V _. in ism,” , H A . .. mausmfflm , ,. . L DEL 7P;Sgrmw _ . x. .. . .. C I. _ Lew :7 : A L :_ . Luminmyma , MPlvmmn‘ : __ » Cull ‘0 0V. _ gsc .1... A . t. V .V .4, . , M L. L . Jwflumhxzfi...» _:1‘.....-..‘.....zxza‘......m...,...n.». z,. ..v. wan..,.;....,.,._..o4. Lu. “HE.VH..N..L.H,.VNM~M: Ly L 5;...2? Em... ...s....‘..;i.z..2.9.: : :3233%....2§.xox ‘ LIBRARY Michigan State University I//I/////7L7I]/7/i/]/f/i/7//YI7/fl7//7i'Y/Q/ZfiQ/i/Zfl/M "" uni $381.}. L, “'16 W!) '9. L I B RA R Y Michigan State University This is to certify that the thesis entitled SORPTION OF METHYLENE BLUE AND 2,h—DICHLOROPHENOXYACETIC ACID BY ISOLATED TOMATO FRUIT CUTICULAR MEMBRANE presented by Ronald Dean Morse has been accepted towards fulfillment of the requirements for Ph .1) o deg-cc in HortiCUlture )6&upnmusn Date July 199 1971 0-7639 uuIlawman]inuunnn’mmmnmmm 3 1293 10594 0781 V3) .r‘ .1 “1/ w L ‘ “In”. 11" '0”. .. .i' l ”’0.“ : ‘ ‘ .’ iLr‘n‘fL—‘ib v») “a; ' . /‘. ‘m‘i'd a" ‘...J " 7"?)5‘1‘27 if]: (T9 W1 LUV .. -fl-l . ABSTRACT SORPTION OF METHYLENE BLUE AND 2,4—DICHLOROPHENOXYACETIC ACID BY ISOLATED TOMATO FRUIT CUTICULAR MEMBRANE BY Ronald Dean Morse Methylene blue (MB+) and 2,4-dichlorophenoxyacetic acid (2,4-D) were used as model organic compounds to study the sorptive properties of the tomato fruit cuti- cular membrane (CM). The CM was isolated enzymatically and was either immersed or exposed unidirectionally to the solution. The quantity of MB+ or 2,4-D sorbed (in umoles/g) was calculated by determining the differ- ence in sorbate present at equlibrium and that present in the original solution. As the pH of the sorption solution was increased, sorption of MB+ increased and that of 2,4-D decreased, with a common inflection point at pH 3.6. Less than 55%of the sorbed MB+ was desorbed in distilled-deionized water (DDW) while approximately 71 to 91 % of the 2,4-D was desorbed with DDW. Sorption of MB+ through the outer surface was limited. After 6 days, sorption through the inner (cell-wall side) surface was 58.8, 10.2, and 7.8 times greater than through the outer surface for nondewaxed, epicuticular Ronald Dean Morse wax removed, and dewaxed CM (both epicuticular and cuticular waxes removed), respectively. Microscopic inspection confirmed the effect of both dewaxing and orientation of the CM on sorption of MB+. Narrow bands, which were lighter in color and appeared to sorb less MB+ than adjacent areas, were present in the center of the cuticular pegs. With MB+ as the sorbate, Langmuir iso- therms were calculated at 5, 15, and 25 C for both non- dewaxed and dewaxed CM. These isotherms exhibited a nonclassical relationship between sorption and temperature-- i.e., sorption increased with a rise in temperature (+AH). The specific sorption area (A) was calculated for the CM at each temperature and the sorption data were adjusted to sorption per unit area (umoles/mz). This adjustment resulted in classical sorption isotherms (-AH). The largest increase in A occurred from 15 to 25 C for non- dewaxed CM whereas dewaxed CM exhibited the greatest increase in A from 5 to 15 C. The marked change in A values corresponded to enhanced permeability observed for naphthaleneacetic acid through nonwaxed and dewaxed isolated pear leaf CM. The effect of temperature on sorption of MB+ was reversible. An increase in temperature from 5 to 25 C resulted in a positive change in entropy (+AS) of the sorption system. This +AS was most likely a reflection of a change in orientation or order of certain chemical constituents within the CM, which Ronald Dean Morse resulted in increased sorption sites. Classical, linear isotherms (constant partioning-type) were obtained for 2,4-D at pH 0.8 whereas nonclassical isotherms (low affinity-type) were obtained for 2,4-D- at pH 5.8. Sorption of MB+ decreased and desorption of MB+ increased with the inclusion of inorganic cations in the sorption and desorption solutions, respectively. Sorption of 2,4-D increased but desorption was not affected by inorganic cations in the sorption and desorption solutions. Inorganic anions had no effect on sorption or desorption of MB+ or 2,4-D. The many distinct differences in sorption and desorption strongly suggest that there are different mechanisms involved in sorption of MB+ and 2,4-D by tomato fruit CM. SORPTION OF METHYLENE BLUE AND 2,4-DICHLOROPHENOXYACETIC ACID BY ISOLATED TOMATO FRUIT CUTICULAR MEMBRANE BY Ronald Dean Morse A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1971 ACKNOWLEDGMENTS The author wishes to express sincere thanks and appreciation to his major professor, Dr. M. J. Bukovac, for guidance and encouragement throughout the develop- ment of this study. I also am grateful for the assis- tance provided by Drs. H. P. Rasmussen, A. A. De Hertogh, M. M. Mortland, and C. J. Pollard, who served on the guidance committee. I am indebted to the Michigan State University Horticulture Department for financial support and to Shegemi Honma who supplied the tomatoes used in this study. For her encouragement and assistance in pre- paring this dissertation, I express my sincere gratitude to my wife, Linda. TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . . . LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . LIST OF APPENDICES . . . . . . . . . . INTRODUCTION . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . Nature of Plant Cuticle . . . . . . . . Definition, Composition and Function . Physiochemical PrOperties of the Cuticle . Methods of Isolating Plant Cuticle . . . . Mechanical . . . . . . . Chemical--With Dilute Acids . . . . . . Enzymatic . . . . . . . . . . . Sorption--Principles and Applications . . . Definition and Physiochemistry of Sorption . The Langmuir Isotherm . . . . . . . . Calculations from Langmuir Plots . . . . Interpretations of Sorption Isotherms . . . Effect of Sorbate on the Sorbent . . . . Factors Affecting Sorption by Plant Cuticle . Inherent Nature of the Cuticle . . . . . Environmental Factors . . . . . . . . Composition and PrOperties of the Sorption Solution . . . . . . . . . . . . Tomato Fruit Cuticle as a Sorbent . . . . Methods of Studying Sorption by Isolated Plant Cuticle . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . Properties of MB+ and 2,4—D . . . . . . . Isolated Tomato Fruit CM . . . . . . . . Cuticular Membrane . . . . . . . . . Source of Tomatoes Used . . . . . . . Isolation Procedures . . . . . . . . General Procedures . . . . . . . . . Abbreviations and Symbols . . . . . Methods of Analysis . . . . . . . . . Surface Morphology of Isolated Tomato Fruit CM iii Page ii V viii 52 53 53 55 55 56 56 57 57 57 62 Effect of Stage of Fruit Ripeness on Sorption of MB+ I O O O O O O O O C O O O 0 Effect of pH on Sorption and Subsequent Desorption of MB+ and 2, 4- -D . . . . . . . . . . Effect of Wax on Sorption and Subsequent Desorp- tion of MB+ and 2, 4- D . . . . . . . . . Effect of Temperature on Sorption and Subsequent Desorption of MB+ and 2, 4- -D . . . . . . . Effect of Chemical Additives on Sorption and Desorption of MB+ and 2,4-D . . . . . . . Reversibility of Sorption . . . . . . . . RESULTS 0 O O O O O O O O O O O C O 0 Surface Morphology of Isolated Tomato Fruit CM . Effect of Stage of Fruit Ripeness on Sorption of MB+ O O O O O O 0 C O O O O O O 0 Effect of pH on Sorption and Subsequent Desorption Of MB+ and 2,4-D o o o o o o o e o o o MB+ O O O O O O O O O O C O C O O 2, 4-D o o o o e o a o o o o 0 Effect of Wax on Sorption and Subsequent Desorp- tion of MB+ and 2, 4- . . . . . . . . . MB+ . . . . . . . . . . . . . . . 2, 4- D . . . . . . . . . . . . . . Effect of Temperature on Sorption and Subsequent Desorption of MB+ and 2, 4- -D . . . . . . . MB+ . . . . . . . . . . . . . . . 2,4-D . . . . . . . . . . . . . . Effect of Chemical Additives on Sorption and Desorption of MB+ and 2,4-D MB+ . . . . . . . 2, 4-D . . . . . . Reversibility of Sorption MB+ . . . . . . . 2, 4-D . . . . . . DISCUSSION 0 O C O O O O O O O C O O 0 SUMMARY C O C C O O C O O C C O C O . Stage of Fruit Ripeness pH . . . . . . . Wax . . . . . . Temperature . . . Chemical Additives . LITERATURE CITED . . . . . . . . . . . . APPENDICES C O O O O O O O O O O O O 0 iv Page 62 62 63 65 67 67 68 68 68 71 71 76 80 82 90 9O 90 113 116 116 127 129 129 132 136 167 167 167 168 169 170 172 187 10. LIST OF TABLES Some chemical and physical properties Of MB+ and 2 ' 4-D O I O O O I O O O Sorption of MB+ by CM isolated from tomato fruit at different stages of ripeness . . Relationship between pH of the sorption solution and the amount of MB+ desorbed from isolated tomato fruit CM . . . . . . Relationship between pH of the sorption solution and the amount of 2,4-D retained by isolated tomato fruit CM after desorp— tion in DDW followed by ethanol, chloroform, or dioxane . . . . . . . . . . . -Effect of different solvents on weight loss of isolated tomato CM . . . . . . . Effect of different solvents on change in weight of CM isolated from green and ripe tomato fruit . . . . . . . . . . Effect of different dewaxing methods on weight loss and subsequent sorption of MB by isolated tomato fruit CM . . . . . + Wax effect--sorption at 25 and 5 C and sub- sequent desorption of MB from isolated tomato fruit CM . . . . . . . . . Effect of epicuticular and cuticular waxes on sorption and desorption of MB+ from isolated tomato fruit CM . . . . . . Effect of epicuticular and cuticular waxes on unidirectional sorption of MB+ by isolated tomato fruit CM . . . . . . . . . Page 54 72 75 79 81 81 83 83 85 85 Table 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. Effect of wax on sorption of 2,4-D by isolated tomato fruit CM . . . . . . . Heat of sorption (AH) of MB+ by isolated tomato fruit CM . . . . . . . . . Influence of temperature on the specific sorption area of nondewaxed tomato fruit CM . Effect of temperature on penetration of NAA through isolated pear leaf upper CM . . Temperature effect--sorption and subsequent desorption of MB+ from nondewaxed and dewaxed isolated tomato fruit CM . . . . . . . Effect of l-hour treatments with DDW at different temperatures on weight loss and subsequent sorption of MB+ by isolated tomato fruit CM . . . . . . . . . . Effect of inorganic cations on sorption of MB+ by isolated tomato fruit CM . . . . . . Effec} of NaCl and Na-acetate on sorption of MB by isolated tomato fruit CM . . . . Effect of CH3OH and CaCl on sorption and desorption of MB+ by 150 ated tomato fruit CM Effect of inorganic anions on desorption of MB+ from isolated tomato fruit CM . . . . Effect of inorganic cations on desorption of MB+ from isolated tomato fruit CM . . . . Relationship between desorption of MB+ from isolated tomato fruit CM and the relative binding strengh (ease of exchange) of various cations . . . . . . . . . . . . . Data for sorption isotherms at 25 C, and sub- sequent effect of inorganic cations on desorption of MB+ from isolated tomato fruit CM. vi Page 90 103 105 105 112 112 117 118 120 122 123 124 125 Table 24. 25. 26. 27. 28. 29. 30. Page Effect of different solvents on desorption of MB+ from isolated tomato fruit CM . . . . 126 Influence of inorganic anions on sorption of 2,4-D by isolated tomato fruit CM . . . . . 128 Influence of inorganic cations on sorption of 2,4-D by isolated tomato fruit CM . . . . . 128 Influence of CH3CH OH, CaClz, and A1C13 on sorption of 2,4-D 6y isolated tomato fruit CM . 130 Relationship between sorption and desorption of MB+ by isolated tomato fruit CM . . . . . 131 Relationship between sorption and desorption of 2,4—D by isolated tomato fruit CM . . . . 133 Evidence in support of multimechanisms of sorption by isolated tomato fruit CM . . . . 166 vii Figure l. 10. ll. 12. LIST OF FIGURES Page The four main types of sorption isotherms . . 18 Morphology of the inner and outer surface of isolated tomato fruit CM. . . . . . . 69 Effect of pH on sorption of MB+ by isolated tomato fruit CM (temp = 20 C) . . . . . . 73 Effect of pH on sorption of 2,4-D by isolated tomato fruit CM (temp = 25 C) . . . 77 Photomicrographs of transverse sections of isolated tomato fruit CM sorbed with MB+ . . 87 Photomicrographs of transverse sections of isolated tomato fruit CM sorbed with MB+ . . 87 Effect of temperature on sorption of MB+ by isolated tomato fruit CM. . . . . . . . 92 Sorption isotherms of MB+ by nondewaxed isolated tomato fruit CM (temp in C) . . . 94 Sorption isotherms of MB+ by dewaxed isolated tomato fruit CM (temp in C). . . . . . . 96 Langmuir sorption isotherms-—sorption of MB+ by nondewaxed isolated tomato fruit CM (temp in C). . . . . . . . . . . . 99 Langmuir sorption isotherms--sorption of MB+ by dewaxed isolated tomato fruit CM (temp in C). . . . . . . . . . . . 101 Sorption isotherms adjusted to unit sorption area, m2--sorption of MB+ by nondewaxed isolated tomato fruit CM (temp in C) . . . 107 viii Figure 13. 14. 15. Page Sorptions isotherms adjusted to unit sorption area, m2——sorption of MB by dewaxed isolated tomato fruit CM (temp in C o o o o o o o o o o o e o o o 109 Sorption isotherms of 2,4-D by nondewaxed isolated tomato fruit CM at pH 0.8 and 5.8 (temp in C) . . . . . . . . . . 114 Desorption isotherms of nondewaxed isolated tomato fruit CM sorbed with 2,4-D (temp in C). . . . . . . . . . . . . . 134 ix Table 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. LIST OF APPENDICES Data for sorption isotherms at 25 and 5 C, and subsequent effect of inorganic cations on desorption of 2,4-D from isolated tomato fruit CM. . . . . . . . . . Data for sorption isotherm at 15 C, and subsequent effect of inorganic anions on desorption of 2,4—D from isolated tomato fruit CM . . . . . . . . . . Effect of time on sorption of MB+ by CM isolated from tomato fruit at different stages of ripeness. . . . . . . Effect of time on sorption of 2,4-D by isolated tomato fruit CM. . . . . . . Wax effect--% desorption of MB+ from isolated tomato fruit CM sorbed at 25 and 5 C O O O I I O O O O O O O . + Temperature effect--% desorpt1on of MB from nondewaxed and dewaxed isolated tomato fruit CM. 0 D O O O O O O O Data--sorption of MB+ versus temperature . + . . Data--MB sorption 1sotherms . . . Data--2,4-D sorption isotherms. . . . Experimental parameters used to collect data in tables and figures . . . . . . Page 188 192 194 195 196 197 198 199 200 201 INTRODUCTION Most progressive horticulturists are aware of the advantages of applying growth regulating chemicals to improve productivity and minimize costs. In doing so, many growers have experienced the unfortunate situation in which their products have been ruined or impaired from application of these compounds. These unfavorable results often occur through misuse or from exceeding the physiological range at the locus of biological response. Misuse can be avoided by more carefully follow- ing the directions of application; however determining the factor(s) that produced an excessive plant response is not a simple task. Before any systemic growth substance can perform its desired biological response, it must be transported from the application medium into the plant tissues. The cuticle (cuticular membrane) is the first barrier a foliar-applied compound must traverse before being absorbed by the epidermal cells. The cuticular membrane forms a continuous layer over the entire surface of leaves and fruits (Esau, 1965; Martin, 1966; Norris and Bukovac, 1968). and M. Of particular interest is the uncertainty as to the presence of pores in the cuticular membrane. Although micrOpores (intermolecular spaces between the macromolecules) are assumed to exist, there is little evidence available that demonstrates the occurrence of macropores in the cuticular membrane. Many investi- gators using electron microscopy have shown it to be a uniform, continuous and poreless membrane (Martin and Juniper, 1970). Absence of macrOpores, together with the knowledge that many compounds of varying size and solubility can pass through the cuticular membrane, support the idea that it is a complex structure with dynamic physiochemical prOperties. Sorption by the cuticular membrane is Viewed as the initial process in the foliar penetration of organic molecules; more knowledge on its sorptive characteristics is needed to more effectively use foliar applied substances. The objective of this study was to obtain a better understanding of some of the factors which affect the sorption of organic molecules by the tomato fruit cuticular membrane. In contrast to the more traditional method of using intact tissues, isolated cuticular membranes were used to demonstrate what factors directly affect cuticular sorption. REVI EW OF LITERATURE Nature of Plant Cuticlar Membranes Definition, Composition and Function All aerial parts of plants are bounded by protective coverings. Some of these coverings are cellular in nature--e.g., the peels or rinds of certain fruits and the barks of trees. Other plant parts such as leaves, stems, petals, and some fruits are bounded by a noncellular lipoidal membrane. Martin and Juniper (1970) have reviewed the nomenclature of this membrane as defined by researchers over the years. In this thesis the nomemclature as proposed by Sitte and Rennier (1963) will be adopted. They suggested the term "cuticle" (proper) should refer to the oldest, outermost layer which never contains cellulose, and that "cuticular layer" should denote the inner layer whether or not it contains cellulose. The cuticle proper and the cuticular layer together represent the cuticular membrane which is usually separated from the epidermal wall by a layer of pectin. The terminology (either "cuticular membrane" or "the cuticle") as used by the authors cited will be maintained throughout the text of this literature review. In most instances, it can be assumed that the authors are referring to a structure as defined by Sitte and Rennier (1963). Both the cuticle proper and the cuticular layer are composed of a cutin matrix embedded with waxes. These embedded waxes are loosely held in the membrane (probably in pockets) as evidenced by the ease with which they can be removed by organic solvent extraction (Martin, 1964) and heat (Hallam, 1964). Phenolic compounds and some complex tannin substances are readily extractable from the cuticle with organic solvents and dilute aqueous alkali, respectively (Baker and Martin, 1966). There is no clear demarcation between the cuticular layer and the cuticle proper (Martin, 1964; Norris and Bukovac, 1968; Sitte and Rennier, 1963). Nevertheless, some variations between the two layers may be observed. Some cuticles differ in behavior when viewed under polarized light (Franke, 1967). The cuticular layer may contain, besides cutin and embedded wax, cellulose and hemisubstances (formerly called hemicellulose)--i.e., polyuronides and glycans (Franke, 1967; Martin, 1964). The amount of polyuronides (e.g. pectin) present in the cuticular layer is not known. Sitte and Rennier (1963) showed that the cellulose content of the cuticle is small and it is present only in the inner lamellae of the cuticular layer. Pectin and cellulose solvents used for isolating cuticular membranes often fail to extract the cellulose and pectic substances from the cuticular layer (Martin, 1964). These failures may be due to the fact that pectin and cellulose may be chemically or physically protecting the cutin from attack by the solvents. These data suggest that pectinaceous and cellulosic materials are closely combined with cutin to form a cutin complex. (Martin, 1964). The cuticle is an important structural component of the plant, forming an envelope which holds the cellular tissues compact and protected from physical abrasions and environmental factors such as wind, frost, and radiation. In most cases, plant cuticle is bounded by a surface layer of wax (epicuticular wax) which plays an important role in regulating water balance and foliar absorption of nutrients and growth regulating substances (Eglinton and Hamilton, 1967). The epicuticular waxes are distinguished from embedded waxes (cuticular waxes) by the ease with which they are removed by short term (about one minute) immersions in chloroform (Martin and Juniper, 1970); and unless otherwise specified, the term "the cuticle" or "cuticular membrane" includes epicuticular wax. Physiochemical Progerties of the Cuticle The chemistry of cuticular membranes has been reviewed by several authors (Franke, 1967; Hull, 1970; Mazliak, 1968). An up-to-date and comprehensive review of this subject is presented by Martin and Juniper (1970). Only a brief summary of the physiochemical properties that apply to the data of this study will be discussed below. The cuticle as a weak cation exchanger.--The main constituent of the cuticle is cutin, which is a polymerization and condensation product of fatty acids and hydroxy fatty acids. Hemisubstances and cellulose are combined with the cutin in the cuticular layer to form a cutin complex (Huelin, 1951; Martin, 1966). The various cutin constituents possess free carboxyl groups which give rise to electrostatic charges; however this negativity is not equally dispersed through- out the cuticle. There is a gradient from low polarity on the exterior to relatively high polarity in the cell-wall side of the cuticular layer (Crafts and Foy, 1962). This gradient exists because (a) the presence of epicuticular wax produces an accumulation of lipophilic groups on the outside rather than the inner cell-wall side of the cuticle; (b) oxygen is required for polymerization of cutin-—hence molecules directly adjacent to the air would be more completely polymerized than those located farther inside (Franke, 1967); (c) the lipophilic methyl groups of cutin molecules are probably oriented toward the inner surface of the cuticle (Frey-Wyssling, 1953). Cuticular membranes may adsorb and transmit cations in a fashion similar to a weak cation exchanger (Crafts, 1964). Martin and Juniper (1970) define an ion exchanger as a substance consisting of a polymeric skeleton, held together by cross—linkages, insoluble in water and organic solvents, and, when in an aqueous medium, imbibing water and exchanging its ions with other ions in the surrounding solution without any structural change occurring in the polymer. As described by many investigators, cutin and cellulose measure up to these criteria and can be classified as weak cation exchangers (Brian, 1967; Martin, 1966; Mazliak, 1968). Pectic materials can sorb cations; but, as defined above, they probably act as polyelectrolytes rather than ion exchangers because of their tendency to disperse in water (Martin and Juniper, 1970). Some of the cuticular waxes and possibly tannin materials may be bound to the cutin. This could influence the degree of ion exchange and transfer of ions through the cuticle (Crafts and Foy, 1962; Martin, 1966; Martin and Juniper, 1970). pH has a pronounced influence on the exchange capacity of a weakly acidic exchanger such as cutin. At low pH values the ionizing groups of the exchanger are undissociated and unable to accept cations. Dynamic character of the cuticle.-—There is evidence that the cuticle structure is dynamic rather than static. Cutin is a hemihydrophilic substance, containing both lipophilic as well as hydrophilic groups; the latter giving the cuticle the capacity to imbibe water and swell. Since the wax components of the cuticle are lipophilic, swelling of the cutin by water will spread the wax components farther apart. This could have the effect of increasing the permeability of the cuticle to water soluble substances (Middleton and Sanderson, 1965; van Overbeek, 1956). The exact spacial configuration of the cutin complex is not known. Cutin is believed to be a polymer possessing intermolecular spaces of sufficient size to accommodate the passage of small molecules. Cuticular waxes are closely associated with cutin and the lipophilic nature of wax could inhibit the penetration of aqueous solutions. However, some large hydrOphilic molecules such as dyestuff, streptomycin, and other antibiotics can penetrate the cuticles of leaves (Goodman, 1962; Kamimura and Goodman, 1964a; 1964b). Sitte (1965) proposed that because the lipid molecules of the cuticle are in constant thermodynamic motion, they are subject to a change in orientation according to their kinetic energy. Therefore, a dynamically changing ultraporosity of the waxy components will result, which could explain the penetration of large hydrOphilic molecules such as streptomycin. Methods of Isolating Plant Cuticle Mechanical Plants with particularly thick cuticles can some- times be stripped from the epidermis; however this is the most precarious method (Franke, 1967; Mazliak, 1968). Chemical--With Dilute Acids Sando (1923) used dilute hydrochloric acid, whereas Huelin and GallOp (1951) employed hot, aqueous solutions of ammonium oxalate (16 %):oxalic acid (4 %) for separating the cuticles from apple peels. The latter reagent reacts by attacking the pectin layer immediately beneath the cuticle; it has been extensively used to isolate cuticles from leaves and fruits (Martin and Juniper, 1970). With this method the cellulose walls of the epidermal cells often remain attached to the cuticle, and the physio- chemical structure of the cuticle may be altered by the heating involved. The controversial data obtained by 10 Kamimura and Goodman (1964b) may be explained because the cuticles were isolated with hot chemicals (Franke, 1967; Martin and Juniper, 1970). Holloway and Baker (1968) isolated cuticular membranes from leaves of several plants by treatment with a zinc chloride-hydrochloric acid solution. This reagent was previously employed to remove the cellulose residue from isolated membranes. Thus, using zinc chloride-hydrochloric acid to isolate cuticular membranes has the advantage of reducing the number of operations required to obtain cellulose-free cuticular membranes. The solution was also effective on a wider range of species than other isolation methods. Isolating the cuticle by dilute acid solutions has the disadvantage of subjecting the cuticular components to possible hydrolysis and/or oxidation (Mazliak, 1968). Enzymatic Skoss (1955) first demonstrated this method by isolating plant cuticle by treatment with an anaerobic culture of the bacterium Clostridium roseum. Orgell (1955) elaborated on this approach by isolating the cuticle from leaf discs, utilizing solutions of commercially available pectic enzymes. A mixture of enzymes derived from snail gut extract was used by Heulin (1959) to separate the epidermis from apple peels. Schieferstein and Loomis (1959) employed a pectinase-cellulase solution to isolate plant cuticle. In reviewing the literature on isolation of plant cuticle, 11 Martin and Juniper (1970) concluded that various pectinase mixtures are used for leaves and fruits in what has become almost standard practice. According to Franke (1967). This method modifies cuticular membranes the least, even though the inner lamellae of the cuticular layer may be destroyed because of its content of pectin. Experiments utilizing isolated cuticles have been criticized on the basis that the isolating methods are likely to induce chemical alterations in the membrane, as well as subjecting it to stresses that could cause structural imperfections (Hull, 1970). With regard to enzymatic isolation, however, these criticisms lack support. Norris and Bukovac (1968) provided convincing evidence that enzymatic isolation did not alter cuticular structure. They compared intact and enzymatically isolated pear leaf cuticles under bright field and polarized light, as well as the surface waxes with electron microsc0py. Both the intact and enzymatically isolated cuticles were similar with regard to intensity and location of birefringence and staining with Sudan III and Sudan IV, and ruthenium red. Although some waxes may have been lost during isolation, the physical arrangement of the epicuticular waxes was not destroyed. 12 Studies which employ isolated cuticle have the advantage of removing the confounding effects of living tissue. On the other hand, data obtained from such an in vitro system can not necessarily be used to interpret results from in 3139 experiments; nevertheless it does provide a basis of studying and understanding the initial phase in the cuticular penetration process--i.e., sorption (Currier and Dybing, 1959; Bukovac, 1970). Sorption--Principles and Applications Definition and Physiochemistry ofFSorption When an aqueous solution is applied to the foliage of plants, the active ingredient is either retained (sorbed) by the foliage or it is lost to the surroundings without being sorbed. The cuticle, being a continuous membrane, is the initial phase or sorbent in which a compound must be sorbed before it can penetrate cellular tissues. There is very little data relating directly to sorption by plant cuticle. Most studies of sorption from liquid solutions have been concerned with equilibrium conditions and predominantly with sorption isotherms (Kipling, 1965). To the author's knowledge, there are no reports in the literature of intact or isolated cuticle being used as the sorbent in these isotherm studies. There are several texts that n u 13 adequately describe the theory and principles of sorption and sorption isotherms (Adamson, 1967; de Boer, 1953; Kipling, 1965; Shaw, 1966). Only a brief discussion of sorption from aqueous solutions as applicable to the cuticle will be reviewed in this thesis. Sorption is a noncommittal term used to describe an accumulation process where the distinction between adsorption and absorption is not clear. Adsorption is the attachment of a molecule onto a surface, whereas absorption refers to partitioning or penetration into a substance (Barrow, 1966). The interpretation of these terms with regard to cuticular membranes is that adsorption is the retention of sorbate molecules on the surface or in pores of the cuticle by electrostatic binding or van der Waals forces, and absorption refers to the acculumation of the sorbate in specific components of the cuticle (Bukovac and Norris, 1968). In most systems, sorption is due to the operation of physical forces only, and this is an implicit assumption in the theoretical treatment of sorption from solution. Physical sorption consists in the binding of molecules to the sorbent surface by essentially van der Waals forces, similar to the forces involved in liquefaction (Shaw, 1966). Solids are known to sorb molecules from solution by a process known as chemisorption. Usually 14 chemisorption occurs at high temperatures; however a few examples have been recorded at room temperature (Kipling, 1965). Chemical is distinguished from physical sorption in that electron transfers (ionic and covalent bonds) take place between sorbent and sorbate in chemisorption, but do not take place in physical adsorption (Barrow, 1966). Hayward and Trapnell (1964) and Barrow (1966) list certain differences in the prOperties of the two types of sorption, which can be used as experimental criteria for distinguishing between them. There is no single criterion that will distinguish the sorption type in all cases. With sorption from solution at low temperatures, generally physical forces are involved or a combination of physical and weak chemical forces. Sorption of organic ions may be governed by van der Waals forces between the uncharged groups of the ion and sorbent as well as by electrical forces between the charged groups of the ion and sorbent. With large organic ions, sorption often is controlled pre- dominantly by van der Waals forces because the ion is large in relation to the magnitude of its charge (Kipling, 1965). The Langmuir Isotherm The amount sorbed per gram of solid depends on the specific area of the sorbent, the equilibrium sorbate Concentration, temperature, and the nature of the solvent 15 and sorbate molecules. From measurements at constant temperature, one can plot the moles sorbed per gram sorbent N against the equilibrium sorbate concentration c. This plot is called a sorption isotherm (Shaw, 1966). Often it is possible to represent experimental data over a limited range by an empirical isotherm equation suggested by Freundlich (Shoemaker and Garland, 1967): where K and a are constants which have no physical significance but can be evaluated by a plot log N versus log c. The Freundlich equation is limited to a narrow concentration range because it fails to predict the observed behavior at low and high sorbate concentrations. In many systems a theory derived by Langmuir can be applied (de Boer, 1953; Shoemaker and Garland, 1967). The Langmuir sorption isotherm is based on the assumptions that (a) only monomolecular sorption takes place, (b) sorption is localized at specific sorption sites, and (c) the heat of sorption (AH) is independent of surface coverage--i.e., homogeneous sorption sites,and mutual forces between sorbed molecules do not affect AH. Sorption from solution is generally localized and forms no multilayers; however, frequently solid surfaces tend to have hetergeneous sorption sites, in which case the sorptive process is less exothermic 16 with increasing surface coverage. Nevertheless, a large number of sorption isotherms fit the Langmuir equation reasonably well (de Boer, 1953; Shaw, 1966). The form of the Langmuir equation appropriate to adsorption from solution is c/N = c/Nm + 1/KNm where c is the equilibrium sorbate concentration, N is the moles sorbate sorbed per gram sorbent, and K is a constant dependent on the temperature, but independent of surface coverage. If the Langmuir isotherm is an adequate description of the sorption process, then a plot of c/N versus c will yield a straight line with slope l/Nm and intercept 1/KNm Calculations from Langmuir Plots Several important quantitative measurements can be derived when sorption isotherm data are applied to the Langmuir equation: (a) The specific sorption area. If the area 0 occupied by a sorbed molecule is known, then the specific sorption area A of the sorbent can be calculated by A = N oN 10"20 m 0 where Nm is obtained from the slope of the Langmuir plot, 0 N is Avogadro's number, and o is given in A2 (Shoemaker O and Garland, 1967). (b) Thermodynamic measurements. 17 When isotherms are determined at two temperatures (T1 and T2), the differential heat of sorption (AH) at a given N value can be calculated with the Clausius-Clapeyron equation: -AH = 2.303 R T1T2(log c2 - log cl) Tz’Tl where AH is the molar heat of sorption, R is the molar gas constant, T is the absolute temperature, and c2 and c are the equilibrium concentrations of the sorbate 1 molecules at T2 and T1' assumes that AH and A remain constant over the temperature respectively. This equation range employed. Sorption from solution involves not only sorption of the sorbate molecules but also the displacement of solvent molecules and other molecules and ions from the sorbent; this may complicate the interpretation of AH (Getzen and Ward, 1969; Shoemaker and Garland, 1967). The change in molar free energy AG of a system resulting from sorption can be calculated from the thermodynamic relationship: AG = R T (In c - In c ) e o where ce is the equilibrium concentration and CO the original concentration of the sorbate prior to sorption (Bailey gtyglg, 1968). If one knows the AH and AG of a process, then the molar entropy TAS at a particular absolute temperature can be calculated by 18 Sorption is always accompanied by a decrease in enthrOpy, and being a spontaneous phenomenon, the free energy of sorption decreases. Therefore, it is evident from the above equation, that the heat of sorption must always be negative--i.e., all sorption processes are exothermic (Shaw, 1966; de Boer, 1953). Since the AH must be exothermic, increasing the tempera- ture will progressively decrease sorption (Figure l). F’ Sorption Equilibrium sorbate concentration Figure 1.--The four main types of solution sorption isotherms (L = Langmuir; T = temperature, where T2>Tl; H = high affinity; S = sigmoid; C = constant partition). ’2- ‘fi '1 ‘- q \ v tlv 19 Interpretations of Sorption Isotherms As mentioned previously, all sorption data do not take the form of a Langmuir isotherm. Giles gE_§l. (1960) have proposed a method of classification of solution isotherms and suggested how different curves can be used to diagnose the sorption mechanism involved. These data can be used to obtain information regarding the physical nature of the sorbate and the morphology of the sorbent, and to measure the specific surface area of the sorbent. At least twenty different isotherms have been encountered, but all can be grouped under four main classes, distinguished by their curvature near the orgin: L = Langmuir, H = high affinity, S = sigmoid shaped, and C = constant partition (Figure l). The initial slope depends on the rate of change of sorption site availability with increase in solute sorbed. As sorption proceeds a bombarding sorbate molecule will encounter increased difficulty in finding suitable sorption sites-- i.e., the external sorbate concentration must be raised in order to realize an additional increment of sorption by the sorbent. This applies to the normal L curves and to the later stages of the H isotherms. The types of systems that give L isotherms are usually characterized by low solvent competition and the sorbate molecules being sorbed flat or as micelles. H curves are special ‘ 11. 20 cases of L curves in which the sorbate has such high affinity that in dilute solutions it is completely sorbed (or at least there is no measurable amount remaining in solution). The initial part of the H curve is nearly vertical; the sorbed species are usually ionic micelles, polymeric molecules, or monomers sorbed chemcially by the sorbent. The opposite condition exists in the initial part of the S curves, where a "cooperative" sorption occurs. With S isotherms, the more sorbate molecules already sorbed, the easier it is for additional sorbate to become fixed. The S isotherms usually occur when three conditions are realized. The sorbate molecule (a) is monofunctional--i.e., it has a fairly large hydrophobic portion, the forces of attraction for the sorbent are localized over a short section of its periphery, and it is sorbed as a single unit and not in the form of a micelle; (b) has moderate intermolecular attraction, causing it to pack vertically in a regular array by end-on or edge-on attachment to the sorbent; (c) meets strong competition for sorbent sites from molecues of the solvent or another sorbed species. The C curve is characterized by constant partitioning of sorbate between solution and sorbent, up to saturation, where an abrupt change to a horizontal plateau usually occurs. The conditions favoring C isotherms are (a) a porous sorbent with flexible molecules and regions of 21 differing degrees of crystallinity, and (b) a sorbate with better penetration power into the crystalline regions of the sorbent than the solvent because of its specific molecular geometry and higher affinity for the sorbent. Linear isotherms indicate that the sorbate is partitioning into regions inaccessible to the solvent. Frequently sorption isotherms will show a second rise and even a second plateau, indicating the complete saturation of the new sites; this second plateau is not always realizable. The new sites on which sorption can occur are as follows: (a) The exposed parts of the layer already present--e.g., surface multilayers and sorption into highly porous areas of the sorbent. (b) New, probably more crystalline regions of the sorbent structure into which the solute begins to penetrate. These new accessible areas may also reflect capillary movement into "ink-bottle" pores which remain completely empty until a critical sorbate concentration is reached and then the entire volume is filled. This may result in a sudden rise in sorption (Adamson, 1967; Shaw, 1966). (c) Part of the original surface exposed by reorientation of the molecules already sorbed. Giles §£_21, (1960) also refers to sorption isotherms of many solutes on wool in which a rise beyond the initial plateau might have been sorption on hetergeneous sites. Sorption of ionic micelles seldom produces 22 a rise in the isotherm beyond the first plateau; this is most likely because of mutual repulsion of the charged sorbed layer and micelles in solution. The length of the initial plateau (monolayer) is indicative of the affinity that the sorbate has for the new sorption sites as compared to the affinity for the original sorption sites. A long plateau indicates that a high energy barrier has to be overcome before additional adsorption can occur on new sites after the surface has been saturated, whereas a short plateau suggests that the new sites have nearly the same affinity for more sorbate as the original sites. Effect of Sorbate on the Sorbent Although in most sorption systems interest is centered around the physiochemical nature of the sorbate, there is every reason to expect sorption to affect the structure of the sorbent; in fact the thermodynamic properties should properly be assigned to the sorbate-sorbent system rather than just to the sorbate (Adamson, 1967). Dunning (1964) has pointed out that the presence of adsorbed gas molecules may alter the energy of heterogeneous adsorption sites. With sorbents that may be swollen by the solvent, penetration by both solvent and sorbates may occur. In this case, the extent of 23 penetration and hence the number of sites for sorption may vary with the sorbate concentration (Kipling, 1965). Adamson (1967) stated that one may expect important structural surface changes to accompany adsorption whenever the adsorption energy is signi- ficant in comparison to the bond energy of the solid. The probable exception would be physical adsorption in high energy rigid solids. Relating to the cuticle as the sorbent, Yamada e£_al, (l965b)have proposed that urea alters the ultraporosity of the cuticle. Urea penetrated isolated cuticular membranes at a velocity higher than one would predict from simple diffusion. Penetration rates exceeded by 10- to 20-fold those for calcium and sulfate ions, and the kinetics of urea penetration were markedly different from those of other substances. Retention of urea to the cuticular membranes was less than for Ca2+ and 8042-. cuticular penetration of urea was not governed by first The data suggested that order kinetics, and some factor other than free diffusion was involved. The authors concluded that perhaps urea was loosening up the membrane structure by alter- ing the cutin and hydrophobic bonds of the cuticle. Besides enhancing its own penetration through isolated Cuticle, urea promoted the passage of other nutrients Simultaneously applied (Yamada et al., 1965a). Enhanced 24 nutrient penetration has also occurred in vivo from foliar applied solutions that contained both nutrients and urea (Okuda and Yamada, 1962; Wallace, 1962; Wittwer, 1964). These data support the idea that sorption of urea alters the ultraporosity of the cuticle. Factors Affecting Sorption by Plant Cuticle Sorption by plant cuticle is influenced by the inherent nature of the cuticle itself, environmental factors, and composition of the sorption solution. All are closely interrelated and constitute variables that must be considered in any sorption experiment. In the remaining pages of this literature review, some of the data will be taken from experiments with intact tissues. In doing so, it is understood that these data are only indirect evidence for describing the factors affecting sorption by plant cuticle. Inherent Nature of the Cuticle Stage of cuticlar develOpment.--Young immature leaves are usually more permeable to plant growth substances than fully expanded or mature leaves (Bukovac, 1965; Currier and Dybing, 1959; Greene, 1969). Weintraub EE_21- (1954) measured the absorption of 2,4-dichlorophenoxyacetic acid (2,4-D) into bean leaves 25 that ranged from one to six weeks of age. During the expansion of each leaf, there appeared to be a relatively brief stage of high permeability followed by a steep decline to a relatively constant amount of 2,4-D penetration. Bukovac and Norris (1968) showed that sorption of naphthaleneacetic acid (NAA) and naphthaleneacetamide (NAAm) by the upper cuticular membrane of pear was greater for young leaves than for old leaves. One possible explanation of the higher absorp- tivity by young leaves is their lack of a well developed cuticle. A cuticle can be separated from young leaves as soon as the bud opens; however chemical isolation of these young cuticles produced cell-sized flakes rather than sheets of cuticle as typical for mature leaves (Martin and Juniper, 1970). Evidence by Schieferstein and Loomis (1956,1959) suggested that the margins of the upper epidermal cells of leaves continue to grow and maintain an immature cuticle for some time. The authors ascribe the greater susceptibility of young leaves to herbicides to the presence of these immature, permeable zones in the cuticle. Sifton (1963) made a detailed study of the development of the cuticles of leaves from Labrador tea. With increasing age of the leaf, progressive polymerization occurred in the cutin complex. 26 Much information has been obtained on the changes in waxiness of leaves and fruits during development (Martin and Juniper, 1970). Wax formation takes place early in the development of the leaf and continues during the period of leaf expansion. Schieferstein and .Loomis (1959) have suggested that, as the plant ages, vvaxes progressively fail to reach the surface due to eunlargement and hardening of the cutin complex and therefore become embedded in regular layers in the aJrusrphous cutin mass. Usually the cotyledonary leaves lieixve thin cuticles with no epicuticular wax, even Vifleen the cuticle is heavy in mature leaves (Martin and CTLlluiper, 1970). The progressive develOpment of wax leaves and stems of young fiber-flax plants has Wax was on bEisenfollowed (Martin and Juniper, 1970). Virtually absent on the seedlings, but increased to 0 - 02 mg/cm2 by 12 days after germination, falling to 0 - 01 mg by 19 days and then rising to 0.02 mg by 31 days. This temporary decrease of wax per unit surface Eirea during leaf expansion was also found on apple Ileaves (Richmond and Martin, 1959; Baker, et al., 1962). 'Young leaves frequently had as much wax and cutin as the fully grown, but during leaf expansion the deposits decline slightly. The authors suggested that this probably occurred because the formation of wax and 27 cutin failed to keep pace with leaf expansion. The cuticle was fully formed soon after the leaf attained its maximum size; thereafter the cutin content remained constant, but the wax levels declined slightly presumably due to the effect of weathering. As a rule, young leaves of a species contain as much wax per unit ssurface area as the mature; however, unlike the apple leeaf cuticle, often the cutin content per unit surface area increases with leaf expansion (Martin, 1966; Martin and Juniper, 1970) . The cuticles of fruits are usually much waxier £1I1<3 thicker than those of the corresponding leaves (EBaaker and Martin, 1967). The young fruits have well- <1§>ansion occurs the cuticles often become heavier E>€213 unit surface area. Little further development (>13 the cuticle occurs after the fruits are fully grown. “Pflus, formation of cuticular substances in both leaves Eirmd fruits is connected with the active growth of the Sub-cuticular tissues. Waxiness of the cuticle.--Sorption and penetra- tion through plant cuticle is controlled ultimately by adhesion between molecules--i.e., the degree of wetting or spreading of the liquid over the surface (van Overbeek, 1956). The degree of wetting can be measured by 28 determining the contact angle between the cuticular surface and the applied liquid. The presence or absence of wax markedly influence the contact angle and consequent wetting and penetration of cuticles (Eglinton and Hamilton, 1967). Fogg (1948) has shown that extracting the fatty components from a Sinapis leaf caused a decrease in contact angle from 96 to 29 degrees. Brushing the cuticle to disturb surface wax increased wettability and cuticular transpiration (Hall and Jones, 1961), and absorption of 3—chlor0phenoxy-d propionic acid (Bukovac, 1965). Removing the wax from plant cuticle by organic solvents increased cuticular permeability to water (Skoss, 1955) and isopropyl- chloroacetamide (Darlington and Barry, 1965). There is little question that both chemical composition and physical structure of epicuticular wax markedly influence wettability. This subject has been reviewed recently by several authors (Bukovac, 1970; Hull, 1970; Martin and Juniper, 1970). Waxes not only influence penetration indirectly via wettability; they may influence it directly in the following three ways: (a) Barrier to adsorption of molecules. Foliar absorption of pesticides is markedly influenced by the quantity of waxes in the cuticle. An inverse relationship apparently exists between overall cuticle thickness and pesticide absorption (Hull, 1970). Removing the wax components from isolated 29 cuticle is known to result in enhanced penetration, even with molecules such as naphthaleneacetic acid (NAA) and various chloroinated phenoxyacetic acids. These molecules should be strongly attracted to the lipophilic waxes and should not pose any wettability problems, yet dewaxing increased the penetration rate through isolated cuticles (Bukovac, ep_gl., 1971; Norris and Bukovac, 1969). (b) Medium of ion exchange. There are dissociable groups at the surface of tomato fruit and pear leaf cuticle (Bukovac and Norris, 1968; Yamada, 1962). Above the pRafor these dissociat- ing groups, the cuticular surface would have a negative charge and could ionically bind cationic substances. (c) Deterrent to ion exchange. Although the waxy surface possesses some ion exchange prOperties, it is small in comparison to the cutin and other constituents of the cuticle. Some of the wax and possibly tannin materials may be bound to the cutin. This could influence the degree of ion exchange and transfer of ions through the cuticle (Crafts and Foy, 1962; Martin, 1966; Martin and Juniper, 1970). Pathways of sorption and penetration.--Various authors have proposed that water-soluble polar compounds may follow an aqueous path and lipid-soluble apolar substances a lipid path through the cuticle (Crafts, 1956; Foy, 1964; Franke, 1967; Pallas and Williams, 30 1962). However, this hypothesis still lacks adequate experimental support. Arguments for this idea are (a) both lipid and water soluble molecules can pass through the cuticle, and (b) polar molecules tend to show different rates of penetration through isolated cuticles when a comparison is made between the rate inside to outside versus that from outside to inside; whereas, NAA, a relatively nonploar molecule, tends to penetrate at equal rates in either direction (Norris and Bukovac, 1969). No general pattern of cuticular pores have been demonstrated that will account for the observed passage of substances through undamaged cuticle. Crafts (1961) proposed that the cuticle is perforated by micropores whose aqueous concentration is dependent on environ- mental conditions. These continuous aqueous phases are thought to accomodate the passage of polar substances moving parallel to non-polar lipid substances which move through lipoidal pathways. No such pore system corresponding to Crafts hypothesis Inns yet been observed, nor are the pectinaceous channels suggested by Roberts, g£_gl, (1948) of sufficient wide occurrence to provide a general aqueous route (Martin and Juniper, 1970; Norris and Bukovac, 1968). Controversy has arisen as to whether or not there are canals in the cuticle serving for excretion of waxes and absorption of lipophilic 31 substances. Hall (1967) and Scott (1966) have provided evidence in favor of the existence of wax canals; however the majority of reports indicate that there are no such canals (Bollinger, 1959; Crisp, 1965; Franke, 1967). An alternative approach to pores or canals as the mechanism of penetration through the cuticle, is proposed by Hallam (1964). He provided valid support for the view that wax lamellae occur within the cuticle. Hallam con- cluded that these wax lamellae could be indicative of cuticular pathways that are anastomosing channels or spaces rather than a large number of pores directly connecting the epidermal cell wall with the exterior. In this connection, Franke (1967) discussed the possibility of intermolecular spaces as well as a dynamically changing ultraporosity existing in the cuticle that might render the penetration of water soluble materials. He also suggested that lipophilic substances penetrate the cuticle by a process of solution or partitioning--the degree of molecular penetration being determined by their solubility, partition, and molecular size. Preferential areas of sorption.--Some regions of undamaged leaves act as preferential sites of absorption. The guard cells and subsidiary cells of stomatous leaf surfaces are reported to be important sites of absorption (Franke, 1967; Sargent and Blackman, 32 1962; Scthherr, 1969). Trichomes are often particularly active in absorption (Dybing and Currier, 1961; Hull, 1964). Finally, the cuticle lying above the anticlinal walls and above, beneath or on both sides of the veins frequently are active sites of penetration (Dybing and Currier, 1961; Franke, 1967; Greene, 1969). Hypotheses and supporting data to explain these preferential permeable regions over the cuticle can be conventiently placed into four categories: (a) Differential cuticle thickness. Martin (1966) stated that the cuticle lying over veins, trichomes and the anticlinal walls tends to be thinner than elsewhere on the leaf. However, with some leaves the cuticle is thicker over the anticlinal walls (Baker and Martin, 1967; Norris and Bukovac, 1968). (b) Modified nature of the wax components. The epicuticular waxes are frequently modified in their density or form around trichomes, stomata, veins and anticlinal walls. Eglinton and Hamilton (1967) and Bystrom eE_3l. (1968) provided evidence that there are more waxes over periclinal walls than the anticlinal walls. Very commonly, as in the pea, cabbage, and many grasses, the number and size of epicuticular projections are markedly reduced or eliminated completely over the guard and subsidiary cells. Other leaves such as some species of the genus Eucalyptus have a higher 33 density of wax over the guard cells than the astomatous portions of the leaf (Martin and Juniper, 1970). Linskens (1950) found maximum wetting of leaves occurred over the veins. There are reports that birefringence is discon- tinuous over anticlinal walls and over veinal tissue (Frey-Wyssling and Hauserman, 1941; Norris abd Bukovac, 1968). This would indicate either a reduced amount of embedded waxes or less highly oriented wax layers or both. (c) Anatomical differences. The basal area of trichome attachment to the epidermis, the thin walled parenchymatous bundle sheath cells between the epidermis and the vein, and the guard and subsidiary cells have various morphological and physiological differences that might provide an important transport route from the cuticle to the veins and other cellular tissues (Esau, 1965). (d) Ectodesmata. Ectodesmata are proposed to be interfibrillar spaces extending through the cell wall to the cuticle (Franke, 1967). These ectodesmata, as detected by the mercuric chloride method, are pre- dominantly found associated with or located beneath trichomes, anticlinal walls, veinal areas and guard cells. Franke (1967,1969) considered ectodesmata to function as pathways of transport in foliar absorption. Schanherr and Bukovac (1970) showed that ectodesmata are not definable cell wall structures, rather that the distribution pattern of ectodesmata is governed by the 34 cuticle and not by structures in the cell wall. Hence, estodesmata are likely a reflection of sites in the cuticle preferentially permeable to mercuric chloride and to other polar compounds. Both surfaces of leaves function in the absorption of chemicals. Usually the abaxial surface is more permeable than the adaxial due to the presence of stomata as well as a thinner cuticle. (Currier and Dybing, 1959; Foy, 1964; Greene, 1969). Most authors agree that the important role of stomata under field conditions is to provide preferential sites of entry via the guard and subsidiary cells (Bukovac, 1970; Martin and Juniper, 1970). Abaxial surfaces do not always possess thinner cuticle. Norris and Bukovac (1968) showed that abaxial pear leaf cuticle was thicker than the adaxial, yet greater penetration of NAA occurred through the isolated abaxial surface than the adaxial surface (Norris and Bukovac, 1969). These authors suggested that discontinuity of birefringent wax and the presence of guard and subsidiary cells in the abaxial surface could explain, at least partially, this difference is permeability. There are little data on the preferential sites of absorption for fruit cuticle. Yamada and co-workers (1964a, 1965b, 1966) used microautoradiography to study ‘the distribution of sorption sites over the inner sur- face of enzymatically isolated astomatous tomato fruit 35 cuticle. The results of their work will be discussed in a subsequent section of this review (see "tomato fruit cuticle as a sorbent"). The preferential absorption sites of fruit cuticle probably differ somewhat from those of leaf cuticle due to some basic anatomical differences (Esau, 1965). Fruits do not have stomata, whereas abaxial leaf surfaces generally do. Irregu- larities in absorption occur over the veins of leaf cutiCle; however this has not been observed over the veinal areas of fruits. The cuticle over the veinal areas of most fruits differ from leaves in that they do not lie adjacent to the surface and therefore have no apparent influence on cuticlar form and thickness. Environmental Factors The effect of light intensity, humidity, and temperature as factors influencing sorption and penetra- tion of chemical compounds through the cuticle will now be reviewed. Light intensity.--Evidence for the effects of light on permeability of cuticles is very limited (Currier and Dybing, 1959; Hull, 1970). The literature contains reports on the effect of light on penetration into leaves and leaf discs (Greene, 1969; Rice, 1948; Sargent and Blackman, 1962; Weintraub, ep_a£., 1954). What influence light has on penetration of isolated cuticles is not known. The data from studies with intact 36 tissues would suggest that light does not alter the permeability of cuticular membranes. Light may promote penetration of substances through the cuticle by stimulating opening of stomata which exposes a more effective sorptive surface associated with the guard and subsidiary cells (Currier and Dybing, 1959; Schonherr, 1969), and by increasing photosynthetic activity, resulting in greater cellular uptake (Brenner, 1969; Greene, 1969) and/or translocation (Currier and Dybing, 1959; Rice, 1948; Sargent, 1966). Humidity.--Although some data suggest that exceptions exist, foliar absorption of both organic and inorganic compounds is facilitated by high humidity (Hull, 1970). High humidity may increase absorption in at least four ways: (a) Stomatal opening is favored, thereby enhancing penetration via this route. (b) The rate of drying of spray droplets is decreased, thus extending the time available for absorption (Bukovac, 1965; Prasad,e§_al., 1967; Rice, 1948). (c) Water stress in the plant may be prevented (Currier and Dybing, 1959; Hull, 1970; Weintraub, e£_a£., 1954). A favorable water balance is important for Optimum translocation, which in turn is considered to be a factor determining the rate of foliar penetration by a chemical (Sargent, 1966). (d) Theedegree of swelling or hydration of the cuticle is incmeased, which in turn may influence permeability to (I) 37 water and water soluble compounds (Martin, 1966; Slatyer, 1960; van Overbeek, 1956). Temperature.—-In most situations, increasing the temperature stimulates penetration of foliar applied substances (Goodman, 1962; Hull, 1970). Since penetration from the ambient solution into the cellular tissues may be governed by a complex combination of both metabolic and physical processes, uncertainity often arises as to the exact nature of the temperature effect on penetra- tion. Temperature may promote penetration by its effects on (a) physiological processes--acceleration of photo- synthesis, phloem translocation, accumulation, proto- plasmic streaming, and growth (Currier and Dybing, 1959; Sargent, 1965), and (b) physiochemical processes—- increased rate of diffusion, lowered solution viscosity, and decreased exchange and adsorption of solutes (Currier and Dybing, 1959; Sutcliffe, 1962). The Q10 of physiological processes is generally in the order of 2-3, whereas for physiochemical processes it is usually about 1.2 (Sutcliffe, 1962; Briggs, SE_31°' 1961). Penetration through the cuticle is considered to be a diffusion process (Franke, 1967), yet there are r6ports of penetration through isolated cuticles where temperature coefficients in excess of 2.0 were obtained ”Morris, and Bukovac, 1969; Silva Fernandes, 1965b). 38 Norris and Bukovac (1969) showed that this temperature effect was completely reversible and the reversibility occurred rapidly. Their data indicated that temperature has an effect on the cuticle other than the physical processes of adsorption and diffusion. Two possible reasons were given to explain the data. The first rationale is based on the rate, as related to the kinetic energy, of molecules diffusing through a lipid membrane of low premeability. In this case Fick's law of diffusion does not apply. The diffusing molecule must acquire sufficient kinetic energy to overcome several energy barriers in passing from the solvent into membrane, through the membrane, and into the inner solution from the membrane. The penetrating molecule can be thought of as being confined to a particular area, moving to a new position as it acquires sufficient energy (activation energy) by collisions with neighboring mole- cules. Such diffusion has a high Q10 (often 2-3) because at higher temperatures more molecules acquire sufficient energy to diffuse in a given time (Jennings, 1963; Sutcliffe, 1962). The barriers preventing passage of diffusates can be visualized as oriented molecules in the membrane which do not have sufficient intermolecular spacing to accomodate penetration. Briggs, ep_a£. (1961) state that the kinetic energy which a molecule must acquire beftme it is able to break the bonds between the oriented 39 molecules is relatively large and is likely to yield temperature coefficients in excess of two for diffusion. The alternative explanation given was that structural changes in the cuticle may have been induced by higher temperatures which altered its permeability. The fact that the temperature effect is reversible implies that the hypothesized change in cuticular structure also would be reversible. Several authors have suggested that temperature may cause a structural change in the cuticle, which enhances penetration of lipOphilic as well as hydrophilic compounds. Van Overbeek (1956) has pointed out that temperature may influence the rate of diffusion of lipophilic molecules through lipid-containing membranes. Lipid molecules are often oriented in layers that have a very low permeability at low temperatures when the layers are highly viscous, but at high temperatures these lipids become less viscous and often less resistant to diffusion of solutes. Hempling (1960), Stadelmann (1969), and Virgin (1953) have proposed that changes in cellular membrane permeability can be explained by alternations in membrane structure. Some insect cuticle have closely packed, oriented wax molecules which are responsible for high resistance to water loss (Beament, 1968). At a precise, critical temperature, which differs lMith the insect, there is a sudden increase in water 40 permeability. The data indicated that at this critical temperature the wax molecules became disoriented. This change produCed a S-fold increase in water permeability. This effect was reversible, for on lowering the temperature the characteristic low permeability was again obtained. Beament proposed that the lipid molecules did not become randomly disoriented, rather their polar groups remained localized while their apolar chains became liquid at the point when the temperature sensitive van der Waals forces were broken. The free apolar segments of the lipid molecules were reoriented in such a fashion that the intermolecular pores increased considerably. The dynamically changing character of the cuticle has previously been discussed. Sitte (1965) suggested that the lipid molecules of the cuticle are in constant thermodynamic motion, perhaps oscillating around a mean position. Increased temperatures might increase the penetration of hydrophilic molecules by altering the ultraporosity of the lipid constituents. Silva Fernandes (l965b) showed that high temperatures enhanced the permeability of isolated apple cuticles to phenyl mercuric acetate, and this is consistent with existence Of labile layers susceptible to temperature within the cuticle. 41 Composition and Prpperties of the Sorption Solution Solvent.--There is generally an inverse relation- ship between the extent of sorption of a solute and its solubility in the solvent used--i.e., the less soluble the material the more strongly it will be sorbed (Adamson, 1967; Leonard, 1958). An oil solvent can increase the penetration of organic molecules into leaves; however the oil component aids in wetting and doubtless other effects that make a comparison with other solvents of little meaning (Currier and Dybing, 1959). Sorbate.-—Penetration through plant cuticle is profoundly influenced by the nature of the sorbate molecule. In general, apolar lipid-soluble compounds are thought to enter leaves more readily than the polar water-soluble compounds (Hull, 1970; van Overbeek, 1956). Molecular size is an important factor in determining penetration, but lipid solubility most often is of more importance than size (Crafts and Foy, 1962; Giese, 1962). In general, ionic compounds penetrate lipid membranes more slowly than non-ionic compounds of similar molecular dimension, and weak electrolytes and cations enter more rapidly than strong electrolytes and anions (Franke, 1967; Giese, 1962; Middleton and Sanderson, 1965). Structural Changes in chemicals can markedly alter their absorption Erroperties (Bukovac, 1970; Hull, 1970; Sargent, 1965). 42 Studies with isolated cuticle have demonstrated that partition and solubility play an important role and molecular size a lesser role in cuticular penetration of lipophilic compounds; while molecular size, charge, and adsorbability are relatively important for organic and inorganic jrnus (Franke, 1967; Haile-Mariam and Wittwer, 1965; Yamada, gE_al., 1964a; 1964b). As the concentration of the sorbate molecules is raised in the external solution, greater penetration occurs. This relationship holds for isolated (Darlington and Cirulis, 1963; Norris and Bukovac, 1969) as well as intact cuticle (Sargent and Blackman, 1962; Middleton and Sanderson, 1965). Bukovac and Norris (1968) showed a linear relationship between external concentration and the amount of NAA and NAAm sorbed by the outer surface of isolated adaxial leaf cuticle. Although sorption is the first phase in the cuticular penetration process, the effects of various factors on sorption do not necessarily parallel their effects on penetration (Orgell, 1957). Yamada, eE_al. (1965) examined the penetration of organic and inorganic molecules through enzymatically isolated astomatous cuticles of tomato fruit. Urea penetrated more readily than anions, yet urea was sorbed to a lesser degree than the inorganic ions (Yamada,e§_al., 1966). The cuticle is permeable to acid substances such as 2,4-D (Bukovac, 1970; Sargent and Blackman, 1962) and NAA (Norris and 43 Bukovac, 1969), but it is quite impermeable to most of the basic dyes (Darlington and Cirulis, 1963). However, the relative sorption of the basic dyes generally exceeds that of the acid substances (Orgell, 1957). Finally, penetration involves sorption, movement through, as well as desorption from the cuticle. The rate of desorption and/or translocation away from the cuticle can have a pronounced effect on penetration rate (Crafts and Foy, 1962; Sargent, 1966; van Overbeek, 1956). pH.--The infleunce of pH on regulating the amount of a chemical entering a leaf or cells is well documented (Giese, 1962; Hull, 1970; Sargent, 1965). The hydrogen ion concentration can influence penetration of chemicals in at least two ways. Firstly, the degree of ionization and lipid solubility of some organic mulecules such as organic acids changes with pH (Giese, 1962). Secondly, the sorption and cation exchange capacity of the cuticle and cellular tissues may be directly influenced by pH (Franke, 1967; Martin and Juniper, 1970; van Overbeek, 1956). Experiments with intact tissues do not clearly differentiate between the pH effect on the cuticle and that on the cells, which may respond somewhat differently to changes in pH (Bukovac, 1970; Hull, 1970). For this reason, the remaining discussion on pH will be limited to studies involving isolated cuticle and synthetic 44 monolayers. Sorption by apricot leaf cuticle was markedly influenced by pH, acid substances decreasing and basic ones increasing in relative sorption with increasing pH (Orgell, 1957). Brian (1952) showed that permeability of lipoprotein monolayers to organic acid anions increased when these layers became less negatively charged. These data were explained on the basis that increasing the pH increased the negativity of the acid constituents of the monolayer, thus promoting sorption of cations and decreasing sorption of anions. Van Overbeek (1956) reported the pKa of the cuticle to be about 5; however Yamada (1962) recorded an apparent pKa value of 2.8 for cuticles from green onion leaves, and 3.2 for those of ripe tomato fruit. A pKa value in the range of 2.8 to 3.2 was reported for both the adaxial and abaxial cuticles from pear leaves (Bukovac and Norris, 1968). These authors showed that sorption of NAA by astomatous pear leaf cuticles was highly pH dependent, being greater below the pKa than above; whereas NAAm sorption was independent of pH. NAAm was uncharged over the range of pH values tested and its lipid solubility was not affected. In contrast, the lipid solubility of NAA increased and both NAA and the cuticle were negatively charged, yet some NAA sorption still occurred. These findings clearly indicate that mechanisms other than 45 electrostatic binding were operative in sorption of NAA at these higher pH values. Inorganic ion additives.--In some situations, the biological response to growth regulators and herbicides has been influenced by the inclusion of certain inorganic salts in the ambient solution (Hull, 1970). In most cases, addition of metallic ions decreased the herbicidal effect of 2,4-D--—Fe2+ and Cu2+ to a greater extent and C02+, Zn2+, and Mn2+ to a lesser extent (Szabo and Buchholtz, 1955; 1956; 1961; Wort, 1964). The effect of the inorganic salt additives was more pronounced at 2+ pH 5 than at pH 3. The addition of Ca (as lime) to NAA sprays was shown to reduce effectiveness of NAA (Avery,et al.,l947; Overholser, et al., 1943). Hard water or 100 ppm CaCl in distilled water decreased 2 absorption of NAA as measured by the leaf angle curvature method (Westwood and Batjer, 1960). Boron, as BO3-, has been found to improve penetra- tion and movement of sugars and 2,4-D (Currier and Dybing, 1959; Hull, 1970). Szabo and Buchholtz (1956, 43- and NH4+ salts increased the biological response of plants to 2,4-D. Hull (1970) 1961) demonstrated that PO reported an investigation in which inorganic salts increased the toxicity of 2,4-D toward various broad- NO KNO Ca NO leaved weeds. NH4 3, (NH4)ZSO4, NH4 2 4, 46 and Ca(H2PO all increased toxicity to varying degrees, 4)2 but NH4NO3 was most effective. Orgell and Weintraub (1957) used bean epicotyl curvature as an index of the influence of various ions on the penetration of 2,4-D into bean leaves. With 2,4-D alone absorption decreased at higher pH values. At neutral or alkaline pH values the response was influenced by the cations present. NH4+ or ethanol-NH4+, supplied either as salts or in the buffer mixture, induced responses approaching . + those found w1th 2,4-D alone. In contrast, Na and + K+ did not manifest this property. Using ethanol-HN4 buffers at pH 8.5 with different anions, greatest curvature occurred in the SO 2- solutions with diminish- 4 ing responses from PO43_, NO3-, Cl-, and 803-. Using various cations in 803- buffers at pH 8.5, n—hexylamine gave 70 degrees curvature, triethylamine slightly less with diminishing response from triethanolamine, . . . + . ethanolamine, morpholine, and Ll+. NH , hydraZine, 4 K+, and Na+ gave much diminished responses, producing less than 30 degrees curvature. These authors inter- preted the data to mean that the ionic additives influenced the rate of 2,4—D absorption by the bean leaves; they did not determine directly whether or not the cation concentration has any effect on the amount of 2,4-D ultimately absorbed. 47 The mechanisms by which inorganic salts influence the effectiveness of growth regulating substances may vary; and, most likely, no one mechanism is responsible in all situations. There are at least three ways inorganic salts could influence the effectiveness of growth regulating substances: (a) Occasional incompat— abilities with the surfactant or active ingredient may occur (Horsfall and Moore, 1962; Hull, 1970). (b) The effect of these salts on enzymatic activity, oxidative processes, and other physiological functions could influence trans- location and/or active uptake of growth regulators (Hull, 1970). In either case, a differential penetration rate would be the ultimate result. (c) These ionic additives might enhance the penetration rate of growth regulating substances through the cuticle. This might be accomplished in the following ways: (i) Increasing the wettability of plant surfaces. Szabo and Buchholtz (1961), however, showed no correlation between the wetting properties of solutions containing various inorganic ions and apparent percent penetration of 2,4—D into bean and sunflower cotyledonary leaves. (ii) Chelation or forming complexes with the active ingredients and/or other adjuvants, thereby creating a more diffusable substance through the cuticle. The enhancing effect of NH + salts on leaf angle curvature was attributed 4 to a synergistic cation-anion combination between the 48 NH4+ configuration =N= and the anion from a strongly ionizing precursor acid (Horsfall and Moore, 1962). Absorption spectra of 2,4-D solutions containing differ- ent inorganic salts showed that only the solution con- taining Fe2+ gave indications of complex formation; however it was believed to be inconsequential with regard to the magnitude of the Fe2+ effect (Szabo and Buchholtz, 1961). (iii) Changing the pH of the foliar applied solution. Although the presence of inorganic salts generally do not significantly influence the pH of solutions containing growth regulators, there is abundant evidence that a pH change near the pka of a substance could be an important factor in determining sorption by the cuticle (Orgell, 1957; see previous discussion on pH). (iv) Neutralizing the negative charge on the cuticle, thereby enhancing anion sorption (Franklin, 1969; Orgell, 1957) or permitting free passage of cations which tend to diffuse slowly because of their poor mobility through narrow pores lined with negatively charged surfaces (Goodman, 1962). (5) Influencing the degree of hydration or "bound water" in the cuticle. Depending upon pH, inorganic ions in the cuticle alter the rate of movement of free water molecules and water soluble substances (see review by Schonherr, 1969, pp. 24-27). There is little information on the influence of inorganic ions on sorption by isolated cuticular 49 membranes. Szabo and Buchholtz (1961) studied the effect of various inorganic salts additives, at pH 3 and pH 5, on penetration rate of 2,4-D through the upper epidermis of the Crassulacean plant ggdum. The Sedum epidermis was obtained by peeling it from the sub-epidermal tissue. The penetration was always less at pH 5 than at pH 3. NH4+ and P043. additives increased penetration, whereas Fe2+ and Cu2+ decreased penetration through Sedum at both pH 3 and pH 5. Zn2+, C02+, and Mn2+ were without effect. Tomato Fruit Cuticle as a Sorbent The tomato fruit cuticle has relatively little epicuticular wax, but a well-developed cuticular membrane; at maturity, the peel contains about l-2.3 mg/cm2 cutin (Bukovac, et_al., 1971; Martin and Juniper, 1970). Tomato fruit cuticle recovered from a processing factory contained approximately 6 to 7 % waxes, 55 to 60 % cutin, and 35 to 40 % cellulose, hemisubstances, and phenolic materials (Brieskorn and Reinartz, 1967a; 1967b). The wax fraction was composed of alkanes, fatty acids, sterols, triterpene alcohols such as alpha and beta amyrin, and p-coumaric acid. The cutin was composed chiefly of 10,l6-dihydroxyhexadecanoic acid, with small amounts of l6-hydroxyhexadecanoic, 9,10,16-trihydroxyhexadecanoic anui9,10:l3¢rihydroxyoctadecanoic acids also present in 50 the cutin complex. Hydrolysis of the polysaccharide fraction produced glucose, xylose, and arabinose (Brieskorn and Reinartz, 1967a; 1967b). The cuticle of tomato fruit contains a yellow pigment, probably a flavonoid, whose devefiopment depends on the same light conditions that regulate flowering and seed germination in certain plants (Piringer and Heinze, 1954). Yamada and coworkers (1962, 1964a, 1964b, 1965a, 1965b, 1966) have demonstrated sorption and penetration of radioactive ions and molecules through isolated tomato fruit cuticle. Retention, after blotting and washing with water, of organic molecules such as urea, maleic hydrazide, and succinic acid 2,2-dimethy1hydrazide (SADH) was essentially the same on either the inner or outer surface; whereas inorganic ions such as Ca2+ and SO42— inner surface than the outer surface. Retention of were retained to a greater extent on the ions against blotting, washing, and exchange was greater 2 for Ca2+ than 8042-. Total removal of 804 _ but not Ca2+ was accomplished by exchange (Yamada, gt_§1,, 1964a). There was no apparent localization of sorption on either the smooth outer or irregular inner surfaces, and urea was sorbed to a lesser degree than Ca2+ (Yamada, §£;§1,, 1966). The rate of penetration of both the inorganic ions (Yamada, et al., 1964b) and the organic molecules (Yamada, et al., 1965b) was greater from the outer to 51 the inner surface than in the opposite direction. Hence, the rate of penetration for inorganic ions, but not organic molecules, was related to the extent of sorption on the surface opposite the site of initial entry. The presence of urea in the treatment solution enhanced the penetration of both Rb+ and Cl- through the tomato cuticle (Yamada, 1965a). When immersed in water, isolated tomato fruit cuticle has a marked capacity to absorb water particularly through the inner surface. The water uptake pattern was not altered by addition of urea or Tween 20 (Bukovac and Norris, 1968). Isolated tomato skins were more permeable to water when the outer surface was in contact with the liquid water phase-~i.e., more water penetrated inward towards the protoplast than outward (Hurst, 1941; 1948). This unequal ratio of water permeability has been shown for astomatous cuticles from ivy leaves (Schieferstein and Loomis, 1959) as well as cuticles from insects and grape fruits (Hurst, 1941; 1948). Hurst (1941, 1948) showed that the penetration of ethyl alcohol through the tomato skin was increased in the presence of kerosene. Bukovac, et a1. (1971) studied the relative effect of epicuticular and cuticular waxes on penetration of 2,4-D through isolated tomato fruit cuticle. Removing the epicuticular wax resulted in an 9.2-fold increase in penetration of 2,4—D, and 52 only a slight further increase occurred when both the epicuticular wax and cuticular waxes were removed. Increasing the number of chlorine groups on the phenoxy moiety of phenoxyacetic acid caused a corresponding increase in penetration rate. This effect was less for cuticle with epicuticular wax removal than for non- dewaxed cuticle. The method of isolating tomato fruit cuticle was shown to influence its permeability to NAA (Norris and Bukovac, 1969). Cuticles isolated mechanically or enzymatically showed no significant difference in perme- ability, but those cuticles isolated chemically with ammonium oxalate:oxalic acid showed an 8- to 10-fold greater permeability. Methods of Studying Sorption _by¥Isolated Plant CutiCle Two basic techniques have been used for studying sorption of isolated plant cuticles. Isolated cuticular discs were either floated on the surface or immersed in a solution of known sorbate concentration (Bukovac and Norris, 1968; Orgell, 1957; Yamada, §E_al., 1964a). The amount sorbed was calculated indirectly by measuring the amount remaining in the ambient solution, or directly by measuring the relative concentration retained by the cuticle. MATE RIALS AND METHODS . + Properties of MB and 2,4-D Methylene blue (MB+) and 2,4-dichlorophenoxyacetic acid (2,4-D) were selected as model organic compounds for studying sorption by isolated tomato fruit cuticular membrane. These sorbates have desirable chemical and physical properties of numerous plant growth substances used in research and commercial horticulture. MB+ is a relatively large water-soluble monovalent cation and upon being sorbed by tissues it can be visually localized and measured. Its steric properties and dimensions are known which are important for calculating the specific sorption area of solids (Kipling, 1965). It has been extensively used to assess the specific sorption area and sorption mechanisms of soil (Bergmann and O'Konski, 1963; Bodenheimer and Heller, 1968; Plesch and Robertson, 1948) and graphite and charcoal (Galbraith, et_al., 1958; Kipling, 1965; Kipling and Wilson, 1960). This might be useful in describing the sorption properties of the cuticular membrane. MB+ (M-225--water soluble) was obtained from Fisher Scientific Company and some of its noteworthy properties are reported in Table l. 53 54 TABLE 1.--Some Chemical and physical properties of MB+ and 2,4-D. Property MB+ 2,4-D Chemical name Tetramethylthionin 2,4-Dichlorophenoxy chloride acetic acid Molecular weight 319.85 221.04 Molecular formula C16H18N3OSC1 C8H6O3Cl Molecular structure O-CH2 — COOH .,Ns -—Cl 113C\ .4 C113 C].— \S/ \N+ H c’ I 3 * CH | 3 c1 Color Blue-green Colorless Charge + charged over a wide pKa = 2.8 range of pH Hygroscopicity Trihydrate Nonhygroscopic Solubility (units when given are in g/100 ml at 25 C) References Methanol. Water . . . . . 4.0 Ethanol lOO % . 1.5 Chloroform. slightly sol. Ethyl ether . . insoluble Bergmann and O'Konski, 1963; Conn, 1961; Gurr, 1960; Merck Inoex, 1960. Ethanol 100 %. .129.9 Dioxane. . . . . . . 87.5 Ethyl ether. . . . . 24.3 Ethanol 50 % . . . . 20.6 Water. . . . . . . . 0.09 Herbicide Handbook, 1967; Marquardt, gt al., 1964; Sargent 33 al., 1969. 55 2,4-D is readily taken up by plants and has been extensively used in commercial agriculture as a herbicide and plant growth regulator. It is a strong auxin and is representative of numerous plant growth substances. Extensive literature is available on its chemistry, meta- bolism, and uptake by plant tissues (Crafts, 1961; Crafts, 1964; Herbicide Handbook, 1967; Marquardt, 1964). 2,4-D is a weak organic acid; hence, by changing the pH of the solu- tion, the effect of charge, and to some extent the solubil- ity, of both the 2,4-D and the cuticular membrane can be studied. Carbon-l4 labeled 2,4-D was obtained from Amer- sham/Searle (specific activity = 29 uc/mmole; labeled in the 2-C position). Various chemical and physical proper- ties of 2,4-D are presented in Table 1. Isolated Tomato Fruit Cuticular Membrane Cuticular Membrane Throughout the remainder of this thesis the cuticular membrane as enzymatically isolated from the epidermal cells of tomato fruit will be abbreviated CM. The CM is composed of a cutin matrix embedded with waxes, hemi- substances, and phenolic substances. The outer surface contains a layer of epicuticular wax and the inner (cell-wall surface) contains cuticular pegs which project down between the anticlinal walls of the epidermal cells from which the CM was isolated. 56 Source of Tomatoes Used The CM was enzymatically isolated from carefully selected fruit of a single tomato cultivar (Michigan-Ohio Hybrid——WR 3 Wilt Resistant) grown in the greenhouse and not sprayed with pesticides or growth regulating chemi- cals. The fruit were harvested at red-ripe, pink, and mature-green stages and only those without irregularities or blemishes were used as a source of CM. Fruit were provided by Dr. Shigemi Honma from varietal studies conducted at Michigan State University, East Lansing, Michigan. Isolation Procedures The CM was isolated enzymatically by cutting each tomato into 4 to 6 equal sections, removing as much pulp from these sections as possible without damaging the CM, and incubating in a 2 % pectinase:0.l % cellulase solution for 2 to 3 weeks at 30 C. The isolation media was buffered at pH 3.8 by combining 185 m1 of l N HCl and 200 ml of l N Na-acetate with distilled- deionized water (DDW) per liter of solution. After enzy- matic separation from the epidermal cells, the CM was rinsed in DDW, carefully cleaned with a rubber policeman to remove any adhering cellular material, re-rinsed in DDW, spread out on paper towels, air-dried for several days, and stored in plastic containers. During the entire iso- lation procedure, care was taken to maintain the structural integrity of the CM. In particular, extreme caution was 57 used in handling the CM to prevent loss or distortion of epicuticular wax. General Procedures Abbreviations and Symbols The following abbreviations and symbols are used throughout the remainder of this thesis: Concn = concentration. E = mean. X:Y = X combined with Y. X/Y = either ratio X to Y, or X treatment followed by Y treatment. When the term tomato fruit CM or CM is used with reference to the experiments herein reported, it will be interpreted to mean isolated tomato fruit CM unless spec- ifically stated otherwise. Methods of Analysis The experiments (a) (b) (C) following conditions were standard for all unless mentioned elsewhere: The CM was isolated from ripe tomatoes. Sorbate solutions were made with DDW and no buffering compounds or chemical additives were used. Inorganic salt solutions were 3 x 10"3 M whether used before sorption as a pretreat- ment, or in the sorption solution as a 58 chemical additive or buffering compound, or in the desorption solvent as a chemical additive. (d) Organic solvents when used for extracting waxy materials from the CM were always 100 %, whereas methanol and ethanol when used as a chemical additive in the sorption solution or the desorption solvent were 50 % by volume. (e) Temperature control was maintained at t 1/2 C on a water-bath shaker (Research Specialities Co.--Mode1 2156). Temperature will be reported for all experiments in the appropriate tables or figures. Many of the important parameters associated with the data appearing in the tables and figures are presented in Table 40 (Appendix). Sorption.--With both sorbates, the concentration sorbed was calculated by measuring the concentration remain- ing in solution at equilibrium and subtracting that from the original amount. MB+ was measured with a Gilford spec- trophotometer at 688 nm and radioactive 2,4-D was measured with a Beckman Low Beta II prOportional counter. Although 2,4-D can be accurately measured spectrophotometrically, during sorption substances (or a substance) were leached 59 out of the CM which absorb UV light in the same region as 2,4-D. These water—soluble substances were leached in varying quantities; therefore a water blank to nullify their effect was not adequate when attempting to distinguish small differences in concentration of 2,4-D. These unknown substances absorb insignificant amounts of light above 500 nm; hence they presented no hindrance to analysis for MB+. Each sorption experiment was conducted basically as follows: Segments of CM were cut into small sections averaging about 9 mmz. After thorough mixing, various subsamples of CM (25 to 50 mg) were weighed on a Metler Gram-atic balance and placed in 13 ml glass vials. The sorbate solution (3 to 5 ml) was added to the vials and screw-on plastic caps were used to prevent evaporation. These vials were then placed in a temperature controlled water-bath shaker until equilibrium was attained. For experiments with MB+, the sorbate solution was pipetted into glass test tubes, stOppered, and allowed to equilibrate several hours at room temperature (generally overnight). This was done because temperature is a variable in measuring the concentration of a solute in a liquid with a spectrophotometer. The optical density (OD) of the sorbate solution was measured at 688 mu and a standard curve of MB+ in DDW was used to convert OD mea- surements to concentration values. A set of vials con- taining DDW as the sorbate was established as a control in 60 each MB+ experiment. After sorption, the solution in these control vials was used as the reference blank to set the spectrophotometer to zero OD. With experiments involving 2,4-D, at sorption equilibrium 0.5 ml of the sorbate solution was plated in aluminum planchets (2.4 cm dia), to which was added 0.5 ml of 0.4 M NaHCO3. The liquid was evaporated off in a drying oven at 50 C, 1 m1 of ethanol was added, and it was evaporated off. NaHCO3 was added to prevent vola- tilization by making the Na salt of 2,4-D; ethanol was added to Spread the 2,4—D more uniformily over the bottom of the planchet. The radioactivity in the planchets was measured and a standard curve of 2,4-D stock solution was used to convert cpm to concentration values. The state of sorption equilibrium was determined by measuring the umoles sorbed per gram CM at various time intervals. This was done for MB+ by pipetting a 3 m1 aliquot from each vial, measuring its concentration with a spectrophotometer, and replacing the solution (Table 33, Appendix). Sorption equilibrium for 2,4-D was established by monitoring the radioactivity of the sorbate at differ- ent time intervals (Table 34, Appendix). Desorption.--The degree and ease of desorption of MB+ and 2,4-D differed dramatically; therefore the pro- cedure used to study desorption was varied. For MB+ 61 experiments, CM equilibrated with MB+ was removed from the vials, rinsed lightly with DDW, spread out on paper towels to remove the adhering DDW, and replaced in dry vials. This procedure removed practically no sorbed MB+ from the CM. Because sorbed 2,4-D was readily removed from tomato fruit CM with DDW, the CM was separated from the sorbate by pipetting all possible sorbate from the vials after sorption. At this point for both MB+ and 2,4-D, the desorption solvent, equal to the volume used for sorption, was added and the vials were replaced in the temperature controlled water-bath shaker and desorption permitted for a prescribed time (usually equal to the sorption time). The quantity desorbed was then measured as described for sorption. The pH of these desorption solvents were as follows: DDW was 5.3 and 50 % methanol and CaCl2 solutions were both 5.8. When other inorganic salts were used as desorption solvents, their pH values are reported in the appropriate table or figure. Statistical analysis.--Where applicable, data were analyzed by analysis of variance and means were compared by Tukey's w procedure (Steel and Torrie, 1960). Unless specifically stated, treatment means were compared at P = 0.05. 62 Surface Morphology of Isolated Tomato Fruit CM Small sections (approximately 4 mmz) of nondewaxed and dewaxed tomato CM were affixed With Haupt's adhesive to polished carbon discs. One section was affixed with its inner surface next to the carbon disc while another was mounted with its outer surface in contact with the discs. They were then air-dried at room temperature and coated in a Varian VE—lO vacuum evaporator with approximately a 50 A layer of carbon followed by a 100 A layer of gold. Using the secondary electron mode of an electron microprobe X—ray analyzer (microprobe; Model EMX—SM, Applied Research Labor- atories), the morphological inner (cell-wall side) and outer CM were studied and photographed. Instrument conditions used were 25 kv accelerating voltage and 0.005 pA sample current. Effect of Stage of Fruit Ripeness on Sorption of MB+ The color of equal size tomato fruit were carefully categorized as being green, pink, or red. The CM was isolated, sectioned, and MB+ sorption was measured after intervals of 21, 45, 70, 120, and 178 hours. Effect of pH on Sorption and Subsequent Desorption of MB16 and 2,4-D Various quantities of HCl and NaOH were added to 4 M MB+ solutions to achieve seven different pH values 2.2x10' ranging from 2.2 to 9.2 (12 replicates per pH treatment). After sorption, desorption of MB+ with DDW, methanol, and 63 CaCl2 (4 replicates per solvent) was measured. Various combinations of HCl, H3PO4, NaH2PO4, and NazHPO4 were added to 2.0 x 10.-4 M 2,4-D solutions to achieve eight different pH values ranging from 0.8 to 7.8. Following sorption the CM was rinsed several times in DDW and placed in 10 m1 of DDW for 3 days. The CM was then removed from the DDW and transferred to 5 m1 of different organic solvents (ethanol, dioxane, and chloroform). The 2,4-D desorbed, as indexed by the radioactivity recovered in the desorbing solution, after 8 days by these organic solvents was measured in the usual way and the radioacti- vity retained in the CM following desorption was mea- sured directly with a Low Beta II counter. Effect of Wax on Sorption and Subsequent Desorption of MB+ and 2,4-D Several experiments were conducted to study the effects of wax on sorption and retention of MB+ and 2,4-D. Two slightly different methods were used to dewax the CM; namely, (a) Soxhlet extraction--two hours with chloroform followed by two hours with methanol——and (b) extraction at room temperature——24 hours with chloroform followed by 24 hours with methanol. The basic difference between these two methods was that Soxhlet extraction exposed the CM to solvents at 50 to 60 C where the second method only at 20 to 22 C. After dewaxing, the CM was spread out on paper towels, air—dried, and 64 stored for future use. Both of these dewaxing methods remove cuticular (embedded) wax as well as epicuticular wax. To remove only the epicuticular wax, segments Of CM were imersed in chloroform for three 20-second intervals; they were air-dried and handled as described above. Using MB+ as the sorbate, the main effects of and interaction between direction of sorption and wax content on sorption were determined. Discs of CM were affixed to the end of heavy—walled glass tubes (7 cm long and inner area of 2.4 cm2) with rubber cement and allowed to dry 4 . - + . overnight. Two m1 of 2.2x10 M MB was placed in these tubes and they were held upright in a stainless steel pan over moistened vermiculite. A tight-fitting lid with equidistant holes was made to hold the tubes in a secure position at approximately 1/2 inch above the moistened vermiculite. This set-up created a water saturated atmos- phere surrounding the tubes and reduced evaporation of water through the CM. At the end of 6 days, 2 ml of DDW was added to each tube and allowed to equilibrate for 30 minutes. The sorption solutions were then removed and the concentration of MB+ was determined. Sorbed MB+ can be visually localized in the CM. Following unidirectional sorption, sections of each cate- gory of CM (nondewaxed, epicuticular wax removed, and dewaxed) were mounted in Tissue-Tek O.C.T. compound ("optimum cutting temperature," -15 to -30 C) and cut at 65 4 um on an International-Harris cryostat. The resulting sections were placed on glass slides and allowed to air- dry. No adhesive was necessary as Tissue-Tek serves also as a stable mounting medium. Photomicrographs were taken with a Wild M20 research microscope using a 35 mm film holder; exposure time was determined with a Wild Photo- automat exposure meter. This same procedure was used to obtain photomicrographs of nondewaxed and dewaxed CM following sorption for 2, 5, 10, and 20 days. With the previously mentioned experiments on the effect of wax on sorption ofbflf in this section (and temperature in the next section), initial pH was 4.8; how— ever the final pH was not measured. An experiment was conducted to determine the change, if any, in pH during sorption. Nondewaxed and dewaxed CM were sorbed at 25 and 5 C with MB+. At equilibrium, the sorption solutions were transferred to test tubes and allowed to equilibrate to room temperature; pH was then measured with a Beckman Zeromatic pH meter. An additional experiment was conducted at 20 C comparing sorption of MB+ by nondewaxed and dewaxed CM from a sorption solution buffered with 0.3 M Na-acetate: 0.2 M HCl. Effect of Temperature on Sorption and Subsequent Desorption of MB+ and 2,4—D Sorption isotherms are frequently used to study . . . + sorptive properties of solids. MB and 2,4—D were used as 66 sorbates and care was taken to maintain identical experi- mental conditions except the ones being tested. To accom— plish this, the following precautions were taken: (a) Sufficient quantities of tomato fruit CM were sectioned and thoroughly mixed to provide the same uniform sorbent material for all isotherms involving the same sorbate. (b) The same sorption solutions and instrumentation were used for each temperature. (c) For each sorbate, isotherms were run consecutively each week in order of decreasing temperature——i.e., highest temperature first and lowest temperature last. With MB+ isotherms, nondewaxed and dewaxed CM were run at the same time under identical con- ditions. A11 MB+ isotherms were determined at the same initial pH, 4.8; the pH of the solution after sorption was not measured. 2,4—D isotherms were conducted at two pH values-~namely, initial pH at 0.8 (final pH at 0.8) containing HCl and NaHZPO and initial pH at 7.3 (final 4, pH at 5.8) containing NazHPO4 and NaHZPO4. Nondewaxed and dewaxed CM were analyzed with a Beckman infrared spectrOphotometer at approximately 25 i 5 C and 10 i 5 C. These temperatures are estimates not measured values. A room temperature of 20 C plus heat from the infrared beam form the basis of the estimated higher value; whereas ice water placed in the chamber holding the cuticular membrane was used to dissipate the heat from the system resulting in the lower estimated temperature value. 67 An experiment was conducted to investigate the reversibility of the temperature effect. Two lOO-mg replicates of nondewaxed and dewaxed CM were held in DDW at 20, 35, 55, 75, or 100 C for 1 hour. After being removed from the DDW with a rubber policeman, the CM was laid on paper towels and allowed to cool to room temperature before further handling. The CM for the two replicates were com— bined for each temperature treatment and prepared for sorp— tion in the usual way. Effect of Chemical Additives on Sorption and Desorption To better understand the nature of the sorptive process CM was exposed to various inorganic and organic compounds either before sorption as a 24-hour soak (pre- treatment), in the sorption solution (in solution), or in the desorption solvent. In most cases, these compounds were used at 3 x 10_3 M. Reversibility of Sorption Desorption isotherms can be used to evaluate the reversibility of a sorption process. Desorption isotherms were determined at 5, 15, and 25 C by plotting the quantity (umoles/g) of 2,4-D retained by the CM after desorption in DDW against the quantity (uM) desorbed. RESULTS Surface Morphology of Isolated Tomato Fruit CM Scanning electron micrographs of the inner and outer surfaces of nondewaxed isolated tomato fruit CM are presented in Figure 2. The outer surface resembled a field of mounds representing the periclinal walls of the epidermal cells from which it was isolated (Figure 2A, 2B). There was little or no pronounced fine- structure apparent in the epicuticular wax. Trichomes containing what appeared to be a small central hole simi- lar to a thick straw were occasionally present on the outer surface (Figure 2A). The inner surface (cell-wall side) was irregular and cuticular pegs projected down between the epidermal cells giving rise to outlines of the epidermal cells (Figure 2C, 2D). There were no distinct differences in surface structure of nondewaxed and dewaxed CM; therefore only photomicrographs of nondewaxed CM are shown. Effect of Stage of Fruit Ripeness on Sorption of MB+ Sorption of MB+ by isolated tomato fruit CM at various time intervals and at three stages of fruit 68 Figure 2.-—Morphology of the inner and outer surfaces of isolated tomato fruit CM. A. Outer surface at 109x Outer surface at 1090X Inner surface at 109x Inner surface at 1090X COED 69 70 3:935 ' " we - W. .. it. an: . ‘9‘ ' i ‘ fl 9‘14. 63?‘ $.99 . 5 71 ripeness is presented in Table 2. Sorption equilibrium was attained in approximately two days for CM isolated from green tomato fruit and in five days for CM iso- lated from pink or ripe fruit. CM from ripe fruit sorbed 24 % and pink fruit 22 % more MB+ (umoles/g) than from green fruit. Effect of pH on Sorption and Subsequent Desorption of MB+ and 2,4-D 6 Increasing the pH of the sorption solution enhanced sorption of MB+. A plot of sorption versus pH shows a sigmoidal relationship between these two variables with an inflection point at pH 3.6 (Figure 3). As sorption pro- ceeded, solution pH decreased except at the lowest pH value, 2.2. At sorption equilibrium, the difference between initial and final pH was progressively greater with each higher pH category. The relationship between pH of the sorption solution and desorption of MB+ with is summarized in Table 3. As the DDW, CH OH, and CaCl 3 2 pH of the sorption solution was increased, the % of the sorbed MB+ that could be desorbed with DDW and CaCl2 attained a maximum; however the quantity of MB+ desorbed by methanol appeared to continue to increase with an increase in pH of the sorption solution. 72 TABLE 2.--Sorption of MB+ by CM isolated from tomato fruit at different stages of ripeness. Duration of sorptionl (hr) Fruit 2 Ripeness 21 45 70 120 178 Mean (umoleS/g) Green 15.1 15.3 15.3 15.3 15.3 15.3a Pink 18.4 18.7 18.8 18.9 18.9 18.7b Ripe 18.6 18.9 19.1 19.2 19.2 19.0c Mean 17.4 17.6 17.7 17.8 17.8 lTemperature = 20 C. 2Means followed by different letters are significantly different at P = 0.05. Figure 3.--Effect of pH on sorption of MB+ by isolated tomato fruit CM (temp = 20 C). 73 45 4O 35 830 O O '2' Z 2 20 .— a. g a) (5 O 74 METHYLENE BLUE o—-o final pH H initial pH L 75 TABLE 3.--Relationship between pH of the sorption solution and the amount of MB+ desorbed from isolated tomato fruit CM. Sorptionl Desorptionl pH + + Initial Final MB sorbed Solvent MB desorbed (umoleS/g) (umoleS/g) (%) 2.2 2.2 5.3 DDW 0.1 l 2.2 2.2 5.3 CH3OH 1.0 18 2.2 2.2 5.4 CaClz 0.2 3 2.6 2.5 6.3 DDW 0.1 1 2.6 2.5 6.3 CH3OH 1.2 18 2.6 2.5 6.2 CaC12 0.3 4 3.2 3.1 11.8 DDW 0.2 l 3.2 3.1 11.7 CH3OH 2.4 20 3.2 3.1 11.8 CaClZ 1.3 11 4.0 3.8 23.1 DDW 0.9 3.8 4.0 3.8 22.8 CH3OH 6.2 27. 4.0 3.8 22.4 CaClz 5.6 24. 4.7 4.0 30.1 DDW 1.3 4. 4.7 4.0 29.7 CH3OH 9.2 31. 4.7 4.0 29.3 CaClZ 7 8 26 6.0 4.4 34.5 DDW 1.4 4. 6.0 4.4 34.4 CH3OH 10.9 31. 6.0 4.4 34.0 CaClz 8.5 25. 9.2 5.4 40.9 DDW 1.3 3. 9.2 5.4 40.6 CH3OH 15.6 38. 9.2 5.4 40.7 CaC12 8.3 20. lTemperature for sorption and desorption = 20 C. 76 There was an inverse relationship between pH of the sorption solution and sorption of 2,4-D (Figure 4). As with MB+, a plot of sorption versus pH shows a sigmoid—like curve with an inflection point at pH 3.6. In four treatments, solution pH at sorption equilibrium varied somewhat from initial solution pH; at pH 3.5, it increased and at the three higher pH values it decreased. In all other treatments, initial and final pH values were the same. The pH of the sorption solution had an effect on the quantity and % retention of 2,4-D after exhaustive desorption by DDW followed by either ethanol, chloro- form, or dioxane (Table 4). Even though 2,4-D was highly soluble in these organic solvents and only sparingly soluble in DDW, the organic solvents desorbed only 1 to 8 % of the 2,4-D sorbed by the tomato CM. The quantity of 2,4-D retained by the CM after desorption with DDW followed with organic solvents varied considerably depending on the pH of the original sorption solution. At pH 0.8, only 5.8 % was retained by the CM; at pH values of 1.4 through 4.0, approximately 20 % was retained and at higher pH values progressively less (on an absolute and relative basis) was retained by the CM. Because self-absorption values were not determined Figure 4.--Effect of pH on sorption of 2,4-D by isolated tomato fruit CM (temp = 25 C). 77 SORPTION (p males lg) l4 l2 IO 78 F’ _ 2.4-D _ o-—o final pH 0 a 0 initial pH 0 l 1 1 ' i 1 J l 2 3 4 5 6 8 79 .0 mm H soHuQHOme 0cm cofludHOm How musumummeB a m.a 00.0 0.0 00.0 Hocmcum Hm.a 0.0 0.5 0.0 no.0 0.0 no.0 En0wonoH£U hm.H 0.0 0.5 m.ma 0H.0 0.5 00.0 wsmxofla 0N.H 0.6 0.0 0.0a 60.0 0.0 00.0 Showonoanu 00.0 0.6 0.6 0.00 05.0 0.0 NH.0 Hosmnum 00.0 H.6 0.0 0.00 00.0 0.H ma.0 mcmxoflo 00.NH 0.m 0.0 N.mm H6.m 0.0 NH.0 Euomouoago 00.6H 6.6 6.H 0.0 00.0 0.H 50.0 Hosmnum 00.6H 0.0 0.0 A00 Am\mMHOEJV A00 Am\mmHOE:v Am\mmHOE:v EU wn cosoo ucm>aom meHOm Hmcflm HafiuHcH pmcflmumu 016.0 Edflnnflaflsvm Q16.m oasmmuo 016.0 mm acoHuQMOmmo Hcowudnom .mcmxoflp Ho .Eu0mouoazo .Hocmzum 0n pmonHom 300 CH coflgmHOmmp Hmumm EU pflsum omeou pmuMHOmH an Umcflmuwu Q|6.m mo unsoaw man man coHusHOm coauduom map mo mm :mm3umn QHQmQOwumHmmil.6 mqmde 80 for the counting procedure, the amount and hence the exact percentage of 2,4-D retained in the CM are not known. The quantity of CM in the planchet was about 5-5 mg/cmz, which according to Wang and Willis (1965) would result in a 15 to 25 % self absorption. However, since the quantity of CM was identical in each planchet, these data provide reliable relative values for retention. The absolute amount retained, however, may be 15 to 25 % higher. Effect of Wax on Sorption and Subsequent Desorption of MB* and 2,4-D Methanol extracted significantly less waxy material from the CM than chloroform but not ethanol,with no signi— ficant difference between the latter two solvents (Table 5). Subsequent extraction of the same CM with a different organic solvent resulted in an additional weight loss that averaged 16 % as much as the first extrac- tion; however the amount was the same irrespective of the organic solvent used. The sum of these two 24-hour extrac— tions showed a slight but significant increase in % weight loss using the combination chloroform/methanol as compared to methanol/chloroform and ethanol/chloroform. The combination DDW/DDW reduced the weight of the CM by 0.39 mg/100 mg which was approximately 8 % of that removed by the organic solvents. Exposing the CM to CaCl2 or A1C13 resulted in an increase in weight compared to DDW which caused a 0.31 % decrease (Table 6). One—minute immersion in 81 TABLE 5.--Effect of different solvents on weight loss of isolated tomato fruit CM. Solvents2 Extractionl DDW/DDW CHCl3/CH3OH CH3OH/CHC1 CH CHZOH/CHCl 3 3 3 (%) lst 0.35a 4.25b 3.95c 4.11cb 2nd 0.04a 0.70b 0.68b 0.59b Total 0.39a 4.95b 4.63c 4.70c lConducted at room temperature (21 C). 2Means within each row followed by different letters are significantly different at P = 0.05. TABLE 6.--Effect of different solvents on change in weight of CM isolated from green and ripe tomato fruit. Fruit Ripenessl Duration of Solvent treatment Temp Green Ripe (min) (C) (%) saw 240 20 —---4 - .31a CaC12(3x10-3M) 240 20 ---—4 + .04b A1C13(3x10-3M) 240 20 ----4 + .05b CHC132 1 20 - 2.55a -4.53c CHClB/CH30H3 120/120 55 - 4.55b -5.75d 1 Means within each column followed by different letters are significantly different at P = 0.05. 2 . . . . . . This one minute extraction is assumed to remove primarily epicuticular wax. 3This treatment removes both epicuticular and cuticular (embedded waxes). 4Not determined. 82 chloroform removed 79 % of the waxy materials asso- ciated with CM from ripe fruit and 56 % of that associated with green fruit. This short-term extrac- tion is assumed to remove primarily the epicuticular wax. More total wax (epicuticular and cuticular waxes) and more epicuticular wax were extracted from CM obtained from ripe than green fruit; however less cuticular wax was recovered from CM from ripe than from green fruit (Table 6). MB+ Different methods of dewaxing resulted in no signi— ficant differences in weight loss; however application of heat either during or subsequent to dewaxing significantly decreased sorption of MB+ (Table 7). There also was a slight but significant difference in MB+ sorbed by the CM that was subjected to organic solvents at 50-60 C compared to CM which was placed in DDW at 55 C after being dewaxed at room temperature. Dewaxed CM sorbed more MB+ at both 25 and 5 C and desorbed less MB+ in methanol than nondewaxed CM (Table 8). The quantity of MB+ desorbed from dewaxed and nonde- waxed CM with DDW and CaCl2 was the same at 25 C but significantly different at 5 C for dewaxed versus nonde- waxed CM. When only the epicuticular wax was removed, the CM sorbed significantly more MB+ than nondewaxed CM and less was desorbed when exposed to 100 % methanol 83 TABLE 7.--Effect of different dewaxing methods on weight loss and subsequent sorption of MB+ by isolated tomato fruit CM. CHCl3/CH3OH CHCl3/CH30H CHCl3/CH3OH 1 (room temp, (room temp)/ (Soxhlet, Measurement 20—22 C) DDW (55 C) 50-60 C) Weight loss (%) 4.90a 4.95a 4.92a Sorption2 (umoles/g) 35.6a 34.2b 33.2c 1 Means within each row followed by different letters are significantly different at P = 0.05. 2Temperature = 20 C. TABLE 8.--Wax effect——sorption at 25 and 5 C and subsequent desorption of MB+ from isolated tomato fruit CM. Desorptionl solvents CH3OH CaC12 CM Sorption DDW CH3OH CaCl2 -DDW -DDW (umoleS/g) 25 C Dewaxed 36.5a 0.9a 5.4 9.7 4.5a 8.8a Nondewaxed 30.0b 1.1a 9.5 9.7 8.4b 8.6a 5 C Dewaxed 32.3a 0.7a 4.0 9.8 3.3a 9.1a Nondewaxed 25.8b 1.6b 9.3 8.7 7.7b 7.1b lTemperature = 20 C. For both 25 and 5 C, means within each column followed by different letters are significantly different at P = 0.05. 84 (Table 9). Similarly, compared to extracting only the epicuticular wax, removing both the epicuticular and cuticular waxes significantly increased sorption of MB+ and the quantity and % of MB+ retained after desorption with methanol. Data on unidirectional sorption of MB+ through the outer and inner morphological surfaces are reported in Table 10. With sorption through either surface, non- dewaxed CM sorbed significantly less MB+ than CM with the epicuticular wax removed or the dewaxed CM (epicuticular and cuticular waxes removed). For both the inner and outer orientation categories, the dewaxed CM showed a slight increase in sorption compared to the CM with only epicuticular wax removed. This difference, however, was not significant. Significantly greater amounts of MB+ were sorbed through the inner than outer morphological surface. This is best demonstrated by calculating the ratio of sorption through the inner side to sorption through the outer side. After six days of sorption, 58.8, 10.2,and 7.8 times more MB+ was sorbed through the inner than outer surface for nondewaxed, epicuticular wax removed, and dewaxed CM, respectively. There was no sig- nificant interaction between wax content and orientation with regard to the actual quantity of sorption by the CM——i.e., the umoles/g increase in sorption from dewaxing was the same irrespective of orientation. However, dewax- ing enhanced sorption 7- to 10-fold Math.the outer side 85 TABLE 9.--Effect of epicuticular and cuticular waxes on sorption and desorption of MB+ from isolated tomato fruit CMl. CM Sorption2 Desorption3 (umoleS/g) (nmoleS/g) (%) Nondewaxed 33.2a 12.2a 37.1a Epicuticular wax removed 35.9b 6.5b 18.0b Dewaxed (epicuticular and cuticular waxes removed) 37.4c 4.9c 12.9c lMeans within each column followed by different letters are significantly different at P = 0.05. 2Temperature = 20 C. 3Temperature = 20 C. Desorption values equal the actual amount of MB desorbed by 100% CH OH minus the amount desorbed 3 by DDW. TABLE 10.--Effect of epicuticular and cuticular waxes on unidirectional sorption of MB+ by isolated tomato fruit CM. Sorptionl . 2 . 2 . CM InSide OutSide Ratio: In/Out (nmoles/2.4 cm2 disc) Nondewaxed 294a 5c 58.8 Epicuticular wax removed 366b 36d 10.2 Dewaxed (epicuticular and cuticular waxes removed 382b 49d 7.8 lTemperature = 20 C. Means followed by different letters are significantly different at P = 0.05. 2The morphological side of the CM in contact with MB+. 86 of the CM facing the sorption solution compared to only a 25 to 30 % increase in sorption with the inner orientation. The inhibitory effect of the cuticular waxes and the effect of orientation of the CM on MB+ can be vividly seen in cross-sections of the CM (Figure 5). When the outer surface of the CM was oriented toward the solution, there was no MB+ visible in the nondewaxed CM (Figure 5A). MB+ was distinctly visible in the outer edge of CM when epicuticular wax or epicuticular and cuticular waxes were removed; however no MB+ could be seen in the inner-side segment of the CM above periclinal walls or the cuticular pegs (Figure 5B). Microscopic inspection of CM WhiCh sorbed MB+ from the inner surface clearly revealed that greater quantities of MB+ were sorbed from the inner than the outer surface (Figure 5C, 5D). There were no con- sistent distinguishable differences in loca- tion of the MB+ sorbed from the inner side by any of the three categories of CM (nondewaxed, epicuticular wax removed, and dewaxed). An experiment was conducted to evaluate the long- term effect of time on unidirectional sorption of nonde- waxed and dewaxed CM. However, problems with evapora- tion and leakage resulted in insufficient replicates to report good quantitative data. Spectrophotometric analysis and microscopic observations of the useable replicates (1-3/treatment) indicated similar localization patterns after 5, 10, or 20 days; however, with the inner Figure 5. --Photomicrographs of transverse sections +of isolated tomato fruit CM sorbed with MB+(A11 photomicrographs were taken under bright field and a blue compensating filter.) A. Nondewaxed CM—-outer orientation. B. Dewaxed CM-—outer orientation. C. Nondewaxed CM--inner orientation. D. Dewaxed CM--inner orientation. Figure 6. -—Photomicrographs of transverse sections+ of isolated tomato fruit CM sorbed with MB+ A. Nondewaxed CM-—inner orientation (phase, blue filter). B. Dewaxed CM--inner orientation (bright field, blue filter). C. Dewaxed CM--inner orientation (bright field, green filter). D. CM with epicuticular wax removed--outer orientation (bright field, green filter). 87 88 89 surface facing the sorbate,there was an increase in visible MB+ sorbed in the CM after five days compared to that at two days. MB+ sorbed after two days tended to be localized in the cuticular pegs (Figure 6B). When transverse sec- tions were prepared of sorbed or nonsorbed CM and observed under the microscope, narrow channels or bands oriented perpendicular to the surface were apparent in the center of the cuticular pegs (Figure 6A, 6B, 6C). These bands were distinctly lighter in color, especially when viewed under phase contrast (Figure 6A) or when using a green compensating filter (Figure 6C). Similar appearing narrow bands were evident for all three categories of CM. Trichomes or epidermal hairs were preferential areas of sorption of MB+. This was particularly evident when transverse sections were prepared from CM sorbed through the outer surface (Figure 6D). Although MB+ penetrated nondewaxed CM through trichomes, there was no apparent lateral movement through the matrix of the CM. A separate study conducted to determine if a change in pH occurred during sorption established that pH changed depending on whether or not the CM had been dewaxed. With sorption at 25 C, final pH was 5.0 for dewaxed and 4.2 for nondewaxed; at 5 C final pH was 5.4 for dewaxed and 4.4 for nondewaxed CM. An additional experiment using buffered MB+ solutions showed that, with initial and final pH at 4.5, dewaxed CM sorbed significantly more MB+ than the non- dewaxed CM (18.1 versus 11.1 umoles/g, respectively). 90 At low concentrations (0.5 and 1.0 x 10.4 M), dewaxing had no effect on sorption of 2,4-D; however there was a trend toward greater sorption with dewaxed CM as the 2,4-D concentration was increased to 2.0 x 10”4 M (Table 11). TABLE ll.--Effect of wax on sorption of 2,4-D by isolated tomato fruit CM.1 Initial Nondewaxed Dewaxed 2,4-D concn CM CM (uM) (umoles/g) 50.0 0.5 0.5 100.0 0.9 0.9 150.0 1.6 1.8 200.0 2.3 3.2 lTemperature = 25 C. Initial pH = 3.7 and final pH = 4.5. Effect of Temperature on Sorption and Subsequent Desorption of MB+ and 2,4-D MB Sorption isotherms were determined at 5, 15, and 25 C to establish the effect of temperature on sorption of MB+ by nondewaxed and dewaxed CM. Five MB+ concentrations (0.6, 1.0, 1.4, 1.8, and 2.2 x 10'4 M) were used at each' temperature. Sorption was also determined with 1.8 x 10-4 91 4 and 2.2 x 10- M MB+ for nondewaxed CM at 10, 30, and 35 C, and for dewaxed CM at 35 C. Data for sorption at 1.8 x 10'4 and 2.2 x 10‘4 M at these various temperatures are presented graphically in Figure 7. The plots of sorption versus temperature differed somewhat depending on whether or not the CM was dewaxed. For the nondewaxed CM, as temperature was increased sorption increased from 5 to 10 C, decreased slightly from 10 to 15, then increased rapidly from 15 to 25, followed by a slight decreasing trend from 25 to 35 C. For dewaxed CM, as temperature increased there was a relatively large increase in sorp- tion from 5 to 15 C, followed by a slight increase from 15 to 25, with a small decrease occurring from 25 to 35 C. Results of sorption of MB+ by nondewaxed and dewaxed CM at 5, 15, and 25 C are given in the form of sorption isotherms in Figures 8 and 9. An increase of 20 C (5 to 25) resulted in greater sorption for nondewaxed than dewaxed CM. Differences were 0, 0.2, 1.6, 1.5, and 1.2 for the five MB+ concentrations tested (Table 38, Appendix). These isotherms diverge from the classical relationship between temperature and degree of sorption at a given sorbate concentration-—i.e., sorption increased as the temperature of the ambient solution was increased; whereas with classical isotherms sorption decreases with a rise in temperature. Nondewaxed CM differed from dewaxed CM in the relative rate of increase Figure 7.--Effect of temperature on sorption of MB+ by isolated tomato fruit CM (original concentra- tions of MB+ were: top = 2.2x10‘4 M and bottom = 1.8x10‘4 M.) 92 SORPTION (p moles l9) 34r- 32 3O 28 26 30 28 26 24 METHYLENE BLUE u:— DEWAXED DEWAXED IO 93 NONDEWAXED NONDEWAXED 1 l I I5 20 25 TEMPERATURE (C) Figure 8.-—Sorption isotherms of MB+ by nondewaxed isolated tomato fruit CM (temp in C). 94 SORPTION (paroles/9) 35 3O 25 20 I5 IO 0 95 i- NONDEWAXED CUTICLE METHYLENE BLUE 250 '5' 50 I I 1 1 1 1 1 1 1 4 0 IO 20 3O 40 50 60 70 80 90 EQUILIBRIUM CONCENTRATION (pH) Figure 9.--Sorption isotherms of MB+ by dewaxed isolated tomato fruit CM (temp in c). 96 SORPTION (paroles/a) 35 30 N U N O O O O 97 r. DEWAXED cuncua 25' METHYLENE BLUE '5' 5. l l l L L l I l J 0 IO 20 3O 4O 50 60 70 BO EQUILIBRIUM CONCENTRATION (I‘M) 90 98 :'L'4-. It. in sorption with a rise in temperature. As temperature was increased, sorption by nondewaxed CM increased relatively little from 5 to 15 C with the largest increase in sorp- tion occurring between 15 and 25 C; however the largest enhancement of sorption for dewaxed CM occurred from 5 to 15 C with only a slight increase from 15 to 25 C. According to Giles et 31. (1960), these MB+ iso- therms fit into the category of L isotherms (Langmuir- type), and the equation c/N = c/Nm + l/KNm can be used to describe the experimental data. If the Langmuir isotherm is an adequate description of a sorption process, then a plot of c/N versus c will yield a straight line with slope 1/Nm. (See Literature Review for a des- cription of these terms.) Based on this criterion, sorp- tion of MB+ by tomato fruit CM can be explained by the Langmuir isotherm equation as evidenced by the straight- line relationship between c/N and c (Figures 10 and 11). The heat of sorption (AH) of a simple sorption process should always be negative (exothermic); however data from these isotherms gave positive AH values which averaged 5.2—5.3 kcal/mole for both nondewaxed and dewaxed CM (Table 12). An implicit assumption in the determination of AH from Langmuir isotherms is that the number of sorption sites (specific sorption area) was not altered by temperature. Apparently in sorption of MB+ by tomato CM there was some temperature-dependent release of sorption potential resulting in enhanced sorption at Figure 10.--Langmuir sorption isotherms--sorption of MB+ by nondewaxed isolated tomato fruit CM (temp in C). 99 (q/Iitor) c/N 3.6 3.2 2.8 2.4 2.0 I.2 0.8 0.4 100 NONDEWAXED CUTICLE METHYLENE BLUE 25" c (1.1M) 50 I5‘ Figure ll.--Langmuir sorption isotherms--sorption of MB+ by dewaxed isolated tomato fruit CM (temp in C). 101 (a/liter) c/N 3.2 2.8 2.4 2.0 I.2 0.8 0.4 102 DEWAXED CUTICLE METHYLENE BLUE o 5. 15° ‘ 25° /.7‘ L I I I » I I I I #1 IO 20 3O 4O 5O 60 7O 80 90 c (11M) 103 TABLE 12.--Heat of sorption (AH) of MB+ by isolated tomato fruit CM1. N (relative Nondewaxed Dewaxed sorption) CM CM (kcal/mole) (umoles/g) Unadjusted Isotherms 10 + 3.53 + 5.72 15 + 4.14 + 4.76 20 + 5.41 + 4.10 25 + 7.94 + 4.50 30 -——- + 7.12 Mean + 5.26 + 5.24 (umoles/mz) Adjusted Isotherms 0.3 - 4.41 - 4.62 0.4 - 2.97 - 4.62 0.5 - 3.31 - 3.68 0.6 - 3.75 - 2.79 0.7 - 3.56 - 2.48 0.8 - 2.96 - 2.68 0.9 - 2.71 - 3.06 Mean - 3.38 - 3.42 1 2No value available at this N level. Calculated at 5 and 25 C using the Clausius-Clapeyron equation. - - ~. , . your.“ W‘m .o_-,.-v _._ _.—.- .o a 104 higher temperatures (+AH). If this released sorption potential has the same properties as the original sorp- tion area, then irrespective of how temperature caused a change in sorption potential it can be measured from the c/N versus c plot of the sorption data for each tempera- ture. The available sorption area (A) at a specific tem- perature can be determined from the formula A = NmNoo 10-20. All three sorption isotherms showed a straight- line plot of c/N versus c; hence Nm from each can be calculated. No is Avogadro's number and o is the area occupied by one MB+ molecule, which is 134 32 assuming the MB+ molecule is orientated flat in a plane parallel to the sorption site of the CM (Giles 22 al., 1960; Kipling and Wilson, 1960). The calculated specific sorption area values for nondewaxed and dewaxed CM are presented in Table 13. The largest increase in A occurred from 15 to 25 C for nondewaxed CM whereas dewaxed CM exhibited the greatest increase in A from 5 to 15 C. These values correspond closely with data on the effect of temperature on penetration of naphthaleneacetic acid (NAA) through isolated pear leaf upper CM (Table 14). Using nondewaxed CM, a QlO of 5.6 was obtained from 15 to 25 C compared to a Qlo of 2.9 from 15 to 25 C. The reverse was true for dewaxed CM——a Q10 of 1.5 was obtained from 15 to 25 C compared to a Q10 of 8.6 from 5 to 15 C. To establish if these specific sorption area values account for the nonclassical order of the Langmuir isotherms 105 TABLE l3.--Inf1uence of temperature on the specific sorption area of nondewaxed and dewaxed tomato fruit CM. Specific sorption Increase Increase CM Temp area over 5 C over 15 C (C) (mz/g) (%) (%) Nondewaxed 5 22.5 15 25.0 11.1 25 33.7 49.8 34.8 Dewaxed 5 26.4 15 33.2 25.8 25 35.6 34.8 7.2 TABLE l4.--Effect of temperature on penetration of NAA through isolated pear leaf upper CM1 (Norris and Bukovac, 1969). Temp Dialyzing membrane Nondewaxed CM Dewaxed CM (C) (nmoles/ Qlo (nmoles/ Q (nmoles/ Q 48 hr) 6 hr) 10 12 hr) 10 5 60.5a 0.4a 6.7a 1.2 2.9 8 6 15 75.8a 1.3a 57.7b 1.0 5.6 l 5 25 72.0a 7.2b 85.0c 1.4 2.4 l 2 35 102.1b 17.4c 99.3c Means within each column followed by different letters are significantly different at P = 0.05. . s ’9”..- _ 1:“.‘~.-" 0‘." 106 obtained, sorption values were adjusted by dividing the sorption value (umoles/g)by its respective A value (mZ/g) to obtain umoles/mz. In this way, sorption was calculated on an equivalent sorption area basis. The adjusted isotherms for nondewaxed and dewaxed CM are illustrated in Figures 12 and 13. On an equivalent sorp— tion area basis, an increase in temperature resulted in a decrease in sorption and thus gave a classical display of sorption data. When adjusted for the temperature effect, negative AH values of 3.4 kcal/mole were obtained for both nondewaxed and dewaxed CM (Table 12). No distinguishable differences were found in the infrared spectra of the CM as a result of dewaxing or exposure to temperatures of 10 and 25 C. An experiment was conducted to evaluate the rela- tive ease of removal (desorption) of the temperature— enhanced sorbed MB+. Twenty-four replicates each of non- dewaxed and dewaxed (at 20-22 C) CM at 25 and 5 C were sorbed for five days. An increase in temperature of 20 C resulted in an increase in sorption of 4.2 umoles/g for a both nondewaxed and dewaxed CM (Table 15). For nondewaxed I CM, differences in desorption between 25 and 5 C were { significantly different using either DDW (-0.5 umoles/g), 2 (+ 1.5 pmoles/g); 3 however, for dewaxed CM, there were significant differences \ methanol (+ 0.7 umoles/g), or CaCl using methanol (+ 1.2 umoles/g) but not using DDW or CaC12. The values using methanol or CaCl2 are the actual Figure 12.--Sorption isotherms adjusted to unit sorption area, m2--sorption of MB+ by nondewaxed isolated tomato fruit CM (temp in C). 107 3011911011 (muons/ma) I.O .0 0 .° 0 .0 .a. 0.2 I' 0.0 O 108 NONDEWAXED CUTICLE METHYLENE BLUE 1 1 1 1 1 1 4 I 1 1 IO 20 3O 4O 5O 60 70 BO 90 EQUILIBRIUM CONCENTRATION (I‘M) Figure l3.--Sorption isotherms adjusted to unit sorption area, m2--sorption of MB+ by dewaxed isolated tomato fruit CM (temp in C). 109 SORPTION 1,. moles/m2) 110 l.2 I. DEWAXED CUTIOLE METHYLENE BLUE I.O _. 5. 0 I5 25, .0 O I .0 m I 0.4 _ / 0.2 0.0 I 4 1 L 1 1 1 1 J 0 IO 20 30 4O 5O 60 70 BO 90 EQUILIBRIUM CONCENTRATION (11M) 111 amounts desorbed by CaCl2 and methanol minus that desorbed by DDW, based on the assumption that both the CaCl2 and methanol solutions removed the DDW fraction also. Thus, the sum of desorption for nondewaxed CM equaled 1.7 umoles/g (-0.5 + 0.7 + 1.5) and for dewaxed CM equaled l.2 umoles/g. These values correspond to 40 % for nondewaxed and 29 % for dewaxed CM of the 4.2 umoles/g MB+ attributed to the temperature effect. Dewaxing at room temperature (20-22 C) and by Soxhlet (50-60 C) resulted in a subsequent differential response to sorption at 25 compared to 5 C. Dewaxing at room temperature resulted in the same increase in subse— quent sorption of MB+ for both temperatures (Tables 8 and 15). At similar concentrations of MB+, the Soxhlet method of dewaxing resulted in 1.2 to 1.5 umoles/g more sorption at 5 than at 25 C (Table 38, Appendix). One-hour treatments with DDW at 20 or 35 C had no effect on weight loss or subsequent sorption by the CM (Table 16). Temperatures of 55 and above had a signi- ficant irreversible effect on sorption of MB+ by both nondewaxed and dewaxed CM. Nondewaxed CM sorbed 2.9, 7.0, and 7.1 umoles/g more MB+ at 55, 75, and 100 c, respectively, and resulted in a progressive loss in weight (0.44, 0.74, and 1.74%) with a rise in temperature of the heat treatment. Dewaxed CM sorbed 1.3, 1.2, and 1.2 umoles/g less MB+ at 55, 75, and 100 c, respectively; but only the 100 C treatment resulted in a significant weight 112 TABLE 15.--Temperature effect——sorption and subsequent desorption of MB+ from nondewaxed isolated tomato fruit CM. Desorption1 solvents CH3OH CaClZ Temp Sorption DDW CH3OH CaCl2 -DDW -DDW (C) (nmoleS/g) Nondewaxed CM 25 30.0a 1.1a 9.5 9.7 8.4a 8.6a 5 25.8b 1.6b 9.3 8.7 7.7b 7.1b Dewaxed CM 25 36.5a 0.9a 5.4 9.7 4.5a 8.8a 32.3b 0.7a 4.0 9.8 3.3b 9.1a lTemperature = 20 C. For both types of CM, means within each column followed by different letters are significantly different at P = 0.05. TABLE l6.--Effect of l-hour treatments with DDW at different temperatures on weight loss and subsequent sorption of MB+ by isolated tomato fruit CM. Temperature of DDW (C) CM1 20 35 55 75 100 Weight loss (%) Nondewaxed 0.04a 0.08a 0.44b 0.74c 1.74d Dewaxed 0.04a 0.02a 0.05a 0.06a 0.80c Sorption2 (umoles/g) Nondewaxed 29.3a 29.2a 32.2b 36.3c 36.4c Dewaxed 36.8a 36.7a 35.5b 35.6b 35.6b lDewaxing was at room temperature (20-22 C) and nondewaxed CM received a 48-hour presoak in DDW. Means within each row followed by different letters are significantly different at P = 0.05. 2Temperature = 20 C. 113 loss (0.80%). Sorption of MB+ by nondewaxed CM following heat treatments of 75 and 100 C was similar to the dewaxed CM at 20 and 35 C. 2,4-D Results of sorption of 2,4-D by nondewaxed tomato fruit CM at 5, 15, and 25 C are presented in the form of sorption isotherms in Figure 14. Four initial concentra— tions were used at each temperature (0.5, 1.0, 1.5, and 2.0 x 10.4 M). The sorption isotherms at pH 0.8 would be classified as C isotherms (constant partitioning) according to the classification of Giles EE.E£° (1960). The isotherms obtained at pH 5.8 would most likely be S isotherms (sigmoid or low-affinity). The isotherms at PH 0-8 are classical——i.e., as temperature was increased sorp- tion decreased. In contrast, the isotherms at pH 5.8 are just the opposite——sorption increased with an increase in temperature. The difference between sorption of 2,4-D at pH 0.8 and pH 5.8 varied considerably. When comparing equivalent initial concentrations at the same temperature, sorption of 2,4-D at pH 0.8 consistantly exceeded that sorbed at pH 5.8. The mean was 13.1 with a range from 4.7 to 23.5 times as much 2,4-D sorbed at pH 0.8 as at pH 5.8 (Table 39, Appendix). Figure l4.--Sorption isotherms of 2,4-D by nondewaxed isolated tomato fruit CM at pH 0.8 and 5.8 (temp in C). 114 SORPTION (p males/o) 115 pH 0.8 5. NONDEWAXED CUTIOLE 2.4-O I5 . l5‘ 25' l2 1. 9 1. 6 .- 3 / 11115.8 .1 / fl . _ 441.7 4—9 o L 1 "T 1 4 1 11 J O 20 4O 60 80 I00 I20 I40 I60 I80 ZOO EQUILIBRIUM CONCENTRATION (11M) 116 Effect of Chemical Additives on Sorption and Desorption of MB+ and 2,4-D Data on the effect of inorganic cations, used either in the sorption solution during sorption or as a pretreatment exposure, on sorption of MB+ by isolated tomato fruit CM is presented in Table 17. Compared to the control, NaCl was without effect on sorption either as a pretreatment or during sorption. CaCl2 in the sorp- tion solution depressed (42.9%) sorption whereas a pre- treatment enhanced MB+ sorption by 17.6 %. AlCl depressed 3 MB+ sorption by 83.3 and 50.0 % when added to the sorption solution and used as a pretreatment, respectively. Na-acetate at 0.2 M was used as a buffer constituent in the isolation of the tomato fruit CM used in these studies. Although the isolated CM was washed many times with DDW before use, possibly some Na+ and, to a lesser extent perhaps, acetate ions and acetic acid molecules remained sorbed after washing. Consequently, the effect of NaCl or Na-acetate:HCl on sorption of MB+ was next determined (Table 18). Compared to the control, NaCl at 3 3 x 10- M had no significant effect on sorption, whereas at 3 x 10.2 and 3 x 10-1 M NaCl caused a progressively significant decrease in sorption. Na-acetatezHC1 at 3 and 2 x 10'3 3 x 10- M enhanced sorption by 10.0 %; however, increasing these substances 10- and lOO-fold, pro— gressively decreased sorption by 4.3 and 62.8 %, 117 TABLE l7.--Effect of inorganic cations on sorption of MB+ by isolated tomato fruit CM. Sorptionl pH Inorganic ion + Difference treatment Initial Final MB sorbed from control2 (nmoleS/g) Control (DDW) 3.9 4.0 21.0 0.0a NaCl: In solution 3.9 4.2 21.5 + 0.5a Pretreatment 3.9 4.3 22.4 + 1.4a CaC12: In solution 3.9 3.8 12.0 - 9.0b Pretreatment 3.9 4.4 24 7 + 3.7c A1C13: In solution 3.8 3.6 3.5 -l7.5d Pretreatment 3.9 4.1 10.5 ~10.5e 4 1Temperature = 20 C. 1.5 x 10- M HCl was added to the MB+ sorption solution. 2Means followed by different letters are significantly different at P = 0.05. 118 TABLE 18.--Effect of NaCl and Na-acetate on sorption of MB+ by isolated tomato fruit CM. Chemicals added to pH Difference sorption Chemical MB+ from solution concn Initial Final sorbedl control2 (m) (umoles/g) Control (DDW) 4.8 4.2 29.8 0.0a NaCl 3x10"3 5.3 4.2 28.7 - 1.1ab 3x10-2 5.3 4.2 22.3 - 7.5c 3x10-1 5.3 4.3 7.6 -22.2d Na-acetate: 3x10‘33 HCl 2X10_3 5.1 5.1 32.9 + 3.1e 3x10-2: 4.5 4.5 28.5 - 1.3b 2x10'2 3x10-1: 4.5 4.5 11.1 -18.7f 2x10-l lTemperature = 20 C. 2Means followed by different letters are significantly different at P = 0.05. 119 respectively. Comparing the NaCl treatments with the Na-acetate:HC1 at each of the three concentration levels shows that in every case the NaCl treatments resulted in less (4.2, 6.2, and 3.5 pmoles/g) sorption than the Na-acetate:HCl. Methanol is an effective solvating compound for 2+ ions have high various organic ions such as MB+, and Ca potential to exchange for other cations on a negatively charged surface. These two chemicals were used in various ways to determine their effect on sorption and desorption of 118+ by tomato fruit CM (Table 19). The effects of CaCl2 in the sorption solution and as a pretreatment were similar to that reported in Table 17. CaCl2 in solution decreased sorption by 5.4 umoles/g (27.8%) whereas CaCl as a pretreatment enhanced sorption of MB+ by 8.4 2 umoles/g (43.3%). Methanol and methanol:CaCl2 decreased sorption of MB+ by 12.6 (64.9%) and 4.2 umoles/g (78.4%), respectively. Following sorption, desorption was carried out with methanol or CaCl2 (Table 19). Methanol desorbed 5.8 umbles/g of MB+ from the control; however this amount was much less than the difference (12.6 umoles/g) in sorption due to methanol in the sorption solution. CaCl2 desorbed 5.4 umoles/g, which was the same as the difference in sorption between the control and the CaCl in-solution treatment. 2 .1. The amount of MB desorbed by methanol and CaCl2 from the CaCl2 pretreatment increased in proportion to the enhance- ment of sorption. Hence, the % desorption was the same for 120 .H0.0 n a pm accumwmap >HDCMOH0Hcmam one muouuoa ucoumwwflp >b UQBOHHOM cEsaoo comm cecbfl3 memo: .300 >2 prHOmop ucsoem 0:0 mscfle bcm>mom oLu >3 omnuomop +02 00 DCDOEm Hmsuom ogu stwm mesam> :oflDQHOme m .000 mom mcoflumcflfilos MSOw :uHB EU 00 muom Umbmmuu >HHonucopfl mpcomwumou HmeEsc cgEom gommm .U om u COHDQHOmmp 0cm COHDQHOm MOM ouswonomEmBH oe.m os.o Naobo o.6 > N m bH.NN oo.H zommo m.e > N.m 6.m Hobo :o so oom.m oe.o Nsobo o.e >H m be.NN om.H :omzo 6.0 >H o.m N.m :o :o be.oH oe.N Naobo N.6H HHH N mm.om om.6 :omzo N.ms HHH N.6 N.m Hobo ”:oHDsHOm CH 6o.bN om.e Naomo N.NN He N 6H.aN so.m momzo m.aN HH m.6 m.e .Hoao usstomouuwsd 6m.eN 66.m Nsooo s.as H 6N.om 60.m :ommo H.ma H N.6 0.6 Hosocoo A00 Am\mofloasv AO\moHOEsv mUmQHOmoo +02 uco>aom UOQuOm +02 Hofluh Assam HmfluflcH ucoEumonh N aboHEmso rm Hcoflumuommo Hcoflumpom .20 bases N N 00680» UTDMHOmH >3 +02 00 COHDQHOmmp 0cm COHDQMOm co Homo saw :0 :U m0 Dowwwm11.ma mqmdh 121 both control and CaCl2 pretreatment CM. The % MB+ desorbed by methanol was the same also for CM sorbed in the presence of CaClZ; however the amount and % of 14B+ desorbed by CaCl2 was approximately one-half of the (zontrol. Although only small amounts of MB+ were desorbed kxy methanol from the CM sorbed in the presence of methanol 811d methanol:CaC12, desorption for both treatments was 2:2 %. CaCl2 desorbed only 0.7 (9.5%) and 0.1 prnoles/g (3.7%) from CM sorbed in the presence of methanol: CéaCl respectively. 2! Calcium salts of C1-, 3042-, and N03- had no signifi- ceant differential effect on desorption of MB+ from the CM (flFable 20). All three calcium salt solutions desorbed aLDproximately 10 times the amount of MB+ compared to (fleasorption with DDW alone. Various inorganic chloride Séilts were used to determine if cations show a differential effect on desorption of MB+ from tomato CM. Compared to DDW alone, more MB+ was desorbed in prOportion to the ease CNE’ exchange of the cations used (Tables 21, 22, and 23). Data on the effect of DDW, chloroform, and methanol Orl desorption of MB+ from tomato fruit CM are presented in Tkik>1e 24. DDW and chloroform desorbed similar quantities (If IWB+ from the CM for nondewaxed, epicuticular wax rennxoved, and dewaxed CM. Methanol, however, progressively + . deSScorbed less MB as more waxes were removed prior to SolifiDtion. Averaged over the three types of CM, there were r") Esignificant differences in desorption between DDW and .H0.0 u m um usmeMMHw >Hucm0fiwflsmflm mum mumpuma quHmMMHv >n Umzoaaom mammz 122 m .umm Hem mmpMOHHmmn snow nufl3 20 mo muwm 00¢mmu0 >Hamoflusmcfl musmmmnmmh Hammad: amEom nommN .0 mm H GOAumnommw 0cm cofiumnom How musumummfimaa £6.00 H.0H n.6 0.0 Namozvm0 0.0m >H be.mm o.oa 0.6 N.m 6ommo m.mN HHH sm.mm H.oa 5.4 m.m Naobo N.mN HH m0.m 0.H 0.0 0.0 Boo 0.00 H A00 Am\mmH081v Am\meoE:v mwmnuommw +02 Hmsfim HMfluHcH ucm>aom wauom +02 mamwue mm asoflumuommo Hc0flumuom .20 uflsum oumEon UquHOmfl Eoum +mz mo coapmnowmw co mcoacm oscmmuosw mo 00000011.0m mamas .H0.0 u m um ucmnmmmew >Hpsmoflmacmww mum mumuema ucmummmww >Q ©m3oHHom mammzm .umm H00 mwumowammu H500 nuHB 20 mo mumm psmfiummuu >Hamoflucmvw mucmmmummu Hmumfiss :mEom A0600 .0 0m u aoflsmuommp 0cm cowumHOm H00 wusuwummfimea 123 00.06 0.6a 6.0 0.0 H03 0.00 HH> we.mo m.m~ m.m N.m maoas m.mN H> em.mm H.0H a.6 m.m Naomo N.mN > o0.0m 0.0 0.0 0.0 0H002_ 5.0m >H n0.0 0.0 0.6 6.0 HOM 0.0m HHH n0.5 0.0 0.0 6.0 H002 6.0m HH 80.0 0.H 0.0 0.0 300 0.0m H A00 Am\moHoEnv A0\mmHOE:0 mcmnuomwc +mz Hmcfim HMfluwsH ucm>aom cmnHOm +mz NHMNHB mm HsoflpQHOmmo Hsowumnom .20 uwsum oumEou pmumaomfl Eonm +mz mo coaumHOmmU co mcowpmo owsmmuocw mo powmmm11.am mqmfia 124 TABLE 22.-~Relationship between desorption of MB+ from isolated tomato fruit CM and the relative binding strength (ease of exchange) of various cations. Cationl MB+ desorbed2 (nmoleS/g) A13+ 19.9 Ca2+ 10.0 Mg2+ 8.2 K+ 2.5 Na+ 2.2 l I a O o Arranged according to decreaSing ease of cationic exchange. 2MB+ desorbed by the corresponding cationic salt solution. 125 TABLE 23.--Data for sorption isotherm at 25 C, and subsequent effect of inorganic cations on desorption of MB+ from isolated tomato fruit CMl. Initial Sorption Desorption2 MB concn Replicate MB+ sorbed Solvent MB+ desorbed (0M) (nmoleS/g) (nmoleS/gl (%) 60.0 1 10.9 DDW 0.1 0.9 2 10.9 NaCl 0.4 3.7 3 10.8 CaC12 1.0 9.3 4 11.0 A1C13 2.8 25.5 X 10.9 1.1 9.8 100.0 1 17.7 DDW 0.2 1.1 2 17.8 NaCl 0.6 3.4 3 17.7 CaClZ 2.8 15.8 4 17.3 A1C13 6.1 35.3 X 17.6 2.4 13.9 140.0 1 24.8 DDW 0.3 1.2 2 24.2 NaCl 0.8 3.3 3 23.8 CaC12 4.0 16.8 4 23.9 A1C13 8.3 34.7 X 24.2 3.4 14.0 180.0 1 29.5 DDW 0.3 1.0 2 29.6 NaCl 0.9 3.0 3 29.9 CaC12 5.8 19.4 4 30.3 A1C13 15.6 51.5 X 29.8 5.7 18.7 220.0 1 33.4 DDW 0.5 1.5 2 34.3 NaCl 1.6 4.7 3 32.8 CaCl3 7.2 22.0 4 33.1 A1C13 5.2 45.9 X 33.4 6.1 18.5 Mean 1 23.3 DDW 0.3 1.1a 2 23.3 NaCl 0.9 3.6a 3 23.0 CaCl3 4.2 16.7b 4 23.1 AlCl3 9.6 38.6C lTemperature for sorption and desorption = 25 C. 2Initial and final pH values for desorption are same as recorded in a similar experiment reported in Table 21. Means followed by different letters are significantly different at P = 0.05. - 126 .000H mum3 mucm>H000 .00.0 n 0 pm ucmHmMMAw >Hucm0000cmflm wum mumppma psmanMHw >b Umzoaaom mammzm soflumHOme 0cm :oHuQHOm new musumuomfimaa be.am e.oa mommo N.em m 6m.N 0.0 maomo N.mm N M0.0 H.H 300 0.60 H News: «.ma 6.0 m00m0 0.00 0 Avm>OEmH mmXMB 0.H 5.0 0H030 H.00 N Hmasoflusoflmmv 5.0 0.H 309 0.00 H Ume3wo H.0N H.m mommo 0.6m m 0.0 0.H 0H0m0 0.60 N Uw>OEmH x03 0.0 H.H 300 0.60 H “masowusowmm o.mm N.NH mommo H.Nm m e.N 0.0 maomo m.Hm N H.6 0.H 300 0.00 H 00x03mwsoz A00 A0\mmHOEJV A0\meoEnv Umbu0m00 +02 mucm>aom UwQHOm +02 mom :0 msoflumHOmwo cowumuom . Z0 uflsnm oumaou vmumHomH Eoum +mz mo coHumHOmmU so mucm>aom namuwmwflw wo pomMMM11.6N mqmse 127 chloroform but methanol desorbed over 10 times more MB+ than DDW or chloroform. 2,4-D The presence of sodium salts of Cl- and SO42— in the sorption solution had no significant effect on sorption of 2,4-D by tomato CM. NaHZPO4 in solution resulted in a significant increase in sorption of 2,4-D accompanied with a decreased final pH compared to the control. However, the difference in sorption between control and NaHZPO4 treatments were not significant when the concentration of MB+ sorbed was adjusted to account for the difference in final pH of the sorption solutions. This adjustment was made by calculating from the pH-sorption curve (Figure 4) the difference in 2,4-D sorption between the control (pH 4.5) and the NaH PO4 treatment (pH 4.1) and subtracting 2 that from the quantity sorbed in the latter treatment. Assuming this adjustment is valid, C1-, 8042-, and H2P04- 42- and PO43-) at the concen- trations tested had no effect on sorption of 2,4-D (Table (as well as fractions of HPO 25). + + 2+ . Compared to DDW alone, Na , K , and Mg in the sorption solution resulted in no significant differences in sorption of 2,4—D by the CM (Table 26). Ca2+ and 3+ Al resulted in decreased final pH values and increased differences in sorption. When these sorption values were adjusted to account for the final pH values, Ca2+ and A13+ still showed significant differences in sorption 128 TABLE 25.--Influence of inorganic anions on sorption of 2,4-D by isolated tomato fruit CM. Sorptionl Ions added pH to sorption 2,4-D Adjusted to solution Initial Final sorbed pH 4.5 (nmoleS/g) Control (DDW) 3.7 4.5 2.0a 2.0a NaCl 3.7 4.5 1.9a 1.9a NaZSO4 3.7 4.4 2.2a 2.0a NaHzPO4 3.7 4.1 2.9b 1.8a 1Temperature = 25 C. Means within each column followed by different letters are significantly different at P = 0.05. TABLE 26.--Inf1uence of inorganic cations on sorption of 2,4-D by isolated tomato fruit CM. Sorptionl Ions added pH to sorption 2,4-D Adjusted to solution Initial Final sorbed pH 4.5 (nmoleS/g) Control (DDW) 3.7 4.5 2.0a 2.0a NaCl 1.9a 1.9a KCl 2.2a 2.2ab MgCl2 2.6a 2.4ab CaCl2 3.9b 2.8bc AlCl3 . 7.5c 3.1c 1Temperature 25 C. Means within each column followed by different letters are significantly different at P = 0.05. 129 of 2,4-D; the difference between the two salts were not significantly different. 2,4-D is highly soluble in ethanol being 229 times more soluble in 50 % ethanol than in DDW. The effect of ethanol as well as CaCl2 and AlCl3 in solution and as a pretreatment on sorption of 2,4-D by tomato CM is reported in Table 27. Compared to DDW alone, ethanol in solution, and AlCl in solu- 2 3 tion and as a pretreatment significantly enhanced sorption; as a pretreatment, CaCl whereas ethanol in solution and CaCl as a pretreatment had 2 no significant effect on sorption of 2,4-D. A similar trend was found after adjusting for the final pH. The data in Tables 31 and 32 (Appendix) clearly establish that compared to DDW alone the anions Cl-, 804-2, and H2P04-, and the cations Na+, Ca2+, and Al3 nificant effect on desorption of 2,4-D from tomato CM. + had no sig- Reversibility of Sorption E Sorption of MB+ by tomato fruit CM was essentially irreversible as evidenced by low desorption in DDW (Table 28). As the initial MB+ concentration was increased from 0.6 to 2.2 x 10-4 M, the % sorption equilibrium values increased from 8.8 to 24.2. When DDW, the solvent used for sorption, was substituted in the vials after removing the sorption solution, less than 1.5 % of 130 TABLE 27.--Inf1uence of CH3CHZOH, CaClz, and AlCl3 on sorption of 2,4-D by isolated tomato fruit Cm. Sorptionl pH Chemical 2,4-D Adjusted to treatment Initial Final sorbed pH 4.5 (nmoleS/g) Control(DDW) 3.7 4.5 2.0a 2.0a CH3CH20H: In solution 4.1 4.4 2.1a 1.9a Pretreatment 3.7 4.2 3.3b 2.6b CaClz: In solution 3.7 4.1 3.9b 2.8bc Pretreatment 3.7 4.5 2.4a 2.4ac A1C13: In solution 3.6 3.5 7.5c 3.1b Pretreatment 3.7 3.8 5.3d 2.7bc lTemperature = 25 C. Means within each column followed by different letters are significantly different at P = 0.05. 131 TABLE 28.--Re1ationship between sorption and desorption of MB by isolated tomato fruit CM. Sorptionl Desorption2 Initial Equilibrium Equilibrium Retained concn Sorbed concn concn in cuticle (uM) (umoleS/g) (uM) (%) (uM) (%) (nmoleS/g) 60.0 10.9 5.3 8.8 0.6 1.1 10.8 100.0 17.7 11.5 11.5 0.8 0.9 17.5 140.0 24.8 15.8 11.3 1.6 1.3 24.5 180.0 29.5 32.4 18.0 1.7 1.2 29.2 220.0 33.4 53.2 24.2 2.4 1.4 33.1 Mean 14.8 1.2 lTemperature = 25 C. The sorption solution was MB in DDW at pH 4.8. 2Temperature = 25 C. The desorption solvent = DDW, and the final pH of all desorbed concentrations = 5.3. 132 + . the MB was desorbed and was practically constant over the entire concentration range. 2’4-D Sorption of 2,4-D by tomato CM was reversible (Table 29)--at least the major portion (pH section, Table 4). As the initial 2,4-D concentration was increased from 0.5 to 2.0 x 10--4 M, the % sorption equilibrium values remained essentially constant. When DDW was sub— stituted for the sorption solution, % desorption values approximated the % sorption equilibrium values (Table 29). Figure 15 depicts the desorption data in the form of desorption isotherms. The lines in Figure 15 were not extrapolated to the base line because, at the highest 411). concentration of 2,4-D used in these studies (2.0 x 10- there was a fraction of the 2,4-D (approximately 1.35 umoles/g) that was fixed in the CM——i.e., a fraction that was not desorbed in DDW (Table 4)1. (There were no data obtained on exhaustive desorption of the CM sorbed with 4 2,4-0 at 0.5, 1.0, or 1.5 x 10' M.) 1The quantity of 2,4-D retained against DDW by the CM was estimated by dividing the actual measured 2,4-D retained in the CM (0.86 umoles/g) by 0.8 (actual minus 20% self-absorption) and adding this value (1.08 umoles/g) to that desorbed in ethanol (0.27 umoles/g) which equaled 1.35 umoles/g. This value equaled 9% (1.35 x loo/14.76) of the original quantity of 2,4-D sorbed by the CM. 133 TABLE 29.--Relationship between sorption and desorption of 2,4-D by isolated tomato fruit CM. Sorptionl Desorption2 Initial Equilibrium Equilibrium Retained concn Sorbed concn concn in CM (uM) (nmoleS/g) (uM) (%) (UM) (%) (umoleS/g) 22.9 50.0 4.0 14.0 28.0 10.8 30.0 2.8 100.0 8.0 28.0 28.0 19.8 27.5 5.8 150.0 12.0 42.0 28.0 28.8 26.7 8.8 200.0 15.3 62.3 31.1 38.7 28.1 .ll.0 Mean 28.8 28.1 _1_5__C_ 50.0 4.1 13.1 26.2 10.8 29.3 2.9 100.0 8.5 23.5 23.5 19.8 25.9 6.3 150.0 12.7 35.7 .23.8 28.8 25.2 9.5 200.0 16.7 49.7 24.8 37.8 25.1 12.5 Mean 24.6 26.4 §_E 50.0 4.4 10.4 20.8 9.9 25.0 3.3 100.0 8.9 19.9 19.9 18.0 22.5 6.9 150.0 13.4 29.4 19.6 26.1 21.6 10.5 200.0 17.8 39.8 19.9 35.1 21.9 13.9 Mean 20.0 22.8 lTemperature = 25 C. The sorption solution was 2,4-D in DDW buffered with HCl and NaH2P04 to pH 0.8. 2Temperature = 25 C. The desorption solvent = DDW, and the final pH of all desorbed concentrations = 1.5. Figure 15.--Desorption isotherms of nondewaxed isolated tomato fruit CM sorbed with 2,4-D (temp in C). (The desorption solvent was DDW. Sorption temperatures were 5, 15, and 25 C and desorption temperature was 25 C for all isotherms. Final pH was 0.8 for sorption and 1.5 for desorption). 134 I4 N 5 m SORPTION (11. moles lg) 135 _ 11011 0511110150 comets 2.4-o b 1 I l L _I O 8 I6 24 32 4O EQUILIBRIUM CONCENTRATION (11M) DISCUSSION Sorption is viewed as the initial step in penetra- tion of foliar-applied compounds through the CM of fruits and leaves. Before entering the subcuticular tissues, however, a substance must penetrate the CM and be desorbed from its inner surface (Sargent, 1966). The results of this study provide additional information on the morphology and chemical constituents of the CM and various factors which influence sorption and desorption of organic mole- cules by the CM. The cuticular pegs of the tomato fruit CM appeared to differ somewhat in density or consistency as evidenced by a narrow band oriented perpendicular to the surface and extending through the central region of the cuticular peg (Figure 6A, 6B, 6C). These narrow bands appeared distinctly lighter in color than the adjacent areas of the CM and were not lost upon removal of the cuticular waxes (Figure 6B, 6C). Microscopic inspection of the CM following sorption of MB+ for two days indicated that the cuticular pegs, except for these narrow bands through the center, were preferential areas of sorption. Various authors (Dybing 136 137 and Currier, 1961; Eglington and Hamilton, 1967; Franke, 1967) have suggested that the area over the anticlinal walls is a preferred pathway for penetration of polar com- pounds through the CM. The importance of bands in the cuticular pegs on sorption and penetration is only specu- lative. They appeared to have a low affinity for MB+ (Figure 6B, 6C). Voisey g£_34. (1970) reported a similar differential labeling of safranin fast green by intact tomato fruit CM. These data, however, could have been an artifact of enhanced transmission of light in the area of the bands which gave the appearance of less staining by MB+ and safranin fast green. Generally, the cuticular pegs are developed relatively late in the life of an organ and are con- sidered to occur when new cells are produced during the develop- ment of the epidermis resulting in interruptions over the anticlinal walls. The concave depressions are subse- quently filled-in with cutin substances (Esau, 1965). Perhaps, the chemical and physical properties of the cutin substances associated with the central portions of the cuticular pegs differ from the adjacent areas and, thus, account for the differences in light transmission and sorptive properties. Sites of trichome attachment to the CM were observed to be preferential areas of sorption of MB+ (Figure 6D). This observation is in agreement with Hull (1964) who has reviewed the literature on preferential absorption of dyes by plant CM. Dybing and Currier (1961) and mitchell 138 g; 34. (1960) have emphasized the importance of trichomes as active areas of absorption of foliar applied substances. Hairs which persist often contain protoplasm that disinti- grates with time, leaving a highly permeable portal of entry for absorbing molecules (Esau, 1965). Further, the CM over hairs, veins, and anticlinal walls is usually thinner than elsewhere over the surface (Martin, 1966). The importance of trichomes in sorption of growth sub- stances would vary depending on the degree of enhancement of sorption and density over the surface. Since few tri- chomes were observed on the tomato fruit, their role in sorption by the CM would probably be of little importance. Orientation of the CM to the sorption solution was a very influential factor affecting sorption of MB+. When sorption proceeded through the outer surface, there was a definite barrier to sorption of MB+ which was only partially overcome by dewaxing (Table 10; Figure 5A, SB). These data clearly establish that one cannot always assume that upon being sorbed a substance will penetrate the CM (Silva- Fernandes, 1965b). Unidirectional sorption of MB+ added yet another chapter to the unexplained reports on differ- ential ratios of sorption and penetration of molecules by plant and insect CM (Bukovac and Norris, 1968; Hurst, 1941; 1948; Schieferstein and Loomis, 1959; Yamada, £4 44., 1964b; 1965b). The dewaxed CM sorbed slightly more 2,4-D than nondewaxed at relatively high solution concentrations; 139 at low concentrations dewaxing had no effect. Dewaxed CM sorbed significantly more MB+ than nondewaxed, especially for unidirectional sorption when the outer surface was oriented toward the sorption solution (Tables 9 and 10). These sorption data confirm data on permeability of isolated tomato fruit CM (Bukovac, 1971) as well as other isolated plant CM (Darlington and Barry, 1965; Hull, 1970; Norris and Bukovac, 1969; Silva-Fernandes, 1965b; Skoss, 1955). These reports, without exception, showed that dewaxing enhanced penetration of both hydrophilic (polar) and lipophilic (apolar) molecules. Apparently, dewaxing enhances both quantitative sorption potential and rate of penetration. However, research is needed to evaluate the relative effect of dewaxing on sorption and penetra- tion using the same compounds. Unidirectional sorption experiments showed that epicuticular wax was a definite barrier to sorption of MB+. This adds further evidence to support the idea discussed by Bukovac (1971) that epicuticular wax partially covers Specialized pathways of penetration for both polar and nonpolar molecules through tomato fruit CM. Such specialized pathways have been proposed by Crafts (1956) and reviewed by Foy (1964), and polar pathways have been experimentally demonstrated in the CM of leaves of Allium and Convallaria by Sch6nherr and Bukovac (1970). Desorption of MB+ with methanol was found to be Enoproximately 2-fold greater for nondewaxed than dewaxed 140 CM (Table 8). Dewaxing appeared to "open-up" the CM resulting in more sorption of MB+ and less subsequent desorption by methanol. This suggests that MB+ was either sorbed into new areas not accessible to the solvat- ing power of methanol or was bound more securily to dewaxed than nondewaxed CM. Heats of sorption (AH), on the average, were the same for nondewaxed and dewaxed CM (Table 12), suggesting that the binding strength was similar. This conclusion is based on the assumption that diSplacement of solvent and other molecules from the CM during the sorption process was the same for both nondewaxed and dewaxed CM (Shoemaker and Garland, 1967). Subsequent desorption of MB+ in CaCl2 was found to be higher for dewaxed than nondewaxed CM with sorption at 5 but not 25 C (Table 8). This is difficult to explain unless the increase in sorption due to dewaxing (6.5 umoles/g for both 5 and 25 C) was not sorbed in the same area of the CM at both sorption temperatures. Perhaps, at 5 C the CaCl -removable MB+ was sorbed at its maximum for dewaxed 2 CM but not for nondewaxed; whereas at 25 C the CaCl - 2 removable fraction was sorbed at its maximum for both nondewaxed and dewaxed CM. Whether or not a portion of the MB+ desorbed by methanol is the same fraction as that desorbed by CaCl2 is not known. However, the fact that desorption by CaCl2 reached a limit at higher pH values, whereas desorption by methanol progressively increased as pH was increased strongly indicates that MB+ desorbed by 141 these two solvents came in part, if not largely, from different fractions or sites within the CM (Table 3). Desorption with different cations indicated that desorption by CaCl2 was via exchange. The methanol-removable MB+ was most likely a fraction that was weakly held by physical forces (van der Waals forces); and, if there was a portion desorbed by both methanol and CaCl it was probably held 2: by both ionic forces operating on the charged moiety and physical forces operating on the apolar groups (Kipling, 1965). Sorption isotherms were used to diagnose the mechanisms of sorption, obtain information on the nature or state of the sorbent (tomato fruit CM), and measure the change in sorption area as influenced by temperature. As the temperature was increased from 5 to 25 C, sorption of MB+ increased. This is in contrast to data using other sorbents such as charcoal and graphite where sorption decreases with an increase in temperature (Kipling, 1965; Shaw, 1966). Various explanations have been given to justify the existence of nonclassical sorption isotherms. The experimental evidence indicated that the nonclassical relationship between temperature and sorption in the tomato fruit CM resulted from an increase in available sorption area with increase in temperature. Before dis- cussing the experimental evidence to support this conclu- sion the following arguments are proposed against some known explanations for previously reported nonclassical isotherms. 142 (a) Differential reduction of MB+ by the CM. CM is considered to be void of enzymes capable of reducing or affecting change in MB+ (Franke, 1967). However, a chemical reduction of MB+ (blue) to MB (colorless) could be misinterpreted as sorption by the CM, since the loss in MB+ in the medium formed the basis for calculating the quantity sorbed. Following sorption of MB+ by nondewaxed and dewaxed CM at 25 C, there was no evidence for the presence of the reduced species (MB) in the sorption solutions. Hence, the CM did not reduce MB+ at 25 C in the aqueous sorption solutions used in this study. (b) Fading or hydrolysisof MB+ at high tempera- tures (Kipling, 1965). Kipling and Wilson (1960) reported that a change in temperature from 20 to 80 C resulted in enhanced adsorption of MB+ by activated charcoal. They showed, however, that this was most likely due to hydroly- sis or fading of the MB+ solutions at 80 C. Control solu- tions were carefully analyzed for fading at the tempera- tures used in this study; there were no differences in OD readings as a consequence of temperature. (c) Negative solubility coefficients for MB+ in DDW (Bikerman, 1958). When similar solutes are compared, sorption usually decreases as the solubility of the solute increases (Bikerman, 1958; Kipling, 1965). In the majority of cases heat is absorbed when an ionic substance dissolves in water; the solubility of that particular substance then increases with a rise in temp- erature in accordance with the requirement of the 143 Le Chatelier principle (Glasstone and Lewis, 1960). The few exceptions to this rule are usually anhydrous salts such as CaSO4 and NaZSO4. Kipling and Wilson (1960) stated that a rise in temperature from 20 to 25 C would likely change the solubility of MB+ in water very little. There is no known evidence to suggest that a rise in temperature over the range used in these studies would decrease the solubility of MB+ in water. (d) Abrupt simultaneous decrease in sorption of the solvent (Bikerman, 1958). The shape of the MB+ sorp- tion isotherms (Langmuir-type) strongly implies that sorption of water did not compete with the sorption of MB+ (Giles 24 44., 1960). Further, DDW desorbed only minute quantities (l to 4%) of MB+ from the CM, indicating that DDW does not compete with MB+ for sites in the CM. (e) Enhancement of diffusion rate (Sutcliffe, 1962; Jennings, 1963). Diffusion of molecules across lipoidal membranes are often markedly influenced by temperature. This, however, is a matter of rate not quantitative sorption potential. No kinetic data were obtained at the different temperatures used,but MB+ solu- tions were carefully analyzed at 5 and 35 C to establish the requirement for equilibrium. values for diffusion Q10 across lipoidal membranes are in the range of 2 to 3 which offers little explanation to justify the high Qlo values of 5.6 and 8.6 reported for penetration of NAA through isolated pear leaf CM (Norris and Bukovac, 1969). 144 (f) Solubilization in lipoidal colloids (McBain and Hutchinson, 1955). The quantity of MB+, as well as most dyestuffs solubilized by lipoidal colloids, increases with a rise in temperature. These colloids are generally large organic micelles such as in soaps, synthetic deter- gents, or suspensions of animal lipoidal materials (Hofstee, 1958; McBain and Hutchinson, 1955; Shaw, 1966). The CM is not composed of such lipoidal colloids rather it is a complex structure composed of various layers of pectin, hemisubstances, and waxes embedded in a hemihydro- phobic matrix of cutin (Franke, 1967). Chloroform extracts only 2.5 % of the sorbed MB+ from the CM (Table 24). When the more lipoidal substances were removed by exhaustive extraction with chloroform and methanol, the CM still sorbed greater quantities of MB+ at 25 than at 5 C (Table 8), indicating that solubilization of MB+ by these lipoidal substances was not related to the enhance- ment effect of temperature on sorption. Enhanced solub- ilization of dyes by lipoidal colloids with rise in temp- erature proceeds in a continuously increasing manner (McBain and Hutchinson, 1955), The curve of temperature versus sorption of MB+ was irregular, even decreasing at certain temperatures (Figure 7). (g) Increased concentration of monomeric MB+ mole- cules (Giles 33 34., 1961; Kipling, 1965). Certain dyes which are highly aggregated in solution have resulted in increased sorption with rise in temperature (Allinghan 145 34 34., 1958; Giles 34 34., 1961). In all such experi- ments the concentration of the dyes was extremely high. There are no reports to the knowledge of the author where MB+ sorption increased with a rise in temperature, except for Kipling and Wilson (1960) who showed this to be caused by fading of the MB+ at the higher temperature (80 C). Allingham 34 34. (1958) and Gailbraith 34 34. (1958) found that micellar adsorption of MB+ by silica and graphite was independent of temperature; the isotherms obtained fit into the category of H (high affinity) isotherms characteristic of sorption of micelles. Isotherms obtained from sorption of MB+ by the CM were L isotherms, as evi- denced by the shape of the isotherm and the straight-line relationship between c/N versus c, which forms the mathe- matical basis of L isotherms (Figures 10 and 11). The theory to explain endothermic sorption (increased sorp- tion with rise in temperature) is based on the assumption that the monomeric molecules are preferentially sorbed and a rise in temperature increases the concentration of the monomeric species (Allingham 33 34., 1958; Shaw, 1966). Robinowitch and Epstein (1941) prOposed a theory of polymerization of MB+ in dilute solutions similar to that reported in this study. Their equations fit the spectrophotometric data in which they assumed MB+ to exist in a monomer-dimer equilibrium at low concentrations rang- 6 ing from 2 x 10- to 2 x 10.3 M. Using their equations at 4 2.2 x 10’ M, 74, 68, and 61 % of the MB+ is in the 146 monomeric form at 25, 15, and 5 C, respectively. However, when the appropriate adjustments were made in the sorption values, the differences in actual concentration of mono- meric MB+ at these different temperatures did not change the nonclassical relationship between sorption of mono- meric MB+ and temperature. There are additional evidences which tend to discredit preferential sorption of monomeric MB+ as the explanation for temperature effect on sorption of MB+ by the CM: (i) MB+ at 4.2 x 10-4 M was added to activated charcoal at 5 and 25 C. At equilibrium, the charcoal sorbed 403 umoles/g at 5 C compared to 396 umoles/g at 25 C; (ii) Figure 7 shows that the relationship between temperature and sorption is irregular--i.e., increasing then decreasing, etc. with a rise in temperature. The dissociation constant (K) for the dimer increases with temperature in a logarithmic relationship (loglOK = 1.9886 - l442/T); (iii) the effect of temperature at the three specific temperatures tested differed considerably for nondewaxed compared to dewaxed CM (Table 13). If endothermic sorption resulted from preferential sorption of monomers, then why the distinct differences between the two types of CM? Increasing the temperature from 5 to 25 C probably resulted in a greater concentration of monomers at 25 than at 5 C, but they apparently did not play an important role in sorption of MB+ by the CM. 147 An implicit assumption in determining heat of reaction (AH) of a sorption process is that the area avail- able for sorption is independent of temperature (Shoe- maker and Garland, 1967). Sorption is an exothermic pro- cess; hence, when heat is absorbed in a system in which sorption occurred, some concomitant endothermic reaction(s) occurred such that the sum of the processes involved in the system were positive (endothermic). The sum of the endo- thermic processes must have been greater than the sorption process because the system absorbed heat. The change in entropy (AS) of the system must have been positive in accordance with the thermodynamic relationship AG = AH - TAS, because sorption involves a decrease in free energy (-AGL The average AH values recorded for MB+ sorption were +5.26 for nondewaxed and +5.24 for dewaxed CM. After adjusting to sorption per m2, the average AH values were -3.38 for nondewaxed and -3.42 for dewaxed CM. Thus, assuming that the latter values are correct, the endo- thermic processes resulting from a rise in temperature were +8.64 for nondewaxed and +8.66 for dewaxed CM. This temp- erature-dependent change in the sorption system was reversible at the temperatures studied (Table 16). Thus, there was no change in free energy (AG = O) and the heat absorbed appeared in the entropy of the system-—i.e., some fraction of the molecular constituents of the CM became more random or less ordered in arrangement. This conclusion is based on the assumption that this 148 temperature-dependent process(s) was (were) associated with the cuticular components. Kipling (1965) and Allingham 3E 34. (1958) explain a +AS associated with endothermic sorptive processes on the basis that displaced solvent molecules gain more translational entropy than is lost by the sorbed solute. This, however, is highly unlikely in this system because water competed little, if any, with MB+ for sorption sites within the CM- Beament (1968) explained a sudden increase in water permeability of insect cuticular membranes on the basis of a change in orientation (+AS) of the outermost layer of lipid molecules. At a critical temperature, which differs among the insects, there is a sudden increase in permeability to water at temperatures within the range 30 to 60 C (Hurst, 1948). Beament (1968) sug- gested that the molecules of this lipid layer are held in a particular orientation by temperature-sensitive van ' der Waals forces (Parsegian and Ninham, 1970) below some critical temperature. As the critical temperature is exceeded they become reoriented. In this new orientation, the polar ends remain fixed whereas the apolar ends become liquid and assume a mean perpendicular position with the Spacing of the polar groups. The free apolar groups of the lipid molecules are reoriented in such a fashion that the intermolecular spaces increase considerably resulting in a 5-fold increase in water permeability. This effect was reversible. 149 There are no data Showing a comparable effect with plant CM; however, there are data which suggest that a similar phenomenon may occur. Silva-Fernandes (1965b) studied the permeability of nondewaxed isolated apple fruit CM to phenylmercuric acetate (PMA) at 25 and 43 C. After 48 hours, 70 pg of PMA (as measured in pg Hg) had passed through the CM at 43 C compared to O Ug at 25 C. Norris and Bukovac (1969) found Qlo values ranging from 1.2 to 8.6 for penetration of NAA through isolated pear leaf CM. The Q10 values varied according to the temper— ature range tested. There appeared to be a critical temperature; and, most important, the critical temper- ature differed with nondewaxed and dewaxed CM and corresponded with the largest % increase in sorption area calculated in this study for nondewaxed and dewaxed tomato fruit CM (compare Tables 13 and 14). Van der Waals forces and similar weak secondary bonds holding large organic molecules together (e.g., lipids, polypeptides, and hemoglobin) can be destabilized with aqueous solutions of urea (Marschner, 1955; Watson, 1965). An ether-soluble fraction can be extracted from plant CM with urea (Yamada, 1962), indicating that there are weak intermolecular forces holding these substances fixed in the CM. Penetration of urea through isolated tomato CM resulted in a progressively enhanced rate of penetration of urea itself (Yamada et al., 1965b) as 150 well as an accelerated rate of diffusion for Rb+ and C1- ions which were Simultaneously applied (Yamada 33_34., 1965a). These data indicate that some hydrophobic bonds between the chemical constituents of tomato fruit (Haweresensitive to urea resulting in an enhanced rate of penetration. There were no distinguishable changes in infrared spectra obtained for nondewaxed and dewaxed tomato fruit CM over the temperature range (10 vs 25 C) where marked sorption and permeability differences have been observed. Thus, neither dewaxing nor temperature difference resulted in measureable alterations in the vibrational levels of the constituent atoms of the CM. This is indirect evidence that the temperature-dependent change in the CM was similar to a phase change involving weak nonspecific forces, because alterations in covalent bonding or hydrogen bonding would have resulted in a marked effect on the group frequencies involved (Rao, 1963). Various workers have proposed that changes in cxfll membrane permeability can be explained on the basis of alterations in membrane structure (Stadelmann, 1969; Virgin, 1953). Hempling (1960) reported that during the transfer of water across Ehrlich ascites tumor cells at different temperatures there was a high positive entrophy change (+AS). Based on the assumption that the diffusion properties of water remained unchanged 151 during the experiment, he suggested that the entropy change reflected a structural alteration of pore geometry permitting a greater diffusion pathway for water. Based on the data obtained and the literature, apparently certain chemical constituents of the plant CM are held together by temperature-dependent van der Waals forces, which at a critical temperature(s) are broken. This results in a positive entropy change, in the form of disorientation or reorientation, of these particular cuticular components to where both sorption and permeability may be increased. If penetration, but not necessarily sorption, iS totally or partially impeded for a particular substance at the more ordered arrangement (below the critical temperature), then extremely high Q10 values for penetration can be calculated at certain temperatures as a result of a positive change in entropy associated with specific constituents of the CM. This enhanced permeability most likely would arise due to enlarged intermolecular Spacing and not to the creation of macropores (Beament, 1968; Franke, 1967; Stein, 1967; van Overbeek, 1956). An unusual relationship developed with regard to AH with increase in sorption. Distinctly for non— dewaxed and partially with dewaxed CM, AH increased with increasing sorption, indicating that perhaps there was an interaction between temperature and sorption coverage 152 to the extent that at 25 C high relative MB+ concentra- tions resulted in more available sorption area due to "concentration pressure" (Adamson, 1970; Kipling, 1965; Shaw, 1966). This would account for the sudden increase of AH at 25 umoles/g for nondewaxed and 30 umoleS/g for dewaxed CM (Table 12). The Langmuir plots of c/N versus c Showed a common intercept for the three temperatures studied. This also suggests that there was a temperature- concentration interaction. At very low concentrations of MB+, sorption was independent of temperature, but as the concentration was increased sorption was progressively more temperature dependent. This, in turn, suggests that the increased MB+ sorption attributed to temperature may have been sorbed into tiny "ink- bottle-like" spaces in the CM (Shaw, 1966). Such Spaces produce what is called an hysteresis effect--i.e., where the sorbate is securily held in the sorbent and is desorbed with great difficulty (Adamson, 1967; Shaw, 1966; Young and Nelson, 1967). Desorption data support this concept; the majority of an equivalent quantity of MB+ attributed to temperature effect could not be desorbed by DDW, methanol, or CaCl2 (Table 15). The Similar shape and characteristics of the isotherms at 5, 15, and 25 C suggest that temperature did not expose a different type of sorption area or establish a new mechanism of sorption. A rise in 153 temperature appeared to release or expose additional area Similar in sorptive properties to those originally present. Adjusting the nonclassical isotherms to sorption per unit area (pmoleS/mz) resulted in a classical relationship between temperature and sorption. The average AH values calculated from these adjusted iso- therms were virtually the same for both nondewaxed and dewaxed CM indicating that the wax constituents had no effect on the apparent binding of MB+ to the CM and the relationship between temperature and sorption. The AH values are indicative of weak ionic binding (Galbraith 3E_34., 1958; Kipling, 1965) and/or van der Waals forces (Barrow, 1966; Hayward and Trapnell, 1964; Shaw, 1966). Most likely all three combinations of sorption could co-exist within the CM--i.e., sorption by electrical forces, sorption by van der Waals forces, and sorption of the same molecule by both electrical and van der Waals forces (Bodenheimer and Heller, 1968; Kipling, 1965). For both nondewaxed and dewaxed CM, there was an apparent decrease in AH values with increas- ing sorption coverage. This indicates that either there were hetergeneous sorption Sites in the CM (de Boer, 1953; Shaw, 1966), or there were mutual forces between the sorbed molecules which resulted in lower energy AH values at higher sorption coverage (de Boer, 1953), or both. 154 Data from this study provide insight into the different mechanisms and properties of sorption and desorption of two basically different classes of compounds--namely, water-soluble (MB+) and lipid- soluble (2,4-D) substances. The quantity of MB+ or 2,4-D sorbed at equilibrium was greatly influenced by pH of the sorption solution. AS pH increased, more MB+ and less 2,4-D was sorbed by the CM. These data concur with the literature (Bukovac and Norris, 1968; Martin and Juniper, 1970; Orgell, 1957). The inflection point in the sorption curves for both sorbates was pH 3.6. MB+ is an organic monovalent cation and remained charged throughout the entire hydrogen concentration range used (Bergmann and O'Konski, 1963); 2,4-D is a weak organic acid with a pKa at 2.8 (Sargent and Blackman, 1969). These data support the many suggestions in the literature that the CM possesses carboxyl groups capable of dissociation (Bukovac and Norris, 1968; Franke, 1967; Orgell, 1957; van Overbeek, 1956). The inflection point can be interpreted to mean that the pKa of the CM would be 3.6; however this is only a first approximation because there is no information on the effect of solution pH on the internal area of the CM as well as on the nonelectrical mechanisms of sorption. Yamada (1962) reported an apparent pKa of 3.2 for the surfaces of ripe isolated tomato fruit CM. 155 At pH 3.6, 86 % of the 2,4-D molecules were ionized, yet there was an inflection point at that pH. This could be explained on the basis of preferential sorption of the undissociated molecules which would be replentished from chemical equilibrium between the two Species of 2,4-D molecules in solution. A comparison of desorption of MB+ and 2,4-D from the CM as influenced by pH of the sorption solution vividly Shows that different mechanisms of sorption are involved with these two sorbates (Tables 3 and 4). Only small quantities of MB+ (1.5 to 4.2 %) were desorbed in DDW, whereas most of the 2,4-D (approxi- mately 71 to 91%) was desorbed in DDW. The pH of unbuffered sorption solutions changed as sorption of MB+ proceeded--decreasing for nondewaxed and increasing Slightly for dewaxed CM. Most likely this change in pH was a result of exchange of hydrogen ions in case of nondewaxed CM and desorption of methanol (sorbed during dewaxing) or some inherent basic com— pounds from dewaxed CM (Getzen and Ward, 1969). Sorption of aqueous MB+ by dewaxed CM no doubt exchanged cations from the CM in accordance with the laws of chemical equilibrium; however there was a net increase in pH at equilibrium. The pH change during sorption of unbuffered 2,4-D solutions varied depending on the initial 2,4-D concentration. 2,4-D is an organic acid 156 with a pH of 3.7 at 2 x 10.4 M in DDW. Therefore, as sorption proceeded the pH increased for solutions with an initial pH of 3.5. Solutions with an initial pH of 5.8 to 7.8 decreased as sorption occurred, indicating that acidic molecules were desorbed during sorption of 2,4-D. One of the most striking differences in sorption of MB+ and 2,4—D by nondewaxed CM appears in the shape or type of isotherms obtained. Sorption of MB+ appears in the form of L isotherms, 2,4-D at pH 0.8 appears as C (constant partitioning) isotherms, and 2,4-D at pH 5.8 most likely fits into the category of S (sigmoid or low affinity) isotherms (Giles 3E_34., 1960). MB+ molecules were probably sorbed flat and there was little competition from concomitant sorption of water (Giles 3E_34.,l960). At pH 0.8, 2,4—D was sorbed by partitioning into regions of the CM that were inaccessible to water molecules. This type of sorption is character- istic of (a) sorbates such as 2,4-D that have more affinity for the sorbent (CM) than the solvent (water), and (b) a sorbent with flexible molecules and differing degrees of crystallinity. Although the CM is an amorphous structure, it is considered to be composed of flexible molecular constituents having different degrees of polymerization and order (Franke, 1967; Sitte, 1965). 157 Classical isotherms were obtained for sorption of 2,4-D at pH 0.8; whereas, at pH 5.8, nonclassical isotherms were obtained. Thus, there appears to be different mechanisms involved in sorption of the undissociated and dissociated molecules of 2,4-D. Bikerman (1958) attributed endothemic sorption (increased sorption with a rise in temperature) to either negative temperature coefficients of the solute or to an abrupt simultaneous decrease in sorption of the solvent. A negative temperature coefficient, in this case, is highly unlikely because 2,4-D is poorly soluble in 3 M); hence, a Signifi- DDW (maximum at 25 C = 4.1 x 10- cant positive temperature solubility coefficient would be expected (Glasstone and Lewis, 1960). DDW desorbed approximately 91 % of the 2,4-D which had been sorbed at pH 0.8 and 88 % at pH 5.8. The isotherms at pH 0.8 were classical and those at pH 5.8 were non- classical. These data indicate that the solvent had a Similar effect on sorption at both pH values and therefore is not a basis for the distinctly different isotherms. Sorption of 2,4-D at pH 5.8 appeared to fit into the S isotherm category. The data in Table 11 illustrate the "cooperative" nature of sorption of 2,4-D at pH 3.7. 158 At 0.5 x 10-4 M, there was very little sorption; but, aS the concentration was increased to 2.0 x 10_4 M, sorption increased 5-fold for nondewaxed and 6-fold for dewaxed CM. Hence, as more molecules were sorbed, it became easier for subsequent molecules to become sorbed in the CM. At pH 3.7, 89 % of the 2,4-D molecules were undissociated compared to 99.9 % at pH 5.8. Assuming the CM has a pKa at 3.6, the dissoci- able groups would be 56 and 99 % undiSSociated at pH 3.7 and 5.8, respectively. Thus, coulombic repulsion would result in considerable resistance to approaching 2,4-D anions (2,4-D-). Sorption of 2,4-D- can be viewed in the following two ways: (a) The 2,4-D- ion was sorbed end-on with the polar end toward the physical forces to an uncharged area of the CM (Giles 3E_34., 1960). (b) An alternative explanation would be that the CM possesses some cationic character which binds 2,4—D-. This latter view is questionable because approximately 88 % of the ions were desorbed in DDW (Figure 4). Assuming the former explana— tion correct, a sorbed 2,4-D- ion would be more tightly held when adjacent to another sorbed ion than isolated; this would account for enhanced sorption at higher concentrations--hence a S-shaped isotherm. From the pH—sorption curve, it can be seen that above pH 4.8 159 there was no decreased sorption of 2,4-D_, suggesting that there is an area or a fraction of the CM that sorbs 2,4-D- irrespective of pH. The presence of chemical additives had a differential effect on both sorption and desorption of MB+ and 2,4-D from the CM. Sorption of MB+ was decreased and desorption was increased with inclusion of inorganic cations in the sorption and desorption solutions, respectively. Sorption was increased but there was no effect on desorption of 2,4—D resulting from inclusion of inorganic cations. Inorganic anions had no effect on sorption or desorption of MB+ or 2,4-D. These results can be explained on the basis of the CM having dissociable groups with a pKa of approximately 3.6. At pH 4.8 to 4.2, cations, but not anions, would compete with MB+ for sorption sites thereby decreasing the amount of MB+ desorbed compared to DDW alone. At pH 3.7, 2,4-D- would be repelled (somewhat) by the negatively charged CM; inclusion of cations would tend to neutralize this charge (Franklin, 1969; Orgell, 1957), thereby enhancing sorption of 2,4-D- by binding of the apolar group of the 2,4-D_ to an uncharged area of the CM. The pH of the desorption solution at equilibrium was 1.5. At this pH only 1 % of the dissociable groups of the CM were ionized; therefore neither inorganic anions or cations affected the quantity of 2,4—D desorbed at equilibrium. 160 When the CM was exposed to a 24-hour CaCl2 or AlCl solution before sorption, subsequent sorption of 3 MB+ was increased by CaCl2 but decreased with A1C13. Although there was a Slight increase in sorption of 2,4-D with the CaCl2 pretreatment, it was not significant. With A1C13, however, there was a Significant increase in sorption of 2,4—D. These data are difficult to explain. AlCl3 pretreatment decreased subsequent sorption + . of MB ; this may have resulted because the CM sorbed AlCl3 (Table 6) and Al3+ was Shown to compete with MB+ for available sorption Sites. CaCl2 was likewise sorbed by the CM and Ca2+ competes with MB+ for sorption sites, yet there was an increase in sorption with the 2+ CaCl pretreatment. Apparently, CaCl (probably Ca ) 2 2 exerted some irreversible effect on the CM which either was inhibited by sorption of MB+ and/or masked by the competing effect of Ca2+ in the sorption solution. The differential effect of CaCl and AlCl pretreatments 2 3 on subsequent sorption of 2,4—D was probably related to their relative neutralizing efficiencies (binding 3+ strength). The binding strenth of Al is known to be higher than that of Ca2+ on soil (Rose, 1966) and cation exchange resins (Dowex: Ion Exchange, 1964). Assuming this same relationship to be true with binding to isolated tomato fruit CM, Al3+ (at the pH tested, 3.5-3.8) would neutralize the negatively charged CM more efficiently than Ca2+. 161 The data on desorption of MB+ and 2,4-0 further illustrate that different mechanisms are involved in sorption of these two sorbates by the CM. MB+ was bound sufficiently to the CM such that only small quantities (1.5 to 4.2 %) were desorbed in DDW (Table 3). Most of the 2,4-D (71 to 91 %) was desorbed from the CM with DDW (Table 4). Subsequent desorption with 100 % ethanol, dioxane, or chloroform removed only minute additional quantities (1 to 8 %) of 2,4-D, yet 2,4-D is 1443 times more soluble in 100 % ethanol than water. These data indicate that most of the sorbed 2,4-D can be desorbed in DDW and that the remaining fraction is held firmly in the CM in an area which is either inaccessible to DDW and these organic solvents or which binds 2,4-D by some chemical means as to render it fixed within the CM. The concentration of 2,4-D used with this particular experiment (Table 4) was 2.0 x 10.4 M. There were no data obtained on exhaustive desorption of the CM sorbed with 2,4-D at 0.5, 1.0, and 1.5 x 10-4 M. The desorption iso- therms for 2,4-D (Figure 15) were linear and the % desorp- tion (Slope of the lines) waS nearly identical with that for sorption at pH 0.8 (Figure 14, Table 29). If the lines in Figure 15 were extrapolated to the base line, desorption would be approximately 100 %, this would indicate that at the lower concentrations of 2,4-D sorption was completely reversible. De Boer (1953) has reported data on sorption of water vapor by Silica gel in which the 162 reversibility of sorption was dependent on the original vapor pressure. Sorption at relatively low vapor pressures was completely reversible, but at higher pressures there were hysteresis effects. Data in Table 29 Show that the average % desorption of 2,4-D was higher than the average % of 2,4-D remaining in solution at equilibrium for sorption at 15 and 5 C. This can be explained on the basis of the relative temp- eratures used for sorption and desorption. Temperatures for sorption were 5, 15, and 25 C; whereas for desorption all three sets of sorbed CM were desorbed at 25 C. At 25 C, the % 2,4-D desorbed was nearly the same (0.7 % difference) as that remaining in the sorption solution. At 15 and 5 C, the % 2,4-D desorbed was greater than that remaining in the sorption solution by 1.8 and 2.8, respectively. The sorption-pH curves for MB+ and 2,4-D indicate an apparent difference in reversibility of sorption of these two sorbates as influenced by pH. Following sorption of MB+ at pH 4.8, aqueous HCl at pH 2.5 desorbed only 14.2 umoles/g, with 14.4 umoleS/g being retained in the CM. (Table 21). This is compared to sorption of only 6.3 umoleS/g at pH 2.5 (Table 3). The quantity of MB+ unaccounted for in desorption by HCl amounted to 8.1 umoles/g (14.4 - 6.3). This value approximates the apparent limit of MB+ that was exchanged by Ca2+, indicat- . . . . + ing that this fraction was not exchangeable Wlth H 163 because it was held securely bound to sites within the CM. This conclusion is only speculative, however, because there was no direct evidence that the portion retained . + . against H was the same fraction desorbed by Ca2+. The relatively large quantity of MB+ desorbed by A13+ was probably a reflection of its relatively high binding strength as well as its capacity to lower the pH of the desorption solution (Table 21). The amount of MB+ that could be desorbed by A13+ reached a limit at approximately 15 umoleS/g (Table 23). Apparently, the final pH of the sorption solution was of greater importance to the extent of sorption of 2,4-D at equilib- rium than the initial pH (Figure 4). This would suggest that sorption of 2,4-D was partially or totally reversible as the hydrogen ion concentration was decreased. Additional research on this subject is needed to clarify this point. Pretreating the CM with ethanol for 24 hours resulted in a significant increase in subsequent sorption of 2,4-D, but there was no difference in sorption by nondewaxed CM of 2,4-D from a 50 % ethanol solution. These data are explained on differential solubility of the sorbate in the solvent, (Bikerman, 1958; Kipling, 1965). At 25 C, 2,4-D is 229 times more soluble in 50 % ethanol than water. Pretreated CM sorbed Slightly more 2,4-D than nondewaxed CM, which is con- sistent with other experimental data (Table 11). 164 Sorption from 50 % ethanol undoubtedly removed some wax which should have resulted in enhanced sorption, p3£'33. Sorption from ethanol solutions, however, was without effect. This probably occurred as a net result of increased sorption from dewaxing combined with a Similar decrease from enhanced solubility of 2,4-D in 50 % ethanol. A Similar Situation as above occurred with sorption of MB+ from a 50 % methanol solution. Methanol is considered to be a better solvent than DDW for MB+. (Bikerman, 1958; Kipling, 1965). This would also explain why less MB+ was sorbed from methanol than from DDW alone. The inclusion of Na-acetate in the sorption solution significantly enhanced sorption of MB+ compared to treatments with identical concentrations of NaCl. At 3 x 10'3 M, NaCl had no significant effect, but 10- and lOO-fold increases in salt concentration progressively decreased sorption of MB+. The latter response can probably be attributed to increased competition at the higher salt concentrations between MB+ and Na+ for sorption Sites. The mechanism responsible for the acetate- induced enhancement of sorption of MB+ is not known. These data point out the importance of knowing the effect of buffers on the particular parameters being considered (Orgell and Weintraub, 1957). 165 The many distinct differences in sorption and desorption of MB+ and 2,4-D by tomato fruit CM strongly suggest that there are different mechanisms involved in sorption by the tomato fruit CM. The most striking examples that support this idea are presented in Table 30. Various authors have proposed that water-soluble (polar) compounds follow an aqueous path and the more lipid- soluble (apolar) substances a lipid path through the CM (Crafts, 1956; Foy, 1964; Roberts, et al., 1948). Since sorption and desorption are integrally related to penetration through the CM, data from these studies support this hypothesis. The dimensions of these path- ways are not known--i.e., are the two general pathways compartmentalized in the CM or iS penetration of both polar and apolar molecules a result of different mechanisms of sorption and desorption in the same area of the CM? Sch6nherr and Bukovac (1970) have experi- mentally demonstrated Specific polar pathways for Hg2+ in the CM isolated from Allium bulb scales and leaves. Additional research is needed to Show the existence of different pathways for hydrophilic and lipophilic molecules in the same CM. 166 03» mo coHumnucmocoo Esflnnflaflsvm u o paw Am\mmHOEJV COHuQMOM u z .mzmmum msu CH .AZJV muMQHOM H .3oo ea soosomoo 0 am .zoo ca eoshomoe soauohom ou H5 >HmumEonumm¢ 0 m away mqu 00 >uflaflnwmuw>mm A00 .UmmmwuocH mmz coflumuom .pmmmmuomw soflumuom name “0H0H411Umuommmm nos ”0H0Hm1100mmmnocH 1ummuwmum m mm 0H0H¢ soaooaom "Nauru soaoosom "Naomo 6:6 Home 00 060000 loo .pmuommmm you was cofiumHOMmc cam .wmmmmnocw coauQHOMwC paw coausHom CH msoflumo 00mmmnocw coflumuom 00mmmuomo coflumuom oacmmuocfl mo pommmm A00 me mm .m we 0 z z 0.0 mm mwd>u EumzuOMH coflumuom any mm mm 2 Z sofludHOM so so so 060000 160 016.0 +02 ucwEHummxm H.20 pflouw oumEou UmpMHOMH >9 coflumHOM mo mEMHcmnomEHuHSE mo uuommsm CH mocmwfi>m11.00 mqmsB SUMMARY + . MB and 2,4-D were used as model organic compounds to study the sorptive properties of the tomato fruit cuticular membrane (CM). Various factors were studied to elucidate these properties. Stage of Fruit Ripeness . . + CM isolated from green fruit sorbed less MB and attained equilibrium more rapidly, possessed more cuticular wax and less epicuticular and total waxes than CM isolated from ripe fruit. pH Increasing the pH of the sorption solution increased sorption of MB+ and decreased sorption of 2,4-D, with an inflection point at pH 3.6 for both sorbates. Desorption with DDW was distinctly different for the two sorbates. Less than 5 % of the MB+ was desorbed while 71 to 91 % of the 2,4-D was desorbed with DDW. As the pH was increased, methanol desorbed progressively more MB+, whereas the quantity 167 168 of MB+ desorbed with CaCl2 attained a maximum value (approximately 8.4 umoleS/g). Wax Dewaxing (Soxhlet chloroform/methanol 50-60 C) resulted in less subsequent sorption of MB+ by the CM than dewaxing at room temperature (20-22 C); however % extractable materials by each method was the same. Removing the epicuticular wax or the epicuticular and cuticular waxes progressively increased the quantity of MB+ sorbed by the CM and decreased the quantity and % of MB+ subsequently desorbed with methanol, with the epicuticular wax being the greater barrier to sorption. There were definite barriers to sorption of MB+ through the outer surface. After 6 days, sorption through the inner surface was 58.8, 10.2, and 7.8 times greater than through the outer surface for nondewaxed, epicuticular wax removed, and dewaxed CM, respectively. Microscopic investigations confirmed the effect of both dewaxing and orientation on sorption. Narrow bands, which were lighter in color and appeared to sorb less MB+ than adjacent areas, were present in the center of the cuticular pegs. These bands were particularly apparent when viewed under phase contrast or when using a green compensating filter. Dewaxing the CM Slightly increased sorption of unbuffered 2,4—D at 2.0 x 10-4 M but not from less dilute 2,4-D solutions. 169 Temperature AS the temperature was increased, sorption of MB+ tended to increase for both nondewaxed and dewaxed CM. This trend was not consistent, however, for at certain temperature ranges sorption decreased with a rise in temperature. With MB+ as the sorbate, non- classical sorption isotherms (Langmuir-type)‘Were obtained at 5, 15, and 25 C for both nondewaxed and dewaxed CM. The Specific sorption area (A) of the CM at each temperature was calculated from the Langmuir isotherms and these values were used to express sorption data on a per unit area basis (umoles/mz). These adjusted isotherms depicted a classical relationship between sorption and temperature with low heats of sorption, averaging 3.4 kcal/mole for both nondewaxed and dewaxed CM. The % increase in the calculated spe- cific sorption area values varied depending on whether or not the CM was dewaxed. The largest increase in A occurred from 15 to 25 C for nondewaxed CM, whereas dewaxed CM exhibited the greatest increase in A from 5 to 15 C. The major portion (60 to 71 %) of the temperature-enhanced sorbed MB+ was retained in the CM after desorption with DDW, CaCl and methanol. One- 2! hour treatments with water at 20 or 35 C had no differential effect on subsequent sorption of MB+, indicating that 170 the effect of temperature on sorption of MB+ was reversible. An increase in temperature from 5 to 25 C resulted in a positive change in entropy (+AS) of the sorption system. It was concluded that this +AS was most likely a reflection of a change in orientation or order of certain chemical constituents within the CM, which resulted in an increased area accessible to sorption of MB+. Classical, linear isotherms (C-type) were obtained for 2,4-D at pH 0.8 whereas nonclassical isotherms (S-type) were obtained for 2,4—D at pH 5.8. Chemicals Additives Sorption of MB+ decreased and desorption of MB+ increased with the inclusion of inorganic cations in the sorption and desorption solutions, respectively. Sorption of 2,4-D was increased but desorption was without effect from inclusion of inorganic cations in the sorption and desorption solutions. Inorganic anions had no effect on sorption or desorption of MB+ or 2,4-D. CaCl2 as a pretreatment increased sorption of MB+ but not of 2,4-D, whereas an AlCl pretreatment decreased 3 sorption of MB+ but increased sorption of 2,4-D. Sorption of MB+ was reduced when the solvent was 50 % methanol, but there was no apparent difference in sorption of 2,4-D from a 50 % ethanol solution. 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APPENDIX 187 188 m.em N.m o.ma m m.om H.m maoam e.HH s ~.om N.m Naomo N.~H m m.mm o.m Homz N.HH N v.m~ s.m zoo m.~a H o.oma m.em N.~ o.m m m.mm ~.~ maoaa m.m v m.m~ H.~ Naomo m.m m m.mm ~.~ Homz m.e m m.e~ H.N zoo B.“ H o.ooa «.mm ~.H o.v m m.mm ~.H maoaa H.¢ q o.mm N.H «Homo «.4 m 0.0m ~.H Homz 0.4 N e.m~ H.H zoo III e.m a o.om omm Amy A@\mmHoE:v Am\mmH081V “Say wmQHOmmc nuv.m ucm>aom omQHOm mumoflammm cocoo ouv.~ o-¢.~ coflumHOmwa coHumHom HMHuHcH N H.20 Danny oumEou owumHOmH Eoum onv.m um QOHDQHOmmc co mcoflumo oflcmmuocfl wo pommmm ucmswwmQSm cum .0 m ocm mm um maumsuomfl cofiumuom MOM mumoul.am mamas 189 o.mN H.H v.¢ m m.hm N.H maUad v.v v o.mm H.H Naomo «.4 m o.m~ H.H Homz 4.4 m b.NN o.H BOD w.v H 0.0m mn.e~ n.~ mfloaa s.m q ma.mm m.~ «Homo m.m m mo.mN m.N HUMZ m.m N ma.nm h.m Boa ~.0H A com: H.m~ m.¢ m.mH m «.mm ~.v maofia m.sa s N.~m 5.4 «Home o.qa m m.mm H.v HUMZ m.mH N N.mN H.v 3CD m.mH H 0.00N va Am\meOEJV Am\mmHOE:V AEJV meMOmmp Duv‘m ucm>aom pmnHOm mumofiammm cocoo DIQ.N le.N mcoflummOmmo coflumuom HMHDHGH .pmscfiucooll.am mamme 190 m.Hm m.m m.eH m m.o~ e.m mHoHa 5.5H a o.- 0.4 NHomo ~.mH m o.Hm m.m Homz 0.5H m m.~m 0.4 zoo m.eH H o.oom m.HN m.m v.MH m m.HN m.~ mHoH< «.mH v H.m~ H.m NHomo v.MH m H.om n.~ Homz v.MH m o.H~ m.~ goo v.mH H o.omH s.m~ o.~ m.m m m.- o.m mHon m.m v e.mm o.~ NHomo m.m m m.mm o.~ Homz m.m m o.- o.~ zoo H.m H 0.00H Awe Am\mmHoenc Hm\mmHOEnc . Hzav UmnHOmmo 0:6.m ucm>Hom chHOm mumowammm cocoa Ib-v.m ou¢.~ NQOHDQHOmwQ coflemuom HmwuHcH .6@5QH#COUIU.Hm mqmHom UmnHOm mumowammm cocoo ou¢.m ouv.m mcofluQHOmwQ coHumHom AmwuwcH .cmscwuc00I|.Hm mqmde 192 m.qm m.m e.mH m o.em ~.m vommmmz o.mH H m.mm o.m Hommmz m.mH m H.HN H.m Homz n.mH m m.em m.m zoo ~.NH H o.omH m.mm ~.N m.m m 4.0N m.m Hommmmz e.m s m.mm o.m sommmz o.m m o.m~ H.~ Homz m.m m m.s~ v.m goo m.m H o.OOH H.mm ~.H H.v m m.m~ H.H vommmmz H.« 4 «.mm H.H H.ommmz m.v m A.Hm m.H Homz H.v m m.mm m.H zoo H.v H o.om Hwy Hm\mmHoEJV Am\mmHOE:v A21v meHOmmo ouv.m ucm>aom vaHOm mumoflammm socoo ous.m ouv.m coHumHOmmo coflumuow HmwuHcH N H .20 pflsum oumEou wouwHOmH Eoum alv.m mo COHDQHOmmU no mcoflcm vacmmuocfl mo pommmm ucmsvmeSm cam .0 ma um EHmSDOmH coHumHOm How mumonl.mm mamae 193 .mo.o u m um ucmHmMMHp zapcMOHMHcmHm mum mumuumH ucmeMMHc an cm3oHHom mammz .0 mm H musumummEoBN .m.H u coHumHOmmp cam m.o u coflummom new mm HmchH . . 6 N . mv mm m m om mmz o OH 6 mH.v~ m.~ vommmz h.cH m mm.mm h.~ H0mz m.oa m om.wm m.m Boo m.oa H com: m.vm m.v 5.0H x H.mm m.« vommmmz m.mH q m.m~ 0.4 vommmz o.kH m >.vm m.v H062 o.hH N m.om v.m zoo m.oH H o.oom 3: 383053 38395: 23 ownHOmmc nuv.m ucm>Hom cmbuom mpmoHHmwm cocoo ouv.m onv.m mcoflumuowmo cowumuom HmwuHcH .UwSGHuGOUII.Nm mqmda 194 maucmoHMHQmwm mum mumuuma pcmHmMMHU .UmcHEHmpmp uoz N u m um ucmHmHHHn we cm30HH0m 30H comm cHsyH3 mcmwz H mm.Hm mo.Hm ~-- ~-- ~-- omm m mmHm mm.sm mm.vm ~-- ~-- ~-- omm mm maHm 20 pmxmsmo mv.mm am.mm ~-- ~-- m-- 0mm m mmHm mo.Hm «9.0m ~-- ~-- N-- omm mm mmHm av.mm am.sm nH.sm nH.mm am.Hm omm om maHm n~.mH am.mH nH.mH gm.mH mm.mH OOH om mem gm.mH am.mH gm.mH Bk.mH as.mH OOH om xch am.mH mm.mH mm.mH mm.mH mH.mH OOH om cmmuu 20 Umxmzmpcoz Hm\meoE:v A21V A0V mnH omH on ms Hm cocoo .mawe mmmcmmHm Hugs aoHHmuom mo coHumuso HmHuHcH uHsum uHsum oumEou .mmmcmmwu mo mmvmum pcmanMHo um Eoum omumHOmH so an +mz Ho coHuauom co meHu mo HumuHm-.mm mHmae 195 TABLE 34.--Effect of time on sorption of 2,4-D by isolated tomato fruit CM. pH Sorptionl 0 Days 14 Days 7 Days 14 Days (nmoleS/g) 0.8 0.8 15.10a 14.76a 1.4 1.4 14.66a 14.88a 5.8 4.7 1.23a 1.28a 7.3 5.8 1.28a 1.37a lTemperature = 25 C. Means within each row followed by different letters are significantly different at P = 0.05. 196 TABLE 35.--Wax effect--% desorption of MB+ from isolated tomato fruit CM sorbed at 25 and 5 C. Desorption1 solvents CH3OH CaCl2 CM DDW CHBOH CaCl -DDW -DDW (%) 25 C Dewaxed 2.5a 14.8 26.6 12.3a 24.1a Nondewaxed 3.6b 31.7 32.5 28.1b 28.9b 5 C Dewaxed 2.2a 12.4 30.3 10.2a 28.1a Nondewaxed 6.lb 35.9 33.1 29.8b 27.0a lTemperature = 20 C. For both 25 and 5 C, means within each column followed by different letters are significantly different at P = 0.05. 197 TABLE 36.--Temperature effect--% desorption of MB+ from nondewaxed and dewaxed isolated tomato fruit CM. Desorptionl solvents Sorption CH3OH CaClz temp DDW CH3OH CaC].2 -DDW -DDW (C) (%) Nondewaxed CM 25 3.6a 31.7 32.5 28.1a 28.9a 5 6.1b 35.9 33.1 29.8b 27.0b Dewaxed CM 25 2.5a 14.8 26.6 12.3a 24.1a 5 2.2a 12.4 30.3 10.2b 28.1b lTemperature = 20 C. For both types of CM, means within each column followed by different letters are significantly at P = 0.05. 198 TABLE 37.--Data--sorption of MB+ versus temperature (see Figure 7). Initial MB+ concn 1.8 x 10'4 M 2.2 x 10’4 M Temp Nondewaxed Dewaxed Nondewaxed Dewaxed (nmoleS/q) 5 23.9 26.7 26.2 29.9 10 25.9 --—1 29.7 ---l 15 25.1 29.5 28.1 32.9 25 28.6 29.9 31.9 34.4 30 27.6 ---1 31.3 ---l 35 26.7 29.6 30.5 34.2 1 Not determined. 199 m.0m m.am v.05 H.0N 0.00 N.wN 0.0NN N.hm 0.0N 0.0m H.mN 0.00 m.m~ 00.00H 0.MN v.MN m.Nm v.HN 0.0m b.0N 0.0vH m.NH v.ha «.mH 0.0H 0.0H 0.0H 0.00H v.v H.HH N.> 0.0a v.0 m.0H 0.0m 0.00 v.vm v.mm m.Nm m.0> 0.0N 0.0NN v.0m 0.0N v.Nm m.mN m.0v 0.0N 0.0ma m.hH m.vN 0.0H m.0N H.MN v.mm 0.0vH N.m N.mH 0.0 N.mH 0.NH 0.5H 0.00H ¢.N m.HH N.v N.HH m.m 0.0H 0.00 20 pmxmzmo A210 Am\meOE:v A220 Am\mmHOE:V A220 Am\mmHoE:v A230 mN ma m cocoo A00 musumuwmfime HmfluHcH . Am UCM w mwhflmflh Mva mEHmSUOmH GOHUQHOm +mzll6#00|l.mm H.HmANH. 200 0.NOH ~0.N N.¢0H >0.H 0.000 00.0 0.00m N.00H N0.H 0.00H 00.0 v.00H 00.0 0.00H 0.00 00.0 0.00 00.0 N.v0 00.0 0.00H H.00 00.0 0.00 00.0 0.00 00.0 0.00 a 0.N0 0N.0H 0.00 N0.0H 0.00 00.00 0.00m H.Nv 00.0H 0.00 00.0H 0.0m 00.00 0.000 0.0m No.0 0.0m N0.0 0.0a 00.0 0.00H 0.0a 00.0 0.NH Na.v N.0H N¢.v 0.00 30 A210 A0\mmH0E20 A210 A0\mmH052v A210 A0\meoE:v A210 0N 0H 0 cocoo A00 musumummEmB HmfiuwcH .Ava musmflm mmmv msnmnDOmH coeumHOm Duv.N-mumo-.00 mamme 201 0 v 0 0.0N0 0N 30 z 00 0 v 0 0.00N 0N 30 z 00 0 v 0 0.000 0N 30 z 00 0 0 0 0.0NN 0N H\H m m- m-- 00H 9 m 0H A0000 .om>oxsm 0cm m0uuozv v0 0 v m um> mm xom mm.em.mH.~H 00 v 0 0.00N 00 a m 00 0 0 N 0.00N om0a Hm> 00 0 0 0 0.00N 0N nm> 0 0 0N 0 0.00N 0N B m 00.00.00.0 0 0 0 0.00N 0N Hm> N 01 011- 000 Hm> 0 Hm> N 0| 0111 000 Hm> 0 0\0 N 0| 0111 000 a m 0 v0 0 0 0.00N 00 3o z 0 0 N0 0 0.00N 0N 3o z 0 Hm> 0 0 0.000 0N B m N 00.0-Mm Hmmmov HHec 12:0 H050 c00umnso Nucmaummup mumo00mmu mumo00mmn monume musm0m \mumoHHmmH .oz \o-v.~ 60 +02 \20 mamezmo no mHnme 0 .mmusm0m 0cm mm0nmu :0 mump uow00oo ou 00m: mumumEmnmm 0mucmE0Hmmxmul.0v m0m49 ) 202 .0000000000 0020 .000HQH00 H00 00:0 00 0000 00 H0QESG 0800 000 000090: 003 CO0HQH000U H00 COHHmHDQ .0009 muc0>0ow COHHQH0000 00 H0085: 0:0 >0 ©0©0>00 COHHQHOm H00 m000000Q0H 00 H00E0: 000 00:00 00H0000QOH 00 HmnEsc 0L0 .000HQH0000 0CH>00>CH muc0EHH0mx0 £003 “>000 COHHQHOm H00 0H0 0000000000 N .0500H0>HH0> uUmxmzmmcoc 2n n “How-omv umexOm n xom “Ho NN-ONV musumummemu EOOH u 9 0H «H « m o.OON m« 30 2 0H 0.0 :0 «H « 0.0 :0 0.00N m« 30 2 «H m « m Hm> mN xom NH 00500:» N 0 m N 0.0HN m- Hm> 0.0 «H m m c.00N om so 2 « 0 NH 0 m.NHN 0N 30 z m m- m- m- m-- m-- e m N 0H5m00 H0> H0> H0> H0> H0> H0> 00 Hm> « m c.00N m« 30 z «m Hm> «-m m Hm> mN x00 00 «H « m Hm> m« 3o z 00.Nm.Hm.0N «H m m c.00N m« 30 z NN.0N.mN m « m o.omN mN Hm> «N m « m Hm> mN 30 z 0N.0N m « m o.OON mN 30 z NN.HN.ON Hmwmov AHEV A: 0 1050 mHnme 000H0H50 NHC0EHm0HH 000000Q0H 00000000H 000008 0H5000 \mmumoHHmmu .oz \o-«.N Ho +02 \20 0cmezwo Ho anme .U®SC0HCOUII.OV mqmfifi ;-4-l[u»? 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