THE SORPTION, PENETRATION, AND UPTAKE 0F SELECTED BIOLOGICALLY ACTIVE COMPOUNDS TH‘RGUGH PLANT CUUCLES AS RELATED TO MOLECULAR .SIRUCTURE ' ?hesis for the Degree of Ph. D. MEMIQAN SEATE UNNERSHY MICE HANSEL PARHAM 2%9 THESIS LIBRARY Michigan State University MSSMM HIGAN STATE UNIVERSITY LIBRARI INN.liilillihliiiifihl{liLHEITIIHIZ“Hill 3 1293 00620 6670 This is to certify that the thesis en .ueu THE SORPTION, PENETRATION, AND UPTAKE OF SELECTED BIOLOGICALLY ACTIVE COMPOUNDS THROUGH PLANT CUTICLES AS RELATED TO MOLECULAR STRUCTURE T presented by Price Hansel Parham has been accepted towards fulfillment of the requirements for Ph.D. Horticulture degree in azw Major professor Date February 14. 196L 0469 PLACE IN RETURN BOX to rerriavo rm'choekom from your record. TO AVOID FINES return on or boron date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative ActionlEqual Opportunity Institution ABSTRACT THE SORPTION, PENETRATION, AND UPTAKE 0F SELECTED BIOLOGICALLY ACTIVE COMPOUNDS THROUGH PLANT CUTICLES AS RELATED TO MOLECULAR STRUCTURE By Price Hansel Parham A series of studies was begun in l967 to evaluate the sorption, penetration, and uptake of selected 1"C-labeled organic compounds as they relate to molecular structure. Sorption and penetration studies were conducted using enzymatically isolated tomato fruit cuticles, and uptake experiments were conducted with green bean leaf disks. The sorption, penetration, and uptake of N,N-dimethyl-2,2- diphenylacetamide, N-methyl-2,2-diphenylacetamide, diphenylacetamide, and diphenylacetic acid increased with time. In general, the de- scending order of sorption, penetration, and uptake was diphenylacetic acid, N,N-dimethyl-2,2-diphenylacetamide, N-methyl-2,2-diphenylaceta— mide, and diphenylacetamide. As the hydrogen ion concentration of the donor solution de- creased, the ionized species of diphenylacetic acid increased, re- sulting in decreased sorption, penetration, and uptake. Undissociated molecules are more lipoidal and more likely to partition into phases such as wax or other lipophilic components of the cuticle. Donor pH 2— Price Hansel Parham of the three diphenylacetamide molecules did not significantly in- fluence sorption, penetration, nor uptake. Polarity had an influence on the processes studied. Addition of a nonionic surfactant resulted in an increased penetration and uptake of compounds of different molecular structure. Sorption into isolated tomato fruit cuticles was reduced with the addition of a surfactant. Non-polar (lipophilic) compounds as those used in these studies may be solubilized in the center of micelles which consist of the hydrophilic portions of nonionic surfactants. Thus, apolar compounds may be permitted to take a polar route through the cuticle. Sorption and penetration of the four different compounds were not influenced by metabolic activity, since respiration of isolated cuticular membranes did not occur. Metabolic activity in the green bean leaf disk may help explain the increased uptake of the four com- pounds used in these studies. THE SORPTION, PENETRATION, AND UPTAKE 0F SELECTED BIOLOGICALLY ACTIVE COMPOUNDS THROUGH PLANT CUTICLES AS RELATED TO MOLECULAR STRUCTURE By Price Hansel Parham A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1969 ACKNOWLEDGMENTS The writer wishes to acknowledge with much appreciation the encouragement and guidance he received from Dr. A. E. Mitchell during the course of this study and in the preparation of this manuscript. The writer also desires to express his thanks and appreciation to Dr. M. J. Bukovac, Dr. G. E. Guyer, Dr. C. M. Harrison, and Dr. A. L. Kenworthy for serving on the guidance committee and for their help and advice throughout the investigation. The writer is indebted to Dr. A. J. Lemin of The Upjohn Company for furnishing the radiolabeled compounds used in this study. The writer is sincerely appreciative of the continued in- spiration, encouragement, and tolerance of his wife, Helen, during the course of graduate training. A special thanks is expressed to Mrs. Hazel Finch for her help in typing the manuscript. ii TABLE OF CONTENTS ACKNOWLEDGMENTS .............................................. LIST OF TABLES .............................................. LIST OF FIGURES .............................................. LIST OF APPENDICES ........................................... INTRODUCTION ................................................ REVIEW OF LITERATURE ..................................... .... Nature of the Cuticular Surface Pathways of Cuticular Penetration Stomata and internal cuticle Canals and intercuticular penetration Leaf pubescence Cuticular structure Environmental Factors Affecting Cuticular Penetration Temperature Moisture Light Surfactants Nature of Chemical and Cuticular Penetration Effects of pH on penetration Size of molecule and penetration MATERIALS AND METHODS ........................................ Definitions Relation of Molecular Structure to Sorption Influence of time on sorption Influence of pH on sorption Influence of surfactant concentration on sorption 0000\10303 Relation of Molecular Structure to Penetration Influence of time on penetration Influence of donor pH on penetration Influence of surfactants on penetration Influence of surface wax on penetration Relation of Molecular Structure to Uptake Influence of time on uptake Influence of pH on uptake Influence of surfactant on uptake Relation of Molecular Structure to Partitioning RESULTS ........................................................ Relation of Molecular Structure to Sorption Influence of time on sorption Influence of pH on sorption Influence of surfactant on sorption Relation of Molecular Structure to Penetration Influence of time on penetration Influence of pH on penetration Influence of surfactants on penetration Influence of surface wax on penetration Relation of Molecular Structure to Uptake Influence of time on uptake Influence of pH on uptake Influence of surfactant on uptake Relation of Molecular Structure to Partitioning Influence of organic solvents on partitioning Influence of pH on partitioning DISCUSSION ..................................................... Cuticular Sorption and Penetration Uptake Influence of pH on Sorption, Penetration, and Uptake Influence of Surfactant on Sorption, Penetration, and Uptake SUMMARY ........................................................ LITERATURE CITED ............................................... APPENDICES ........................................... . ......... iv Table LIST OF TABLES Radiolabeled compounds used in this investigation and the relevant prOperties of each ................... Scintillation solvent mixture used in the isolated tomato fruit cuticle penetration exoeriments .......... Citric acid-phosphate buffer solution used to maintain stated pH level ....................................... Trade names and a summary of the relevant physical and chemical properties of the surface active agents used in this study .................................... Scintillation solvent mixture used for counting samples from the bean leaf uptake exoeriments ......... Page 22 23 24 34 Fiqure TO LIST OF FIGURES Diagrammatic representation of the structure of an upper leaf cuticle, not necessarily drawn to scale. (From Norris and Bukovac, 1968) ................ Apparatus used to study the relation of molecular structure to cuticular penetration ..................... Set of assembled apparatus for the cuticular penetration studies .................................... Comparative rates of cuticular sorption of different compounds through isolated tomato fruit cuticles as influenced by time at different pH levels .............. Influence of donor pH on the sorption of different compounds into isolated tomato fruit cuticles, with and without a surfactant, 24 hours after treatment ..... The concentration influence of the nonionic surfactant Tergitol l5-S-9 on the sorption of different compounds into tomato fruit cuticles, 24 hours after treatment Penetration of different compounds through cuticles enzymatically isolated from tomato fruit, as influenced by time .............................................. . The influence of donor pH on penetration of different compounds, with and without a surfactant, through enzymatically isolated tomato fruit cuticles, 24 hours after treatment ........................................ Influence of different nonionic surfactants (0.l% v/v) on penetration of N,N-dimethyl-2,2-diphenylacetamide through isolated tomato fruit cuticles ................. Influence of concentration of the nonionic surfactant Tween 20 on penetration of N,N-dimethyl-2,2-diphenyl- acetamide through isolated tomato fruit cuticles, l8 hours after treatment .................................. vi 44 ll 12 l3 l4 TB 16 T7 TB 19 Influence of concentration of the nonionic surfactant Tergitol l5-S-9 on penetration of N,N-dimethyl-2,2- diphenylacetamide through isolated tomato fruit cuticles, 24 hours after treatment .................... Influence of the nonionic surfactant Tergitol l5-S-9 (0.3% v/v) on penetration of different compounds through isolated tomato fruit cuticles, 24 hours after treatment ....................................... Influence of surface wax on the penetration of different compounds through isolated tomato fruit cuticles, 24 hours after treatment .................... Comparative rates of uptake of different compounds (at pH 4.0) into green bean leaf disks as influenced by time ............................................... Relation of molecular structure of 1L*C-carbonyl labeled compounds into green bean leaf disks 24 hours after treatment. Donor solution was distilled water without a surfactant ............................ Influence of donor pH on uptake of 4 different compounds into green bean leaf disks, 6 and 24 hours after treatment ................................. Influence of nonionic surfactant (0.3% v/v) Tergitol 15-5-9 on uptake of different compounds into green bean leaf disks, 12 hours after treatment ............. Influence of concentration of the nonionic surfactant Tergitol l5-S-9 on uptake of N,N-dimethyl-2,2-diphenyl- acetamide into green bean leaf disks, 6 and 12 hours after treatment ....................................... Relation of molecular structure and pH of 1‘*C-carbonyl labeled compounds to partitioning into different organic solvents from aqueous donor solutions, 24 hours after treatment ................................. vii 54 59 61 64 67 69 72 74 77 LIST OF APPENDICES Appendix Page A Influence of donor pH on the sorption of different compounds into isolated tomato fruit cuticles, 24 hours treatment time .................. 102 B The concentration influence of the nonionic surfactant Tergitol 15-8-9 on the sorption of different compounds into tomato fruit cuticles, 24 hours after treatment ........................... l03 C Penetration of different compounds through tomato fruit cuticles as influenced by time ........ 104 D The influence of donor pH on penetration, 24 hours after treatment .................................... l05 E Influence of surfactant Tergitol 15-5-9 on uptake of N,N-dimethyl-2,2-diphenylacetamide into green bean leaf disks, 6 and l2 hours treatment time ..... 106 F Relation of molecular structure and pH to partitioning into different organic solvents from aqueous donor solutions, 24 hours after treatment .. l07 viii INTRODUCTION One of the major points regarding chemicals used for the control of insects, diseases and weed pests of economically important crops is the ability of these pesticides to reach the target site systemically. This is also true for those compounds used to regulate growth response or to correct a nutrient deficiency. Investigations on the absorption of inorganic and organic molecules into living cells have been con- ducted for many years and are continuing. In both plants and animals a non-living protective or barrier membrane must be traversed before chemicals can penetrate living cells. This membrane is the first vital biological interface in terms of cellular activity and it is the plant's contact with its external environment. As reported by Bukovac and Norris (ll,72), Jyung (56), van Overbeek (98), Scott (85), and Crafts and Fay (19), it is the cuticle that is considered the first barrier to penetration into the plant, but there is no question that bioactive chemicals and other substances penetrate it. Skoss (92) reports this is also true for excretion. The most convenient way to make application of most pesticides is by aqueous spray to aboveground parts. This requires a) that the material become attached to the cuticular surfaces of the plant, b) the material actually transgress the cuticular membrane, and c) that 2 it be delivered to some reaction site. Without this sequence of events, it is very difficult to get a uniform type response from a plant system. Since wax may be either superimposed on or interspersed in the plant cuticle, it may form a barrier to penetration of hydrophilic substances. Most pesticides are studied to observe a biological response by the plant or pest and to determine the metabolic fate of the chemi- cal within the plant. Very little is known about the absorption and translocation of a biologically active compound as related to molecular structure. Because little information is available regarding pene- tration of exogenously applied organic compounds as they relate to molecular structure, a study was developed to determine: a) the sorption of carbonyl-1“C labeled diphenylacetic acid and its dimethyl-, methyl-, and diphenylacetamide analogs into cuticles enzymatically isolated from ripe tomato fruit; b) the penetration of these molecules through isolated tomato fruit cuticles; c) uptake of these 1“C-labeled molecules by excised bean leaf disks; and d) the effect of donor pH and surface active agent concentrations upon sorption, penetration, and uptake of these different molecules. REVIEW OF LITERATURE Nature of the Cuticular Surface. In both plant and animal a non-living protective or barrier mem- brane must be traversed before exogenously applied compounds can penetrate the living cells. External membranes of leaves which are considered the first barriers to penetration are called cuticles, (van Overbeek, 98). The generalized structure of the plant cuticle has been reported by Eglinton and Hamilton (29), Franke (37), and Norris and Bukovac (72). The cuticle is built up of several alternating layers of cellulose, pectin, cutin, and wax (Figure l). The cutin it- self is believed to be composed of polymerized long-chain fatty acids and alcohols. This is bound by epicuticular waxes toward the outside and pectin substances toward the cellulose cell wall. According to Eglinton and Hamilton (29), surface waxes are complex mixtures of long-chain alkanes, alcohols, ketones, aldehydes, acetals, esters, and acids. The picture is complicated by the positioning and number of functional groups, the degree of chain branching and un- saturation, and the increasing number of other types of constituent. However, the wax composition of a species may differ for different parts of the same plant and may vary with season, age of the plant, locale, humidity, temperature and other internal and external factors. 3 Figure l. Diagrammatic representation of the structure of an upper leaf cuticle, not necessarily drawn to scale. (From Norris and Bukovac, 1968). .343 fine r so uozuoziuuza 9.532 r @ muozfimnzm 0:8; r mm 2.858. r 9 xii: 2.50 r so 3232;:qu maroon r 9 2:3 2. 3835 is 532535 r so x; «53.52% r 5 . ® .H,,...w... arr/x , . w - in. , x . _ .n... .4 5.... f " so $3 mm :0 mm: 6 Minute wax protuberances, termed wax rodlets, are observed on the cuticle and according to Mitchell, gt_gl, (66) these often occur in various forms as granular, platelike, crystalline-granular, and rod- shaped. These protuberances can act as a barrier to penetration of exogenously applied aqueous solutions without a surface active agent. Rich and Horsfall (80) observed similar results with toxicants used for the control of fungi. The region directly beneath the epicuticular wax contains wax platelets embedded in the cutin which form the framework and general body of the cuticle. Pathways of Cuticular Penetration. Several excellent reviews have been written within the past few years concerning the penetration of exogenously applied compounds into plant cells. Reviews by van Overbeek (98), Crafts and Foy (19), Mitchell (66), Franke (37), Currier and Dybing (23), Wittwer (104), and Sargent (83) have contributed greatly to the understanding of cuticular penetration. Stomata and internal cuticle. For several years it was thought that hydrOphilic molecules entered the leaves mainly through stomatal pores. This hypothesis was substan- tiated by many investigators such as Boynton (7), Foy (35), Sargent and Blackman (83). Wittwer and Teubner (105), Wittwer gt_al, (104), Jyung 91g. (56). and Teubner 2.29.- (97) who found that upper surfaces with- out stomates had less solute uptake than lower surfaces. Most workers agree with Norman gt_al, (71) and van Overbeek (98) that aqueous solu- tions do not penetrate through stomatal pores unless the surface tension 7 is very low. The size of the stomatal pore (3—6/4 when fully Open) is such that the surface tension of the leaf will prevent the water droplet from gaining entrance into it. Stomatal entry of aqueous solutions must depend, therefore, upon an adequate surface active agent to reduce surface tension. Investigations conducted by Wittwer gt_al, (104,105), Currier and Dybing (23), Currier §t_al, (24), and Norris and Bukovac (72) concluded that once the molecule enters the sub-stomatal cavity it must still transgress a lipid membrane called the internal cuticle. The molecule can be considered in the leaf but not in the cell. The cuticular membrane of the stomatal cavity is thinner, more hydrated, and easier to penetrate than the epicuticular surface of the leaf. Stomatal frequency per unit of leaf area was highly correlated with the rate of rubidium absorption in bean and tomato leaves, (Jyung and Wittwer, 55). The entrance into a stomatal cavity does not pre- clude the necessity for cuticular penetration. Several investigators (Bukovac and Norris, 11,72; Jyung et al.,56; Wittwer et 1., 104; and Yamada et al., 108) have demonstrated that substances applied in aqueous solutions can penetrate intact and isolated astomatous cuticles. Canals and intercuticular penetration. Scott gt_al, (86,87) were of the opinion that canals may exist in the cuticle, but efforts to Show that these canals actually exist have not been successful. However, Hall and Donaldson (43) claimed that cuticular pores, approximately 6-7 myrin diameter, were revealed in epidermal cells of clover (Trifblium Pepens) and cauliflower (Brasatca 8 oleraceae) from which wax is extruded. USing microscopy, Bukovac and Norris (11) and Norris and Bukovac (72) showed the pear cuticle to be a uniformly continuous and poreless membrane. If canals were present, they might facilitate the movement of non-polar substances through the cuticle to the hypodermal cells. Scott et_al, (86) and Kamimura (57) reported that breaks, fissures, or punctures made by insects or me— chanical means are sometimes found in the cuticular membrane. The passage of solutes through these imperfections has been termed inter- cuticular penetration by Wittwer and Teubner (105). Leaf’pubeecence. Ennis §t_al, (32) and Mitchell _t__l, (66) reported that pubes- cence on the leaves increased cuticular penetration of exogenous aqueous solutions. Since most plant hairs are covered with a cuticle, this possible mode of entry does not eliminate the cuticle as the first barrier to penetration. Harley _t__l, (47) did not find pubescence to be a factor in NAA penetration of apple leaves. While working with 32P-Systox, Tietz (96) found that hairy leaves of primrose (Primula obconica) absorbed three times as much as did the smooth leaves of Cy~ clamen (Cyclamen persicum). Cuticular Structure. The behavior of the cuticular framework composed of cutin which exhibits moderate hydrophilic properties is significant. Neintraub (102) and van Overbeek (98) reported that after absorbing water the cuticle swells and spreads apart the embedded wax platelets (wthh ex- hibit hydrophilic properties). This increases permeability of the 9 cuticle to water and to certain organic substances that tend to move with water. Conversely, low moisture content would move the wax plate- lets closer together and therefore reduce water and solute movement through the cuticle. If wax platelets alternate with cutin lamellas, they should prevent the penetration of aqueous solutions because of the apolar lipophilic properties of wax. Investigations conducted by Orgell (73) indicated that the plant cuticle was characterized by an imbricate arrangement of lipoid platelets cemented together by hydrophilic pectin- aceous materials. Roberts gt_gl, (81) concluded that "intercuticular penetration” of aqueous solutions should be possible because the pectin layers beneath the epicuticle extend to the middle lamellas of the tissues and should provide a hydrophilic pathway to the vascular bundles close to the epidermal tissue of the leaves. Direct evidence of this has not been clearly demonstrated. ‘Environmental Factors Affecting_§yticular Penetration. Cuticular penetration is influenced by several factors other than those discussed above. Such factors as temperature, moisture, light, surface active agents, and chemical formulation may have an influence on cuticular penetration of aqueous solutions. Temperatare. It is believed that warm temperatures, (10° to 40°C) will promote penetration of solutes through the cuticle. Currier and Dybing (23) and Sargent (84) concluded that temperatures of 10° to 37°C indirectly influenced the penetration rate of aqueous solutions by influencing cy— toplasmic viscosity, accumulation, binding, metabolic conversion, and 10 translocation of the penetrant, i.e., by regulating processes which in- fluence the concentration gradient across the surface layers. Warm temperatures directly influenced the rate of diffusion of lipophilic substances through lipoid—containing membranes. Barrier and Loomis (2), Rice (79), and Sargent and Blackman (84) demonstrated that increased temperatures increased the penetration of 2,4-dichlorophenoxyacetic acid (2.4-0) into plant parts. Luckwill and Jones (63) and Harley (47) showed a direct relationship of increased napthaleneacetic acid ab- sorption to increased temperature. Luckwill and Jones (63) showed that over the range of 5° to 25°C there was a linear relationship using Bramley apple leaves (18% to 45% absorption). Jyung gt_al, (55) re- ported that rubidium absorption by green tobacco leaf cells was tempera- ture dependent. It has also been demonstrated that temperature in- fluences the rate of absorption of systemic insecticides. While working with Vicia faba (broad bean), Bennett and Thomas (4) found that over a 7-hour period 32P-schradan was absorbed in greater quantities at 26° than at 15°C (64.4% vs. 100%). After 72 hours the absorption was essentially the same. Mbleturc. It has been reported by Palmquist (77) that when a scraped leaf of a water-stressed plant was immersed in a fluorescein solution, the dye moved rapidly through the leaf. Went and Carter (103) reported that sucrose uptake by tomato leaves was independent of the humidity level. While Pallas (76). working with 2,4-dichlorophenoxyacetic acid (2.4-0), found that high humidity increased foliar absorption. Similarly, Volk 11 and McAuliffe (101) reported increased penetration of urea under high humidity conditions. Clor gt_al, (12,13) demonstrated that the rate and extent of translocation of 2,4-D and amitrol in cotton leaves was enhanced when the plants were placed in polyethylene bags to produce a high humidity atmosphere. Crafts and Foy (19) suggested that under low atmospheric humidity the leaf was under tension and air blocks pre- vented union of the spray with the water continuum. Under this con- dition, the aqueous route of penetration was unavailable but penetra- tion may still have occurred by the lipoidal route. Prasad et_al, (78) demonstrated that more dalapon was absorbed and translocated under conditions of high (88 :_5%) than at low (28 :_3%) post-treatment rela- tive humidity. Luckwill and Jones (63) found that NAA uptake in apple leaves was 50% and 90% respectively at 37% and 100% relative humidity. Light. The effect light has upon penetration of foliar applied solutes is not clear. According to Sargent (82) light may promote absorption by causing an increase in the export of carbohydrates with which growth regulators appeared to be associated during translocation from the leaf. Several investigators have reported an increase in absorp- tion with increased light intensity. Jyung and Wittwer (53) reported a rapid increase in absorption of phosphate and rubidium as the light intensity was increased up to 320 ft-c. While working with green and albino leaves, Juniper (52) reported an increase in the thickness of the cuticle with a rise in light intensity. Sargent and Blackman (83,84) reported a greater increase in the rate of 2,4—dich1orophenoxyacetic 12 acid penetration in Phaseolus vulgaris and Ltgastrum ovalcjbltum in the light than in the dark. However, Bennett and Thomas (4) found that light was apparently not very important in the absorption of “P—schradan over a 72 hour period. Total absorption at 27°C was 79.8% in the dark, and 88.7% under fluorescent light. With Coleus, broad bean, and runner beans, darkness post-application also reduced the rate of absorption. Shrfactants. Surface active agents (surfactants, wetters, detergents, etc.) are materials which are used at very low concentrations to lower the surface tension of water. They are included extensively in the formulation of pesticides to increase wettability which in turn may influence penetra- tion or distribution of aqueous solutions. An effective surface active agent is composed of molecules containing an alkane-type grOup which 15 oil soluble and one or more polar groups which are water soluble. These agents are composed of molecules whose structures prOVide a hydrophilic part called the "head", and a hydrophobic part called the ”tail”. In 1890 Gillette (40) found that part of the leaf surface of plum was destroyed when sprayed with a four-ounce-per-gallon concentration of whale oil. An inherent phytotoxicity of several species of plants to sulfonated alcohols in insecticides was reported by Cory and Langford (l4). Blackman gt_gl, (2) reported that lowering the surface tension reduced the volume of spray retained by species not readily wetted. These data agreed with those reported by Moore (36) who concluded that some surface active agents increased spreading on waxy or non-waxy surfaces, but not necessarily both. Numerous investigators such as Buchanan (10), 13 Crafts (18), Currier (22), Ennis (31), Daines et_gl, (25), Ilnicki gt_al, (50), Laning and Aldrich (60), Leonard and Crafts (61), Luepschen and Rohrbach (62), McWhorter (64), Mitchell _t_al, (66), and Prasad gt_al: (78) have reported the enhancement of pesticide action by the addition of surface active agents. Other investigators, (Behrens, l; Jansen _t_gl,, 51; Hauser, 48; Sargent and Blackman, 83; Staniforth and Loomis, 94; Skoss, 92; Weintraub gt_gl,, 102) reported that 2,4-dichlorophenoxyacetic acid penetration was increased by the inclusion of a surface active agent_ While using a fluorescent dye in studying penetration in Zebrtna peniula, Dybing and Currier (28) found that surface active agents enhanced foliar penetration. In all cases where the surfactant was present, penetration of the lower surface was more rapid than for the upper surface. It was concluded that solutions containing surface active agents penetrated mainly through open stomata. Enhanced stomatal penetration by surfac- tants has already been discussed on Page 6. Brown (8) reported that surface active agents increased the action of defoliants. Increased fungicide penetration with surface active agents has been reported by Daines et_gl, (25) and Luepschen and Rohrbach (62). Several reports where surface active agents enhanced the penetration of insecticides were reviewed by Mitchell, Smale, and Metcalf (66). Bukovac and Norris (11) reported that the water uptake pattern in immersed isolated tomato fruit cuticle was not altered by the addition of polyoxyethylene-ZO-sorbitan monolaurate (Tween 20). Sargent and Blackman (83) reported that Tween 20 increased the rate of 14 penetration of 2,4«D in the dark into the abaxial and the adaxial sur- faces of PhaseoZus vulgaris. Other investigators as well as Teubner gt_al, (97) found that Tween 20 was ineffective in enhancing the pene- tration of labeled phosphorus (32P) into bean leaves. However, Neely and Phinney (70) reported that Tween 20 did effectively promote pene- tration of foliar applied gibberellic acid in maize. Freed and Montgomery (38) concluded that although reduction of surface tension was important, the relationship of molecular interaction between sur- factant and the herbicide was of equal or greater importance. They sug- gested a highly specific requirement for surfactant formulation to fit the herbicide in order to achieve maximum effectiveness. Buchanan (10) reported that it was not possible to classify surfactants as toxic or non-toxic on the basis of ionogenic grouping as there were various levels of toxicity within the ionogenic groups. Nature of ghemical and Cuticular Penetration. According to Crafts and Foy (19), there are at least two routes for penetration of exogenously applied materials into plant leaves. These are a polar or aqueous route, and a non-polar or lipoidal route. There is a gradient of polarity from the interior of the cuticular layers and of the cellulose wall which exhibits high polarity. Crafts (15) reported that apolar compounds usually penetrated the cuticular membrane much more rapidly than polar compounds. According to van Over- beek (99) and Veldstra (lOO) penetration of growth regulators may be improved when the molecule exhibits the proper polar-apolar balance. Middleton (65) reported that many non—polar organic solutes 15 were absorbed by plant foliage more rapidly than the highly polar in- organic salts. In studies conducted by Yamada gt_al, (111), it was consistently demonstrated that the dialyzing membranes were more per- meable than cuticular membranes to both organic compounds and in- organic ions. These investigators found that permeability of urea through tomato fruit cuticles was twice that of N,N-dimethylamino- succinamic acid and six times that for maleic hydrazide. Urea also penetrated the tomato fruit cuticle 10 to 20 times more than inorganic ions as Rb+, Ca++, Cl', and 504'“. Urea was believed to have pene- trated by a process of facilitated diffusion. Franke (37) concluded that lipophilic substances may penetrate by a process of solution and the rate determined by the solubility, partition coefficient, and molecular size. Effects of pH on penetration. There have been many conflicting results regarding the part played by the pH of donor solutions on the penetration of weak acids into plant parts. In mid-1947, pH was shown by Hamner _t__l: (45) to in- fluence the regulating compounds of weak acids. Simon gt_al, (90) reported that the penetration of 3,5-dinitro-O-cresol into leaves was not influenced by the pH of the external solution. Albert (1), Crafts (16,20), and Swanson and Whitney (95) reported that many weak acids penetrated plant cells more readily in the undissociated form, i.e., at pH values below their pK. Crafts (18), Orgell and weintraub (75), and Sargent and Blackman (83,84) reported that 2,4-dichlorophen- oxyacetic acid (2,4-D) penetration was greater in acid solutions than 16 in neutral solutions. Over the lower range of pH, it was shown that penetration of 2,4-D decreased as the hydrogen—ion concentration of the donor solution decreased. It has been shown by Kuiper (58) that the effect of decenylsuccinic acid and a few of its mono-amides on growth retardation of beans depended on pH level. Jyung and Wittwer (53) reported a depression in absorption of phosphate with tris-phthalate and the sodium—acetate systems into bean leaves buffered at pH 4.7. They reported that "specific absorption" was greatest at a pH of 3.7. Shindo and co-workers (88) found that the penetration rate of S-benzoyl-thiamine into red blood cells diminished markedly with de— creasing pH of the medium. While working with enzymatically isolated pear cuticle, Bukovac and Morris (11) found that greater retention of napthaleneacetic acid (NAA) occurred at pH levels below than above its pK. There was no significant change in retention of napthaleneacetamide (NAAm) over a wide range of pH levels. Their data indicated that NAA retention by the pear leaf cuticle was pH dependent whereas NAAm was not. Van Overbeek (98) suggested that the pH effect from penetrating chemi- cals was on plant protein rather than on the donor substance applied. Experiments by Volk and McAuliffe (101) suggested that sodium hydrox- ide increased the permeability of the cuticle. Orgell (74) reported that the cuticle does not have a charge at a pH lower than 5.0, while at a pH above 7.0 the cuticle is negatively charged. This contrasted to the work by Yamada (106) who reported that cuticles from green onion 17 leaves have a pK value of 2.8, and from ripe tomato fruits, a pK value of 3.2. Bukovac (personal communication) found that the pK value of pear cuticle was 2.8-3.0. Therefore, it seems that pK regulated elec— trostatic repulsion and attraction phenomena affect cuticular sorption. and, consequently, the rate of foliar uptake of ions. However, Bukovac and Norris (11) found that NAA binding took place in cuticle of pear leaves at pH values greater than its pK of 4.2. They implied that mechanisms other than electrostatic binding were involved in chemical binding to cuticular surfaces. Size of'moZecuZe and penetration. Franke (37) reported the cuticle was negatively charged and these charges were first neutralized by cations. Goodman (41) reported that application of cations as Ca++ and Mn++ brought about dehydration and this increased the entry of larger cationic molecules. Cations such as Ca++ and Mn++ penetrated rapidly because of their small ionic radius. Franke (37) reported that the rapid penetration of small ions facili- tated the passage of larger cationic molecules. Craig (21) sug- gested that molecular size and structure of solutes could be corre- lated with diffusion rates through calibrated dialyzing membranes. Harley gt_al, (46) reported that certain 2,4—D formulations gave in— creased effectiveness in the following order: sodium salt, ammonium salt, amine, and then the ester formulation. Dorschner and Buchholtz (27), Holly (49), Muir and Hansch (68), and Veldstra (100) studied the structure-activity relationship of N-substituted alpha-chloroacetamides. 18 The influence of slight structural changes on activity was often striking. Penetration may have been a controlling factor in these changes, but there is little direct evidence that this was true. MATERIALS AND METHODS Because little information is available regarding sorption, penetration, and uptake of exogenously applied compounds as they re— late to molecular structure, this study was conducted to evaluate this effect using isolated tomato fruit cuticles and excised bean leaf disks. Four radiolabeled (carbonyl-1“C) compounds having the general chemical structure were donated by The Upjohn Company, Kalamazoo, Michigan. These com- pounds, along with the relevant chemical properties, are shown in 9 CH3 Table l. The compound where R = -C-N: is a well-known herbicide CH3 named diphenamid (N,N-dimethyl-2,2-diphenylacetamide). The other 3 are derivatives of diphenamid and not commercially available as herbi- cide products. Tomato (Lycoperaicon escuZentum L. cv. Michigan-Ohio Hybrid) plants were fruited in the greenhouse at Michigan State University. The astomatous cuticular membranes were enzymatically separated from ripe tomato fruits using the method described by Norris and Bukovac (72). 19 20 _s\me mm_.o econ oebaoapscaeaeo 4N.N_N Iomu-u-: Pa\ms o__.o aa_5abaoa_seacaeo mN.__N nuzmwuu-z m _s\me Nmo.o mneEabmua_sca;awu-N.~-.sgbas-z mm.mNN :mvz-u- -1 mvPEmumom mzu/ _e\me om~.o -_»=a;aea-u.m-.sgpaseu-z.z .m.mm~ mzuxzmum .: goam a boom: asaz .aoweagu “capo: mcsbuacum cw xuw__a=pom capaumpoz gmpzumpo: .gumm we mmwugoaoga u=m>mpmg mcu new cowummwemm>cp mega cw now: mucaoaeou umpmnmpo_umm .F mpnwp 21 A liquid scintillation spectrometer, Packard Tri-Carb Model 314EX1 was used to detect radioactivity. Thin layer chromatography procedures were used to identify N,N-dimethyl-2,2-diphenylacetamide which pene- trated isolated tomato fruit cuticle. Developing solvent was benzene- chloroform-acetic acid mixture 85:10:5 (v/v); the absorbant was silica gel GF. Defim' tions . In this study sorption refers to the compound that is bound to the cuticular components, either by absorption or adsorption, and is not removed by washing with distilled water. Penetration refers to the process of a compound traversing the cuticle. Uptake refers to the compound that penetrates the cuticle and enters the metabolically active cells and may also involve some movement from cell to cell. Relation of Molecular Structure to Sorption. Before cuticular penetration of a compound can take place, it must first pass through the various wax and cutin components of the cuticle. It is reasonable to assume that some of the compound applied to the cuticular surface would be retained by the cuticle. A series of experiments was therefore designed to determine the influence of molecular structure on cuticular sorption. Cuticular disks (10 mm in diameter) were cut from isolated ripe tomato fruit and placed in glass vials. One milliliter donor solution was added to each vial. All disks were completely submerged in the 1Packard Instrument Company, Inc., LaGrange, Illinois. 22 donor solution. Each treatment was replicated six times and held at 25°C. After a predetermined length of time, each cuticle was removed from the donor solution, blotted and rinsed three different times in distilled water. After the third rinse, each cuticular disk was dried at ambient temperature and placed in a 20 ml Wheaton glass scintil- lation vial. Fifteen milliliters solvent mixture (Table 2) was added. These vials were then placed in a liquid scintillation spectrometer for a counting time of 30-90 minutes. An internal standard consisting of 50 ,ul 1“C-Toluene was added to the sample and recounted to deter- mine counting efficiency and disintegrations per minute (0PM). Counting efficiency obtained this way was approximately 68 percent. Table 2. Scintillation solvent mixture used in the isolated tomato fruit cuticle penetration experiments. Solvent Amount Source Napthalene (mp 80-81°C) 73 gm Matheson, Coleman & Bell Cincinnati, Ohio Liquifluor (25x) 50 ml Pilot Chemicals, Inc. Watertown, Mass. 1,4-Dioxane 350 ml Fisher Scientific Company Fair Lawn, New Jersey Toluene 350 ml Allied Chemical, Industrial Division Morristown, New Jersey Methyl Alcohol (anhydrous) 210 ml Mallinckrodt Chemical Works New York, N. Y. 23 Influence of_time on sorption. A time course study was conducted to determine the amount of com- pound retained by the cuticle as influenced by time. Concentration of the donor solution for each of the four compounds was 1.05 x 10'5 M/l. Each donor solution was buffered to pH 3.0, 4.0, or 5.0 (Table 3). After the predetermined length of time had elapsed (3-24 hours), each cuticle was removed from the donor solution and the DPM was determined as above for each sample. Table 3. Citric acid-phosphate buffer solution used to maintain stated pH level. Citric Acid NA Hpot pH Level (0.1 M) (0?2 M) 3.0 39.8 mi 10.2 ml 4.0 30.7 ml 19.3 mi 5.0 24.3 mi 25.7 mi 6.0 17.9 mi 32.1 ml 7.0 6.5 mi 43.6 mi Influence of pH on sorption. A series of experiments was conducted to determine the influence of donor solution pH on sorption into isolated tomato fruit cuticles. Disks from tomato fruit cuticle were placed in vials containing 1 ml donor solution at 1.05 x 10'5 M/l concentration. The cuticular disks remained submerged in the donor solution for 24 hours. Retention of each compound by the cuticle was first determined without a surfactant Table 4. 24 Trade names and a summary of the relevant physical and chemical properties of the surface active agents used in this study. Surface Active Agent Tergitol 15-5-9 Tween 20 Surfactant DFl6 Source Ionogenic Class Union Carbide Corp. New York, N.Y. nonionic Appearance clear liquid Viscosity 68.8 c.p.s. (cps. 0 25°C) Specific Gravity 1.000 0 25°C Density 999.29 g/l Chemical Ethoxylated Classification Alcohol Atlas Chemical Industries nonionic yellow liquid 363 c.p.s. 1.10 1098.74 g/l Ethoxylated fatty acid ester Rohm & Haas Co. Philadelphia, Pa. nonionic clear liquid 35 c.p.s. 0.987 987.31 g/1 Ethoxylated & Propylated Al- cohol (linear alcohol) 25 and then with Tergitol 15-S-9 added at 0.3% (v/v). After 24 hours, each cuticular disk was removed from the donor and radioactivity determined. Influence of surfactant concentration on sorption. Tomato fruit cuticle disks (10 mm diameter) were placed in donor solutions containing 0.1, 0.2, 0.3, or 0.4 percent Tergitol 15-5-9 surfactant (Table 4). Sorption into these was compared with disks from standard donors which did not contain a surfactant. Donor solution concentration was 1.05 x 10'5 M/l and was buffered to pH 4.0. After 24 hours, each disk was removed from the donor solution, blotted and rinsed three times, and allowed to dry at ambient temperature before radioactivity was determined. Relation of Molecular Structure to Penetration. A series of experiments was conducted to evaluate the relation- ship of molecular structure to penetration. Disks, 15 mm in diameter, were obtained from astomatous ripe tomato fruit cuticle which had been removed enzymatically. The apparatus used (Figures 2 and 3) was constructed using two L-shaped components from glass tubing of 5 mm inside diameter. The overall length of each component was approximately 12 mm. A flange was made on one of the tubes to which a 1 oz. polyethylene bottle cap was affixed by inserting the tube through a hole punched in the cap. This tube served as the donor tube and will hereafter be referred to as the donor tube. The neck (male part) of a 1 oz. polyethylene bottle was fused onto the second L-shaped component by heating the glass and then 26 Figure 2. Apparatus used to study the relation of molecular structure to cuticular penetration. Receiver side Donor side Tomato fruit cuticle A B C D Teflon washers 28 Figure 3. Set of assembled apparatus for the cuticular penetration studies. 30 forcing the polyethylene neck onto it. Two teflon washers (Figure 2), which had previously been dipped in melted petroleum jelly, were placed on each side of the tomato disk. The teflon washers had 14 mm outside diameters and 9 mm inside diameters. The 2 L-shaped tubes were joined with the cuticle fastened between the donor and receiver parts of the apparatus. Outer surface of the cuticle was always in contact with the donor solution. Each treatment was replicated six times and held in an air-conditioned laboratory at 25 :_l°C. After a predetermined time, an aliquot was removed from the receiver side of the tube. Each aliquot was placed in a 20 ml Wheaton glass scintillation vial. To this was added 15 m1 of a scintillation solvent mixture (Table 2). Counting time in a liquid scintillation spectrometer was 30-90 minutes for each sample, depending upon the level of radioactivity found; those samples with high levels were counted for the shorter period of time. An in- ternal standard of 1“C-Toluene (5.10 x 10’5 dpm/g :_3%) was added to the counted sample and recounted to determine the efficiency of counting. For these experiments an efficiency of 60 :_1 percent was obtained. Influence of time on penetration. A series of experiments was conducted to compare the rate of pene- tration of diphenylacetic acid and its 3 diphenylacetamide derivatives as influenced by time. The concentration of the donor solution of the compounds was 1.05 x 10"5 M/l. Each solution was buffered to pH 4.0, the pK of diphenylacetic acid being 3.94. A surfactant (0.3% Tergitol 15-5-9) was added to each donor solution. Four milliliters (ml) of the 31 concentration was used as the donor solution and 4 ml distilled water served as the receiver solution. After the predetermined time (3-24 hours), 0.5 m1 aliquots were removed from the receiver and donor and the radioactivity determined. Influence of donor pH on penetration. Several experiments were conducted to determine the influence of donor pH on the penetration of different molecular structures through tomato fruit cuticles. Donor solutions were used at a concentration of 1.05 x 10‘5 M/l and buffered to pH 3.0, 4.0, or 5.0 with a citric acid-phosphate buffer as listed in Table 3. The influence of donor pH on cuticular penetration was compared first without a surfactant, and then with Tergitol 15-3-9 surfactant at 0.3% (v/v). After 24 hours, a 0.5 m1 aliquot sample was taken from the receiver solution and the amount of radioactivity determined as above. Influence of surfactants on penetration. The influence of surfactants on the penetration of the 4 com- pounds through isolated tomato fruit cuticle was determined using several different nonionic surfactants (Table 4). Technical data were obtained either from brochures provided by the respective companies, or from personal communication with formulation chemists. In all ex- periments, the concentration of surfactant added to the donor solution ranged from 0.05% to 0.4% (percentages were based on a volume to volume ratio). All donor solutions of the compounds were used at a concentration of 1.05 x 10'5 M/l in distilled water or buffered to 32 pH 4.0. After a predetermined period of time, usually 24 hours, a 0.5 ml aliquot sample was taken from the receiver solution and radio- activity determined. Influence of surface max on penetration. Surface waxes were removed from tomato fruit cuticles to deter- mine whether these influence penetration of the compounds under in- vestigation. Cuticular disks of 15 mm diameter were allowed to remain submerged in chloroform for 24 hours. They were then rinsed in dis- tilled water, blotted with a Kimwipe and allowed to dry at ambient temperature in the laboratory. The apparatus and experimental pro- cedures used were the same as described earlier (Page 25). Penetration was permitted to proceed for 24 hours and the level of radioactivity was determined and compared to the donor solution. Relation of Molecular Structure to Uptake. In order to compare a metabolically active system with isolated tomato fruit cuticles, bean plants (Phaseolus lunatus cultivar Henderson Bush) were grown in vermiculite in the greenhouse. Disks 15 mm in diameter were cut from smooth, fully expanded primary leaves immediately upon removal of a leaf from the plant. Care was exercised to avoid removing disks containing part of the midrib or large veins. Immediately after cutting, 6 leaf disks were placed on Whatman No. 4 filter paper in a 9 centimeter petri dish. One milliliter of distilled water was used to moisten the filter paper and maintain cell turgidity. At no time were the leaf disks permitted to become flaccid. Glass 33 cylinders 13 mm high and of 9 mm inside diameter were affixed to the bean leaf disks by placing a small bead of petroleum jelly on the cylinder end in contact with the disk. A donor solution (0.1 ml) was added to each cylinder. All treatments were replicated six times and held at approximately 25°C. After a predetermined length of time (1 to 24 hours), the cylinders were removed and the excess donor solution washed off with distilled water. Each leaf disk was cleansed with a cotton ball saturated with xylene (mixed isomers) to remove epicuticular waxes and any 1L‘C-labeled donor material binding to the surface waxes. Remaining chemical would be that in the cuticle and which had been taken up by the living cells of the bean leaf disk. After cleansing with xylene, the disks were allowed to dry at ambient temperature for 10-15 minutes. The entire leaf disk was then placed in a glass Wheaton scintillation vial containing 15 ml solvent mixture (Table 5). Samples were then placed in a Packard Tri-Carb liquid scintillation spectrometer and counted for a period of 30-90 minutes each. Fifty microliters of 1‘iC-Toluene internal standard (5.10 x 10'5 dpm/g :_3%) was added and the sample recounted to obtain counting efficiency and disintegrations per minute. The figure obtained for counting efficiency was approximately 60%. Influence of time on uptake. A time course study was conducted to determine the relationship between time and uptake of the four compounds. The concentration of the donor solution for each compound was 1.05 x 10"5 M/l, buffered to [H 4.0. After the predetermined times of 1, 3, 6, 12, or 24 hours 34 had elapsed, the amount of radioactivity was determined for each bean leaf disk. Table 5. Scintillation solvent mixture used for counting samples from the bean leaf uptake experiments. Solvent Amount PPOl POPOP2 Source Liquifluor 25x 63 ml 7.3 gm 93.75 mg Pilot Chemicals, Inc. Watertown, Mass. Toluene 937 ml - - Allied Chemical, Industrial Division Morristown, New Jersey 12,5-Diphenyloxazole 2l,4-Bis-2-[5-phenyloxazolyl]benzene Influence of'pH on uptake. A series of experiments was conducted to determine the influence of donor pH on uptake of compounds by green bean leaf disks. The donor concentration for each compound was 1.05 x 10"5 M/l, buffered to pH 3.0, pH 4.0, or pH 5.0. Tergitol 15—S-9 nonionic surfactant (0.3% v/v) was added to each donor solution. The influence of donor pH was com- . pared 6 and 24 hours later by determining the radioactivity in the leaf disks. Influence of surfactant on uptake. Disks of 15 mm diameter were cut from fully expanded green primary leaves of bean plants. These were then placed on moist filter paper in a 9 cm petri dish. Donor solutions of 1.05 x 10'5 M/l 35 concentration were applied to six green bean leaf disks for each treat- ment. A nonionic surfactant (Tergitol 15-S-9) was added to each com- pound at a concentration of 0.1, 0.2, 0.3, or 0.4 percent (v/v). After a predetermined period of time (usually 6 or 12 hours), each leaf disk was placed in a scintillation solvent mixture and the disintegrations per minute determined as described above. Relation of Molecular Structure to Partitioning. The plant cuticle is primarily a layer of cutin composed of cross-linked hydroxy fatty acid bound by a layer of wax. According to Eglinton and Hamilton (29) the surface waxes are complex mixtures of long-chain alkanes, alcohols, ketones, aldehydes, acetals, esters, and acids. The solubility of externally applied compounds in these cuti- cular substances is involved in sorption. This experiment was con- ducted to evaluate the degree of partitioning of N,N-dimethyl-2,2-di- phenylacetamide, N-methyl-2,2-dipheny1acetamide, diphenylacetamide, and diphenylacetic acid from an aqueous solution into two organic sol- vents. Chloroform and oleic acid were used as the organic solvents. Donor aqueous solutions were buffered to pH 4.0 or pH 7.0 with citric acid-phosphate buffer solution (Table 3). Equal portions (1.5 m1) of the donor and solvent were placed in a 2 ml Kimax volumetric flask and then sealed with a ground glass stopper. Each treatment was replicated 5-6 times. At the beginning of the ex- periment, each flask was agitated for 5 seconds using a $8223 Vortex Genie Mixer set at speed No. 9. Another agitation, identical to the first, was given 22 hours later. A 0.5 m1 aliquot was taken from both 36 the water and organic phases 24 hours after the beginning of the ex- periment. The upper phase was always sampled first. An aliquot from the lower phase was obtained by carefully inserting a 0.5 ml Kimax volumetric pipette through the upper phase while forcing air through the pipette to minimize contamination. Each pipette was wiped with a Kimwipe prior to placing the aliquot in a 20 m1 Wheaton glass scin- tillation vial. Fifteen milliliters scintillation solvent (Table 2) was added to each vial. Samples were then placed into a Packard Tri- Carb liquid scintillation spectrometer and counted for 30-90 minutes. Background counts were obtained using a scintillation solvent sample containing 0.5 m1 aliquot chloroform, oleic acid and water, depending upon the phase being counted. An internal sample of 50 /ul 1”C- Toluene (5.10 x 10’5 dpm/g :_3%) was added to each sample before re- counting to determine efficiency. smug? RESULTS Relation of Molecular Structure to Sorption. Influence of time on sorption. The quantity of diphenylacetic acid sorbed into enzymatically isolated tomato fruit cuticles is shown in Figure 4-IV. There was a rapid increase in sorption for the first 6 hours, followed by a leveling-off or decline with increasing time. The latter character- istic was more evident at pH values of 4.0 or pH 5.0 than when di- phenylacetic acid was buffered to pH 3.0. A summary of the sorption of N,N-dimethyl-2,2-diphenylacetamide, N-methyl-2,2-diphenylacetamide, and diphenylacetamide into tomato fruit cuticles is shown in Figures 4-1, 4-II, and 4-III. In general, sorption of the three amide molecules increased with time. The sorp- tion was rapid during the first 3 to 6 hours and increased 3-fold by the end of the 24-hour treatment. The descending order of sorption of the amide molecules was N,N-dimethyl-2,2-diphenylacetamide, N-methyl-2,2-diphenylacetamide, and diphenylacetamide respectively. Influence of pH on sorption. The influence of donor pH on the sorption of the 4 different compounds into tomato fruit cuticles is shown in Figure 5. The amount of N,N-dimethyl-2,2-diphenylacetamide, N-methyl-2,2-diphenylacetamide, 37 38 Figure 4. Comparative rates of cuticular sorption of different compounds through isolated tomato fruit cuticles as influenced by time at different pH levels. I. N,N-dimethyl-2,2-diphenylacetamide II. N—methyl-2,2-dipheny1acetamide III. Diphenylacetamide IV. Diphenylacetic acid ——-—_—..__ 39 --- pH 4.0 - - pH 5.0 U Q. I N ny— c _ C _ H I \ G o o. 3 oumm0m hzmummm 2.0 F 24 12 l2 TIME IN HOURS Figure 5. 40 Influence of donor pH on the sorption of different compounds into isolated tomato fruit cuticles, with and without a surfactant, 24 hours after treatment. I. II. III. IV. N,N-dimethyl—2,2-dipheny1acetamide N-methyl-2,2-diphenylacetamide Diphenylacetamide Diphenylacetic acid 41 O T , mu , um“ I cc MM RHR I U” 0. . ll . ww Wu 1. H . . A... . __ . _ pm . - _.__ ©é© . H" p r b h b .4 a a _ , _ . . . . . . a 1 & I a. a. . , W W . H" 7L , I x mm x x . , W m . I OI OI _ 0 ”Jo .. H4 0 Q... 9.. n or p b b b - E n u \\ b h b o. o. o. o. o. o o. 0. o.//o. 0. 0. ea 4. .5 9. .I 7. .0 so as 9. .l ommmow hzwomum 5!) 41) 3x1 51) 4x1 31) pH 42 or diphenylacetamide sorbed into the cuticle was not affected by the different pH levels in these experiments (Figures 5-I, 5-II, and 5-III). However, there was a highly significant difference (Appendix A) in the amount of diphenylacetic acid sorbed into the tomato fruit cuticles from donors buffered at different pH levels (Figure 5-IV). More diphenylacetic acid was sorbed into the cuticular membrane at pH 3.0 and 4.0 than at pH 5.0. The amount of diphenylacetic acid sorbed was inversely proportional to the pH levels used; e.g., 6.8% at pH 3.0, 4.9% at pH 4.0, and 1.8% at pH 5.0 (Appendix B). Similar sorption curves were obtained at the different pH levels for each molecular structure regardless of whether or not a surfactant was used. Influence of surfactant on sorption. Significantly less radiolabeled compound sorbed into the tomato fruit cuticles when a surfactant was used (Figure 6 and Appendix B). All concentrations (0.1% to 0.4% v/v) of the nonionic surfactant Tergitol 15-S-9 decreased retention of the 4 different compounds com- pared to the quantity sorbed without the addition of a surfactant. The amount sorbed by the cuticles 24 hours after treatment with a sur- factant was one and one-half times less for N,N-dimethyl-2,2-diphenyl- acetamide, and approximately two and one-half times less for N-methyl- 2,2-diphenylacetamide, diphenylacetamide, and diphenylacetic acid than from donors without a surfactant. 43 Figure 6. The concentration influence of the nonionic surfactant Tergitol 15-S-9 on the sorption of different compounds into tomato fruit cuticles, 24 hours after treatment. I. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-diphenylacetamide III. Diphenylacetamide IV. Diphenylacetic acid PERCENT SORBED 51) 41) 31) 21) 'o C’ 5’ o z“ o 04 O 21) L0 44 PERCENT SURFACTANT ' I P 11 ‘ 11-0365:wa - "-0.“ EN’" (1) CH3 G \cn3 . N _ \ V—N v .__1_. 1 1 1 1 __1_, 1 1 1 III . 11 \ ' rec-"411:: t H—C-‘é’gou . . 9 . \ F i K/‘N b ,___4‘ 1 1 1 1 ___J_ 1 1 1 1 NONE 0.1 0.2 0.3 0.4 NONE 0.1 0.2 0.3 0.4 45 Relation of Molecular Structure to Penetration. Influence of time on penetration. The penetration of N,N-dimethyl-2,2-diphenylacetamide, N-methyl 2,2-diphenylacetamide, diphenylacetamide, and diphenylacetic acid through enzymatically isolated tomato fruit cuticles is illustrated in Figure 7. Penetration of all compounds increased with time. At first there was a gradual increase in penetration rate for all com- pounds, and then a more rapid linear increase from 6 to 24 hours. In all instances, significant quantities (Appendiin) of diphenylacetic acid molecule penetrated at a more rapid rate than diphenylacetamide, N-methyl-2,2-diphenylacetamide, or N,N-dimethyl-2,2-diphenylacetamide. The descending order of penetration for these compounds was diphenyl- acetic acid, N,N-dimethyl-2,2-diphenylacetamide, N—methyl-2,2-diphenyl- acetamide, and diphenylacetamide respectively. Influence of pH on penetration. There was a highly significant difference (Appendixll) in the i amount of diphenylacetic acid which penetrated isolated tomato fruit 5‘ cuticles from donor solutions buffered to different pH levels (Figure 8). Greater amounts of diphenylacetic acid penetrated the cuticular mem- brane at pH values below than above the pK value (3.94). There was a 2- to 4-fold increase in penetration when a sur- factant was added to the donor solution (Figure 8). Still, the in- fluence of pH was evident regardless of whether or not a surfactant was used. However, the pH level used in these experiments appeared to Figure 7. 46 Penetration of different compounds through cuticles enzymatically isolated from tomato fruit, as in- fluenced by time. I. II. III. IV. N,N—dimethyl-2,2-diphenylacetamide N-methyl-2,2-diphenylacetamide Diphenylacetamide Diphenylacetic acid 36 24 .b N (3:0 §c: ‘: ,mouzs x 10‘5 01 a: 24 12 42 47 9 30- I l O I O I Z / I I T l l l l I 3 6 l2 24 3 6 l2 24 TIME IN HOURS Figure 8. 48 The influence of donor on penetration of different compounds, with and wit out a surfactant, through en- zymatically isolated tomato fruit cuticles, 24 hours after treatment. I. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-diphenylacetamide III. Diphenylacetamide IV. Diphenylacetic acid 42- 36 30- 24 a: O 111101.53 x 10“5 A N OI as 8 24 l2 N U # V T 49 30 41) 50 pH -— WI SURFACTANT --- W/O SURFACTANT 0 ----- 0-----0 1 1 1 II a? H-C-C-OH 0‘ \\\ ‘\ \ \ \ x \0 1 1 1 30 41) 5O 50 have little to no effect on penetration of the N,N—dimethyl-2,2-di- phenylacetamide, N-methyl-2,2-diphenylacetamide, and diphenylacetamide. At a pH level of 4.0, the amount of diphenylacetic acid which pene- trated was twice that for N,N-dimethyl-2,2-dipheny1acetamide and N-methyl-2,2-diphenylacetamide, and three times that for diphenylaceta- mide. Influence of surfactants on penetration. The addition of the nonionic surfactant Tergitol 15-S-9 to the donor solution promoted penetration of all four compounds through iso- lated tomato fruit cuticles. This was independent of the pH level oi the donor solution. Note that the penetration pattern of each com- pound was the same for all pH levels tested with the variation being amount, as influenced by the addition of the surfactant. Several nonionic surfactants were evaluated to determine their influence on penetration of N,N-dimethyl-2,2 diphenylacetamide through tomato fruit cuticles. The results are illustrated in Figure 9. These data show that after 24 hours all surfactants increased penetration. However, Tergitol 15-S-9 gave the greatest increase in penetration; Tween 20, the least; while surfactant DF-l6 was intermediate. This order of penetration was similar for both the 6 and 24-hour treatments. Tergitol 15-S-9 surfactant (0.1% v/v) resulted in penetration of N,N- dimethyl—2,2-diphenylacetamide after 6 hours being equal to 24 hours without a surfactant (Figure 9). The influence of concentration of the surfactants Tween 20 and Tergitol 15-S-9 is shown in Figures 10 and 11. These data again show 51 Figure 9. Influence of different nonionic surfactants (0.1% v/v) on penetration of N,N-dimethyl-2,2-diphenylacetamide through isolated tomato fruit cuticles. hzIVldfl lNBOUEd 68 Influence of donor pH on uptake of 4 different com- pounds into green bean leaf disks, 6 and 24 hours after treatment. Figure 16. I. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-diphenylacetamide III. Diphenylacetamide IV. Diphenylacetic acid 69 50 41) 31) m m 4 U U _ o O _ HUN _ x 4.6 s 2 _ s . _ x _ . 1. .. .. flu . . mu . 3 . . H” n . Hun» . M. u m o. nqu MC _ _ ©c© Qwé m n r p p p p p p - pI LI P n n n 4 1 4 a . I — . . . . . .4 I . m . . . 23 a. . H H _ C C . H H . xNx _ sz . . ll . hdflv OW? _ . Qc Q... _ _ H” “H p n n n p n b b .I b L n p L A. 11 .6 .0 9. 00 no .3 nu 4. .l n. :0 9. .0 9. 9. l. l. I. _| .I .I mx> Varenna, 5-10 Settembre. Clor, M. A., A. S. Crafts, and S. Yamaguchi. 1962. Effects of high humidity on translocation of foliar applied labeled compounds in plants. Part I. Plant Physiol. 37: 609-617. . 1962. Effects of high humidity on translocation of foliar applied labeled compounds in plants. Part II. Translocation from starved leaves. Plant Physiol. 38: 501-507. Cory, E. N. and G. S. Langford. 1935. Sulfate alcohols in in- secticides. Jour. Econ. Ent. 28: 257-260. Crafts, A. S. 1948. A theory of herbicidal action. Science 108: 85-86. 1953. Herbicides. Ann. Rev. Plant Physiol. 4: 253-282. ° 1956. The mechanism of translocation: Methods of study with C-labeled 2.4-0. Hilgardia 26: 287-334. 1956. Translocation of herbicides. I. The Mechanism of translocation: Methods of study with 1L*C labeled 2,4-0. Hilgardia 26: 287. , and C. L. Foy. 1962. The chemical and physical nature of plant surfaces in relation to the use of pesticides and to their residues. Res. Rev. 1: 112-139. , and H. G. Reiber. 1945. Studies on the activation of herbicides. Hilgardia 16: 487-500. Craig, L. C. 1964. Differential dialysis. Science 144: 1093- 1099. Currier, H. B. 1954. Wetting agents and other additives. Proc. 6th Calif. Weed Conf., Sacramento. pp. 10-15. , and C. B. Dybing. 1959. Foliar penetration of herbicides - Review and present status. Weeds 7: 195-213. , E. R. Pickering, and C. L. Foy. 1964. Relation of stomatal penetration to herbicidal effects using fluorescent dye as a tracer. Weeds 12: 301-303. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 95 Daines, R. H., R. J. Lukens, E. Brennan, and I. A. Leone. 1957. Phytotoxicity of captan as influenced by formulation, environ- ment, and plant factors. Phytopathology 47: 567-572. Dickinson, S. 1960. The mechanical ability to breach the host barriers. In Plant Pathology, An Advanced Treatise. Chap. 6.2: 203-232. Horsfall, J. G. and Diamond, A. E., Eds., Academic, New York. Dorschner, K. P. and K. P. Buchholtz. 1956. Wetting ability of aqueous herbicidal sprays as a factor influencing stands of alfalfa seedlings. Agron. J. 48: 59-63. Dybing, C. 0., and H. B. Currier. 1959. A fluorescent dye method for foliar penetration studies. Weeds 7: 214-222. Eglinton, G., and R. J. Hamilton. 1967. Leaf epicuticular waxes. Science 156: 1322-1334. Elworthy, P. H., A. T. Florence, and C. B. Macfarlane. 1968. Solubilization by Surface-Active Agents. Chapman and Hall Ltd. London. 335pp. Ennis, W. B., Jr. 1951. Influence of different carriers upon the inhibitory properties of growth-regulatory sprays. Needs 1: 43-47. , R. E. Williamson, and K. P. Dorschner. 1952. Studies on spray retention by leaves of different plants. Weeds 1: 274-286. Foy, C. L. 1961. Absorption, distribution, and metabolism of 2,2-dichloropropionic acid in relation to phytotoxicity. I. Penetration and translocation of 36Cl- and 1L*C-labeled dalapon. Ann. Review Plant Physiol. 36(5): 688-697. . 1961. Absorption, distribution, and metabolism of 2,2-dichloropropionic acid in relation to phytotoxicity. II. Distribution and metabolic fate of dalapon in plants. J. Plant Physiol. 36(5): 698-709. . 1962. Absorption and translocation of dalapon—2-1“C and 36C1 in Tradescantia fluminensis. Needs 10: 97-100. . 1964. Review of herbicide penetration through plant surfaces. Agr. Food Chem. 12: 473-476. Franke, W. 1967. Mechanisms of foliar penetration of solutions. Ann. Rev. Plant Physiol. 18: 281-300. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 96 Freed, V. H., and Marvin Montgomery. 1959. The effect of sur- factants on foliar absorption of 3-amino-l,2,4-triazole. Weeds 6: 386-389. Furmidge, C. G. L. 1959. Physico-chemical studies on agricul- tural sprays. J. Agric. and Food Chem. 10: 274. Gillette, C. P. 1890. Experiments with arsenites. Iowa Agri- cultural Experiment Station Bull. 10: 401-420. Goodman, R. N. 1962. Antibiotics jg Agriculture. 165-168 (Woodbine, M., Ed.), Proc., Univ. Nottingham, Butterworths, London. Halevy, A. H., and S. H. Wittwer. 1965. Foliar uptake and trans- location of Rubidium in bean plants as affected by root absorbed growth regulators. Planta (Berl.), 67: 375-383. Hall, 0. M., and L. A. Donaldson. 1962. Secretion from pores of surface wax on plant leaves. Nature 194 (4834): 1196. Hamm, P. 0., and A. J. Speziale. 1957. Effect of variations in the acyl moiety on herbicidal activity of N-substituted alpha- chloroacetamides. J. Agric. and Food Chem. 5: 30-36. Hamner, C. L., Lucas, E. H., and H. M. Sell. 1947. The effect of different acidity levels on the herbicidal action of the sodium salt of 2,4-Dichlor0phenoxyacetic acid. Harley, C. P., L. 0. Regeimbal, and H. H. Moon. 1956. Absorption of nutrient salts by bark and woody tissues of apple and sub- sequent translocation. Proc. Amer. Soc. Hort. Sci. 67: 47-57. ., H. H. Moon, and L. 0. Regeimbal. 1957. Effects of the additive Tween 20 and relatively low temperatures on apple thinning by napthaleneacetic acid sprays. Proc. Amer. Soc. Hort. Sci. 69: 21-27. Hauser, Ellis W. 1955. Absorption of 2,4-dichlorophenoxyacetic acid by soybean and corn plants. Agronomy Journal 47: 32-36. Holly, K. 1956. Penetration of chlorinated phenoxyacetic acids into leaves. Ann. Appl. Biol. 44: 295-299. Ilnicki, R. 0., W. H. Tharrington, J. F. Ellis, and E. J. Visinski. 1965. Enhancing directed post-emergence treatments in corn with surfactants. NE Weed Cont. Conf. Proc. 19: 295-299. Jansen, L. L., W. A. Gentner, and W. C. Shaw. 1961. Effects of surfactants on the herbicidal activity of several herbicides in aqueous spray systems. Weeds 9: 381-405. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 97 Juniper, 3- E- 1960. Growth, development, and effect of the environment on the ultra-structure of plant surfaces. J. Linn. Soc. (Bot.), 56: 413-419. Jyung, W. H., and S. H. Wittwer. 1964. Foliar absorption - An active uptake process. Amer. Jour. Bot. 51(4): 437-444. 1965. Pathways and mechanisms for foliar absorption of mineral nutrients. Agric. Sci. Rev. 3(2): 26-36. , and M. J. Bukovac. 1965. Ion uptake by cells enzymically isolated from green tobacco leaves. Plant Physiol. 40(3): 410-414. . 1965. The role of stomata’in the foliar absorption 0f'Rb by leaves of tobacco, bean and tomato. Amer. Soc. Hort. Sci. 86: 361-367. Kamimura, S., and R. N. Goodman. 1964. Influence of foliar characteristics on the absorption of a radioactive model compound by apple leaves. Physiol. Planatarum 17: 805-815. Kuiper, P. J. C. 1967. Surface-active chemicals as regulators of plant growth, membrane permeability, and resistance to freezing. Meded. Landbouwhogeschool Wageningen 67(3): 1-23. Kylin, A. 1960. The accumulation of sulfate in isolated leaves as affected by light and darkness. Bot. Notiser. 113: 49-81. Laning, E. R., Jr., and R. J. Aldrich. 1951. Increasing the effectiveness of herbicides by the addition of wetting agents. Proc. NE Weed Control Conf. 5: 175-180. Leonard, 0. A., and A. S. Crafts. 1956. Uptake and distribution of radioactive 2,4-0 by brush species. Hilgardia 26: 366-415. Leupschen, N. S., and K. G. Rohrbach. 1968. The effect of sur- factants on Botran applications in controlling Rhizopus decay of peaches. Colorado State University Experiment Station Progress Report PR 68-3. Luckwill, L. C., and C. P. Lloyd-Jones. 1962. The absorption, translocation and metabolism of l-Na thaleneacetic acid applied to apple leaves. J. Hort. Sci. 37(3): 190-206. McWhorter, C. G. 1963. Effects of surfactants on the herbicidal activity of foliar sprays of diuron. Weeds 11: 265-269. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 98 Middleton, L. J. 1959. The absorption of fertilizer by leaves. New Biology 30: 91-102. Penguin Books, Harmondsworth, Middlesex. Mitchell, J. W., B. C. Smale, and R. L. Metcalf. 1960. Absorp- tion and translocation of regulators and compounds used to control plant diseases and insects. Advances in Pest Control 3: 359-436. Moore, W. 1921. Spreading and adherence of arsenical sprays. Univ. of Minnesota Technical Bulletin 2. Muir, R. M., and C. Hansch. 1955. Chemical constitution as re- lated to growth regulator action. Ann. Rev. Plant Physiol. 6: 157-176. Mysels, K. J. 1968. Personal communication. R. J. Reynolds Tobacco Company, Winston-Salem, N. C. Neely, P. M., and B. 0. Phinney. 1957. The use of mutant dwarf-l of maize as a quantitative bioassay for gibberellin acti- vity. Plant Physiology Meetings, Stanford University, Stanford, California Proceedings 32: XXXI. Norman, A. G., C. E. Minarik, and R. L. Weintraub. 1950. Herbi- cides. Ann. Rev. Plant Physiol. 1: 141-168. Norris, R. F., and M. J. Bukovac. 1968. Structure of the pear cuticle with special reference to cuticular penetration. Amer. J. Bot. 55(8): 975-983. Orgell, W. H. 1955. The isolation of plant cuticle with pectic enzymes. Plant Physiol. 30: 78-80. . 1957. Sorption properties of plant cuticle. Proc. Iowa Acad. Sci. 64: 189-198. ., and R. L. Weintraub. 1957. Influence of some ions on foliar absorption of 2,4-D. Bot. Gaz. 119: 88. Pallas, J. E., Jr. 1960. Effects of temperature and humidity on foliar absorption and translocation of 2,4-dichlorophenoxyacetic acid and benzoic acid. Plant Physiol. 35: 575-580. Palmquist, E. M. 1939. The path of fluorescein movement in the kidney bean, Phaseolus vulgaris. Am. J. Bot. 26: 665-667. Prasad, R., C. L. Foy, and A. S. Crafts. 1967. Effects of rela- tive humidity on absorption and translocation of foliarly applied Dalapon. Weeds 15: 149-156. Rice, E. L. 1948. Absorption and translocation of ammonium 2,4-dich10r0phenoxyacetate by bean plants. Bot. Gaz. 109: 301-314. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 99 Rich, 5., and J. G. Horsfall. 1952. The relation between fungi- toxicity, permeation, and lipid solubility. Phytopathology 42: 457-460. Roberts, E. A., M. D. Southwick, and D. H. Palmiter. 1948. A micro-chemical examination of McIntosh apple leaves showing re- lationship of cell wall constituents to penetration of spray sOlutions. Plant Physiol. 23: 557-559. Sargent, J. A. 1965. The penetration of growth regulators into leaves. Ann. Rev. Plant Physiol. 16: 1-12. ., and G. E. Blackman. 1962. Studies on foliar penetration. I. Entry of 2,4-dichlorophenoxyacetic acid. J. Exp. Bot. 13(39): 348-368. . 1965. Studies on foliar penetration. II. The r01e oleight in determining the pene- tration of 2,4-dich1orophenoxyacetic acid. J. Exp. Bot. 16(46): 24-27. Scott, F. M. 1950. Internal suberization of tissues. Bot. Gaz. 11: 378-394. ., B. G. Brystron, and E. Bowler. 1962. Carcidium floridium seed coat, light and electron microscooic study. Amer. J. Bot. 49: 821. ., K. C. Hamner, E. Baker, and E. Bowler. 1957. Ultrasonic and electron microscope study of onion epidermal wall. Science 125: 399-405. Shindo, H., Okamoto, K., and Jun-ichi Totau. 1967. Transport of organic compounds through biological membranes. 1. Accumulative uptake of S-Benzoylthiamine by human erythrocytes. Chem. Phar. Bull. 15(3): 295-302. Silva Fernandes, A. M. S. 1965. Studies on plant cuticles. IX. The permeability of isolated cuticu1ar membranes. Ann. Appl. Biol. 56(2): 305-313. Simon, E. W., Roberts, H. A., and G. E. Blackman. 1952. Studies in the principles of phytotoxicity. III. The pH factor and the toxicity of 3,5-dinitro-0-cresol, a weak acid. J. Exp. Bot. 3: 99. Sitte, P., and R. Remier. 1963. Untersuchungen an cuticularen Zellwandschichten. Planta 60: 19-40. Skoss, J. D. 1955. Structure and composition of plant cutic1e in relation to environmental factors and permeability. Botan. Gaz. 117: 55-72. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 100 Smith, L. W., C. L. Foy, and D. E. Bayer. 1966. Structure- activity relationships of Alkylphenol ethylene oxide ether non- ionic surfactants and three water-soluble herbicides. Weed Res. 6(3): 233-242. Staniforth, D. W., and W. E. Loomis. 1949. Surface action in 2.4-0 sprays. Science 109: 628-629. Swanson, C. A., and J. B. Whitney, Jr. 1953. Studies on the translocation of foliar-applied 32P and other radioisotopes in bean plants. Amer. J. Bot. 40: 816-823. Tietz, H. 1954. Hofchen-Briefe 7: l. Tuebner, F. G., S. H. Wittwer, W. G. Long, and H. B. Tukey. 1957. Some factors affecting absorption and transport of foliar-applied nutrients as revealed by radioactive isotopes. Mich. Agric. Exp. Sta. Quarterly Bulletin. 39: 398-415. van Overbeek, J. 1956. Absorption and translocation of plant regulators. Ann. Rev. Plant Physiol. 7: 355-372. 1956. Studies on the relation between molecular structure and penetration of growth regulators into plants. In: Wain, R. L., and F. Wightman (editors), The Chemistry and Mode of Action of Plant Growth Regulators. Pages 205-210. Veldstra, H. 1953. The relation of chemical structure to bio- logical activity in growth substances. Ann. Rev. Plant Physiol. 4: 151-198. Volk, B., and C. McAuliff. 1954. Factors affecting the foliar absorption of 15N-labeled urea by tobacco. Proc. Soil Sci. Am. 18: 308-312. Weintraub, R. L., J. N. Yeatman, J. W. Brown, J. A. Thorne, J. D. Skoss, and J. R. Conover. 1954. Studies on entry of 2,4-D into leaves. Proc. 8th N.E. Weed Control Conference 5. Went, F. W., and M. Carter. 1948. Growth response of tomato plants to applied sucrose. Amer. J. Bot. 35: 95-106. Wittwer, S. H., W. H. Jyung, Y. Yamada, et g1, 1965. Pathways and mechanisms for foliar absorption of mineral nutrients as re- vealed by radioisotopes. Reprint from "Isotopes and Radiation in Soil - Plant Nutrition Studies". Vienna, 1965. Journal Article No. 3621, Mich. Agric. Exp. Station. , and F. G. Teubner. 1959. Foliar absorption of mineral nutrients. Ann. Rev. Plant Physiol. 10: 13-27. 106. 107. 108. 109. 110. 111. 101 Yamada, Y. 1962. Studies on foliar absorption of nutrients by using radioisotopes. Ph.D. Thesis, Kyoto University, Kyoto, Japan. ., M. J. Bukovac, and S. H. Wittwer. 1964. Ion binding 5y surfaces of isolated cuticu1ar membranes. Plant Physiology 39(6): 978-982. ., W. H. Jyung, S. H. Wittwer, and M. J. Bukovac. 1965. The effects of urea on ion penetration through isolated cuticu1ar membranes and ion uptake by leaf cells. Amer. Soc. Hort. Sci. 87: 429-432. ., H. P. Rasmussen, M. J. Bukovac, and S. H. Wittwer. 1966. Binding sites for inorganic ions and urea on isolated cuticu1ar membrane surfaces. Amer. J. Bot. 53(2): 170-172. ., S. H. Wittwer, and M. J. Bukovac. 1964 . Penetration of ions through isolated cuticles. Plant Physiol. 39: 28-32. 1965. Penetration of organic compounds through isolated cuticular membranes with special reference to 1“C urea. Plant Physiol. 40(1): 170-175. APPENDICES 102 Appendix A. Influence of donor pH on the sorption of different compounds into isolated tomato fruit cuticles, 24 hours treatment time. 0000? Compound 1! pH 1 II III IV Percent Sorbed* 3.0 3.7 3.2 2.5 6.8 a 4.0 4.2 3.1 2.4 4.9 b 5.0 4.5 3.9 2.4 1.8 c * Numbers followed by unlike letters are significantly different, H50 (95%). 1] Compounds: I. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-diphenylacetamide III. Diphenylacetamide IV. Diphenylacetic acid 103 Appendix B. The concentration influence of the nonionic surfactant Tergitol 15-S-9 on the sorption of different compounds into tomato fruit cuticles, 24 hours after treatment. fi Percent Compound I! Surfactant I II III IV Percent Sorbed* None 3.1 a 3.1 a 2.1 a 4.8 a 0.1 2.4 b 1.3 b 0.9 b 2.5 b 0.2 2.4 b 1.1 b 0.7 b 1.4 c 0.3 2.4 b 1.4 b 0.9 b 1.7 c 0.4 2.3 b 1.6 b 0.8 b 1.5 c *Numbers followed by unlike letters are significantly different, H50 (95%). 1] Compounds: I. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-dipheny1acetamide III. Diphenylacetamide IV. Diphenylacetic acid 104 Appendix C. Penetration of different compounds through tomato fruit cuticles as influenced by time. Time in Compound 1/ “°“”5 I II III IV ,liMoles x 10'5* 3 0.25 c 1.63 C 0.08 b 2.19 c 6 2.61 c 2.64 be 1.02 b 6.55 c 12 8.26 b 7.76 b 4.36 b 20.25 b 24 23.06 a 17.85 a 15.27 a 44.21 a *Numbers followed by unlike letters are significantly different, H50 (95%). 1] Compounds: I. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-diphenylacetamide III. Diphenylacetamide IV. Diphenylacetic acid 105 Appendix D. The influence of donor pH on penetration, 24 hours after treatment. Donor Compound 1] pH I II III IV ‘,MM01es x 10'5* 3.0 17.51 17.86 10.26 41.07 a 4.0 16.79 18.06 12.55 37.09 a 5.0 17.54 15.19 12.15 30.15 b *Numbers followed by unlike letters are significantly different, H50 (95%). 1] Compounds: I. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-diphenylacetamide III. Diphenylacetamide IV. Diphenylacetic acid 106 Appendix E. Influence of surfactant Tergitol 15-S-9 on uptake of N,N-dimethyl-2,2-diphenylacetamide into green bean leaf disks, 6 and 12 hours treatment time. Percent Time in Hours Surfactant 6 12 Percent uptake* None 5.58 a 7.10 a 0.1 8.65 b 10.31 b 0.2 8.19 b 11.83 b 0.3 7.25 b 10.31 b 0.4 8.56 b 9.01 b *Numbers followed by unlike letters are significantly different, H50 (95%). 1] Compounds: 1. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-dipheny1acetamide III. Diphenylacetamide IV. Diphenylacetic acid 107 Appendix F. Relation of molecular structure and pH to partitioning into different organic solvents from aqueous donor solutions, 24 hours after treatment. Organic Compound l/ Solvent 0” I II III IV Percent* Chloroform 4.0 99.16 94.98 95.21 94.47 b 7.0 99.01 97.61 96.88 66.17 a Oleic Acid 4.0 93.54 94.85 94.80 92.89 b 7.0 98.51 95.77 97.50 97.79 b * Numbers followed by unlike letters are significantly different, HSD (95%). 1/ Compounds: I. N,N-dimethyl-2,2-diphenylacetamide II. N-methyl-2,2-dipheny1acetamide III. Diphenylacetamide IV. Diphenylacetic acid ‘- -. h.- .I- 1. I1. - L —'-.F IL; 0311110 . 'o 31 131110 II cup 11. 2 1.10 mm same (IO/W OJ. 13.10991 JnoA 11101; 31102001113 31X08 1080.138 NI BOV'Id 1 131533111111“ 93933 uofigqagw _ Awash MICHIGAN STATE UNIV. LIBRARIES IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 31293006206670