INFLUENCE of HLB of POLYSORBATE SURFACTANTS on MOBILITY of METHAZOLE in PLANTS and SOIL by Tsuneyuki Takeno Thesis Submitted To

Home , Methazole

INFLUENCE OF HLB OF POLYSORBATE SURFACTANTS ON

MOBILITY OF METHAZOLE IN PLANTS AND SOIL

Tsuneyuki Takeno

Thesis submitted to the Graduate Faculty of the

Virginia Polytechnic Institute and State University

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Plant Physiology

APPROVED:

------.-~------,--~------c. L. Foy, Chairman

..,....,_ ____ ------~--7------__ ------L. H, Aung J. L. Hess

August, 1973

alacksburg, Virginia ACKNOWLEDGMENTS

The author expresses sincere appreciation to.his major professor,

Dr. C. L. Foy, Department of Plant·Pathology and PhysiolOgy, not only for suggestions and criticisms Jn the research and writing .of this investigation, but for providing an opportunity to study at this university. Thanks go to Drs. L. H. Aurtg, arid J. L, Hess for serving on the author's graduate connnittee.

Dr. J, M. Byrne, Department of Biology, was consulted on aspects of interpretation of scanning electron photo micrographs. The author thanks , Forestry and Wildlife Science Division, for assistance in operating a scanning electron microscope.

The author thanks the Velsi.col Chemical Corporation, Chicago,

Illinois, and Atlas Chemicals Division, !CI-America Inc.; Wilmington,

Delaware for supplying the radioactive methazole and surfactants,· respectively.

·The author thanks the Department of Plant Pathology and

Physiology for financial support and the,opportunity to conduct this

investigation,

Thanks are also extended to the KAO-Atlas Company Ltd., Tokyo,

for permission to enter the graduate study.

Thanks go to for various technical assistances

in conducting this investigation,

Appreciation is expressed to Mrs. Sherry Williams for typing

this manuscript.

ii The author is particularly indebted to his wife, not only for drawing the figures but for various assistances, under- standing, and encouragement during his graduate studies.

iii TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ii

TABLE OF CONTENTS iv LIST OF TABLES . . . . vi LIST OF FIGURES ...... viii

I. INTRODUCTION . • • 1 II. EFFECT OF HLB OF POLYSORBATE SURFACTANTS ON

TRANSLOCATION OF METHAZOLE- 14c 3

Introduction 3

Materials and Methods 6

Radiolabeled methazole .. • 6 Surfactant solutions with different HLB 6

Radiolabeled surfactant solutions 6

Culture of plants 7

Treatment of plants 7

Assay for 14c 8

Results and Discussion 9

Absorption and translocation of 14c

with or without polysorbate surfactant

in methanol and/or ~ater •... 9

Effect of methanol concentration ~ . • .. • 16

Effect of HLB of surfactant-methanol

solution . 24 14 Distribution of po1ysorbate surfactant- C • 29

iv Page

Effect of HLB of polysorbate surfactant 42

Acknowledgments . • 45

Literature Cited 46

III. EFFECT OF POLYSORBATE SURFACTANTS ONTHE

ULTRASTRUCTURE OF LEAF SURFACES 47

Introduction 47

Materials and Methods • . 50

Results and Discussion 52

Literature Cited 60

IV. EFFECT OF HLB OF POLYSORBATE SURFACTANTS ON

LEACHING OF METHAZOLE- 14c IN SOIL 78

Introduction 78 Materials and Methods . . . 79 Results and Discussion· 81

Literature Cited 88

V. SUMMARY 89

VITA .• . 93

v -·- .~ LIST OF TABLES

Table Page

II•l. Disti:'i.bution of 14c in primary leaves of 30- day.,.old cotton plants following 20-µl drop applicati:on of heterocyclic ring•labeled · methazole-14c (O .1 JlCi) over the midrib.· Radioactivity data are averages of four repl.ications · •. .• • •· • . • ·• • .•. ·• • • • • • •. 15 .. ·. 14 . . .· n-2. Di.stributiOn of ·· · C in the fourth leaves of 45-day-old prickly· sida pl~nts following 20-)11 drop applieation of hete:rocyclic ring-labeled methazole-14c (O .1 pCi) over the midrib. Radioactivity data are averages •of four replications • • . • • ...... 17 . . . 14 11~3. Distribution of C in· primary leaves of 30-. day-old ~otton plants following 25-µl application of heterocyClic ring-labeled methazole-14c (O.l µCi) methanol or. methanol-aqueous solutions over the midrib. Radioactivity data are averages of five replications . • . • . ..•••. ~ • .. ·,. . . 22 II-4. Distribution of 14c in the fourth leaves of 45-day-old prickly s.ida plants following 25-pl application .of heterocyclic dng- labeled inethazole-14c (O. l µCi) methanol or methanol-aqueous solutions over the midrib. Radioactivity data are averages of five replications • • • • . • • • • • • • • 23

Il-5. Distribution of 14c in primary leaves of 30.,. day-old cotton plants following 20-)ll drop application of heterocyclic ring-labeled methazole-14c (O .• l µCi) methanol solution over the midrib~ Radioactivity data are averages of four :replications . • • • . • • . • • 30. ·

II-6. Distribution of 14c in the fourth leaves of 45-clay-old prickly sida plants following 20-µl drop application of heterocyclic ring- labeled methazole-14c (O.l µCi) methanol solution over the midrib .. Radioactivity data are a~erages of £our replications . • • • • • 31

vi ',' •.

Table Page

II-7. · Distribution of 14c in treated lea:f after . treatment of 30-day-old cotton plants with approximately 0~1 )lCi of fatty acid-labeled polysoibate surfactant over the midrib of a primary leaf. Radioactivity data are averages of four replications. Applied volume of treatment solution varied because of different specific activities of Span 80-14c and Tween ao-14c ...... 40

II-8. .Distribution of 14c in treated leaf after treatment of 45-day-old.prickly sida plants with approximately 0 .1 pCi of fatty acid- labeled polysorbate stirfactant over the midrib of the fourth leaf. Radioactivity data are averages .of four replications. Applied volume of treatment solution varied because of different specific activities of Span 80-14c and Tween 8o-14c •••.•.•..

vii LIST OF FIGURES

Figure Page

II- l. Time course of translocation of radioactivity in ~Qtton plants treated with Q.l pCi of · heterocyClic ririg-labeled methazole-14c with 1%(v:,/v)· polysorbate s~rfactant in methanol or methanol-aqueous solution in a 20-µl drop on a, single primary leaf.· The treated spot was cut out. Autoradiograms right; plants left. HLB of surfactant. used:· A, E, and I -- 4.3; B, F, and J -- 8.0; C, G, and· · K -- i2.0; D, H, and L •- 15.0. Time. period after treatment: A, B, C, a.nd D 3 hr; E, F, G~ and H -- 24 hr; I, J, K,· arid L -- 72 hr. Methanol content in treatment ·solutions (%, v/v) : . A, E, and I -- 100; B, F, and J 82.5;. C, G, artcl K -- 64; D, H, and L .... _ SO • • • • • • • , ·, .• • 10

II- 2. Time course of translocation of radioactivity in prickly sida plants treated with 0.1 ~Ci of heterocyclic ring-labeled methazole•l4c with 1% (w/v) surfactant in methanol or methanol:.. aqueous s.olutions in a· 20"'.')ll drop on the. fourth leaf. The treated spot was·.. cut out. Autoradiograms right; plants left~ HLB of surfactant used: A, E, and I -- 4.3; B, F, and J 8.p; C, G, and K -- 12.0;: . D, H, and L --15.0. Time period after treatment: A, B, Cf and D -- 3 hr;E, F, G, and H -- 24 hr; I, J, K;. and L -- 72 hr. Methanol content iri. treatment solutions (%, · · v/v): A, E; and I -- 100; B, F, and J -- 82. 5; C, G,. and K _.:., · 64; D, H, and L --· .50 12

II- 3. Time course of translocat,ion of radioactivity in cotton plants treated with 0.1 pCi of heterpcyclic ring-labeled. methazole-14c · wit.bout surfactant in a 25-:)ll drop on a '_; single primary leaf. The treated spot was cut out. Autoradiograms above; plants below. Methanol concentration in treatnient solutions (%, v/v) :. A,· iOO; B, 82. 5;. · C, 64 ; D, 50 ; E, 40 • • • . . . . ~ . ; . • • • • 19

·.viii Figure Page

II- 4. Time course of translocation of radioactivity in prickly sida plants treated with O.1 J:LCi of heterocyclic ring-labeled methazole-I4c without surfactant in a 25-pl drop on the fourth leaf. The. treated spot was cut out. Autoradiograms above; plants below. Methanol concentration in treatment solutions(%, v/v): A, 100; B, 82.5; C, 64; D, 50; E, 40 . . . 20

II- 5. Time course of translocation of radioactivity in cotton plants treated with 0.1 pCi of heterocyclic ring-labeled methazole-14c dissolved in 100% methanol with polysorbate surfactant in a 20-pl drop on a single primary leaf. The treated spot was cut out. Auto radiograms above; plants below. ID..B of polysorbate surfactant used: A, no surfactant used (control); B, 4.3; C, 8.0; D, 12.0; E, 15.0 ...... 25

II- 6. Time course of translocation of radioactivity in prickly sida plants treated with 0.1 pCi of heterocyclic ring-labeled methazole-14c dissolved in 100% methanol with polysorbate surfactant in a 20-pl drop on the fourth leaf. The treated spot was cut out. Auto radiograms above;' plants below. HLB of polysorbate surfactant used: A, no surfactant (~ontrol); B, 4.3; C, 8.0; D, 12.0; E, 15.0 ...... 27

II- 7. Time course of translocation of radioactivity in cotton plants treated with approximately. 0.1 pCi of fatty acid-labeled polysorbate surfactant (1%, w/v) in methanol and/or aqueous-methanol solution on a single primary leaf. The treated spot was cut out. Autoradiograms right; plants left. Methanol content in treatment solutions (%, v/v) and (HLB) of surfactant used: A, E, and I -- 100 (4.3); B, F, and J -- 65 (8.0); C, G, and K -- 28 (12.0); D, H, and L -- 0 (15.0). Time period after . treatment: A, B, C, and D -- 3 hr; E~ F, G, and H -- 24 hr; I, J, K, and L -- 72 hr ....•...... •. . 32

ix Figure Page

II- 8. Time course of translocation of radioactivity in cotton plants treated with approximately 0.1 pCi of fatty acid-labeled polysorbate surfactant (1% w/v) in methanol solution on a single primary leaf. The treated spot was cut out. Autoradiograms right; plants left. HLB of surfactant used: A, 4.3; B, 8.0; C, 12.0; D, 15.0 ...... 34

II- 9. Time course of translocation of radioactivity in prickly sida plants treated with approximately 0.1 pCi of fatty acid-labeled polysorbate surfactant (1%, w/v) in methanol and/or aqueous-methanol solution on the fourth leaf. The treated spot was cut out. Autoradiograms right; plants left. Methanol content in treatment solutions (%, v/v) and (HLB) of surfactant used: A, E, and I -- 100 (4.3); B, F, and J -- 65 (8.0); C, G, and K -- 28 (12.0); D, H, and L -- 0 (15.0). Time period after treatment: A, B, C~ and D -- 3 hr; E, F, G, and H -- 24 hr; I, J, K, and L - - 7 2 hr...... 36

II-10. Time course of translocation of radioactivity in prickly sida plants treated with approximately 0,1 pCi of fatty acid-labeled polysorbate surfactant (1%, w/v) in methanol solution on the fourth leaf. The treated spot was cut out. Autoradiograms right; plants left, HLB of surfactant used: A, 4.3; B, 8.0; C, 12.0; D, 15.0 ...... 38

II-11. Influence of HLB of polysorbate surfactant on absorption and translocation of heterocycl ic ring~labeled methazole~ 14 c in the primary leaf of 30-day-old cotton plants 43

II-12. Influence of HLB of polysorbate surfactant on absorption and translocation of heterocyclic ring-labeled methazole-14c in the fourth leaf of 45-day-old prickly sida plants 44

x Figure· Page

III-1. SEM micrographs of G. hirsutum leaf surfaces: (1) Adaxial surfa~e of untreated leaf; (2) Abaxial surface of the same leaf; (3) Adaxial surface of leaf 3-h after dipping in 5% (w/~) ethanol-aqueous solution; (4) Abaxial surface of the same leaf; (5) .Adaxial surface of leaf 72-h after dipping in 5% (w/w) ethanol-aqueous solution; (6) Abaxial surface of the same leaf; (7) Adaxial surface of leaf 3-h after dipping in ethanol; (8) Abaxial surface .of the same leaf. Scale represents 10 )l • • • • • • • • 62

III-2. SEM micrographs of G. hirsutum leaf surfaces treated with polysorbate surfactant (IU..B 4.3) solution: (1) Adaxial surface of leaf 3-h after dipping in 1% (w/w) surfactant solution; (2) Abaxial surface of the same leaf; (3) Adaxial surface of leaf 72-h after dipping in 1% (w/w) surfactant solution; (4) Abaxial surface of the same leaf; (5) Adaxial surface of leaf 3-h after dipping in 0.1% (~/w) surfactant solution; (6) Abaxial surface of the same· leaf;· (7) Adaxial surface of leaf 72-h- after dipping in 0.1% (w/w) surfactant solution; (8) Abaxial surface of the same leaf. Scale represents 10 p 64

III-3. SEM micrographs of G. hirsutum leaf surfaces treated with polysorbate surfactant (IU..B 8.0) solution: (1) Adaxial surface of leaf . . 3-h after dipping in 1% (w/w) surfactant solution; (2) Abaxial surface of the same leaf; (3) Adaxial surface of leaf 72-h after dipping in 1% (w/w) surfactant solu-. · tion; (4) Abaxial surface of the same leaf; (5) Adaxial surface of leaf 3-h after dipping in 0.1% (w/w) surfactant solution; ( 6) Abaxial surface of the same leaf; (7) Adaxial surface of leaf 72-h after dipping in 0 .1% (w/w) surfactant i:;olution; (8) Abaxial surface of the same leaf. Scale represents 10 p ...... ~ . . . . 66

xi Figure Page

III-4. · SEM micrographs of .Q. hirsutum leaf surfaces treated with polysorbatesurfactaht (HLB 12,0) solution: (1) Adaxial surfac'e of leaf 3-h after dipping in 1% (w/w) surfacta.nt solution; (2) Abaxial surface of the sa.me · leaf; (3) Adaxial surface of leaf 72~h after dipping in 1% (w/w) surfactant solution; (4) Abaxial surface of the same leaf; (5) Ada.xial surface of lea£ 3~h after dipping· in 0.1% .· .. (w/w) surfactant solution; (6) Abaxial surface of the same leaf; (7) Adaxial ·surface of leaf · 72-h after dipping in 0.1%(w/w) surfactant solution; (8) Abaxial surfac.e of the same leaf. .... 68' Scale reptesents 10 p • ...... • . • .• . . ·.. Ill-5. SEM micrographs 0£ G.· hirsutum lea£ surf~ces. · treated with poly-;orbate surfactant (HLB 15. 0) solution: (1) Adaxial ·surface of leaf 3-h after dipping in 1% (w/w) surfac.tant ·solution; (2) Abaxial surface of the same le~f; (3) Adaxial surface of leaf 72-h after . dipping in 1% (w/w) surfactant solution; (4) Abaxial surface of the same leaf; (5) Adaxial surface of leaf 3-h after dipping in 0;1% · (w/w) surfactant solution; (6) Abaxial surface of the same leaf;. (7) Adaxial surface of leaf 7.2-h after dipping in 0.1% (w/w)' surfactant solution; ( 8) Abaxial surface of the same leaf. Sc.ale represents 10 p ...•...••• ~· •• ~ . . . 70

UI-6. SEM micrographs of S. spinosa leaf surfaces: .. (1) Adaxial surface of untreated leaf; (2) .· Abax.ial surface of the same leaf; (3) · Adaxial surface of leaf 3-h afte'r dipping in 5% (w/w) .· ethanol-aqueous solution; (4).Abaxial surface of the same leaf; (5) Adaxial surface of leaf 3-h after dipping in ethanol; (6) Abaxial surface of the same leaf; .. (7). Adaxial surface of leaf 72-h after dipping in ethanol; (8) Abaxial surface of the same leaf. Scale'.. represents 10 p • . . . . • ...... 72

xii Figure Page

III-7. SEM micrographs of ~· spinosa leaf surfaces treated with 1% (w/w) polysorbate surfactant solution: (1) Abaxial surface of leaf 3-h after dipping in surfactant (HLB 4.3) solution; (2) Adaxial surface of leaf 72-h after dipping in the same solution; (3) Adaxia1 surface of leaf 3-h after dipping in surfactant (I:il..B 8.0) solution; (4) Abaxial surface of the same leaf; (5) Adaxial surface of leaf 3-h after dipping in surfactant (I:il..B 12.0) solution; (6) Adaxial surface of leaf 72-h after dipping in the same solution; (7) Adaxial surface of leaf 72~h after dipping in· surfactant (I:il..B 15.0) solution; (8) Abaxial surface of the same leaf. Scale represents· 10 µ. • • .. • • • • • • • • • . • • • • • • 74

III-8. SEM micrographs of leaf surfaces treated with herbicide: (l) Adaxial surface of G. hirsutum leaf 3-h after dipping in 1.1% (w/w) methazo1e.wettable powder suspension; (2) Adaxial surface of G. hirsutum leaf 72-h after dipping in the same suspension; (3) Adaxial surface of G. hirsutum leaf 72-h after dipping in o-:-8% (w/w) methazole ethanol solution; (4) Abaxial surface of the same leaf; (5) Adaxial surface of~· spinosa leaf 72-h after dipping in 1.1% (w/w) methazole wettable powder suspension; . ( 6) Abaxial surface of the same leaf; (7) Adaxial surface of ~· spinosa leaf 3-h after dipping in 0.8% (w/w) methazole ethanol solution; (8) Adaxial surface of ~· spinosa leaf 72-h after dipping in the same solution. Scale represents lOp 76 . I. INTRODUCTION

One of the major economical problems in agriculture is weeds.

Chemicals have been used to control these pests for several years and during this period many new herbicides with diverse chemiCal properties have been developed. The compound 2-(3,4-dichlorophenyl)~

4-methyl-l, 2 ,4-oxadiazol idine-3, 5-:-dione (methazole) has, reportedly,

.shown promising selective herbicidal activity against many broad- leaved weed species, especially prickly sida (Sida spinosa L.) in cotton (Gossypium hirsutum L.) and several other important crops at low rates of application (2 to 4 lb/A preemergence; 2 to 3.3 lb/A postemergence)~ The extent and scientific bases of herbicidal. selectivity between cotton and prickly sida have not yet been.determined .. Field evaluation of the compound is being continued. Basic information concerning the possible uptake, mobility and persistence of methazole in relation to its practical selective use against priekly sida in cotton is much needed but still lacking,

Meanwhile, poiysorbate surfactants, nonionic polyoxyethylated derivatives ·of sorbitan fatty acid esters, have been used as additives

to herbicidal formulations and are sometimes found effective to enhance their herbicidal activity. Properties of nonionic surfactants are known to be changed according to the hydrophilic-lipophilic balance (HLB); however, few systematic studies have been conducted

on the influence of HLB on the translocation of herbicides in plants and soils. The effect of polysorbate surfactants on the herbicidal

action and selectivity of tnethazole is unknown.

1 The objectives of this inve·stigation were as follows:

1. . To evaluate the influence of polysorbate surfactants with . . ' . . · different lil..B on. the absorption, translocation and distri-

butional fate of methazole in cotton and prickly sida. ··

2. To: evaluate the influence of lil..B of polysorbate surfac.tants

on the activity of methazole upon the leaf.cuticle of cotton

·and.· prickly sida by· scanning electron microscopy~

3. . To evaluate the influence of polysorbate surfactants with

different lil..B on the mobility of methazole in soils. II. EFFECT OF HLB OF POLYSORBATE SURFACTANTS ON

TRANSLOCATION OF METHAZOLE- 14c

Introduction

The absorption of chemicals by the leaf of a plant is a problem of major importance in chemical weed control.. In order to niodify the uptake, distribution, and persistence of chemicals, numerous types of adjuvants have been employed. Among them, surface active agents or surfactants are perhaps most widely accepted for this purpose. Despite the increasing use of surfactants and other additives in agricultural chemical sprays, this area has connnanded very little coordinated research effort thus far. Acceptance of various surfactants in agricultural practice has been based too much in the past on the results of empirical testing rather than fundamental scientific principles.

Foy and Smith (3) considered the role of surfactants in modifying the activity of herbicides. Hull (5) discussed the activity of surfactants in enhancing foliar absorption of the active toxicant. It has long been recognized, in a general way, that surfactants may facilitate and accentuate the emulsifying, dispersing, wetting, solubilizing and/or surface modifying properties of herbicidal formulations to bring about enhancement of foliar penetration and herbicidal action. Surfactants presumably accomplish this by virtue of their combined polar and apolar properties in the molecule, rendering compatible aqueous and lipoidal phases (3). Surfactants normally

reduce surface tension of aqueous systems, improve wetting, and may

3 4 favor both stomatal and cuticular penetration. Herbtcide-surfactant- plant surface interactions subtler and more specific than mere surface tension lowering and increased wetting are suggested. Minimum surface tensions and contact angles occurred at 0,1 to 0.5% concentration.for most surfactants. However, maximum herbicidal activities were often observed at ten times these levels or greater (2). The final influence of a surfactant, then, will be determined by the nature of the surfactant [ibnogenic class, hydrophilic-lipophilic balance (HLB), chemical structure, and concentration], the herbicide, the solvent, the plant surface, and the physical environment.

HLB of noniQnic surfactants represents a fundamental property of the surfactant and may greatly affect their performance. The optimum surfactant HLB requirement will,. however, change from .system to system. 1 Umoessien et al. reported that absorption and general phytotoxicity of linuron (3-(3,4-dichlorophenyl)-l-methoxy-l-methylurea] and prometryne [ 2 ,4-bis ( isopropylamino) -6- (methyl thio)-s-triazine] . - were enhanced by surfactants within the HLB range 5.4 to 15. Morton 2 and Coombs found greatest herbicidal enhancement of picloram(4-amino-

3,5,6-trichloropicolinic acid)-2,4,5~T [(2,4,5-trichlorophenoxy)

1 . . . Umoessien, S. N., F. M. Ashton, and C. L. Foy. 1967; Effects of hydrophilic-lipophilic balance (HLB) of nonionic surfactants on phytotoxicity of linuron and prometryne to carrots. Weed. Soc. Amer. Abstr., p. 15. 2 Morton, H. L. and J. A. Coombs. 1969. Influence of surfactants on phytotoxicity of a picloram.;2,4,5-T spray on three woody plants. Weed Sci. Soc. Amer. Abstr., p. 65. 5

acetic acid] with surfactants within the HLB range of 13.3 to 15.4. . 3 Hull and Shellhorn treated the seedlings of mesquite [Prosopis

juliflora (Schwartz) DC].with the butoxyethanol ester of 2,4,5.:.T in

various combinatiohs of phytotoxic and nontoxic oils and nonionic

surfactants of HLB 8.6 and 1.8 and found that when sorbitan

monolaurate (HLB 8.6) was used with phytotoxic oils, subsequent

apical epinasty and growth repression was greater than with other

combinations.

Methazole [ 2-(3 ,4-dichlorophenyl) -:4..,.methyl-l, 2 ,4.. oxadiazolidine-

. . . ' 3,5-dione] has shown promising herbicidal activity against prickly

sida (Sida spinosa L.), as well as many other broadleaf species,· in

. cotton (Gossypium hirsutum L.) and several other important crops at 4 5 . relatively low rates of application (1). Connel e.t al. reported

excellent control of prickly sida greater than 10 cm in cotton .with

a postemergence application of methazole. The absorption, trans-

location, and metabolic fate of methazole have been reported by

Jones and Foy (6, 7).

The experiments described herein were conduct:ed to evaluate the

influence of HLB of polysorbate surfactants. on the absorption and

3 Hull, H. M. and S. J. Shellhorn. 1967. Foliar absorption of 2,4,5-T from emulsions and straight oil carriers in combination with oil-soluble surfactants. West Weed Contr. Conf., Res. Prag. Rept. p. 123.

4velsicol Chemical Corporation. 1971. Probe (VCS 438) herbicide development bulletin. Velsicol Chemical Corporation. 8 p.

5connel, J., L. S. Jeffery, T. C. Mccutchen and J. R. Overton. 1971. Control of prickly sida in cotton. Proc. S. Weed Sci. Soc. Abstr. 24:107. 6 translocation of methazole-14c in cotton and prickly sida plants; also, to evaluate the absorption and translocatiori of 14c-labeled polysorbate surfactants with different HLB in these species.

Materials and Methods

14 Radiolabeled methazole. The methazole- C used was heterocyclicring- · labeled in the number 3 carbon (specific activity, 7.66pC:i./mmole). 14 . A stock solution of methazole- C was prepared by dissolving 100 µCi 14 of methazole- C in 10 ml methanol. Various treatment solutions were made by dilution of the stock solution with surfactant solution, methanol, or distilled water.

Surfactant solutions with different HLB. Stock solutians of Span 80

(nonionic surfactant containing sorbitan monooleate, HLB 4.3) and Tween

80 (nonionic surfactant containing polyoxyethylene sorbitan monooleate,

HLB 15.0) were prepared by dissolving 200 mg of each in methanol and distilled water, respectively. Surfactant solutions with HLB 8.0 were prepared by mixing 65 parts of Span 80 stock solution and 3.5 parts of

Tween 80 stock solution; surfactant solutions with HLB 12.0, by mixing

28 parts of Span 80 stock solution and 72 parts of Tween 80 stock solution. 14 Radiolabeled surfactant solutions. Stock solutions of Span 80- C la- 14 beled in the fatty acid moiety (1 )lCi/mg) and Tween 80- · C la.be1ed in

the fatty acid moiety (O. 25 µCi/mg) were prepared by dissolving 100 pCi

of Span 8o-14c and 25 pCi of Tween 8o-14c in 10 ml methanol and distilled water, respectively. Radiolabel~d surfactant solutions with HLB 8 and 12

were prepared in the same manner as were nonlabeled surfactant solutions. 7

Culture of plants. Prickly sida and cotton ('Deltapine 16') were ' 6 sown in 15-cm plastic pots containing Weblite , covered with 5 mm peat and 10 mm W~blite, respecti,vely, and allowed to grow in the greenhouse. Prickly sida and cottonplants, washed free of Weblite

18 to 20 days and 8 to 10 days after seeding, respectively~ were selected for uniformity in both root and top·growth, then transplanted into wide mouth jars wrapped with aluminum foil. Each jar contained two plants in one-half strength Hoagland and Arnon's nutrient solution (4) supplemented with 5 ppm Sequestrene 138 Fe7 as the source of iron. The plants were grown under greenhouse conditions until cotton and prickly sida were 30 days old and 45 days old, respectively. At the time of treatment cotton plants were 18 to 22 cm in height with the two primary leaves fully expanded, and prickly sida plants were 15 to 20 cm· in height at the 7th- to 8th-leaf stage.

Treatment of plants. In treatments to study the influence of HLB of surfactant, foliar absorption and translocation in 30-day-old cotton plants was determined by applying a 20- or 25;..)ll drop of heterocyclic ring-labeled methazole-14c (O.l .).lCi, 3.4 )lg) dissolved in methanol or methanol-aqueous solution with 1.0% (w/v) surfactant with different

HLB on the midrib of one primary leaf; foliar absorption and translocation in 45-day-old prickly sida plants was determined in the same manner as with cotton plants. In prickly sida, however, the

6Expanded shale, Weblite Corporation, Roanoke, Va. 24011.

7sodium ferric ethylenediamine di•(o-hydroxyphenyl acetate), a product of CIBA-Geigy Corporation, Ardsley, New York 10512. · 8

fourth primary leaf wa:s treated •. In the· treatments for studying

foliar absorption of labeled surfactants, approximately 0.1 µGi . . . ' (see precise amounts l~ter) of labeled; surfactant in methanol or

methanol•aqueous solution were applied in the· same way... The treat-

ment was loca~ized by confining the solution in lanolin rings.

Duplicate plants were harvested 3, 24, and 72 hr after treatmen.t and ·

processed for aut:oradiOgraphy. The treatments were replicated :four

times unless otherwise indicated •. At the end of the treatment

periods the lanolin paste, was removed and the treated spot was cut

out with a cork .borer.

Assay for 14c. At harvest, roots were. rinsed in distilled water and

blotted free of excess moisture with absorbent paper towels •. After

the treated portion of thEL leaf was removed, the plants were sect:ioned

into roots, stem, and leaves; they were then moun~ed, pressed for.24

hr, and oven-dried for 48 hr.at 70 C. The dried mounted plants were

then exposed-to GAF X-ray .film for 14 days, at which. time the film

was dev~eloped. The treated laminas, minus the treated portion, and

.. their petioles were ground in polyethylene scintillation vials sus-

pended· in Aquasol Universal L.S.C. ·cocktail, and counted.using a .3000

Series Packard Tri•Ca'rb Liquid Scintillation Spectrometer. The in-

·. fluence of lILB of polysoi:bate surfactant can be determined in two ways··.

in this· investigation. One way is by subtracting the value for the . . distrib~tion of 14c with methanol and/or distilled ~ater.solution . . frcim those -for the distribution of 14c with polysorbate surfactant,

· methanol and/or distilled water.. Another is by subtracting. the - 9

value for the distribution of 14c with methanol from those for the

distribution of 14c with polysorbate surfactant and methanol.

Results and Discussion

Absorption and translocation of 14c with or without polysorbate

surfactant in methanol and/or water. The autoradiograms of 3, 24,

and 72-hr foliar treatment of cotton and prickly sida showed only

apoplastic movement of 14c in spite of the 1% (w/v) addition of

polysorbate surfactant in treatment solution (Figures II-1 and II-2).

Jones and Foy (7) demonstrated that methazole in cotton tissue was

rapidly metabolized to 1-(3,4-dichlorophenyl)-3-methylurea (DCPMU)

and 1-(3,4-dichlorophenyl)urea (DCPU). Patterns of translocation of

radiolabeled materials were not altered by the lll..B of polysorbate

surfactants in either cotton or prickly sida; that is, they trans~

located only from the treated area toward the end of the treated

leaf in the direction of the transpiration stream. Jones and Foy (6)

reporte· d simi· · 1 ar· o b servations· wit· h met h azo 1 e-14c in· cotton. Methazole

and its 14c-labeled metabolites are apparently unable to surmount the

plasmalemma barrier; rather, they remain in the apoplast and move

only in an acropetal direction as is characieristic of the substituted

urea, synnnetrical triazine and uracil herbicides. Short treatment

showed rapid accumulation of 14c in the veins and tissue closely

associated with the veins and as the treatment time increased the . .· 14 tissue between the veins were permeated with methazole- C and/or

14c-labeled metabolites. In the. same treatment time, the distribution

of 14c in the veins and closely associated tiSsue increased with the 10

Figure II-1. Time course of translocation of radioactivity in cotton

plants treated with 0.1 pCi of heterocyclic ring-labeled methazole-

14c with 1% (w/v) polysorbate surfactant in methanol or methanol-

aqueous solution in a 20-pl drop on a single primary leaf. The

treated spot was cut out. Autoradiograms right; plants left. HLB of

surfactant used: A, E, and I -- 4.3; B, F, and J ... - 8.0; C, G, and

K -- 12.0; D, H, and L -- 15.0. Time period after treatment: A, B,

C, and D -- 3 hr; E, F, G, and H -- 24 hr; I, J, K, and L -- 72 hr.

Methanol content in treatment solutions(%, v/v): A, E, and I --

100 ; B, F, and J 82.5; C, G, and K -- 64; D, H, and L -~ 50. 11

• • •

•

Ill

• • • 12

Figure II-2. Time course of translocation of radioactivity in prickly

sida plants treated with 0.1 pCi of heterocyclic ring-labeled

methazole-14c with 1% (w/v) surfactant in methanol or methanol-

aqueous solutions in a 20-pl drop on the fourth leaf. The treated

spot was cut out. Autoradiograms right; plants left. HLB of sur-

factant used: A, E, and I -- 4.3; B, F, and J -- 8.0; C, G, and

K -- 12.0; D, H, and L -- 15.0. Time period after treatment: A, B,

C, and D -- 3 hr; E, F, G, and H -- 24 hr; I, J, K, and L ... - 72 hr.

Methanol content in treatment solutions(%, v/v): A, E, and I --

100; B, F, and J 82.5; C, G, and K -- 64; D, H; and L -- 50. 13

•

• • 14

concentration of methanol 'in the treatment solutions arid also

increased as HLB of pol.ysorbate surfactant became 1owet'. or more

1 iphophil iC.

A single 20-pl drople.t of 100% methanol solution (HLB 4. 3)

required 30 min to disappea".r from the laminas of both plants whereas

. 50% (v/v) methanol solution (HLB 15.0)" required sometitµ.es more. than

3 hr because o:I; the lower volatiiity of water compared with methanol. ·

At harves.t, 24 hr artd 72 ·hr after treatment, contact injury wa.s

observed in the area of applicatiOn for both cot.tori and prickly sida

· with all treatments but the exten.t of injury was· greater in proportion

to. the increasing content of methanol. Quantitatively, 14c in the treated cotton lamina out.side of the treated area was ~ximum with the surfactant with HLB 8.0 in 82.5%

(v/v) methanol at each treatment time, 3, 24_, ·and 72 hr (Table II:-1). . . . The surfactant with HLB 4.3 in 100% methanol was second and followed

in order by the surfactant withHLB 12.0 in 64% (:v/v) methanol and

HLB 15.0 in ;50% (v/v) methanol. ·As the treatment time increased,

radioactivity in the treated leaf increased except in the case of

the surfactant with HLB '15.0 in 50% (v/v) methanol. Distribution of

14c from .the treated area to the end of the treated l.eaf was more

rapid at the lower HLB of surfactant and higher methanol content

than at the higher HLB of surfactant and lower methanol content.·

The treatment with'.Span 80/Tween 80 (65/35, v/v) mixture (HLB 8.0)

was most effective in the.translocationof heterOcycl:i;c ring-labeled 14 . .··.· ·.· . . methazole- C in 30-day-old cotton plants. Table II-1. Distribution of 14c in primary leaves of 30-day-old cotton plants following 20-)ll

drop application of heterocyclic ring-labeled methazole- 14c (O .1 pCi) over the midrib. Radioactivity data are averages of four replications.a

Surfactant compositionb (HLB)

Span 80/Tween 80 ratio

Treatment Plant Span 80 (65 :35) (28:72) Tween 80

time .tissue (4. 3) (8.0) (12.0) (15.0)

I-'" (hr) (dpm) (dpm) (dpm) (dpm) IJI

3 laminae 19298 ab 21824 ab 6998 b 6097 b petiole 99 k 101 k 106 k 163 jk

24 laminae 28880 ab 33396 ab 17809 ab 9477 b petiole 126 jk 174 jk 130 jk 171 jk . c 72 1amina 34574 a 39572 a 22479 ab 8967 b petiole 159 jk 257 j 185 jk 181 jk

aValues followed by the same letter do not differ at the 5% level using Duncan's Multiple Range Test .

. bSurfactant content was 1% (w/v) in each treatment solution. c . The treated spot was cut out and discarded. 16

14 In the distribution of c in the treated. . prickly. . sida. leaf

ot,ttside of the treated area the more distinct difference among

treatments was observed in spite of the smaller absolute value than I cotton according to the statistical analysis; 14c was maximum with·

the surfactant having HLB 4. 3 in 100% In.ethanol a.t each treatment

time and decreased in proportion to the methanol content .in the . . . . 14 • .· . .. •. . treatment solution. The amount of C-labeled material in the .

treated prickly sida lamina, howeve:r, was small and ~t rangedbetween ·. 14 ·.· 320 and 8200 dpm in 72 hr after treatment, while the. amount ()f. · C- ·

labeled material in the treat.ed cotton lamina ranged between 6100 . .: ' . . - : . . .. ·· .. and 39600 dpm in 72 hr, Th·e difference was most reniarkaple with the

surfactant having HLB 8 .• 0 and 82. 5% (v/v) methanol-aqueous solution.

These observations suggest that the mobility of heterotyclic ring-

labeled methazole-14c varies based. on plant species as well as th~ .

composition of the. formulation.

Radioactivity in the petioles of the treated leaves was also

determined for be.th plants (Tal>les. II-1 and II-2). The pattern of

ac~umulation of 14c-labeled material was similar to that observed. in

the treated laminas, but. the. levels of radioactivity were much

smaller and significant differences were not observe~ among.

treatments. ·

Effect of methanol concentration.. ~ecause of the low water solubility of methazole-14cand Span 80 (HLB 4.3), the treatment solutions were prepared with methanol or methanol-aqueoussolut.ion. Auto.,.

· radiograms of one primary leaf of. 30-day-old. cotton plants arid Table II-2. Distribution of 14c in the fourth leaves of 45-day-old prickly sida plants following 20-pl drop application of heterocyclic ring-labeled methazole- 14c (O.lpCi) over the midrib. Radioactivity data are averages of four replications.a

Surfactant compositionb (HLB)

Span 80/Tween 80 ratio

Treatment Plant Span 80 (65 :35) (28: 72) Tween 80 time tissue (4. 3) (8.0) (12.0) (15.0)

I-"' (hr) (dpm) (dpm) (dpm) (dpm) ...... c 3 1 amina 2362 cd 1061 d 769 d 320 d petiole 26 .mn 45 lmn 33 lmn 21 n . c 24 1 amina 6263 a:b 4212 be 1988 cd 1116 d petiole 97 jk 114 jk 72 klm 38 lmn c 72 lamina.. 8165 a 6503 ab 5346 b 2639 cd petiole 133 j 111 jk 102 jk 78 kl aValues followed by the same letter do not differ at the 5% level using Duncan's Multiple Range Test. bSurfactant content was 1% (w/v) in each treatment solution. c . The treated spot was cut out and discarded. 18 the fourth leaf of the 45-day-old prickly sida plants treated with

O.lpCi of heterocyclic ring-labeled methazole-14c dissolved in various methanol-aqueous solutions are shown in Figures II-3 and

II-4. The concentration of methanol.was adjusted to the same level as in the previous experiments (Tables II-1 and II-2) ·not only to examine the effect of methanol.concentration; but also to compare purely the effect of HLB of surfactants; they were 100, 82.5, 64,

50, and 40% (v/v). The influence of methanol concentration on the absorption and transloca.tion of methazole-14c wa:s similar in the two . 14 . species. The autoradiograms show that the translocation of C of

100% methanol solution frbm the treated area to the remainder of the leaf was very rapid during the first 3 hr in both species. The effect of 100% methanol solution was more remarkable than those of other methanol concentrations. These phenomena a:re distinctive in the autoradiograms 72 hr after treatment. With 100% methanol, at

72 hr after treatment, radiolabeled substance{s) in the leaf of prickly sida had spread from the vein of the treated leaf into the closely associated tissue; whereas, at all other methanol concentrations, 14c remained almost entirely in the veins. In the cotton leaf, radiolabeled material was spread more effectively from the vein to the surrounding area when 100% methanol was used rather tha:n other methanol concentrations. Translocation of radiolabeled substance(s) appeared to be more rapid in cotton than in prickly. sida. 19

•

(3 hr treatment) (72 hr treatment)

Figure II-3. Time course of translocation of radioactivity in cotton

plants treated with 0.1 µCi of heterocyclic ·ring-labeled methazole-

14c without surfactant in a 25-)ll drop on a single primary leaf.

The treated spot was cut out. Autoradiograms above; plants below.

Methanol concentration in treatment solutions (%, v/v): A, 100;

B, 82.5; C, 64; D, 50; E, 40. N 0 A ?.D E 0 !! ~A (3 hr treatment) (72 hr treatment)

Figure II-4. Time course of translocation of radioactivity in prickly sida plants treated with 14 0.1 pCi of heterocyclic ring-labeled methazole- c without surfactant in a 25-pl drop on the

fourth leaf. The treated spot was cut out. Autoradiograms above; plants below. Methanol

concentration in treatment solutions (%, v/v): A, 100; B, 82.5; C, 64; D, 50; E, 40. 21

Quantitatively, .·the effect of methanol con~entration on the translocation of heterocyclic ring-labeled methazole.:.. 1 4c~as determined by.counting the radioactiv:i;ty in the treated leaf which moved outside of.the treated area. The data confirmed the observations mad.e from the autoradiograms. . The .effect of methanol increased slightly up to 82.5% (v/v) methanolconcentrationandachieved the maximumradioaciivity at 100% methanol for both cotton: and prickly sida. The difference in 14c translocated when l00% methanol was employed, rather than .. other methanol concentrations from. 40 through

82 .5% (v/y), was remarkable for both species·; differences among the

latter group became evident only 72 hr after foliar treatment. These

observations suggest that addition of 17.5% (v/v) or more water to

the treatment _sol,ution negated· the ef feet of methanol considerably

on the uptake and trartslocation of methazole-14c in both cotton and

. . prickly sida. The radioactivity in the cotton lamina treated with .

0.1 pCi of methazole-14.c in 100% methanol solution dropped 72 hr

after treatment. The reason for this phenomenon is not ¢xplained.

In prickly sida, translocation of _radiolabeled·material·from the

treated area to the.tip of the leaf i~creased in proportion to the

treatment. time. The radiolabeled material trans.located from the

treated area to other parts of the leaf more quickly in cotton: than

prickly sida, as was observed in the previous experiment. At the time . · ·

of counting radioactivity in cotton and prickly·sida laminas, the

radioactivity of the petioles of treated leaves of both plant species Table II-3. Distribution of 14c in primary leaves of 30-day-old cotton plants following 25-pl application of heterocyclic ring-labeled methazole- 14c (0.1 pCi) methanol or methanol-aqueous solutions over the midrib. Radioactivity data are averages of five replications.a

Methanol (%, v/v) Treatment Plant content

time tissue 100.0 82.5 64.0 50.0 40.0

(hr) (dpm) (dpm) (dpm) (dpm) (dpm) b 3 lamina 20245 c 5799 fg 7135 fg 6006 fg 4893 g petiole 188 kl 253 jkl 255 jkl 190 kl 194 kl N N b 24 lamina 37 508 a 11860 efg 12524 ef 10618 efg 9806 efg petiole 263 jkl 382 j 293 jk 131 1 177 kl b 72 lamina 31398 b 19541 cd 13470 cdef 10365 efg 15474 cde petiole 220 kl 179 kl 146 1 189 kl 292 jk

aValues followed by the same letter do not differ at the 5% level using Duncan's Multiple Range Test. bThe treated spot was cut out and discarded. Table II-4. Distribution of 14c in the fourth leaves of 45-day-old prickly sida plants following

25-pl application· of heterocycl ic ring-labeled methazole- 14c (O .1 pCi) methanol or methanol- aqueous solutions over the midrib. Radioactivity data are averages of five replications.a

Methanol (%, v/v) Treatment Plant content

time tissue 100.0 82.5 64.0 50.0 40.0

(hr) (dpm) (dpm) (dpm) (dpm) (dpm) b 3 lamina 4023 c 615 g 363 g 278 g 360 g

petiole 46·lmnop 28 nop 23 op 18 p 18 p N w 24 1 amina . b 7400 b 840 fg 649 f g 359 g 507 g petiole 100 k 36 lmnop 34 lmnop 30 mnop 31 mnop b 72 lamina 12293 a 2344 d 1756 d 1048 de 1493 ef petiole 138 j 51 lmno 62 1 59 lm 54 lmn

aValues followed by the same letter do not differ at the 5% level using Duncan's Multiple Range Test. bThe treated spot was cut out and discarded. 24

was also determined. tevels·of•radioactivity in the·petioles were

low for both.plant; species and among treatments, and no significaµt

differences existed •

.Effect of HLB of surfactant-methanol solut::i_on., The effect of methanol

on the absorption and 'translocation of methaz.ole-14c and/or its

metabolite~ was clear in the preceding'exper~ments.· The autoradio-

granis of the primary leaves of 30-day..;,old cotton plants.and the

fourth leaves of 45-d~y-old prickly sida plants treated with 9.1

pCi heterocyclic ririg-labeled :metha:zole-14c dissolved in 100% .

methanol wit.h or without 1% (w/v) polysorbate surfactant are shown

· in Figures II-5 and II-6. In comparison with Figures II-1 a:nd II-2,

short treatment times, up to 24 hr, showed greatest accumulation of .1·. radiolabeled material in the veins and tissues closely associated

with the veins. This was particularly evident in· the autoradiograms of cotton leaves. . These accelerated accumulations of 14c among all ·treatments of this experiment suggest that they are du.e to the. effect

'of 100% methanol solution itself rather than the. combined effeet of

. surfactant and methanol because visible differences could not be

observed between the autoradiograms of plants treated with surfactant·

and those without surfactant~

The differences of accumulation of l4c seen in Figures II-1 and

II-2 based on the difference of HLI~. of surfactant and methanol concentra-

tion were not observed in Figures II-5.and II-6, either. 'It. is presumed

that by alteration of .the waxy component of the cuticle, methanol 25

Figure II-5. Time course of translocation of radioactivity in cotton

plants treated with 0 .1 pCi of heterocyclic ring-labeled methazole-

14c dissolved in 100% methanol withpolysorbate surfactant in a

20-pl drop on a single primary leaf. The treated spot was cut out.

Autoradiograms above; plants below. HLB of polysorbate surfactant

used: A, no surfactant used (control); B, 4.3; C, 8.0; D, 12.0;

E, 15.0. !., CJ·5•

CB·S· Cl lJ

N 0\

A B' c , A

....., 'f...... ,n• H. "/411• ...• • • h .I Cl·S. ..Ct ·S, ~ /• • Cl ·S• ~ ~ • D E I ~ D ~ E. D :I E cl;, (3 hr treatment) (24 hr treatment) (72 hr treatment) 27

Figure II-6. Time course of translocation of radioactivity in prickly

sida plants treated withO.lpCi of heterocyclic ring-labeled

methazole-14c dissolved in 100% methanol with polysorbate surfactant

in a 20-pl drop on the fourth leaf. The treated spot was cut out.

Autoradiograms above; plants below. HLBof polysorbate surfactant

used: A, no surfactant (control); B, 4.3; C, 8,0; D, 12.0; F,:·, 15.0. 28

(3 hr treatment)

( 24 hr treatment)

(]2 hr treatment)

>H ' .

29 was apparently· able to increase the penetration .and the stibsequent ·. ·

tr ans location of met.hazole-14c and/or its 14c...;metabol:i;tes. .• Methanol . . used in th.is experiment masked the effect of polysotbate .·surfactants . themselves as well as the effect of HLB of these surfactants.

Quantitatively, the dat~· (Tables II-5 and ~I-:-6). confirmed what

. -, . was observed in the autoradiograms (Figures It-5 and· II""'.6). The ... '.· -:;·· amount of 14c-labeled I!laterial in the treated leaf increased as the time elapsed in all the treatments with surfactant i:nboth species.

In treatment of prickly sida~ polysorbate surfactant showed the . . . tendency to restrict the influence of. 100% meth~nol s~lution. - ·. . . Distribution of polysorbate. surfactari.t-14c •. · B.ecause o·f the different

specific activities .of Span' 8o-14c and Tween ao..:14c, the amount of

radioactivity applied for each treatnient was somewhat varied: Span

ao~ 14 c (HLB 4.3) 0.1 pCi; Span 80-:-14c/Tween 8o-14c G5/35·(v/v)· (HLB

8.0) 0.1025 pCi; Span .8o-14c/Tween. 8o-14c 28/72 (v/v) (HLB 12.0)

0.158 µCi; Tween 80~ 1 4c (HLB 15.0) 0.1 pGi. 'The concentration .of -. ·.. . the surfactant in each treatment solution wa.s prepared atl%' (w/v)

to correspond to the other experiments. Autoradiograms (Figures II-7,.II-8, II-9, and II-10) showed no distribution of 14c_:fatty add-labeled polysorbate .surfactant in

either cotton or prickly sida; no visua.l distr.ibuti~n of l4c was

observed with any methanol concentration, any HLB of surfactants; or

at.any treatment time. Quant:j..tative data {Tables II-7 and II-8)

support these visual observations. Smith arid F.oy · (8) arid Norris8

8Norris, R. F. 1971. Surfactant and cuticular penetraticm. Weed Sci. Soc. Amer. Abstr. '· p. 11. Table II-5. Distribution of 14c in primary leaves of 30-day-old cotton plants following 20-pl drop application of heterocyclic ring-labeled methazole- 14c (O.l pCi) methanol solution over the midrib, Radioactivity data are averages of four replications.a.

Surfactant compositionb (HLB)

Span 80/Tween 80 ratio

Treatment Plant Span 80 (65: 35) (28:72) Tween 80

time tissue (4.3) (8.0) (12.0) (15.0) w (hr) (dpm) (dpm) (dpm) (dpm) (dpm) 0 . c 3 lamina 19298 a 13989 b 12916 b 12058 b 20245 a petiole 99 m 94 mn 87 mn 98 mn 188 kl . c 24 1 amina 28880 a 35315 a 31797 a ·22900 a 37 508 a petiole 126 lmn 85 mn 150 klm 57 n 263 j . c 72 lamina 34574 a 43453 a 39496 a 44925 a 31398 a petiole 159 klmn 120 lmn 96 mn 145 lm 220 jk aValues followed by the.same letter do not differ at the 5% level using Duncan's Multiple Range Test. bSurfactant content was 1% (w/v) in each treatment solution. cThe treated spot was cut out and discarded, Table 11-6. Distribution of 14c in the fourth leaves of 45-day-old prickly sida plants following 20-pl drop application of heterocyclic ring-labeled methazole- 14c (O.l pCi) methanol solution over the midrib. Radioactivity data are averages of four replications.a

Surfactant compositionb (HLB)

Span 80/Tween 80 ratio

Treatment Plant Span 80 (65:35) (28:72) Tween 80 time tissue (4. 3) (8.0) (12.0) (15.0) w (hr) (dpm) (dpm) (dpm) (dpm) (dpm) ......

3 laminae 2362 ef 3694 ef 1775 f 1569 f 4023 e petiole 26 m 27 ID 22 ID 27 ID 46 lm

24 laminae 6263 cd 4321 de 6475 cd 3736 e 7400 c petiole 97 kl 75 klm 104 kl 82 klm 100 kl

72 laminae 8165 be 7290 c 7482 c 9431 b 12293 a petiole 133 jk 92 kl 129 jk 170 j 138 jk aValues followed by the same letter do not differ at the 5% level using Duncan's Multiple Range Test. bSurfactant content was 1% (w/v) in each treatment solution. cThe treated spot was cut out and discarded. 32

Figure II-7. Time course of translocatioi:J. of radioactivity in .cotton

plants treated with approximately 0 .1 pCi of fatty acid-labeled

polysorbate surfactant (1%, w/v) in methanol and/or aqueous•methanol

solution on a single primary leaf. The treated spot was cut out.

. . . Autoradiograms right; plants left. Methanol content in treatment

solutions (%, v/v) and (HLB) of surfactant used: A, E, and I -- 100

(4.3); B, F, and J -- 65 (8.0); C; G, and K -- 28 (12.0); D, H, and

L -- 0 (15.0). Tiine period after treatment: A, B~ C, and D .;.._ 3 hr;

E, F, G, .and H - - 24 hr; I, J, K, and L -- 7 2 hr. 33

. - •

•

( -·•~--- ••

• • •

.• • • 34

Figure II-8. Time course of translocation of radioactivity in cotton

plants treated with approximately 0 .1 pCi of fatty acid-labele.d

polysorbate surfactant (1%, w/v) in methanol solution on a single

primary leaf. The treated spot was cut out. Autoradiograms right;

plants left. HLB of surfactant used: -~, 4.3; B, 8.0; C, 12.0;

D, 15.0. 35

(3 hr treatment)

/ · c / • •· D ' I. CB·/,

I· A / · B " (24 hr treatment)

c D •

C:. I·/, .,.

(72 hr treatment) 36

Figure II-9. Time course of translocation of radioactivity in prickly

sida plants treated with approximately 0.1)1Ci of fatty acid-labeled

polysorbate surfactant (1%, w/v) in methanol and/or aqueous-methanol

solution on the fourth leaf. The treated spot was cut out. Auto-

radiograms right; plants left. Methanol content in treatment solu-

tions (%, v/v) and (HLB) of surfactant used: A, E, and I 100

(4. 3); B, F, and J -- 65 (8.0); C, G, and K -- 28 (12.0); D, H, and

L -- 0 (15.0). Time period after treatment: A, B, c, and D 3 hr;

E, F, G, and H -- 24 hr; I, J, K, and L ~- 72 hr. 37

( /

~·, ,~ •••••.,, ...• ••••• • UI I

.,,,... .. o.~ ~, .... •• ·~ • • • ••1•, •••••• .. •.~.:...... ~~· ' ··•.. : 38

Figure II-10. Time course of translocation of radioactivity in prickly

sida plants treated with approximately 0.1 pCi of fatty acid-labeled

polysorbate surfactant (1%, w/v) in methanol solution on the fourth

leaf. The treated spot was cut out. Autoradiograms right; plants

left. HLB of surfactant used: A, 4.3; B, 8.0; C, 12.0; D, 15.0. 39

• •

(3 hr treatment)

- ~ ft • A l D (24 hr treatment)

!14 !!I !!c, !!D1 (72 hr treatment) Table II-7. Distribution of 14c in treated leaf after treatment of 30-day-old cotton plants with approximately 0 .1 pCi of fatty acid-labeled polysorbate surfactant over the midrib of a primary leaf. Radioactivity data are averages of four replications. Applied volume of treatment solution varied because of different specific activities of Span 8o- 14c and Tween 80- 14c.a

Surfactant-methanol aqueous solution compositionb 0 (methanol %, v/v) 14 14 . 14 Span 8o- 14c Span 80- C/Tween 80- C ratio Tween 80- C Treatment Plant (65: 35) (28: 72) time tissue (100) (65) (28) (O) ~ (hr) (dpm) (dpm) (dpm) (dpm) 0 d d 3 laminae 4002 ab 3414d ab 2266d b. 2652 ab petiole 179 jk 131 k 76 k 139 k . c d 24 lamina 3333 ab 3665d ab 2214: b 5083 a petiole i61 jk . 156 k 89 k 131 k . c d 72 1 amina 3324 ab 3699d ab 1562: b 9845 a petiole 185 jk 278 j 61 k 188 k aValues followed by the same letter do not differ at the 5% level using Duncan's Multiple Range Test. bSurfactant content was 1% (w/v) in each treatment solution. cThe treated spot was c~tout and discarded. d . Data were corrected for 0 .1 pCi application. Table II-8. Distribution of 14c in treated leaf after treatment of 45-day-old prickly sida plants with approximately 0 .1 pCi of fatty acid-labeled polysorbate surfactant over the midrib of the fourth leaf. Radioactivity data are averages of four replications. Applied volume of treatment solution varied because of different specific activities of Span 8o-14c and Tween 8o- 14c.a

Surfactant-methanol aqueous solution compositionb (methanol %, v/v)

14 14 . 14 Span 8o- 14c Span 80- C/Tween 80- C ratio Tween 80- C

Treatment Plant (65 :35) (28: 72) time tissue (100) (65) (28) (O) .p... I-' (hr) (dpm) · (dpm) (dpm) (dpm) d . 3 laminae 227 a 121: b 233d a 153 ab petiole 2G 1 26 1 17 1 25 1 d 24 laminae 223 a 202d a 182d a 184 a petiole 19 1 80 j 27d 1 20 1 d 72 laminae 233 a 123: b 184d a 119 b petiole 25 1 56 k 37 kl 25 1 aValues followed by the same letter do not differ at the 5% level using Duncan's Multiple Range Test. bSurfactant content was 1% (w/v) in each treatment solution. cThe treated spot was cut out and discarded. dData were corrected· for 0.1 pCi application.

I 42

reported similar observations on the movement of labeled polysorbate ..

surfactants. Foy and Smith,9 however, reported the movement of 35s~

sodium laurylstilfate and 14c--polyoxyethylene polyols in cotton and

barley (Hordeum vulgare L.). These observations suggest that the

movement. of surfactants and/or their metabolites in plants depends

upon the size and shape of the complex molecule or mixture.of com-

pounds. It may be informative to investigate the limits of molecular .• size and shape which penetrate. into plant surfaces. Effect of HLB. of polysorbate surfactant. In the fir.st part of this paper it was described. that the influence of HLB of polysorbate

surfactant could be· determined in two ways. But becaus·e of the

extraordinary effect of 100% methanol· solution on the absorption and

t~anslocation of heterocyclic ring-labeled methazole-14c, the effects

of polysorbate surfactants themselves as well as their HLB effect

were nullified when they were used· by d:i,.ssolving in 1-00% methanol.

Therefore; it is considered not appropriate to judge the effect of ·

HLB of polysorbate surfactant by stibfracting the value for the

distribution of 14c with methanol from those for the distribution of

14c with polysorbate surfactant and methanol {seconcl way). Figures·

II-11 and II-12 show the. results, with.,both plant species, of sub-

tracting the value for the. distribution of 14c with methanol and/or distilled water solution. from those for the distribution of l4c with

9Foy, G. L. and L •. W. Smith. 1964. Tracer studies with radio-- labeled surfactants and dalapon. Weed Soc. Ainer. Abstr.; p. 79. 43

3 24 72 TREATMENT TIME (HR)

-I

(HLB 4.3) (HL8 8.0) ( HLB 12.0) ( HLB 15.0)

Figure II-11. Influence of HLB of polysorbate surfactant on absorption 14 and translocation of heterocyclic ring-labeled methazole- c in the

primary leaf of 30-day-old cotton plants. 44

(HLB 4.3) (HLB 8.0) ( HLB 12.0) ( HLB 15.0)

4 - l0 "b 2 > !: 0 ~ --3--24--72- ~-3--24--72- 3 24 72 3 1_2 TREATMENT TIME (HR)

-4

Figure II-12. Influence of HLB of polysorbate surfactant on absorption 14 and translocation of heterocyclic ring-labeled methazole- c in the

fourth leaf of 45-day-old pric~ly sida plants. 45

polysorbate surfactant, methanol, and/or distilled water (first way).:

In these figures, distributions of l4c in the treated lamina outside

the treated area were plotted because inthe petiole significant

difference was not observed among treatments. The difference of the

IiLB effect of polysorbate surfactant shown in Figures II-11 and

II-12 appears to be true for both plant species studied. The effect

of surfactant having HLB value of 8 was greatest in the translocation

of heterocyclic ring-labeled methazole-14c, and it was particularly

distfo.ctive in the primary leaf of 30-day-old cotton plants.

The plots below zero obtained at HLB 4.3 for both SJ;>eci.es are

.actually_ the result of the above mentioned calculations, in which . . : .. the effect of 100% methanol have been subtracted. But by the same

reason that we considered the second way of calculation to be rather

unreasonable in this investigation, we still have something uncle~r •.

Because 100% methanol solution was used at HLB 4.3. Although difficult

to interpret the effect of HLB of polysorbate surfactant from these.

figures only' this is one of the conceivable methods to determine the ..

effect of surfactants.

Acknowledgements

The surfactants and herbicide used in this St\ldY were generously

provided by Atlas .Chemicals Division, ICI-America Irie., Wilmington,

Delaware 19899 and Velsicol Chemical Corporation, Chicago, Illinois

60611, respectively. 46

Literature Cited

1. Ballard, J., H. A. L. Greer, and P. W. Santelmann. 1972. Pre..;. and post-em.ergence activity of VCS-:438 herbici:de. Proc. s. Weed Sci. Soc. 25:167-174.

2. Foy, C. L. and L. W. Smith. 1965. Surface tension lowering, wettability of paraffin and c:orn leaf surfaces, and herbicidal · enhancement of dalapon by seven surfact:ants. Weeds 13:15-19.

3. Foy, c. L. and t. w. Smith. 1969. The role of surfactants in . modifying the _activity ef herbicidal sprays. Pesticidal fomu- lations research. Physical and colloidal chemic:alaspects. Adv. Chem. Series 86:55-95.

4. Hoagland, D. R. and, D. I. Arnon. 1950. The water-culture method for growing' plants without soil. .Califom,ia Agr. Exp. Sta., Berkeley, Circ. 347. 32 p.

5. Hull, H. M. 1970. . Leaf structure as related to absorption of · pesticides arid other compounds. Residue Rev. 31:1-150. ·

6.. Jones, D. W. and C. L. Foy. 1972. Absorption and translocation of bioxone in cotton. Weed Sci. 20: 116--120,

7. Joneli'!, D. W. and C~ L. Foy. 1972. Metabolic fate of bioxone in cotton. Pee tic. Bio chem. Physiol. 2: 8-26.

8. Smith, t. W. and C. L. Foy. 1966. Penetration and distribution studies in bean, cotton, and barley from foliar and root applications of Tween 20-14c, fatty acid and oxyethylene labeled. J. Agr. and Food.Chem. 14:117-122. · III. EFFECT OF POLYSORBATE SURFACTANTS ON THE ULTRA-

STRUCTURE OF LEAF SURFACES

Introduction.

Herbicides and plant growth reglllators are sprayed onto plant surfaces. Plant epidermal waxes are connnonly the first barrier to penetration encountered by these chemicals. The amount and distribution of these chemicals deposited on the plant surface are greatly influenced by the degree of wetting of plant surfaces. The effect of cuticle characteristics bn the deposition, distribution, and retention of chemicals applied to foliage has been discussed (3, 5, 12, 13, 14). ·

Measurement of the contact angle of a droplet applied to foliage is one method of determining wettability o.f the plant surface by the solution; the angleof contact between solution and plant surface .is. governed by a number of cuticular factors including hairiness, cor- rugation of the surface, and chemical and physical nature of the cuticular waxes. Though both polar and non-polar substances can pass through cutinized epidermal walls, the mechanism of this pene.- tration is not fully explained. Crafts (2) supposed that there are two routes by which exogenous molecules may transverse·the distance from the cuticle surface into the living inner cells, a lipoid and an aqueous pathway.

The stomata would appear to provide obvious routes of penetration but Furmidge (9) observed that injury by surfactants to apple (Malus

syl vestris L.) and plum (Prunus domestica L.) leaves bore little

relation to the distribution of stomata, and concluded that entry

47 48 was principally other than stomatal. Schonherr and Bukovac (16) reported that liquids having a surface tension less thah 30 dyne -1 cm gave zero contact angle on the leaf surface and infiltrated stomata spontaneously, whereas liquids having a surface tension -1 greater thari 30 dyne cm did not wet the leaf surface and failed to infiltrate stomata. They experimented with several surfactants in reaching the above conclusion.

Some regions of the leaf act as preferential sites of adsorption.

Trichomes, especially the basal portion of trichomes, provide an important route of entry (13). Trichomes are usually cutinized, therefore, absorption is cuticular. The cuticles of the guard cells, subsidiary cells of stomata, and of the regions immediately adjacent to the mid-vein of leaves ate also important sites of absorption.

Despite the increasing use of surfactant.s and other additive~ in agricultural chemical sprays, this area has commanded very little coordinated research effort thus far. Acceptance of various·surfactants in agricultural practice has been based too much in the past on the results of empirical testing rather than fundamental scientific principles. Hull (12) discussed the activity of surfactants in enhancing foliar absorption of the active toxicant. Foy and Smith

(8) considered the role of surfactants in modifying the activity of herbicides. Surfactants normally reduce surface tension of aqueous systems, improve wetting, and may favor both stomatal and cuticular . penetration. Herbicide-surfactant-plant surface interactions subtler and more specific than mere surface tension lowering and increased wetting are suggested (7). Minimum surface tensions and contac.t 49

...... · .· ... ·. .·. ·...... ·. .·. . .. . ·. angles occurred·. at 0.1 to 0 .5% concentration for tnost surfactants •...... - . . : . . However, maximum herbic.idal activities were often observed at ten

times these levels or greater (7). The final influ~nce of a surfactant, then, will be determined by the nature of the surfactant.

[ ionogertic class, hydrophilic-1 ipophilic balance (HLB) , chemical

structure, and concentration], the herbicide; the solvept, the plant

surface·, and the physical environment.· Darlington and Barry ,(4)

. suggested that surfactants .can modify the permeability of the cuticle

and that this effect could be more fully exploited in the formulation

of commercial spray chemicals •. Our experiments (See Section II)··

sho:wed that foliar-appiied 14c-fatty acid labeled pol:y:sorbate .sur-

factants did not. move appreciably from th.e site of application in.

either Gossypium .hirsutum L. or Sida.spinosa L. Smith and Foy (18).

reported similar observations on the movement of labeled surfactan:ts.

Amelunxen, Morgenroth, and Picksak (1) _reported the use of

stereoscan electron microscopy in biological investigation, and

showed the multiple fo.rm.s of wax excretions from leaves, shoots, and

fruits by photographs. Hall_~ al. (10) combined contact angle

. . determinations· with electron microscopy and found that contact angles

in excess of 145° were given on leaves when numerous wax· rods or

plates covered the surfaces. Silva FernaJides (16) studied water-

repellency of surface waxes with electron microE;1copy and classified .· . . . . . the surface. waxes of several plants to be water-repellent and non-

water-repellent; a crystalline or semicrystalline wax repelled water,

·while a non-crystalline wax did aot repel water~ 50

Wortmann (21) made ele.ctron microscopy stµdies of the changes·.

in the surfac.es of leaves caused by pesticides and a wetting agent •.

MCPA (4-chloro-2-inethyl phenoxyacetic acid) and parathion (diethyl

4-nitrophenyl phosphorothionate) changed the wax ~tructures ·of these.

plants. When treated with a surfactant at 0.02%, the structures

(particularly the 'rods) were largely destroyed, but regenerated in

an-entirely new multi-branched form. Ong, Falk, and Bayer (15)·

·. observed leaf ·surfaces upon which herbicid.e was sprayed, by using

a scanning electron. microscope (SEM) equipped to detect the fluores~ .

cence and reported a new~ rapid method for.spatially localizing.·

herbicides on leaf surfaces.

The experiments descl;'ibed herein were conducted by using SEM to

investigate the possible infl~ence of polysorbate surfactal;lts having

different HLB value~ on the plant cut.icular structure. and/or how

polysorbate surfactants were deposited on the. leaf surface.

Materials and Metho.ds

Span 80 (nonionic surfactant· containing sorbitart monooleate,

HLB 4.3, Atlas.Chemical.s.Division; ICI-Arnerica Inc., Wilmington, DE),

Tween 80 (nonionic surfactant containing polyoxyethylene.sorbitan

tnonooleate, HLB 15.0; Atlas Chemicals Division, ICI00 Arnerica Inc.,

Wilmington, DE) and their mixtures of 65/35 (w/w) Span 80/Tween 80

(HLB 8.0), 28/72 (til/w) ·.Span 80/Tween 80 (HLB 12.0) were used in this

investigation. Both 0.1 and 1.0% (w/w) surfactant solutions (all

·HLB values) were prepared by first dissolving the surfactants in 5 g

of ethanol and then diluting to.100 g with distill.ed water. Also,

' .. ~ . . ,· 51 an ethanol-aqueous ·solution (5%, .w/w) and undiluted ethanol were included to evaluate the solvent effect.

Methazole [2-(3,4-dichlorophenyl)-4-methyl-l,2,4-o:x;adiazoiidin,e-

3,5-dione, 99.4% (w/w) active, Velsicol Chemical Corporation, Chicago,

IL] solution was prepared by dissolving 0. 8 g in 99. 2 g ethanol.

Methazole 7 5% wettable powder (1.1 g) was also diluted to 100 g with distilled water.

G. hirsutum (var;i.ety, 'Deltapine') and~· spinosa were sown in

5-cm plastic pots containing Weblite (co1IDDercial grade of expanded shale, Weblite Corporation, Roanoke, VA), covered with 5 mm peat and 10 mm Weblite, respectively, and allowed to grow in the greenhouse. g. hirsutum and ~· Spinosa washed free of WebLite 8 to 10 days and 18 to 20 days after seeding, respectively, were selected ' for uniformity in both root and top growth, then transplanted int<;> wide mouth jars wrapped with aluminum foil. Each jar contained two plants in one-half strength Hoagland and Arnon's nutrient solution

(11) supplemented with 5 ppmw Sequestrene 138 Fe [sodium ferric ethylenediamine·di-(_£-hydro:x;yphenyl acetate), Geigy Agricultural

Chemicals, Ardsley, NY] as the source of iron. The plants were grown under greenhouse conditions until g. hirsutum and S. spinosa were 30 and 45 days old, respectively. At the time of treatment

G. hirsutum was 18 to 22 cm in height with the two primary leaves

fully expanded; ~· spinosa was 15 to 20 cm in height at 7th- to 8th..

leaf stage.

In treatments to study the influence of polysorbate surfactant

having different.HLB values, solvent, and methazole, the .primary leaf 52 of 30-day-old Q. hirsutum and the fourth leaf of 45-day-old 2_. spinosa were dipped for 1 minute in 100 ml of 0.1 and/or 1.0% (w/w) polysorbat.e surfactant solution, solvent, or methazC>le spray solution.

The treated leaves were cut at the petiole 3 ... h and 72-h after treatment and two discs of approximately 6 nnn in diameter were punched . out·from each species; one was for study of the adaxial surface, another for observation of the abaxial surface, Leaf discs were fixed on metal holders with silver enamel and pl.aced into a vacuum chamber. A uniform coating of palladium-gold was evaporated onto the surface (19). The samples were fully prepared within the day of harvest and observed by use of an AMR Model 900 high resolution

SEM the next day ..

Results and Discussion

The SEM micrographs of the leaf surfaces of G. hirsutum are ·. shown in Figures III-1 through III-5. Figu_re III-1(1) and III-1(2) are natural leaf surfaces, which did not receive any treatment. The cuticle in cotton covered the epidermal cells smoothly and it was · difficult to locate the boundary between the cells as reported by

Troughton and Donaldson (20). Stomata were somewhat more numerous on the abaxial than the adaxial side of the leaves. A feature of

the stomata of Q. hirsutum is the pronounced lip at the entrance to

the stomata! pore. This is an extension of the cuticle and is connnon

in many species (20). 'l'he wax deposit of the adaxial. surface appeared

thicker than that of the abaxial· surface. The adaxial surface also

appeared more fibrous than the abaxial surface. 53

Figure III-1(3) through 1(6) illustra.te the results of the treatment of G. hirsutum with 5% (w/w) ethanol-aqueous solution which was used as a solvent for polysorbate surfactants. Figure III-1(3) shows the blocky adaxial surface 3-h after treatment, which resembles the SEM micrograph of adaxial surfa6e of G. hirsutum leaf taken by Troughton and Donaldson (20). Their specimens were prepared by freeze-drying.

The epidermal cells [Figure III-1(3)] were beginning to shrink c;ind the surface was becoming wrinkled. The abaxial surface showed a smaller

' . blocky pattern than the adaxial surface of the same leaf. The stomata of the abaxial surface after treatment with 5% (w/w) ethanol-aqueous solution were opened wider than those· of the adaxial surf ace [Figure

III-1(4) and 1(6)]. The blocky structure of both surfaces of the leaf 3-h after treatment turned back to a more fibrous appearance

· 72-h after treatment [Figure III-1(5) and 1(6)].

Figure III-1(7) and 1(8) illustrate the leaf waxes of both surfaces of G. hirsutum treated with 100% ethanol. Both surfaces were dehydrated with ethanol and exhibited complicated fibrous wax structures. ·The louver structure of leaf waxes was observed outside of the guard cells on the adaxial surface. Thes SEM micrographs suggest that the ethanol solubilized and removed some of the waxes

from the leaf surfaces, so that they were dehydrated and shrunken.

Figure III-2(1) through 2(8) illustrates the SEM micrographs

of G. hirsutum treated with 0.1 and 1.0% (w/w) Span 80 (HLB 4.3)

solutions. Of the leaf surfaces of G. hirsutum treated with poly-

sorbate surfactant solutions, those treated with 1.0% (w/w) Span 80

solution showed the most distinct results [Figure III-2(1) through 54

2(4)]; Span 80 was accumulated in the troughs of irregular blocky corrugations of the leaf surface 3-h after treatment [Figure III-2(1) . · and 2.(2)] and then severely eroded leaf wax structure 72-h after treatment [Figure III-2(3) and 2(4)]. The stomatal bpening was masked with Span 80 and/or eroded leaf waxes [Figure III~2(3)J. The accumulation of the surfactant in the troughs of irregular blocky corrugations was also observed in the treatment with 0.1% (w/w)

Span 80 solution [Figure III-2(5)]. The abaxial surface of the leaf was etched with Span 80 [Figure III-2(6)]. The SEM micrographs of the leaves 72-h after treatment with 0.1% (w/w),Span 80 solution showed wrinkled surfaces but the severe erosion of the surface waxes was not observed [Figure III-2(7) and.2(8)]. The stomata of the abaxial surface of the leaf were opened wider than those of the adaxial surface. The erosion and etching of the surface waxes of . the leaves suggest that Span 80 has an affinity for leaf waxes of g. hirsutum to some extent and can solubilize them. Reported con~ stituents of leaf surface waxes of G~ hirsutum were triacontane, dotriacontane, hexatriacontane, octacosanol, triacontanol and dotriacontanol (14).

Figure III-3(1) through 3(8) illustrates the leaf surfaces treated with polysorbate surfactant. (HLB 8.0) solution. Our study

(See Section II) .showed that polysorbate surfactant with HLB 8.0 . . . . 14 enhanced the transloca tion of heterocycl ic ring-labeled methazole- · C most effectively in g. hirsutum. The erosion and the etching of the leaf surfaces due to the surfactant were not observed but micro- shrinkage appeared on both sides of the leaf treated with 1.0% (w/w) 55

polysorbate surfactant solution {Figure III-3(1) through 3(4)]. T):ie

micro-shrinkage appeared also on the leaf surfaces treated with O.l%

(w/w)·polysorbate surfactant solution [Figure III-3(5) and 3(6)).

These SEMinicrographs suggest that the leaves pecame slightly water-

stressed, but it was not so severe as seen in Figure III~l(7) and 1(8).

Figure III-4(1) through 4(8) illustrates· the results obtained.

by the treatments with polysorbate surfac.tant (HLB 12.0) solutions.

The SEM micrographs of the leaf 3-.h after treatment with l .0% (w/w) . . . polysorbate surfactant (HLB 12.0) solution [Fig1,1re III-4(1) and 4(2)]

showed a network of cracked cuticle which covered the epidermal cells.

The stomata of the leaf 72-h after treatment with the same solution ·

had not closed at this stage, but other epidermal cells shrank and the

surface became wrinkled [Figure III-4(3) and 4(4)] .: Although this

surfactant is water soluble, these observations showed that it was .

·nevertheless able to influence the' surface waxes of the leaf of .Q.

hirsutum.. The etched epidermal (!Uticl~ was obs.erved in the adaxial

surface of the leaf .treated with 0 .1 % polysorbate surfactant (HLB 12. 0) . . solution 3-h after treatment [Figure III-4(S)] and the surface then

became wrinkled 72-h aft~r treatment [Figure III-4(7)). At both con-

centrations of the polysorbate_ surfac:tant solutions, epidermal cells

adjacent to the guard cells began to .shrink first and then other·

epidermal cells became wrinkled. These observations suggest that t.he

epidermal cells. close to. the guard cells' become water-stressed faster .

than other parts of ·the leaf.

Figure III-5(1) through 5(8) illustrates the results of the . . treatment with Tween 80 (HLB 15.0) solutions. Both sides of the 56 leaf treated with 1.0% (w/w) Tween 80 solution [Figure III-5(3) and

5(4)] revealed the cracked surface of the lea:ves wh.ich resembled the leaf surfaces treated with 1.0% polysorbate surfactant (HLB 12.0) solution [Figure III-4(1) and 4(2)]. The reticulated pattern may have been water soluble surfactants themselves, which spread over the leaf, dried, and then cracked because of low affinity for the leaf waxes. The study with 14c-labeled metha:zole support this hypothesis; the uptake and transloi;:ation of 14c were less affected by more hydrophilic polysorbate surfactants than by more lipophilic surfactants.

The adaxial surface of the leaf treated with 0.1% (w/w) Tween 80 solution showed wrinkled epidermal cells. The abaxial surface of the same leaf showed an etched surface. But the epidermal cells recovered to the smoother appearance within 72-h after treatment [Figure III•5(7) and 5(8)] which were similar to the control [Figure III-1(5) and

1 (6)]. This observation indicates· that 0 .1% polysorbate surfact.ant

(HLB 15.0) solution did not significantly influence the superficial conformation of waxes on G~ hirsutl,lm leaves.

The SEM micrographs of the leaf surfaces of S. spinosa are shpwn

in Figures III-6 and III-7. This leaf appears smooth to the naked

eye. but numerous trichomes were observed on both sides of the leaf

by SEM. These trichomes have a narrow, needle-like upper part and

some of them showed the branched, multicellular hairs which were

stellate [Figure III-6(2), 6(6), and 6(8)]. Figure III-6(1) and

6(3) show the trichome arising from the leaf surface. The cutic;le

covers the trichome and leaf surface continuously, and it is difficult 57 to explain from SEM micrographs above how the trichome. arises from ordinary epidermal cells (6).

Figure III-6(1) and 6(2) illustrate the untreated, leaf surface of .§. spinosa. Both sides of the leaf showed fibrous cuticle covering the epidermal cells.

Figure III-6(3) and 6(4) are the SEM micrographs of leaf surfaces treated with 5% (w/w) ethanol solutie>n which was used as a solvent for polysorbate surfactants. These micrographs were similar to the untreated leaf [Figure III-6(1) and 6(2)) suggesting that 5% ethanol solution did not significantly influence the cuticle of the leaf.

When the leaf of _§. spinosa was dipped in 100% ethanol, it curled and browned in 72-h after treatment and the SEM roicrographs

[Figure III-6(5) through 6(8)] showed that the leaves were dehydra.ted severely as seen in the treatment of G. hirsutum [Figure III-1(7) and·

1(8)), but ethanol did not influence the trichomes. It is suggested that ethanol solubilized and removed some of the waxes from the leaf surfaces of _§. spinosa.

Figure III-7(1) through 7(8) illustrates the leaf surfaces of

S. spinosa treated with 1.0% (w/w) polysorbate surfactant solutions.

About 3-h after treatment with Span 80 (HLB 4.3) solution, numerous small brown spots appeared on both sides of the leaf of S, spinosa and then turned black within 72-h. SEM micrographs to follow were taken over these discolored regions of the leaf. Figure III-

7(1) and 7(2) show that Span 80 was deposited on the leaf surfaces like oil drops. The abaxial surface became slightly water-stressed 58

72-h after treatment but the trichomes were not influenced. These

SEM micrographs suggest that Span 80 does not have a special affinity

for the leaf waxes.of_§.. spinosa and, therefore, the leaf wax com-

position of _§.. spinosa is different .from that of G. hirsutum.

Figure III-7(3) and 7(4) illustrate the leaf surfaces 3-h after

treatment with polysorbate surfactant (HLB 8.0) solution. Our study

(See Section II) showed that polysorbate surfactant (HLB 8.0) enhanced

the translocation·of heterocyclic ring-labeled methazole-14c most

effectively in _§.. spinosa as well as G. hirsutum and the SEM micro-

graphs revealed .that this leaf was most dehydrated among the leaves

treatedwith polysorbate surfactant.solutions having different HLB

values. These observations suggest that the'surfactant (HLB 8.0)

influenced the surface waxes of the leaf most effectively and enhanced

the penetration and translocation.of methazole-14c in_§.. spinosa.

Figure III.;..7(5) through 7(8) are the SEM micrographs of the leaf

· of E_. spinosa treated w~th water soluble polysorbate surfactant

solutions. No remarkable changes in leaf cuticle were observed.

Trichomes were apparently not affected by these surfactants.

The SEM micrographs of the leaf surfaces of G. hirsutum treated

with a wettable powder suspension of methazole are shown in .Figure

III-8(1) and 8(2); micrographs of·comparably treated_§.. spinosa leaves·

are shown in Figure III-8(5) and 8(6). Wettable powder deposited on

the leaf surfaces of both plant species were readily observed under

. the microscope. The epidermal cells of G. hirsutum were beginning

to shrink and the surface became. wrinkled at both sides of the ·leaf.

The adaxial surface of G. hirsutum [Figure III-8(1)] resembled the 59

leaf treated with 0 .1% Tween 80 solution [Figure. III-5(5)] but the

leaf surfaces Of ~·. Spinosa Were not .influenced noticeably Wit.h the

same suspension. Crys.tall ized methazole observed on the leaf surfaces

of both plant species treated with 0.8% (w/w) methazole ethanol solu-·

tion [Figure III-8(3), 8(4), and 8(8)]. Figures III-1(7). ~nd 1(8)

. ' illustrated the severely dehydrated leaf surfaces of .Q. hirsutum

. treated with ethanol but the dehydration of the leaf treated with

0. 8% (w/w) methazole ethanol solution wa:s not so severe [Figure IIi-

8(3) and 8(4)]. The lea:f surfaces of ~· spinosa were n:ot affected

with the same. solution. The amount of chemicals deposited on the

leaf of S. spinosa was greater than that of G. hirsutum. 60

1. AMELUNXEN, F., K. MORGENROTH, und T. PICKSAK. 1967. Unter:.. .·. . suchungen an der Epidermis mit dem Stereoscan~Elektronenmikroskop. · ··· z. Pflanzenphysiol. 57 :79-95. ·

2. CRAFTS~ A. S. 1961.. 'l'ranslocation in plants. ·Holt, Rinehart and Winston, Inc., New York~ 182'p.

3. CRAFTS, A. s., and C. L. FOY~ 1962. ·The chemical and physical. nature of plant surfaces in relation to the use of pesticides. and to their residues. Residue Rev. 1:112.. 139.

4. DARLINGTON, .w.· A., and J. B. BARRY. 1965. Effects of chloroform . . and surfactants on perineability of apricot leaf. cuticle~ J. Ag·r •· Food Chem. 14:76-78.

5. .EBELING, W. 1963. Analysis of the .basic processes involved in the deposition, degradation, persistence; and· effectiveness of pesticides. Residue Rev. 3:35-163.

6. · FAHN, A. 1967. Plan.t anatomy. Pergamon Press, Oxford. p. 154. ·

7. FOY, c. L., and L. W. SMITH. 1965. Surface tension lowering wettability of paraffin and corn leaf surfaces, and herbicidal enhancement of dalapon by several surfactants.· Weeds 13:],.5-19.

8. FOY, C. L., and L. W•. SMITH. 1969. The role of surfactants in modifying the activity of herbicidal sprays. Pesticidal formulations research. Physical .and colloidal chemical aspects •. Adv. Chem. Series 86:55-6_9.

9. FURMIDGE, C. G. L. 1959 •. Physicochemical studies ori agricultural sprays. Part 1, 2, 3. J. ~ci. Food Agr. 10:267-282, 419-425.

10. HALL, D. M., A. I. MATUS, J . .A. LAMBERTON, and H. N. BAR.BER. 1965. Infra-specific variation in wax on leaf surfaces. Aust. J. Biol. ·Sii; 18:323-332.

11 •. HOAGLAND, D. R.' and i:>. I. ARNON. .1950. 'l'he·water-culture method for growing plants without soil. California Agr. Exp. Sta., Berkeley~ Circ. 347. 32 p.

12. HULL, H. !1. 1970. Leaf structure as related to absorption of ·.pesticides and other compounds. Residue Rev. 31:1... 150.

13. LINKENS, H. F., W. HEINEN, and A. L. STOFFERS. .1965 .. Cuticu.U of leaves and the residue problem,. Residue Rev. 8:136-178.

. . . . · 14. MARTIN, J. T., and B. E. JUNIPER. 1970. T.he cuticles of plants. St. Martin's Press, New York. 347 p. 61

15. ONG, B. Y., R. H. FALK, and D. E. BAYER. 1973. Scanning electron microscope observations.of herbicide dispersal using cathodoluminescence as the detection mode. Plant Physiol. 51: 415-420. . .. 16. SCHONHERR, J. and M. J, BUKOVAC. 1972. Penetration of stomata by liquids. Plant Physiol. 49: 813.;.819.

17. SILVA FERNANDES, A. M. S, 1965. VIII. Surface waxes in relation to water-repellency, Studies on plant cuticle. Ann. Appl. Biol.. 56:297-306.

18. SMITH, L. W., and C. L. ·FOY. 1966, Penetration and distribution studies. in bean, cotton, and b~rley from foliar and root appli- .· cation of Tween 20-14c, fatty acid and,oxyethylene labeled. J. Agr. Food Chem. 14:117-122.

19. STILL, G. G., D. G. DAVIS, and G. L. ZANDER. 1970. Plant epicuticular lipids. Plant Physiol. 46: 307-314.

20. TROUGHTON, J., anc:l L.A. DONALDSON. 1972. Probing plant struc.,.. ture. McGraw-Hill Book Co., New York. p. 26. ·

21. WORTMAN, G. B. 1965. Elektronenmikroskopische Untersuchengen der Blattoberflache und deren Veranderungen durch Pflanzenscutzmittel. z. Pflanzenkr. Pflanzenpathol. Pflanzenschutzber. 72:641-670. 62

·Figure III-1. SEM micrographs of .Q. hirsutum leaf surfaces: (1)

Adaxial surface of untreated leaf; (2) Abaxial surface of the same

lea~; (3) Adaxial surface of leaf 3-h after dipping in 5% (w/w)

ethanol-aqueous solution; (4) Abaxial surface of the same leaf;

(5) Adaxial surface of leaf 72-h after dipping in 5% (w/w) ethanol ..

aqueous solution; (6) Abaxial surface of the same leaf; (7) Adaxial

surface of leaf 3.,h after dipping. in ethanol; (8) Abaxial surface

of the same leaf. Scale represents 10 µ. 63 64

Figure III-2. SEM micrographs of Q. hirsutum leaf surfaces treated

with polysorbate surfactant (HLB 4.3) solution: (1) Adaxial surface

of leaf 3-h after dipping in 1% (w/w) surfactant solution; (2)

Abaxial surface of the same leaf; (3) Adaxial surface of leaf 72-h ·

after dipping in 1% (w/w) surfactant solution; (4) Abaxial surface

of the same leaf; (5) Adaxial surface of leaf 3-h after dipping in

0.1% (w/w) surfactant solution; (6) Abaxial surface of the same

leaf; (7) Adaxial surface of leaf 72-h after dipping in 0.1% (w/w)

surfactant solution; (8) Abaxial surface of the same leaf. Scale

represents 10 p. 65 66

Figure III-3. SEM micrographs of Q. hirsutum leaf surfaces treated

with polysorbate surfactant (HLB 8.0) s.olut·ion: (1) Adaxial surface.

of l~af 3-h after dipping in 1% (w/w) surfactant solution; (2)

Abaxial surface of the same le~f; (3) Adaxial surface. of leaf 72-h

after dipping. in 1% (w/w) . surfactant. solution; (4) Abaxial. surface

of the same leaf;_ (5) Adaxial surface· of leaf 3-h after dipping in

0 .1 %. (w/w) surfactant solution; (6) Abaxial surface of the same

leaf; (7) Adaxial surface of leaf 72-h after dipping in 0.1% (w/w)

surfactant solution; (8) Abaxial surface of the same leaf. .Scale

repr~sents 10 p. 67 68

Figure III-4. SEM micrographs of Q. hirsutum leaf surfaces treated

with polysorbate surfactant (HLB 12.0) solution: (1) Adaxial surface

of leaf 3-h after dipping in 1% (w/w) surfactant solution; (2)

Abaxial surface of the same leaf; (3) Adaxial surface of leaf 72-h

after dipping in 1% (w/w) surfactant solution; . (4) Abaxial surface

of the same leaf; (5) Adaxial surface of leaf 3-h after dipping in

0.1% (w/w) surfactant solution; (6) Abaxial surface of the same

leaf; (7) Adaxial surface of leaf 72-h after dipping in 0.1% (w/w)

surfactant solution; (8) Abaxial surface of the same leaf. Scale

represents 10 p. 69 70

Figure III-5. SEM micrographs of Q. hirsutum leaf surfaces treated

with polysorbate surfactant (HLB 15.0) solution: (l) Adaxial surface

of leaf 3-h after dipping in 1% (w/w) surfactant solution; (2)

Abaxial surface of the same leaf; (3) Adaxial surface of leaf 72-h

after dipping in 1% (w/w) surfactant solution; (4) Abaxial surface

of the same le.;tf; (5) Adaxial surface of leaf 3-h after dipping in

0.1%. (w/w) surfactant solution; (6) Abaxial surface of the same

leaf; (7) Adaxial surface of leaf 72-h after dipping in 0.1% (w/w)

surfactant solution; (8) Abaxia'.l surface df the same leaf. Scale

represents 10 p. 71 72

Figure III-6. SEM micrographs of ~· spinosa leaf surfaces: (1)

Adaxial surface of untreated leaf; (2) Abaxial surface of the same .

leaf; (3) Adaxial surface of leaf 3-h after dipping in 5% (w/w)

ethanol-aqueous solution; (4) Abaxial surface of the same leaf;

(5) Adaxial surface of leaf 3-h after dipping in ethanol; ( 6)

Abaxial surface of the same leaf; (7) Adaxial surface of leaf 72-h

after dipping in ethanol; (8) Abaxial surface of the same leaf.

Scale represents 10 p. 73 74

Figure III-7. SEM micrographs of~· spinosa leaf surfaces tre~ted

with 1% (w/w) polysorbate surfactant solution: (1) Abaxial surface

of leaf 3-h after dipping in surfactant (HLB 4. 3) solution; (2)

Adaxial surface of leaf 72-h after dipping in the same solution;

(3). Adaxial surface of leaf 3-h after dipping in surfactant (HLB

8.0) solution; (4) Abaxial surface of the same leaf; (5) Adaxial.

surface of leaf 3-h after dipping in surfactant (HLB 12.0) solution,;

(6) Adaxial surface of leaf 72-hafter dipping in the same solution;

(7) Adaxial surface of· 1eaf 72~h after dipping in surfactant (HLB

15.0) solution; (8) Abaxial surface of the same leaf. Scale

. repre~ents 10 µ~

I I 75 76

Figure IH-8. SEM micrographs of.leaf surfaces treated with herbi;.. . . cide: (1) Adaxial surface of Q. hirsutum leaf 3-h after dipping in

1.1% (w/w) me.thazole wettable powder suspension; (2) Adaxial surface

of G. hirsutum leaf 72-h after dipping in the saine suspension; (3) . - . . . : ' Adaxial surface of.Q. hirsutum leaf .72-h after dipping in 0.8.% (w/w)

methazole ethanol solution; (4) Abaxial surface of the same leaf;

· (5) Adaxial surface of E.~ spinosa leaf 72-h after dipping in 1.1%

(w/w) methazole wettable powder suspension; (6) Abaxial. swtface of .

the same leaf; (7) Adaxial surface of E_. Spinosa leaf 3-h after . .· . ' .. dipping in O_. 8% (w/w) methazole ethanol solution; (8) Adaxial

surface of E_. · spinosa leaf 72-h after dipping in the same solution.

Scale represents 10 p. 77 IV~ EFFECT OF HLB OF POLYSORBATE SURFACTANTS ON LEACHING

OF METHAZOLE-c 1 ~ IN SOIL

Numerous herbicides are currently recommended and used.for weed

eoi:itrol in agricultural, industrial and recreational areas. It is·.

not unexpected that chemical.additives (primarily surfactants)

applied to plant or .so;i.l before, with or after herbicide. treatment.

can modify adsorpt.ion., absorption, mobility, leaching, diffusion,

. . accumulation atid metabolism of the compound in such a way as to

increase or decrease herbicide residues. However, relatively little

information is available concerning the modifying effects of chemical

adjuvants on the distribution, availability, and. persistence of

herbicides. in soil (2, 4., 8).

·Methazole [2-(3,4-dichlorophenyl)-4-methyl-l,2,4-oxadiazolidine-

3,5-dione] has shown.promising herbicidal activityagainst.prickly

sida (Sida Spinosa L.), as well as many Other broadleaf species, ;i.n

cotton (Gossypium hirsutum·L.) and .several other important crops at

relatively low rates of appl;i.cation (1, 3, 9). The absorpt:ion, trans-.

location ap.d metabolic :l;ate of methazole in cotton have been reported.

by Jones and Foy (5, 6). This compound is suggested for use as a

pre- and post-emergence herbicide; i.t is presently formulated as , ' • . . I either a 75% wettable powder or a 5% granular material. It is reported

that .when the methazole is applied to the. soil surface, it remains

.predominantly in. the top layers of sandy and clay loam soils· (9).

But the movement of the formulated methazole containing some

surfactants in soil is not known.

. , 79

The hydrophilic-lipophilic balance (HLB) of nonionic .surfactants represents.a fundamental property of the surfactant and may g:t'~~tly affect their performance. Experiments were conducted to evaluate the influence of HLB of polysorbate surfactants on the depth of leaching of methazole-c14 in Landisburg-Greendale silt loam soil.

Materials and Methods

The methazole-cl4 used was heterocyclic ring-labeled in.the number 3 carbon {specific activity, 7.66p.c/r.OM). A stock solution of

· methazole-c14 was prepared by dissolving 100 pc of methazole-c14 . in

10 ml acetone. Var.ious treatment solutions were made by dilution of

the stock solution with rtonradiolabeled methazole, surfactant and acetone.

Plastic straws 6 nnn in diameter and 21 cin long were used as soil

columns. The bottom of each column was packed with a small amount of

glass wool. The columns were filled as uniformly as possible with

4.5 g of Landisburg-Greendale silt loam soil to within 3 cm of the top.

The surfactants used were Span 80 (nonionic surfactant containing

sorbitan monooleate, HLB 4.3) and Tween 80 (nonionic surfactant con-

taining polyoxyethylene sorbitan monodleate, HLB 15.0) manufactured

by Atlas Chemicals Division, ICI-America Inc., Wilmington, DE 19899.

The surfactants were mixed 65 parts of Span 80 and 35 parts of Tween

80 to make a surfactant with HLB 8.0; also, 28 parts of Span 80 and

72 parts of Tween 80 to make a surfactant with HLB 12.0. 80

Landisburg-Greendale silt loam soil (organic matter 1.41%) was

dried in a 70°C oven for 10 days and sieved to pass a 40...:.mesh screen

but not pass a 100-mesh screen. 2 .· Methazole (0.22 g/m ) and surfactants at three concentrations

were applied in 20pl acetop.e solution to the surface of the soil, so

that there was.1/4, 1, and 4 times as much surfactant as methazole

present on a weight basis. Each 20-pl treatment solution contained

·1. 9 pg (O .1 µc) of heterocyclic ring-labeled methazole~cl4 and 3 .1 pg

of nonradiolabeled methazole.

A total of 10 ml of distilled water was used to leach the

herbicide into the soil 30 minutes after treatment, when the treated

soil seemed dried. Water was dropped continuously onto the soil

surface to maintain a water level of at least 5 nnn depth on the

surface. After all water on the soil surface had disappeared into

the soil, the soil columns were kept at the same position for 4 hours

and then placed at the horizontal position in a 70°c oven for 24

hours. The straws were then sectioned in 1-cm lengths from the top

of the soil; placed in.polyethylene scintillation vials manufactured

by Hruden Laboratory Products and dried for 2 days in a 70°C oven.

The straw shells were removed and discarded, leaving the soil in the

vials, to which was added Aquasol Universal L.S.C. Cocktail manufac-

tured by New England Nuclear. The vials ~ere then shaken for 1 hour,

allowed to settle in refrigerator over night,. and counted using. a ·3000 series Packard Tri-Carb Liquid Scintillation Spectrometer

manufactured by Packard Instrument Company. The treatments were

replicated three times. 81

Results and Discuss icin

Th~ purpose of .this experiment was to compare the· movement of

heterocyclic ring-labeled methazole-c14 when polysorbate surfactant.s

with different HLB were used with the herbkide rather than how the

. herbicide w.ould move under field conditions.

The degree to which soil adsorbed methazole-c14 when polysorbate

surfactant was present, is shown in Figures IV-1, IV-2, IV-3, and

IV-4. The influence of polysorbate surfactant was observed in each

of the soil columns sectioned at relatively shallow deptJ;i.

In preliminary experiments without using p()lysorbat~ .surfactants,

Landisburg-Greendale silt.loam.soil sieved to pass a 40:..meshscreen

and also.a 100-mesh screen were examined. However, ln both soils

more than 90% of the applied radiolabeled material was fo.und within

1 to 2 cm of the surface of the soil regardless.of the amount of

leaching water and. the leaching t_ime.; approximately 4 inl of· water

took 24.hours for leaching 0.1 )lC of heterocycliC ring-labeled

methazole-c14 into a 17 cm soil columii. Therefore, soil was s·ieved

to pass a 40-mesh screen but not to. pass a 100-mesh screen. By using

this soil,, 7 .6 to 8.0 ml of leached water was recovered out of .10 ml

distilled water applied; it t.ook 2 to 2 1/2 hours for the water applied,

on .the top of the soil surface to be absorbed into the soil.

All.treatments showed the greatest amount of residue of hetero.,. 14 cyclic ring-labeled methazole-C in the ·s·oil within the firs.t l cm

depth. The amount of the residue decreased· in proportion to the depth 82

ADSORPTION OF f4c, ~ 10 20 30 40

/ // I I 1// / I / 1 I I I I I I I I

-----\ \ \ \ \ \ \\ \ 2

2 4 I 1111111 /1 1111 Ill/I, u ,\\\\ \\\ \\ \ \ \ \\\\ z. 2 5 ::> _J 0 u 6 _J 0 (/) 7 HLB 4.3

~ 0 CJ NO SURFACTANT 8 x VllJ.a I 14 SURFACTANT/HERBICIDE ~ a.. - I/ I SURFACTANT/HERBICIDE ~ 9 0 ~ 411 SURFACTANT/HERBICIDE

LEACHED WATER

Figure IV-1. Influence of the amount of Span 80 (HLB 4.3) on leaching 14 depth of heterocyclic ring-labeled methazole-c , expressed as 14 percent of adsorption methazole-C on Landisburg-Greendale silt

loam soil. 83

1 ADSORPTION OF 4c. % 10 20 30 40

'/////////////////////II/ //I'/// // //// ////// ·'''''''' ' ''' ,,,, ' ,, ,, ,, ''''''" 2 // ////////// / //'/ ////////h/1////1// ,,,,,,,,,,,,,,,,, ,,,,,,,,, ''"'''' 3

'I I/Ill/// /1///111//////' 4 ~ (.) •'"'\\'·''"''" '''''"''" z ~ :::> ....J 0 (.) ....J 0 H LB 8.0 V> u.. CJ NO SURFACTANT 0 IV/IA 114 SURFAClANTI HERB ICI DE I ~ Cl. - I/ I SURFACTANT/ HERB ICIDE LAJ 0 ~ 411 SURFACTAN T/ HERB ICIDE

Figure IV-2. Influence of the amount of 65/35 (w/w) Span 80/Tween 80

mixture (HLB 8.0) on leaching depth of heterocyclic ring-labeled 14 14 methazole-C , expressed as percent of adsorption of methazole-C

on Landisburg-Greendale silt loam soil. 84

ADSORPSION OF 14c, % 10 20 30 40

~///////////////////////////////////// // //////////. ,,,, '' ,, ' '""'' ,,,, ,, ,,,,,,,,\\\\,\\\\' 2

3 '/11/////// ////////////////////////I ~'''"'''"''''''''""'""\\' '. "/////////////////////,, ~ 4 0 . ·''''''''''''''''"'""' z ~ 5 :::> ...J 0 0 6 ...J HLB 12.0 0 CJ) 7 t:::l NO SURFACTANT ~ 0 8 FIA I /4 SURFACTANT /HERB IC I DE ~ - I/ I Sl.RFACTANT/HERBIC IDE ....Cl. w ~ 4/1 SURFACTANT/HERBICIDE 0 9

WATER ------~--~~~--~~~~~~~--

Figure IV-3. Influence of the amount of 28/72 (w/w) Span 80/Tween 80

mixture (HLB 12.0) on leaching depth of heterocyclic ring-labeled 14 14 methazole-C , expressed as percent of adsorption of methazole-C

on Landisburg-Greendale silt loam soil. 85

14 AOSORPT I ON 0 F c. % 10 20 30 40

"///////////////////////////////////////'//////////////////// "''''''''''''''''''''''''''''''''' ,,,,,,,,. 2 ////ll//llll//lll/l/ll/////////l///////1/////// '''' ,,,_ -, ,, ,,,,,,,,,,,,,,,,,,,,,, 3 '/II //1I11IIII/I/111II111111II/I11' ~--..;:, '''''''''''''"''''''''''''''''' ~ 'llll//11//////////////h u 4 . ~''''''''''''''''"'''''''' z ~ 5 ::::> _J 0 u _J 0 HLB 15. 0 CJ) LL. D NO SURFACTANT 0 f7llA I 14 SURFACTANT /HERBICIDE I..... a.. - 111 SURFACTANT/HERBIC IDE w 0 ~ 41 I SURFACTANT I HERBIC IDE

WATER

Figure IV-4. Influence of the amount of Tween 80 (HLB 15.0) on 14 leaching depth of heterocyclic ring-labeled methazole-c , expressed 14 as percent of adsorption of methazole-c on Landisburg-Greendale

silt loam soil. 86 in the soil column. These results .indicate that the herbicide remain,s predominantly in the top layers of the soil.

When methazole was applied with 1/4 times as much surfactant, approximately 85% of radioactivity out of the total dosage was determined in the soil within 4 cm of the surface of the soil regardless of HLB of polysorbate surfactant used. The degree of the adsorption of radiolabeled material in the same depth of soil decreased slightly with the increase of surfactant, while the radio- ' activity of the treatment with the methazole-c14 without. surfactant was approximately 76% in the same depth of soil column. However; approximately 96% or more of radioactive material was determined in·

the soil within 7 cm in depth at all treatments; at this level, not only HLB of polysorbate surfactant but also polysorbate surfactant

itself did not show any influence on the leaching of heterocyclic

ring-labeled methazole-c14

When the ratio of polysorbate surfactant and the herbicide was

the same, the influence of HLB of polysorbate surfactant was minimal.

The results of the study on soil adsorption of substituted urea

herbicides as influenced by surfactants show that nonionic and.anionic

surfactants have either no effect or decrease adsorption of these

herbicides (2, 8). Bayer (2) reported that Tween 20 (nonionic

surfactant containing polyoxyethylene sorbitan monolaurate, HLB

16.7) increased the leaching depth of d{uron [3-(3,4-dichlorophenyl)-

1,1-dimethylurea] by increasing the concentration of the surfactant. 87

. . 14 Our results suggest that adsorption of methazole-C onto the

· soil surface was different· from that of diuron. The reason why

relatively low amounts of polysorbate surfactant restrict the movement of the herbicide more than greater amounts of the surfactant is

not explained.

Koren..!:.:!: al. (7) reported that the movement of the thiocarbainate

herbicides was directly related to the water solubility of the herbi-

cide and inversely related to the organic matter content of the soil.

Our experimental results confirm this concept; water solubility of 0 . methazole is 1.5 ppm at 25 C and it was adsorbed on the soil in the

top layers of the column. Tween 80 (HLB 15,0) is a water soluble

. . surfactant and used as a solubilizer in numerous industrial fields.

However, even when methazole was applied with 4 times as much Tween

80, significant difference in adsorption of c14 was not observed in

comparison with the addition of same amount of lower water soluble

polysorbate surfactant. 88·

References

1. Ballard, J., Greer, H. A. L~ and Santelmann,. P. w.· 1972. .Pre- and post:""emergence activity of VCS-438 herbicide. Proc. S. Weed Sci. 25:167-174.

2. Bayer, D. E, 1967. Effect of surfactants of le?ching of substituted urea herbicides in soiL W~eds 15:249-252.

3. Connel, J., Jeffery, L. S., McCutchen, T. C. and Overton, J. R. 1971. Control of prickly sida in cotton. Proc •. S .. Weed Sci. Soc, Abs tr.· 24: 107 ..

4. Howarth, R. · 1962.· Grower finds advantages in wetting agent. Southern Florist and-Nurseryman 74:12-13.

_5,. Jones, D. W. and Foy, C. L. 1972. Absorption and trans location of bi_oxone: in cotton. Weed Sci. 20: ll6-120.

6. Jones, D•. W. and Foy, c. L. 1972. Metabolic fate of bioxone in cotton. Pest~ Biochem. Physiol. 2!8-26,

7 ~ Koren, E., ·Foy, C. L, and Ashton, F. M. 1969; Adsorption, _ volatility, and migration of thiocarbamate herbicides in s.oil. · Weed Sci~ 17:148~153.

8. Smith, L •. W. and Bayer, D. E. 1966. Soil adsorption of diuron ·as influenced by surfactants. Soil Sci. 103: 328-330.

9. Velsicol Chemical Corporation. · 1971. Prove (VCS-438) herbicide development bulletin.· _Velsicol Chemical Corporation. p. 8.

I I I I V•. SUMMARY

The primary objectives of these studies were to investigate the

influence· of lil..B of polysorbate surfactants on the mobility of 14c-

labeled methazole.in cotton, priCkly sida, and soil, and also. to

evaluate the effect of polysorbate surfactants and methazole on the

epicuticular wax of cotton and prickly sida by scanning electron· microscopy. 14 . ' 14 Methazole- · C and/or its ·. C":'labe.led metabol'ites moved aero-

petally in the treated leaves of both species following foliar appli~

cation. No basipetal movement was detected. The translocation

pattern was characteri$tic of compounds which move only in the

apoplast. The mobility pattern was n:ot alte'ted with polysorbate

surfactants having different lil..B values in either cotton or prickly .

sida. 14 The accumulation of C in the veins and tissue closely asso- .

ciated with the veins was rapid, and permeation of interveinal tissue

wit; h 14c·. i ncrease d wit· h t i me. Tota ·1 · upta k e an. d · d i.stri.· · · b uti.on· · o f 14c

· increased with increasing concentratio·n of methanol in the treatment

·solutions and also decreasing HLB vaJues of p_olysotbate surfactants•

More 14c was trarislocated in cotton than in prickly sida.

The influence of methanol concentration without surfactant on the

absorption and tra:nslocation of 14c was qualitatively similar but

quantitatively different in the two species. With 100% methanol solu- 14 tion at 72 hr after treatment, C spread fi:om the vein of the treated

leaf into the closely associated tissue, whereas, at all other methan,ol •.

89 90

concentrations from 40 through 82.5% (v/v), 14c remained alniost

entirely in the veins.

The effects of polysorbate surfactants. were masked when 100% methanol was used in both species.

No distribution of 14c.,...fatty acid-labeled polysorbate surfactants wer.e observed by· use of autoradiography with any .methanol concentra-

tion, andHLB of surfactants, or at any treatment.time in both species.

The influence of HLB of polysorbate surfactant was determined

by subtracting the values for the distribution of 14c with methanol

and/or distilled water solution from those for the distribution of

14c with polysorbate E"urfactant, methanol and/or distilled water.

The surfactant having an HLif value of 8 caused greatest enhancement

of translocation of heterocyclic ring-labeled methazole-14c and/or

its metabolites iriboth 30-day-old cotton and 45-day'."'"old prickly. sida·

leaves.

The possible influ~nce of polysorbate surfactants having differ-

ent HLB values, ethanol, methazole wettable powder suspension, and

methazole-methanol solution on the ultrastructure of the leaves of

cotton and prickly sida were investigated using a scanning· electron

microscope (SEM) •.

The cuticle in cotton covered the epidermal cells smoothly and

it was difficult to locate the boundary between the cells. Both

surfaces of cotton leaves treated with 100% ethanol were dehydrated·

and appeared fibrous. The ethanol appeared to solubilize and remove

or redistribute some of the waxes from the leaf surfaces. The SEM 91 micrographs showed blocky surfaces of both sides of the cotton leaf treated with 5% (w/w) ethanol solution which was used as a solvent for polysorbate surfactants.

The leaf waxes of cotton were severely eroded in 72 hr after treatment with 1% (w/w) polysorbate surfactant (HLB 4.3) solution and etched with 0.1% (w/w) polysorbate surfactant solution. The surfac.tant appeared to have an affinity for leaf waxes of cotton and to. solubilize them.

The cotton leaf became water-stressed with 0 .1 and 1.0% (w/w) polysorbate surfactant (HLB 8.0) solutions. The cotton leaves treated with water soluble polysorbate surfactant (HLB 12.0 and 15.0) revealed similar surfaces. Polysorbate surfactant solutions at 0.1 and 1.0%

(w/w) appeared to cause etching and cracking of the epicuticular wax.

The reticulate pattern observed on leaf surfaces.may have been

the water soluble surfactants themselves, which spread over the leaf, dried, and then cracked because of low affinity for the leaf waxes. . . 14 The study with C-labeled methazole supports this hypothesis; the ·. . 14 uptake and translocation of C were less affected by more hydro-

philic polysorbate surfactants than by more lipoph:i.lic surfactants.

Polysorbate surfactant (HLB 4.3). eroded cotton leaf surfaces severely.

The 1,1.ptake and translocation of 14c-labeled methazole was most affected

by the surfactant (HLB 8.0) when the methanol effect was subtracted 14 from the total uptake of C and by the surfactant (HLB 4.3) when

the methanol effect was disregarded. The SEM study seems to support

the latter approach. · 92

The SEM micrographs of the leaf of prickly sida appeared smooth

· . to the naked eye but numerous "trichomes were observed. The leaves

of prickly sida were dehydrated with 100% ethanol but less affected

than those of cotton by polysorbate surfactants.· Trichomes were not

influenced with these surfactants.

Crystalline deposits when technical methazole-ethanol solution

was appli,ed, amorphous deposits when wettable powder suspension was

applied, were observed on the leaves.

Polysorbate surfactants having different HLB values were examined

for their possible influence on the leaching of heterocyclic ring--

labeled 14c-methazole in Landisburg-Greendale silt loam soil. The

herbicide was applied with 0 (control), 25, 100, and 400% (w/w) as

much polysorbate surfactant onto the soil surface and leached with

approximately 35.7 cm distilled water. In all instances, the greatest

amount of 14c remained within the first 1 cm depth of soil; 14c

.decreased in proportion .to the depth in the soil column· for all

experiments. When the herbicide was applied with surfactant (1:4,

w/w, all HLB values), leaching of methazole was not affected.

However, ratio of 1:1 and 4:1 (herbicide and surfactant) caused slight

and significant retardation of movement, respectively. The influence

of HLB of polysorbate surfactants was minimal. The vita has been removed from the scanned document INFLUENCE OF HLB OF POLYSORBATE SURFACTANTS ON MOBILITY OF METHAZOLE IN PLANTS AND SOIL

Tsuneyuki Takeno

(ABSTRACT)

. ~- ~ . . - . Methazole;. C and/or its C-labeled metabolites moved aero-

petally in the trea_ted leaves. of· cotton (Gossypium hirsutum L. ,

. 'Deltapirie') and prj,ckly sida (Sida Spinosa L.). The mobility pattern

was not altered with_polysorbate surfactants with different IIl.B

(Hydrophilic-Lipophllic Balance) values. Total uptake and distribution of 14c increased.with increasing concentration.of methanol as a -14 . solvent and decreasing Ill.B.values of surfactants. More C was trans-

located in cotton than. in prickly sida. The effects of surfactants

wel:'e masked by the drastic solvent action of 100% methanol.· When the

solvent effect was subtracted, the surfactant with ·m.B 8 caused great-

est enh ancement o f . trans 1 ocation . o f 14·C . in b.ot. h species.

Scanning electron photomicrographs showed that poly~orbate

. ; . . ' . . si.lrfactant.(IIl.B 4.3) eroded cotton leaf surfaces severely at the ~%

(w/w). level. Reticulated and etch.ed patterns were observed on cotton

leaf surfaces treated.with water soluble surfactants. Trichomes on

the leaves of prickly sida appeared not to be affected. by the sur-

factan~~. The.leaves of prickly sida were less affected than those

.of cotton by the surfactants. S~rface deposits of formulated methazole

were different in appearance from those of technical methazole. · The influence of polysorbate surfactants on the leaching of methazole-14c in Landisburg-Greendale silt loam soil was examined.

When the herbicide was applied with surfactants (1:4, w/w, all IIl.B values), leaching of methazole was not affected. However, ratios of

1:1 and 4:1 (herbicide/surfactant) caused slight and significant retardation of movement, respectively. The influence of lll..B of polysorbate surfactants was minimal.