AC CE ENNI , Am DEL 80m: c 4». 1,? .. .w. A? H HHIHN u . , Ar. ATAAA LUREA NQSA H71§727H fArHA-‘Dé or mf . MICHIGAN: _ AA DAF m C HAN EN- ,..:..: It"... 5. . . .y 4 01.52! 5.2511)?! «4.5% .2, n.5,: , .. 2;. . t, V‘.’ N a: true"? ‘ ‘ .c, 3.1.1:; .32... .r...‘ ...~...‘>.£ , z .. ..t-\...A . .. 4.. 7.1. :, . .....c__.....,..._h..u.... ; , . . . , ‘ ....\.....v.x.,.i “.7 .\J¢f. ,lo. 3.34 v-xv w _ r w- ” .‘vrfii llHMU/lllfilflflfflflHHHIHII LIBRARY 2434 Mfihigan §flte ‘ mVCrSlty This is to certify that the thesis entitled ENHANCEMENT OF PHENYLUREA HERBICIDE ACTIVITY WITH CARBARYL AND DIAZINON presented by Dafrosa Arganosa Del Rosario has been accepted towards fulfillment of therequirements for Ph . D . degree in Horticulture / , / / ‘/ y// 1 ’1’." ”(142,411 l/ U" fi ' v ~ Major professor Date February 23, 1972 0-7639 MINES 1999 ABSTRACT ENHANCEMENT OF PHENYLUREA HERBICIDE ACTIVITY WITH CARBARYL AND DIAZINON By Dafrosa Arganosa Del Rosario Numerous herbicide-insecticide combinations were applied to the foliage of tolerant and susceptible plants to detect synergisms which might occur. More injury was obtained with a combination of the herbicide linuron (3- [3,H-dichlorophenyl] al-methyl, l-methoxyurea) and the insecticide carbaryl (naphthyl figmethylcarbamate) in tol- erant carrot (Daucus carota L. cv. Chantenay Long Type). Respiration of leaf tissue was not appreciably affected by either chemical, but photosynthetic rate in the presence of both chemicals was greatly reduced when compared to tissue receiving the herbicide or carbaryl 14 only. Carbaryl enhanced the rate of C~linuron uptake by carrot leaves and parsnip (Pastinaca sativa L. cv. Hollow Crown) leaf discs. The rate of linuron metabo- lism was also decreased in the presence of carbaryl. The foliar activity of diuron (3[3,4-dichloro- phenle-l, l-dimethylurea) and monuron (3[3-chloro- phenylj-l, l-dimethylurea) was also enhanced by carbaryl Dafrosa Arganosa Del Rosario in tolerant cotton when the insecticide was applied to the leaves or to the roots. Diazinon (Q) Q—diethyl‘Q-Z-isopropyl-6-methyl-4- pyrimidinyl phosphorothioate) enhanced the toxicity of the 4 analogous phenylureas, linuron, monolinuron (3 [3-chlorophenyl]—l-methyl, l-methoxyurea), diuron and monuron in carrots, cotton (Gossypium hirsutum L. cv. Polymaster) and corn (Z§£.E§X§ L. cv. Harris Gold Cup). The foliar uptake of the lLia-phenylurea herbicides was increased up to 5 fold in the presence of diazinon for- mulated as a wettable powder. Uptake was also consi- derably increased in the presence of pure diazinon. The synergism obtained with these chemicals was appar- ently caused by increased herbicide uptake induced both by diazinon and its carrier surfactant. Metabo- —lism of the 4 phenylureas in cotton leaves was unaf- fected by the presence of diazinon. The dimethylureas were more easily degraded by cotton than the methoxymethylureas and the monohalo- genated analogs were more rapidly metabolized than the dihalogenated analogs. The selectivity of phenylurea herbicides is related to both differential uptake and metabolism in different plant species. Selectivity can be partially lost if other pesticides are added which either enhance uptake or inhibit degradation of these herbicides. ENHANCEMENT OF PHENYLUREA HERBICIDE ACTIVITY WITH CARBARYL AND DIAZINON By Dafrosa Arganosa Del Rosario A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1972 ACKNOWLEDGMENTS The author expresses her sincere thanks to Dr. Alan R. Putnam for his guidance and assistance during the conduct of the work and preparation of the manus- cript. Appreciation is also expressed to members of my guidance committee, Dr. S. K. Ries, Dr. D. P. Penner, Dr. D. R. Dilley and Dr. M. J. Zabik, for their guidance during the course of study and helpful comments and suggestions on the preparation of the manuscript. The technical assistance of Martha van Buskirk and Paul Love is highly appreciated. Thanks is also extended to Gregory Pagano for taking the photographs. Special thanks is extended to Dr. S. K. Ries for the use of his laboratory equipment and guidance so essential for the completion of the work. Appreciation is also expressed to the E. I. Du Pont Company, ClBA-Geigy Company and American Hoechst Corporation for supplying the isotopes and other chemicals for this study. The moral support and patience of my family are gratefully acknowledged. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . vii LIST OF APPENDICES . . . . . . . . . . . . . . viii INTRODUCTION . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . 3 General Review of Research with Pesticide Combinations . . . 3 Evaluation of Pesticide Combinations. . 7 Substituted Urea Herbicides . . . . . . . . 9 Absorption and Movement . . . . . . . . 11 Metabolism . . . . . . . . . . . . . 12 Mechanism of Action . . . . . . . . . . l3 Carbaryl . . . . . . . . . . . . . . . . . 14 Diazinon . . . . . . . . . . . . . . . . . 16 MATERIALS AND METHODS . . . . . . . . . . . . . 18 Initial Screening of Pesticide Combinations for Enhanced Toxicity. . . . . . . . . . . 18 Toxicity of Phenylurea Herbicides and Herbicide—Insecticide Combinations . . . . 20 Effect of Carbaryl and Diazinon on Herbicide Activity . . . . 21 Effect on Respiration and Photosynthe- sis of Leaf Tissue . . . . . . . . . . 22 Efffict of Insecticides on the Uptake of 1 C-Phenylurea Herbicides . . . . . . . . 24 iii Page Application and Sampling . . . . . . . 24 Assay of Radioactivity . . . . . . . . 26 Effect of Insecticides on the Metabolism of lLAC-Phenylurea Herbicides . . . . . . 27 Application and Sampling . . . . . . . 27 Extraction and Analysis . . . . . . . 28 RESULTS AND DISCUSSION . . . . . . . . . . . 30 Initial Screening of Pesticide Combi- nations for Enhanced Toxicity. . . . . . 3O Toxicity of Phenylurea Herbicides and Herbicide—Insecticide Combinations . . 32 AEffect of Carbaryl and Diazinon on Herbicide Activity. . . . . 34 Effect on Respiration and Photo- synthesis of Leaf Tissue . . . . . 49 Effect of Car aryl and Diazinon on the Uptake of 1 C-Phenylurea Herbicides. . . 51 Effect of Carbaryfi and Diazinon on the Metabolism of C-Phenylurea Herbicides. 64 SUMMARY AND CONCLUSIONS . . . . . . . . . . . 7O Insecticide Induced Enhancement of Phenyl- urea Herbicide Activity. . . . . . . . . 70 Effect of Insecticides on the Uptake of Phenylurea Herbicides . . . . . . . . . 72 Effect of Insecticides on Metabolism of Phenylurea Herbicides. . . . . . . . . . 73 Implications for Field Situations. . . . . 74 LITERATURE CITED. . . . . . . . . . . . . . . 76 APPENDICES. . . . . . . . . . . . . . . . . . 83 iv Table 10 LIST OF TABLES Specific activity of the carbonyl- labeled phenylurea herbicides . . . . Concentration of phenylurea herbicides (mgfl) required to produce 50% fresh weight reduction in 4 species The effect of 4 phenylurea herbicides and combinations with insecticides on the growth of cotton. . . . . . . . The effect of 4 phenylurea herbicides and combinations with insecticides on the growth of carrots The effect of 4 phenylurea herbicides and combinations with insecticides on the growth of corn. The effect of 4 phenylurea herbicides and combinations with insecticides on the growth of soybean The effect of foliar application of insecticides on the toxicity of root- applied phenylurea herbicides on cotton. . . The effect of root—applied insecticides on the toxicity of foliar applied phenylurea herbicides on cotton The effect of both herbicides and in- secticides applied to the nutrient solution on the growth of cotton Summary of the interactions which occurred with insecticides and phenyl- urea herbicides in cotton Page 24 33 35 36 39 41 42 44 45 48 Table Page 11 Oxygen uptake of parsnip leaf discs treated with linuron alone or in combination with carbaryl (% of Control) . . . . . . . . . . . . 5O 12 Oxygen evolution of parsnip leaf discs treated with linuron alone or in com- bination with carbaryl (% of Control). . . 52 13 The uptake of lLHCnlinuron, monolinuron and diuron (cpm) as affected by pure diazinon on cotton leaves. . . . . . . . . 62 14 The uptake of 14C—linuron (cpm) as affected by carbaryl in carrot leaves and parsnip leaf discs . . . . . . . . . . 63 15 The effect of diazinon on the metabolism of linuron by cotton leaves . . . . . . . 64 16 The effect of diazinon on the metabolism of monolinuron by cotton leaves . . . . . 65 17 The effect of diazinon on the metabolism of diuron and monuron by cotton leaves 5 days after treatment . . . . . . . . . . 66 18 The effect of carbaryl on the metabolism of linuron by carrot leaves . . . . . . . 68 vi Figure LIST OF FIGURES Page Toxicity obtained with combinations of diazinon and the 4 phenylurea herbicides on (a) carrot (b) corn (c) cotton . . . . . . . . . . . . . . . 38 a) Toxicity obtained with combinations of carbaryl and the 4 phenylurea her- bicides applied on the roots of cotton. b) Toxicity obtained with foliar appli- cation of linuron and carbaryl on carrots . . . . . . . . . . . . . . . . 47 A comparison of uptake of 14C-linuron (wettable powder) with or without dia- zinon (wettable powder) on (a) cotton (b) soybean . . . . . . . . . . . . . . 54 A comparison of uptake of l)J'C-monoli- nuron (wettable powder) with or without diazinon (wettable powder) on (a) cotton (b) soybean. . . . . . . . . . . . . . . 56 A comparison of uptake of 14C-diuron (wettable powder) with or without dia- zinon (wettable powder) on (a) cotton (b) soybean . . . . . . . . . . . . . . 58 A comparison of uptake of lb’C-monuron (wettable powder) with or without dia- zinon (wettable powder) on (a) cotton (b) soybean . . . . . . . . . . . . . . 51 Vii Appendix A LIST OF APPENDICES Pesticide interaction from foliar application on carrots . . Pesticide interactions from foliar application on cucumber. . . . . Pesticide interactions from foliar application on soybean . The effect of 4 substituted urea her— bicides alone and in combination with insecticides on the growth of soybean. The effect of 4 substituted urea her- bicides alone and in combination with insecticides on the growth of corn The effect of 4 substituted urea her- bicides alone and in combination with insecticides on the growth of cotton viii Page 83 84 86 88 89 90 INTRODUCTION The application of pesticide combinations either together or at intervals is a common practice in modern crop production. There are many possible combinations of herbicides, insecticides and fungicides which may be uti- lized on a single crop. The activity of a combination of pesticides may be synergistic, antagonistic, additive, or similar as either material used independently. Interac- tions resulting from the use of combinations of pesticides may be potentially harmful or beneficial. Harmful inter- actions may result from increased toxicity toward desira- ble plant species or non-target species, increased resi- dual life in the soil, or inactivation of the pesticide. On the other hand, beneficial interactions may result from increased or prolonged toxicity toward undesirable plant species or enhanced degradation of pesticide residues. The use of combination treatments might make it possible to decrease the rates of pesticides needed. Identifica- tion of potentially harmful combinations will make it pos- sible to avoid them in commercial crop production. The objectives of this study were to determine the effect of common carbamate and organophosphate insecti- cides on the activity of 4 analogous phenylurea herbi- cides with varying halogen and alkyl substitution. The interactions were further investigated in an attempt to elucidate their bases, and to determine if they were char- acteristic of a family of compounds or specific for certain chemical structures. LITERATURE REVIEW General Review of Research with Pesticide Combinations Many of the reports regarding herbicide-insecticide interactions have resulted from studies with cotton (Gos- sypium hirsutum L.). The combination of diuron (3 [3,4- dichlorophenle—l, l-dimethylurea) and phorate (Q, 97 diethyl-Sf[(ethylthio)methyl]-phosphorodithioate)resulted in severe loss of cotton seedlings reported in Texas in 1962 (62). Synergistic phytotoxicity was also observed in oats (Avena sativa L.) using this same combination (55). Retarded seedling growth and reduced yield of cot- ton were also obtained by a combination of phorate with either monuron (3 [p—chlorophenyl]-l, l-dimethylurea) or diuron (32). In contrast, a combination of phorate or disulfoton (g, Q-diethyl—S: [2-(ethylthio)—ethyl]—phospho- rodithioate) with trifluralin (oc,ac,ac,trifluro-2, 6-dini- troefl,‘N-dipropyl-pftoluidine) resulted in higher cotton yield as compared to trifluralin alone (3,43). Hassaway and Hamilton (34) reported that soil application of tri— fluralin and phorate did not affect cotton seed germina- tion. However, in the presence of phorate, the triflu— ralin treated plants produced more secondary roots. Severe leaf burn and subsequent yield loss were evident in rice (Oryza sativa L.) after application of 'propanil (3, 4—dichloropropioanilide) and carbaryl 3 (l—napthyl meethylcarbamate) (8). Linuron (3- [3, 4- dichlorophenyl]-l-methoxy, l—methylurea) or chloramben methyl ester (methyl 3-amino-2, 5-dichlorobenzoate) ap- plied in combination with phorate or methomyl (methyl- IN[(methylcarbamoyl)oxy] thioacetimidate) produced sig- nificantly lower seedling vigor in soybean (Glycine max (L.) Merr.) than when each pesticide was used alone (45). Recent findings of Hamill (33) showed synergistic interaction of carbofuran (2, 2-dimethyl-2, 3-dihydro- benzofuranyl-7-N9methylcarbamate) with alachlor (2- chloro—2, 6—diethyl-N:methoxy-methyl acetanilide), bu- tylate (Seethyl diisobutylthiocarbamate) or chlorbrom- uron (3- [4—bromo-3-chlorophenyl]-l-methoxymethylurea) in barley (Hordeum vulgare L.). Combinations of herbicides are often utilized to broaden the control of annual monocotyledonous and dicotyledonous weeds in agronomic and horticultural crops. Herbicide combinations may also be synergistic on a single weed species. A mixture of simazine (2- chloro-4, 6-bis[ethylamino]-§-triazine) and paraquat (l, l'—dimethyl-4, 4'—bipyridinium ion) was more effect- ive in controlling quackgrass (Agropyron repens Beauv.) than either simazine or paraquat alone (63). Similar results have been obtained with a mixture of ammonium thiocyanate and amitrole (3-aminoas-triazole) on quack- grass (19). Enhanced effects of mixtures of 2, 4-D (2, 4-dichlorophenoxyacetic acid) and picloram (4—amino- 3,5,6-trichloropicolinic acid) on the control of several perennial weeds have also been reported (9). Enhanced injury to soybean was also reported with combinations of chloroxuron (3 [pg(pfchlorophenoxy) phenyl]—l, l-dime- thylurea) and nitralin (4fmethylsulfonyl]—2, 6-dinitro- N, N—dipropylaniline) (45). In some cases, antagonistic responses have been observed. Picloram plus bromacil (5-bromo-3-sgngutyl- 6-methy1uracil) appeared less effective in controlling grasses than either of the herbicides alone (72). Car- bamate and growth regulator herbicides were antagonistic both in whole plants and plant segments (44). When com- binations of chlorpropham (isopropyl mechlorocarbanilate and 2, 4—D were applied to the foliage of either pigweed (Amaranthus retroflexus L.) or smartweed (Polygonum lapathifolium L.) the severe twisting effects of 2, 4-D were greatly reduced. The induced elongation of soy- bean hypocotyl section by 2, 4-D, dicamba (3, 6-dichlo- ro-gfanisic acid), or picloram was inhibited in the presence of either chlorpropham or EPTC (Seethyl diproe pylthiocarbamate). Several mechanisms have been offerred to eXplain the nature of pesticide interactions. Three possible sites of pesticide interaction have been proposed by Nash (55): first, at the site of absorption where one pesticide affects the penetration of the other; second, within the plant where one pesticide affects a primary pathway and the other affects the secondary pathway and third, where both pesticides affect the same pathway. Davis and his co-workers (18) showed that paraquat in- creased the uptake of picloram but reduced its trans- port in some plant species. The uptake and transport of 2,4,5-T (2,4,5-trichlorophenoxy-acetic acid) in mesquite (Prosopis juliflora Swartz) decreased in the presence of picloram but the uptake and transport of picloram increased in the presence of 2,4,5-T. Agbakoba (2) found that picloram increased transloca- tion of 2, 4-D in field bindweed (Convolvulus arvensis L.) but the reverse was not demonstrated. Synergism with certain pesticide combinations may occur because the insecticide inhibits the meta- bolic processes in the plant that are active in the degradation of the herbicide. The tolerance of rice plants to propanil is correlated with the metabolism of the chemical by the presence of the propanil hydro— lyzing aryl acylamidase (51). The insecticide car— baryl inhibits the activity of this enzyme and when applied with propanil, selectivity is lost. Likewise Swanson and Swanson (70) showed that carbaryl inhibited the degradation of monuron by cotton plants. Other in_yitrg studies have since demonstrated that an enzyme called demethylase is inhibited by carbaryl (24). Demethylase was isolated from a number of plants and was believed responsible for the successive removal of the methyl group in substituted dimethylurea herbicides. Chang et_al, (13) recently reported inhibition of the metabolism of herbicides from several chemical groups by some organophosphate and carbamate insecticides. An explanation for the antagonism observed in one pesticide combination is an alteration in nucleic acid metabolism (56). 2, 4-D applied in combination with EPTC reversed the EPTC inhibition of growth and caused an increase in nucleic acid content. Analyses showed that the EPTC-2, 4-D combination caused an in— creased synthesis of ribOSOmal RNA and t-RNA over the levels observed with EPTC alone. Evaluation of Pesticide Combinations. Gowing (29) employed probit analysis in evaluating herbicide mixtures. This procedure consisted of plotting the logarithm of the concentration against the percent response on a probability scale and fitting a weighted regression line to the data. From this graph, a pre- diction of the amount of chemical for a given response can be made. Reliability of the measurement is maxi- mum near the 50% response level. Mathematical methods are available for testing results of pesticide combinations. The expected res- ponse for a given combination of two herbicides can be calculated as follows (30): X = the percent inhibition of growth by herbi- cide A at p lb/a Y Z the percent inhibition of growth by herbi— cide B at q lb/a E : expected percent inhibition by the two herbicides at p lb/e and q lb/a EZX+Y—%—6 When the observed response is greater than the expected, the combination is synergistic; when less than the ex— pected, it is antagonistic, and if the observed and ex- pected responses are equal the combination is additive. Colby (15) modified this formula by converting the ori- ginal data to percent of control rather than percent inhibition. By doing so, the number of arithmetic operations required to obtain E is reduced. He pro- posed the following formula: >< EZ%6 where X is the growth as a percent of control with her- bicide A at p lb/a, Y is the growth as a percent of control with herbicide B at q lb/a and E is the expected growth as a percent of control with herbicides A and B at p lb/a and q lb/a. Synergism is indicated when the observed growth as a percent of control is less than the expected, and antagonism when the observed growth is greater than expected. Instead of calculating the expected response of a combination, Bovey et_al. (9) set a definite value to describe herbicide combinations. If a combination of 2 herbicides gave 20% or more defoliation than a single herbicide at equivalent rates, the mixture was consi— dered synergistic and if 20% or less, the mixture was antagonistic. -Tammes (74) formUlated a graphic representation of the effect of two pesticides or a pesticide and ad- juvant applied jointly. Each of the components of a combination is expressed on a coordinate. In the graph, a quantitatively defined effect, for example 50% mor— tality, is inserted. The values are obtained by inter- polation of the data of the combination on log/probit paper. The line which connects the points in the graph is called an isobole. From the isobole presentation, one can determine if the combination is synergistic, antagonistic or additive. Some workers have analyzed pesticide combinations similar to single treatments and used tests such as Duncan's Multiple Range Test to compare the means (12. 34, 36)° Substituted Urea Herbicides The herbicidal properties of the substituted ureas were discovered by Thompson and his co-workers (75) and 10 by E. I. du Pont (10). The general structure of the phenylurea herbicide is: The analogous compounds utilized in this study have the following substitution and solubility properties (38): Solubility in Herbicide R1 R2 X1 X2 water at 25 C (ppm) Linuron OCH3 CH3 Cl Cl 75 Monolinuron OCH3 CH3 Cl H 75 Diuron CH3 CH3 01 Cl 42 Monuron CH3 CH3 01 H 230 The substituted urea herbicides are widely em- ployed as selective pre—emergence or post-emergence treatments for annual weeds and at higher rates as soil sterilants for the control of all annual and perennial weeds. Linuron selectively controls germinating and newly established broadleaves and grasses in crops such as cotton, corn, potatoes, carrots, parsnip and soybean. Monolinuron is used on potatoes, dwarf beans, vines and other crops. At lower rates, diuron and monuron ll selectively control germinating broadleaf and grass weeds in cotton, sugarcane, pineapple, asparagus and citrus. Absorption and Movements. Substituted urea herb- icides are easily absorbed by the roots but they are less readily taken up by the aerial portion of the plants. Studies with monuron and diuron indicated that these herbicides moved primarily in the apoplast and moved laterally and acropetally in the transpiration stream (17). - When applied to leaf surfaces, the substituted ureas are able to penetrate the cuticular and epidermal layers. Foliar absorption of the newer methoxymethyl ureas is significantly greater than that of the dime- thylureas. No basipetal movement was observed when diuron was applied to the leaves indicating that there is little or no translocation occurring in the phloem (35). Absorption into the leaf surfaces is greatly facilitated by the addition of suitable surfactants (4,39). Surfactant WK (dodecyl ether of polyethylene glycol) enhanced the deposition of high concentrations of linuron and diuron on some grasses. Environmental factors such as high temperature, high relative humi- dity and moisture stress increase the penetration of these chemicals into the leaves (39). 12 Rogue (42) demonstrated that in coriander (Corian- drum sativum L.) and tomato (Lycopersicon esculentum Mill.), the monohalogenated compounds like monuron and monolinuron were more toxic than their dihalogenated counterparts. He attributed this difference to the fact that plants took up less dihalogenated than monohalogenated herbicide. In post—emergence application, the dihalogenated herbi- cides were generally slightly more toxic than their mono- halogenated counterparts. Metabolism. Fang (22) reported the formation of a monuron complex in bean leaves after foliar application of radioactive monuron. Sweetser (69) showed that monu- ron complexed with flavin mononucleotides and upon illu- mination was inactivated. Other studies indicated that 15 to 25% of linuron absorbed may be bound to a protein (56). The ability of diuron to form a complex with pro- tein in_yitrg_was also demonstrated using bovine serum albumin (11). Another means of metabolic breakdown is by deals kylation (27). Demethylation is the initial step in the degradation scheme, followed by hydrolysis. In the case of the methoxymethylurea, linuron, the major meta- bolite found in plants was the demethylated derivative, 3- (3, 4—dichloropheny1)—l-methoxyurea (56). Further hydrolysis of the demethylated ureas pro- duces their corresponding anilines. Onley et al. (58) 13 reported the possibility of 3, 4-dichloroaniline trans- formation into 3, 4-dichloronitrobenzene. Ring substi- tution and ring rupture or dehalogenation have not been reported. Mechanism of Action. The substituted urea herb- icides are known to be potent inhibitors of photosyn- thesis, particularly the Hill reaction. Wessels and van der Veen (78) and Cooke (16) found that several substituted urea herbicides were very efficient inhibi- tors of the Hill reaction in isolated chloroplasts. Monuron at a concentration of l x 10-6 M completely inhibited the Hill reaction (16). Duysens gt a; (21) reported that two pigment systems are involved in cytochrome oxidation during photosynthesis. Light of 560 mu.causes a reduction of the cytochrome while light of 680 mu oxidizes the cytochrome. Diuron at l x 10'6 ll inhibited the reduction at 560 my and oxidation of the cytochrome took place instead. The photosynthetic mechanism in green plants is believed to include two light reactions (7): OXIDATION-REDUCTION POTENTIAL (VOLTS) Carbaryl Carbaryl (l-napthyl Nemethylcarbamate) is one of O O O I O O O .61 l4 [] UREA HERBICIDE INHIBITION /// LIGHT égg SYSTEM II P 7‘ <7'700 / W / / / //-— I \ // A J l// ( ATP <\/7 / LIGHT / ADP YH SYSTEM I F— NADPH .27 FERREDOXIN a$7 // H/ /’ XH Although the site of action of substituted ureas is be- lieved to be in light system II, the enzymatic events associated with this process and the specific molecular sites inhibited by the ureas are still unknown (61). the most widely used carbamate insecticides for fruits, vegetables, forage, cotton and many other economic crops. 15 The insecticidal properties and characteristics were reported by Haynes et 31. (37). Carbaryl is soluble in most organic solvents and has a water solubility of less than 0.1%. It is stable in light, heat and acids but is easily hydrolyzed in alkalies. The metabolism of carbaryl has been investigated in beans (Phaseolus vulgaris L.), peas (Pisum sativum L.), pepper (Capsicum frutescens L.) and corn (Zea mays L.) (53). The comparison of the mass and ultraviolet spectra of isolated metabolites with the spectra of synthetic compounds confirmed the presence of N-hydro- xymethylcarbaryl, 4-hydroxymethylcarbaryl and 5-hy— droxymethylcarbaryl. Each plant possessed essentially the same metabolites in varying amounts. The metabolic fate of carbamate insecticides is often similar in both plants and insects (20, 47). However, differences exist in their rate of formation, their conjugating moieties and their ultimate distri- bution within the organisms. Besides hydroxylation in the 4 and 5 positions, carbaryl also forms the 5, 6Adihydro-5, 6-dihydroxy derivative which conjugates in plants. Hydrolysis of the carbamate-ester linkage occurs slowly. Little is known about the plant enzymes responsible for carbamate metabolism, but in insects a mixed function oxidase system(s), present in the 16 microsome, appears to be the main catalyst (47). The carbamate insecticides are considered to kill insects and mammals entirely by cholinesterase inhibition (57). The symptoms accompanying their action in intact animals are typically cholinergic, involving lachryma— tion, salivation, myosis, convulsions and death. Diazinon Diazinon (Q, Q-diethyl 972—isopropyl-6-methyl-4- pyrimidinyl phosphorothioate) is extensively applied to soil and crops to control phytophagous insects. It is effective for controlling resistant soil insects such as corn rootworm, wireworm and cabbage maggot and also used against many foliage insect pests of fruits, veg- etables, forage, field crops and ornamentals. Diazinon is available as wettable powder, emul- sifiable and oil solutions, dust and granular formula- tions. It is miscible with alcohol, ether, petroleum ether, cyclohexane, benzene and similar hydrocarbons and soluble in water at .004%. Gasser (25) first reported on the biological pro- perties of diazinon and its effectiveness as a contact or vapor insecticide. Gunner gt al.(3l) showed that diazinon was absorbed and translocated in plants after foliar application and later appeared in roots and root exudates. Diazinon was absorbed and accumulated in 17 higher quantities in the roots of bean plants than in any other regions (46). Translocation to the leaves occurred, but only small amounts of diazinon were pre- sent in the primary leaves after two days. Systemic activity has occurred in leaves of sugar beet seedlings grown in treated soil indicating that diazinon may accumulate in the foliage after continuous exposure of roots (59). Root treatment of cabbage (Brassica ole- racea L.) and tobacco (Nicotiana tabacum L.) with water preparations of diazinon also resulted in translocation and residues in the foliage (52). Pea plants grown in diazinon treated sand also accumulated the insecticide in the leaves, especially at higher rates of treatment and shorter harvest times (49). Metabolism of diazinon in plants has been shown to involve hydrolysis of the phosphorous pyrimidyl ester bond and subsequent metabolism of the 2—isopro- py1—4-methy1-6-ol to carbon dioxide (46). Small amounts of diazoxon, (Q, Ogdiethyl 97 [2-isopropyl-4- methyl-6-pyrimidinyl] phosphate)were also detected in field grown crops (64). It is widely accepted that organophosphate in- secticides kill animals, both vertebrate and inverte- brate, by inhibiting cholinesterase activity with sub- sequent disruption of the nervous activity caused by accumulation of acetylcholine at the nerve endings (57). MATERIALS AND METHODS Initial Screening of Pesticide Combinations for Enhanced Toxicity Combinations of several common herbicides and insecticides were initially screened as foliar sprays to identify synergisms which might occur. The plant spe- cies used were carrots (Daucus carota L. cv. Chantenay Long Type), cucumber (Cucumis sativus cv. Spartan Pro- gress), soybean (Glycine max (L.) Merr. cv. Harris), cotton (Gossypium hirsutum cv. Polymaster) and corn (§§a_may§ L. cv. Harris Gold Cup). Seeds of each spe- cies were planted in 11 x 16 cm styrofoam flats filled with muck soil. Upon emergence the seedlings were thinned to an equal number of plants in each pot. Car— rots were sprayed at the 2 to 4 leaf stage, cotton, corn and cucumber at the 2-leaf stage and soybean at the first trifoliate leaf stage. The chemicals and their rates of applications for each species are indicated in the appendices. All the formulations used were wettable powders. The amount of herbicide or herbicide-insecticide mixture required for each treatment was mixed thoroughly in a quart milk bottle. Spraying was done at 40 psi using 18 19 C02 as a source of pressure and water volume was equiva- lent to 100 gallons per acre. The potted plants were passed under the nozzle on a conveyor which moved at a fixed rate of speed. Before spraying, the soil was covered with vermiculite to prevent the herbicides from reaching the soil. Afterwards the vermiculite was re- moved and the treated plants were transferred into the greenhouse and arranged in a randomized block design on benches. AThe greenhouse was maintained at 25*5 C with a daylength of 16 hr. Sunlight was supplemented with fluorescent lights. All the treatments were replicated 3 to 6 times and each experiment was conducted at least 2 times. Injury ratings were obtained at intervals using a scale of 1 to 9 where 1 indicated no injury, 2 to 3 slight injury, 4 to 6 moderate injury, 7 to 8 severe injury and 9 death. The plants were harvested after 2 weeks and fresh weight was also obtained. The data were subjected to analysis of variance and the combi— nations were compared using the ISD test and Colby's formula (15). If the expected response was greater by 15% or more the combination was considered synergistic by Colby's method. 2O Toxicity of Phenylurea Herbicides and Herbicide~Insec- ticide Combinations Seeds of cotton, carrot, corn and soybean were ger— minated in wooden flats containing vermiculite. After 7 to 10 days, the seedlings were transplanted into % strength Hoagland's solution (40). The solution was con- tained in 180 ml plastic containers wrapped with alumi- num foil to exclude light. The nutrient solution was changed every other day. A circular sponge which fit~ ted the top of the container was used to hold the plants in place. Cotton was treated when the first 2 leaves were fully expanded. Corn and carrots were treated at the 3—leaf stage and soybeans were treated at the first trifoliate stage. The following substituted urea herbicides were used: linuron (Lorox 50% WP), monolinuron (Aresin 50% WP), diuron (Karmex 80% WP) and monuron (Telvar 80% WP). The leaves were dipped in suspensions of commer- cial formulations of the above herbicides at concentra— tions ranging from 300 to 4800 mg/l. The leaves were allowed to dry at room temperature before the plants were transferred to the greenhouse. After 10 days, visual ratings and fresh weight of the shoot were ta- ken. The data were subjected to analysis of variance and a ISD was used to compare the treatment means. 21 Effect of Carbaryl and Diazinon on Herbicide Activity. The 4 substituted ureas used in the above experiment were also applied to leaves singly and in com- bination with carbaryl (Sevin 50% WP) or diazinon (50% WP). The plants were grown in a similar manner as des- cribed above. Treatment was accomplished by dipping the leaves in a suspension of the herbicide alone and the herbicide—insecticide mixture. The concentration of the 4 herbicides used for cotton, corn and carrot was 1200 mg/l while for soybean, linuron and monolinuron were applied at 300 mg/l and diuron and monuron 600 mg/l. Carbaryl and diazinon were applied at a concen- tration of 2400 and 1200 mg/l respectively. The treat- ments were replicated 3 times and the experiments were repeated 2 times. After treatment, the plants were kept in a growth chamber with a night temperature of 20C3and day temperature of 25(1 The growth chamber was maintained with a 16-hour daylength and a light inten- sity of 5 watts/cm2 (red region) and 4 watts/cm2 (blue region). After one week, visual injury ratings were obtained and fresh weights of the shoots were recorded. Data were subjected to analysis of variance, converted to percent of control and the combinations were assessed using Colby's formula (15). 22 Tests were also conducted in which one component of the combination was applied to the foliage, the other to the root media and both components applied to the root media. Six day old cotton seedlings were transplanted into nutrient solution with % strength Hoagland's solu- tion. The herbicides were dissolved in the nutrient solution at a concentration of 1.0 x 10"6 M. After 2 days, the shoots were dipped in suspensions of wettable powder formulations of either carbaryl or diazinon at a concentration of 2400 and 1200 mg/l respectively. In another experiment, the insecticide was applied in nutrient solution at a concentration of 1.0 x 10"5 M. After 2 days, the Shoots were dipped in suspensions of wettable powder formulations of the 4 herbicides at a concentration of 1200 mg/l. In the third experiment, the herbicide was applied to the nutrient solution at a concentration of 1.0 x 10'6 M and the insecticide at 1.0 x 10"5 M. In all cases, visual injury ratings were taken and fresh weights of the shoots were re- corded 10 days after treatment. Effect on Respiration and Photosynthesis of Leaf Tissue. The oxygen uptake and evolution of isola- ted leaf discs treated with linuron alone in combination 'with carbaryl was followed using the method of Umbreit _§§_al. (76) in a Gilson Differential Respirometer. 23 Discs (8.0 mm) were obtained from recently expanded pars- nip (Pastinaca sativa L. cv. Hollow Crown) leaves using a cork borer. The leaf discs were incubated for 1.5 hr in solutions containing 1.0 x 10'5 M linuron alone or combined with 1.0 x lo-l’r M carbaryl. At the end of the incubation period, the leaf discs were removed from the solution, rinsed 3 times with distilled water and dried with absorbent paper. They were then transferred into a respirometer flask containing 1.0 ml of 0.05 M phosphate buffer pH 7.0. The center well contained 20% KOH and a folded filter paper. Oxygen uptake was fol- lowed in the dark for a duration of 5 hr after which dry weight of the leaf discs was obtained. To measure photosynthesis, the same procedure of preparation and treatment of plant material was followed. ‘ Instead of KOH buffer, the center well contained a 002 buffer which maintained a 0.3% C02 concentration in the flask (60, 70). The buffer system was made up oflIDmlcfi‘ 60% diethanolamine, .5 ml 6 N HCl, 4.5 ml distilled water and 3 gm KHCO3. The temperature was maintained at 25(3throughout the 5 hr run. The tissue was exposed to alternate periods of light (30 min) and darkness (25 min) and readings were obtained at each interval. In these tests, the treatments were replicated 4 times and the experiments were conducted twice. 24 Effect of Insecticides on the Uptake of luC-Phenylurea Herbicides The effect of diazinon on the uptake of the 4 phenylureas in tolerant cotton and susceptible soybean was determined. Application and Sampling. The plants were treated in the same growth stage as in the previous experiments. The concentration of the herbicides and diazinon used was 1200 mg/l. The treating suspensions were spiked with l”c.1abeled herbicides to give a final activity of 0.0005 uc/fil. The specific activity of the labeled herbicides is presented in Table 1. All the radioactive herbicides were maintained in absolute alcohol in the freezer prior to use. Table 1. Specific activity of the carbonyl-labeled phenylurea herbicides. Herbicide Specific Activity .uC/ms Linuron 6.8 Monolinuron 9.4 Diuron 4.0 Monuron 0.204 . 25 Twenty microliter droplets (.01 Juc) of the herbi- cide alone and the herbicide-insecticide combination were applied at the midrib of the leaf. Also 20 pl aliquots of each solution were counted to determine the total amount of radioactivity applied. The droplet was allowed to dry in the laboratory at 25(3before the plants were transferred to a growth chamber. The growth chamber was maintained with a l6-hour daylength with a light inten- sity of 5 watts/cm2 (red region) and 4 watts/cm2 (blue region) and a temperature of 25 0 Three or 4 replicates were sampled at 6, 12, 24 and 48 hr after treatment. Upon sampling, the treated leaf was cut from the plant and washed 5 times with 95% ethyl alcohol to re- move the herbicide remaining on the leaf surface. After washing, the leaves were frozen in a dry ice-+ acetone bath and dried in an oven. The uptake of technical grade herbicide in the presence or absence of technical grade diazinon was also followed in cotton leaves. The cotton leaves were treated at the same growth stage and the manner of treat- ment and sampling were similar to that of the above experiments. The concentration of linuron was 4.0 x lO'LL M (.5 uc), monolinuron 2.0 x lO‘u'M (.5 uc). diuron 3.0 x lO‘u'M (.25 MC) and monuron 6.0 x lO'u'M (.05 uc). Diazinon was used at 6.6 x 10'5 M. 26 In other experiments with carrots, the uptake of linuron in the presence or absence of carbaryl was deter- mined. The 20 M1 (.01 pc) treating solution was distri- buted as droplets over the divided leaf surfaces. The treated leaves were handled in the same manner as in the above experiments. Assay of Radioactivity. Total radioactivity in the treated leaves was recovered by combustion in a Model 3151 Nuclear Chicago Combustion Apparatus equipped with a light source and stirring equipment. The dried leaf samples were placed inside cellophane bags and inserted in a platinum wire basket. The flask was flushed with oxygen and the sample was completely oxi- dized. The flask was allowed to cool for about 5 minutes before adding 15 ml of trapping solution which consisted of ethanol: ethanolamine at a ratio of 2:1 (v/v). The solution was stirred for 10 minutes. A 1.0 m1 aliquot was removed and placed in a scintilla- tion vial containing 15 ml of cocktail mixture. The cocktail mixture was prepared by dissolving 4 grams of BBOT (2,5«bis [5-33133 butylbenzoxezolyl (2A )] thiopen) with 1 liter of toluene and 400 m1 of Triton X—100. Quantitative determination of radioactivity was ob- tained with a Packard Tricarb Scintillation Spectro- meter equipped with external standardization. 27 14 Effect of Insecticides on the Metabolism of urea Herbicides C-Phenyl- Application and Sampling. The rate of metabolism of the 4 phenylureas in the presence or absence of dia- zinon was followed in cotton. Plants were grown in the same manner as the previous experiments. The concentra- tion of the herbicides used were linuron 3.0 x lO‘LL M (0.5 no), monolinuron 5.0 x 10'LL M (1.0 uc), diuron 5.0 x 10"LL M (0.5 uc) and monuron 6.0 x 10‘)‘L M (.05 uc). The herbicides and diazinon were applied to the first true leaf of cotton with a microsyringe. The solution was allowed to dry in the laboratory at 25 C and then the plants were placed in a growth chamber under the same conditions previously described. The treated leaves were removed 2 and 5 days after treatment. In other experiments, the metabolism of linuron in the presence or absence of carbaryl was studied in carrots. The plants were treated at the 3—leaf stage. )1 Linuron was applied at a concentration of 3.0 x 10- M (0.5 MC) and carbaryl at 3.0 x 10'“ M. Twenty micro- liters of solution was applied with a microsyringe on each leaf. Eight leaves were used for each treatment and upon sampling 4 leaves were combined to insure high activity in the extract. The plants were placed in a growth chamber with the same condition as the above experiments and sampling was done 2 and 5 days after treatment. 28 Extraction and Analysis. The leaves treated with linuron and monolinuron were washed with 95% ethyl alco- hol and extraction was done immediately. The leaves were macerated thoroughly using mortar and pestle and ethyl alcohol (10 ml/g sample) as solvent. The extracts were filtered using Whatman No. 1 filter paper and the residue was washed 3 times with the extracting solvent. The filtrate was then evaporated to dryness in a rota- ting evaporator. The residue was saved for combustion to determine the alcohol insoluble metabolites. After evaporation to dryness, the residue was redissolved in 1.0 ml of absolute ethyl alcohol. The extracts were refrigerated overnight prior to chromatography. The procedure was essentially the same for washing and extracting the diuron and monuron from leaves except methyl alcohol was utilized as a solvent. Chromatography of the leaf extracts was done on glass plates coated with silica gel H with a thickness of 250 microns. The plate was divided into three sec- tions, one portion for the standard lLLC-compound, the second portion for the extract from plants treated with herbicide alone and the third portion for the herbicide-insecticide treatment. Fifty “1 from each leaf extract was spotted in a band at the origin. In the case of linuron and monolinuron, the plates were 29 developed in benzene: acetone (2:1; v/v) (71). After developing to a distance of 15 cm on the plate, the plate was divided into 1.0 cm sections, the silica gel was scraped, and the scrapings were placed in scintilla— tion vials and counted for radioactivity. Rf values were calculated and used as a reference for identifying the parent compound and metabolites. RESULTS AND DISCUSSION Initial Screening of Pesticide Combinations for Enhanced Toxicity Several herbicide-insecticide combinations were synergistic when applied as foliar Sprays to carrot, soybean, corn, cotton and cucumber (Appendices A to F). Combinations of linuron and carbaryl enhanced toxicity on carrot as illustrated by fresh weight and injury ratings (Appendix A). Since linuron is widely used for weed control in carrots and carbaryl is used to control insects in that crop, this interaction was investigated in more detail. The tests with other species indicated inter- actions between substituted urea herbicides and carba- ryl, diazinon and malathion. In susceptible cucumber, diuron and chloroxuron toxicity was slightly enhanced with carbaryl, diazinon and malathion but the herbi- cide rates were probably too high to obtain maximum differences (Appendix B). Interactions were also evident with some of these combinations in soybean (Appendix C). 30 31 Since diazinon and carbaryl were the compounds which most consistently increased the activity of the substituted urea herbicides, they were applied in combi- nation with 4 analogous ureas (linuron, monolinuron, diuron and monuron) on soybean, corn and cotton. In soybean, combinations of diuron with diazinon or car- baryl produced synergistic interactions (Appendix D). In corn, the presence of carbaryl and diazinon caused an increase in toxicity of linuron and monolinuron (Appendix E). Synergistic interactions were obtained in cotton with combinations of the 4 phenylurea herbi- cides and carbaryl (Appendix F). -The major problem encountered in the greenhouse screening tests was variability in results between ex— periments. The amount of enhancement in herbicide activity caused by the added insecticides varied con- siderably from test to test. This may have occurred because of difference in environmental conditions prior to or after spraying the plants. At this time, the greenhouse soil mix was also unsatisfactory for growing uniform plants. Insect control caused a pro- blem in the greenhouse and the use of fumigants or other insecticides introduced a third chemical for possible interaction. Another factor may have been variable exposure of foliage to the sprays. 32 To overcome variability, uniform seedlings were selected and transplanted to nutrient culture. Instead of spraying the chemicals, the shoots were dipped in the chemical suspension. With this method, uniform wetting of the upper and lower leaf surfaces was obtained. Fresh weight was utilized because there was no difference in relative toxicity using fresh or dry weight. Toxicity of the Phenylurea Herbicides and Herbicide- Insecticide Combinations Cotton, carrot, corn and soybean responded dif- ferently to foliar application of the 4 phenylurea herbicides (Table 2). Carrot was the most tolerant to the 4 phenylureas whereas corn was tolerant to diuron, monuron and linuron and slightly more suscept- ible to monolinuron. Cotton was relatively tolerant to the dimethylureas but more susceptible to the me- thoxymethylureas. Soybean was the most susceptible species to all of the phenylureas. Chlorosis of leaves which ultimately resulted in necrosis and. death of the plant was observed in the affected species. 33 Table 2. Concentration of phenylurea herbicides (mg/l) required to produce 50% fresh weight reduction in 4 species. fifi Herbicide Plant Linuron Monolinuron Diuron Monuron Soybean >4800 > 4800 Carrot > 4800 > 4800 > 4800 > 4800 ED50 values (effective dose required to reduce fresh weight by 50%) for oat, soybean, corn and cotton 11 days after exposure to monuron and diuron in culture solution revealed that the order of susceptibility of the species to both herbicides was: oat > soybean > corn > cotton (68). Foliar application of diuron and monuron, as indicated in this experiment, showed the following order of susceptibility: soybean > cotton > corn. The tolerance of carrot to linuron is consist- ent with previous results (48). Sublethal concentra- tions of linuron by root uptake were previously re- ported as 0.57 ppm for corn and 0.28 ppm for soybean (56). This experiment further demonstrated the rela- tive tolerance of corn and susceptibility of soybean to foliar application of linuron. 34 Effect of Carbaryl and Diazinon on Herbicide Activity. Cotton was relatively tolerant to both diuron and monuron, however, when they were combined with the insecticides carbaryl and diazinon, toxicity was greatly increased (Table 3). Diazinon had a more pronounced effect than carbaryl as indicated by injury ratings and fresh weights. Linuron plus carbaryl and monolinuron plus carbaryl produced no additional toxicity over that obtained with the herbicides applied alone. However, the toxicity of linuron and monolinuron was enhanced by diazinon. In tolerant carrots, the combination of linuron or monuron with carbaryl was synergistic as indicated by the fresh weight (Table 4). Monolinuron or diuron plus carbaryl mixtures were about additive. Diazinon interacted with the 4 phenylureas regardless of their substitution and provided synergism in all cases (Figure 1). Corn was also tolerant to the 4 phenylurea her— bicides. Combinations of linuron or monolinuron with carbaryl both resulted in synergism (Table 5). Again, the combination of diazinon with all 4 herbicides gave extremely pronounced synergistic effects. A slight burning of the corn leaves at the margin was observed with treatments receiving diazinon alone. 35 mo.mma oopmsflpmo mcams czac> cm>aompo Scam pompoMMHc .Ammv aaaSsm cc Sassooaoacwam * .oosoos a_soaoo so copsaooaso a mm a.a em as oma moa o.a coma cocawoam - - moa m.a ooam assesseo - - *oo om o.o ooma cocaaoao ooma coaocoz *mm mm s.m ooam assessao ooma dosages Sm o.m u u ooma Cognac: *moa a: s.m coma cocanaam coma cognac moa as s.: ooam asssosso ooma coaoao ooa b.a : : ooma noHSHQ *ms a: m.m ooma sedansao coma cosocaaocoz ms cc o.s ooam aassosmo coma cosssaaocoz as m.c A - ooma coaoqaaocoz *mm mm o.m coma cooauaao ooma cosscaa mm mm m.m ooam asasosso ooma coaooaa am w.b u u ooma GOMSCHQ ooa o.a u u : xomso aoopoomxm co>aompo daoaono wo Rq. mcflpcm Aa\wev ocaoap Aa\wev aEmHmaochm unwaoz Smoam amsmfl> docoo voomcH Scooo ooHoHQHmm .coppoo mo szosm 639 Go mocaoapoomcfl SpHB mcoapmcflpfioo cam mocfloapaos coasahcogm : mo poommm one .m canoe 36 mm c.a em as cca cca o.a coma cooaamac - - mo o.a ccam asaaosoc - - *mc cm o.a coma sodassac coma sconce: cs a: s.m ccam asasosmc coma cosocoz mm s.a - - coma cosccoz *cm aa m.w coma cocaasac coma cocoac as ms s.m occm asseoasc coma coscao cm o.a . - coma coacac mm mm o.a coma cocawsac coma coaosaaocoz cm co m.: cc:m asssoasc coma cosocaaosoz :m o.m u : ooma CcascHaoccz *mc am o.a coma oosaaeac coma coaocaa cs cm o.a cccm asssoasc coma cosocaa mm m.a . - coma cosccaa cca o.a - - - Rocco oopoomxm co>ammno Aaoapzoo mo Rq wcflpmm Aa\mav ocaoap Aa\wev Smawaocmm pzmaoz gmoam acSma> Cuzco :comQH Cocoo ocaoapsom .mpoaaco wo spSOMm 63p Co mocaoapoomca SpHB msoapmcapaoo cam mocfioflnaos coasahcosm : mo pooeeo one .3 canoe Figure l. 37 Toxicity obtained with combination of diazinon and the 4 phenylurea herbicides on (a) carrot, (b) corn, (c) cotton. *UOZZHWC—AHCEQ'IjtJ-jUOtIib Check Linuron (1200 mg/l) Linuron-+ Diazinon (1200 4-1200 mg/l) Diazinon (1200 mg/l) Check Monolinuron (1200 mg/l Monolinuron 4-Diazinon (1200 4-1200 mg/l) ‘Diazinon (1200 mg/l) Check Diuron (1200 mg/l) Diuron-+-Diazinon (1200 +-l200 mg/l) Diazinon (1200 mg/l) Check Monuron (1200 mg/l) Monuron +—Diazinon (1200 4-1200 mg/l) Diazinon (1200 mg/l) 38 ‘ wa lawn: 39 a: c.c em as cma cc m.m coma cooauoac - - cca o.a ccam assaosac - - m *ac sa s.s coma socassac coma ceases: as cca m.a ccam asaoossc coma cosocoz mm m.a u u coma cognac: c *cc am s.c coma cosaasac coma coaoac cca mca c.a ccam asaspasc coma cosoac mca o.a - . ccma coacac c cc cm s.s coma cocauoac coma cosocaaocoz c mm c: m.m ccam asssosso coma cosooaaosoz as s.a, . - coma cosooaaocoz m *ss cm s.c coma cocaaaac coma coaodaa c mc SS s.m ccam aSacosac coma coaocaa cc m.a - - coma sosocaa cca o.a - - - roocc cmpommxm co>ammpo Aaospcoc co ev mdaasm Aa\wsv ooaoaa Aa\msv Samamamchm pgwaoz Smoam amsma> Cuzco ucmmcH Cuzco ocaoanaom .ano mo spacaw ogp Go mocHOHpomea Spas mcoachHQEoo UQc mocfloapacs coasazcogm : Cc pommmo mQH .m manB 40 Soybean was the most susceptible species to the 4 phenylureas. In this experiment, diazinon at a con- centration of 600 and 1200 mg/l and carbaryl at 2400 mg/l were also phytotoxic to soybean (Table 6). Since toxicity occurred from both the herbicides and insecti- cides, the combinations of diazinon with linuron, mono- linuron and monuron were all about additive in their effects. Carbaryl plus linuron or monolinuron also produced additive responses. The only synergisms ob- tained were with diuron in combination with both in- secticides. It should be noted that the toxicity ob- tained with low rates of herbicides applied alone was severe. Other synergisms may have been evident if lower rates were utilized. Split application experiments were conducted only with cotton plants. In one study, the phenyl— ureas were applied in nutrient solution at a concen- tration of 1.0 x 10*6 M; and the shoot was dipped in suspensions of commercial preparations of insecti— cides after 2 days. Foliar applied carbaryl had little or no effect on the fresh weight reduction obtained with the 4 phenylurea herbicides by root uptake (Table 7). No significant difference was observed in the visual rating of these combinations. With the possi- ble exception of monuron, foliar applied diazinon did not produce enhancement of the phenylureas when 41 aa :.c em as cca a: c.c coma cocaaaac - - ac c.c coc cocaaoac - - cs o.a coam asacoasc - - am am o.a coma cocaaoac coc coaccoz mm mm m.c ccc cosaasac ccc cognac: a: ac m.s ccSm assapaac coc dosage: 3m o.c u u ooc Couscoz a: cm c.c coma oceansac coc cognac m *sc cm c.c coc socaaaac coc cognac cs cc m.c ccam asaoosec ccc cognac mm s.: - - ccc cognac mm mm c.c coma sosausac com sosooaaosoz mm mm m.c coc cocaasac com conceaaosoz a: s: o.a ccam aaaeossc com soacsaaocoz ac c.s - - com coaocaaosoz cm am c.c coma socausac com cososaa am mm c.c coc cocaasac com cosscaa ma c: s.s ccam assaosac com coscsaa cc o.a - - com coaccaa cca o.a - - - Rocco cmpommxm cc>aomno Aaoapcoo mo RV mqapcm Aa\mev mcaoap Aa\wev Emamaochm pnwaoz gmoam acSma> Cuzco nommcH accoo ocaocnaom .cmmnzom Mo szoam map co mmcaoapoomsa Spas macflpcflaneoo UQM mmcHOHnamL moasahcmnm : mo poommo one .c oaoae 42 cm a.a mm as cca cma o.a coma cosaacac - - aaa o.a ccam asscossc - - m aaa *as o.a ocma cocascac c-oaxa coaccoz cc ac o.a cccm aesaoaac c-caxa soaccoz cw o.a I u cloaxa monsooz cc cca o.a coma cocaueac c-caxa coscac am cm o.a ccam assaoaac c-caxa cognac as m.a - - c-caxa coscac m cm sc c.m coma cocaaaac c-caxa coaccaaocoz m as cm c.m ccam assscaoc c-caxa cosscaaocoz mm c.m u u QIOHXH Seascaaocoz sc ms o.a coma cocassac c-caxa cososaa cm s: m.m ccam asseossc c-caxa sosccaa mm c.m - - c-caxa coaocaa ooa o.a u u u Mocso copoomxm co>aomno aaosacoo mo ev mcacom Aa\msv ocaoap sz Emamaoczm pcmacz QmQMm acSma> Cuzco uocmcH Cuzco ccaoapaom .coppco Go mmcaoansoz mmSDHSQCsm coaammc poop so hpaoaxcp cSp so mmcaoapoomca mo Coapmoaagmm acaace so poowmo CQB .s oapme 43 applied to roots. There was a slight stimulation in growth when either carbaryl or diazinon was applied to foliage of cotton. In the second study, the insecticide was applied in nutrient solution and the shoot was dipped in herbicide suspensions 2 days after insecticidal treatment. Carba— ryl alone was extremely toxic to the roots of cotton (Tables 8 and 9). There was no further growth of the roots after treatment and the root tips turned brown. The plant was stunted in growth although the leaves re— mained green. Carbaryl applied in nutrient solution produced enhanced toxicity with diuron and monuron only. Application of carbaryl to the roots of corn and peas has also been reported to reduce the growth of these plants (50). The roots grown in carbaryl-treated sand were shorter and thicker than roots grown in untreated sand and the lower portion of the root system had a dark purplish color. Diazinon was only slightly toxic to the cotton root system (Tables 8 and 9). When diazinon was applied in solution and the herbicides were applied through the foliage, no interactions were observed (Table 8). When both the herbicides and insecticides were applied to the roots, synergisms were obtained with com- binations of either diuron or monuron with carbaryl (Table 9 and Figure 2). Root application of the 4 44 Table 8. The effect of root-applied insecticides on the toxicity of foliar applied phenylurea herbicides on cotton. Herbicide Insecticfde Visual Fresh Weight (1200 mg/l) (1.0xl0f M) Rating (% of Control) Observed Expected Check — 1.0 100 Linuron - 8.0 15 Linuron Carbaryl 7.3 17 6 Linuron Diazinon 8.0 20 12 Monolinuron - 4.0 54 Monolinuron Carbaryl 5.0 34 43 Monolinuron Diazinon 4.6 44 43 Diuron — 1.0 73 Diuron Carbaryl 5.6 29 28 Diuron Diazinon 1.3 90 58 Monuron - 3.3 59 Monuron Carbaryl 6.6 29 26 Monuron Diazinon 5.3 46 47 - Carbaryl 3.0 39 — Diazinon 1.6 80 ISD at 5% 0.8 20 45 Table 9. The effect of both herbicide and insecticide applied in solution on the growth of cotton. Herbicidg Insecticide Visual Fresh Weight Syner— (l.0x10' M) (l.0x10-5 M) Rating (% of Control) gism Observed Expected Check — 1.0 100 Linuron - 8.7 11 Linuron Carbaryl 6.7 21 6 Linuron Diazinon 9.0 11 ll Monolinuron - 3.0 35 Monolinuron Carbaryl 4.7 33 20 Monolinuron Diazinon 4.3 37 34 Diuron - 2.3 63 Diuron Carbaryl 7.7 13 35 S Diuron Diazinon 1.0 83 60 Monuron - 2.0 50 Monuron Carbaryl 8.0 15 28 Monuron Diazinon 1.3 92 48 - Carbaryl 2.7 56 - Diazinon 1.0 96 ISD at 5% 1.1 31 Figure 2. 46 (a) Toxicity obtained with combination of carbaryl and the 4 phenylurea herbi— cides applied on the roots of cotton. V A SEQ 'Iltlj U OUJ cAH II II Check 6 Linuron (1.0 x 10' M) . 6 Linuron + Carbaryl (1.0 x 10‘ +— 1.0 x lo-5M Carbaryl (1.0 x 10-5M) Monolinuron (1.0 x 10‘6 M) 6 Monolinuron +~Carbary1 (1.0 x 10‘ -+ 1.0 x 10-5 M) Diuron (1.0 x 10“6 M), 6 Diuron +-Carbaryl (1.0 x 10‘ -+ 1.0 x 10-5 M) Monuron (1.0 x 10"6 M), 6 Monuron +—Carbaryl (1.0 x 10‘ -+ 1.0 x 10‘5 M) Toxicity obtained with foliar appli- cation of linuron and carbaryl on carrots. A = Check B = Linuron (1200 mg/l) C = Linuron-+ Carbaryl (l200-+ 2400 mg/l) D = Carbaryl (2400 mg/l) ‘ ‘ 3* 1“ ' ’ ‘ . 2, . HX ' )3; 11%., r i; I 1‘ ' ..f( ' 33 ‘- ' " . I O ." 48 phenylureas and diazinon again did not interact, in con- trast to results from foliar application of both chemicals. In general, the dimethylureas (monuron and diu- ron) were more affected by carbaryl than the methoxy- methylureas (linuron and monolinuron) regardless of the site of application (Table 10). The inhibition of N—demethylation of monuron in cotton leaf discs by carbaryl was previously demonstra- ted by Swanson and Swanson (70). Carbaryl is an ef— fective inhibitor of demethylase, an enzyme believed to Table 10. Summary of the interactions which occurred with insecticides and phenylurea herbicides on cotton. Site of Site of Phenylurea Herbicide Insecticide Application Application Foliage Root Carbaryl Foliage Monuron, Diuron No inter- action Root Monuron, Diuron Monuron, Diuron Diazinon Foliage Monuron, Diuron, No inter- Linuron, Mono- action linuron Root No interaction No inter- action 49 be responsible for the demethylation of the phenylureas (24). In these experiments, carbaryl enhanced the toxi- city of diuron and monuron in cotton but did not have a pronounced effect on linuron and monolinuron. It appears that demethylase is quite specific for the methyl group and that the cleavage of the methoxy group may be brought about by another enzyme apparently not affected by carbaryl. In contrast, diazinon increases the toxicity of the dimethylureas and methoxymethylureas to cotton only if the chemicals are applied together on the foliage. This may indicate that diazinon affects some processes involved in foliar uptake only. It may also indicate that diazinon is not translocated to the leaves of cotton or it is rapidly metabolized to other compound(s). Effect on Respiration and Photosynthesis of Leaf Tissue. These studies were conducted to determine if enhancement of linuron activity by carbaryl was a re— sult of altered respiration or photosynthesis. Pars— nip was chosen as the test plant because of the larger leaf area and the ease by which uniform leaf discs could be prepared. Furthermore, parsnip and carrot be- long to the same family and respond similarly to linuron. 50 Linuron at a concentration of 1.0 x 10-5 M and carbaryl at 1.0 x 10-h M caused only a slight reduction in oxygen uptake (Table 11). In the presence of both chemicals, there was no difference in respiratory rate as compared with the control. The negative effect of linuron on respiration is in agreement with reports that substituted urea herbicides did not affect respi- ration of leaf tissues and algae (28, 66). Various in- secticides such as lindane, dieldrin, DDT, methoxychlor as well as carbaryl have been reported to cause a Table 11. Oxygen uptake of parsnip leaf discs treated with linuron alone or in combination with carbaryl (% of control). Chemical Concn (M) Time (Hr) l 3 5 Carbaryl 1.0x10‘)Jr 78 81 87 Linuron 1.0x10'5 82 88 96 Linuron-+ Carbaryl 1.0x10‘5+ Lexie-LA 95(64)a 97(71) 104(84) ISD at 5% n.s n.s 10 aExpected values calculated according to Colby's method. 51 significant reduction in the rate of respiration of root tips of corn, oats, peas and cucumber (50). The respi- ratory pathway does not appear to be the site of action affected by these interacting chemicals. Linuron at a concentration of 1.0 x 10‘5 M or carbaryl at 1.0 x 10‘)‘L M initially caused a slight re- duction in oxygen evolution (Table 12). The linuron plus carbaryl mixtures caused a synergistic reduction in oxygen evolution throughout the duration of the experi- ment. Since the primary mode of action of the substi- tuted phenylurea herbicides is considered to be an in— hibition of photosynthesis, any factor which brings about an increase in the amount of herbicide reaching the Site of action would have a significant effect on the toxicity of the herbicide. Carbaryl may have caused more movement of linuron to the site of action, in this case, the chloroplasts. Carbaryl might also increase membrane permeability allowing more linuron to be taken up by the leaves. Another possibility is that carbaryl may inhibit the metabolic processes in the plant that are active in the degradation of the herbicide. Effect of Carbaryl and Diazinon on the Uptake of l‘*C-Pheny1urea Herbicides 14 The foliar uptake of C—phenylureas alone and in combination with diazinon was monitored in cotton 52 Table 12. Oxygen evolution of parsnip leaf discs treated with linuron alone or in combina— tion with carbaryl (% of control). ~ a Chemical Concn (M) Time (Hr) 2 3 4 5 Linuron l.0x10‘5 85 99 97 88 Linuron 1.0x10‘6 96 96 95 104 Carbaryl 1.0x10'4 82 92 97 113 Linuron-+ 1.0x10'5—F 6 Carbaryl 1.0x10‘ (70) (91 S) (84*S) (88*S) Linuron-t 1.0xlo-64- 47 73 68 75 Carbaryl 1.0x10‘ (78*S) (88 S) (94 s) (117*S) LSD at 5% 36 36 34 38 aTime represent hours after the initial 1.5 hour treatment. bExpected values calculated according to Colby's formula. S indicates synergism. *Significantly different from observed value using estimated LSD 05 0f Hamill (33). and soybean. In both species, diazinon caused a signi- ficant increase in uptake of the 4 phenylureas. In cotton, the uptake of 14C—labeled linuron and monoli- nuron in the presence of diazinon was rapid after 6 hr and then uptake leveled off slightly (Figure 3 and 4). Diuron was poorly absorbed by cotton and soybean leaves but the addition of diazinon caused a signifi- cant increase in the uptake of the chemical (Figure 5). 53 Figure 3. A comparison of uptake of l“Cnlinuron (wettable powder) with or without diazi- non (wettable powder) on (a) cotton (b) soybean. F value for the interactions of chemical x time significant at 5% level. ABSORPTION (CPM) x 100 54 COTTON o lINURON o lINURON 30 + DIAZINON 35 25 20 15 10 0/0 SOYBEAN 35 23 /\/ 15 10 5 ‘0 o o 6 12 24 43 TIME (Hours) Figure 4. 55 A comparison of uptake of 1LLC-monolinuron wettable powder with or without diazinon wettable powder on (a) cotton (b) soy- bean. F value for the interactions of chemical x time significant at 5% level. ABSORPTION (cm) x 100 56 COTTON .\./"” /°\°/ 35 3O 25 20 15 o MONOLINURON + DIAZINON 10 O MONOLINURON SOYBEAN 35 30 . 25 \ _, a . 0/ L4” I 20 15 10 o 6 12 24 48 TIME (Hours) Figure 5. 57 A comparison of uptake of lU’Cadiuron (wettable powder) with or without diazi- non (wettable powder) on (a) cotton (b) soybean. F value for the interactions of chemical x time significant at 5% level. ABSORPTION (CPM) x 100 35 30 58 COTTON o DIURON + DIAZINON O DIURON SOYBEAN 12 24 48 TIME (Hours) 59 Even less monuron was absorbed by cotton and soybean leaves and uptake was again enhanced by the presence of diazinon (Figure 6). This experiment also demonstrated that the me- thoxymethylureas were more readily taken up by the leaves than the dimethylureas which may explain their relative toxicity. In general, both species absorbed similar amounts of the herbicide with the exception of linuron being more readily absorbed by cotton than soybean. Since uptake of the dimethylureas in the two species is similar, the superior tolerance of cotton suggests that it metabolizes these compounds. In other experiments, pure 14C-herbicides and technical grade insecticides were applied to ascertain that there was a direct interaction of the chemical and not merely a surfactant effect. Significant in- creases in the uptake of luC-linuron, monolinuron and diuron was obtained in the presence of pure diazinon (Table 13). With monuron, no uptake was observed with or without the insecticide. The basis for the observed interaction between the 4 phenylurea herbicides and diazinon appeared to be caused by increased herbicide uptake induced partly by the insecticide and partly by its surfactant. 60 14 Figure 6. A comparison of uptake of C—monuron (wettable powder) with or without dia- zinon (wettable powder) on (a) cotton (b) soybean. ABSORPTION (cpm) x 100 61 COTTON o MONURON + DIA ZINON o MONURON \ / a o .44 1 1 4 6 12 24 48 TIME (Hours) 62 Table 13. The uptake of lLLC—linuron, monolinuron and diuron (cpm) as affected by pure diazinon on cotton leaves. Chemical Time (Hr) 6 12 24 48 . 1 Linuron 514 a 743 a 679 a 1046 a Linuron-+ Diazinon 821 b 888 b 919 b 1530 b Monolinuron 1735 a - 1475 a 1310 a Monolinuron-t Diazinon 2270 b - 2035 b 2030 b Diuron - — 320 a 240 a Diuron—+ Diazinon — — 365 a 525 b iMeans with uncommon letter are significantly differ— ent at 5% level. The rate of uptake of lLLC-linuron (with wettable powder) was enhanced in the presence of carbaryl (wet— table powder) in carrot leaves and parsnip leaf discs (Table 14). However, after 48 hr there was no dif- derence in the total amount accumulated in carrot. In this case, the interaction between linuron and carbaryl may also be brought about by other factors such as de- creased metabolism or more herbicide movement into the chloroplasts. 63 .sm pm pacacmmac hapqccamacwam mam acppca Coaeooss Spas comm: a I p caam c mwcm I n smsa I assmosoc .+ Seascaa I c amsa m mmwa I c mom I Seascaq Qanamm c owsm Q owm: Q msmm I Q coca ahscpamo + Coascflq c swzm c comm c cwom I am mmaa GOMSCHQ poaaco m: cm :m ma w c Aasv mEaB acoaecso pacdm .momac Emma madmacm ch wo>cma possmo Ca abscpaco an copommwc mm AEmov Coassaan mo mxcpmo msa .aa manB ca 64 Effect of Carbaryl and Diazinon on the Metabolism of i4C-Phenylurea Herbicides Diazinon had no apparent effect on the metabo— lism of the 4 phenylureas by cotton leaves. The parent compound was identified by a comparison with the Rf of the standard lac-herbicide. A major portion of 14C- linuron still existed as the parent compound after 5 days (Table 15). Plants receiving linuron alone had 81% linuron still unaltered as compared to 79% in the linuron plus diazinon treatment. There was essentially no change in distribution of metabolites in the two treatments. Table 15. The effect of diazinon on the metabolism of linuron by cotton leavesa. Rf Two Days Five Days Linuron Linuron +— Linuron Linuron +— Diazinon Diazinon 0.0 7.5 8.5 8.0 8.0 0.1 0.2 1.5 2.0 3.0 3.0 0.3 2.0 2.5 5.0 1.0 0.4 0.5 1.0 1.0 1.0 1.0 0.6 0.7 0.8 84.0 83.5 81.0 79.0 0.9 1.0 8Percent of total spotted. 65 14 After 2 days, about 82 to 84% of the C-monoli- nuron was still not metabolized by the cotton leaves and 5 days later 66% of the total 140 remained as mono- linuron (Table 16). There was also an increase in the quantity of metabolite(s) remaining at the origin. After 5 days, metabolites appeared at Rf 0.2 and 0.3 but there was no difference in the amount present between the treatments. Because of the poor rate of absorption observed 14 with C-diuron and monuron, it was decided to sample only at 5 days after treatment. Cotton leaves meta— bolized diuron and monuron at a relatively rapid rate 14 (Table 17). After 5 days, only 34% of the total C Table 16. The effect of diazinon on the metabolism of monolinuron by cotton leavesa. Rf Two Days Five Days Mono— Monolinuron—t Mono— Monolinuron-+ linuron Diazinon linuron Diazinon 0.0 9.0 8.5 21.0 22.0 0.1 0.2 — - 2.0 2.5 0.3 — — 1.5 1.5 0.4 0.5 3.0 3.0 3.0 4.0 0.6 0.7 0.8 82.0 84.0 65.5 66.0 0.9 1.0 aPercent of total Spotted. 66 remained as unchanged diuron. However, it should be noted that the quantity taken up was also less as com— pared to the methoxymethylureas. About 18% of the ra— dioactivity remained at the origin. A metabolite with an Rf of 0.3 accounted for 24% and 22% for both treat- ments indicating again that there was no difference in metabolism caused by diazinon. Table 17. The effect of diazinon on the metabolism of diuron and monuron by cotton leaves 5 days after treatment. Rf Diuron Diurona+ Diazinon Monuron Monuron-t Diazinon 0.0 18.0 19.5 0.1 1.5 1.0 18.0 15.0 0.2 49.0 48.0 0.3 24.0 22.0 13.0 10.0 0.4 14.0 13.0 0.5 7 9.0 5.010 8.0 0.6 33 5a 3 .5 0.7 0.8 0.9 1.0 a 14C—diuron b lLLC—monuron Monuron was most rapidly metabolized by cotton leaves (Table 17). After 5 days only 5% remained as the unaltered herbicide. A major percentage appeared at 67 Rf 0.2 but the distribution was similar for monuron alone and monuron plus diazinon. The metabolite was not positively identified, however, comparison with Rf value already published (70) showed that this Rf (0.2) corresponded to the demethylated derivative of monuron. The relative tolerance of cotton to diuron and monuron as shown by the capacity of the plant to meta— bolize these dimethylureas was once again demonstrated in this experiment. As mentioned previously, deme— thylase is believed responsible for the N—demethyla- tion of these compounds. The results of this experiment confirm previous reports that monuron was more rapidly metabolized than diuron by cotton leaves (70). It also appears that the monohalogenated ureas as a group may be metabo- lized more rapidly than the dihalogenated ureas. Dimethylureas are more easily degraded by cotton leaves than the methoxymethylureas. It seems likely that the tolerance of cotton to the dimethylureas and susceptibility to the methoxymethylureas is due to both differential absorption and metabolism. Metabolism of Linuron in Carrots. Carrot is tolerant to linuron. Two days after treatment with 14 linuron alone, only 24% of the total C was still the parent herbicide (Table 18). When linuron and 68 Table 18. The effect of carbaryl on the metabolism of linuron by carrot leavesa. Rf Two Days Five Days Linuron Linuron-t Linuron Linuron-t Carbaryl Carbaryl 0.0 50.0 33.5 57.5 50.5 0.1 5.0 5.5 0.2 7.0 5.5 4.0 3.0 0.3 0.4 3.5 2.5 4.5 2.5 0.5 6.0 4.5 6.0 3.0 0.6 0.7 0.8 24.0 42.5 16.5 23.0 0.9 1.0 8Percent of total spotted. carbaryl were applied together, 42.5% still existed as the parent herbicide after 2 days. The decreased rate of metabolism may, in part, explain the effect of car- baryl in enhancing the toxicity of linuron in carrots. The major metabolite(s) remained at the origin and there was a considerable reduction in lLLC-compounds present due to the combination treatments. Similar trends were evident after 5 days although the differ- ences were not as pronounced after that period of time. The carbaryl induced inhibition of monuron and propanil metabolism demonstrated by other workers (51, 71) was hypothesized to be the basis for the increased 69 toxicity of these herbicides. Tolerant plants appar- ently have or can synthesize the particular enzyme res— ponsible for the degradation of the herbicide. With monuron and carbaryl, synergism was evident in tolerant cotton and carrot but no interaction occurred in sus— ceptible soybean (Tables 3,4,6). In cotton, demethyla— tion occurs rapidly to produce nontoxic derivatives 'whereas in soybean, demethylation occurs much more slowly with the resulting accumulation of the toxic derivative, the monomethylurea (68). Demethylase iso- lated from cotton showed a much higher specific acti- vity than that isolated from soybean (24). This indi— cates that plant Species with more of that enzyme pre- sent will be more susceptible to phenylurea herbicides when carbaryl is introduced into the tissue. SUMMARY AND CONC LUS I ONS Insecticide Induced Enhancement of Phenylurea Herbicide Activity Four analogous substituted urea herbicides, li— nuron, monolinuron, diuron and monuron were applied alone and in combination with carbaryl and diazinon on tolerant and susceptible plant species. In toler- ant carrots, carbaryl enhanced the toxicity of linu- ron and monuron. In tolerant corn, mixtures of car- baryl with linuron and monolinuron were synergistic. When carbaryl was applied with diuron and monuron, increased toxicity was obtained in moderately toler— ant cotton. In susceptible soybean, toxicity occurred both from herbicide and insecticide treat— ments and all combinations appeared about additive. In all species, the addition of diazinon to foliage caused a significant increase in the foliar toxicity of the 4 phenylurea herbicides. The methoxymethyl- ureas were more toxic to all plant species than the dimethylureas. Foliar application of carbaryl had little ef- fect on the growth of cotton treated with herbicides through the roots. 0n the other hand, root appli- cation of carbaryl enhanced the toxicity of diuron 7O 71 and monuron applied to cotton leaves but did not have a pronounced effect on the activity of linuron and monolinuron. Similar results were obtained when both carbaryl and diuron or monuron were applied to the roots. The relative differences in the responses ob- tained with dimethylureas or methoxymethylureas and carbaryl may be attributed to the ability of the in- secticide to inhibit the activity of the enzyme demethylase. In contrast to carbaryl, diazinon increased the activity of the 4 phenylurea herbicides regardless of their substitution and in both tolerant and suscepti— ble plants. However, the response was obtained only if the herbicide and insecticide were applied together on the foliage. This suggested that diazinon or a com— ponent of its wettable powder formulation increased the foliar penetration of the herbicides. The nature of the linuron and carbaryl inter— action was investigated in parsnip leaf discs. Respi— ratory rate was not significantly affected by the com— bination of herbicide and insecticide. However, pho— tosynthesis, measured by oxygen evolution, was greatly reduced by the mixture of linuron and carbaryl as com- pared to linuron alone. This response indicated that more linuron may have reached its site of action when carbaryl was present. 72 Effect of Insecticides on Uptake of Phenylurea Herbicides Formulated carbaryl enhanced the rate of uptake of 14C-linuron in carrot leaves and parsnip leaf discs. In carrots, there was no significant difference in total uptake after 48 hr. It could not be ascertained from these tests if the increased rate of uptake was caused directly by carbaryl or another component of the wettable powder formulation. Increased uptake may be at least a partial cause for the enhancement of li- nuron activity by carbaryl. In tolerant cotton and susceptible soybean, the uptake of lb’C-linuron, monolinuron, diuron and monuron (with wettable powder) was greatly enhanced by the pre- sence of diazinon (wettable powder). Linuron and mo— nolinuron were much more readily absorbed by the leaves of both species than diuron and monuron. This may account for the difference in toxicity between dimethylureas and methoxymethylureas. Cotton and soy- bean leaves absorbed comparable amounts of herbicides, except linuron which was shown to be more readily ab- sorbed by cotton. Therefore tolerance of different species to phenylureas is not necessarily related only to the amount of herbicide absorbed. A significant increase in uptake of pure 140— linuron, monolinuron, and diuron was obtained with the 73 application of pure diazinon, although the magnitude of increase was not as big as that brought about by the formulated chemicals. It appears therefore, that the increase in uptake is caused partly by diazinon and partly by its surfactant. Effect of Insecticides on Metabolism of Phenylurea Herbicides Carbaryl caused a decrease in the rate of meta- bolism of linuron as shown by the presence of more parent herbicide in the carrot leaves treated with both compounds. There was a considerable difference in the amount of 14C-metabolite(s) present depending on treatments. Synergism from the combination of linuron and carbaryl, therefore, may be due to both increased uptake and a decreased rate of metabolism. Other workers have demonstrated that carbaryl inhi- bits the activity of demethylase obtained from cot- ton. The demethylated products are less toxic than the parent herbicide. Since carbaryl applied either to roots or leaves enhanced the activity of diuron and monuron applied to leaves, inhibition of herbi— cide metabolism is a logical eXplanation for the observed synergism. Diazinon had no apparent effect on the meta- bolism of the 4 phenylurea herbicides in cotton. 74 The basis for the observed synergism between diazinon and these herbicides is more likely a result of increased uptake brought about by diazinon and its surfactant. The dimethylureas (monuron and diuron) are more easily degraded by cotton than the methoxymethylureas (linuron and monolinuron). Likewise the monohalogenated ureas (monolinuron and monuron) are metabolized more rapidly than their dihalogenated counterparts (linuron and diuron). The relative tolerance of cotton to the dimethylureas and susceptibility to the methoxymethyl- ureas may be due to both differential absorption and metabolism. The selectivity of phenylureas in other plant species are likely also controlled by these two processes. Implications for Field Situations The pesticide interactions reported here were obtained from either tank mixes or applications made at short time intervals under greenhouse conditions. The study indicates that carbaryl and diazinon may enhance the activity of phenylurea herbicides, causing excessive damage in certain tolerant plants. Where insect and weed problems warrant the use of these, or related chemicals on a crop within short time intervals, further field tests should be con— ducted to ascertain that the applications do not 75 decrease the yield or quality of the crop. Some of these combinations at lower rates of application may be useful for enhancing herbicide activity on weed species without damaging crops. LITERATURE CITED 10. 11. LITERATURE CITED Abdel-Wahab, A.M., . J. Kuhr and J. E. Casida. 1966. 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