SELECTIVHY AND FATE OF 2 ~ CHLORO =- N - (I '- MUHYL - 2 - PROPYNYL) 'ACEI'ANHJDE WWGHLOR) 3N ONION AND PROSO MILLS? Thesis for the Degree of M. 8. MICHiGAN STATE [NW-176W EDWARD LOUIS 31% 1973 - , ‘ ABSTRACT SELECTIVITY AND FATE OF 2-CHLORO-Nf(l-METHYL-Z-PROPYNYL) ACETANILIDE (PRYNACHLOR) IN ONION AND PROSO MILLET By Edward Louis Silvia Preemergence applications of 2-chloro-Nf(l-methyl-Z—propynyl) acetanilide (prynachlor) effectively controlled annual grasses and selected broadleaved weeds up to 6 weeks in muckland onions (Allium £323_L. 'Downing Yellow Globe'). All rates provided commercially acceptable control for 3 weeks and longer term weed control was obtained at higher rates. Prynachlor was highly effective in con- trolling all weeds present with the exception of Pennsylvania smart- weed (Polygonum pensylvanicum L.). Preemergence or postemergence applications at the loop, flag, and one leaf stages at rates as high as 6.72 kg/ha produced no significant reduction in onion dry weights. Greenhouse rate studies showed that on muck soil, prynachlor inhibited growth of onion less than 50% at rates as high as ll.2 kg/ha, whereas onions were killed at 6.72 kg/ha on a mineral soil mix. In contrast, susceptible proso millet (Panicum miliaceum L.) was killed at l.l2 kg/ha on muck soil and 0.56 kg/ha on mineral soil mix. Studies were conducted to determine the basis for selectivity in these two monocotyledonous plants. Root and shoot uptake of IAC- prynachlor was assayed utilizing emerging seedlings and a micro split- pot technique. After l8 hours, uptake by proso millet roots exceeded that of onion roots by 58%. However, over longer time periods, onion absorbed more than proso millet. Root uptake was greater than shoot Edward Louis Silvia uptake in both Species when equal amounts of herbicide were applied to each zone. The amount transported from root to shoot in proso millet was up to 20 fold that which occurred in onions. Germinating onion seedlings metabolized prynachlor more rapidly than proso millet seedlings, although similar metabolites were found in both Species. In 3 week old plants, uptake by onion exceeded that of proso millet and majority of the activity remained in the roots of both species. Onion roots reached maximum uptake in 24 hours, whereas activity in proso millet roots continued to increase throughout 96 hours. Rapid metabolism of prynachlor was accomplished by root and shoot of onion. Over 96% was converted to non- toxic, 2-oic-Nf(l-methyl-Z-propynyl) acetanilide. Less than 2% of the activity was identified as parent compound. A similar distribution of metabolites was observed in root and shoot of proso millet. Inc-prynachlor was applied preemergence to onions growing on muck soil to follow residue levels until harvest. At the flag stage, nearly IO fold higher concentrations of ll'C-compounds were detected in the onion shoots than in the roots. At maturity, the concentration of ”4C- compounds detectable in the onion bulbs was about 1 part per billion expressed on a dry weight basis. SELECTIVITY AND FATE OF 2-CHLOR0-Nf(l-METHYL-Z-PROPYNYL) ACETANILIDE (PRYNACHLOR) IN ONION AND PROSO MILLET By Edward Louis Silvia A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture I973 ACKNOWLEDGEMENTS The author wishes to express his sincere thanks to Dr. A. R. Putnam for his guidance and assistance during the course of this study and the preparation of this thesis. Appreciation is also expressed to Drs. N. F. Meggitt and H. C. Price for their guidance and suggestions in editing the manuscript. The assistance of Paul Love and Martha van Buskirk is also gratefully acknowledged. Special thanks to my wife for her help and understanding during my intense course of study. TABLE OF CONTENTS ACKNOWLEDGEMENTS . LIST OF TABLES . LIST OF FIGURES CHAPTER I: LITERATURE REVIEW Chemical Weed Control in Onion (Allium cepa L.) Action of the 2-chloroacetamides General PrOperties and Structure . Uptake . Metabolism . Selectivity Mode of Action . Prynachlor . . . . . Structure and Properties . Metabolism . CHAPTER 2: HERBICIDAL ACTIVITY OF PRYNACHLOR ON MUCKLAND ONIONS Materials and Methods Results and Discussion CHAPTER 3: BASIS FOR THE SELECTIVITY OF PRYNACHLOR IN ONION AND PROSO MILLET . Abstract Page Table of Contents (Cont.) Introduction Materials and Methods Results and Discussion Literature Cited CHAPTER A: FATE OF PRYNACHLOR IN MATURING ONION PLANTS Materials and Methods Results and Discussion CHAPTER 5: SUMMARY AND CONCLUSIONS LIST or REFERENCES . Page IA IS 20 32 3A 3A 39 A5 A7 LIST OF TABLES Table CHAPTER 2 l. Response of weeds and onions to preemergence applications of prynachlor . CHAPTER 3 l. Uptake of 1[IO-prynachlor by roots and shoots of onion and proso millet . 2. Transport of llIC-prynachlor in onion and proso millet after root or shoot exposure . CHAPTER A l. Total 1[IE-residue in onion plants after a preemergence application of prynachlor on a Houghton muck soil. Page 10 2A 2A 39 LIST OF FIGURES Figure Page CHAPTER 2 l. Response of onion seedlings at four stages of growth to prynachlor . . . . . . . . . . . . . . . . . . . . . . . . . l3 CHAPTER 3 I. Micro split-pot technique utilized to assay uptake of IhC- prynachlor by shoots or roots of onion and proso millet . . I8 2. Response of a.) onions and b.) proso millet growing on two soil types to preemergence application of prynachlor . . . . 22 3. Distribution of ll‘C-~prynachlor (parent), 2~oic-N-(l-methyl- 2-pr0pynyl) acetanilide, 2-oxo-Nf(l-methyI-Z-propynyl) acetanilide, and Nfisobutynyl aniline in onion and proso millet after l8 hours . . . . . . . . . . . . . . . . . . . 26 A. DeveIOpment of 2-oic-Nf(l-methyI-Z-propynyl) acetanilide in a polar solvent system . . . . . . . . . . . . . . . . . . . 29 5. Distribution of IhC-prynachlor (parent), 2-oic-Nf(I-methyl- 2-propynyl) acetanilide, 2-hydroxy-N-(l-methyl-Z-propynyl) acetanilide, 2-oxo-N-(I-methyl-2-pr5pynyl) acetanilide, and Nfisobutynyl aniling in onion and proso millet after 96 hours . . . . . . . . . . . . . . . . . . . . . . . . . . . 3i CHAPTER A l. Extraction procedure to remove Itic-prynachlor and metabolites from onion and proso millet seedlings . . . . . . . . . . . 38 2. Uptake of Inc-prynachlor from nutrient culture by 3 week old onion and proso millet seedlings . . . . . . . . . . . . . . A2 3. Distribution of Inc-prynachlor (parent), 2-oic-Nf(I-methyl- 2-propynyl) acetanilide, 2-hydroxy-N-(l-methyl-Z-propynyl) acetanilide, 2-oxo-Nf(l-methyl-Z-propynyl) acetanilide in onion and proso millet after 96 hours . . . . . . . . . . . AA vi CHAPTER I LITERATURE REVIEW Chemical Weed Control in Onions (Allium cepa L.) Weeds are a major problem in onion production. They may smother relatively slow growing seedling onions, and compete for nutrients and water. On high organic matter soils, weed control is made more difficult by the numerous flushes of weeds throughout the season and the short-lived activity of most herbicides. Dense populations of weeds and diverse species also amplify the problem. On Michigan mucklands, the major annual weed species are large crabgrass (Digitaria sanguinalis (L.) Scop.), yellow foxtail (Setaria glauca (L.) Beauv.), barnyardgrass (Echinochioa crusgallis (L.) Beauv.), witchgrass (Panicum capillare L.), redroot pig- weed (Amaranthus retroflexus L.), common purslane (Portulaca oleracea L.), common chickweed (Stellaria media (L.) Cyrillo), Pennsylvania smartweed (Polygonum pensylvanicum L.), common lambsquarters (Chenopodium 21222 L.), and mustards (Brassica 32:). The earliest method of combatting weeds in onions was hand weeding and cultivation. On muck soils, this is extremely difficult without reducing onion stands and also is extremely costly. It was estimated that hand weeding of onions may require as much as I70 hours of labor per acre (2A). In hopes of reducing the high labor requirements for hand weeding, chemical weed control was initiated in the I9A0's. It was recognized that morphological features of onions such as waxy leaves, and their tendency to grow erect might lend tolerance to the cr0p. One of the first chemicals to be utilized based on this type of selectivity was sulfuric acid. It could be sprayed over the tops of onions with minimum damage to crop and burn off most of the emerged weeds (l2). Although control was effective, sulfuric acid was harsh on men, equipment, and perhaps soils. Several oils such as diesel oil and stoddard solvent were also found to be effective, but caused yield reductions. Less crOp damage was obtained with amine formulations of (2, A-dichlorophenoxy) acetic acid (2, A-D) than with oils (I2). Today, these early postemergence herbicides have been replaced by 2, A-dichloro- phenyl -pfnitr0phenyl ether (nitrofen), and 3-[§f(pfchlor0phenoxy) phenyl]-l,l-dimethylurea (chloroxuron). Applied postemergence to weeds, nitrofen and chloroxuron effectively control many broadleaved weeds and selected annual grasses (8, 22, 30). The value of preemergence herbicides has not been overlooked in onion production. During the l950's, two of the more effective pre- emergence chemicals were 3-(chhlor0phenyl)-l, l—dimethylurea (monuron) and iSOprOpyl mfchlorocarbanilate (chloroprOpham) (I2). ChloroprOpham is still recommended for use today (26). Several of the 2-chloroacetamide herbicides have shown promise for selective preemergence use in vegetable cr0ps (A, 7, 23). The first to be commercially introduced was NJNfdially-2-chloroacetamide (CDAA). It was found to provide good control of annual grasses in particular, and is still in use (7, 26). In I965, 2-chloro-NfisoprOpylacetanilide (propachlor) was introduced as a preemergence herbicide for control of a broad spectrum of broad- leaved weeds and grasses. PrOpachlor was found superior to several herbicides in controlling weeds in onions (23, 30). Long-season control on onions was obtained when propachlor was applied preemergence and when chloroxuron was applied postemergence (30). Unfortunately, registration for propachlor as a herbicide for onions was never obtained. A newer member of the 2-chloroacetamide group, 2-chloro-Nf(l-methyl- 2-propynyl) acetanilide (prynachlor) has been reported to be highly toxic to both broadleaved weeds and annual grasses, while onion is highly tolerant (A, 27, 29). Action of the 2-chloroacetamides General Properties and Structure. The 2-chloroacetamide herbicides belong to a class of herbicides generally known as the amides. They are also known as acetanilides if one substituent group is a phenyl ring. Their properties are varied depending on their substitution. They have a general structure of the following configuration: R] 0 \\ H ‘ \ \ //// N-C-CHZCL R2 Uptake. Researchers have shown that the 2-chloroacetamides are readily taken up by several plant species (I, A, 9, l0, ll, l6). Smith g£_al: (3l) attempted to describe the possible relationship between amount of uptake of 2-chloroacetamide derivatives and suscept- ibility. Germinating seeds of corn (Eea_m§y§_L.) and soybean (Glycine max L.) represented tolerant species, whereas oats (Avena sativa L.) and cucumber (cucumis sativus L.) represented susceptible species. All of the test plants absorbed the 2-chloroacetamide derivatives, but to different extents. Corn absorbed the least and soybean the greatest amount of the chemicals. It was concluded that susceptibility was not determined by the amount of the chemical absorbed. Split-pot experiments showed that site of uptake of 2-chloro-2', 6'-diethyI-Nf(methoxymethyl) acetanilide (alachlor) in cotton (Gossypium hirstum L.) was directed toward the root zone rather than the shoot zone. Cotton was treated with alachlor at the root zone, shoot zone, and both root and shoot zone. When absorption of the chemical was in the root zone, dry weight of the cotton plants were reduced considerably. Only a slight reduction in dry weights occurred when soil in the shoot zone was treated, and placing the chemical in both root and shoot zones only slightly increased phytotoxicity (ll). Knake £5.21: (l8) reported that when giant foxtail (Setaria faberii Herrm.) was treated with alachlor in the shoot or root zone, it was the shoot zone treatment which effected the plant most noticably, leading one to believe that the major site of uptake in monocotyledons is the shoot zone. Armstrong (I) showed that application of alachlor to the roots of yellow nutsedge (Cyperus esculentus L.) did not reduce plant growth, whereas application in the shoot zone gave excellent control. Metabolism. Metabolism of the 2-chloroacetamides has been studied by several researchers (l0, l5, l9, 3i, 32). Studies of CDAA metabolism in corn and soybean showed that CDAA was completely metabolized in both species within four days (I6). Co-chromatographic analysis of the extracts of corn confirmed glycolic acid as the major metabolite. In soybean seedlings, glyoxylic rather than glycolic acid was found to be the major product of CDAA metabolism. Research by Zelitch and Ochoa showed glyoxylic acid to be in equilibrum with glycolic acid, both of which function in plant respiration (35, 36). In corn and soybean, propachlor has been found to be rapidly metabolized to a highly polar metabolite (l7). Through hydrolysis and vapor-phase chromatography the metabolite was found to be a water-soluble acidic compound with a structure similar to the parent compound, minus the chloro group. This is believed to have been displaced by some nucleophillc endogenous substrate in the plant (25). Lamoureux g£_al, (l9) found propachlor to be metabolized by corn to both the glutamyl- cysteine conjugate and the glutathione conjugate of propachlor. With 3',A'-dichlor0propionanilide (propanil), Stroller gt_al, (33) reported that the parent compound is first converted to arylamines and then complexed with glucose. Selectivity. Smith t l. (3i) studied the rate of metabolism of 2-chloroacetamide derivatives by both tolerant and susceptible plants. He found that both susceptible and tolerant test plants could absorb and metabolize the chemical in A8 hours, but in 6 hours tolerant species were able to metabolize larger amounts, whereas susceptible species metabolized very little. It was concluded that the degree of tolerance of various seedlings to 2-chloroacetamides seems to be related to the length of time required to metabolize the chemical. Those species which metabolized the chemical as soon as it entered or within a short time thereafter, had only small amount of parent compound present. In contrast, susceptible species incapable of rapid metabolism accumulated higher and therefore, lethal concentrations. The mechanism of selectivity for prOpanil was pr0posed by Still gt 21: (32). He suggested that the tolerance of rice is correlated with the activity of acylanilide hydrolase an enzyme which deactivates propanil. Mode of Action. The 2-chloroacetamides are believed to inhibit growth by stopping cellular elongation and cell division. Cavin £5.31: (5) showed that when CDAA was applied to susceptible barley (Hordeum vulgare L.) and peas, (Pisum sativum L.) cell division was inhibited in germinating seedlings, and the amount of inhibition was directly propor- tional to the concentration of CDAA. Propachlor possesses similar cellular inhibition characteristics (3, 9, l0). Dhillon §t_§l: (9) reported that cell division in onion root was totally inhibited by propachlor at I6 ppm. A possible mode of action of CDAA was postulated by Jaworski (16) who reported that CDAA reduced the respiration of germinating rye grass (Lolium multiflorum Lam.) by inhibiting sulfhydryl containing enzymes involved in respiration. Studies with thiol compounds and the 2-chloro- acetamides showed an interaction with sulfhydryl groups and various enzyme system (20). Mann _£_§l, (2i) treated barley coleoptiles with CDAA and found 5l to 70% inhibition of IllC-leucine incorporation into protein at 2 and 5 ppm respectively. He concluded that CDAA was either inhibiting the uptake of certain amino acids or causing inhibition of protein synthesis. In work with propachlor, Duke 35.31: (l0) reported inhibition in cucumber roots and correlated this with the inhibition of protein synthesis in root tips. He further suggested that the primary site of action is at the level of protein formation and is due to the prevention of the transfer of aminoacyl-sRNA to the polypeptide chain. Prynachlor Structure and Properties. Prynachlor is a member of the 2-chloro- acetamide group and has a phenyl ring and a (l-methyl, 2-pr0pynyl) group fulfilling the N-substitution. It has the following structure: 0 II H3C-CH CECH Prynachlor has a molecular weight of 22l.7, is highly soluble in benzene, and its solubility in water and ethanol at 20°C is 0.50 and 239.2 g/L., respectively (3A). Its melting point is A0-A70C. Acute toxicity tests indicated an oral L050 of ll77 mg/kg for rats and a dermal L050 of I926 mg/kg for rabbits (2). Metabolism. Prynachlor metabolism studies in corn and soybean have found the majority of the metabolite present to be the conjugated acid, 2-oic-Nf(l-methyl-Z-propynyl) acetanilide (28). Metabolism involves hydroxylation at the chlorine position followed by several oxidation steps. The metabolic degradation scheme for prynachlor in corn as proposed by Hazelton Laboratories is given on page 8. o H -N-C-CH2-Cl ECH C-H /\ CH3 C Z-chloro-N-(I-methyl- 2-propynyT) acetanilide o H -N-C-CHO I C-H /\ CH CECH 3 2-oxo-Nf(l-methyl- 2-propynyl) acetanilide O H -€> -N-C-CH2-OH -%> conjugate I C-H 2-hydroxy-N-(I-methyl- 2-pr0pynyIT-acetanilide 00 -—a» -T-C-C-OH -—e> conjugate C-H /\ CH3 C 2-oic-Nf(I-methyl- 2-pr0pynyl) acetanilide CH CHAPTER 2 Herbicidal Activity of Prynachlor on Muckland Onions Materials and Methods Preemergence tests. Field studies were conducted during l97l and I972 at the Michigan State University Muck Experimental Farm, on a Houghton muck (80% organic matter) with a resident population of common purslane, redroot pigweed, Pennsylvania smartweed, large crabgrass, barnyardgrass, witchgrass, and yellow foxtail. The field was plowed, fertilized, disk harrowed, and floated after which onions 'Downing Yellow Globe' were seeded on May l9, I972. The rows were spaced 30 cm apart with 50 seeds per meter. Prynachlor was applied on plots l.22 X 7.63 m with a C02 powered plot sprayer regulated at 30 psi. Prynachlor (AEC) was applied preemergence at rates of 0, 2.2A, 3.36, A.A8, 6.72 and 8.96 kg/ha. Each treatment was replicated four times in a randomized complete block design. Plots were evaluated for both broadleaved weed and annual grass control on a l to 9 scale where l=no control and 9: complete control. Weed ratings were obtained 3 and 6 weeks after initial seeding. Control plots were hand weeded once a week until harvest. Eight weeks after treatment, prynachlor treated plots were hand weeded along with control plots. At maturity, the crop was hand-harvested and total fresh weights of bulbs from each plot recorded. Postemergence tests. CrOp safety studies were conducted to determine the tolerance of onions to prynachlor at early stages of crop growth. This study was also conducted at the Michigan State University Muck Experimental Farm during the summer of l97l. Onions were seeded into l.22 X 7.63 m plots at one week intervals for A weeks. Plots were hand lO weeded until application of the herbicide on the fourth week. Treatments were replicated 3 times in a split-plot design. The herbicide was applied at rates of 0, 2.2A, 3.36, A.A8, and 6.72 kg/ha as previously described. Two weeks after herbicide application, shoot growth from one meter sections of row was cut from each plot. Shoots were oven dried at ASOC and weights recorded to the nearest mg. Results and Discussion Preemergence applications of prynachlor effectively controlled annual grass and selected broadleaved weeds up to 6 weeks (Table I). Table l. Response of weeds and onions to preemergence applications of prynachlor. 21 Day A2 Day Prynachlor rate BroadleafI Grass Broadleafl Grass Yield (kg/ha) (weed control rating) (kg/ha) O l.O l.O l.0 l.0 21,138. 2.2A 7.7 8.0 6.3 6.0 28,865. 3.36 8.3 8.3 7.5 7.0 29,678. A.A8 9.0 9.0 8.5 7.7 32.93l. 6.72 9.0 9.0 9.0 8.0 36,l83. 8.96 9.0 9.0 9.0 9.0 37,AO3. HSD AT 5% LEVEL .6A .A7 l.28 l.28 ll,312. IPennsylvania smartweed not included in rating. II All rates provided commercially acceptable control for 3 weeks, and longer term weed control increased at higher rates. Although an unusually dense weed p0pulation (l6l7 weed/m2) existed, prynachlor was highly effective in controlling all weeds present with the exception of Pennsylvania smartweed, which consisted of 3% of the population. An increase in onion yields occurred with increasing rates of the herbicide up to 8.96 kg/ha. This may have resulted because of l.) the inability to maintain the hand weeded control plots as free of weeds as chemically treated plots, 2.) damage to weeded plots by hoeing and cultivation and 3.) the higher densities of Pennsylvania smartweed at lower rates of prynachlor. Preemergence or postemergence applications at the loop, flag, and one leaf stages at rates as high as 6.72 kg/ha showed no significant reduction in onion dry weights (Figure I). This data indicated that prynachlor can be applied safely to emerged onions without injury. However, application on emerged weeds are also ineffective, so fields would have to be cultivated and/or weeded prior to application of prynachlor. 12 Figure l. Response of onion seedlings at four stages of growth to prynachlor. DRY WT. (my) p 01.! LEAF IFLAG 4|.OOP VPREIIERGENCE \M‘ 2.24 3.33 4.48 6.72 RATE (Ital In) CHAPTER 3 Basis for the Selectivity of Prynachlor in Onion and Proso Millet Abstract. On a Houghton Muck Soil, 2-chloro-N:(l-methyl-2-pr0pynyl) acetanilide (prynachlor) inhibited growth of onion (Allium cepa L. 'Downing Yellow Globe') less than 50% at rates as high as ll.2 kg/ha. In contrast, onions were killed at 6.72 kg/ha on a mineral soil. Susceptible proso millet (Panicum miliaceum L.) was killed at l.l2 kg/ha on muck soil and 0.56 kg/ha on mineral soil. Root and shoot uptake of ll‘C-prynachlor was assayed utilizing emerging seedlings and a micro split-pot technique. After l8 hours, uptake by proso millet roots exceeded that of onion roots by 58%. However, over longer time periods, onion absorbed more than proso millet. Root uptake was greater than shoot uptake in both species when equal amounts of herbicide were applied to each zone. The amount transported from root to shoot in proso millet was up to 20 fold that which occurred in onions. Germinat- ing onion seedlings metabolized prynachlor more rapidly than proso millet seedlings. Introduction Herbicide selectivity may be due to physical, morphological and/or physiological factors. Physiological selectivity is determined by rates of uptake, trans- location, and biochemical alteration of the herbicide by the plant (5, IA). It is probably the most important type of selectivity for preemergence IA 15 herbicides. Researchers have correlated differential uptake and trans- location with susceptibility of certain plant species (A, l6). Plants can metabolically alter herbicide activity. Classic examples are the differential dealkylation of 2-chloro-A-(ethylamino)-6-(iSOpropylamino)- s-triazine (atrazine), demethylation of 3-(Efchlorophenyl)-I,I- dimethylurea (monuron), and conjugation of 5-amino-A-chloro-Z-phenyl-3 (2H)-pyridazinone(pyrazon) (6, 8, l0, ll, I2). Smith §£_§l: (9) observed uptake of the 2-chloroacetamides by both tolerant and suscept- ible species, but he could not relate this to selectivity. Absorption of the 2-chloroacetamides is effected by the plant zone of uptake. Root zone of cotton was reported to be the primary site of uptake of 2-chloro-Nfisopropylacetanilide (prOpachlor), while giant foxtail (Setaria faberii Herrm.) was effected most noticably when propachlor was placed in the shoot zone (3). The 2-chloroacetamides are metabolized by both tolerant and susceptible plants, but selectivity may be determined by the rate of deactivation (2). Metabolism must be rapid enough to assure that concentrations capable of inhibiting growth are not accumulated. Prynachlor has demonstrated excellent herbicidal activity on annual grasses and selected broadleaved weeds with selectivity in several crops. It is particularily effective on soils with a high organic matter content. The objective of this study was to compare uptake, transport, and metabolism of prynachlor in a tolerant and a susceptible monocotyledonous species to determine the basis for selectivity. Materials and Methods Tolerance. Comparative tolerance of onion and proso millet was determined by applying increasing rates of prynachlor to plants growing on two soil l6 types. Fifty seeds of each species were seeded l.5 cm deep into l5 by I0 by 7 cm styrofoam flats containing either a Houghton muck soil or mineral soil mix ( I loam: l peat: l sand). After planting, prynachlor (AEC) was applied with a laboratory sprayer operated at 30 psi to onion at rates of 0, 2.2A, A.A8, 6.72, 8.96, and ll.2 kg/ha, and to proso millet at rates of 0, 0.lA, 0.28, 0.56, and l.l2 kg/ha, with each treatment being replicated A times in a randomized complete block design. Plants were maintained at a greenhouse night temperature of 2l0C and day temperatures ranged from 21 to 32°C. After 2 weeks, aerial growth was collected and dry weights recorded to the nearest 0.l mg. Uptake. In order to facilitate study of seedling uptake and translocation of ll‘C-prynachlor, a micro split-pot was devised (Figure l). Notches l cm deep were cut in plastic nitrogen analyzer vials (I0 ml) at the highest end. Two vials were glued together so the cut ends met. Floral putty was placed at the botton of the cut. Nine grams of sterile silica sand was placed in each side. Plants of both species were seeded in IS by ID by 7 cm styrofoam boxes containing vermiculite. The seeds were germinated in a growth chamber (30°C). When plants penetrated the media surface, seedlings were removed for placement in the micro split-pots. A seedling was placed on the floral putty with root portion in one vial and the shoot portion in the other vial after which the notch was filled with putty to provide a barrier between the vials. The seedlings were covered with A grams of silica sand. Two ml of distilled water containing 0.2] pc of ring-labelled Inc-prynachlor (A.8 pc/p mole) was pipetted into either the root or shoot portion of the experimental chamber. Two ml of distilled water was also l7 IA Figure I. Micro split-pot technique utilized to assay uptake of C- prynachlor by shoots or roots of onion and proso millet. 18 l9 placed on the untreated side of the test container. Each treatment was replicated A times, and the tests were repeated. After treatment, the micro pots were transferred to high humidity glass chambers to reduce evaporation. Plants were harvested at 18 and 96 hours. Roots of harvested plants were rinsed in distilled water for 30 seconds to remove the adhering chemical. Plants were separated into root and shoot and immediately frozen with dry ice and acetone. After drying at ASOC, weights were obtained. Samples were combusted utilizing a Nuclear Chicago Model 315] Oxidizer unit equipped with magnetic stirrers. One liter flasks were purged with oxygen and stoppered with rubber septum caps prior to combustion. Upon cooling of the flasks, lO ml of ethanol: ethanolamine (2:l V/V) was injected and they were stirred for 15 minutes. One ml of the C02 trapping solution was added to 10 ml of liquid scin- tillation fluid (Ag BBOT/liter of toluene) and counted. The scintillation fluid had 63% efficiency as determined by External Standardization on a Packard Tricarb Scintillation Spectrometer. All cpm (counts per minute) data were converted to dpm (disintegrations per minute). Transport was determined by dividing the amount of activity from on the untreated side of the seedling by the total activity in the plant. Metabolism. Metabolism was also studied by applying IL'C-prynachlor to germinating onion and proso millet seedlings. One hundred and fifty seeds were planted into styrofoam cups filled with silica sand. When the seedlings started to emerge, 10 ml of distilled water containing 1 pc of ring-labelled prynachlor was pipetted over the surface of each cup. Cups were placed in a water saturated chamber to reduce evaporation. Plants from each species were harvested l8 and 96 hours after treatment and handled 20 as described in the previous study. Dried plant portions (IOO mg) were ground in a tissue homogenizer and extracted with 6 ml of methanol. Extracts were spun in a clinical centrifuge at 7000 rpm for 20 minutes. The supernatant was then care- fully decanted and saved. The pellet was washed with 2 ml of methanol and respun. This procedure was repeated 3 times and the supernatants combined. The methanol extracts were concentrated to 0.5 ml and 100 pl applied as a streak to 250 micron silica gel H thin layer plates. The plates were developed in non-polar and polar solvent systems which were benzene:methanol (97:3 V/V) and methanol:ethyl ether: acetic acid (6:3:1), respectively. The solvent fronts were allowed to run 15 cm. Parent compound and metabolites were co-chromatographed with standards supplied by BASF Wyandotte Corporation, Parsippany, New Jersey. Standards were detected by a bromocresol green-bromophenol blue-potassium permanganate spray reagent. After detection and determination of Rf values for standards, I cm sections were scrapped into scintillation vials contain— ing lO ml of scintillation fluid and counted. Results and Discussion Tolerance. In onion, 50% inhibition was reached at A.A8 kg/ha and complete kill was observed at 6.72 kg/ha on mineral soil (Figure 2). On muck soil, a reduction in growth occurred with increasing rates, but onion did not reach 50% inhibition at rates as high as ll.2 kg/ha. Proso millet on mineral soil showed 50% inhibition at 0.28 kg/ha. Complete kill was accomplished at 0.56 kg/ha on mineral soil and l.l2 kg/ha on muck soil. 2i Figure 2. ReSponse of a.) onions and b.) proso millet growing on two soil types to preemergence application of prynachlor. 120‘ i DRY WT. OF ONION (mg) pm In. or P. mun (my) 22 uucx sou. -muenAL sou. 2.24 4.4. 0.12 a a 11.2 HAT! (kg/h.) Maven SOIL -mulnAI. SOIL 0.14 0.22 o.“ 1.12 RATE (kg/ I'll) 23 At all rates, both species growing on the muck soil were injured less than those growing on mineral soil. This is probably due to binding of the herbicide by the organic matter which reduces its activity, and greater leaching which occurs in mineral soils moving the herbicide more rapidly to the root zone. Although organic matter provided protection for both species, great differences exist between species in inherent tolerance to the herbicide. Onion can tolerate 10 to 20 times the rate which is tolerated by proso millet. Uptake. When the herbicide was made readily available to germinating seedlings, rates of uptake by roots were greater than by shoots of both species (Table 1). After 18 hours, uptake by proso millet roots exceeded onion roots. However, after 96 hours onion had absorbed a greater quantity of the herbicide. Shoots of onion more effectively absorbed prynachlor than proso millet shoots. In proso millet, a higher percentage of that absorbed by the root was transported to the shoot (Table 2). In both species, basipetal transport was limited. Although the increased rate of accumulation and greater transport of prynachlor in proso millet may contribute to susceptibility, they may not be the only factors explaining the basis for selectivity. Metabolism. In plants exposed l8 hours to IllC-prynachlor, onion roots and shoots had metabolized 96% of the radioactivity to non-toxic 2-oic-Nf(I-methyl-Z-propynyl) acetanilide (Figure 3). Only 2% of the activity recovered was identified as parent prynachlor. Proso millet roots also rapidly metabolized the herbicide, but after 18 hours, proso millet shoots still contained 2A% unaltered prynachlor. 2A Table l. Uptake of l[IO-prynachlor by roots and shoots of onion and proso millet. Total uptake Plant Portion I exposed 18 hr. 96 hr. Mean (dpm) Onion Root 908 2153 l531 Shoot 595 I838 l2l7 Meanl 752 I996 Proso millet Root lA3l l8l6 I623 Shoot 37A 1280 827 MeanI 903 15A8 1Interaction of species X portion and species X time is significant at at the 5% level. Table 2. Transport of IllC-prynachlor in onion and proso millet after root or shoot exposure. % Transported Plant Portion exposed l8 hr. 96 hr. Onion Root O.A 5.A Shoot I.l A.2 Proso Millet Root 5.8 ll.6 Shoot l.7 l.6 25 Figure 3. Distribution of l[TC-prynachlor (parent), 2-oic-N-(l-methyl- 2-propynyl) acetanilide, 2-oxo-Nf(l-methyl-Z-prgpynyl) acetanilide, and Nfisobutynyl aniline in onion and proso millet after 18 hours. 26 ONION EROOT SHOOT 02-2 h2m8(m. e:— a: - ROOT SHOOT R MILLET >h_>_.—.OXOIO>IIN e... 2: >:>:.uX2¢>=ufl PENHHH. 2. 0.0 .N All” (Aillll O m w w m m o >.=>_._.u<0_a<¢ fl CHAPTER 5 Summary and Conclusions The herbicidal activity, uptake, transport and metabolism of prynachlor by onion and proso millet may be summarized as follows: 1. Preemergence application of prynachlor at rates of A.A8 kg/ha or higher effectively controlled many annual grasses. and selected broadleaved weeds on a Houghton muck soil for 6 weeks and did not reduce yield. 2. Preemergence or postemergence applications at the 100p, flag, and one leaf stages at rates up to 6.72 kg/ha did not reduce plant weights. 3. On muck soil, onion showed less than 50% reduction in growth at 11.2 kg/ha, but complete kill was achieved at 6.72 kg/ha on mineral soil. This indicates that organic matter imparts some protection. A. Proso millet, a susceptible species, was killed at 1.12 kg/ha on muck soil and 0.56 kg/ha on mineral soil. 5. Root uptake exceeded shoot uptake in onion and proso millet seedlings when the herbicide was equally available in each zone. 6. The rate of uptake by proso millet root exceeded that of onion roots. 7. Seedlings of proso millet transported a higher percentage of Il'C-prynachlor from root to shoot than onion seedlings. A5 11. A6 Onion seedlings metabolized llIC-prynachlor more rapidly than proso millet seedlings. After treatment on muck soil, onions in the flag stage contained nearly 10 fold higher concentrations of 1tic-prynachlor in the shoot than in the root. In 3 week old plants, uptake by onion exceeded proso millet and the majority of the activity remained in the roots of both species. Both proso millet and onion at 3 weeks of age, metabolized over 90% of parent prynachlor to non-toxic 2-oic-N:(I-methyl-2- propynyl) acetanilide after 18 hours of uptake. Mature onion bulbs contained about 1 part per billion '“c- prynachlor or metabolites after a single preemergence application of A.A8 kg/ha. LIST OF REFERENCES Armstrong, T. F. 1972. Factors affecting the response of yellow nutsedge to alachlor. Ph.D. thesis, Michigan State Univ. E. Lansing. 81 p. BASF Wyandotte Corporation. 1971. Technical data sheet for basamaize (BAS 2903-H). A pp. Baird, D. D., R. F. Husted, C. L. Wilson. 1969. Pre and post- emergence herbicide activity of N-isOpropyl-Z-chloroacetanilide on echinochloa crusgalli, zea mays and glycine max in the greenhouse. Proc. Northeast. Weed Contr. Conf. 23:653-655. Binning, L. K. 1970. Postemergence weed control in muck grown onions. Research Report North Centr. Weed Contr. Conf. 27:11. Canvin, D. T. and G. Frieson. 1959. Effects of CDAA and IPC on germinating barley and peas. Weed Sci. 7:153-159. Chandler, J. M., E. Basler and P. W. Santleman. 1971. Uptake and translocation of IllC-labelled alachlor. Weed Sci. Amer. Abstr. No. 103. Chesalin, G. A. and N. V. Yurina. 196A. The effectiveness of chemical weed control in some vegetable crops. Agrobiologiija pp. 599-608. Cialone, J. E., D. A. Braden, and N. J. Smith. 1970. Studies on the use of chloroxuron and other herbicides for weed control in onions. Proc. Northeast. Weed Contr. Conf. 2A:157-l65. Dhillon, N. S. and J. LaMar Anderson. 1972. Morphological, anatomical and biochemical effects of prOpachlor on seedling growth. Weed Res. 12:182-189. Duke, W. B., F. W. Slife, and J. 3. Hanson. 1967. Studies on the mode of action of 2-chloro-NfiSOpropylacetanilide. Weed Sci. Soc. of Amer. Abstr. p. 50. Eshel, Y. 1969. Phytotoxicity, Ieachability and site of uptake of 2-chloro 2', 6'-diethyl-Nf(methoxymethyl) acetanilide. Weed Sci. l7:AA1-AA3. A7 12. 13. 20. 21. 22. 23. 2A. 25. A8 Guzman, V. L. and E. A. Wolf. 195A. Weed control in onions in the organic soil of the Florida Everglades. Weeds. 3:66-7A. Hamm, P. C. and A. J. Spezialle. 1956. Relation of herbicidal activity to the amide moiety of N-substitutedId-chloro- acetamides. J. Agr. Food Chem. A:518. Hoagland, D. R. and D. I. Arnon. I938. The water-culture method for growing plants without soil. Univ. of Calif. Agr. Exp. Sta. Circ. 3A7. Jaworski, E. G. 1969. Analysis of the mode of action of herbicidal °(-chloroacetamides. J. Agr. Food Chem. 17:165-170. Jaworski, E. G. 1956. Biochemical action of CDAA. Sci. 123-8A7. Jaworski, E. G. and C. A. Porter. 1965. Uptake and metabolism of 2-chIoro-NfisopropyI-acetanilide in plants. Amer. Chem. Soc. 1A9th Meeting. Knake, E. L. and L. M. Wax. 1968. The importance of the shoot of giant foxtail for uptake of preemergence herbicides. Weed Sci. 16:393-395. Lamoureux, G. L., L. E. Stafford, and F. S. Tanaka. 1971. Metabolism of 2-chloro-N:jsopropyl-acetani1ide in the leaves of corn, sorghum, sugarcane, and barley. J. Agr. Food Chem. 19:3A6-350. Lindle, N. 1962. The reaction of thiol compounds and chloroacetamides. Biochem. J. 82:Al8. Mann, J. E., L. S. Jordan, and B. D. Day. 1965. Factors controlling the synthesis of natural and induced lignins in Phyleum and Elodea. Plant Physiol. A0:8AO-8A7. Noll, C. J. 1965. Chemical weeding of onion grown from sets. Proc. Northeast. Weed Contr. Conf. 19:56-57. Noll, C. J. 1970. Chemical weeding of onions grown from transplants. Proc. Northeast. Weed Contr. Conf. 2A:155. Nyland, R. 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Chem. 201:719. .L_‘__ 'l¥__ i’a'ILi HIGAN STATE Ulll‘v EPSITI LIB”: 1" mm Igiiiyg Iv : .ll. 1' III. | I II 4 I’ I 31293 03174 808 if- S