b u f f e r . During t h i s e x t r a c t i o n , 55% o f the 'bound' r a d i o a c t i v i t y was evolved and recovered in e i t h e r ethanolamine o r NaOH s o l u t i o n s . Almost all o f the r a d i o a c t i v i t y in the NaOH 'trap' was i d e n t i f i e d as Na2^C03. I t was thus concluded t h a t approximately h a l f o f the 'bound' r a d i o a c t i v i t y in GFC was probably present as -carbonate, d e r i v e d from f i x a t i o n o f ^ C C ^ evolved a f t e r h y d r o l y t i c cleavage o f the l^C-carbamate e s t e r under flooded c o n d i t i o n s . As expected, a s i m i l a r amount o f r a d i o a c t i v i t y (approx 50%) remained in the soil residue a f t e r
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NaOH e x t r a c t i o n . The remaining r a d i o l a b e l e d residues were a l k a l i e x t r a c t e d , and a f t e r a c i d i f i c a t i o n were recovered from the f u l v i c a c i d f r a c t i o n . Acetone:water r e f l u x i n g e x t r a c t e d only 20% o f the GFC 'bound r e s i d u e s , o f which three quarters was i d e n t i f i e d as pirimicarb. During a c i d e x t r a c t i o n o f GFR, l e s s than 2% o f the r a d i o a c t i v i t y was evolved i n t o the a i r stream. Instead, almost all o f the 'bound' C c o n s t i t u e n t s behaved as those in BAR and GAR, being e x t r a c t e d w i t h a l k a l i and s o l u b l e in a c i d s . From acetone: water e x t r a c t i o n , 10% o f the 'bound' r a d i o a c t i v i t y in GFR was i d e n t i f i e d as p i r i m i c a r b and 25% as 5,6-dimethyl-2-dimethylamino4-hydroxypyrimidine. A f u r t h e r 20% was e x t r a c t e d but not characterized. It has thus been p o s s i b l e t o i d e n t i f y between 20% and 65% o f what was o r i g i n a l l y b e l i e v e d t o be the 'bound' r a d i o a c t i v e residues o f p i r i m i c a r b r e s u l t i n g from i n c u b a t i o n o f soil under 'aerobic' and flooded c o n d i t i o n s . The 'bound' -residues o f p i r i m i c a r b remaining unidentf i e d are a t present under i n v e s t i g a t i o n using chromatographic and isotope d i l u t i o n techniques. 1
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