11 Classification and Analysis of Pesticides Bound to Plant Material J. WIENEKE Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 2, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0029.ch011
Radioagronomy, Kernforschungsanlage, Julich 517 Julich, Postfach 1913, West Germany
The use of radiotracer technology to follow pesticide metabolism yields a precise accounting of the radiolabel distribution, but provides very little information about the chemical structure of the metabolic products. Since most residue analysis methods are designed to assay the parent compound, the more polar metabo l i t e s require additional steps for their isolation and p u r i f i cation. After removal of the nonpolar and polar pesticide and pesticide metabolites the remaining plant residue i s thought to consist of insoluble material. However, during the course of a study on the metabolism of the insecticide, azinphos i n bean plants, the nature of the bond residues became questionable. However, the results of the following preliminary investigations with C-azinphos w i l l contribute to a better understanding of the extraction and c l a s s i f i c a t i o n of bound pesticide residues in plant material. Methods: The f i r s t t r i f o l i a t e leaves of bean plants were sprayed with the C-azinphos. At the time of sampling, the treated leaves were stripped with benzene (2X) and frozen at -18°. The frozen plant material was crushed and homogenized i n an "Ultra Turrax" blender with the following solvents: 1. acetone, 2. acetone:water (3 : lv/v) and 3. two volumes of chloroform. The volume of each extracting solvent was 5 - 6 ml/gr fresh weight. Each solvent was removed from the homogenate by vacuum f i l t r a t i o n . After chloroform extraction, the residue was washed 3 times with acetone. Further Soxhlet extraction with acetone for 28-48 hrs yielded negligible radioactivity (0.02-0.05% of the applied radioactivity or 1-2% of the radiolabel present i n the extracted residue). Radioactivity i n the unextractable residue fraction increased with time. Four weeks after application of the radiolabeled insecticide, 5-11% of the radioactivity was in the bound residue fraction. The nature of the azinphos- C bound residues was questioned. Was the parent molecule incorporated or was the bound C-residue a fragment of the original insecticide which was somehow incorporated into the plant polymeric structure? To answer this 14
14
14
14
166
In Bound and Conjugated Pesticide Residues; Kaufman, Donald D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 2, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0029.ch011
WIENEKE
Pesticides Bound to Plant Material
AZINPHOSMETHYL (GUTHION ) CH O 3
P
s
w
P - S - C H , - N / CH3O
If
1
1
\ v
S - ( 3,4 - dihydro - U - oxo - benzo [d]-[1.2,3]-triazin-3-ylmethyl ) 0,0 - dimethyl phosphorodithioate Figure 1. Chemical structure and name of azinphos ClabeUed in the carbonyl position U
In Bound and Conjugated Pesticide Residues; Kaufman, Donald D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
168
BOUND AND CONJUGATED PESTICIDE RESIDUES
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 2, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0029.ch011
Table 1:
Distribution of radioactivity i n different fractions a f t e r subsequent e l u t i o n of t h e u n e x t r a c t a b l e residue of bean leaves t r e a t e d w i t h ^ C - a z i n p h o s , w i t h v a r i o u s s o l v e n t s at room temperature
ELUTION SOLVENT
VOLUME ELUTED ML
Acetone Ethylacetate Methanol Methanol/H 0 (4:1) Acetonitril/EthylAcetate/Methanol H 0 (65/15/10/10) Butanol/Acetic Acid/H 0 (4/1/1) 6 N Acetic Acid H0 2 N NH OH H 0
28 27 31 33
9
COLUMN I 749013 DPM/ 304.9 MG DR.M. %
COLUMN I I 919169 DPM/ 374.2 MG DR.M. %
38 37 32 44
0.5 0.2 5.2 22.2
0.4 0.1 4.5 23.9
32 - 33
1.7
0.8
33 - 36
24.3
38.9
16 29-30 4-5 12 - 20
18.9 0.2 6.1 3.2
7.2 0.1 4.4 3.2
82.5
83.5
-
l
2
2
2
4
2
T o t a l Soluble
In Bound and Conjugated Pesticide Residues; Kaufman, Donald D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 2, 2016 | http://pubs.acs.org Publication Date: June 1, 1976 | doi: 10.1021/bk-1976-0029.ch011
11.
WIENEKE
Pesticides Bound to Plant Material
169
question, a l i q u o t s of the unextracted residue f r a c t i o n were treated as f o l l o w s : 1. Hydrolysis with 25% formic a c i d (known to cleave h e m i c e l l u l o s e and o l i g o s a c c h a r i d e s e t c . ) , 2. d i s s o l u t i o n of c e l l u l o s e i n the remaining residue (procedure a f t e r Waksmann, 1931). Radioactive m a t e r i a l was d i s t r i b u t e d i n the c a t i o n i c , a n i o n i c and n e u t r a l f r a c t i o n s , i n the c e l l u l o s e f r a c t i o n and a l s o i n the f r a c t i o n corresponding to l i g n i n . There was no i n d i c a t i o n that the parent compound or a major p o r t i o n of the parent compound was present i n these f r a c t i o n s . A second approach to the problem of the e x t r a c t i o n of bound l^c-azinphos residues was to f i l l a chromatography column with the unextractable residue f r a c t i o n and e l u t e the m a t e r i a l with d i f f e r e n t solvents and solvent mixtures. The r e s u l t s of these studies (Table 1) demonstrate that greater than 80% of the r a d i o l a b e l i s extracted by t h i s method. T h i n l a y e r chromatographic a n a l y s i s of the various eluted f r a c t i o n s revealed that the r a d i o l a b e l was d i s t r i b u t e d i n s e v e r a l compounds. The s i g n i f i c a n c e of bound p e s t i c i d e residues i s p r i m a r i l y a c o n s i d e r a t i o n of the amount and the character of the substances analyzed as bound r e s i d u e s . The r e s u l t s reported i n table 1 suggest that the removal of bound materials from plant t i s s u e i s dependent upon the solvent e x t r a c t i o n procedure. These r e s u l t s challenge us to examine c r i t i c a l l y the various commonly used e x t r a c t i o n methods f o r the i s o l a t i o n of f r a c t i o n s reported as "bound p e s t i c i d e r e s i d u e s " . The question a r i s e s as to whether all the substances that are not d i s s o l v e d by a s p e c i f i c solvent method may be c l a s s i f i e d as bound r e s i d u e s , or should only those portions that remain a f t e r e x t r a c t i o n with e i t h e r a c i d or a l k a l i n e solvent mixtures be considered bound p e s t i c i d e residues? Furthermore it seems questionable to analyze for bound residues i n p l a n t materials i f the concentration i s low, such as with azinphos which was only 5-10% of the t o t a l found i n treated bean leaves. Even when c a l c u l a t e d as azinphos equivalents the t o t a l concentration i n the unextractable residue i s f a r below the residue tolerance l e v e l s f o r the parent product. I f , however, the concentration of the unextractable residue r a d i o a c t i v i t y i s high, the nature of the r a d i o a c t i v i t y a s s o c i ated with t h i s f r a c t i o n should be determined. I f these residues can be d i s s o l v e d , then the s o l u b i l i z e d r a d i o a c t i v i t y should be c h a r a c t e r i z e d to determine whether it i s the parent compound, or a metabolic fragment.
Waksman, S.A. and Tenney, F.G.: Soil Sci. 24, 275 (1931) cited in: Bodenkundliches Praktikum, edited by E. Schlichting and H.P. Blume, Parey-Verlag, Hamburg, Berlin.
In Bound and Conjugated Pesticide Residues; Kaufman, Donald D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1976.
