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Picloram residues in potatoes and carrots and picloram photodecomposition. Soniassy, Ranjit Nunderdass 1970

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PICLORAM RESIDUES IN POTATOES AND CARROTS AND PICLORAM PHOTODECOMPOSITION by RANJIT NUNDERDASS SONIASSY .Sc. (Hons.) University of B r i t i s h Columbia 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Plant Science We accept th i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 197° In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f • P l a n t Science The U n i v e r s i t y o f B r i t i s h Co lumb ia V a n c o u v e r 8. Canada Date D e c . 1 1 . 1 9 7 0 - i i i -ABSTRACT Residues of picloram i n four v a r i e t i e s of potatoes, given pre-emergence treatments with picloram at 2 oz per acre and picloram at 2 oz + linuron at 2k oz per acre were determined by electron capture gas chromatography. Average residue l e v e l s of 3*9 and 2.7 ppb (fresh weight) were obtained f o r the picloram and picloram + linuron treatments. This difference was s i g n i f i c a n t at the 1$ l e v e l . Tuber injury, ranging from formation of corky tissues on the surface to s p l i t t i n g , was observed with the picloram treatments. No such injury was observed with the picloram + linuron treatments. Yields were noticeably higher i n the picloram + linuron treatments. The reduced picloram residues could thus be associated with the d i l u t i n g effect of higher y i e l d s which resulted from the addition of linuron. A s i m i l a r experiment using four v a r i e t i e s of carrots given either pre-emergence or post-emergence treatments with picloram at and 2 oz per acre respectively gave no detectable 1^ picloram residues. Using C carboxyl labeled picloram t h i s f i n d i n g was further investigated and i t was shown that picloram was absorbed by the f o l i a g e and roots and translocated throughout the whole plant. The leaves accumulated four times more r a d i o a c t i v i t y than the taproots. The r a d i o a c t i v i t y i n the leaves and taproots was i n the form of the parent picloram molecule. The picloram present i n the taproot was located mainly i n the _ i v _ xylem. A study of the s t a b i l i t y of picloram, i t s potassium s a l t and i t s methyl ester under short wave u l t r a v i o l e t l i g h t (253»7 nm) revealed that a l l three compounds were degraded into several photoproducts. The methyl ester was the least stable, being 85$ degraded a f t e r one hour exposure. Picloram and i t s potassium s a l t were more stable, each being 50$ decomposed a f t e r one hour exposure. P a r t i a l polymerisation of a l l three compounds may also have taken place. TABLE OF CONTENTS Page Introduction 1 Review of L i t e r a t u r e 3 Physical and Chemical Properties 3 Uses as a Herbicide 3 Herbicidal Action 5 Picloram as a Substitute f o r Auxin 6 Absorption as Undissociated Molecules 7 Translocation and D i s t r i b u t i o n 8 Excretion by Roots 9 H i s t o l o g i c a l Abnormalities 9 Ethylene Production 9 Carbohydrate Synthesis 10 L i p i d Synthesis 10 Respiration and TCA Cycle 11 Enzyme Synthesis 11 Nucleic Acid Metabolism and Protein Synthesis 12 Animal T o x i c i t y 13 Fate i n Animals Ik Decomposition by Microorganisms Ik Fate i n S o i l 15 Fate i n Plants 16 Residues of Picloram 17 - v i -A n a l y t i c a l Methods 17 Colorimetric Method 18 U l t r a v i o l e t Spectroscopy 19 Infrared Spectroscopy 19 Bio-assay 19 Thin Layer Chromatography 20 Gas Chromatography 21 Extraction 21 Clean-up 23 Methylation 25 Detection and Estimation 27 A r t i f a c t s i n Gas Chromatography 29 Newer Trends i n Analysis 29 On-column Decarboxylation 29 Alkaline Pre-column 30 Trimethyl S i l y l Derivatives 31 Photodecomposition 31 Materials and Methods 3^ F i e l d T r i a l 3^ Sample Preparation 35 Extraction 35 CIean-up 37 Scraping and E l u t i o n 40 Liquid-Liquid P a r t i t i o n i n g 40 Column Preparation 4 l Operating Conditions 4-3 _ vi:L-Absorption and Translocation 43 D i s t r i b u t i o n of Radioactivity 45 Determination of Metabolites 45 L o c a l i z a t i o n of Picloram 46 Photodecomposition 46 Results 48 Potatoes 48 Recovery of Picloram from Potatoes 50 Carrots 50 Leaf Treatment 52 Root Treatment 53 D i s t r i b u t i o n 55 Metabolism 56 L o c a l i z a t i o n 57 Photodecomposition 59 Discussion 62 Potatoes 62 Carrots 67 Photodecomposition 70 Summary 73 Bibliography 75 Appendix - v i i l -LIST OF TABLES Table I. Energy d i s t r i b u t i o n i n u l t r a v i o l e t range 32 Table I I . Bond energies 32 Table I I I . Residues of picloram i n potatoes 49 Table IV. Recovery of picloram from potatoes 50 Table V. D i s t r i b u t i o n of picloram i n 2-month old carrot plants 55 Table VI. R values of control and i r r a d i a t e d picloram, i t s potassium s a l t and i t s methyl ester 59 Table VII. Decomposition of picloram, i t s potassium s a l t and i t s methyl ester 60 Appendix I. Composition of Appendix I I . Composition of Appendix I I I . Chemical names complete nutrient solution l i q u i d s c i n t i l l a t i o n f l u i d of pesticides used i n text - ix:.: -LIST OF FIGURES Figure 1. Figure 2. Figure 3* Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9» Figure 10. Figure 11. Figure 12. Figure 13. Flash evaporation assembly 36 TLC streaker with t e f l o n t i p 38 TLC plate showing separation of methyl ester picloram from some natural products occurring i n potatoes 39 Gas chromatography assembly 42 Gas chromatographic peaks of methyl ester picloram 51 Mounts and radioautograms of 3-week old carrot plants, l e a f treated with labeled picloram 52 Mounts and radioautograms of 3-week old carrots plants, root treated with labeled picloram 54 Radioautograms of extracts of carrot l e a f and taproot 56 Radioautograms of longitudinal sections of carrot 57 Mounts of longitudinal sections of carrot §8 Photoproducts of picloram, i t s potassium s a l t and i t s methyl ester 6l Injury on potato tubers, due to picloram 63 Potato l e a f malformation, due to residual effect of picloram 65 Figure 14. Photoproducts of methyl ester picloram 72 - X -ACKNOldEDGEMENTS I wish to thank Dr. A.J. Renney, Assistant Dean, Faculty of A g r i c u l t u r a l Sciences, f o r the supervision of t h i s thesis. Much appreciation i s extended, f o r the review of th i s thesis, to the other members of my graduate committee: Dr. V.C. Brink, Department of Plant Science Professor T.L. Coulthard, Department of Mechanical Engineering Dr. V.C. Runeckles, Chairman, Department of Plant Science Dr. P.M. Townsley, Department of Food Science I would l i k e to thank the following persons f o r various assistance: Mr. I. Derics, Department of Plant Science; Dr. G.¥. Eaton, Department of Plant Science; Dr. B. Mullick, Faculty of Forestry; Mr. I. Williams, Canada Department of Agriculture. The very capable typing of t h i s thesis by Miss Ingrid Muehlthaler i s deeply appreciated. F i n a n c i a l support from the Canadian International Development Agency and study leave from the Government of Mauritius are g r a t e f u l l y acknowledged. INTRODUCTION A new era i n chemical weed control was ushered i n i n 1944 with the appearance of* the auxin-like, phenoxy herbicides, namely, 2,4-D, 2,4,5-T and MCPA (34). These were the f i r s t synthetic herbicides, active at rates of a few pounds per acre rather than hundreds of pounds per acre, as was the case with the older herbicides the arsenicals and borates. The announcement of a s t i l l more potent auxin-like growth regulator, picloram (4-amino -3 ,5»6-trichloro-picolinic-acid) i n 1963 (32) was another forward step towards getting greater k i l l and greater s e l e c t i v i t y i n weed control, while lowering rates of application to a few ounces per acre. Both i n h i b i t i o n and promotion of growth are involved with auxin-like herbicides, depending on the concentration used, the plant species and the type of application. Apart from t h e i r p l a n t - k i l l i n g capacities, herbicides also have b e n e f i c i a l effects on plants. Reports on stimulation of plant growth, leading to increased y i e l d , by application of sublethal concentrations of 2,4-D are numerous. One recent example of th i s b e n e f i c i a l effect of herbicides, was reported by Luck w i l l (65) when i t was found that an application of 3 mg simazine per plant was equivalent to that of 570 mg nitrogen, applied as ammonium n i t r a t e , i n enhancing growth and nitrogen content of corn. Some of these newer chemicals, though used at rates of a few ounces per acre, are very persistent and by acting at 2 -the c e l l u l a r l e v e l can induce profound changes i n an organism or i t s progeny. Greater sophistication i n a n a l y t i c a l methods coupled with instances where some of the chlorinated hydrocarbons have been found i n animal tissues have l e d to a greater awareness that some of these chemicals, or t h e i r breakdown products, are passing on to man i n his d i e t . The concept of "Pesticide Residues'* was thus born. This awareness has been manifested i n several ways, including studies of uptake by, and translocation and d i s t r i b u t i o n i n the organism concerned; residues and persistence i n the environment at large i n c l u d i n g target and non-target organisms; and metabolism and degradation by b i o l o g i c a l and non-biological systems. S c i e n t i f i c c u r i o s i t y , a willingness to know and the fear of the unknown have forced s c i e n t i s t s to aim at following these man-made molecules as c l o s e l y as possible once they have been sprayed on a target organism. The objectives of t h i s study were to: 1. become f a m i l i a r with techniques useful f o r picloram residue studies. 2. f i n d whether residues of picloram could be found i n a tuber crop, potato, and a root crop, carrot. 3. determine whether picloram residue l e v e l s were affected by the presence of a second herbicide, linuron. k. study the s t a b i l i t y of picloram i n acid, s a l t and ester form when subjected to u l t r a v i o l e t r a d i a t i o n . - 3 -REVIEW OF LITERATURE Physical and Chemical Properties of Picloram Picloram has the molecular structure: CO OH (4-amino -3 ,5 ,6-trichloropicolinic acid; M.¥.= 241.5) and i s a white c r y s t a l l i n e powder, with a f a i n t c h l o r i n e - l i k e odor (32,92). I t s vapor pressure at 35°C i s 6.16 x 10- 7 mm Hg. I t i s very s l i g h t l y soluble i n the non-polar organic solvents and quite soluble i n the polar ones, as shown below: Solvent S o l u b i l i t y i n ppm Acetone 19,800 Ethanol 10,500 A c e t o n i t r i l e 1,600 Diethyl ether 1,200 Water 430 Benzene 200 Carbon d i s u l f i d e 50 Kerosene 10 Hexane insoluble I t i s commercially available f o r weed control, under the trade name of Tordon (Dow Chemical Co.). Uses as a Herbicide Picloram was f i r s t used to control weeds on non-crop areas l i k e highways, r a i l r o a d s and around farm buildings and fences. Presently i t i s used f o r the control of brush (103*107), woody range species (23) and deep- rooted perennial weeds (57,89,102). Rates of \ to 1 ounce per acre are e f f e c t i v e against seedling broad-leaf weeds. Perennial weeds may be controlled by 1 - 4 l b per acre. S o i l applications of 6 - 8 l b per acre control most woody plants. It has a r e l a t i v e l y long r e s i d u a l l i f e i n s o i l , estimated at about 13 months (29). Because of i t s good translocation properties, i t i s ef f e c t i v e both v i a f o l i a r and s o i l applications. Mixtures of picloram and other herbicides, p a r t i c u l a r l y 2,4-D and 2,4,5-T are currently used i n weed control (3,4,6o). Herbicide mixtures may not only broaden the spectrum of weeds to be controlled but also reduce the rates of application, by making use of t h e i r s y nergistic properties. Chuma (14) reported that a mixture of picloram and 2,4-D each at 0.01 and 0.1 l b per acre respectively k i l l e d tops of f i e l d bind weed fa s t e r than 2,4-D alone or picloram alone at the same rates. This enhanced a c t i v i t y i s due to the greater translocation rate when applied i n combination than when applied singly. Bovey e_ a l ( l l ) reported the antagonistic properties of picloram and other herbicides. They found that the addition of paraquat to picloram decreased the effectiveness of picloram on huisache, (Acacia  farnesiana (L.) and mesquite plants, Prosopis j u l i f l o r a (Swartz). Davis e_ a l (17) showed that i n mesquite, huisache and bean plants, the transport of picloram was reduced, when used i n combination with paraquat. Whether differences i n translocation alone can explain synergistic and antagonistic properties of picloram, when used with other herbicides, cannot be answered yet. Picloram has a long residual l i f e i n s o i l and has been removed from the United Kingdom market f o r t h i s reason. However i t i s licensed f o r use i n B r i t i s h Columbia. Herbicidal Action  General Van Overbeck (100) defined a herbicide as "a chemical that deranges the physiology of a plant over a period long enough to k i l l i t " . A few of these ph y s i o l o g i c a l processes are: photosynthesis, r e s p i r a t i o n , c e l l d i v i s i o n and nucleic acid metabolism. Bach of these processes i s the o v e r a l l expression of a multitude of sub-processes which can be i n h i b i t e d by herbicides. A few of these sub-processes are: 'a) I n h i b i t i o n of l i g h t system I, [b) i n h i b i t i o n of l i g h t system I I , ) Photosynthesis c) i n h i b i t i o n of photophosphorylation. 'd) Uncoupling oxidative phosphorylation, e) i n h i b i t i n g oxidative phosphorylation,) Respiration ,f) i n h i b i t i n g oxygen uptake. fe) I n h i b i t i o n of spindle formation, ) C e l l d i v i s i o n clumping of chromosomes. ) (i ) Interference with nucleic acid metabolism, (j) interference with protein synthesis. Picloram The p h y s i o l o g i c a l basis f o r the h e r b i c i d a l action of picloram i s e s s e n t i a l l y unknown. Several workers have delved into a few aspects of i t s mode of action. A major advance was made when picloram was shown to possess auxinic properties (32, 49). This was an aid to understanding i t s mechanism of action because the physiological a c t i v i t y of another auxin-like herbicide - 6 -namely 2,4-D has already benefited from two decades of research. The various aspects of the mode of action of picloram are dealt with i n the succeeding pages. Picloram as a Substitue f o r Auxin Picloram at low concentration can be substitued f o r ind o l e - 3 - a c e t i c acid (iAA) and 2,4-D as an auxin source i n synthetic media. Goodwin (28) reported that 1 - 2 mg picloram per l i t e r of synthetic medium gave optimum ca l l u s growth. In our laboratory root elongation of cress seedlings was obtained with concentrations of 0.001 - 0.005 Ppm while growth retardation was observed at about 0.05 PPm concentration and above. Kefford et a l (49) d i d a comprehensive study to test the potency of picloram as an auxin. The b i o l o g i c a l systems tested and the res u l t s are given i n totoj Tests 1. Extension of wheat and oat c o l e o p t i l e sections* 2. Extension of pea stem sections. 3» Extension of sectioned pea root t i p s . 4. Growth of i n t a c t roots from y - i r r a d i a t e d and non-i r r a d i a t e d wheat grain, ( y - i r r a d i a t i o n prevents c e l l d i v i s i o n but not growth). 5. Development of parthenocarpic tomato f r u i t s . 6. Induction of c e l l d i v i s i o n i n tobacco stem p i t h . Results ( i n same order as tests)? 1. On the basis of the concentrations producing h a l f maximal extension, picloram had about one t h i r d the a c t i v i t y of IAA i n - 7 -wheat and oat c o l e o p t i l e sections, but the maximum section elongation obtained with picloram and IAA were the same. 2. For the extension of pea internode sections picloram was about 100 times more active than IAA; 2,4-D had a c t i v i t y i n t e r -mediate between picloram and IAA. 3. The growth of pea root t i p s was stimulated by low concentration and i n h i b i t e d by high concentration of picloram. Least growth was obtained with picloram followed by IAA and 2,4-D. 4. As i n h i b i t o r s of root elongation on non-irradiated seedlings IAA, 2,4-D and picloram showed approximately equal e f f i c i e n c y ; but f o r y - i r r a d i a t e d seeds, picloram was l e s s i n h i b i t o r y than IAA or 2,4-D. 5. When compared to 2-naphthoxyacetic acid i n the induction of growth of f r u i t s from emasculated tomato flowers, picloram was much l e s s active, but nonetheless i t showed some a c t i v i t y . 6. In absence of a k i n i n source neither IAA nor picloram induced c e l l d i v i s i o n . In presence of k i n i n , picloram at 10 M was as e f f e c t i v e as IAA at 10~^M, the IAA concentration found optimum f o r t h i s t e s t . Absorption as Undissociated Molecules Baur et a l (8) showed that pH had a s i g n i f i c a n t e f f e c t on the uptake of picloram by potato tuber d i s c s . The molecule i s absorbed mostly i n the undissociated form but uptake i s not dependent on the concentration of the undissociated molecule. No report, however, has been made as to whether there - 8 -i s a difference i n the p h y t o t o x i c i t i e s of the dissociated and undissociated picloram molecule. Vapors of the potassium s a l t of picloram have been found to be h e r b i c i d a l l y active on bean plants (Phaseolus  vulgaris) (25). Since the s a l t was at ambient temperature, i t can be reasonably assumed that the herbicide molecule was i n the undissociated state. Translocation and d i s t r i b u t i o n Horton jet a l (44) using agar blocks containing carboxyl labeled picloram, showed that transport through petioles of bean and coleus plants and stems of pea plants i s b a s i p e t a l . Again they showed that picloram i s transported f a s t e r then 2,4-D, under the same conditions. Bovey est a l (12) showed that translocation of picloram from f o l i a g e to roots, i n huisache plants, was increased by the presence of leaves. Chuma elb a l (l4) c l a r i f i e d the s i t u a t i o n better by showing that older leaves appear to play a more s i g n i f i c a n t r o l e i n the absorption and subsequent movement of both picloram and 2,4-D, than the young leaves. He found no difference i n the amount of picloram absorbed by i n t a c t plants and plants i n which the upper leaves and growing t i p s were removed. However the removal of lower leaves or complete d e f o l i a t i o n reduced the absorption of picloram. In f i e l d bind weed the presence of leaves i s important i n the d i s t r i b u t i o n of picloram, i n huisache however the d i s t r i b u t i o n i s the same with d e f o l i a t e d and undefoliated plants. In huisache, i n presence of the leaves, more picloram - 9 -was translocated to the roots than to the shoots. Removal of a l l the leaves, except the treated l e a f , reversed the d i r e c t i o n of f o l i a r applied picloram. Excretion by Roots Hurtt et a l (46) showed that picloram was excreted by roots of bean plants, a f t e r application of the herbicide to the f o l i a g e . I t was released from the roots of donor plants, into the nutrient solution, within 24 hours; the excreted picloram was taken i n by the roots of untreated plants and since the l a t t e r developed symptoms s i m i l a r to that of picloram t o x i c i t y , i t was assumed that the molecule was excreted unchanged. Excretion of picloram or other herbicides from the roots i s not considered a major pathway of the d e t o x i f i c a t i o n mechanism of plants. H i s t o l o g i c a l Abnormalities Kreps (53) studied the h i s t o l o g i c a l abnormalities induced by picloram i n Canada t h i s t l e roots. Swelling and s p l i t t i n g were observed throughout the entire root system. Some c e l l s i n the exodermis and subexodermis areas had deteriorated, leaving a i r spaces i n the cortex. Parenchyma c e l l s , found i n the centre of the cortex, were p a r t i a l l y or completely destroyed. The cambium layer had disintegrated leaving the xylem s t i l l i n t a c t but detached from the rest of the root t i s s u e . The greatest damage observed was complete destruction of cortex, phloem, cambium while xylem and periderm remained i n t a c t . Ethylene Production Baur et a l (7) found a tenfold increase i n ethylene - 10 -production a f t e r treatment of huisache and mesquite seedlings with picloram. There i s some controversy i n the l i t e r a t u r e as to whether auxin-like herbicides induce ethylene production i n res i s t a n t species, susceptible species or both. Morgan et, a l (79) showed that 2,4-D promoted ethylene production from susceptible dicotyledonous plants and not from the r e s i s t a n t monocotyledons. Abeles (2) however showed that 2,4-D increased ethylene production i n both r e s i s t a n t (corn) and susceptible (soybean) plants. The rate of ethylene production was greater i n soybean than i n corn. Ethylene i t s e l f i s a growth regulator and auxin-induced ethylene production i s believed to be mediated through the production of s p e c i f i c enzymes, which i n turn enhance the sythesis of ethylene. Carbohydrate Synthesis Increases i n t o t a l carbohydrates and reducing sugars have been shown (56). Root exudates from corn seedlings treated with 50 - 500 ppm picloram gave increases i n t o t a l carbohydrates ranging from 170 to 430$, while the increase i n reducing sugars ranged from 190 to 270$. This increase i n carbohydrate exudation i s detrimental to the plant by causing increased root rot i n cereal seedlings (95). No explanation f o r t h i s observation has yet been postulated. L i p i d Synthesis The effect of picloram on l i p i d synthesis has been reported (69). L i p i d synthesis was followed by incubating sesbania hypocotyls (Sesbania punicea) with malonic acid - 2-^C, - l i -the l i p i d s were extracted and the incorporation measured by amount of radioactive l i p i d s produced. An average increase of about 45$ above the control was obtained with concentrations of 1 - 2 0 mg of picloram per l i t e r . However there was no co r r e l a t i o n between increase i n l i p i d synthesis and concentration of picloram used. Respiration and the T r i c a r b o x y l i c Acid Cycle Mitochondria are heavily involved i n r e s p i r a t i o n and i s o l a t e d mitochondria have proved useful i n studying i n h i b i t o r s of r e s p i r a t i o n . Poy et a l (22) tested the ef f e c t of several herbicides on the substrates of the t r i c a r b o x y l i c acid cycle of i s o l a t e d cucumber mitochondria. Picloram at 10""^ M strongly i n h i b i t e d the oxidation of succinate and moderately i n h i b i t e d the oxidation of <F—keto-glutarate. The i n h i b i t i o n was measured by the amount of oxygen consumed. For succinate and keto-glutarate the oxygen consumptions, above the contro, were 10 and 43$ respectively a f t e r 1 hour. Enzyme Synthesis During germination the de novo synthesis of p r o t e o l y t i c enzymes, leading to an increase i n the p r o t e o l y t i c a c t i v i t y , i n squash cotyledons has been reported. Ashton et a l (6) tested the e f f e c t of several herbicides on the p r o t e o l y t i c a c t i v i t y of squash cotyledons. Picloram at 10""^  and 10~^ M concentrations i n h i b i t e d p r o t e o l y t i c a c t i v i t y by 38$ and 78$ respectively. At 10-3M concentration the i n h i b i t i o n f o r 2,4-D was 45$. On the other hand West et a l (105) have shown that i n h i b i t i o n of - 12 -p r o t e o l y t i c a c t i v i t y by 2,4-D i s not responsible f o r i n h i b i t i o n of growth. Moreland (78) reported an h i b i t i o n i n d~-amylase induction of 60$ i n barley seeds as a r e s u l t ot treatment with picloram. Nucleic Acid Metabolism and Protein Synthesis Malhotra et a l (68) investigated the re l a t i o n s h i p between species s e n s i t i v i t y to picloram and changes i n nucleic acid metabolism. For the 5 species of graded s e n s i t i v i t y used -barley (very r e s i s t a n t ) , wheat, com, cucumber and soybean (very sensitive) - he found a p o s i t i v e c o r r e l a t i o n between induction of RttA synthesis and h e r b i c i d a l s e n s i t i v i t y . The sensit i v e plant species responded to picloram treatment with an increase i n RNA content whereas the RNA content of the res i s t a n t species changed very l i t t l e . Since the sensi t i v e plant species have high DNA and RNA contents, compared to the res i s t a n t ones, i t was thought that the resist a n t species may have some kind of "block1* which prevents accumulation of nuclear material. One such block might be i n the l e v e l of deoxyribonuclease and ribonuclease. Malhotra also showed that the endogenous l e v e l of deoxyribonuclease and ribonuclease i n the res i s t a n t species was much higher than i n the sensitive ones. The s p e c i f i c a c t i v i t i e s of bound ribonuclease correlated inversely with h e r b i c i d a l s e n s i t i v i t y . Resistant plants were higher i n bound nucleases than sensitive ones. No such c o r r e l a t i o n f o r the free enzyme was found. I t i s possible that picloram may have a si m i l a r effect i n promoting nucleic acid synthesis i n both - 13 -r e s i s t a n t and sensitive species. However the presence of* higher l e v e l s of native bound nucleases i n the r e s i s t a n t species prevent accumulation of n u c l e i c acids. The nucleic acids may be degraded as soon as they are synthesised. The r o l e of nucleases, however, i n the metabolism of the c e l l i s s t i l l l a r g e l y unknown. Key (50) showed that ribonucleic acid synthesis and protein synthesis are essential processes f o r c e l l elongation. Working with 2,4-D, Key et a l (51) showed that nucleic acid metabolism was suppressed when growth was suppressed and was accelerated when growth was accelerated. No such work has yet been reported f o r picloram. Moreland et^ a l ( 7 8 ) investigated the incorporation of a few precursors into protein and RNA and found that picloram had: 1. no effect on incorporation of ATP -8- l i fC into RNA. 2. no effect on incorporation of 1 - l e u c i n e - l - ^ C into protein. 3. an i n h i b i t o r y effect on the incorporation of o r o t i c ac±d-6-^C into RNA. Animal T o x i c i t y Picloram i s of low t o x i c i t y to humans, live s t o c k , w i l d l i f e and f i s h . Jackson ( 4 7 ) reported that single o r a l doses of 540 and 720 mg per kg body weight were not toxic to c a t t l e and sheep respectively. The following o r a l f o r s m a 1 1 animals was reported by Lynne ( 6 2 ) : - 14 -Animal LV^* (mg per kg) Rat 8,200 Mouse 2,000 - 4,000 Guinea p i g 3,000 Rabbit 2,000 Chicken 6,000 Hardy (37) found that the presence of 1 ppm picloram i n water did not a f f e c t the development, behaviour and reproduction of daphnia, guppies and algae. Fate i n Animals Fisher et a l (21) analysed the feces, milk and urine of dairy cows, fed with 5 ppm piclorara-treated fodder. He found that no residue was present i n the milk and feces and most of i t was eliminated i n t a c t i n the urine. On the other hand Kutschinski (54) found that the average residue l e v e l s of picloram i n cow's milk were 0.05 and 0.2 ppm when c a t t l e were fed with 300 and 1,000 ppm picloram respectively i n forage. He also found that the residues disappeared within 58 hours a f t e r withdrawal of the picloram from the d i e t . Decomposition by Microorganisms Picloram i s apparently neither an important substrate f o r , nor an i n h i b i t o r of growth of microorganisms. Youngson et a l (110) found that only small amounts of picloram, ranging from 0.24 to 1.21$ were decomposed i n cultures of bacteria * L D C J Q ; The s t a t i s t i c used to indicate the degree of t o x i c i t y . I t i s expressed as the number of milligrams of a toxicant per kilogram of body weight of an animal, s u f f i c i e n t to k i l l f i f t y per cent of such animals. - 15 -and fungi. The decomposition was followed by evolution of co 2. Goring jet a l (30) showed that concentrations of picloram as high as 1,000 ppm did not i n h i b i t the growth of microorganisms. Fate i n S o i l Picloram p e r s i s t s i n a l l types of s o i l , under d i f f e r e n t temperature and moisture l e v e l s f o r more than one growing season. Studies by Herr (kQ) and Herr et a l (hi) indicated that s o i l organic matter i s most i n f l u e n t i a l i n preventing the leaching of picloram. Hamaker jet a l (33) reached the same conclusion, adding that "the sorption of picloram i s primarily caused by organic matter and hydrated metal oxides, with clays probably playing a minor r o l e " . In spite of being sparingly soluble i n water, picloram i s leached r e l a t i v e l y e a s i l y i n a l l types of s o i l , though more so i n sandy s o i l s , (7*0 . Hartley (39) suggested that hydrolysis and oxidation reactions might be accelerated by the adsorption of herbicides by s o i l c o l l o i d s . Armstrong et a l (5) and Harris (38) have published evidence supporting t h i s non-biological breakdown i n the case of the c h l o r o t r i a z i n e herbicides. Hance (35,36) investigated t h i s process f o r picloram. He found that f o r a constant amount of a i r - d r i e d s o i l , the rate of breakdown of picloram was not s i g n i f i c a n t l y affected by the amount of water added during the incubation period. - 16 -This eliminated the assumption that the extent of adsorption on s o i l p a r t i c l e s could influence the decomposition. Using d i f f e r e n t amounts of s o i l , he showed that the rate of breakdown of the herbicide was dependent on the amount of s o i l present. Since the rate of decomposition i s related to the amount of s o i l and not to the extent of adsorption i t would appear that the decomposition occurs at s p e c i f i c s i t e s i n the s o i l . Whether these s p e c i f i c s i t e s are s o i l c o l l o i d s as suggested by Hartley or on carbon and/or hydrated oxides of i r o n and aluminium as suggested by Hamaker i s not known. Fate i n Plants Radioautography of wheat seedlings, grown i n s o i l containing carboxyl labeled picloram, showed the presence of radioactive residues i n a l l the organs. Redemann (87) showed that i n mature wheat grain, most of the residues existed as the parent molecule, with small amounts of breakdown products. The l a t t e r have been i d e n t i f i e d as: oxalic acid 8$ 4-amino -3 ,5-dichlorohydroxypicolinic acid 5$ 4-amino - 2 ,3 ,5-trichloropyridine 4$ unchanged picloram 83$ In cotton plants, however, in t a c t picloram with no other metabolite was found. Meikle et a l (71) found that 95$ of the r a d i o a c t i v i t y was d i s t r i b u t e d i n the leaves and stems and 5$ i n the roots. - 17 -Residues of Picloram Most of the l i t e r a t u r e on. "Residues of Picloram" deal with methodology. Only a l i m i t e d amount of information i s available on residues per se found i n treated crops and dairy products. Crops Samples of wheat and barley treated with picloram at rates of 0.2 to 1 ounce per acre, applied as post-emergence sprays, were analysed by Bjerke et a l (10). Seventy-five per cent of the samples were reported to contain residues l e s s than 0.05 ppm. Residues as high as 0.22 ppm i n wheat grain, 0.44 ppm i n wheat straw and 0.64 ppm i n barley grain were also reported. Dairy Products Kutschinski et a l (55) reported that steers fed d a i l y , over a period of two weeks, with 200 - l,600.ppm picloram i n t h e i r diet had the following amounts of picloram residues i n t h e i r tissues; Fat and muscle le s s than 0.05 - 0.5 Ppm blood and l i v e r 0.12 - 2.0 ppm kidney 2.0 - 18.0 ppm A n a l y t i c a l Methods The choice of a n a l y t i c a l method depends on several fa c t o r s , such as the amount of compound present, the l e v e l of detection (whether milligram or nanogram range), the nature of b i o l o g i c a l or other materials from which the compound under in v e s t i g a t i o n i s to be i s o l a t e d and l a s t l y the a v a i l a b i l i t y of - 18 -equipment. Broadly two types of analyses are encountered: f o r m u l a t i o n and r e s i d u e . I n f o r m u l a t i o n a n a l y s i s a l l o f the f o l l o w i n g methods can be a p p l i e d : 1. C o l o r i m e t r i c 2. U l t r a v i o l e t s pectroscopy (UV) 3. I n f r a r e d spectroscopy (IR) h. Bio-assay 5. T h i n l a y e r chromatography (TLC) 6. Gas l i q u i d chromatography (GLC) The f i r s t f i v e methods are p r e f e r a b l e because of r a p i d i t y of measurement. I n r e s i d u e a n a l y s i s , where v e r y s m a l l amounts of the compound are to be s t r i p p e d from l a r g e amounts of b i o l o g i c a l o r o t h e r m a t e r i a l s , TLC and GLC are the standard techniques used. C o l o r i m e t r i c Method A c o l o r i m e t r i c method s u i t a b l e f o r the a n a l y s i s of p i c l o r a m i n commercial f o r m u l a t i o n s o r i n s o i l s c o n t a i n i n g h i g h r e s i d u e l e v e l s , has been d e s c r i b e d (13)• The method c o n s i s t s o f e x t r a c t i n g the sample w i t h s o l u t i o n s of IM ammonium a c e t a t e o r 2M potassium c h l o r i d e c o n t a i n i n g s u f f i c i e n t potassium hydroxide to g i v e an e x t r a c t o f pH 7.0. The p i c l o r a m , as the potassium s a l t , i s t r e a t e d with 3«5M s u l f u r i c a c i d and 0.1M sodium n i t r i t e . The c o l o r o f the d i a z o compound i s allowed to develop at room temperature i n the dark, as i t i s u n s t a b l e t o u l t r a v i o l e t l i g h t . The absorbance at - 19 -405 nm* i s measured on a spectrophotometer. T h i s method i s p r e f e r r e d where speed r a t h e r than s e n s i t i v i t y i s the c r i t e r i o n . U l t r a v i o l e t Spectroscopy Hummel (4-5) has used the UV absorbance o f p i c l o r a m i n i t s e s t i m a t i o n . The m a t e r i a l i s d i s s o l v e d i n 6M h y d r o c h l o r i c a c i d and the absorbance at 285 nm measured. I n f r a r e d Spectroscopy T h i s method was developed by Melcher (72). The sample i s e x t r a c t e d w i t h a c i d i f i e d acetone, the s o l v e n t evaporated to dryness and the r e s i d u e d i s s o l v e d i n dimethyl formamide and t r a n s f e r r e d i n t o an i n f r a r e d c e l l . The absorbance at 11.15 u** i s due to p i c l o r a m . Bio-assay The s u s c e p t i b i l i t y o f c e r t a i n p l a n t s to extremely small q u a n t i t i e s o f p i c l o r a m has made p o s s i b l e the use of b i o -assay methods f o r i t s d e t e r m i n a t i o n . Bio-assay methods may d e t e c t q u a n t i t i e s l e s s than 1 ppm, however they are not q u a l i t a t i v e . A bean b i o - a s s a y t e s t had been d e s c r i b e d by Leasure (59). The l i m i t s o f d e t e c t i o n were r e p o r t e d to be 0.5 ppb*** and 1 ppb i n sand and s o i l r e s p e c t i v e l y . Goodwin (27) claimed t h a t b i o -assay w i t h s e s b a n i a s e e d l i n g s c o u l d d e t e c t p i c l o r a m i n presence of phenoxy h e r b i c i d e s , as s e s b a n i a i s i n s e n s i t i v e to be l a t t e r . The lowest l i m i t of d e t e c t i o n was r e p o r t e d to be 0.004 ppm. * 1 nanometer (nm)= 1 x 10~^ meter * * 1 micron (u) = 1 x 10"° meter * * * 1 p a r t p e r b i l l i o n (ppb)= 1 p a r t i n 109 p a r t s - 20 -Thin Layer Chromatography The t h i n layer chromatography of picloram had been described by several workers. Whitenberg (106) reported the detection of picloram i n the 1 to 0.1 ug range on s i l i c a gel and alumina plates, developed with the solvent system: petroleum ether + chloroform + 95$ ethanol, 7 + 2 + 1 (v/v) and sprayed successively with: (a) 1$ t e r t i a r y butyl hypochlorite i n cyclohexane and (b) 1$ potassium iodide + 1$ soluble starch i n d i s t i l l e d water. No R^ . values were reported. Abbot et a l ( l ) reported the separation of twelve herbicides, including picloram by TLC. The supports consisted both of single and mixed absorbents of s i l i c a gel and keiselguhr and out of the eight solvent systems t r i e d , only one was successful i n moving picloram, namely: chloroform + acetic acid, 19 + 1 (v/v) with an Rj. of 0.21; the support was s i l i c a gel. Meikle (7l) described four solvent systems f o r the paper chromatography of picloram. The solvent systems are l i s t e d below: Solvent system R f picloram 1. 1-butanol saturated with 1.5M ammonium 0.47 hydroxide 2. tert-amyl alcohol + 15M ammonium hydroxide + 0.25 water, 1 0 + 1 + 5 (v/v) 3. benzene + propionic acid + water, 0.39 2 + 2 + 1 (v/v) 4. toluene + propionic acid + water, 0.35 2 + 2 + 1 (v/v) - 21 -Paper chromatography has now been la r g e l y replaced by thi n layer chromatography on c e l l u l o s e support, as i t gives better resolution, lower l i m i t s of detection and a reduction i n the development time. Gas Chromatography The presence of three chlorine atoms and two nitrogen atoms i n the picloram molecule makes i t s detection by gas chromatography, equipped with an electron capture detector, possible. Organic acids when gas chromatographed as such give large t a i l i n g peaks with long retention times, (21). When e s t e r i f i e d however, the esters give sharp peaks, making qu a n t i t i z a t i o n possible. In the residue analysis of pesticides by gas chromatography three broad steps are to be considered: (a) extraction or '"stripping" of the residue from the sample (b) clean-up or i s o l a t i o n of the pesticides from the contaminants and (c) q u a l i t a t i v e and quantitative measurement of the residue by gas chromatography. Extraction Extraction i s necessary i n any analysis where the compound under in v e s t i g a t i o n i s present i n extraneous materials. Depending on the material from which the pes t i c i d e i s to be stripped o f f , two extraction procedures have been currently used, namely: - 22 -1. blending with suitable solvents 2. soxhlet extraction with appropriate solvent, with or without thimble. The need f o r a "universal" or broad spectrum extracting solvent i n p e s t i c i d e residue analysis has l e d to the development of the solvent propylene carbonate (93). Both polar and non-polar compounds can be extracted with i t . The extraction procedures described f o r picloram divide into two main groups v i z : 1. extraction with d i l u t e a l k a l i , (10,58) 2. extraction with a c i d i f i e d polar organic solvents, (21,73,92). In the f i r s t method of extraction, the material i s blended with i c e cold 0.1M potassium hydroxide i n a 10$ potassium chloride solution. A f t e r f i l t r a t i o n , the extract i s a c i d i f i e d with mineral acid and the picloram p a r t i t i o n e d i n t o d i e t h y l ether. The p r i n c i p l e of thi s method i s that potassium hydroxide converts picloram into i t s highly water soluble potassium s a l t . Addition of potassium chloride decreases the amount of coextractives. When a c i d i f i e d , the potassium s a l t i s converted into picloram acid. Since the s o l u b i l i t y of picloram i n d i e t h y l ether i s 1,200 ppm compared to 430 ppm i n water, i t goes p r e f e r e n t i a l l y i n t o the ether layer. In the present study t h i s method of extraction was not found to be convenient. When fresh carrots and potatoes are extracted with d i l u t e potassium hydroxide solution a c o l l o i d a l suspension i s obtained, which immediately clogs a f i l t e r paper - 23 -and takes a considerable time to f i l t e r over glass wool. When t h i s f i l t e r e d solution i s a c i d i f i e d a gelatinous l i k e p r e c i p i t a t e i s obtained which again presents f i l t r a t i o n d i f f i c u l t i e s . On extraction with ether an emulsion i s obtained which can only be broken down by centrifugation. A l l these problems make an alkaline extract inconvenient f o r f r e s h carrots and potatoes. In the second extraction procedure the material i s blended with a c i d i f i e d acetone or a c i d i f i e d d i e t h y l ether. Acetone i s more currently used because of the higher s o l u b i l i t y of picloram i n t h i s solvent. ( S o l u b i l i t y i n acetone 19,800 ppm, i n ether 1,200 ppm). In the present study extraction with a c i d i f i e d acetone of both carrots and potatoes gave a cl e a r solution which did not present any f i l t r a t i o n problem, and was subsequently adopted. Clean-up After solvent extraction the compound or compounds under in v e s t i g a t i o n are i n the presence of coextractives and a clean-up step i s often essential i f gas chromatography i s to be the f i n a l step i n the analysis. Several workers have reported that t h i s step may not be necessary i n the extraction of pesticides from s o i l ( 1 0 , 9 2 ) . The following clean-up procedures are currently used: 1. Column chromatography, with alumina, s i l i c a gel, f l o r x s i l or activated charcoal as adsorbent. 2. Gel f i l t r a t i o n using sephadex (LH 20) or other ion exchange resins. - 24 -3. Thin layer chromatography (TLC) or another v a r i a t i o n of t h i s technique, channel layer chromatography (CLC). Both single and mixed layers of alumina, s i l i c a gel, f l o r i s i l and c e l l u l o s e have been used. 4 . Cosweep d i s t i l l a t i o n . 5. Thin layer electrophoresis (TLE) or high voltage paper electrophoresis (HVE). The thin layer electrophoresis and high voltage paper electrophoresis of ionisable herbicides have been described (86,101). The p r i n c i p l e s are e s s e n t i a l l y the same: the material i s applied to a support, either a c e l l u l o s e t h i n layer plate or f i l t e r paper, the support i s buffered and a high voltage (about 100 v o l t s per cm) applied to the two ends. An e f f i c i e n t cooling system i s necessary to prevent drying of the support. Purkayastha (86) showed that the following herbicides can be separated from one another by t h i n layer electrophoresis: 2,4-D, 2,4,5-T, MCPA, 2,4-DB, fenoprop, dicamba, amiben and picloram. For picloram, two spots described as strong and medium, with opposing p o l a r i t i e s (cationic and anionic) and d i f f e r e n t migration rates were obtained. No further work was car r i e d out to f i n d out i f either one was the unchanged picloram molecule. Bjerke*s (10) method of clean-up of picloram residues present i n small grain and straw has been adopted by other workers. The method consists of passing an ethereal extract of grain or straw containing picloram through an alumina column, - 25 -and e l u t i n g the picloram with 0.25M sodium bicarbonate solution. Picloram (acid) i s obtained by a c i d i f y i n g the bicarbonate extract. In the present study t h i s method was not adopted because of the u n a v a i l a b i l i t y of a s u f f i c i e n t number of chromatographic columns equipped with t e f l o n stop cocks. Improvised chromatographic columns drawn from glass tubing with tygon tubing at the ends gave spurious peaks on the chromatogram and were rejected. A clean-up method, described l a t e r , using a mixture of 15$ s i l i c a gel +• 85$ c e l l u l o s e was used. Methylation Organic acids are polar and d i f f i c u l t to chromatograph. Conversion into t h e i r esters, which are l e s s polar, removes t h i s d i f f i c u l t y . Methylation i s also desirable to decrease the retention time i n the column, to decrease t a i l i n g and to increase the s e n s i t i v i t y of the detection. This increase i n s e n s i t i v i t y i s very desirable when dealing with trace amounts of the p e s t i c i d e . Most e s t e r i f i c a t i o n procedures were i n i t i a l l y developed f o r f a t t y acids, (24,75,84,91). Some of these procedures have been successfully applied to herbicides containing the -COOH group. The phenyl acetic a c i d herbicides have been e s t e r i f i e d by diazomethane (94). Marquardt et a l (70) used diazomethane and boron trifluoride-methanol mixture f o r the methylation of 2,4-D. - 26 -Wool son et a l (108) compared the e f f i c i e n c y of methylation of several herbicides with the following methylation reagents: 1. Thionyl chloride 2. Oiazomethane ( s o c i 2 ) (CRgN,,) k. Sodium n i t r i t e - s u l f u r i c acid 6. Dimethyl sulfate 3. Boron trifluoride-methanol 5. Perchloric acid (BFyMeOH) (NaN0 2-H 2S0 4) (HCIO^) (CH 3) 2S0 4 In a l l cases diazomethane gave the best y i e l d s of esters. This reaction i s i d e a l because the co-product i s gaseous nitrogen which offers no separation problem. The reaction can be written as: fluoride-methanol and diazomethane were t r i e d . Dimethyl sulfate gave a brown viscous l i q u i d when warmed with the carrot and potato extracts. This viscous l i q u i d formed a gummy la y e r and did not dry e a s i l y when streaked on a TLC plate. Boron t r i -fluoride-methanol mixture when used with Bjerke's method of clean-up and electron capture gas chromatograph did not prove to be sa t i s f a c t o r y because of the r e l a t i v e l y large amount of solvent required to wash a l l the f l u o r i d e ions away. Otherwise the f l u o r i d e ions give i n t e r f e r i n g peaks on the chromatogram. Moreover boron t r i f l u o r i d e etches glass vessels slowly, giving them a f r o s t y appearance. Diazomethane, though highly toxic and RCOOH In the present study dimethyl sulfate, boron t r i -- 27 -needing to be f r e s h l y prepared, was found to be most convenient and was adopted. The advantages are that the reaction i s almost instantaneous, takes place at room temperature and gives a quantitative y i e l d . Detection and Estimation Gas chromatography i s a useful and powerful technique f o r the q u a l i t a t i v e and quantitative analysis of trace amounts of organic compounds, both natural and synthetic. I t i s especially suitable f o r residue work because of the r e l a t i v e l y large number of samples that can be analysed, once the laborious processes of extraction, clean-up and concentration have been achieved. A gas chromatographic retention time, as a single parameter f o r q u a l i t a t i v e and quantitative analysis, can introduce serious errors, but coupled with one or more other methods, i t can give p o s i t i v e i d e n t i f i c a t i o n of a compound. Confirmation of a compound i s obtained by f i n d i n g the retention times on at l e a s t two columns with l i q u i d phases of d i f f e r i n g p o l a r i t y or by c o r r e l a t i n g the gas chromatographic data with data obtained from other physical methods l i k e : TLC, UV, IR, NMR and MS. The detection of picloram by gas chromatography has been described by several workers. Since the column i s the heart of a gas chromatograph a few recorded column packings are given below: - 28 -1. Fisher et a l (21) 10$ carbowax 20M on Anakrom ABS, 90-100 mesh 2. Merkle et a l (73) 1.5$ SE 30 on Chromasorb W, 80-100 mesh 3. Bjerke et a l (10) 1$ LAC-2R-466 + 0.5$ H-jPO^ (w/w) on Gas Chrom Z, 80-100 mesh h. Leahy et a l (58) 2.5$ Neopentyl gly c o l adipate on Chromasorb W, 80-100 mesh 5. Sana et a l (92) 2$ Versamid 900 on Chromasorb W, 80-100 mesh A few of the above l i q u i d phases have been t r i e d as substrates i n the GLC of the methyl ester of picloram i n the presence of natural products from carrots and potatoes that cannot be separated by the clean-up method used. Versamid 900 i s reported to be a high temperature l i q u i d phase, but conditioning of the column at 150°C under a flow of nitrogen gas, produced a browning of the l i q u i d phase. Several t r i a l s were made with the same re s u l t and the Versamid column was rejected. Because of long retention times, 23 and 20 minutes, both carbowax 20M and neopentyl gly c o l adipate are not suitable when a large number of analyses are to be performed. The best l i q u i d phase was found to be 0V-1, which i s a methyl s i l i c o n e s i m i l a r to 3E-30 but having greater thermal s t a b i l i t y . - 29 -A r t i f a c t s i n Gas Chromatography I t has been suggested that the term a r t i f a c t be confined to interference which arises from the technique i t s e l f and to use the term "naturally i n t e r f e r i n g component" f o r such components a r i s i n g form the sample per se, (90). In the l i t e r a t u r e , however, peaks a r i s i n g from nat u r a l l y i n t e r f e r i n g components are s t i l l being termed " a r t i f a c t s " . Most of these a r t i f a c t s were reported i n the i n s e c t i c i d e analysis of green plants (26), wild l i f e (k2) and s o i l ( 8 3 ) . Polychlorinated biphenyls were reported as a r t i f a c t s i n t e r f e r i n g with the detection of the organochlorine i n s e c t i c i d e s such as DDT (52), A l d r i n , and BHC ( 6 l ) . To date no a r t i f a c t , of a n a l y t i c a l or sample o r i g i n , has been reported i n the gas chromatographic detection of herbicides. Newer Trends i n the Analysis of Picloram ..." On-column decarboxylation Several of the most promising herbicides have free a c i d i c groups and t h e i r detection by GLC has several d i s -advantages, including cumbersome extraction procedures, handling of toxic reagents f o r methylation and predominance of i n t e r f e r i n g peaks. H a l l (31) described an on-column decarboxylation of picloram. This makes use of a p y r o l y s i s column connected to the main column i n such a way that a f t e r i n j e c t i o n the components pass f i r s t through the p y r o l y s i s column then into the main column. Pyrolysis of the acid ( i ) leads to the decarboxylated - 30 -product ( i i ) which i s then detected. 0 ( I ) ( II ) The e f f i c i e n c y of the decarboxylation reaction was reported to be about 95$. Two other advantages of t h i s method, i n the case of picloram are that the compound ( i i ) can be detected at a lower temperature than the methyl ester, and impurity peaks obtained as a r e s u l t of e s t e r i f i c a t i o n are no longer present i n the pyrolysi s method. The thermal decaroxylation method can be used i n the analysis of other herbicides (31)• This decarboxylation technique has been described to be superior to the available methods f o r the analysis of picloram residues. Alkaline pre-column The alkaline pre-column described by M i l l e r et a l (76) has been devised to remove "troublesome crop peaks" i n the analysis of pe s t i c i d e residues, from crops. The alkaline pre-column, as the name suggests, i s an alk a l i n e couinn made up of 25$ KOH/NaOH on Gas Chrom Q connected p r i o r to the main column. During operation, the pre-column i s heated just as i s the main column. The advantage of t h i s a l kaline pre-column i s that no lengthy clean-up steps are required. I t s p o t e n t i a l f o r - 31 -removing i n t e r f e r i n g peaks i n carrots was c l e a r l y shown (76). The disadvantage of t h i s method i s that some pesticides are destroyed completely by i t . Trimethyl S i l y l Derivatives Though methylation with diazomethane i s a "clean reaction", the usual way of preparing i t from "Diazald" (N-Methyl-N-nitroso-p-toluene sulfonamide) (18, 19) gives several peaks on a chromatogram. Trimethyl s i l y l ethers (TMS) of b i l e acids were f i r s t prepared by Makita (67). Later H o r i i et a l (43) showed that the TMS derivatives of carboxylic acids can be ea s i l y prepared and gas chromatographed. This method may prove useful i n preparing the TMS derivatives of the a c i d i c herbicides p r i o r to gas chromatography. Photodecomposition  Introduction In the past decade photochemistry has blossomed dramatically into a f i e l d of i t s own. After an i n i t i a l break-through i n 1952 (80), the cry of organic chemists around the world seemed to be " i f you have some of i t , i r r a d i a t e i t " . The presence of pesticides i n the s o i l , water and a i r , the p o s s i b i l i t y of t h e i r undergoing a va r i e t y of reactions by UV l i g h t and the search f o r new methods of decreasing the pest i c i d e burden of the environment have added another dimension to the pic t u r e . The usefulness of short wave UV l i g h t to break down chemical bonds may be seen from the following Tables ( l 6 ) : - 32 -TABLE I Energy D i s t r i b u t i o n  wavelength (nm) Energy (Kcal/mole) 200 143 300 95 420 68 TABLE I I  Bond Energies  Bond Energy (Kcal/mole) C-C f i n ethane) 88 C-H ( i n methane) 98 0-H ( i n water) 119 C-N ( i n HCN) 73 C-Cl(in CCl^) 81 Table I gives the re l a t i o n s h i p between the wavelengths of UV l i g h t and the energy associated with each wavelength. Table I I gives the bond energies of a few commonly encountered bonds i n organic compounds. I t i s seen that the 200 - 300 nm range has enough energy to break most of the commonly occurring bonds i n organic p e s t i c i d e s . Herbicide Photodecomposition The photodecomposition of several herbicides has been reported. B e l l (9) reported the formation of 5 degradation products of 2,4-D. One major product of 2,4-D sodium s a l t was shown to be 2 ,4-dichloro phenol ( l 5 )» Jordan tst a l (48) studied the photolysis of the urea and t r i a z i n e herbicides. Diquat and - 33 -paraquat have also been photolysed (96,97) under laboratory conditions. M i t c h e l l (77) reported a study on the photo-decomposition of l 4 l pesticides (herbicides, i n s e c t i c i d e s and fungicides) spotted on chromatography paper. Because of the complexity of the problem of e l u c i d a t i n g molecular structure of organic molecules, only a few instances are recorded where the photoproducts were i d e n t i f i e d . Redemann (88) reported that picloram was rapid l y photolysed by UV r a d i a t i o n and by sunlight. The photoproducts were not i d e n t i f i e d . Plimmer et a l (85) found that the methyl ester of picloram was converted into a single major product, with one chlorine atom les s than the parent molecule. Merkle et a l (74) reported that 60$ picloram spread either on a glass surface or on a s o i l surface was degraded by UV l i g h t i n 48 hours. Under the same experimental conditions 35$ was reported to be broken down by sunlight. Our observations on the decrease i n concentration of standard solutions of methyl ester of picloram kept on a bench top, and the reported decrease i n phytotoxicity of picloram i n sunlight, under f i e l d conditions, l e d to an inves t i g a t i o n of the photodecomposition of picloram, i t s potassium s a l t and i t s methyl ester. The s a l t form i s commercially available as a herbicide and the methyl ester i s used i n the gas chromatographic analysis of picloram. - 34 -MATERIALS AND METHODS F i e l d T r i a l  Potatoes Between May and August I969 four v a r i e t i e s of potatoes (SPlanum tuberosum L. ) , namely Kennebec, Netted Gem, Norland and Pontiac were grown i n the f i e l d i n a completely randomized design. The f i e l d 18.0 meters x 9.0 meters was divided along i t s length into 16 plots each 0.6 m x 9«0 m with 0.45 m spacings between the plots and a clearance of 0.82 m at the edges. The potatoes were planted 0.3 n» apart, i n single rows i n the middle of the p l o t s . Picloram was applied at 2 oz per acre* alone, or i n combination with 24 oz linuron per acre. Linuron was used i n combination with picloram because linuron i s registered f o r use i n weed control on potatoes as well as on carrots. The required amounts of the commercial herbicides (as the active ingredients) were sprayed at the rate of 400 gallons of water c a r r i e r per acre. This high volumne spray was used to reduce d r i f t to a minimum. A l l sprayings were done one week a f t e r planting i . e . as a pre-emergence treatment. After a growth period of 100 days samples of tubers were collected, by digging h i l l s i n the centre of each treatment, leaving the edges to act as guards. Carrots An attempt was made to study the l e v e l of residues of picloram i n carrots (Daucus carota L . ) . Four v a r i e t i e s , namely: *1 oz per acres 70 g per hectare - 35 -Royal Chantenay, Gold Pack, Touchon and Imperator, were grown i n the f i e l d between May and August 1969, i n a completely randomized design s i m i l a r to that described previously. Picloram was applied at the rates of -§• and 2 oz per acre as follows: 1. Picloram \ oz per acre (post-emergence). 2. Picloram •§• oz per acre (post-emergence) + linuron 24 oz per acre (pre-emergence). 3. Picloram 2 oz per acre (pre-emergence) + linuron 2k oz per acre (pre-emergence). Sample Preparation The carrot taproots and potato tubers were washed thoroughly and surface dried. About 4 - 8 pounds were cut into small cubes, mixed thoroughly and 2 pound samples taken by quartering. These samples were then stored i n a deep-freeze u n t i l analysed. Extraction A 100 g sample was blended with 100 ml methanol + 25 ml N s u l f u r i c acid f o r 5 minutes. Addition of mineral acid to the extraction solvent i s essential i f the picloram i s to be subsequently e s t e r i f i e d (99). I t has been suggested that a trace of the mineral acid enters the ether phase and catalyses the d i a z o t i s a t i o n process (10). The extract was f i l t e r e d twice i n the same funnel containing a pad of glass wool, into a 500 ml ® round bottomed f l a s k . This aqueous al c o h o l i c extract was f l a s h evaporated f o r 3/4 - 1 hour to remove most of the methanol. For s o l u b i l i t y reasons, removal of methanol i s imperative i f the picloram i s to - 36 -be partitioned into d i e t h y l ether. The f l a s h evaporation assembly i s shown i n Figure 1. F i g . 1. Flash evaporation assembly The aqueous concentrate was transferred into a 100 ml separatory funnel containing 10 ml saturated sodium chloride solution. This was extracted by gentle shaking with 3 x 15 ml di e t h y l ether. After each extraction the ether layer was dried - 37 -over anhydrous sodium sul f a t e and co l l e c t e d together into a 100 ml b o i l i n g tube. The ether extract was evaporated to dryness on a kO°C water bath. A l l operations were done i n a fume cupboard. The dried residue was cooled to room temperature and treated with 2 ml diazomethane-ether reagent, prepared as described by De Boer (18,19). The reagent was added down the side of the tube so as to concentrate a l l the product at the bottom of the tube. The reaction was allowed to proceed f o r 10 minutes, at room temperature, a f t e r which excess diazomethane was decomposed by returning the tube to the water bath. Care was taken not to remove any undecomposed diazomethane out of the fume cupboard at any time because of i t s highly toxic and p o t e n t i a l l y explosive nature (l9)» The reagent was f r e s h l y prepared before use because of i t s short l i f e ( 2 - 3 hours) at room temperature. A l l sharp edges, l i k e ends of glass tubings i n the d i s t i l l i n g apparatus, were f i r e - p o l i s h e d to eliminate spots where explosions are most l i k e l y to occur. Clean-up The residue was dissolved i n a few drops of methanol and streaked on a TLC pla t e . This process was repeated three times. The plate consisted of a mixed layer of 85$ 1 a v i c e l * c e l l u l o s e and 15$ s i l i c a gel G, dried at room temperature. Cellulose powder, containing a binder, when spread on a TLC plate gives a hard coating which can be streaked without damaging the surface. S i l i c a gel does not have t h i s property. On the other - 38 -hand s i l i c a gel gives a better separation of plant pigments than c e l l u l o s e . Mixtures of various compositions of c e l l u l o s e and s i l i c a gel were t r i e d and the mixture: 85$ ce l l u l o s e + 15$ s i l i c a gel was adopted because i t gave a hard surface making repeated streaking possible and also giving good separation of the plant products ( v i s i b l e undcrUV l i g h t ) from the methyl ester of picloram. The streaker was made by drawing the small end of a pasteur pipette i n a flame and attaching a short length of a very small diameter t e f l o n tubing to i t as shown i n Figure 2 . F i g . 2 . Streaker showing t e f l o n t i p on l e f t With this apparatus i t was possible to streak a band about 2 mm width. The right hand corner of the TLC plate was spotted with a solution of methyl ester of picloram to serve as a reference. The plate was developed in the solvent system: hexane acetone + methanol, 5 0 + 7 + 5 ( v/ v) to a previously marked - 39 -15 cm l i n e . This solvent system was found to be the best to effect the separation. The R^ . of the methyl ester was about 0.75. L o c a l i z a t i o n of the known ester was obtained by shi e l d i n g the TLC plate with another glass plate and spraying only the zone containing the reference spot. The spray reagent consisted of a 0.005% solution of acridine i n ethyl alcohol. Viewing the plate under short wave UV l i g h t showed the pyridine r i n g of picloram as a v i o l e t spot on a blueish green background. This was marked with a p e n c i l . The TLC plate i s shown i n Figure 3» F i g . 3. TLC plate streaked with potato extract (left) and spotted with methyl ester picloram ( r i g h t ) . The plate was photographed under short wave UV l i g h t . The natural products separated into bands: a,b,c and d. The o r i g i n i s denoted by 0. - 4o -Scraping and E l u t i o n A band 2 cm above and 2 cm below the reference spot was scraped o f f . A pasteur pipette, containing a plug of glass wool, was used both as a c o l l e c t i n g and e l u t i n g device. A vacuum l i n e connected to the small end was used to suck the c e l l u l o s e -s i l i c a gel powder into the pipette. E l u t i o n was accomplished by disconnecting the vacuum l i n e , clamping the pipette v e r t i c a l l y , and e l u t i n g with 3 x 10 ml methanol, at the rate of about 1 ml per minute. This rate was obtained by gently compressing the glass wool plug. L i q u i d - l i q u i d P a r t i t i o n i n g The methanol extract was transferred into a separatory funnel, d i l u t e d with 200 ml d i s t i l l e d water and extracted successively with 15 and 5 ml benzene. The benzene extracts were dried over anhydrous sodium sulfate, c o l l e c t e d into a 25 ml volumetric f l a s k and made up to volume. L i q u i d - l i q u i d p a r t i t i o n i n g was necessary because benzene, which could be injected d i r e c t l y i n t o the gas chromatograph, f a i l e d to elute the methyl ester from the TLC substrate. A more polar solvent l i k e methanol was required. Since the methyl ester i s more soluble i n methanol (22.34 g/100 ml) than i n benzene (12.60 g/100 ml) (92), addition of water was necessary f o r e f f i c i e n t p a r t i t i o n i n g . - 4 l -Gas Chromatography  Column Preparation 0.4-5 g of the l i q u i d phase, OV-1, was dissolved i n 75 ml of chloroform i n a 500 ml 'S round bottomed f l a s k , 15 g of Chromasorb w" was added and the sl u r r y refluxed f o r 1 hour. The chloroform was evaporated to dryness on a rotary evaporator, under vacuum. This was then packed into a 1.8 m long and 6 mm od s p i r a l pyrex tube by means of a vacuum pump. Uniform packing was obtained by repeatedly tapping the tube f i r m l y along i t s entire length u n t i l no further s e t t l i n g accurred. The column was then conditioned at 24o°C f o r 48 hours under a nitrogen gas flow of 60 ml/min. The column was not connected to the detector during this conditioning phase. Gas Chromatograph The gas chromatograph used was a PYE SERIES 104 CHROMATOGRAPH, equipped with a radioactive (10 m i l l i c u r i e ) ^ N i electron capture detector. The output was fed into a Honeywell 12-inch recorder. The assembly i s shown i n Figures 4A and B. - k2 -F i g . h. Gas Chromatography Assembly F i g . F i g . 4B. Honeywell Recorder - h3 -Operating Conditions Column temperature 225°C Injector block temperature 250°C Detector temperature ' 265°C Ca r r i e r gas, p u r i f i e d nitrogen at a flow rate of 60 ml per minute. Chart speed 30 inches per hour. 5 u l samples injected with a Hamilton 701 N, 10 jul syringe. Under these operating conditions the methyl ester appeared as a sharp peak with a retention time of 1 minute. Greenhouse Experiments  Absorption and Translocation Three week-old plants of Royal Chantenay carrots of uniform si z e were removed from vermiculite and transferred to nutrient solution (see Appendix i ) , contained i n 100 ml test tubes, at the rate of one plant per tube, by the technique of Crafts and Yamaguchi (109)• A gentle stream of a i r was bubbled continuously through the solution by means of a c a p i l l a r y tube. Transpirational losses were made up d a i l y with d i s t i l l e d water. After two days, when the plants had recovered from traumatic effects, they were treated with the labeled herbicide. Three experiments, each r e p l i c a t e d twice, were conducted. The f i r s t was a control. In the second and t h i r d the herbicide was applied to the oldest l e a f and to the nutrient solution respectively. The rate was 1 uCi ( l uM) per treatment. To prevent - hk -run-off from the l e a f , the solution (50 ul) was applied with a micro syringe. For the root treatment the 1 uCi (50 ul) was injected into the nutrient solution. The plants were removed at i n t e r v a l s of 1 and 3 days. The roots and treated leaves were washed with 0.01 N ammonium hydroxide solution, followed by d i s t i l l e d water. The water was blotted o f f and the plants were pressed f l a t under sheets of b l o t t i n g paper and quickly frozen under a layer of dry i c e . The plants were then freeze dried, mounted and radioautographed. A three week exposure gave s a t i s f a c t o r y images. D i s t r i b u t i o n . Metabolism and L o c a l i z a t i o n Two month-old carrot plants, with taproots about 2 cm i n diameter and 8 - 10 cm i n length were removed from vermiculite and transferred to nutrient solution contained i n 1 l i t e r beakers, at the rate of 5 plants per beaker. Care was taken to preserve the secondary roots during the uprooting and washing processes. Aeration and other procedures were s i m i l a r to that described perviously. After 2 days s t a b i l i s a t i o n 5 mg radioactive picloram dissolved i n 5 ml of 50$ ethanol was added to the nutrient solution i n each beaker. After a feeding period of 5 days the plants were harvested and the storage and secondary roots washed thoroughly with 0.01 N ammonium hydroxide solution, followed by d i s t i l l e d water. The di f f e r e n t parts of the plants were sectioned and the leaves, taproots and secondary roots c o l l e c t e d together and weighed separately. This provided the materials needed f o r the d i s t r i b u t i o n , metabolism and - 45 -l o c a l i z a t i o n studies. D i s t r i b u t i o n of Radioactive Picloram within the Whole Plant Two grams of leaves and 5 grams each of the tap and secondary roots were ground with s i l i c a sand i n 10 ml acetone -methanol ( l : l ) mixture i n a mortar. The extracts were centrifuged at 2,000 revolutions per minute. The supernatants were collected, and the p e l l e t s extracted twice with 3 ml aliquots of the solvent mixture. The supernatants f o r each tissue were pooled together and made up to 20 ml. One ml of each extract was mixed with 10 ml of l i q u i d s c i n t i l l a t i o n f l u i d (see Appendix II) i n a v i a l , and the counts per minute recorded on a l i q u i d s c i n t i l l a t i o n counter (Model Mark I, Nuclear Chicago). The experiment was re p l i c a t e d twice. The average counts per minute per gram of tissue and the t o t a l cpm f o r the leaves, taproots and secondary roots were computed. Determination of Metabolites i n Leaves and Taproots Twenty-five grams of leaves or 100 grams of taproots were blended f o r 5 minutes with 100 ml acetone - methanol (1:1) mixture and centrifuged. The extract was f l a s h evaporated to about 10 ml and t h i s was extracted with 3 x 5 ml ether. The ether layers were pooled together and concentrated to about 5 ml. An aliquot of t h i s extract (10 - 40 ul) was spotted on a ce l l u l o s e TLC plate. Solutions of picloram and i t s methyl ester (only other derivative of picloram available) were also spotted on the same plate and developed i n the solvent system hexane + acetone + methanol + acetic acid, 5 0 + 7 + 5 + 2 (v/v). - 46 -Radioautograms of the developed TLC plates were made. L o c a l i z a t i o n of Picloram i n Carrot Longitudinal Section Longitudinal sections, about 1 ram thick, were obtained by shaving the carrot with a metal planer. The sections were selected f o r uniformity, washed and freeze dried. The dried sections were mounted on b l o t t i n g paper and radioautographed. Photodecomposition Studies  Stock Solutions 1. Picloram l i fC carboxyl labeled, a c t i v i t y 1 uCi/uM, 2 mg dissolved i n 2 ml methanol. 2. Potassium s a l t picloram 2 mg picloram dissolved i n 2 ml 0.005M KOH i n methanol. 3. Methyl p i c o l i n a t e 2 mg picloram methylated with 1 ml diazomethane-ether reagent. The ether evaporated to dryness and the residue dissolved i n 2 ml methanol. 4. U l t r a v i o l e t source short wave length, 253*7 nm. Batches of three 5 u l aliquot each of the solution 1,2 and 3 were spotted separately on three TLC plates. The solvent was evaporated to dryness and the three spots on each plate exposed to 0,24 and 48 hours respectively, to the UV source, enclosed i n a "Chromatovue" box. The plates were then developed i n solvent system: water + methanol + acetic acid, 8 0 + 2 0 + 2 (v/v), to a previously marked 15 cm l i n e . They were then dried at low temperature (40 - 50°C) with a continuous current of a i r to remove the acetic acid. The plates were then taken to the dark room, where each one was pressed i n contact with an X-ray f i l m . - 47 -The f i l m was kept i n p o s i t i o n by another glass plate and the edges taped together. The whole operation was done under safety l i g h t conditions. The plates were stored i n a l i g h t proof drawer, l i n e d with lead sheets, to prevent stray r a d i a t i o n reaching them. Af t e r one week exposure the films were removed and processed i n the standard way f o r ordinary negatives. The R^ . values of the unexposed and exposed compounds and t h e i r degradation products were computed. The extent of degradation of each compound was obtained by c a r e f u l l y scratching the corresponding spot from the TLC plate, t r a n s f e r r i n g the c e l l u l o s e into a v i a l containing 10 ml of the s c i n t i l l a t i o n f l u i d , (see Appendix I i ) . Radioactive counts were obtained by the use of a l i q u i d s c i n t i l l a t i o n counter. The extent of decomposition was computed from the counts per minute. - 48 -RESULTS Potatoes A l l four v a r i e t i e s of potatoes contained detectable amounts of picloram. For each var i e t y the picloram + linuron treatments consistently gave lower amounts of picloram residues than the picloram alone treatments. No picloram residue was found i n the controls. The means of the picloram alone treatments ranged from 3.5 to 4 .2 ppb with a grand mean of 3*9 PP*> of picloram residues. The means of picloram + linuron treatments ranged from 2.4 to 2.9 ppb with an o v e r a l l mean of 2.7 ppb of picloram residues. The difference of 1.2 ppb between these two o v e r a l l means was s i g n i f i c a n t at the 1 $ l e v e l . Since there was no s i g n i f i c a n t v a r i e t y or var i e t y x treatment i n t e r a c t i o n , there i s l i t t l e evidence that the v a r i e t i e s responded d i f f e r e n t l y to the treatments. An average recovery of 73$ was obtained when untreated potatoes were f o r t i f i e d with known amounts of picloram. The picloram peaks (as methyl ester) are shown i n Figures 5A, B and C. The residues of picloram i n the treated potatoes ( i n parts per b i l l i o n f resh weight) are given i n Table I I I . - 4 9 -TABLE II I Residues of Picloram i n ppb Fresh Weight Potatoes Variety Treatment Picloram @ 2 oz Picloram @ 2 oz + Linuron @ 24 oz Netted Gem 4 . 0 4 . 2 3 . 8 4 . 2 4 .0 + 0 .2 2 . 4 2 . 6 4 . 0 ) 2 . 4 ) 2 . 8 + 0 . 8 Kennebec II II II 3 . 3 3 . 6 3 . 6 3.5 3.5 + 0 . 2 2 . 8 2 . 9 1 . 9 2 . 1 2 . 4 + 0.5 Norland 4 . 9 4 . 5 2 . 7 4 . 9 4 . 2 + 1 . 0 2 . 7 3 . 6 2 . 1 3 . 3 2 . 9 + 0 . 7 Pontiac II II it 3 . 3 4 . 7 4 . 7 2 . 7 3 . 8 + 1 . 0 2 . 9 2 . 8 2 . 1 2 . 7 2 . 6 + 0 . 3 Grand mean picloram treatment f o r a l l v a r i e t i e s 3 . 9 Grand mean picloram + linuron treatment f o r a l l v a r i e t i e s 2 . 7 Difference of means 1 . 2 * * ** s i g n i f i c a n t at the 1 $ l e v e l - 50 -Table IV gives the percentage recovery of picloram from untreated potatoes, f o r t i f i e d with known amounts of the herbicide. TABLE IV Recovery of Picloram from Potatoes average  $ recovery recovery added found 0.02 0.017 0.02 0.015 0.02 0.015 0.04 0.031 0.0k 0.026 0.0k 0.028 85 75 75 ) 73$ 75 65 70 ) Carrots No residues of picloram were detected i n either the pre-emergence or the post-emergence treatments, though an average recovery of 70$ was obtained with carrots spiked with picloram. - 51 -A B C F i g . 5. Gas Chromatographic peaks of potato tuber e x t r a c t s (A) C o n t r o l s p i k e d with p i c l o r a m b e f o r e a n a l y s i s (B) Pre-emergence treatment w i t h p i c l o r a m @ 2 oz/acre (C) C o n t r o l V e r t i c a l arrows show peaks given by methyl e s t e r of p i c l o r a m . - 52 -Absorption and Translocation  Leaf Treatment Radioautographs f o r the l e a f applications are shown i n Figures 6A and B. These c l e a r l y demonstrate that basipetal translocation of picloram and r e d i s t r i b u t i o n throughout the whole carrot plant had taken place. An increase i n the amount of r a d i o a c t i v i t y i n the leaves was noticed from day 1 to day 3« No such increase was noticed i n the roots. F i g . 6A. Results of treatment of 3-week old carrot plants with labeled picloram. Radioautographs (right) and mounted plants ( l e f t ) . Dosage 1 uCi applied on compound l e a f (shown by arrow). Treatment time: 1 day. - 53 -F i g . 6B. Results of treatment of 3-week old carrot plants with labeled picloram. Radioautographs (right) and mounted plants ( l e f t ) . Dosage 1 uCi applied on compound l e a f (shown by arrow). Treatment time: 3 days. Root Treatment Radioautographs f o r the root applications are shown i n Figures 7 A and B. These demonstrate that acropetal translocation i n the carrot plant had taken place. On day 1 more r a d i o a c t i v i t y was concentrated i n one l e a f . On day 3 the r a d i o a c t i v i t y was d i s t r i b u t e d throughout the entire plant. The absence of secondary roots i n t h i s treatment was very prominent. Untreated plants did not show any darkening on the X-ray f i l m . - 5k -F i g . 7A  1 day F i g . 7« R e s u l t s of treatment of 3-week o l d c a r r o t p l a n t s w i t h l a b e l e d p i c l o r a m . Radioautographs ( r i g h t ) and mounted p l a n t s ( l e f t ) . Dosage 1 u C i a p p l i e d to n u t r i e n t s o l u t i o n . Treatment times: (A) 1 day, (B) 3 days. - 55 -D i s t r i b u t i o n o f P i c l o r a m i n 2 month.-old C a r r o t P l a n t s The d i s t r i b u t i o n o f r a d i o a c t i v i t y i n the p l a n t s i s shown i n T a b l e V. TABLE V D i s t r i b u t i o n of R a d i o a c t i v i t y T i s s u e cpm/g T o t a l cpm f r e s h t i s s u e jo D i s t r i b u - $ D i s t r i b u - t i o n / g f r e s h t i o n w i t h i n  t i s s u e whole p l a n t Leaves Storage r o o t s Secondary r o o t s 15,500 3,800 1,280 558,000 456,000 20,480 75 19 54 44 On a f r e s h weight b a s i s the l e a v e s accumulated f o u r times more r a d i o a c t i v i t y than d i d the storage r o o t s . The weight of the storage r o o t s was about f o u r times t h a t o f the l e a v e s . Thus on a whole p l a n t b a s i s t h e r e was roughly the same amount of r a d i o a c t i v i t y i n the l e a v e s as i n the storage organs. Very l i t t l e r a d i o a c t i v i t y was found i n the secondary r o o t s . - 56 -Metabolism of Picloram i n Carrot Leaf and Taproot The radioautogram of the le a f and carrot extracts i s shown i n Figure 8. Fi g . 8. Radioautogram of carrot taproot and le a f extracts. A) picloram, (B) methyl ester picloram, C) carrot taproot, (D) carrot l e a f . Because of the low l e v e l of r a d i o a c t i v i t y din the tissues i t was necessary to spot the maximum amount of extracts on the TLC plates. This maximum amount was li m i t e d by the fact that a f t e r a few m i c r o l i t e r s had been spotted (d i f f e r e n t f o r le a f and root extracts), the spot became greasy and dried out with great d i f f i c u l t y . For thi s reason the amount of ra d i o a c t i v i t y i n the taproot and l e a f extracts i s q u a l i t a t i v e and not quantitative. The spreading observed on the radioautogram - 57 -was due to overloading. The centre of the spots given by both extracts corresponded to the parent picloram. L o c a l i z a t i o n of Picloram i n Carrots Sections Radioautograms of the longitudinal carrot sections are shown i n Figure 9 » F i g . 9. Negative radioautograms of longitudinal carrot sections. The radioautograms from l e f t to right correspond respectively to the 5th, 6th and 7th sections i n the mount shown i n Figure 10. The r a d i o a c t i v i t y i s l o c a l i s e d mostly i n the xylem tissue. The r a d i o a c t i v i t y i n the cortex might be due to accumulation of the radioactive solution i n the holes, cracks and folds present i n the cortex. Some of these radioactive spots (a and b i n Figure 9) are located where the secondary roots joined to the taproot. I t has been shown however (Table - 58 -that the secondary roots did not accumulate more r a d i o a c t i v i t y than the taproot. F i g . 10. Mounted longitudinal sections of carrot. The sections from top l e f t to bottom right are from the outside to the middle of one carrot. The negative radioautograms of the 5th, 6th and 7th section, from top l e f t , are shown i n Figure 9» - 59 -PHOTODECOMPOSITION The R f values of the photoproducts of picloram, i t s potassium s a l t and i t s methyl ester are given i n Table VI, TABLE VI R f Values of Control and Irradiated Compounds  Compound R f (control) Rf (irradiated) Picloram ( r * 0 .53 0 .79 ( 0 .60 0 .69 0.80 Potassium s a l t ( r * picloram ( 0.53 0.76 ( 0.60 ( 0.69 ( 0.77 Methyl ester 0.58 (major) ( r * picloram 0.69 ( 0.58 0.82 (very weak) ( 0.69 ( 0.85 r * residual spot at o r i g i n of chromatogram - 60 -The extent of photodecomposition a f t e r 24 and 48 hours i s given i n Table VII. TABLE VII Extent of Decomposition a f t e r 2k and 48 hours Exposure to UV l i g h t Compound Amount decomposed ($>) 24 hours 48 hours Picloram 50 65 Potassium s a l t 50 55 Methyl ester 85 90 For a l l three compounds, both the 24 and 48 hours exposure gave "residual spots" at the o r i g i n of the chromatogram. The unexposed picloram and i t s potassium s a l t gave only one spot each when chromatographed. The methyl ester, however, gave three spots, one of which (R f 0.82) i s very weak and barely v i s i b l e as shown i n Figure l 4 . Each of the three compounds gave several radioactive products on exposure to UV l i g h t . The molecular structures of these have not been i d e n t i f i e d , but compounds with i d e n t i c a l Rf values are designated by i d e n t i c a l l e t t e r s . Picloram and the potassium s a l t gave three photo-products each, and a l l three have the same R f values (0.53» 0.60, O .69). The methyl ester gave two photoproducts one of which (R f O.69) corresponds to one of the above mentioned three compounds. - 61 -Table VII shows that picloram and i t s potassium s a l t are 50% decomposed within the f i r s t hour, whereas the methyl ester i s 85$ decomposed i n the same time. The radioautograms of the thin layer chromatograms are shown i n Figures 11A, B and C. A B C Picloram Potassium s a l t Methyl ester F i g . 11. Radioautograms of thin layer chromatograms spotted with: picloram (A), potassium s a l t picloram (B), and methyl ester picloram (C). Each plate was exposed to UV l i g h t (253.7 nm), f o r 0,24 and 48 hours ( l e f t to right) and developed i n the solvent system: water + methanol + acetic acid, 8 0 + 2 0 + 2 (v/v). Spots given by the unexposed compounds are designated by P (picloram) KP (potassium s a l t picloram) and MeP (methyl ester picloram). Residual spots at the o r i g i n were obtained f o r a l l three compounds, f o r both 24 and 48 hour exposures. Photoproducts having the same R values are indicated by i d e n t i c a l l e t t e r s . - 62 -DISCUSSION Potatoes The main effect of picloram was s i g n i f i c a n t at the 1$ l e v e l , but there was no s i g n i f i c a n t v a r i e t y or v a r i e t y x treatment i n t e r a c t i o n . Thus there i s l i t t l e evidence that the v a r i e t i e s responded d i f f e r e n t l y to the treatments. The picloram treatment consistently gave higher amounts of residues, i n a l l four potato v a r i e t i e s , compared to the picloram + linuron treatment. The r a t i o of residues i n the picloram to the picloram + linuron treatment i s about 1.5 s 1» Y i e l d data were not recorded but were markedly higher i n the picloram + linuron treatments. The reduced picloram residues could thus be associated with the d i l u t i n g effect of higher y i e l d s which resulted from the addition of linuron. D i l u t i o n of pesticides i n plants as a r e s u l t of plant growth has been suggested (20). The y i e l d s of the treatments were not actually measured because the rate of 2 oz picloram per acre, when applied alone, k i l l e d about 25$ of the plants. This amount i s evidently approaching the l e t h a l rate f o r potatoes, when the higher y i e l d of the picloram + linuron treatment i s considered i t would appear that, on a plant basis, more picloram i s accumulated i n the picloram + linuron treatment than i n the picloram alone treatment. This could be due to there being more p h y s i o l o g i c a l l y active plants r e s u l t i n g from the former treatment. - 63 -At harvest time the plants i n the picloram + linuron treatment had a normal root system, and had no v i s i b l e injury symptom either on the f o l i a g e or on the tubers. On the other hand the plants i n the picloram treatment were stunted, and had l i t t l e f o l i a g e . Most tubers were small, and developed a corky tissue on the surface. S p l i t t i n g of some tubers was also observed. These i n j u r i e s are shown i n Figures 12A nd B. F i g . 12. Injury on surface of potato tubers due to picloram. (A) Formation of corky tissues. ( 3 ) S p l i t t i n g of tuber i n addition to formation of corky tissues. The only residue study of picloram i n crops under f i e l d conditions was described by Bjerke e_t a l (10) where he obtained about 0.03 ppm picloram i n wheat grain. In the present study the residues f o r the picloram and picloram + linuron treatments, f o r a l l four v a r i e t i e s , averaged 3.9 and 2.7 PPb respectively. - 64 -Assuming an 80$ moisture content i n fr e s h potatoes (104), residue l e v e l s of 0.02 and 0.014 ppm on a dry matter basis are obtained f o r the two treatments. No tolerance* has yet been set f o r picloram i n food. On the basis of Jackson's (47) and Hardy 1s (37) t o x i c o l o g i c a l studies of picloram i t may be concluded that t h i s l e v e l i s not injurious to any of the organisms tested. The l e v e l of picloram residues present i n the tubers at harvest may be so small as not to cause any acute t o x i c i t i e s to humans and animals. However the picloram residue l e v e l present i n the s o i l , may be s i g n i f i c a n t to the point of adversely a f f e c t i n g a subsequent crop. No picloram residues were determined i n the s o i l i n the present study. Figure 13 shows a l e a f of a potato plant growing i n the experimental p l o t , i n the spring following treatment. The l e a f c l e a r l y shows the deformation t y p i c a l of auxin herbicides. Since the plants were growing from tubers l e f t over from the previous season, i t i s not possible to say whether the deformation i s due to the picloram residue present i n the s o i l alone or to picloram residues present i n the tubers alone or to a combination of these two f a c t o r s . •Tolerance: the permitted concentration of a residue i n or on a food. - 65 -Studying the effect of linuron on the uptake of inorganic ions (81), i t has been shown that when linuron was incorporated into the nutrient solution i t increased the uptake of Ca + + , Mg + +, N0^_, SO^ ions and there i s a decrease i n the uptake of water. I t was suggested that t h i s could be brought about by changes i n membrane permeability. Whether incorporation of linuron increases membrane permeability leading to any change i n picloram uptake cannot be answered yet. On the other hand, Davis _et a l (17) showed that i n mesquite, huisache and bean plants, the transport of picloram was reduced when used i n combination with paraquat. - 66 -Onsager et_ a l (82) showed that the residues of organo chlorine i n s e c t i c i d e s i n mature sugar beets were d i r e c t l y proportional to the residues i n the s o i l at the time of planting. In the present study, whether picloram was sprayed alone or mixed with linuron, i t was applied at a constant rate of 2 oz per acre, giving two d i f f e r e n t residue l e v e l s . Therefore, f o r t h i s herbicide, i t seems that the p r o p o r t i o n a l i t y f a c t o r d i f f e r s depending whether the herbicide i s applied alone or i n combination with linuron. Lichtenstein j|t a l (6k) showed that even d i f f e r e n t v a r i e t i e s of the same plant (carrot) could absorb d i f f e r e n t amounts of i n s e c t i c i d e s from s o i l . In our study i t was shown that a l l four v a r i e t i e s of potatoes had the same capacity f o r accumulating picloram from s o i l . I t should be mentioned that the residue accumulated by the tuber at harvest does not depend only on one process, namely uptake from s o i l . I t i s the amount that has been absorbed by the roots, translocated into the plant and l a t e r transported into the tuber a f t e r metabolism by the leaves and shoots ( i f any) and leakage by the roots into the s o i l have taken place. Excretion of f o l i a g e applied picloram by roots of bean plants has been confirmed (k6). Picloram may not be metabolised by potato plants because they are very susceptible to the herbicide, as are bean plants. One gas chromatographic peak as such, i n the q u a l i t a t i v e and quantitative determination of p e s t i c i d e residues extracted from plants or animals, may lead to serious errors i f not - 67 -confirmed by an independent method. In residue analysis the method of choice i s t h i n layer chromatography. In t h i s study two independent methods were used, namely thin layer chromatography and gas chromatography. The purpose of the TLC was twofold: 1. to remove i n t e r f e r i n g plant products and 2. to i s o l a t e the methyl ester by making use of i t s Rj. value. The ensuing gas chromatography then provided a confirmatory test as well as a quantitative evaluation. Carrots The observation that no residues of picloram were found i n carrots was i n t e r e s t i n g because, throughout the l i t e r a t u r e on p e s t i c i d e residues, root crops are mentioned as the greatest absorbers of organochlorine compounds. Lichtenstein et a l (63,64) found that carrot translocated and accumulated more residues of organochlorine i n s e c t i c i d e s than any other root crop, with the exception of parsnip. As the absorption and translocation, d i s t r i b u t i o n , metabolism and l o c a l i z a t i o n studies were a l l done to cast some l i g h t on the absence of picloram i n the carrot taproots, they are a l l discussed together here. Absorption and translocation of picloram i n a wide spectrum of plants have been shown by several workers (12 ,14, 17 , 4 4 , 46 , 49 ) . However i t has not been reported whether the carrot plant can absorb and translocate picloram. The r e s u l t s - 68 -of the absorption and translocation experiments c l e a r l y show that young carrot plants can absorb and translocate picloram, when applied both to the f o l i a g e and to the roots. This would indicate that both pre-emergence and post-emergence treatments would be e f f e c t i v e i n getting the picloram into the young carrot plant. Further i t was shown that 2-month old carrot plants fed with radioactive picloram i n the nutrient solution absorbed and translocated varying amounts of radio-a c t i v i t y i n a l l of i t s tissues. Since picloram has a h a l f - l i f e of the order of 13 months i n s o i l (29), and since there was enough picloram i n the s o i l to cause l e a f deformation i n the succeeding year, i t may be reasonably assumed that the carrot plant could absorb piclorm present i n the s o i l over i t s entire growth period. Since picloram was not detected i n the treatments and since about 70$ of the added picloram was detected i n recovery studies, i t may be suggested that the picloram was broken down i n the carrot plant. The taproot and l e a f metabolism experiment may not have been sensitive enough to detect i t because only one carbon atom i n the picloram molecule, namely the -C00H group, was labeled. I f p a r t i a l decarboxylation as a mechanism of d e t o x i f i c a t i o n was operating, r a d i o a c t i v i t y would not be detected by radioautography as the labeled CO2 would be l o s t through the stomata. Redemann (87) found that 83$ of the r a d i o a c t i v i t y i n mature wheat grain was found as the unchanged picloram. - 69 -Meikle (71), however, found that i n cotton plants a l l the r a d i o a c t i v i t y was present as the parent picloram. Different plant species and even d i f f e r e n t v a r i e t i e s of the same plant species can degrade a foreign molecule to d i f f e r e n t extents depending on whether the plant i s r e s i s t a n t or susceptible to the foreign molecule (66). To what extent the picloram molecule i s degraded i n the carrot plant cannot be answered yet. Prom Table V i t i s seen that, either on a weight basis or on the whole plant basis, more picloram was translocated and probably accumulated into the leaves than into the roots. This could probably be one mechanism by which the carrot plant overcomes the h e r b i c i d a l effect of picloram i n the taproot. Carrots must also be more resistant to the chemical than are potatoes as no symptoms of picloram i n j u r y were noticed on the treated carrots. The l o c a l i z a t i o n study indicated that within the carrot taproot some r a d i o a c t i v i t y was found i n the xylem. This could represent that amount of radioactive material being transported through the xylem at harvesting time. Lack of r a d i o a c t i v i t y i n the other tissues of the taproot may indicate that the xylem i s transporting the material into the leaves which may be acting as a sink. - 70 -Photodecomposition This experiment was not performed under the i d e a l , conditions of a photolysis experiment. However i t approaches more clos e l y the conditions that exist when herbicides are present on or around s o i l p a r t i c l e s . Again on s o i l or plant surfaces, the presence of trace amounts of m e t a l l i c oxides, serving as catalysts, may greatly accelerate the rate of photochemical reactions. The pH of the medium also influences the breakdown of organic molecules (9)« Exposure to UV l i g h t of a l l three compounds under in v e s t i g a t i o n produced an intense res i d u a l radioactive spot at the o r i g i n of the developed chromatogram. A s i m i l a r phenomenon was obtained by Smith (98), working with diquat. Further Smith showed that h i s residual spot was not diquat and had no phytotoxic properties. Whether these residual spots are due to polymerisation of the compound, by the action of UV l i g h t , has not been investigated yet. Again i t i s not known whether they have phytotoxic properties. The chromatography of the unexposed methyl ester gave more than one spot but, to date, no isomer of t h i s compound has been reported. On the other hand the chromatography of the unexposed picloram gave only one spot. Purkayastha (86) reported that the thin layer electrophoresis of picloram produced a main anionic and an additional c a t i o n i c spot. The i d e n t i c a l R f values produced by the three compounds indicate that they may a l l have common photoproducts; probably the - 71 -same pathway of degradation may be involved. Identity of a few of these products i s essential i n suggesting any degradation pathway. The methyl ester i s more rapidly degraded than either picloram or the potassium s a l t , and the l a t t e r two compounds are degraded at nearly the same rates. A probable explanation i s that the methyl ester absorbs UV l i g h t more strongly than either picloram or i t s potassium s a l t . Picloram and i t s potassium s a l t behave very much a l i k e i n t h e i r decomposition rates as well as i n the pattern of photoproducts formed. Plimmer and Hummer (85) reported that the methyl ester i s converted into a single major product by the loss of one chlorine atom. He postulated that either the 3-hydroxy-4-amino-5,6-dichloro-2-picolinic acid ( l ) and/or the 4-amino-5»6-dichloro-2 - p i c o l i n i c acid ( i i ) may be formed: COOH ( I ) Cl ^ N ^ C O O H NH 2 ( II ) The present experiment suggests the formation of at leas t two products marked "c" and "d" i n Figure 11C. Again the spot Md" could be due to two compounds, unresolved by the solvent system. The formation of several photoproducts i s - 72 -also supported by the chromatogram shown i n Figure 14. The photoproducts are shown by arrows. Here also more than one photoproducts i s observed. Radioautogram of thin layer chromatogram spotted with methyl ester picloram and exposed to UV l i g h t (253.7 nm) f o r 0,24 and 48 hours ( l e f t to r i g h t ) . Solvent system: hexane + acetone + methanol + acetic acid, 5 0 + 7 + 5 + 2 , (v/v). The unexposed compound ( l e f t ) gave four weak spots below the main spot. The photoproducts f o r the 48 hour exposure are indicated by arrows. Note progressive decrease i n i n t e n s i t y of main spot with increased exposure. Visual comparison of the t o t a l i n t e n s i t i e s of a l l spots f o r the 48 hour exposure and 0 hour exposure may indicate that some r a d i o a c t i v i t y i s l o s t . Since there i s only one /l4 labeled carbon ( C 0 0 H ) on the molecule, i t may be concluded that degradation by a decarboxylation mechanism may be involved. Photodecomposition may be a s i g n i f i c a n t process by which the picloram herbicide, both i n the form of acid and potassium s a l t , may be broken down on s o i l surfaces. U l t r a v i o l e t radiation may, however, be a very important fa c t o r i n degrading the methyl ester and may have to be watched f o r when standard solutions of i t are stored f o r some time. F i g . 14. - 73 -SUMMARY P i c l o r a m used as a pre-emergence spray at a sub-l e t h a l r a t e on f o u r v a r i e t i e s of potatoes was found to l e a v e r e s i d u e s of the h e r b i c i d e i n the t u b e r s . A p i c l o r a m + l i n u r o n mixture was found to l e a v e lower amounts of p i c l o r a m r e s i d u e s than the p i c l o r a m alone treatments. A d i l u t i o n f a c t o r , r e s u l t i n g from h i g h e r y i e l d s w i t h the h e r b i c i d e combination, may be i n v o l v e d . The d i f f e r e n c e between the means of the p i c l o r a m and p i c l o r a m + l i n u r o n treatments was s i g n i f i c a n t at the 1$ l e v e l . However t h e r e was no s i g n i f i c a n t v a r i e t y or v a r i e t y x treatment i n t e r a c t i o n . Pre-emergence and post-emergence treatments of p i c l o r a m on f o u r v a r i e t i e s of c a r r o t s were found to l e a v e no d e t e c t a b l e amounts of p i c l o r a m i n the t a p r o o t s of a l l v a r i e t i e s . T h i s was i n v e s t i g a t e d f u r t h e r and i t was found t h a t young c a r r o t p l a n t s can absorb and t r a n s l o c a t e p i c l o r a m , when i t i s a p p l i e d both to the f o l i a g e and to the r o o t s . F o l l o w i n g the d i s t r i b u t i o n of r a d i o a c t i v e p i c l o r a m i n 2-month o l d c a r r o t p l a n t s i t was found t h a t , on a weight b a s i s , the l e a v e s accumulated about f o u r times more r a d i o a c t i v i t y than the t a p r o o t s . Chromatographic evidence showed t h a t the r e s i d u a l r a d i o a c t i v i t y , both i n the f o l i a g e and i n the t a p r o o t s , was p r o b a b l y i n the form of the parent molecule, with no o t h e r m e t a b o l i t e . - 74 -Vhen picloram i s present i n carrot taproot i t i s more l i k e l y to be present i n the xylem than i n any other tissue. Investigation of the s t a b i l i t y of radioactive picloram, i t s potassium s a l t and i t s methyl ester under short wave u l t r a v i o l e t l i g h t (253«7 nm) showed that the methyl ester i s quite unstable, being 85$ degraded into several photoproducts a f t e r one hour exposure. Picloram and i t s potassium s a l t are somewhat more stable, each being 50$ decomposed into several photoproducts a f t e r one hour exposure. One common feature of a l l three compounds i s the formation of an intense radioactive spot, at the base of the chromatogram. This was not resolved by the solvent system and may have been due to a polymerisation product. - 75 -BIBLIOGRAPHY 1. Abbott, D.C. and P.J. Wagstaffe 1969. Thin layer chromato-graphic i d e n t i f i c a t i o n of the active ingredients of mixed herbicide formulations. J . Chromatog. 43: 361-367. 2. Abeles, F.B. I968. Herbicide - induced ethylene production! Role of the gas i n sub-lethal doses of 2,4-D. Weed S c i . 16:498-500. 3. Al l e y , H.P. I967. Some observations on Tordon-2,4-D Herbicide combinations. Down to Earth 2 3(l ) : 2 4. Alley, H.P., G.A. Lee and A. Gale I967. Weed control b r i e f s , Crops Section, University of Wyoming. 5. Armstrong, D.E., G. Chesters and R.F. Harris 1967. Atrazine hydrolysis i n s o i l . Proc. S o i l . S c i . Soc. Am. 31:61-66. 6. Ashton, F.M., D. Penner and S. Hoffman 1968. E f f e c t of several herbicides on p r o t e o l y t i c a c t i v i t y of squash seedlings. Weed S c i . 16:169-171. 7. Baur, J.R. and P.W. Morgan I969. 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Day and W.A. Clerx I963. E f f e c t of u l t r a v i o l e t l i g h t on substituted urea and t r i a z i n e compounds. Proc. Western Weed Control Conf. p. 78. 49. Kefford, N.P. and O.H. Caso I966. A potent auxin with unique chemical structure: 4-amino-3,5,6-trichloro-p i c o l i n i c acid. Bot. Gaz. 127:159-163. - 79 -50. Key, J.L. 1964. Ribonucleic acid and protein synthesis as essential processes f o r c e l l elongation. Plant Physiol. 39:365-370. 51. Key, J.L., C.Y. L i u , E.M. G i f f o r d J r . and R. Dengler 1966. Relation of 2,4-D-induced growth aberrations to changes i n nucleic acid metabolism i n soybean seedlings. Bot. Gaz. 127:87. 52. Koeman, J.H., M.C. Ten Noever De Brauwand R.H. De Vos I969. Chlorinated biphenyls i n f i s h , mussels and birds from the River Phine and the Netherlands Coastal Area. Nature 221:1126-1128. 53. Kreps, L.B. and H.P. A l l e y 1967. H i s t o l o g i c a l abnormalities induced by picloram on Canada t h i s t l e roots. Weeds 15:56-59. 54. Kutschinski, A.H. 1969. Residues i n milk from cows fed with 4-amino-3»5 » 6-trichloropicolinic acid. J . Agr. Food Chem. 17:288-290. 55. Kutschinski, A.H. and Van Riley 1969. Residues i n various tissues of steer fed with 4-amino-3,5,6-trichloro-p i c o l i n i c acid. J. Agr. Food Chem. 17:283-287. 56. L a i Ming-Tan and G. Semeniuk 1970. Picloram-induced increase of carbohydrate exudation from corn seedlings. Phytopath. 60:363-364. 57« Laning, E.R. J r . I963. Tordon f o r the control of deep-rooted perennial herbaceous weeds i n the Western United States. Down to Earth 19(l):3-5. 58. Leahy, J.S. and T. Taylor 1967. A gas-chromatographic determination of residues of picloram. Analyst 92:371-374. 59* Leasure, J.K. 1964. Bio-assay method f o r 4-amino-3, 5 , 6 - t r i c h l o r o p i c o l i n i c acid. Weeds 12: 232-233. 60. Lee, G.A., A.K. Dobrenz and H.P. A l l e y I967. Preliminary investigations of the effect of Tordon and 2,4-D on l e a f and root tissue of Canada t h i s t l e . Down to Earth 23(2):21-23. 61. Lee, D.F., J . Britton, B. Jeffcoat and R.F. M i t c h e l l 1966. Interference with the determination of a l d r i n and benzene hexachloride residues by gas chromatography. Nature 211:521-522. - 80 -62. Lynne, G.E. 1965. A review of t o x i c o l o g i c a l information on Tordon herbicide. Down to Earth 21(4):6-8. 63. Lichtenstein, E.P. and ICR. Schulz 1965. Residues of a l d r i n and heptachlor i n s o i l s and t h e i r trans-l o c a t i o n into various crops. J . Agr. Food Chem. 13:57-63. 64. Lichtenstein, E.P., G.R. Myrdal and K.R. Schulz I 9 6 5 . Absorption of i n s e c t i c i d e residues from contaminated s o i l s into f i v e carrot v a r i e t i e s . J. Agr. Food Chem. 13:126-131. 65. Luckwill, L.C. I968. In "Recent Aspects of Nitrogen Metabolism i n Plants" (Ed. Hewitt E.J. & C.V. Cuttings). Academic Press, London, N.Y. 280 p. 66. Luckwill, L.C. and C P . Lloyd-Jones i960. Metabolism of Plant Growth Regulators. Ann. Appl. B i o l . 4 8 : 6 1 3 -636. 67. Makita, M. and W.M. Wells 1963. Quantitative analysis of f e c a l b i l e acids by gas l i q u i d chromatography. Anal. Biochem. 5:523* 68. Malhotra, S.S. and J.B. Hanson 1970. Picloram s e n s i t i v i t y and nucleic acid i n plants. Weed S c i . 18:1-4. 69. Mann, J.D. and L. Minn Pu I 9 6 8 . I n h i b i t i o n of l i p i d synthesis by certa i n herbicides. Weed S c i . 16:197-198. 70. Marquardt, R.P., H.P. Burchfield, E.E. Storrs and A. Bevenue 1964. In "Analyti c a l Methods f o r Pesticides, Plant Growth Regulators and Food Additives Vol. 5". (Ed. G. Zweig). Academic Press, N.Y. 450 p. 71. Meikle, R.W., E.A. Williams, and C.T. Redemann 1966. Metabolism of Tordon herbicide i n cotton and decomposition i n s o i l . J . Agr. Food Chem. 14:384-387. 72. Melcher, R.G. 1965. Unpublished work. Spectroscopy Service Laboratory, The Dow Chemical Company, Midland, Michigan. 73. Merkle, M.G., R.W. Bovey and R. H a l l I 9 6 6 . Determination of picloram residues i n s o i l using gas chromato-graphy. Weeds 14:161-164. - 81 -74. Merkle, M.G., R.W. Bovey and F.S. Davis 1967. F a c t o r s a f f e c t i n g the p e r s i s t e n c e of p i c l o r a m i n s o i l . Agron. J . 59:413-415. 75. M e t c a l f e , L.D. and A.A. Schmitz I 9 6 I . The r a p i d p r e p a r a t i o n of f a t t y a c i d e s t e r s f o r gas chromatographic a n a l y s i s . A n a l . Chem. 33s363-366. 76. M i l l e r , G.A. and C.E. Wells 1969. A l k a l i n e pre-column f o r use i n gas chromatographic p e s t i c i d e r e s i d u e a n a l y s i s . J o u r n a l A.O.A.C. 52:548-553. 77. M i t c h e l l , L.C. I 9 6 I . The e f f e c t o f u l t r a v i o l e t l i g h t (2537 A.) on l 4 l p e s t i c i d e s chemicals by paper chromatography. J o u r n a l A.O.A.C. 44:643-712. 78. Moreland, D.E., S.S. Malhotra, R.D. Gruenhagen and E.H. S h o k r a i i I969. E f f e c t s o f h e r b i c i d e s on RNA and p r o t e i n s y n t h e s i s . Weed Res. 17:556-563. 79. Morgan, P.W. and W.C. H a l l 1962. E f f e c t of 2,4-D on the p r o d u c t i o n of ethylene by c o t t o n and g r a i n sorghum. P h y s i o l . P l a n t . 15:420-427. 80. Mustafa, A. 1952. D i m e r i s a t i o n r e a c t i o n s i n s u n l i g h t . Chem. Rev. 51:1-23. 81. Nashed, R.B. and R.D. I l n i c k i 1967. E f f e c t of l i n u r o n on i o n uptake i n corn, soybean and c r a b g r a s s . Weed S c i . 15:188-192. 82. Onsager, J.A., H.W. Rush and L . I . B u t l e r 1970. Residues of a l d r i n , d i e l d r i n , chlordane and DDT i n s o i l and sugar beets. J . Econ. Ent. 63:Il43-ll46. 83. Pearson, J.R., F.D. A l d r i c h and A.W. Stone I967. I d e n t i f i c a t i o n o f the a l d r i n a r t i f a c t . J . Agr. Food Chem. 15:938-939. 84. Peterson, J . I . , H. De Schmertzing and K.J. Abel 1965. Trans e s t e r i f i c at i o n of l i p i d s with BCl-j. J . Gas Chromatog. 3:126-130. 85. Plimmer, J.R. and B.E. Hummer 1967. P e r s o n a l communication i n "Degradation of H e r b i c i d e s " (Ed. P.C. Kearney and D.D. Kaufman). Marcel Dekker Inc. N.Y. 394 p. 86. Purkayastha, R. I969. D i r e c t d e t e c t i o n o f i o n i s a b l e h e r b i c i d e s by e l e c t r o p h o r e s i s . B u l l . E n v i r o n . Contam. & T o x i c o l . 4(4) 246-255. - 82 -87. Rederaann, C.T., R.W. Meikle, P. Hamilton and V.S. Banks 1968. The fate of 4-amino-3,5 *6-trichloropicolinic acid i n spring wheat and s o i l . B u l l . Environ. Contam. & To x i c o l . 3:80-96. 88. Redemann, C.T. I967. Personal communication i n "Degradation of Herbicides". (Ed. P.C. Kearney and D.D. Kaufman). 89. Renney, A.J. and E.C. Hughes I969. Control of knapweed, Centaurea species i n B r i t i s h Columbia with Tordon herbicide. Down to Earth 2k(k);6-8. 90. Robinson, J., A. Richardson and K.E. Elgar I966. Paper presented at the 152 nd Am. Chem. Soc. National Meeting. Abst. 75, D i v i s i o n of Agr. and Pood Chem. N.Y., Sept. 11-16. 91. Rogozinski, M. 1964. A rapid quantitative e s t e r i f i c a t i o n technique f o r carboxylic acids. J . Gas Chromatog. 2:136-137. 92. Saha, J.G. and L.A. Gadallah I967. Determination of the herbicide Tordon i n s o i l by electron capture gas chromatography. Journal A.O.A.C. 50(3):637-641. 93. Schnorbus, R.R. and W.F. P h i l l i p s I967. New extraction system f o r residue analysis. J . Agr. Pood Chem. 15(4) -.661-666. 94. Segal, H.S. and M.L. Sutherland 1964. Comparison of flame i o n i s a t i o n and electron capture detectors f o r the gas chromatographic evaluation of herbicide residues. Residue Reviews Vol. 5 (Ed. P.A. Gunther). Academic Press, N.Y. 95. Semeniuk, G. and T.B. Tunac 1968. Tordon increase i n root rot severity i n wheat and corn. South Dakota Acad. S c i . Proc. 4?:346. 96. Slade, P. and A.E. Smith 1967. Photochemical degradation of diquat. Nature 213:919. 97. Slade, P. I965. Photochemical degradation of paraquat. Nature 207:515-516. 98. Smith, A.E. 1967. Personal communication i n "Advances i n Pest Control Research Vol. 8". (Ed. R.L. Metcalf) Interscience Publishers. - 8 3 -99. S t a n l e y , C.¥. 1 9 6 6 . D e r i v a t i z a t i o n o f p e s t i c i d e - r e l a t e d a c i d s and phenols f o r gas chromatographic d e t e r m i n a t i o n . J . Agr. Food Chem. 14:321-323. 1 0 0 . Van Overbeck, J . 1 9 6 4 . In "The P h y s i o l o g y and Bioc h e m i s t r y of H e r b i c i d e s " . (Ed. L . J . Audus). Academic P r e s s , N.Y. 555 P. 1 0 1 . ¥alker, J.R.L. and J.E. Thompson I 9 6 7 . T h i n l a y e r e l e c t r o p h o r e s i s of p h e n o l i c compounds. J . Lab. P r a c t i c e 1 8 : 6 2 9 - 6 3 1 . 1 0 2 . Warden, R.L. 1 9 6 4 . Tordon f o r the c o n t r o l of f i e l d bindeweed and Canada t h i s t l e i n the North C e n t r a l U n i t e d S t a t e s . Down to E a r t h 2 0 : 6 - 1 0 . 1 0 3 . Watson, A.J. and M.G. W i l t s e I 9 6 3 . Tordon f o r brush c o n t r o l on u t i l i t y r i g h t - o f - w a y i n the E a s t e r n U n i t e d S t a t e s . Down to E a r t h 19:11-14. 104. Watt, B.K. and A.L. M e r r i l l 1 9 6 3 . Composition of food s . A g r i c u l t u r e Handbook # 8 . U n i t e d S t a t e s Department of A g r i c u l t u r e . 1 0 5 . West, S.H., J.B. Hanson and J.L. Key i 9 6 0 . E f f e c t o f 2,4-D on n u c l e i c a c i d and p r o t e i n content o f s e e d l i n g t i s s u e . Weeds 8 : 3 3 3 . 1 0 6 . Whitenberg, D.C. 1 9 6 7 . D e t e c t i o n o f N - c o n t a i n i n g h e r b i c i d e s on t h i n l a y e r chromatograms. Weeds 15:182. 1 0 7 . W i l t s e , M.G. 1 9 6 4 . Tordon h e r b i c i d e as a s o i l treatment f o r brush c o n t r o l . Down to E a r t h 19:3-6. 108. Woolson, B.A. and C.I. H a r r i s I 9 6 7 . M e t h y l a t i o n of h e r b i c i d e s f o r gas chromatographic d e t e r m i n a t i o n . Weeds 15:168-170. 1 0 9 . Yamaguchi, S. and A.S. C r a f t s 1 9 5 8 . A u t o r a d i o g r a p h i c method f o r s t u d y i n g a b s o r p t i o n and t r a n s l o c a t i o n of h e r b i c i d e s u s i n g C l a b e l e d compounds. H i l g a r d i a 28:161 -191. 1 1 0 . Youngson, C R . , C.A.I. Goring, R.W. Meikle, H,H. S c o t t and J.D. G r i f f i t h I 9 6 7 . F a c t o r s i n f l u e n c i n g the decomposition o f Tordon h e r b i c i d e i n s o i l s . Down to E a r t h 23:3 -11 . - 84 -Appendix I COMPOSITION OF NUTRIENT SOLUTION The following amounts of stock solutions are added to 1,500 ml d i s t i l l e d water and the volume brought up to 2,000 ml. Stock solution Volume 1 M Ca ( N 0 3 ) 2 10 1 M KNO^ 10 1 M MgSO^ 4 1 M KH2P0^ 2 FeEDTA* 2 Mi c ronut r i en t s ** 2 (ml) * 1 ml stock solution contains 5 mg of Fe. ** Micronutrient stock solution contains 2,86 g of H3BO.J (boric acid), 1.81 g of MnCl 2.4H 20 (manganese chloride), 0.11 g of Zn C l 2 (zinc chloride}, 0.05 g of CuCl 2.2H 0 (copper chloride), and 0.025 g of Na2Mo0^.2H20 (sodium molybdate) per l i t e r . - 85 -Appendix II COMPOSITION OF SCINTILLATION FLUID Dioaxane 800 ml Toluene 200 ml Ethanol 30 ml PPO (2,5-diphenyloxazole) 7 g POPOP (2,2'-paraphenylene bis-5-phenyloxazole) 200 mg Naphthalene 50 g Cab-O-Sil 36 g - 86 -Appendix I I I CHEMICAL NAMES OF PESTICIDES USED IN TEXT 1. A l d r i n 1.2,3,4,10,10-hexachloro-l,4,4a,5,8,8a-hexa-hydro-l,4-endo, exo-5, 8-dimethanonaphthalene 2. Araiben 3-amino-2, 5-dichlorobenzoic a c i d 3. BHC 1,2,3,4 ,5,6-hexachlorocyclohexane 4. 2,4-D 2,4-dichlorophenoxyacetic a c i d 5. 2,4,5-T 2 ,4,5-trichlorophenoxyacetic a c i d 6. 2,4-DB 4-(2,4-dichlorophenoxy) b u t y r i c a c i d 7. DDT l , l , l - t r i c h l o r o - 2 , 2 b i s (p-chlorophenyl) ethane 8. Dicamba 3,6-dichloro -2-methoxybenzoic a c i d 9. Diquat 1,1 '-ethylene-2, 2 ' - d i p y r i d y l i u m dibromide 10. Fenoprop 2- (2 ,4,5-trichlorophenoxy) p r o p i o n i c a c i d 11. L i n u r o n 3 - ( 3 ,4-dichlorphenyl) - 1-methoxy-l-raethyl u r e a 12. MCPA 2-methyl-4-chlorophenoxyacetic a c i d 13. Paraquat 4 , 4 , - b i p y r i d y l i u m - l , 1»-dimethyl d i c h l o r i d e 14. P i c l o r a m 4 - a m i n o - 3 , 5 , 6 - t r i c h l o r o p i c o l i n i c a c i d 15. Simazine 2-chloro-4, 6-bis ( e t h y l a m i n o ) s - t r i a z i n e 16. Tordon 4 - a m i n o - 3 , 5 , 6 - t r i c h l o r o p i c o l i n i c a c i d (picloram) 

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