ABSTRACT ALTERATIONS IN CHLOROXURON SELECTIVITY INDUCED BY CHEMICAL AND ENVIRONMENTAL FACTORS BY William D. McReynolds, Jr. Foliar applications of 3-[pf(pfchlorophenoxy)phenyll- l ,l-dimethylurea (chloroxuron) with p-chlorophenyl-Ef methyl-carbamate (PCMC) or isoprOpyl mfchlorocarbanilate (chlorprOpham) resulted in synergistic toxicity on onion (AZZium cepa L.), soybean (Glycine max (L.) Merr.), and white mustard (Brassica hirta Moench.). Their respective carriers (solvents and emulsifier) also enhanced the activity of chloroxuron indicating a direct effect of the formulation as well. Laboratory studies with the three species revealed a two to five fold enhancement in foliar penetration of l4C-chloroxuron in the presence of chlorpr0pham. Soybean leaves treated with chlorprOpham lost water more rapidly than control leaves. This indicated a possible disruption of cuticular barriers, which may explain the increase in penetration of chloroxuron. No increases in l4C-chlorprOpham uptake were observed when combined with chloroxuron. Root William D. McReynolds, Jr. treatment with both herbicides to onions did not produce the enhanced injury, indicating the interaction is a foliar phenomenon. Solutions containing the two technical grade herbicides applied to onion foliage resulted in more injury, which was not as severe as that obtained with the combina- tions of the commercial formulations. The increased uptake of chloroxuron in the presence of chlorprOpham resulted in a reduced rate of 14 C02 fixation in intact onion plants. The two herbicides applied to preparations of isolated chloroplasts did not result in a synergistic effect on the Hill reaction. Thus, the in- crease in uptake of chloroxuron in the presence of chlor- propham is possibly the main factor responsible for the synergism. Decreased rates of herbicide metabolism have been hypothesized to account for other carbamate and phenylurea interactions. In this study, no differences in metabolism of either 14C-herbicide in the combination was observed over periods of 3-5 days. The alteration of metabolism apparently is not an important factor in this interaction. Onions sprayed at different growth stages varied in tolerance to chloroxuron. Generally, the tolerance to chloroxuron increased with increasing age and size. Onions in the two to three leaf stage grown on muck soils were not injured by chloroxuron at rates up to 4 lb/A. William D. McReynolds, Jr. Exposing onions to different light intensities prior to chloroxuron treatment altered selectivity. As light intensity increased, tolerance to chloroxuron was increased. Surface extractable waxes also increased as light intensity increased, and may be a morphological factor responsible for onion tolerance to chloroxuron. ALTERATIONS IN CHLOROXURON SELECTIVITY INDUCED BY CHEMICAL AND ENVIRONMENTAL FACTORS BY 1‘“ William ijMcReynolds, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1972 (9 ACKNOWLEDGMENTS The author wishes to thank 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. W. F. Meggitt, S. K. Ries, M. J. Zabik and D. Penner for their guidance and suggestions in editing the manuscript. The technical assistance of Martha van Buskirk and Paul Love is also gratefully acknowledged. Special thanks to my wife for her understanding and immense patience during my intense course of study. ***** ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . Chloroxuron . . . . . . . . . . . . . . . . . . General Preperties and Characteristics . . . Foliar Uptake and Movement in Plants . . . . Root Uptake and Movement in Plants . . . . . Action of Chloroxuron and Other Phenylureas in Plants . . . . . . . . . . Degradation of Chloroxuron in Plants . . Chlorpropham . . . . . . . . . . . . . . . . General Characteristics and Properties . Uptake and Movement in Plants . . . . . Chlorpropham Degradation in Plants . . . . . Action of Chlorpropham and Other Carbamates in Plants . . . . . . . . . . . . . . . . Phenylurea and Carbamate Interactions . . . . . Factors Affecting Foliar Applications of Phenylureas and Carbamates . . . . . . . . . . Stage of Growth at Time of Application . . . Nature of Plant Surfaces . . . . . . . . . . Chemical Alteration of Plant Surfaces . . . METHODS AND MATERIALS . . . . . . . . . . . . . . . Toxicity of Pesticide Combinations Containing Chloroxuron . . . . . . . . . . . Greenhouse Studies . . . . . . . Weed Control . . . . . . . . . . . . . . . . Factors Affecting Chloroxuron Toxicity to Onions . . . . . . . . . . . . . . . . . . . . Stage of Growth . . . . . . . . . . . . . Light Regime Prior to Treatment . . . . . . iii Page vi viii U) bwww coqqqmb 11 11 12 l4 l6 Laboratory Studies . . . . . . . . . . . . . . . General Plant Growing Procedures . . . . . . Preparation of 14C- Labeled Solutions . . . . Radioactivity Assay . . . . . . . . . . Plant Responses to Chloroxuron and Chlorpropham Onion Growth Studies . . . . . . . . . . . . Fixation of 14C02 in Onions . . . . . . . . Effects of Chlorpropham and Chloroxuron on the Hill Reaction . . . . . . . . . . . Herbicide Influence on Water Loss from Leaves . . . . . . . . . . . . Uptake of 14C- -Chlorpropham by Leaves . . . . Uptake of 14C- -Ch1050xuron by Leaves . . . . Translocation of C— —Chloroxuron and 4C- -Chlorpr0pham in Onions . . . . . . . The Effect of Chlorpropham on Metabolism of 14C- -Chloroxuron . . . . . . . . . . . . The Effect of Chloroxuron on Metabolism of l4C-ChlorproPham . . . . . . . . . . . RESULTS AND DISCUSSION . . . . Toxicity of Pesticide Combinations Containing Chloroxuron . . . . . . Greenhouse Screening Studies . . . . . . . . weed contrOl O O O O O O I O I O O O O O O 0 Factors Affecting Chloroxuron Toxicity to Onion Stage of Growth Studies . . . . . . . . . The Effects of Light Regime on Chloroxuron TOXiCity O I O O O I O I O O O I O O O O 0 Plant Responses to Chloroxuron and ChlorprOpham Onion Growth Studies . . . . . . . . . . . . Fixation of 14C02 in Onions . . . . . . . . Effects of Chlorpropham and Chloroxuron on the Hill Reaction . . . . . . . . . . . Herbicide Influence on Water Loss From Leaves . . . . . . . . . . . . . . . Uptake of 14C- -Chlorpropham in Leaves . . . . Uptake of 14C- Labeled Chloroxuron by Leaves Movement of l4C-Chloroxuron and 14C- Chlorpropham in Onion Leaves . . . . . . . The Effect of Chlorpropham on Metabolism of 14C-Chloroxuron . . . . . . . . . . . . The Effect of Chloroxuron on Metabolism of l4C-Chlorpropham . . . . . . . . . . . iv Page 20 20 21 21 22 22 23 24 26 27 28 30 31 32 34 34 36 38 38 43 45 45 46 48 48 52 57 64 66 69 SUMMARY AND CONCLUSIONS . . . . . . . . . . . LITERATU APPENDIX A. B. RE CITED 0 O O O O O O O O O O O O O O Chloroxuron and ChlorprOpham Interaction on Soybeans . . . . . . . . . . . . Chloroxuron Interaction with ChlorprOpham and PCMC on onions . . . . . . . . . Chloroxuron Interaction with ChlorprOpham, PCMC and Their Solvent Carriers on Onions Chloroxuron Interactions with Chlorpropham, PCMC, and Their Carrier Solvents on White Mustard . . . . . . . . . . . Toxicity from Split and Combined Applications of Chloroxuron with Chlorpropham or PCMC on White Mustard Page 72 76 84 85 86 87 88 Table 10. 11. LIST OF TABLES Visual weed control ratings of chloroxuron and chloroxuron plus Chlorpropham combinations in onion field plots . . . . . Fresh weight of onions treated with chloroxuron (4 lb/A) after growing in different light intensities . . . . . . . . . Effect of light intensity on chloroform extractable waxes from onion leaves . . . . . Fresh weight of onion plants treated with chloroxuron and Chlorpropham in nutrient solution for 7 days . . . . . . . . . . . . . Fresh weight of onion shoots dipped in solutions prepared from technical herbicides after 12 days 0 O O O O I O O O O O C O O O O The fixation of 14C02 in onions 7 days after spraying with chloroxuron, Chlorpropham and the combination . . . . . . . . . . . . . . . Inhibition of the Hill reaction of the isolated spinach chloroplasts by chloroprOpham and chloroxuron . . . . . . . . Uptake of l4C-chloroxuron alone and in combination with Chlorpropham by intact soybeans after 5 days . . . . . . . . . . . . Uptake of l4C-chloroxuron by onion leaves after 5 days . . . . . . . . . . . . . . . . Uptake of l4C-chloroxuron by pre-dipped soybean leaves . . . . . . . . . . . . . . . Uptake of l4C-chloroxuron by excised soybean leaves pre-dipped in technical Chlorpropham . vi Page 37 43 44 45 46 47 49 57 60 63 63 Table 12. 13. 14. 15. 16. 17. Page The movement of l4C-chlorprOpham in onion leaf sections after 30 hr . . . . . . . . . . . . . 64 Volatilization of l4C-chlorpropham from onion leaves 0 O O O O O O O O O O I O O O O O O O O 65 The movement of l4C-chloroxuron in onion leaf sections after 48 hr . . . . . . . . . . . 66 Metabolism of l4C-chloroxuron by soybeans . . . 67 Metabolism of 14C-chloroxuron by onions . . . . 68 Summary of metabolism of l4C-chlorpropham by onions and soybeans . . . . . . . . . . . . 7O vii LIST OF FIGURES Fresh weight of onions grown on two soil types and sprayed at different stages of growth with chloroxuron . . . . . . . . . Fresh weight of onions grown on muck soil in the field and sprayed at different growth stages with chloroxuron . . . . . . Weight loss of soybean leaves dipped in chloroxuron (1/8 lb/A) and chlorprOpham (1/8 lb/A) treatments . . . . . . . . . . Uptake of l4C-chlorpropham by excised soybean leaves . . . . . . . . . . . . . . Uptake of l4C-chlorpropham by onion leaves 0 O O O O O O O O O O O O O O O O O Uptake of l4C-chloroxuron by excised soybean leaves . . . . . . . . . . . . . . Uptake of l4C-chloroxuron by intact white mustard leaves which were pre—dipped with Chlorpropham . . . . . . . . . . . . . . . viii Page 40 42 51 54 56 58 61 INTRODUCTION The substituted urea, 3-[pf(pfchlor0phenoxy) phenyl]-l ,l-dimethylurea (chloroxuron) is currently used as a postemergence herbicide in soybeans (Glycine max (L.) Merr.), carrots (Daucus carota L.), onions (Allium cepa L.) and strawberries (Fragaria virginiana L.). At recommended rates, chloroxuron selectively controls seedling broadleaved weeds including smartweed (Polygonum pensylvanicum L.), common purslane (Portulaca oleracea L.), jimsonweed (Datura stramonium L.) and the annual grasses, barnyardgrass (Echinochoa crusgalli (L.) Beauv.), large crabgrass (Digitaria sanguinalis (L.) Scop.) and lovegrass (Eragrostis cilianensis (A11.) Lutati). The selectivity of a herbicide is controlled by many factors and it may be altered by a change in just one of the factors involved. Chloroxuron selectivity has been variable with incidents of crop damage resulting (12, 29, 48). During 1969, several Michigan growers reported that recommended rates of chloroxuron caused severe injury to onions in the two to four leaf stage. The only common denominator that could be determined was that applications of chloroxuron had been made at or near the time of applica— tions of other pesticides. Several common pesticides used in Michigan onion production were applied with chloroxuron in greenhouse and field tests. Chemical interactions were observed which resulted in increased toxicity to onions. The objectives of this study were to examine the nature of the chemical interaction between chloroxuron and isopropyl mfchlorocarbanilate (ChlorprOpham), and to iden- tify some parameters that influence chloroxuron toxicity to onions. This knowledge may allow more effective use of chloroxuron and reduce the possibilities of crop damage. L I TERATURE REVIEW Chloroxuron General Properties and Characteristics. Pure chloroxuron is white, crystalline, odorless and low in water solubility (2.7 ppmw). The chemical is formulated as a 50% wettable powder and its usual carrier is water (40). Chlo- roxuron is utilized both as a preemergence or postemergence herbicide. When used preemergence, much of the chemical is strongly adsorbed to soil particles and therefore is not as effective as other substituted ureas (30). The visual symptoms of phytoxicity to chloroxuron are similar to other substituted ureas. The first signs are a loss of turgor and death of the leaf tips, followed by a chlorotic appearance, necrosis and death of other portions of the leaf (51). Foliar Uptake and Movement in Plants. Chloroxuron penetrates cuticular and epidermal layers to varying degrees in different plant species. Morningglory (Ipomoea spp. L.) a susceptible species, absorbed more l4C-chloroxuron than soybean, a tolerant Species (26). Although movement within the leaf is restricted, some movement in the xylem has been noted and the rate of acrOpetal movement is determined by factors that affect the rate of transpiration (26, 31). Surfactants enhanced the foliar activity of chloroxuron on weeds, but increased phytoxicity to crops was also noted (26, 85). Chloroxuron activity was greatly enhanced when applied to foliage in an acetone carrier (35). The foliar activity of other substituted ureas is also increased by surfactants (7, 42, 55). Root Uptake and Movement in Plants. Many reports have shown rapid root uptake of substituted ureas from nutrient and soil solutions. Uptake is followed by rapid translocation to stems and leaves via the xylem or tran- spiration stream (8, 18, 39, 58, 70, 76). In contrast, chloroxuron is readily absorbed by roots, but translocation out of the roots is restricted and varies in different plant species (31). Additional root uptake experiments have been carried out with tolerant soy- beans and susceptible morningglory. The soybeans absorbed more chloroxuron from nutrient solutions than morningglory. However, more than 90% of the chloroxuron remained in the soybean roots compared to 62% for the morningglory (26). Action of Chloroxuron and Other Phenylureas in Plants. Chloroxuron and the other substituted ureas are known to interfere with photosynthesis (10, ll, 17, 57, 84). The ISO value for chloroxuron to inhibit the Hill reaction in isolated spinach (Spinach oleracea L.) chlorOplasts was 1.75 x 10'7 M (26). A general scheme for photosynthesis, commonly describes two light reactions (I and II) involved in pro- duction of energy to reduce CO The substituted ureas are 2. thought to be involved in light system II, the oxygen evolu- tion pathway (10, 81, 82). The effect at the site of action of substituted ureas may involve inhibition of light emis- sion from a "specialized" pigment in light system II (44). Although, inhibition of photosynthetic reactions is a general phenomenon with substituted ureas, plants can also be killed in the dark (79) indicating herbicide action on more than one physiological system. Degradation of Chloroxuron in Plants. The degrada- tion of chloroxuron in plants has been studied in detail and a pathway postulated (32). The parent compound is deme- thylated and hydrolyzed to (pfchlorophenoxy)aniline. This pathway has been confirmed in studies with similar pheny- lureas (61, 62, 70). It has been postulated that the differential rate of translocation and subsequent metabolism by demethylation and hydrolysis is a major factor in selec— tivity of chloroxuron and other phenylureas. Foliar treatment of chloroxuron in two different plant species showed differential uptake, but little or no metabolism of the chemical. It was concluded that rate of uptake was more important to selectivity than degradation (26). Other mechanisms for metabolism of various substituted ureas have been reported. Monuron (3-[37 chlorOphenle-l,l-dimethylurea) formed a complex in the leaves of red kidney beans (Phaseolus vulgaris L.). Upon acid hydrolysis, this complex yielded unaltered monuron which accounted for about 20% of the total recovered (25). Formation of a monuron-flavin mononucleotide complex occurred in viva in Chlorella. Formation of this complex was light dependent and inactivated the monuron (80). Other substituted ureas may undergo a similar type of complex formation or binding to natural plant components (61, 62). In studies with other substituted ureas, attempts were made to balance the amount of uptake with amounts of metabolites recovered after extraction and chromatography. The recovery of known metabolites did not account for the amount of chemical taken up. Therefore, it is probable that additional pathways of degradation have yet to be discovered (33). The enzyme systems involved in the breakdown of the substituted ureas are generally unknown (33). One enzyme (Efdemethylase) has been isolated from cotton seedlings and is effective in demethylating monuron (27). ChlorprOpham General Characteristics and Properties. Chlorpropham is a low melting solid with a vapor pressure of 1 X 10-5 mm Hg at 20°C. It has a water solubility of 78 ppm and is commercially formulated as a emulsifiable concentrate (4 lb/gal) (40). ChlorprOpham is used as a highly selective pre- emergence herbicide that effectively controls many annual grasses and broadleaved weeds. It is selective to such crops as soybean, snap bean (Phaseolus vulgaris L.), pumpkin (Cucurbita pepo L.), onions and carrots (40). Although chlorprOpham is used most commonly as a preemergence treatment, several researchers have reported postemergence activity on young weeds (24, 71, 83). Uptake and Movement in Plants. ChlorprOpham uptake was assayed using three species of varying susceptibilities to the herbicide. Foliar application of chlorprOpham showed little or no movement from the site of application in any species. In root applications, Chlorpropham moved to all plant parts in each species in a short period of time (64). Chlorpropham has been reported to cause two types of phytoxicity from postemergence treatments. A fast action (2 days) on redroot pigweed (Amaranthus retroflexus L.) was accompanied by a reduction in both photosynthesis and chlo- rcphyll content and an increase in respiration. A slow action on pale smartweed (Polygonum lapathifolium L.) was not evident until 2 weeks after treatment (24). Postemergence activity of chlorprOpham was greatly enhanced by applying the herbicide in an iSOparaffinic oil carrier rather than water. A four to eight fold increase in penetration was observed in two different species when using the iSOparaffinic oil carrier (6). Barban (4-chloro-2—butynyl mfchlorocarbanilate),an analog of chlorprOpham,was applied as droplets to 13 differ- ent plant species. Little or no movement from the site of application was observed in any species (67). ChlorprOpham Degradation in Plants. Foliar treat- ment with chlorprOpham and other similar phenyl carbamates to 13 different species resulted in the formation of water soluble, 3-chloro-aniline containing substance (X). Up to 60% of the aniline moiety of the various carbamates was formed into X. Since some of the carbamates tested did not have any biological activity, it was concluded that forma- tion of X had nothing to do with herbicidal activity (67). Application of Chlorpropham to three species of varying susceptibility resulted in formation of various water soluble substances. These substances were hypothe- sized to be conjugates with natural plant components and not products due to hydrolysis of the chlorprOpham (64). Further identification of these water soluble,polar metab- olites indicated they were/e-glucosides of a modified chlorprOpham molecule with no cleavage of the carbamate bond. It was concluded that the metabolites resulted from modification of the 2-pr0panol ester portion of the ChlorprOpham molecule (45). Recent work with soybeans adds support to these observations (75). Hydrolysis of Chlorpropham and other carbamates apparently is not a major pathway of degradation, at least, after foliar application to plants. Several workers have failed to find the hydrolysis products that would be eXpected if this pathway was operative (41). In contrast, studies of Chlorpropham breakdown by various soil micro- organisms has shown that hydrolysis is a major degradation pathway in soils (41). Swep, methyl 3,4-dichlorocarbanilate was metabolized to a stable lignin complex in rice (41) and hydrolysis of the herbicide was limited (14). Action of Chlorpropham and Other Carbamates in Plants. The carbamates may cause mitotic poisoning and inhibition of cell division (23), inhibition of primary root growth (68, 77) and chromosomal aberrations in meristimatic regions (69). At higher concentrations than other photosynthetic inhibitors such as phenylureas, chlorprOpham inhibits the Hill reaction (56, 72, 84). Inhibition of amino acid utilization was reported in a susceptible species but not a tolerant species treated with Chlorpropham. This inhibition was postulated to be a primary cause of selectivity and perhaps is involved in the mode of action of the herbicide (52). 10 Phenylurea and Carbamate Interactions Mixtures of herbicides, and herbicides with other pesticides are common in crOp production today. When the chemicals are applied either together or consecutively, it is reasonable to expect that two or more pesticides will exist in the plant where they may interact. There are several recent reports concerning combinations of pheny- lureas and carbamates and their interactions. Simultaneous treatment with certain carbamate insecticides inhibited metabolism of monuron in cotton (Gossypium hirsutum L.) leaf discs. Monuron degradation was strongly inhibited by l-napthylmethylcarbamate (carbaryl) in this eXperiment. The data suggest that certain carbamates can prevent degradation of phenylureas in plants, thereby causing synergistic effects (78). A recently reported interaction between a carbamate insecticide, 2,2-dimethyl-a,3-dihydrobenzofurany1-7-E7 methycarbamate (carbofuran) and a herbicide, 3-(4-bromo- 3-chlor0phenyl)-l-methoxy—l—methylurea (chlorbromuron), was observed in corn (Zea mays L.) and barley (Hordeum vulgare L.). The inhibition of herbicide degradation by the insecticide was postulated to cause the synergistic response (37). Applications of a mixture of 3,4-dichlor0pro-. pionanilide (propanil) and carbamate or phosphate insec- ticides resulted in injury to rice (Oryza sativa L.) plants 11 that did not occur when the herbicide was applied alone (9). Several of these compounds inhibit the activity of the hydrolase enzyme responsible for metabolism of propanil (53). Combinations of 9,97diethyl-gf(ethylthio)methyl phOphorodithioate (phorate) with 3-(3,4-dichlor0phenyl)-1, l-dimethylurea (diuron) and monuron reduced seedling growth and yield of cotton (36). Chloroxuron and 4-(methyl-sulfonyl)-2,6-dinitro §,§fdipropylaniline (nitralin) when used in combination were reported to enhance injury to soybean (47). Combinations of other herbicides and pesticides have produced interactions on whole plants or plant parts (2, 3, 15, 16, 21, 46, 59, 60, 65, 73). The bases for some of these interactions appear to be changes in uptake and move- ment of chemicals (2, 21, 37) and/or changes in the degrada- tion pathways (9, 13, 27, 37, 53, 78). Factors Affecting Foliar Applications of Phenylureas and Carbamates Stagepof Growth at Time of Application. Generally, as stage of growth increases, the tolerance of a plant species to a herbicide increases. Feeny (26) reported that chloroxuron toxicity to morningglory decreased as stage of growth increased, especially past the fifth leaf stage. The uptake of chloroxuron was less in older than in younger plants. Soybeans treated at several stages of growth responded differently after chloroxuron application. Two 12 week old soybeans were more susceptible than 1 or 3 week old soybeans. Differential uptake of chloroxuron did not account for the difference. In another study, the phytotoxicity of chloroxuron generally decreased as the stage of growth of five weed species at treatment time increased. Soybeans in the early unifoliate or first trifoliate stages were most sensitive to chloroxuron injury in this test (12). McWhorter (55) reported that diuron, another sub- stituted urea was also less phytotoxic to weeds as stage of growth increased. Similar results have been reported for carbamate herbicides. Wild oats (Avena fatua L.) became more tolerant to is0propy1 carbanilate (propham) as growth increased (28). The postemergence activity of chlorprOpham decreased as stage of growth of three weed species increased (24). A general review of the influence of plant age on susceptibility to herbicides is discussed in a report by Aberg (1). Nature of Plant Surfaces. The nature of plant surfaces is important in relation to the effectiveness of foliar-applied herbicides. Penetration of the herbicide through plant surfaces is very complex and many of the phenomena involved such as retention, absorption, accumu- lation and persistence of herbicides are not well understood. Plant surfaces are known to be highly complex and variable, 13 having protuberances of many forms, wax crystals and rodlets of numerous types, and cutin layers of varying thicknesses and composition which include wax platelets of a crystalline nature and pectic and cellulose components (19). The plant cuticle is continuous and not cellular. The penetration of herbicides depends on such factors as cuticle thickness, the nature of wax deposits, hydration of the cutin and the presence of ectodesmata. Of these, the amount and orientation of wax may be the chief factor in- hibiting penetration of non-polar compounds. Usually younger leaves will absorb more herbicide than mature leaves. Consequently, factors that influence cuticle thickness may influence penetration of herbicides. Within a plant species, environmental factors such as humidity, light, temperature, insect damage, wind abrasion and many others become involved in herbicide penetration through leaf surfaces (19, 20, 38). Penetration of both chloroxuron and Chlorpropham into leaf tissue has been demonstrated (24, 26). Surfact- ants or organic solvents increase the foliar activity of both chemicals (6, 35). Environmental factors which are documented to enhance toxicity after application of these chemicals are high humidity and temperature (12, 64). Other than these reports, the literature contains little regarding modification of plant surfaces and factors affecting pene- tration of these two herbicides. 14 Chemical Alteration of Plant Surfaces. The wide use of surfactants to increase effectiveness of postemergence herbicides is well documented but not fully understood. Various hypotheses on their effects are found in the lit- erature (19, 20). Another related area that is not well documented is the effect of various formulated pesticides on the foliar activity of the respective component chemicals. Changes in physical and chemical properties of the combina- tions may cause alterations in activity of the respective components. A split application of crop oil and 2-chloro-4- (ethylamino)-6-(isoprOpylamino)-s-triazine (atrazine) gave better results than when the two were together. The crOp oil was postulated to modify the plant surface to enhance penetration (5). Chlorpropham uptake was similarly in- creased in crop oil emulsions and the hypothesis of a modified cuticle was proposed (6). When acetone was used as a solvent carrier for chloroxuron, activity was increased. It is possible the acetone had some effect on the plant's surface barriers thereby increasing chloroxuron activity (35). Chloroxuron activity on weeds and crops was also enhanced by use of a crop oil. PCMC (pfchlorophenyl—Efmethylcarbamate) is an adjuvant applied with Chlorpropham to increase soil resid- ual life by inhibiting microbial breakdown (50). Whether 15 or not this combination affects postemergence activity by extending residual activity on or within leaves has not been determined. Chlorpropham pre-applied to the roots of pea (Pisum sativum L.) plants provided protection from foliar Sprays of prOpanil. In contrast, Sf(2,3—dichloroallyl)-diiSOpropyl- thiolcarbamate (diallate) pre-treatment altered deposition of surface waxes and enhanced prOpanil injury (74). Root treatment with other herbicides also alter plant surfaces. TCA (trichloroacetic acid» dalapon (2,2- dichloroprOpionic acid) and dinoseb(2-sec-butyl-4,6-dini- trOphenol) were observed by several workers to reduce leaf waxes and alter susceptibility to other herbicides (22, 63). EPTC (S-ethyldiprOpylthiocarbamate) inhibited the deposition of external foliage wax on cabbage (BraSSica oleracea L.). Dinoseb toxicity was increased after modifi- cation of the plant's surface wax by EPTC (34). It is apparent that the use of various pesticides simultaneously or sequentially on crops will have to be examined for possible modifications in activities of each pesticide. The activities of foliar applied herbicides may possibly be altered to improve crop selectivity and/or weed susceptibility, by modifying various factors that influence herbicidal action. METHODS AND MATERIALS Toxicity of Pesticide Combinations Containing Chloroxuron Greenhouse Studies. Several experiments were conducted to determine the toxicity of chloroxuron in combination with PCMC and Chlorpropham applied at various rates. These chemicals had been reported to interact with chloroxuron (54, 66). Onions (var. Trapp's Yellow Globe), soybeans (var. Amsoy), and white mustard (Brassica hirta Moench.) were used as test Species. Seeds of each species were germinated in non-sterilized Houghton muck soil in styrofoam flats (12 by 16 cm) and thinned to an equal num— ber before treatment. All plants were grown in the green— house at 25 t5°C with a daylength of 16 hr. Supplemental fluorescent light was supplied in addition to natural sunlight. At the time of pesticide applications, onions and mustard were in the three to four leaf stage and soybeans in the one to two trifoliate stage. All chemicals were applied as tank mixes in quart bottles. A movable belt sprayer, utilizing CO2 pressure at 30 psi and delivering a water volume equivalent to 100 gpa, was used for pesticide applications. 16 17 The chemical treatments used and rates of application for each species are listed in Appendix A-E. The carrier solvent system and emulsifiers for Chlorpro- pham and PCMC were also obtained from the Pittsburgh Paint and Glass Company and tested at equivalent rates of the commercial formulation without the active ingredient. One split application of the chemical combinations was compared to combinations applied together. The time period between treatments was 4 days. Visual injury ratings were obtained 7 days after treatment and fresh and dry weights of shoots were measured after excising them at ground level either 7 or 14 days after treatment. All tests were repeated and treatments were replicated four to six times per test. Weed Control. Combinations of chloroxuron and chlorprOpham were evaluated for postemergence activity on a Miami loam soil at the Horticulture Research Center. Plots were 12 X 15 ft with three replications arranged in a randomized block design. Emerged weeds, less than 6 inches high, were present at the time of application. The main weed Species present in the plots were redroot pigweed, barnyardgrass, sheperdspurse (Capsella bursa-pastoris (L.) Medic.) and common purslane. Plots were sprayed on June 30 with chloroxuron at 2 and 4 lb/A, with and without chlor- prOpham at 2 lb/A and appropriate checks. Herbicides were applied in tank mixes with a small plot CO pressure sprayer 2 18 at 30 psi in a volume equivalent to 36 gpa. Ten days later, visual injury ratings were obtained from each plot. Factors Affecting Chloroxuron Toxicity to Onions Stage of Growth. Field and greenhouse tests were conducted to evaluate the relationship of stage of growth with susceptibility to chloroxuron treatment. The field test was conducted at the Muck Experimental Station in the summer of 1971. Onions were seeded in rows 1 ft apart on June 29th and at weekly intervals for 5 weeks. A split block design was employed with planting dates split for chemical treatments. Plot size was 3 X 6 ft and each treatment was replicated three times. The test area had been treated with 2-chloro-Nf(l-methyl-Zépropynl)acetanil- ide (prynachlor) at 3 lb/A before onion seeding for pre- emergence weed control. Irrigation water was applied twice a week from the time of the first seeding. Herbicide treatments were applied on July 29th, when onions were in the following stages of growth; two to three leaf, one to two leaf, flag, 100p and before emergence. The shoots from each planting were harvested after a growth period of 6 weeks from time of seeding. Fresh and dry weights, plant counts, and visual injury were recorded for each treatment. Two greenhouse eXperiments were conducted in a similar manner, but included two soil types. Fifty onion 19 seeds were planted in styrofoam flats filled with either Houghton muck soil or a greenhouse mix (sand:peatzloam, 1:1:1) at weekly intervals for 5 weeks. The chemical treatments were applied at similar stages of development as reported for the field tests. The onions were sprayed and maintained under greenhouse conditions previously described. The treatments were arranged in a randomized block design and each treatment had five replications. Light Regime Prior to Treatment. Onions were seeded in styrofoam flats filled with Houghton muck soil, thinned to 15 per flat and allowed to develop to the two to three leaf stage. At this time, plants were transferred to a growth chamber in which the shelves were adjusted to vary light intensity. Three ranges of light intensity were used; low (960-2,690 lux), medium (6,100-15,000 lux), and high (ll,000-21,500 lux). Light intensity was measured in the leaf canopy region. Flats were re-randomized daily to lessen the variability within each light intensity range. After 8 days of conditioning under the three light regimes, plants were sprayed with chloroxuron at 4 1b/A. After Spraying, the treated and check plants were put in the greenhouse. Two weeks after treatment, the shoots were harvested and fresh weights determined. Another group of plants grown in the three light regimes was used to measure chloroform extractable waxes 20 from the leaves. Plants were cut off at soil level and dipped (10 seconds) in two successive washes of chloroform (50 ml). The washes were combined and poured into pre- weighed flasks and dried and reweighed. The extracted substances in the flasks were assumed to be chloroform extractable surface waxes and the data were expressed as ug wax/leaf. The experiment was repeated to verify toxicity results, but no wax measurements were obtained in the second experiment. Laboratory78tudies General Plant Growing Procedures. Seeds of onions, soybeans and white mustard were germinated in a flat of vermiculite in the greenhouse. After reaching a suitable transplanting stage (first leaf for onion and mustard and cotyledon stage for soybean), plants were transferred to 180 ml plastic containers wrapped in aluminum foil. Half strength Hoaglands solution (43), (150 ml per cup) was used as a growth media and changed every third day during the course of each experiment. A circular sponge which fitted the top of the container was used as a support for the plants. After transplanting, the plants were kept in the laboratory for 24 hr to prevent leaf desiccation before moving them into a growth chamber or greenhouse. The growth chamber was maintained at 20°C with a 16 hr daylength. 21 Light intensity was approximately 22,500 lux at the leaf canOpy. The greenhouse was maintained at 20 t5°C with a daylength of 16 hr supplemented by fluorescent light. Preparation of l4C-Labeled Solutions. The radio- active stock solutions used in uptake and metabolism were prepared in the following manner. Chloroxuron (Tenoran- 50% WP) and Chlorpropham (Chloro IPC-4 lb/gal) were mixed with water to produce suspensions equivalent to 1 1b/A in a delivery system of 100 gpa. Aliquots of these suspensions were pipetted into scintillation vials. 14C-chloroxuron (carbonyl labeled-1.18 mC/mM) or l4C-chlorprOpham (ring labeled-1.29 mC/mM) in absolute ethanol was added to the suspensions and stored in a refrigerator until use. Aliquots of the 14 C-spiked commercial formulations were applied as droplets on leaves using a Hamilton micro- syringe. The radioactivity of each stock was assayed to quantify amounts of 14C added to each leaf during exper- imentation. Radioactivity Assay, Total radioactivity from the dried plant samples was determined by combustion in a Model 3151 Nuclear Chicago Combustion Apparatus equipped with a burning lamp and stirring equipment. The dried leaf samples or representative 50 mg fractions were placed inside cello- phane bags or cigarette papers and inserted into a platinum wire basket. The flask was flushed with oxygen after which 21 Light intensity was approximately 22,500 lux at the leaf canOpy. The greenhouse was maintained at 20:tS°C with a daylength of 16 hr supplemented by fluorescent light. Preparation of l4C—Labeled Solutions. The radio- active stock solutions used in uptake and metabolism were prepared in the following manner. Chloroxuron (Tenoran- 50% WP) and Chlorpropham (Chloro IPC—4 lb/gal) were mixed with water to produce suspensions equivalent to l lb/A in a delivery system of 100 gpa. Aliquots of these suspensions were pipetted into scintillation vials. 14C-chloroxuron l4C-chlorprOpham (ring (carbonyl labeled-1.18 mC/mM) or labeled-1.29 mC/mM) in absolute ethanol was added to the suspensions and stored in a refrigerator until use. Aliquots of the 14C-spiked commercial formulations were applied as droplets on leaves using a Hamilton micro- syringe. The radioactivity of each stock was assayed to quantify amounts of 14 C added to each leaf during eXper- imentation. Radioactivity Assay. Total radioactivity from the dried plant samples was determined by combustion in a Model 3151 Nuclear Chicago Combustion Apparatus equipped with a burning lamp and stirring equipment. The dried leaf samples or representative 50 mg fractions were placed inside cello- phane bags or cigarette papers and inserted into a platinum wire basket. The flask was flushed with oxygen after which 22 the sample was completely oxidized. After a 5 minute cooling period, 15 m1 of trapping solution (ethanol: ethanolamine 2:1 v/v) was added with a syringe pushed through the rubber septum cap. The solution was stirred for 10 minutes, and a 1.5 or 5.0 m1 aliquot was removed and placed in a scintillation vial containing 10 m1 of scintil- lation solution. The solution was prepared by dissolving 4 g of 2,5-bis-[2-(S-tert-butylbenzoxazolyl)]-thiophene (BBOT) in a liter of toluene and 400 m1 of Triton X-100. Quantitative determination of radioactivity was obtained with a Packard Tricarb Scintillation Spectrometer equipped with external standardization. All data were subjected to analysis of variance and where applicable, mean differences were evaluated with Tukey's HSD Test. Plant Responses to Chloroxuron and.Chlorpr0pham Onion Growth Studies. Onions (two to three leaf stage) grown in nutrient culture were used as test species. Technical chloroxuron and chlorprOpham were added to the 6 M and 9.3 x 10'6 root media at 6.8 X 10- M, respectively. After 6 days whole plants were harvested and fresh and dry weights determined. This experiment was repeated in a similar manner. In another experiment, leaves were treated with solutions of chemicals containing 1 X 10'.6 M chloroxuron 23 and 1.4 X 10-6bdchlorpropham. Onions at the third leaf stage were dipped in these solutions and allowed to air dry. After drying, the plants were transferred to the greenhouse. There were five replications containing two plants each for every treatment. After 12 days, whole plants were harvested and fresh and dry weights recorded. The experiment was repeated using onions grown to the fourth leaf stage to verify earlier results. Fixation of 14C02 in Onions. Onions grown to the fourth leaf stage in nutrient culture were sprayed with chloroxuron at 2 1b/A, Chlorpropham at 2 lb/A and the com- bination at 2 and 2 lb/A. After spraying, the plants were returned to a growth chamber at 22°C under continuous light. Seven days after herbicide treatments, the plants were exposed to 14CO2 to measure relative photosynthetic activity. A clear plastic box (18 X 18 X 36 in) was used as the photosynthesis chamber. After the plants were placed in the chamber, the end was sealed to prevent loss of radio- active COZ' Bal4CO with a specific activity of l mC/39.5 mg 3 was utilized as the source of 14C02. A mg of Bal4CO3 was placed in a stopped flask with two vent tubes. The flask was placed adjacent to the chamber and one vent tube was inserted through a small slit in the plastic. The chamber was covered with a black plastic cloth for a 5 minute equilibrium period. During this period, 4 ml of 50% lactic acid was added to the flask through the other vent tube. 24 The resulting 14CO2 was driven from the flask into the chamber by gentle heat. A small fan was blowing inside the chamber to mix the influx of 14CO2 into the existing atmo- Sphere. At the end of the equilibrium period, the black cloth was removed and the plants were allowed to fix 14CO2 for a 25 minute period. The plants were exposed to fluo- rescent light in addition to natural sunlight during the fixation period. At the end of the fixation period, the plants were immediately removed from the chamber and excised at the meristem region and quick frozen in dry ice and acetone. Afterwards, the samples were 1y0philized and pulverized into fine particles. A homogeneous 50 mg sample of the pulverized leaves was assayed for radioactivity. This experiment was repeated a second time using onions sprayed with chlorprOpham at l lb/A, chloroxuron at 3 lb/A and the combination. Fixation, freezing, drying, sampling and assay techniques were identical to the first experiment. Effects of ChlorprOpham and Chloroxuron on the Hill Reaction. The methods used in this study were adopted from the techniques of Moreland et al. (56, 57) to measure herbi- cide effects on the Hill reaction in isolated chloroplasts. Ferricyanide is used as an artificial electron acceptor in‘ the electron transport process of isolated chloroplasts. The reduction of ferricyanide is measured spectrophotomet- rically and herbicides that prevent reduction are considered to be photosynthetic inhibitors. 25 Spinach purchased from a local grocery was used as the source of chloroplasts. Approximately 50 g of spinach leaves were blended in 100 ml of 0.5 M sucrose. The homogenate was filtered through four layers of cheese cloth and the filtrate was centrifuged for 5 minutes at 1000 X g. The pellet was saved, resuspended in 10 ml of 0.5 M sucrose and recentrifuged. This washing process was repeated sev— eral times, after which, the pellet was resuspended in 10 ml of 0.5 M sucrose and placed in an ice bath until used in the reaction mixture. The chlorOphyll content of the suspended chloro- plasts was determined by the method of Arnon (4). The original suspension was diluted to approximately 360 pg chlor0phyll/ml of suSpension. The 10 m1 reaction mixture had the following 4 M ferricyanide, l X 10-2 2 composition: 5 X 10- M potassium chloride, 0.17 M sucrose, 5 X 10- M potassium phosphate buffer (pH 6.8), 0.2 ml of herbicide and 1.0 m1 of the chlorOplast suspension. The herbicides were added to the reaction mixtures in 0.2 ml ethanol to give final concentrations of l X 10-4 M to l X 10-7 M. The amount of ethanol added to the reaction mixture resulted in 2% by volume and had no measurable effect on chlorOplast activity. The control tubes contained ethanol in an equal concentration. 26 The reduction of ferricyanide was measured using a Beckman DBG Grating SpectrOphotometer. The ferricyanide had a maximum absorbance at 420 nm. Light for the reaction was supplied by a flood lamp placed in the growth chamber. The test tube containing the reaction mixture was placed in a beaker of water to prevent excessive heat from the lamp. The reaction mixture was allowed to stand in the water beaker for a 2 minute equi- librium period in the dark after which absorbance readings were taken. Chloroplasts were added and the lamp turned on for a 6 minute reaction period. Absorbance was again mea- sured and the differences were expressed as umoles ferri- cyanide reduced/g chlorophyll/hr. All treatments were repeated twice and the control reaction was repeatedly checked to make sure the chloroplasts were active. Herbicide Influence on Water Loss from Leaves. The objective of this study was to quantitate by water loss possible alterations of the cuticular barrier induced by herbicides. Soybean plants in the first trifoliate stage were used as the test species. The leaves were dipped in sus- pensions of formulated Chlorpropham, chloroxuron and the combination at 1/8 lb/A rates (assuming 100 gpa). After the leaves had dried, leaflets of the first trifoliate leaf were excised and weighed immediately. After weighing, the leaves were placed on paper towels and moved under fluorescent 27 lights on the lab bench at 25°C. At hourly intervals after the initial weighing, fresh weights were again recorded. Weight losses were eXpressed as a percent of the initial weighing. The weight losses were presumed to be primarily water losses. The excised end of the leaf petiole had been dipped in lanolin to prevent water loss at the cut. A second study was conducted in essentially the same manner to verify technique and results. Uptake of l4C-ChlorprOpham by Leaves. The primary leaf of the first trifoliate of greenhouse grown soybeans was excised and placed in a petri dish in distilled H20. Twenty pl (0.004 uC) droplets of spiked suspensions equiv- alent to l lb/A Chlorpropham and chloroxuron were placed on the center of each leaf over the main vein. After droplet application, leaves were allowed to air dry in the lab (about 1 hr) and were then transferred to a growth chamber under continuous light at a temperature of 25°C. Uptake was assayed at 12, 24, and 48 hr. At harvest, each leaf was washed in three successive washes of 80% ethanol (10 ml). Immediately, after washing, the leaves were frozen in dry ice and acetone and lyophilized. The dried samples were stored until use for radioactive assay of leaf uptake. Another similar eXperiment was conducted with soy- beans except that the harvest times were 2, 4, and 8 hr and the drOplets contained 0.01 uC/20 ul. 28 Similar experiments with some modifications were repeated with white mustard. The third leaf was used as the site for droplet applications. The treatment area was not washed at harvest, but was excised with a number 8 cork borer and discarded. The rest of the leaf was used for radioactive assay. Other uptake tests were carried out on onions grown in nutrient culture to the early fourth leaf stage. A lanolin ring was applied 100 mm from the tip of the third leaf with a syringe. The leaves were placed at an approx- imate 49° angle and a 10 ul droplet (0.022 uC) was placed next to the lanolin ring which prevented runoff. After drying, the plants were placed in a growth chamber under continuous light at 20°C. Harvest times were 12, 24, and 48 hr. Leaf tips were cut off 10 mm above the lanolin ring and frozen in dry ice and acetone and lyophilized. After drying, the tips were stored in an oven at 40°C until radio- active assay. The rest of the leaf including the lanolin ring area was discarded. Uptake of 14C-Chloroxuron by Leaves. Uptake of 14 . . . C-chloroxuron was measured 1n exc1sed and 1ntact soybean leaves. The procedures were essentially the same as the experiment described for l4C-chlorpropham uptake. The specific activity of the droplets was 0.005 uC/20 ul. Harvest times were 2, 4, and 8 hr after the droplets had dried. 29 A different method of studying uptake was employed with white mustard and onions. White mustard plants grown in nutrient culture to the fourth leaf stage were used. Half of the plants were randomly selected and dipped in an emulsion of formulated Chlorpropham at a rate equivalent to 0.5 lb/A in a 100 gpa delivery system. After dipping, the leaves were allowed to air dry and fixed in a horizontal position. 14C-chloroxuron was applied as a 20 ul droplet (0.01 uC) to the central vein region of the third leaf. 14C—herbicide but were not dipped in Check plants received chlorprOpham. After the drOplets had dried, the plants were placed in a growth chamber at 22°C under continuous light and harvested at 12, 24, and 48 hr by excising the treated leaves. The wash procedure, freezing and lypholizing were identical to eXperiments already described. A similar experiment was conducted on onions grown in nutrient culture to the third leaf stage. Half of the plants were dipped in emulsified chlorprOpham (equivalent to l lb/A in 100 gpa). After drying, l4C-chloroxuron drOplets were suspended at the juncture where the third leaf had emerged from the leaf sheath of the second leaf. The 14C- chloroxuron was added in two 10 ul drOplets applied within a half hr of each other. A droplet had a specific activity of 0.005 uC/lO ul. The plants were transferred to a growth chamber at 22°C under continuous lighting for a 5 day period. The onions were harvested by excising the plant at the 3O meristem region with a razor blade. Leaf washing, freezing and lyophilizing procedures have already been described. The dried plants were stored and later assayed for radioactivity. Two additional uptake experiments were conducted using soybeans which were pre-dipped. In the first experi- ment, intact soybeans were dipped in Chlorpropham emulsions equivalent to 1/8 lb/A and the Chlorpropham carrier solvent system at an equivalent rate. After the plants had dried, the primary leaves of the first trifoliate leaf were excised and herbicide applied. Four 10 ul droplets were placed on each leaf. Each droplet had a specific activity of 0.0025 uC/lO ul. One harvest was carried out after a 48 hr uptake period. In the other eXperiment, excised soybean leaves were floated (adaxial surface to the solution) on technical chlor- prOpham at 3.7 X 10.4 M for a 3 hr period. The leaves were turned over and transferred to moist petri dishes where the leaf surface was allowed to dry. Afterwards, 14C-chloroxuron drOplets were placed over the central vein region. Two 20 ul droplets (0.01 uC) were added per leaf and radioactiv- ity was assayed after a 4 hr uptake period. Translocation of 14C-Chloroxuron and 14C-Chlorpropham in Onions. In the first experiment, onions grown in nutrient culture to the fourth Ieaf stage were used as the test species. A lanolin ring was applied 150 mm from the tip 31 of the third leaf. Two 10 ul (0.01 uC) droplets containing l4C-chlorprOpham alone and in combination with chloroxuron were placed on the ring and allowed to dry. After treatment, the plants were placed in a growth chamber at 22°C under continuous light. After a 30 hr uptake period, the third leaf was excised and divided into three sections 40 mm long beginning at the leaf tip. The application area was also harvested by cutting 10 mm below it and the remainder of the leaf was saved to check for basipetal movement. After the sectioning process, the leaf parts were quick frozen, lyophilized and saved for radioactive assay. l4C-chloroxuron was added in two 10 ul drOplets (0.01 uC) to onions pre-dipped in chlorprOpham and non- dipped checks. After 48 hr, leaves were sectioned into two 70 mm sections starting at the tip, a 20 mm section at the site of application, and the remaining leaf portion. The sections were quick frozen, lypholized and saved for radio- active assay. The Effect of Chlorpropham on Metabolism of 14C- Chloroxuron. The metabolism of l4C-chloroxuron was studied in excised and intact soybean leaves and intact onion leaves. Herbicide droplets were applied and plants were handled as previously described in the uptake experiments. Harvests were conducted at l, 2 and 3 days with excised soybean leaves, 3 days for intact soybean leaves, and 5 days for onion leaves. Leaf washing and lyophilizing methods were 32 similar to previous experiments. The dried leaf samples were placed in 25 ml test tubes and extracted four times with 2 ml of boiling ethanol at 80°C for a 5 minute period. The extracts were decanted and combined. The residue was saved and dried for detection of non-extracted radioactivity by combustion technique. The combined extracts were fil- tered through Whatman No. 1 filter paper and evaporated by a gentle stream of filtered air to a volume of approximately 1 ml. A 100 ul aliquot of the concentrated extract was spotted on glass plates coated with silica gel H with a thickness of 250 microns. Plates were developed in the following solvent systems; ethyl acetate:chloroform (1:1 v/v), benzene:acetone (2:1 v/v) and benzene:acetone (1:1 V/V) . Plates were developed to a distance of 15 cm. The plates were divided into 1 cm sections and scraped into vials containing 10 m1 of scintillation solution. 14C- standards were run in all systems to use as reference points for the parent compound in the extracts. The Effect of Chloroxuron on Metabolism of 14C- Chlorpropham. l4C-labeled Chlorpropham was applied to excised soybean leaves and intact onion leaves using the methods previously described in the uptake experiments. Harvest times were 2 days for soybeans and 3 days for onions. 33 Dried plant material was ground with a mortar and pestle in 5 ml of 80% ethanol and filtered through Whatman No. 1 filter paper. The residue was rinsed repeatedly with 80% ethanol and saved for determination of non-extracted radioactivity by the combustion technique. The filtrate was evaporated to dryness in a gentle stream of filtered air. The evaporated extracts were then partitioned several times in equal volumes of chloroform and water. The two fractions were then evaporated to about 1 ml and a 100 ul aliquot was spotted on silica gel H plates for assay. The solvent system used for both experiments was acetone: benzene:water (85:25:10 v/v/v). RESULTS AND DISCUSS ION Toxicity of Pesticide Combinations Containing Chloroxuron Greenhouse Screening Studies. Initial field screening trials conducted on onions resulted in inter- actions that reduced fresh weights and yield (54, 66). Two compounds that enhanced toxicity with chloroxuron were selected for further study in several greenhouse tests. Chlorpropham and PCMC, an experimental adjuvant which inhibits its microbial degradation, may soon be used together in commercial onion and soybean production. Since chloroxuron is also utilized on these crops, the interac- tions observed may be of a practical nature. The greenhouse tests involved three species; onions (tolerant), soybeans (tolerant) and white mustard (suscep- tible). Interactions were observed, using the combinations at varying rates, in all species (Appendices A—E). Data from these tests are reported as visual rating means (1 no damage, 2-3 slight, 4-6 moderate, 7-8 severe, 9 dead), and fresh weight means expressed as a percent of control. The percent of control values were used to interpret the inter- actions by the method of Colby (15). The statistical eval- uation of Colby's method was taken from the thesis of Hamill 34 35 (37). No dry weight values are reported since, in this type of experimentation, changes in dry matter production are small because of the short time period between spraying and harvest. Soybean was the most tolerant species to chloroxuron, but when combined with Chlorpropham, higher rates resulted in interaction effects (Appendix A). The respective chlor— prOpham carrier (solvent + emulsifier minus active ingredi- ent) did not alter the toxicity of chloroxuron appreciably. On onions, both PCMC and chlorprOpham interacted with chloroxuron (Appendix B). The respective carriers in combination with chloroxuron also caused fresh weight reductions compared to chloroxuron alone (Appendix C). The PCMC carrier enhanced chloroxuron toxicity as much as the active ingredient, indicating that this interaction is caused by the solvent and emulsifier in the formulation. The Chlorpropham carrier also enhanced chloroxuron activity, but when the active ingredient was included, toxicity was considerably increased. These results suggest that there is not only an interaction between herbicides, and between other components of the formulation as well. Additional evidence for the interactions of herbi- cides and carriers was obtained in a test on susceptible white mustard (Appendix D). Combinations of both the com- mercial formulations or carriers with chloroxuron caused interactions that paralleled the results obtained in the onion tests. 36 The relative importance of applying the chemicals in close sequence to one another was tested on white mustard (Appendix E). As a check, the same treatments were applied in tank mixes to a similar set of plants. Chlorpropham applied 4 days prior to or with chloroxuron caused increased toxicity. In contrast, PCMC interacted with chloroxuron only when the two were applied together. These results demonstrate the importance of both toxicity of sequential applications as well as combination tank mixes of herbicides. Weed Control. The advantages of combinations of herbicides in chemical weed control are well known (15). Although the combination of Chlorpropham and chloroxuron is injurious to crOps such as onions and soybeans, it may be useful in weed control. Visual injury ratings from a field weed control trial indicated that the two herbicides com- bined were more toxic to broadleaved weeds and grasses than the chemicals applied alone (Table 1). Although, the rat- ings were significantly different, complete control of large weeds and grasses was not obtained. It may be possible to achieve complete control by applying the treatments to smaller weeds. Some of the weeds in this test were up to 4 in high at time of treatment. 37 Table 1. Visual weed control ratings of chloroxuron and chloroxuron plus Chlorpropham combinations in onion field plots, Ratingsa Broadleaved Herbicide Rate Weeds Grasses (lb/A) Check -- 1.0 a 1.0 a Chloroxuron 2 3.5 ab 1.8 ab Chloroxuron 4 5.0 bc 2.0 ab Chloroxuron plus Chlorpropham 2 + 2 6.7 cd 2.7 bc Chloroxuron plus Chlorpropham 4 + 2 7.7 d 3.5 c Chlorpropham 2 3.7 b 2.0 ab aMeans followed by unlike letters are significantly different at the 5% level. 38 Factors Affecting Chloroxuron Toxicity to Onion Stage of Growth Studies. In the greenhouse study, onions were planted at weekly intervals for a 5 week period on two soil types, Houghton muck and sand:loam:peat mix. At the time of chloroxuron application, the stage of onion development was similar for both soil types. However, onions grown on muck soil were larger and more vigorous than those grown on mineral soil. The fresh weights 42 days after seeding were expressed as percent of controls for each stage of growth. In general, the results indicated that older plants (two leaves) were the most tolerant to chlo- roxuron in both soil types and as the rate of herbicide increased, the tolerance decreased (Figure 1). In addition, onions grown on muck soils were larger and more tolerant than onions grown on mineral soils, especially in the earlier stages of develOpment. Although preemergence activity was noted on both soil types, it was appreciably greater on the mineral soil. The stand count and dry weight data followed similar trends and are not presented. A corresponding field trial conducted on mudk soil also demonstrated that emerged onions became more tolerant as they got larger (Figure 2). At the two leaf stage, there was no significant reduction in fresh weight when 4 lb/A was applied. Chloroxuron again showed little preemergence activity on muck soil. These results agree with reports 39 Figure 1. Fresh weight of onions grown on two soil types and sprayed at different stages of growth with chloroxuron. F value for the interaction of chemicals X dates X soils is significant at the 5% level. 4O .<\n.: 2083x080azu 80:2 ._<¢wz.1 a . o u l O“ I 0' I. 00 I on 8— Lnaou >3 GGUMSHm>o m>fluwopm muw3 mosam> pmuoomxmm av av mm oloa + Ioa cousxouoHno v mafia Emcmoumuoaflu mm om mm onoa + 10H sousxouoHno m moam EmnmoumuoHco mm 0H mm on ooa mo bloH cousxouoacu mm nv vloa om ow mloa OOH mo mica EmnmoumuoHru ooa mo II Houucou Ammaosnv ASL mcmuoomxm pm>ummno H£\Hawnmouoano mfi\pmosoom cofiumuucmocoo mpfioflnuom opflcmwofiuumm Houucoo m0 w .cousxouoHno cam EmsmoumuoHno >Q mummHmouoHco nomcfimm woumHomfi mnu mo Goduomwu Hafiz can no cowuwnflnsH .5 magma Figure 3. 50 Weight loss of soybean leaves dipped in chloroxuron (1/8 lb/A) and Chlorpropham (1/8 lb/A) treatments. FRESH WEIGHT (% OF CONTROL) 51 o connm y =95; - 6.1,. A cmonnorum y = 91.1 -10.1x o cmonnormm + cmonoxunou yam—9.4x 40 ° 1 2 3 4 s rm: ma ) 52 Uptake of l4C-Chlorprppham in Leaves. There was no difference in uptake of l4C-chlorpropham alone or in combi- nation with chloroxuron in excised soybean leaves after 12, 24, and 48 hr (Figure 4). A similar experiment was con- ducted with shorter harvest times. The uptake with both treatments was again similar at 2 and 4 hr, but at the 8 hr harvest the chloroxuron plus l4C-chlorpropham combination was significantly (5% level) lower than l4C-chlorpropham alone. It appears that uptake of l4C-chlorpropham is rapid in soybean leaves and probably was completed by the time the treatment drOplet had dried, under the conditions of these experiments. The synergism observed between chloroxuron and Chlorpropham cannot be explained by an increase in uptake of Chlorpropham in soybeans. The uptake experiment on intact mustard leaves resulted in counts that were low. The technique of excising the treatment area was faulty, since 14C-chlorpropham was not readily translocated from the site of application and was lost when the treatment area was excised and discarded. The uptake study on onion leaves again resulted in low recoveries of 14C-labeled Chlorpropham. There was no difference in uptake due to treatment, but there was a sig- nificant increase in uptake with time (Figure 5). In this test l4C-chlorpropham that was taken up and translocated at least 10 mm above the lanolin ring area was also assayed. 53 Figure 4. Uptake of l4C-chlorpropham by excised soybean leaves. 54 38:. m5..— uv on 9N ZO-SXOsn: 1U d 2(1508n80a203 2(2308m80azuno «— 801 x avaI/waa 53 Figure 4. Uptake of l4C-chlorpropham by excised soybean leaves. 54 38...: m2: at on QN 2083x080; :U a S\>\> oaummummv o muosmumcmnumcouoom mm3 Emumwm ucw>aomm nu H mm I: m cousxouoano msam surmoumuoazu mcoflco II H mm I: m Emnmoumuoanu mcoaco H H mm I: m cousxouoHco moan Emnmonmuono cmobwom m a no II N EmcmoumuoHnu cmwn>om Am>mpv 0vH w ocoflmmm owH EuomouoHno 0mm mafia mpfloflnuom mofloomm womxcmq bwuomuuxocs «o w pcsomaoo ucoumm w mnemonxom can mcoflso wn EmamoumuoHsolova mo EmHHonmumfi mo humassm .hH magma 71 demonstrated that Chlorpropham is rapidly lost due to volatilization which would partially explain the lack of metabolism. Another possibility is the concentration of chemicals at herbicidal levels was sufficient to stOp any degradation at the site of application due to the death of the tissue. SUMMARY AND CONCLUS IONS The tolerance or susceptibility of crOps and weeds to the herbicide chloroxuron is controlled by many factors. Three factors that can influence the selectivity of chlo- roxuron were studied: stage of growth, light intensity and interaction with other pesticides. As the stage of growth and develOpment increased in onions, tolerance to foliar applications of chloroxuron was increased. In both the greenhouse and field, onions were injured from chloroxuron applied before the two to three leaf stage. Thus, tolerance of onions to chloroxuron is probably due to morphological and/or physiological changes occurring approximately at the two to three leaf stage of growth. Epicuticular wax formation may be occurring at this stage which could prevent chloroxuron sprays from being retained or prevent penetration of that which is retained. Also in larger onions, the increased dry matter production may cause a dilution of the herbicide. Onions that were pre-treated for one week under low light intensity and sprayed with chloroxuron were severely damaged compared to onions grown at higher light intensities. Surface wax deposition increased as light intensity increased. 72 73 Whether these results can be extrapolated directly to a field situation is uncertain. Certainly, light intensities are lower during periods of cloudy weather but may still be higher than those produced in the growth chamber. One possibility is that surface wax deposition is decreased under cloudy weather periods. Therefore, one barrier to chloroxuron penetration into the leaf is reduced and chloroxuron injury is increased. The interaction of chloroxuron with PCMC and chlorpr0pham has been demonstrated to be of considerable importance on crops or weeds. Increased toxicity from the combination of chloroxuron and Chlorpropham appears to be caused by an increase in the amount of chloroxuron taken into the leaf tissue with a subsequent decrease in rate of photosynthesis. There appears to be alteration of some barrier to chloroxuron penetration caused by the chemicals Chlorpropham, PCMC and/or their respective carrier solvent systems. In the case of chlorpr0pham, both the herbicide and carrier solvent system are involved. Water loss studies on soybean leaves add support to the hypothesis of cuticular disruption caused by chlorpr0pham. The PCMC, carrier solvent system is as effective with or without the active ingredient. Enhancement of chloroxuron activity with surfactants and oils has been well documented. 74 Application of Chlorpropham 4 days prior to chloroxuron application also produced enhanced toxicity. This did not occur using PCMC. The fact that interactions can occur from applications several says apart and/or together is a factor that will have to be considered in commercial pesticide spraying programs to prevent potential injury to crops. There were no apparent differences in metabolism of chloroxuron or Chlorpropham in the presence or absence of the other compound. Some carbamate insecticides inhibit activity of the degradation enzymes for phenylureas herbicides. This was not evident in the chloroxuron and chlorpr0pham interaction where applications were made at herbicidal rates, similar to a field treatment. Severe tissue necrosis and the fact that both herbicides are not readily transported from the site of application could have prevented degradation processes from operating. Another factor which may be involved is the rapid volatilization of significant amounts of Chlorpropham from the surfaces of leaves after treatment. When applied in combination with chloroxuron, volatility losses are reduced. The inhibition of the Hill reaction in isolated chloroplasts was not exaggerated in the presence of the combination of chemicals, although both herbicides exhib- ited inhibitory properties. No interaction at this site 75 indicates that increased chloroxuron uptake is probably the most significant factor resulting in the observed interaction. The photosynthesis rate is greatly reduced when both chemicals are applied to intact plants. While the enhanced toxicity from Chlorpropham and chloroxuron combinations may be potentially harmful to crops such as onions and soybeans, beneficial values may accrue from an increase in activity on weed species. An exact understanding of the causes of interactions of pesticides may lead to successful uses of synergistic combinations of compounds without injury to crops. 10. 11. LITERATURE CITED Aberg, E. 1964. Susceptibility: factors in the plant modifying the response of a given species to treatment. 401-422 p. In The Physiology and Biochemistry of herbicides. L. J. Audus (ed.). Acad. Press. London and New York. 555 p. Agbakoba, C. S., and J. R. Goodin. 1969. Picloram enhances 2,4-D movement in field bindweed. Weed Sci. 17:19—21. Arle, H. 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Comparative tests on the uptake and distribution of labeled herbicides by Zebrina pendula and Tradescantia fluminensis. Hilgardia 27:421-424. Crafts, A. S., and C. L. Foy. 1962. The chemical and physical nature of plant surfaces in relation to the use of pesticides and their residues. Pes. Rev. 1:112-139. Currier, H. B., and C. D. Dybing. 1959. Foliar penetration of herbicides. Review and present status. Weeds 7:195-213. Davis, F., S. R. Bovey and M. G. Merkle. 1968. Effects of paraquat and 2,4,5—T on the uptake and transport of picloram in woody plants. Weed Sci. 16:336-338. Dewey, O. R., P. Gregory and R. K. Pfieffer. 1956. Factors affecting the susceptibility of peas to selective dinitro herbicides. Proc. Brit. Weed Control Conf. 1:313-327. Ennis, W. B., Jr. 1949. Histological and cytological responses of certain plants to some aryl carbamic esters. Amer. J. Bot. 36:823. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 78 Eshel, Y., and G. F. Warren. 1967. Postemergence action of CIPC. Weeds 15:237-241. Fang, S. C., V. H. Freed, R. H. Johnson and R. D. Coffee. 1955. Absorption, translocation and metabolism of radioactive 3-(p-chlorophenyl)-1,l-dimethylurea (CMU). J. Agr. Food Chem. 3:400-402. Feeny, R. W. 1968. Selective action of chloroxuron on soybean and morningglory. Ph.D. Thesis, University of Maryland. Frear, O. S., H. R. Swanson and F. S. Tanaka. 1969. N-demethylation of substituted 3-(pheny1)-1- methylureas. Isolation and characterization of a microsomal function oxidase from cotton. Phytochem. 8:2157-2169. Freed, V. H. 1951. Some factors influencing the herbicidal efficacy of isopropyl H-phenyl carbamate. Weeds 1:48-49. Freeman, J. F., P. Frields and C. Slack. 1965. Herbicide combinations and methods of treatment for control of weeds in soybeans. Proc. NCWCC 23:104. Geissbuhler, H., C. Haselback and H. Aebi. 1963. The fate of N-(4-chlorophenoxy)-phenyl-U,N-dimethylurea (C-1983) in soils and plants. I. Absorption and leaching in different soils. Weed Res. 3:140-153. Geissbuhler, H., and L. Ebner. 1963. The fate of N-(4-chlorophenoxy)-phenyl-N,N-dimethylurea in soils and plants. II. Uptake and distribution within plants. Weed Res. 3:181-194. Geissbuhler, H., C. Haselback, H. Aebi and L. Ebner. 1963. The fate of N-(4-chlorophenoxy)-phenyl-N,U- dimethylurea in soils and plants. III. Breakdown in soils and plants. Weed Res. 3:277-297. Geissbuhler, H. 1969. The Substituted Ureas. In Degradation of Herbicides by P. C. Kearney and D. D. Kaufman. Marcel Dekker Inc. New York. 394 p. Gentner, W. A. 1966. The influence of EPTC on external foliage wax deposition. Weeds 14:27-31. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 79 Gentner, W. A. 1966. Influence of acetone on the herbicidal prOperties of chloroxuron. Weeds 14: 95-96. Hacskaylo, J. J., K. Walker and E. G. Pires. 1964. Response of cotton seedlings to combinations of preemergence herbicides and systemic insecticides. Weeds 12:288-291. Hamill, A. S. 1971. The bases for the interaction of alachlor, butylate, or carbofuran on barley and corn. Ph.D. Thesis. Michigan State University. 126 p. Hammerton, J. L. 1967. Environmental factors and susceptibility to herbicides. Weeds 15:330-336. Haun, J. R., and J. H. Peterson. 1954. Translocation of 3-(p-chlorophenyl)-l,-dimethylurea in plants. Weeds 3:177-187. Herbicide Handbook of the Weed Science Society of America. 2nd edition. 1970. 367 p. Herrett, R. A. 1969. Methyl- and Phenylcarbamates. In Degradation of Herbicides by P. C. Kearney and D. D. Kaufman. Marcel Dekker Inc. New York. 394 p. Hill, G. D., I. J. Belasco and H. L. Ploeg. 1965. Influence of surfactants on the activity of diuron, linuron, and bromacil as foliar sprays on weeds. Weeds 13:103-106. Hoagland, D. R., and D. I. Arnon. 1938. The water- culture method for growing plants without soil. Univ. of Calif. Agri. Exp. Sta. Circ. 347. Jagendorf, A. F., and E. Uribe. 1966. ATP formation caused by acid-base transition of spinach chloroplasts. Proc. Natl. Acad. Sci. 55:170-177. James, C. S., and G. N. Prendeville. 1969. Metabolism of chlorpr0pham (iSOpropyl m-chlorocarbanilate) in various plant Species. J. Agr. Food Chem. 17:1257- 1260. James, C. S., G. N. Prendeville, G. F. Warren and M. M. Schreiber. 1970. Interactions between herbicidal carbamates and growth regulators. Weed Sci. 18: 137-139. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 80 Johnson, B. J. 1970. Effects of nitralin and chloroxuron combinations on weeds and soybeans. Weed Sci. 18:616-618. Johnson, B. J. 1971. Effects of repeated applications of herbicides on soybeans. Weed Sci. 19:548-550. Juniper, B. E. 1960. Growth, development and effect of environment on the ultra structure of plant surfaces. Linnean Soc. London J. Botany 56:403-406. Kaufman, D. D. 1970. Pesticide metabolism. In Pesticides in the soil. Sym. Michigan State University. 146 p. Klingman, G. C. 1963. Weed Control: As a Science. John Wiley and Sons, Inc. New York and London. 411 p. Mann, J. D., L. S. Jordan, and B. E. Day. 1965. The effect of carbamate herbicides on polymer synthesis. Weeds 13:63-66. Matsumaka, S. 1968. Propanil Hydrolysis: Inhibition in rice plants by insecticides. Science 160:1360- 1361. McReynolds, W. D., Jr., and A. R. Putman. 1971. The influence of added pesticides and their carrier solvents on chloroxuron toxicity to onions. Weed Sci. Soc. Am. Abst. No. 169. McWhorter, C. G. 1963. Effects of surfactants on the herbicidal activity of foliar sprays of diuron. Weeds 11:265-269. Moreland, D. E., and K. L. Hill. 1959. The action of alkyl N-phenylcarbamates on the photolytic activity of isolated chloroplasts. J. Agr. Food Chem. 7:832-837. Moreland, D. E., and K. L. Hill. 1962. Interferences of herbicides with the Hill reaction of isolated chloroplasts. Weeds 10:229-236. Muzik, T. J., H. J. Cruzado and A. L. Loustalot. 1954. Studies on the absorption, translocation, and action of CMU. Bot. Gaz. 116:65-73. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 81 Nash, R. H. 1967. Phototoxic pesticide interactions in soil. Agron. Jour. 59:227-230. Nash, R. H. 1968. Synergistic phytotoxicities of herbicide-insecticide combinations in soils. Weed Sci. 16:74-78. Nashed, R. B., and R. D. Ilnicki. 1970. Absorption, distribution and metabolism of linuron in corn, soybean and crabgrass. Weed Sci. 18:25-28. Nashed, R. B., S. E. Katz and R. D. Ilnicki. 1970. The metabolism of l4C-chlorbromuron in corn and cucumber. Weed Sci. 18:122-125. Pfeiffer, R. K., O. R. Dewey and R. T. Brunskill. 1957. Further investigation of the effect of preemergence treatment with trichloroacetic acid and dichloro- propionic acids on the subsequent reaction of plants to other herbicide sprays. Fourth Int. Cong. Crop Prot. 1:523-525. Prendeville, G. N., Y. Eshel, C. S. James, G. F. Warren and M. M. Schrieber. 1968. Movement and metabolism of CIPC in resistant and susceptible species. Weed Sci. 16:432-435. Putnam, A. R., and S. K. Ries. 1967. The synergistic action of herbicide combinations containing paraquat on Agropyron repens (L.). Weed Res. 7:191-199. Putnam, A. R., F. Hess and W. McReynolds. 1970. Weed control research in vegetable, fruit, and ornamental crops.> Hort. Report No. 16. Michigan State University. 43 p. Riden, J. R., and T. R. Hopkins. 1962. Formation of a water soluble, 3-chloroaniline-containing sub- stance in barban treated plants. J. Agr. Food Chem. 10:455-458. Roberts, J. R. 1965. Comparative tolerance of some dicotyledons to Chlorpropham. Weed Res. 5:61-67. Scott, M. Z., and B. E. Struckmeyer. 1955. Morphology and root anatomy of squash and cucumber seedlings treated with isoprOpyl N-(3-chlorophenyl) carbamate (CIAC). Bot. Gaz. 117:37-45. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 82 Smith, J. W., and T. J. Sheets. 1966. Uptake, distribution and metabolism of monuron and diuron by several plants. J. Agr. Food Chem. 15:577-581. Smith, R. J., Jr., and W. C. Shaw. 1966. Weeds and their control in rice production. U.S.D.A. Agr. Handbook No. 292. 64 p. St. John, J. B. 1971. Comparative effects of diuron and Chlorpropham on ATP levels in Chlorella. Weed Sci. 19:274-276. Sterrett, J. P., J. T. Davis and W. Hurtt. 1971. Antagonistic effects between picloram and bromacil with oats and wheat. Weed Sci. Soc. Amer. Abst. No. 12. Still, G. G., D. G. Davis and G. L. Zander. 1970. Plant Epicuticular Lipids: alteration by herbi- cidal carbamates. Plant Physiol. 46:307-314. Still, G. G., and E. R. Mansager. 1971. Metabolism of iSOprOpyl 3-chlorocarbani1ate by soybean plants. J. Agr. Food Chem. 19:879-884. Strang, R. H., and R. L. Rogers. 1971. A micro- radioautographic study of l4C-diuron absorption by cotton. Weed Sci. 19:355-362. Swanson, C. R., W. C. Shaw and J. H. Hughes. 1953. Some effects of iSOpropyl N-(3-chloropheny1) carbamate and an alkanolamine salt of dinitro ortho secondary butyl-phenol on germinating cotton seeds. Weeds 2:178-189. Swanson, C. R., and H. R. Swanson. 1968. Inhibition of degradation of monuron in cotton leaf tissue by carbamate insecticides. Weed Sci. 16:481-484. Sweetser, P. B., and C. W. Todd. 1961. The effects of monuron on oxygen liberation in photosynthesis. Biochim. et BiOphys. Acta 51:504-508. Sweetser, P. B., C. W. Todd and R. T. Hersh. 1961. Effect of photosynthesis inhibitors on light re- emission in photosynthesis. Biochim. et BiOphys. Acta 51:509-518. 81. 82. 83. 84. 85. 83 Treharne, R. W., T. E. Brown and L. P. Vernon. 1963. Separation of two light-induced electron spin resonance signals in several algal species. Biochim. et BiOphys. Acta 27:205-206. Van Overbeek, J. 1962. Physiological responses of plants to herbicides. Weeds 10:170-173. Warren, G. F. 1954. Weed control in onions with CIPC. Proc. NCWCC 11:71. Wessels, J., and R. van der Veen. 1956. The action of some derivatives of phenylurethane and 3-phenyl-l,1-dimethylurea on the Hill reaction. Biochim. et Biophys. Acta 19:548-549. Wheatly, L. R., R. R. Wilson and R. H. Cole. 1966. Chloroxuron, a possible postemergence herbicide for soybeans. Proc. NEWCC 20:345-351. APPENDICES 84 4 Appendix A. Chloroxuron and Chlorpropham interaction on soybeans Fresh Weight Visual (% of Control) Rating a Chemical Rate (7 Days) Observed Expected (lb/A) Control 4 1.0 100 Chloroxuron 4 2.7 94 Chloroxuron plus Chlorpropham 4 +0.5 4.7 90 84 Chloroxuron plus Chlorpropham 4-+1 5.7 79 77 Chloroxuron plus Chlorpropham 4-+2 6.0 73 76 Chloroxuron plus Chlorpropham 4-+4 7.7 54 75* Chloroxuron plus chlorpr0pham carrier 4-+0.5 2.0 94 Chloroxuron plus Chlorpropham carrier 4-tl 2.7 93 Chloroxuron plus chlorpr0pham carrier 4-+2 2.7 90 Chloroxuron plus Chlorpropham carrier 4-+4 3.7 92 Chlorpropham 0.5 3.0 89 Chlorpropham 1 3.7 82 Chlorpropham 2 5.7 81 Chlorpropham 4 6.7 80 LSD at 5% 0.9 11 aExpected values calculated by Colby's formula. *Significantly different from observed value using estimated LSD at the 5% level. Appendix B. onions 85 Chloroxuron interaction with chlorpr0pham and PCMC on Fresh Weight Visual (% of Control) Rating a Chemical Rate (7 days) Observed Expected (lb/A) Control 3 1.0 100 Chloroxuron 3 2.2 90 Chlorpropham 1.5 2.2 112 Chlorpropham 3 2.0 98 PCMC 1 1.8 110 PCMC 2 1.8 110 Chloroxuron plus Chlorpropham 3-+l.5 2.2 108 101 Chloroxuron plus Chlorpropham 3-+3 2.6 63 88 Chloroxuron plus chlorpr0pham 3-+3 2.6 63 88 Chloroxuron plus PCMC l 2.8 75 99 Chloroxuron plus PCMC 2 2.6 58 99* LSD at 5% 0.9 30 a Expected values calculated by Colby's method. *Significantly different from observed value using estimated LSD at the 5% level. 86 Appendix C. Chloroxuron interaction with Chlorpropham, PCMC and their solvent carriers on onions Fresh Weight Visual (% of Control) Rating a Chemical Rate (7 Days) Observed Expected (lb/A) Control -- 1.0 100 Chloroxuron 3 1.0 95 PCMC l 1.0 84 Chlorpropham 2 1.0 82 Chloroxuron plus PCMC 3-+1 5.4 51 80* Chloroxuron plus PCMC carrier 34-1 6.6 51 Chloroxuron plus Chlorpropham 3-(2 6.4 43 78* Chloroxuron plus Chlorpropham carrier 3-+2 2.7 64 LSD at 5% 0.8 21 aExpected values calculated by Colby's method. *Significantly different from observed value using estimated LSD at the 5% level. 87 Appendix D. Chloroxuron interactions with Chlorpropham, PCMC, and their carrier solvents on white mustard Fresh Weight Visual (% of Control) Rating a Chemical Rate (7 Days) Observed Expected (lb/A) Control -- 1.0 100 Chloroxuron 0.5 5.2 41 Chlorpropham 1 2.7 102 Chlorpropham carrier 1 2.7 102 PCMC l 2.5 87 PCMC carrier 1 2.7 114 Chloroxuron plus Chlorpropham 0.5~+l 7.0 5 42* Chloroxuron plus Chlorpropham carrier 0.5-+1 5.5 26 42 Chloroxuron plus PCMC 0.5-+1 6.0 4 36* Chloroxuron plus PCMC carrier 0.5-+1 6.0 5 47* LSD at 5% 0.5 23 aExpected value calculated by Colby's method. *Significantly different from observed value using estimated LSD at the 5% level. 88 .Ho>ma wm map um own cmumEHumm moans moam> cw>ummno Eouw uanoMMHp hauCMOHchmHmi .cocumfi w.>naoo an cocoauoamo mosam> couommxmm gm hm m.o n.o wm um omq .ao am am pm S.a 6.6 H1.H ozom mafia cousxouoano on mm on as m.s o.m H.+m.o ozom mafia cousxouoano No ov 4N0 mm o.m n.m H1.H Emcmoumuoaco moan cousxouoHcO on ma on as m.o v.6 H1.m.o surmoumuoHro mafia cousxouoaro om om m.m H 020m mm mm m.m a Emcmonmnoacu mm mm H.m a cousxouoHcO mm mm o.m m.o cousxouoacu mm mm o.H In Houucou la\nav mcouoomxm co>uomoo mcvoommxm oo>nomoo cocflnsoo peamm mpmm Hmoflsoco peamm mcflumm Hmsma> laouuaoo Go we prmnmz amass cumumse ovens co 020m Ho Emcmoumuoaco cufl3 cousxouoHno mo mcoflumoflammm cmcflnfioo cam uflamm EOHM mufioflxoa .m xflccmmm< (“Willis 3169 2506 1' 930 u H N”! 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