BASES FOR THE INTERACTION OF: magma; f , ‘ i ‘ " 311mm on 95139330me WIT-H CARBQFUVRAN; ON BARLEY-AND com - -» Thesis for thefi'egree-of‘f’h. D. V fl I MSCHiGAN STATE Una/mm : ‘ ‘ ALLAN. STEWARTHAMILL _ WW \ WWW 3 1293 01008 8130 LIB R A R Y Michigan 5? tat: Universi y '*“""‘l‘*"- - I." This is to certify that the thesis entitled BASES FOR THE INTERACTION OF ALACHLOR, BUTYLATE OR CHLORBROIVIURON WITH CARBOFURAN ON BARLEY AND CORN presented by Allan S. Hamill has been accepted towards fulfillment of the requirements for PhD. Crop and Soil fie ein gre Science ‘m y/j) - ’/ f ’7 I —-\ (ch-14:: - "re, 7'L"?’ Major professor Date July 9, 1971 0—7639 FEB 1 3 20m .C. anhkrnw v. U- u. q 7 A h \ vs.‘ ( 5 .v «\w A V c no - tn .. .x W s Av «so \ «n‘ Y. I a L w ABSTRACT BASES FOR THE INTERACTION OF ALACHLOR, BUTYLATE OR CHLORBROMURON WITH CARBOFURAN ON BARLEY AND CORN By Allan S. Hamill Numerous herbicide-insecticide combinations were screened on barley (Hordeum vulgare L. var. Larker) and corn (gga mays L. var. Michigan A00) for phytotoxic interactions. The herbicides, 2—chloro-2',6'—N-(methoxymethyl) acetanilide (alachlor), g-ethyl diisobutylthiocarbamate (butylate) and 3-[4-bromo-3-chlorophenylJ-l—methoxy-l-methylurea (chlorbromuron) were combined with the insecticide 2,2- dimethyl-2,3-dihydrobenzofuranyl-7—N-methylcarbamate (carbofuran) for respiration, photosynthesis and metabolism studies in the growth chamber. The herbicides were applied preemergence and the insecticide as a seed treatment. A germination experiment indicated all three herbicides synergistically reduced the radical length of barley seed- lings and the alachlor—carbofuran treatment synergistically reduced germination. A statistical procedure was developed for the calculation of the significant difference between the calculated expected value and the observed value, as proposed by Colby, for pesticide combinations in experiments with a completely randomized design. no. 4.. fl; flu. w u my . . BC . 0 . ‘ A r . Y. ad .14 AC AG M. r. a . lllljlls . ‘ o . I A W. flu hfi Alv 9! * hs “1% 11‘ , av w. o.‘ w. by 4. 1 . y a 1i sq ‘u 8 AL “L; o NV 0 c a .fla + .. a : . .r.N a S s k e . i r 3 OH. .0; 4.. u k e e .T C n a h t w C a .. i P b + o. r e r e C t O y... P C .a . J 1 . L V C .1. .W .i. a 9;» as e S i «-u any 4. S Q» ad. u P u e C I a S a i 0 Fe 9 r. a n. ma 3 n. e C 5. no .3 C 0.. mu 5 n. c e .. a 5 he C .‘g a» e .h» a. a . Ad hi «Q g c e A ”w . a 3. r“ n... L u .d “4 kw he at; Ru 3 AL a: a: h. ad to n c F c L a 2. z. n» s . .nu ~m Aw a» a» c. a he. A» cl. 3. 2. a. we r“ a . rd. no a» Q. A. r... h. L.” «.0 w4 0 . no. .n... H. flv ” S. n. .«l r... :u u. _..u n. A.» 2 . my. a . Lu k... F- «C s. g. slv C oi s O y a .n. d s u \ 3o 7. h». “I; V! \ RU Allan Stewart Hamill Carbofuran interacted with alachlor to synergistically reduce barley but not corn germination and growth. Alachlor was found to increase the respiration rate of barley. luc accumulation from l“Cnalachlor was greater in the plant roots than in the shoots. The basis for the observed interaction appeared to be greater alachlor uptake by barley plants which had received the carbofuran seed treatment. Specifically the increased accumulation of alachlor in the roots was accentuated by the reduced rate of alachlor metabolism in the roots. Carbofuran interacted with butylate to synergistically reduce barley, but not corn root and shoot growth. The combination of these two carbamate pesticides synergistically increased respiration in barley. luc from luC-butylate preferentially accumulated in barley shoots and corn roots. The bases for these interaction effects in barley appeared to be increased absorption and decreased metabolism of butylate, as well as increased respiration in the presence of carbofuran. Although the same metabolism trend was apparent in corn, the butylate level was much lower since the absorption of butylate was reduced by the carbofuran treatment. Carbofuran interacted synergistically with chlorbromuron to reduce the height and weight of Al-day-old corn, the root length of 3-day-old barley and the leaf area and dry weight of 7-day-old corn grown in sand culture. The W. G. 1n.— r u ya” u e m” c e V. e . t C r” a. a n“ M. "xiv - . 1 . CW .5 S n. l. w h n . Av 29 w .. L y A... s . Lao A a a: u . A V A... D. .r.. .r” r” n .c w . r. u v u. I. . pub L a II II |. ill-I‘ll l‘lv I y... S O a K e w .. a i A... Ti a: by 0. Ac 1. A a. .n“ h. C S n... f. m. S C n e .s i r... . C n S n; S .3 e s n,“ V; 4 a H . O H... M . ha ‘1. 3 . C. 0. fly he be flu» . . s L... n 5 a. . a; A: n. “a a. a. .5 a. . r“ A,” a: .un 9‘ .r.. a. u. . C» s a .r u b v no. . t 4 v. . ‘1 ‘ AV 4 ‘ J ‘ e n». RM to S Q... n he G» ran a. C» my 5 e a. .C . . n. . a. 2. We. Allan Stewart Hamill chlorbromuron-carbofuran combination reduced net photosynthesis in barley and corn and increasedrespiration in barley. 1“C from luC—chlorbromuron preferentially accumulated in barley and corn shoots. The carbofuran seed treatment reduced the level in barley shoots and corn roots and increased the content of 1”C in barley roots and corn shoots. The basis for this interaction appeared related to the increased accumulation of chlorbromuron in corn and barley shoots, due to reduced chlorbromuron metabolism, thus increasing the parent chlorbromuron content. These factors contributed to an extended period of exposure of the corn leaf to the herbicide causing the physiological responses measured. Thin layer chromatography indicated a number of different herbicide metabolites in the root and shootof each species. In many instances, these metabolitesuere altered in quality and/or quantity when carbofuran was present in the treatment medium. In BASES FOR THE INTERACTION OF ALACHLOR, BUTYLATE OR CHLORBROMURON WITH CARBOFURAN ON BARLEY AND CORN By Allan Stewart Hamill A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences E .C e . Pr. :2 . . . a . w. C. .C 1-. y D» 9‘ a: can. Co a» {A l .H r.“ i h. .T. 7. a r _ . O C C t W G a a 4 ... S 4. 3 F. at I T: L S . .1 2 To 3... S V. n. w. 2. t n. :. C . . a . .u .. 2. u-... 3.. 3“: “4-381;; 1 v“. I'll'llllllll [.[[([r[[ l‘l [ [II-[t ,,/ .p "‘1 K ~ f," x I , . I . . v ACKNOWLEDGEMENTS The author wishes to express his sincere gratitude to Dr. Donald Penner for his guidance and constructive criticism during the investigation and preparation of this manuscript. The assistance of Drs. N. C. Leeling, w. F. Meggitt, A. R. Putnam and E. C. Rossman is also gratefully acknowledged. The technical assistance of Miss Pamela Agren, Miss Gail Berke, Mrs. Karen Dolega, Mrs. Terry Rice and Mr. Roy Early is also sincerely appreciated. Last, but certainly not least, the author gratefully thanks his wife Anne, for willingly typing the manuscript and her constant support. 11 ... ... ; . r c t. .L , v. n . ~z. my Q. “If «U «.n T. v. . a; 1; h; u ml. d H“ 1mm «b a. m . b. m i 3.. l .t O a. n3 .1 e .1 .1 e r. n v n V 6a 8 fl . Pu v... n W; P. A» Dr. . D r... v. .7. m . P i. u .0 rd 9 e .1 .l Tn Av my 5.4 .4” a» La A» h. r a e t +0 #0 Nu m o n .. . r-” a a .1 D“ h. «L S S n... v .. a . m . .1 .1 E t . . .n e r. C tr. 9 e a u.“ a; a: . . . . ad a... 1.. fi . Q.» 3. D. n a v . v . u. \. v .. n. J. I5” - a v. a v . v u III. ll... illrll .IJ. TABLE OF CONTENTS ACKNOWLEDGEMENT. LIST OF TABLES .LIST OF FIGURES. INTRODUCTION LITERATURE REVIEW. General Features of Pesticide Combinations . Statistical Analysis of Combinations . Alachlor, Butylate, Chlorbromuron and Carbofuran. . . . MATERIALS AND METHODS. Screening of Pesticide Combinations for Altered Phytotoxicity Pesticide Combination Effects on Germination Pesticide Combination Effects on Photosynthesis and Respiration . . . . . . . . . . . . . . Treatment of Plants with lac-Herbicides. Extraction and Analysis of Radioactive Material. . . . . . . . . . . . . . . Thin Layer Chromatography of Radioactive Extract RESULTS AND DISCUSSION Greenhouse Screening of Pesticide Combinations Germination Effects. Statistical Analysis Method. Effect of Alachlor on Growth, Photosynthesis and Respiration . . . . . . . . . iii Page ii vi ix 2U 26 28 28 31 33 33 I 1" U V . . .. v: 1. v. an. t. u. C. n. r\ “v I. "A C E O "J C t n. .x.. a; To an E S 3 i r a .38 an 2.. Z t D“ t O 9 .5 9 a . e P Fe To C C t .1.“ C at V. .K O V. D. Ti H . 9 .3 o. C a a a +. a 1 Dr. A e x. . _ 0.. w” no. .2 cu . . so .3“ +« .n.. .L... HA n. n. a 0,. C. 3.5.. 3.3.. DA V n... .. TS a.» T. T. u» n. um; "7.. «xy 2. u... .v c T. 5». a.“ as» . d t... TABLE OF CONTENTS (Cont.) Page RESULTS AND DISCUSSION (Cont.) Effect of Butylate on Growth, Photosynthesis and Respiration. . . . . . . . . . . . . . 36 Effect of Chlorbromuron on Growth, Photosynthesis and Respiration . . . . . . . . . “9 Uptake, Translocation and Metabolism of luc— Alachlor . . . . . . . . . . . . . . . . . . . . 58 Uptake, Translocation and Metabolism of luc- Butylate . . . . . . . . . . . . . . . . . . . . 72 Uptake, Translocation and Metabolism of luc- Chlorbromuron. . . . . . . . . . . . . . . . . . 87 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . 10“ LIST OF REFERENCES. . . . . . . . . . . . . . . . . lll APPENDICES. . . . . . . . . . . . . . . . . . . . . 117 iv LIST OF TABLES Table l. The effect of carbofuran seed treatment on the height and dry weight of 37—day~old barley which received various preemergence herbicide applications. The effect of carbofuran seed treatment on the height and dry weight of Ul-day-old corn which received various preemergence herbicide applications. . . . . . . The effect of carbofuran seed treatment on seed germination and maximum root length of 3-day—old barley seedlings which received various preemergence herbicide treatments The effect of alachlor, carbofuran and the combination of alachlor and carbofuran on the growth of lU-day-old barley The effect of alachlor, carbofuran, and the combination of alachlor and carbofuran on respiration and photosynthesis on lu-day— old barley. . . . . . . . . . . . . . . The effect of alachlor, carbofuran and the combination of alachlor and carbofuran on the growth of 7-day-old corn. The effect of alachlor, carbofuran and the combination of alachlor and carbofuran on respiration and photosynthesis of 7-day- old corn. . . . . . . . . . . . . . . . . . The effect of alachlor, carbofuran and the combination of alachlor and carbofuran on the growth of 7-day-old corn. The effect of alachlor, carbofuran and the combination of alachlor and carbofuran on respiration and photosynthesis of 7-day— old corn. . . . . . . . . . . . . . . Page 29 3O 35 37 38 39 U0 U1 .m “In” T “12?... c.f.r.. fr: fr}: 1).-.. . . . .. e .1 r: E i ... t e i .1 E .1 r: e i .1 a e .1 my 8 .1 .1 C E C .C e C E .C E C .3 t .C C. .C C. w... .3 .C C. ‘C .A .C C. .C r .C «I a: e I 9 e I S .3 e T. S C e K a. E 7.. E .3 e T. O E f S .3 e e T e e .3 e e P 3 Po .5 C .3 .3 3 a. .. .2 C e n. h C .F. .1. O a. 7. .5 O P 5.. C e t. L... f. a .5. .7. ....‘ 5...: a E . a." .C5. ”.3? Q “.3 Poi Mi CZ ~.C TC «.2 3: ~.C PC ~.gcC m..: CC mig.C..« . < «xv uhlv 0 I O o I O I I I I .. i .3 l u .2 u. 2., T.[{ I III I l[!'\."llt:rl|’l\[|[\[lll\[ll\y{ll|il\r ll I'll/It‘ll ([{l l (flllll‘ll‘ [III [ [I LIST OF TABLES (Cont.) Table 10. ll. l2. 13. 14. 15. 16. 170 18. 19. The effect of butylate, carbofuran and the combination of butylate and carbofuran on the growth of lA-day-old barley. The effect of butylate, carbofuran and the combination of butylate and carbofuran on respiration and photosynthesis of 14—day— old barley . . . . . . . . . . . The effect of butylate, carbofuran and the combination of butylate and carbofuran on - respiration of excised barley leaf tips from lu-day-old barley plants. The effect of butylate, carbofuran and the combination of butylate and carbofuran on the growth of 7-day-old corn . The effect of butylate, carbofuran and the combination of butylate and carbofuran on respiration and photosynthesis of 7- -day- old corn . . . . . . . . . The effect of butylate, carbofuran and the combination of butylate and carbofuran on the growth of 7-day-old corn . . . . . . The effect of butylate, carbofuran and the combination of butylate and carbofuran on respiration and photosynthesis of 7-day- old corn . . . . . . . . . . . . The effect of chlorbromuron, carbofuran and the combination of chlorbromuron and carbofuran on the growth of lO-day-old barley. The effect of chlorbromuron, carbofuran and the combination of chlorbromuron and carbofuran on respiration and photosynthesis of 10—day- old barley . . . . . . . . . . . . . . . . The effect of chlorbromuron, carbofuran and the combination of chlorbromuron and carbofuran on respiration of excised barley leaf tips from lO-day-old barley plants. vi Page 42 “3 U6 “7 A8 52 53 LIST OF TABLES (Cont.) Table 20. 21. 22. 23. 2A. 25. 26. 27. 28. 29. The effect of chlorbromuron, carbofuran and the combination of chlorbromuron and carbofuran on the growth of 7-day-old corn. The effect of chlorbromuron, carbofuran and the combination of chlorbromuron and carbofuran on respiration and photosynthesis on 7-day-Old corn. . . . The effect of chlorbromuron, carbofuran and the combination of chlorbromuron and , carbofuran on the growth of 7-day—old corn The effect of chlorbromuron, carbofuran and the combination of chlorbromuron and carbofuran on respiration and photosynthesis of 7-day—old corn. . . . . . . . The partitioning and distribution of 1“C- alachlor in lu-day-old barley and 7-day—old corn grown in nutrient solution containing alachlor, 10 and 5 days respectively,after luc- alachlor application . . . . . . . The partitioning of 1”C as a percent of the 1“C present in the shoot or root of lA-day- old barley and 7-day-old corn grown in nutrient solution containing alachlor, lO and 5 days respectively, after l“C-alachlor application. . . . . . . . . . . . . . . The separation on TLC of shoot and root extracts from 14- -day- -old barley 10 days after luc- alachlor treatment . . . The percent extractable lL‘C-alachlor remaining in lA-day-old barley and 7-day- old corn shoots and floots, 10 and 5 days respectively,after 1 C-alachlor treatment. The separation on TLC of shoot and root efitracts from 7-day-old corn 5 days after C-alachlor treatment . . . . . . . . The partitioning and distribution of luc- butylate in lA-day-old barley and 7—day—old corn grown in nutrient solution containing butylate, 10 and 5 days respectively,after luc- butylate application . . . . . . vii Page 55 56 57 59 65 66 68 7O 71 79 LIST OF TABLES (Cont.) Table 30. 31. 32. 33- 3A. 35. 36. 37. 38. The partitioning of 1“C as a percent of the 140 present in the shoot or root of lU-day- old barley and 7- -day- -old corn, grown in nutrient solution containing butylate,l and 5 days respectively, after 1 C butylate application. . . . . . The separation on TLC of shoot and root extracts from lA- -day- -old barley, 10 days after 140- butylate treatment . . . . . . . The percent extractable luC-butylate remaining in lU-day-Old barley and 7—day- old corn shoots and roots, 10 and 5 days respectively, after lL‘C-butylate treatment. . . . . . . . . . . . The separation on TLC of shoot and root extracts from 7-day-old corn 5 days after lL‘C—butylate treatment The partitioning and distribution Of luc- chlorbromuron in lO-day-old barley and 7-day-old corn, grown in nutrient solution containing chlorbromuron, 6 and 5 days respectively, after luC-chlorbromuron, application. . . . . . . . . . . . . . . . The partitioning of 1“C as a percent of the 1”C present in the shoot or root of 10-day- old barley and 7-day—old corn, grown in nutrient solution containing chlorbromuron 6 and 5 days respectively,after 19C- chlorbromuron application. . . . . . . The separation on TLC of shoot and root extracts from lO—day-old barley, 6 days afterlMC-chlorbromuron treatment. . . . The percent extractable lL‘C-chlorbromuron remaining in lO-day-old barley and 7-day- old corn shoots and roots, 8 and 5 days respectively, after ltic-chlorbromuron treatment............ The separation on TLC of shoot and root extracts from 7-day-old corn, 5 days after lac-chlorbromuron treatment. . . . viii Page 75 81 82 85 88 9A 96 97 100 TII\(-\.fl\.[ I'll" Ill l.[)l|\)!lll Sf.ll|[l\f.'l‘.fl‘l[zt\(l)llll\l’n|l|\’lflll l.ll.ll\{'l.l[ ({{I‘ ll" [olfl‘lll .‘1 (III (II LIST OF FIGURES Figure Sealed plastic chamber used for photosynthesis and respiration studies. The inside of a growth chamber containing the microchambers and trapping mechanism used in the growing of barley and corn treated with lac-butylate Statistical equations used for the calculation of significant difference between the observed and expected values' for plant response to chemical combinations Barley plants (left) and radioautographs (right) of barley plants harvested 10 days after l“C-alachlor was placed in the nutrient solution. Upper: luc- alachlor treated plants. Lower: l14C— alachlor treated plants in the presence of carbofuran as a seed treatment . . . . Corn plants (left) and radioautographs (right) of corn plants harvested 5 days after l”C-alachlor was placed in the nutrient solution. Upper: luC-alachlor treated plants. Lower: luC-alachlor treated plants in the presence of carbofuran as a seed treatment. Barley plants (left) and radioautographs (right) of barley plants harvested 10 days after luc-butylate was placed in the nutrient solution. Upper:l”C-butylate treated plants. Lower: ltic-butylate treated plants in the presence of carbofuran as a seed treatment. ix Page 18' 21 33 6O 62 76 3|", I‘ll! lilli'llllll‘ [.ll‘ 'l‘ ‘I l l LIST OF FIGURES (Cont.) Figure 7. Corn plants (left) and radioautographs (right) of corn plants harvested 5 days after l“C-butylate was placed in nutrient solution. Upper: lL‘C-butylate treated plants. Lower: luC-butylate treated plants in the presence of carbofuran as a seed treatment. Barley plants (left) and radioautographs (right) of barley plants harvested 6 days after lL‘C-chlorbromuron was placed in the nutrient solution. Upper: 1A0- chlorbromuron treated plants. Lower: l”C- chlorbromuron treated plants in the presence of carbofuran as a seed treatment. Corn plants (left) and radioautographs (right) of corn plants harvested 5 days after l“C-chlorbromuron was laced in the nutrient solution. Upper: 1 C— chlorbromuron treated plants. Lower: l“C- chlorbromuron treated plants in the presence of carbofuran as a seed treatment. Page 78 89 91 INTRODUCTION There have been many reports in recent years of agricultural chemicals interacting to give both desirable and undesirable responses on plants (l,2,5,A3,57,6l,67). In agriculture at least two or more chemicals, either insecticides, herbicides, fungicides or fertilizers are generally present in the plant microenvironment at the same time. Registration of pesticides requires information on the action and fate of the chemicals in the environment. However, little information is available concerning the interaction among the various agricultural chemicals that could be present simultaneously in the environment. In several instances severe damage has been reported in economic crOps following the simultaneous or sequential application of pesticides (8,21,28,35). In other situations chemical combinations promoted the growth of the crop plant and still adequately controlled the weeds (2,3,15,52). Social pressures have dictated that pesticide residues in foodstuffs be kept at minimum or zero levels. Social concern for environmental quality has been reflected in the rejection of persistant pesticides and new criteria for registration of existing and new compounds. At present, adequate information concerning the fate Of pesticides in combination with other pesticides is unavailable. It would be beneficial to know in advance the effect of potential combinations on their action and persistance before widespread use . !.l'l[‘|l|..'llv'li“ llllilll'll The purpose of this study was to investigate possible herbicide-insecticide combinations with respect to changes in phytotoxicity. Several combinations that increased or decreased phytotoxicity were selected for further investigation to obtain at least in part, the basis for the interaction. Literature Review General features of pesticide combinations: Pesticide combinations have become one of the more important components of weed control. Herbicide combinations have been employed for well over a decade because of the ineffectiveness of even a so-called broad spectrum herbicide to adequately control all weed species (27). Other early applications of herbicide mixtures stemmed from the need for more effective weed control in areas where other crOps dictated the limited use of 2,U- dichlorOphenoxy acetic acid (2,A-D) (59). Two or more narrow spectrum herbicides now may take the place of a broad spectrum herbicide lengthening the time of a weed-free environment and permitting lower application rates of the chemicals involved (16). Mixtures of agricultural chemicals have been used to a large extent on sugar beets (Beta vulgaris L.), cotton (Gossipium hirsutum L.), beans (Phaseolus vulgaris L.) and corn (Zea mays L.) (9,29,32,3A,UU,U5,58,59). Combinations synergistic in their action have been found by Putnam gt a1. (56) and Nash (“9). Synergistic combinations may not always be beneficial. Bowling at al. (8) found that some organOphosphate or carbamate insecticides applied to rice (Oryza sativa L.) seedlings interacted synergistically with 3,h-dichlorOpropionanilide (propanil) to kill the rice. The herbicide propanil.was used for the control of barnyard grass (Echinochloa spp.) and other weeds [t[{l‘l‘({{»{ (I‘ll A in rice. This interaction occurred even when the chemicals were not applied at the same time. Certain pesticide combinations have shown an antagonistic response (5,55,61). In this situation, the chemicals interacted in a manner to reduce phytotoxicity. Numerous explanations for the deviation from the predicted results have been offered. Davis gt a1. (20) determined that l,l—dimethyl-U,U bipyridinium salt (paraquat) increased A-amino-3,S,6— trichlorOpicolinic acid (picloram) uptake in some plant species while it reduced transport in others. Uptake and transport of 2,A,5-trichlorOphenoxy acetic acid (2,A,S-T) decreased in the presence of picloram, but the uptake and transport of picloram was increased by 2,U,5-T. Agbakoka §£.§l- (1) reported that picloram enhanced 2,4-D movement in bindweed (Convolvulus arvensis L.). Arle (3) incorporated the insecticides g,g-diethyl §-[(ethy1thio)-methy1]— phosphorodithioate (phorate) or g,g-diethyl S—[2(ethylthio)- ethyl]-phosphorodithioate (disulfoton) in «,¢,«-trifluoro- 2,6-dinitro-N,N—dipropyl-p-toluidine (trifluralin) treated soil which resulted in increased cotton seedling growth when compared to the trifluralin alone. He concluded that the greater number of secondary roots in the zone of incorpora- tion accounted for the results. Smith (60) found similar results on germinating corn and wheat (Triticum spp.) with trifluralin and organic phosphate insecticides. Il.‘ l[[l.’l![{[w“[ Swanson and Swanson (62) found that simultaneous treatment of cotton leaf discs with certain carbamate insecticides inhibited the further degradation of l-(p— chlorophenyl)-3-methylurea (monomethylmonuronh but not the step from 3-(p-chlor0phenyl)—l,l-dimethylurea (monuron) itself to monomethylmonuron. Chang gt gt. (12) reported insecticide inhibition of the degradation of a number of different herbicides in isolated leaf tissues. The tolerance of rice to prOpanil is believed to be related to metabolic degradation of propanil (U3). However, in the presence of an organophosphate or organothiOphosphate insecticide, rice becomes susceptible to propanil. These insecticides inhibit the enzyme which further metabolizes the first metabolic breakdown product of propanil. This enzyme is believed to be similar to acetylchlolinesterase. Another method postulated as a mechanism for the interaction of pesticide combinations was that of Beste gt gt. (6). They determined that following §~ethyl dipropylthiocarbamate (EPTC) plus 2,A-D treatments, sorghum (Sorghum vulgare Pers.) respired normally while respiration of EPTC treated plants was inhibited. These workers also postulated an interaction effect on nucleic acid metabolism as a basis for the antagonism between 2,4-D and EPTC (7). This seems logical as the carbamate inhibits cell division (A,51) and the phenoxyacetic acid promotes cell division (Sl). .llll It'll-‘1 lll[.t[’ll [l[1[[.[{llll Statistical analysis of combinations One of the most difficult problems confronting a researcher involved in working with pesticide combinations is expression of the data, correctly indicating interactions observed. Gowing (26) applied the concepts of probit analysis to herbicide research. This involved a sequential expression of data, in graphical form, from the simple percent inhibition versus concentration to percent inhibition versus log concentration, and finally to percent inhibition in a probability distribution against the log concentration. This method permitted obtaining a value for fifty percent inhibition level for a chemical. This level was designated as a toxic unit. The mixtures of chemicals were then made up in various ratios of toxic units and the results plotted on a graph involving percent inhibition against toxic units. The expected value for the combination was a line connecting points giving a one to one ratio. Chase gt gt. (l3) expanded on the probit analysis method by determining the percent inhibition as a percent of control and then using the difference between this and one hundred before plotting against log concentration. In another report, Gowing (27) compared three methods of data expression: percent response versus concentration of herbicide, percent response with log concentration, and reciprocals of response and concentration. He pointed out that all methods may be useful but caution must be used with any of them. Tammes (63) interpolated data for two chemicals at five rates sprayed in all combinations on the same logarithm- probit diagram. He then took the fifty percent mortality probit level and plotted it on regular graph paper with the rate of one chemical required on the Y axis and that of the other on the X axis. The pictorial presentation of the isobole determined synergism or antagonism. For accurate isobole plots, a considerable amount of data must be generated. The influence of two or more different factors can be tested by a factorial arrangement of the treatments in a suitable experimental design. Inherant in the analysis of variance of a factorial is the interaction term (A2). A significant interaction term in the analysis of variance would imply that this combination was different from single treatments. This method indicates, however, that each of the treatments alone contributed in an additive manner to the combinations. Some researchers have used this approach with an accompanying Duncan's multiple range.test to Show that a significant interaction occurred (10,30,36). 'Others simply analyzed combinations, as if they were Just a single treatment and applied a comparison test to the means (3, 31,35). The quadratic equation which contains an interaction term to estimate the regression coefficients has been used by other workers (A8,5A,66). This equation necessitates II 'I ll' [tr . L ll! II" I. .. IIII.‘[ l-[Il ([.([ {(.[ the use of at least three levels for each treatment which lends itself to the factorial experimental design (1A). The regression line established for the two components indicates the effect of one compound on changing levels of the other. The interaction term in this equation, however, as in the factorial, is additive in nature with each term contributing in that manner. Colby (l7) approached the interaction term with the assumption that interactions occurred in a sequential fashion rather than in an additive manner. His method involved calculating an "expected" or predicted response for the combination. The easiest method for calculation of the expected value is: (percent of control for A) (percent of control for B) 1100 This expected value for the combination is then compared to the observed percent of control value obtained for the treatment where the two chemicals were combined. Synergism is stated to occur if the observed value for compound A plus B is less than the calculated "expected" value while antagonism occurred if the observed value is greater. This formula can be extended to include a third compound if necessary. Colby (17) has attempted to determine statistical significance of the difference between the expected and Observed values by Chi square analysis and analysis of variance on the logarithmically transformed percent of control values. The Chi square analysis has been II I .IllIll-I‘.f.‘.."n._l‘ir l[.l[{[ ([{[.[(‘l 9 shown to be not valid for this type of data (18). The other test is not applicable for a completely randomized design experiment. Alachlor, Butylate, Chlorbromuron and Carbofuran: 2-Chloro-2L6Ldiethyl-N-(methoxymethyl) acetanilide (alachlor), is recommended for control of most annual grasses and certain broadleaf weeds in corn and soybeans. It is applied preemergence either to the soil surface or preplant incorporated at one to four pounds per acre. Alachlor is primarily absorbed by the germinating plant shoots and secondarily by the roots followed by translocation through— out the vegetative parts of the plant (68). Following application of alachlor to the soil, high concentrations are found in the initial emerging plant shoot and relatively smaller amounts in the reproductive sections, or younger more actively growing points. In contrast to soil application of alachlor, foliar applications to susceptible species showed some downward movement with fairly uniform distribution. Resistant species displayed significant accumulation in the cotyledons and young growing points (11). Cotton plants exposed to soil and nutrient solution containing alachlor showed stunting and inhibition of lateral root growth, but no foliar chlorosis (2A). Chandler (11) found that a susceptible species, wheat, took up more alachlor via the root system than did a tolerant species, soybean. Alachlor is readily leached in lighter sandy soils but is adsorbed to colloidal particles in the soil. llall'l‘rtr 1l[[[["[[{rll [ll-Ill 10 The mechanism of action for this herbicide is not known, however, the compound is closely allied to 2-chloro—N- isoprOpylacetanilide (propachlor) for which some mechansim of action information is available. Jaworski (33) reported work done by Duke with cucumbers (Cucumis spp.) which showed that propachlor inhibited nucleic acid synthesis and protein synthesis. The protein synthesis step was hindered first. , It was shown that propachlor prevented the activation of amino acids and the transfer of the aminoacyl—tRNA to the polypeptide. Duke (23) later reported the prevention of l“C"leucine incorporation into protein as an early step in the mode of propachlor action. Dhillon (22) substantiated this work with the same results for germinating squash (Cucurbita spp.) seedlings. Propachlor was shown to be metabolized to at least three water-soluble metabolites within twenty-four hours in each of four different species. One of the metabolites has been identified as a glutathione conjugate of the herbicide (Al). The structural resemblance of alachlor and prOpachlor point to a similar mechanism of action and possibly metabolism. §-ethyldiisobutylthiocarbamate (butylate) is a selective incorporated preemergence herbicide for control of seeded perennial grasses and annual grasses in corn. Some broad-leaf weeds are controlled by 3 to A lb/A of butylate under favorable conditions. It is rapidly taken up by plant roots and transported acropetally to the whole plant. Sandy soils permit butylate leaching, which Ill I'll-$.(IIIII‘I' ll'l.‘ ({[I (1{([..[[.Il 11 decreases as the clay and organic matter increase. Volatilization from moist soil and microbiological breakdown substantially contribute to butylate loss from the soil and are factors contributing to a half life of one and a half to three weeks in certain soils (68). Threewitt (65) found the loss of activity of butylate for rates up to 10 lb/A to follow a parabolic curve. The length of time required for disappearance was shortened for an early spring application compared to a late winter application. However, while biological activity was reduced in the above manner, a colorimetric test showed the remaining butylate to be in a physiologically inactive state. The mechanism of action for this herbicide is unknown. It does appear to inhibit growth in the meristematic region of the leaves of grass plants (68). A report of increased weed seed germination induced by butylate vapors perhaps by the breaking of semi-dormancy has also been postulated as a mechanism of action (6A). There is little persistance of butylate in corn as it is degraded to CO2 and natural plant compounds within 7 to 1A days (68). Most annual grasses and broad-leaf weeds are controlled by 3-[A-bromo-3-chlorophenyl]-l-methoxy—l-methylurea (chlorbromuron) at l or 2 lb/A as a preemergence broadcast, banded spray or as a directed postemergence spray before the weeds are two to three inches high (68). It is reasonable to assume that the mode of action of chlorbromuron is similar to other substituted urea herbicides 12 and as such is a potent inhibitor of photosynthesis (38). The metabolic pathway of dimethyl substituted urea herbicides degradation in plants has been shown to proceed by demethylation to the monomethyl derivative then to phenyl urea and occassionally to aniline. The monomethyl derivative is phytotoxic. However, for the methoxy methyl urea 3-(3,A-dichlorophenyl)-l-methoxy-l-methyl urea (linuron) the first product, the methoxy derivative, is relatively nonephytotoxic. Nashed gt_gt. (50) obtained evidence for the formation of the non—phytotoxic methoxy derivative and for "binding" of chlorbromuron in corn shoots and roots. Three other metabolites were also present in 3-week-old corn treated for eight days prior to harvest; the methyl, the phenylurea, and the aniline derivatives. Metabolism of chlorbromuron in corn was evident two days after treatment with the former two metabolites, first detected at four days. No metabolites of chlorbromuron could be detected in the susceptible 3-week—old cucumber treated four days prior to harvest. The carbamate insecticide 2,2-dimethyl-2,3—dihydrobenzo- furanyl-7-N—methylcarbamate (carbofuran) in soil treatments not only controls root-attacking pests but also foliar feeders as well. The active ingredient is absorbed by the roots and translocated throughout the plant. 'It is to be used as foliar contact spray at 1/8 to l lb/A or as a soil applied plant systemic, at 1/2 to 8 lb/A depending on the crop and formulation (53). Carbofuran is recommended for 13 use in corn at 7 1/2 to 10 lb applied in a 7 inch band, per 13,000 linear feet. In plants carbofuran is metabolized by hydroxylation and hydrolysis to at least four metabolites all of which, as well as existing freely, conjugate to form glucosides (39,AO,A6). The cereal leaf beetle which was once considered a menace to small grain production in the United States is now considered a critical threat. The use of carbofuran as a seed treatment for small grains has successfully protected these crOps for about one and one-half months after planting. The seed treatment has reduced the amount used and cost of insect control and decreased danger Of environmental contamination through reduced application rates and lack of drift. For these reasons and because carbofuran has demonstrated promise for use as a seed treatment it was included in this study (A7). Alachlor, butylate, and chlorbromuron are all relatively new herbicides recommended for corn. Their demonstrated potential as herbicides and the limited residue period indicate greater future use. MATERIALS AND METHODS Screening of Pesticide Combinations for Altered Phytotoxicity Numerous pesticide combinations were tested in the greenhouse in completely randomized design experiments at temperatures of 25 C to 35 C and 16 hours day length. Ten barley (Hordeum vulgare L. var. Larker) or corn (Zea mays L. var. Michigan A00) seeds were planted in greenhouse potting soil (peat:loam:sand; l:l:l) in 32 oz waxed cottage cheese containers with drainage holes in the bottom. The pots were initially watered with approximately one inch of water. Thereafter, the pots received water as needed and fertiliza— tion once a week with a 28-18-8 fertilizer applied at the rate of 100 lbs actual N per acre. The herbicides were applied at the time of seeding with a movable table sprayer. The pressure system was activated with a CO2 cylinder to 30 pounds pressure and the water volume was equivalent to 100 gallons per acre. The insecticides were applied either as a granular, flowable, or seed treatment at the time of seeding or as a wettable powder 7 to l0 days after seeding. Insecticide seed treatment was done with a sticker, glycerol:95 percent ethanol:water (l:l:l; v:v:v) at the rate of 0.5 ml for 100 g of corn seed and 2.0 ml for 100 g of barley seed. The seed, sticker and insecticide were shaken together in a container for a minimum of A minutes 1A 15 and then dried under the hood. Granular insecticide was applied with the seed at seeding. The pots were thinned to 5 plants per pot within 10 days after planting. The pots were re-randomized on the greenhouse bench on a regular basis. Barley experiments were terminated at 37 days, and corn at Al days. The pesticide combination effects on plant growth were measured by recording individual plant height and dry shoot weight per pot. Data were subjected to analysis of variance, converted to percent of control and combinations were evaluated according to the method of Colby (17). All values stated in the tables and appendix are the means of two experiments with two or more replications per experiment. Pesticide combination effects on germination Alachlor, butylate and chlorbromuron each at 10‘5 M, alone and in combination with carbofuran as a seed treatment at A oz/lOO lb of seed were studied for combination effects on germination and seedling vigor. The herbicide solutions were made up in sterile water at pH 7.0 containing 20 ppm Of streptomycin sulfate. Glass Petri dishes with two sheets of Whatman No. 2 filter paper were autoclaved and received 10 ml of the various solutions. Three replicates with 10 seeds per dish of each treatment were placed in the dark at 20 C for 3 days. Radical length and germination number were noted. Data were subjected to tests previously described. Pesticide combination effects on photogynthesis and respiration Ten corn or barley seeds were planted on washed sand in l6 6 oz styrofoam cups and covered with vermiculite. The styrofoam cup had numerous holes in the bottom and was placed in a 10 oz waxed cup with a large hole half way up the side for drainage. The three herbicides, alachlor, butylate and chlorbromuron, at 10-6, 10-5, and 10‘6 M, respectively, were prepared in modified Hoagland's solution, pH adjusted to 6.8 and added daily to the cups. Preliminary trials showed these concentrations to elicit a response similar to that Observed in soil in the greenhouse. After the excess solution had drained out, the cups were re-randomized and returned to the controlled environment chambers. The insecticide carbofuran was applied as a seed treatment at A oz/lOO lb of seed or at l0"l5 M in modified Hoagland's solution (Appendix A). Three replicates of each barley and corn treatment were germinated and grown at 25 C and 30 C respectively. The photoperiod was a 16 hour day and an 8 hour night with the tOp of the cups receiving 2000 ft candles. Two to four days after planting, the seedlings were thinned to 5 plants per cup. Photosynthesis and respiration measurements were made lA days after seeding for alachlor and butylate treated barley, 10 days after seeding for chlorbromuron treated barley and 7 days after seeding for all the corn treatments. The cups were placed one at a time in a sealed clear plastic test chamber (Figure l). The plastic chamber was located in a growth chamber similar to that in which the plants 17 were grown, and attached to a Beckman Model IR 2l5 CO2 infrared gas analyzer. Air from a compressed air tank was passed through the chamber at a rate of 500 cc per minute measured at the outlet from the analyzer. The analyzer was connected to a Sargent Model SR recorder. The analytical system was adjusted to zero on the recorder with nitrogen and to fifty percent deflection with compressed air without plants in the chamber. The plants were then placed in the plastic test chamber, the lights turned off allowing the plants to respire; thus increasing the CO2 content of Of the effluent gas until a straight horizontal line response was obtained on the recorder indicating that equil- ibrium had been obtained. The lights were then turned on and the plants were permitted to photosynthesize lowering the CO2 content of the effluent gas until the recorder again gave a horizontal straight line response. A one unit change on the recorder paper was equivalent to 3.23 pg of CO2/min. (Appendix B). Respiration of the excised apical 1.5 cm of the leaf tips of barley plants receiving butylate and chlorbromuron with or without carbofuran was also determined using a YSI Oxygen Polarograph. Plant height was measured from the surface of the vermiculite to leaf or shoot apex. The foliage was cut off at the vermiculite surface and all aerial portions traced for’determination of total area with a planimeter. The trwiced material was oven dried and weighed. Data were SubJocted to analysis of variance and Duncan's Multiple l8 Figure 1. Sealed plastic chamber used for photosynthesis and respiration studies. ‘H.—_‘—-———-\’—_wa—_-_C—— ~“_’_._ L‘MH"A wag—\MF—‘m “fly-K.” b‘ .’ .’ '5’ x I J /. o/ ' f — u 19 20 Range Test. Statistical significance of the interaction was determined by obtaining an estimated LSD value between the observed and expected combination values, expressed as a percent of control. 1A Treatment of plants with C—herbicides Twenty-five ml per cup of the lL’C—alachlor or chlorbromuron were applied A days after planting barley and 2 days after planting corn. The solutions were not allowed lLAC treatments to run out the hole of the outer cup after the had been made. Equal volumes of non-labelled herbicide were added to the cup as necessary thereafter. The radioactive solutions were prepared at the same molar concentrations from ring-labelled alachlor (1.02 mc/ mmole) and carbonyl-labelled chlorbromuron (6.5 uc/mg) as the non-labelled treatment, but contained 1 uc/l of lAC_ herbicide. The volitility of butylate dictated greater caution in the l“Cubutylate treatments. Small glass enclosed growth chambers were constructed in an effort to trap any escaping labelled compound(S) (Figure 2). The bottoms were removed from two liter sulfuric acid bottles and the tops were fitted with three holed rubber stoppers. Glass tubing was placed in two of the holes; one piece bent and reaching to the bottom to serve as a gas exit port and the other with a serum cap seal on the outside descended downward to the cup and permitted administration of the 1{AC-butylate to the 21 Figure 2. The inside of a growth chamber containing the microchambers and trapping mechanism used in the growing of barley and corn treated with lac-butylate. ’—.,—_'—u...—..__ “.H —JMA WI—‘H 22 23 cup. The third hole served as an air inlet. The outlet tube from each microchamber was connected with glass tubing to separate gas scrubbers containing an ethanolamine: ethylene glycol monomethyl ether solution (l:2,v:v). Tygor® tubing served as connectors from the scrubbers to brass fish-tank needle valves which regulated the flow of air separately from each scrubber. A small vacuum pump was connected via a manifold to the needle valves to draw the air from the growth chamber through the system. The temperature in the growth chamber was adjusted to maintain 25 C and 30 C in the glass chambers in separate barley and corn experiments, respectively. The cups were seeded as described previously, but with 7 barley or 5 corn seeds and placed in two-quart plastic bags. The plastic bags were sealed to thebottom of the glass chambers with masking tape. Wooden frames with holes out large enough to catch the top of the cup in the plastic bag also served as tables for the microchamber; thus the top of the cup and the bottom of the chamber were on the same level. After the initial treatment, the addition of 25 ml of 10-5 M butylate with or without alkyl chain luC-butylate (2.9 uc/umole) to the cups was made with a syringe through the serum cap on the center glass tube of the microchamber. The radioactive solution contained 5 uc/l. At the date of harvest photosynthesis and respiration data were obtained and analyzed as previously described. 2A The plants were then removed from the cups and the roots washed free of sand and vermiculite. One or two plants from each replicate were chosen for freeze drying and radioautography according to the methods of Crafts and Yamaguchi (19). The remaining plant roots and shoots were separated, measured as before, and then freeze—dried for further analysis. Extraction and analysis of radioactive material All extraction procedures were done in duplicate on shoot and root samples from two separate experiments. The samples were ground in a Wiley mill using a no. 60 mesh screen and stored in glass scintillation vials. The ground plant material was extracted for lLAC—labelled materials for 6 hours with 10 ml of 80 percent acetone in each vial. The vials were placed on their side in a reciprocating shaker at a speed sufficient to give complete agitation of the entire sample. Following extraction the vials were centrifuged at A55 x g for 5 minutes in a swinging bucket Sorval GLC-l centrifuge. The supernatant was removed with a disposable pipette and stored in a scintillation vial. The pellet was extracted as before for 8 hours, the extract again centrifuged and the corresponding supernatants combined. The supernatant volume was reduced under nitrogen with the vial on a steam bath at 30 C. Upon completion of a third extraction with 10 ml of 100 percent acetone for 10 hours the homogenate was filtered through No. l Whatman filter 25 paper. The filtrate was added to the reduced supernatant from before, reduced in the same manner to approximately 10 ml and placed in 10 ml conical graduated centrifuge tubes. The acetone-insoluble residue was air dried under a hood and stored in a drying oven. The volume of the combined reduced filtrate-supernatant was reduced under nitrogen to lAc was lost A ml. A preliminary experiment indicated no with the acetone. One ml of hexane was added to the remain- ing water portion and mixed in with a spatula and then with a Vortex test tube stirrer. The mixture was centrifuged at A55 x g for 10 minutes, the test tubes sealed with parafilm and then placed in the freezer for about 1 hour to insure good layering of the hexane and water. After the hexane layer was removed from the test tube, one half was used for Spotting on thin layer plates and the other half was counted for radioactivity content in a Packard Tri-carb Scintillation Spectrometer. One-half ml of the water fraction was added to 15 ml of a scintillation fluid consisting of 0.1 g l, A-bis 2-(A-methyl-S-phenyloxazolyl)-benzene (dimethyl POPOP), 5 g 2,5 diphenyloxazole (PPO), 50 g naphthalene, 380 ml toluene, 380 ml 1,A dioxane, and 2A0 m1 absolute ethanol. This scintillation solution was used for counting all samples. A small portion of the residue was weighed and combusted by the Schoeninger combustion method of Wang and Willis (67), to determine the amount of 1MC incorporated into acetone- insoluble residue. All radioactive samples counted in the 26 liquid scintillation counter were corrected for quenching and volume, then converted to disintegration per minute per gram dry weight of sample initially ground. Thin layer chromatography of radioactive extracts Preliminary work showed luC-labelled compounds could be separated on silica gel H thin layer plates with a thickness of 250 microns. Only those samples which contained at least 100 counts per minute inthe entire sample were spotted. The hexane-soluble fractions were spotted in small circles whereas the water-soluble fractions were spotted in 1 inch wide bands. Plates Spotted with 1AC_ alachlor or lLAC-chlorbromuron or their metabolites were developed in petroleum ether:chloroform:95 percent ethanol (7:2:l;v:v:v), 1“ C-butylate and metabolites in chloroform: methanol:pyridine (lOO:1:lO;v:v:v). After develOpment for 15 cm on the plate, the plates were scraped in 1 cm bands. The scrapings were placed in scintillation vials and counted for radioactivity as previously described. Two scintillation counters were used. One counter was a Packard Tri-carb Scintillation Spectrometer with settings on the red and green channels - gain 9 percent and 9 percent, window A-B and C-D and discriminators at 23-70 and 30—1000, respectively. The other counter was a Nuclear Chicago Mark 1 liquid scintillator with settings on the B and C channels of E 500 for both attenuators, a window width setting of 1.2- 9.9 for Channel B and 0.5-9.9 for Channel C. Quenching was corrected by the channels ratio method. An efficiency curve 27 was calculated for each machine and all counts were converted to disintegrations per minute per gram dry weight as before. RESULTS AND DISCUSSION To date most published reports dealing with pesticide interaction specifically herbicide-insecticide interactions, are those resulting from the application of such combinations to rice and cotton (8,28,29,A3). It would seem unlikely that such combination effects do not occur in other crops, for this reason we tested numerous pesticide combinations for possible interactions on barley and corn grown in the greenhouse (Appendix C and D). The chemical combinations chosen for further study are shown in Tables 1 and 2. Barley was used in the study because of its similarity to monocotyledonous weeds in corn. The combination of carbofuran seed treatment with alachlor or butylate on barley gave a synergistic interaction (Colbynuflflnxi)for the former on dry weight and for the latter on plant height (Table l). Chlorbromuron and carbofuran interacted antagonistically on barley dry weight. An additive response was obtained with alachlor or chlorbromuron in combination with carbofuran on plant height and for butylate with carbofuran on dry weight. The terms synergistic and antagonistic have been extended to include interactions observed between an insecticide and a herbicide even though the insecticide is not phytotoxic in itself. Greater phytotoxicity than expected in the presence of the insecticide as defined by 28 l .ll’nllll'll'.)' In." I‘ltll-Ill.‘ ill‘rlllllnll‘ol‘l‘llllllv’lr‘" .ll'\. 'lllll l (I, (III! .llIll'l-‘f .‘I‘.(Ir‘.lll 29 .ooom no DA ooa\mo : mm: pcoEpmopp mo mums one .como Ca mosoad m sea; mCOHomOHHoop : pom pcmHQ\Ew mm.o mo ozam> unwfioz zap owmpo>m cm mucommpoop osam> maca o .como CH mommao m spa: mCOflpmoaadop : pom Eo m.:m no ozam> unwed: Osman owmpo>m cm mucomopoop OSHm> maze .EmficomMpcm mfimsoo < .Emwwpocmm wamsqo m mcofipmsvo m.>oaoo Eonm oo>fipoo M a mm mm mm mm m.a :osontntoHco mm om m mm mm m.a oooasesm m moa mm em om o.H Loanoma< Fm mm Hopucoo o pcospmomp ooom ampsmoopmo as HHH m.a copsEopnhoaso em m: m.H sesamesm moa mm o.H hoanoma¢ o 00H 9 ooa Hoppcoo pcospmopp Aflopucoo AHOApcoo OUHOHuoomCH oz mo psoohomv no pcooaomv Adxoav COHpom osaw> pnwfioz coapom Ozam> pcmfion comp lamch woouoooxm has mILOpCH oompooaxm pcmam coameHHoom unoEpmonB moaoanhom .mCOHpOOHHQQm onaoaopmc mocmwpoEoopQ mSOHmw> oo>Hoomp QOfinz moammn vac Immolsm mo pnwfioz zap one unwfioc on» co ucoEpmopu omom ampsmonpmo mo poommo one .H OHOOB 3O .ooom mo OH ooa\mo : mm: pcospwonu mo pump one o .nomo :fi mpcmaq m Sufi: mcoHOmOHHQop : pom pcmHQ\Ew ow.o no unwaoz who owmpo>m cm mqummLQOL msam> mace .nomo ca mpcmaq m spas mQOHumOHHQOL : pom Eo ©.m: mo Ozam> pswfioz panda o®dpo>m cm mucomopoop osam> mace o .Echommpcm mamsvo < .Ewfiwpocmm mamzvo m mCOHpmSGO m.moaoo Eopm oo>fipoo m m mm mm m Hoa mm 0.: COLSEoanoaco m am as mm mm o.m oemaapsm am am Hopscoo o psoEpmomp comm nonsmonmmo om OHH 0.: copzsopnpoano moa em o.m cecasosm o ooa e ooa Hopscoo pcoEpmonp Aaoppcoo V Afloapsoo OchoapoomCH 02 mo pcoonomv no unwound» n<\pa~ coapom oxam> usmfioz cofipom osam> ucmfion pump lemuCH umpooaxm ago mIHOuCH mompoooxm ucmfim COHpOOHHQQm psoEummne mvwofinpom .mcoameHaoam Ochoanpoc monommeoopQ mSOHnm> UO>HOOOA moan; shoe oHo Izmonaz mo unwfioz ago one unwaon one no pcoEumOLp comm cmhzmonpmo mo poommo One .N canoe 31 Colby (22) is designated as synergism while reduced phytotoxicity is designated antagonism. In preliminary experiments, the carbofuran seed treatment showed no interaction with alachlor on corn and no data was recorded. Synergistic reduction of corn dry weight was obtained with the butylate and carbofuran combination while both weight and height of the corn were reduced synergistically by the chlorbromuron and carbofuran combination (Table 2). The carbofuran seed treatment combined with butylate gave only an additive effect on corn height. To determine the influence of soil on these results a short term germination test on carbofuran treated barley seed with and without alachlor, butylate or chlorbromuron was done in the absence of soil. The germination was synergistically reduced by the alachlor and carbofuran combination as shown in Table 3. Although the other pesticide combinations did not affect the germination, all three herbicides combined with carbofuran showed a synergistic reduction of radical growth. This data indicated that the combination effects observed in the greenhouse studies did not necessarily occur in the soil. Since germination was not completely inhibited by any of these combinations, their interaction appeared to influence certain physiological functions of the plants, therefore, their photosynthesis and respiration effects were the subject of further investigation. 32 .ooom do oH coaxso : moz pcoEuoopp go opma one .Eo m.mm no zpwcoa poop wcaaoooo mucomopooh osHm> mace w .Emflcowopcm maozvo < .Emfiwpocmm mamsvo m mCOHuozdo m.>oaoo Eopm oo>fiuoo o .o om um xpoo on» CH mcoapfiocoo oHHLopm pops: oouocHEpow opoz mooom one o m mm om mm mm H.m :0L550Lopoaco m ow >2 mm . Hm m.m opoazpsm m mm ma m mm H: H.H poanooa< mm mm Homecoo oucoEooohp ooom copsmonpoo mm OOH H.m copzaoaOLoano mm woa m.m opoamusm om 2m H.H Loanooa< o ooa ooa Hopucoo ucoEpmohp ooHOHuoomCH oz Hoppcoo Aaonpcoo A: mica xv no pcoopomp mo pcoopomv coapoo osao> npwcoa coauom oSHo> COApoppcoocoo ucospoope InoucH oopoooxm poom owhoch poopooaxm coameHEpoo oofioanpom o.mp:o8poopu ooHOanon oocowLoEooLQ mSOHho> oo>Hooom Soap: wwcfiaooom moanoo UHOIzoolm mo SuwCoH poop Ezefixoe poo coapoCHELow ooom co pcosuoopp ooom nonsmonpmo mo uoommo one .m oanoe 33 The greenhouse experiments had been carried out in a completely randomized design. This design was deemed best for growing the plants in the growth chamber. It was pointed out earlier that Colby's equation makes a desirable model depicting how interactions may occur in the plant and yet is not suited to this type of design. Only the mean value for the individual treatments can be used in calculating an accurate expected value for comparison with an observed combination mean. It is not possible to determine, by analysis of variance procedures, whether the observed and expected value were significantly different. The formulas found in Figure 3 were developed to specify the difference necessary in order to label interactions antagonistic or synergistic. The first step involves the determination of an upper and lower confidence limit for the observed combination mean. The confidence limit is then substituted into the Least Significant Difference (LSD) equation to estimate the LSD between the observed and expected combination value expressed on a percent of control basis. Should the expected value lie outside this 5 percent level LSD value, the combination can be considered significantly different at the 5 percent level. Observation of the values will dictate whether it is synergistic or antagonistic. This method was used on data obtained from the growth chamber, photosynthesis, and respiration studies. Alachlor significantly reduced the dry weight, height and leaf area per plant of lA-day-old barley plants (Table A) but not the leaf area on a per pot basis. The Where R* = 3A Confidence limits for the observed combination mean. Upper limit Ll CR* +\V/(C-1) (OH,2 + l) CR* - v(c-l) (CR*2 + 1) Lower limit L2 Estimated LSD between observed and expected combination values expressed as percent of control. LSD = tas d = tas gd 2 % [\JIC-l) (CR*2 + l) J (V/E) (lOO) ><|| ><| H Observed combination mean N NI |._: Control mean > noHnoanEoo Uouooaxo Ono Oo>nomno one soozeoo Own coooEHOoo so an Ho>oH seaHaoooosa me. one so oososonoao ecooanacwam o .ooom go nH OOH\No : mo opon on» no no: pnoEpoonp Uoom nonsmonnmo one n .pmoe ownom oHQHpHsz m.noonso on mnHonoooo Ho>oH szHHnononq mO. onp no sznoOHmHanm noOMHo won on nonpoH oEom on» an ooonHom nEOHoo m CH mnooz o * :O m: * OO m: ox mm osHo> oopooaxm o mm m w: o ON o mm O NO unoEpoohu Uoom nonsmonnoo 5 + E OIOH 3. anOHnooH¢ n mm o w: O OO o N: 9 mm 2 IOH .LOHn oHn o HOH n OOH o OOH n :O n :O npnoEpoopp ooom nonsmonhoo Aesond\so ANS Aeeoad\ms smv Aeoo\me mama Asccadxmso moo Aood\NEo :smv o OOH n OOH o OOH n.OOH on OOH Honunoo panom panoz aha mono moon unoEuooLE Honpnoo mo unoonom .onnon OHOIzonszH mo npzopw on» no nonsmonnoo Ono LOHnoon mo COHpoanEoo on» can nonsmonnoo .LOHnooHo no poohmo one .: oHnoB 36 carbofuran treatment was not different from the control; however, when combined with alachlor, the reduction in leaf area and dry weight per pot and plant height was synergistic.' This indicated that the plants receiving the combination treatment were shorter. This information supports the combination effects observed in the greenhouse and germination studies. Photosynthesis was not inhibited but respiration was increased by alachlor compared to the control (Table 5). The carbofuran treatment increased barley respiration when combined with alachlor significantly above that of the control; however, the result was not significantly different from the alachlor treatment by itself. Corn growth, photosynthesis or respiration were not affected by alachlor or the insecticide seed treatment- herbicide combination (Tables 6 and 7) as indicated from the preliminary greenhouse experiments. However, when carbofuran was applied in the nutrient media at 10‘5 M, corn growth was significantly increased (Table 8), resulting in a synergistic reduction for leaf area per plant and per pot. Net photosynthesis differed between the alachlor and carbofuran treatments but neither differed from the control (Table 9). The butylate or carbofuran treatment did not differ from the control for any of the measured parameters on barley plants (Tables 10 and 11). The combination of the two pesticides resulted in a synergistic reduction of the 37 .pmoe ownom mO. onp no ernOOHMHanm nommHo non .Ooom mo nH OOH\NO a no opon one no was nnoEumonp noon nonnmonnoo one oHaHpan m.noonno on wnHonoooo Ho>oH muHHHnononQ n on nonpoH oEmm on» an ooonHom nESHoo o nH mnooz o HonpnoO mo pno onom eHH HOH Ozm osz> Oopooaxm o me o MHH n OOH unoEpoonp Ooom nonsmonnoo + z OIOH .nOHnooH< o OOH o OO O Hmm : OIOH .noHnooHn o OOH o mOH no :mH npnonoonp noon nonsmonnoo “Nao\sas\moo we OH:.oO Am5o\eas\moo we mem.ov Asm\eaexmoo ma omv o OOH o OOH .om OOH Honpnoo mHmonnnmmOponm, .mHmonpnNmOponQ nOHponHmmom HMpoe poz pnoEuoone .onnon OHouemvqu no mHmonpnzmouonQ Ono nOHpmnHamop no nonsmonnoO Ono nOHnooHo mo nOHpoanEoo on» ono .nonnmonnoo anOHnooHo no poommo one .m oHnme 38 .Ooom mo nH OOH\No : mo ouon on» no nos unoEpoonu Ooow nonsmonnmo one o .umoe ownom oHQHpHnE m_nmonna on wnHUnoooo Ho>oH epHHHnonona mO. onp no ernoOHMHanw nommHO non 0O nonpoH oEom on» en OoonHom nESHoo o nH mnmoz m em em Hm mm Om oSHo> Oopooqu pnoEpmonn Ooow o mm o mm o mm o mm o NO nonsmonnoo + S OIOH .nOHnooHn o mm m OO O :O m mm O OO 2 otOH .noHeooH< n HOH o em m mm m HOH O HO npnoEnmonp noon nonsmonnoo ApnoHQ\Eo mmv ApnOHQ\wE may Apoa\we eezv AunMHQ\mEo MOO Apoa\mEo Hmzv m OOH m OOH m OOH w OOH om OOH . Hoepnoo pano: pnwwvz anO mono moon pnoEuoone Honnnoo mo pnoonom .nnoo UHOIemOIe mo nuzonw one no nonnmonnoo Ono nOHnooHo mo nOHpoanEoo one ono .nmnnponnmo .noHnomHm mo poommo one .O oHnme 39 .Ooom mo nH OOH\NO : no opmn on» no no; pnoEpmonu noon nmnnmonnmo one n .nmoe ownom oHQHpan m.nmon:O on wnHOnoooo Ho>oH OpHHHnonona mO. one no.>HnnoOHmHanm nommHO pon on nounoH oewm on» an OoSOHHoe nESHoo o nH mnooz o mmH OOH mm osHo> Oopoomxm unoEpoonp Ooom o OOH o mm o OO nonsmonnmo + Z olOH .noHnooHn o mHH o OOH o me E OIOH .LOHnooHn m OOH m mm . o OO npnoEpmonp Uoow nonsmonnoo Amao\eHE\moo ms eoe.ov ANEo\saa\moo we moe.ov Aew\saexmoo ms ommv o OOH o OOH ow OOH . Honpnoo meonunzmouonml mHmonunmwononm. nOHponHQwom pnospmone Houoe poz Honnnoo mo unoonom .nnoo OHoneovle mo mHmonpnemouonQ Onm nOHnonHQmon no nmnnmonnmo Onm nOHnooHo mo nOHuoanEoo one Ono nonsmonnmo anOHnooHo mo poommo one .e oHnoe A0 mO. on» no eHnnOOHmHanm noMMHO non on nounoH oEOm on» an UoZOHHom nESHoo o nH mnooz .Honnnoo mo nnoonoa o no commonaxo monHo> nOHnoanEoo nonooaxo Ono no>nomno one coosooe Own poocEHOmo so an HosoH seaHaoooond me. one so ooeooonnao osooanacmam o .pmoe ownom oHQHanz m.nmonno on wannoooo Ho>oH enHHHnonona m om moH mHH * OHH ow OHH osHos ooecodxm z mIOH o OO o NO no HOH o OO o OO nonneonnoO + z IOH .noHewcH< O OO O OO m :0 O mm O OO 2 OIOH .LOHnooH< n mHH n HmH n OmH n emH n mmH E mIOH nonnmonnoo AnanQ\Eo mmv AnnoHQ\OE OOV Anoa\me eeav AnnoHa\mEo mmv Anoa\mso Hmzv n OOH o OOH no OOH o OOH om OOH Honnnoo uanom nanoz znO mono moon unoEpoone Honnnoo mo pnoonom .nnoo OHOIeonle no nnzonw onn no nonsmonnoo ono LOHnooHo mo noHnoanEoO on» Ono nonsmonnoO .noHnomHo mo uoommo one .O oHoce Al .nmoe ownom oHQHans w_noon:O on wnHOnoooo Ho>oH euHHHnononQ mO. onn no mHnnoOHmHanm nommHo non on nonnoH oEom onn an noonHom nESHoo o nH mnooz o :O we Hm ozHo> oonooqu z mIOH no OOH no OO o 3O nonsmonnoo + S OIOH .noHeooae n mHH n OOH o me 2 IOH .noHe one o HO o He o ON E m|OH nonsmonnoo HmEo\nHE\mOO O: :O0.0V AmEo\nHE\mOO w: NO:.OO Hew\nHE\mOO w: ommv no OOH . no OOH oo OOH Honnnoo mHmonnnNmononm mHmonnnmmOponml nOHnonHmmom Honoe noz unonnoone HOcHUCOO .HO Uflmohwmml .nnoo oHOIzooue mo mHmonnnzmononQ ono nOHnonHQwon no nonsmonnoo ono nOHnooHo mo nOHnoanEoo on» ono nonsmonnoo .nOHnooHo mo noommo one .O oHnoe A2 .Honnnoo mo nnoonom o no oommonaxo mosHo> nOHnoanEoo nonooaxo ono oo>nomno one gooseoo Own ooSeEnomo so as Ho>oH aeaHaoooond mo. one so monotonoao osooaoasoam O .ooom mo nH OOH\NO : no onon on» no mo: nnoEnoonn ooom nonsmonnoo one .nmoe ownom oHQHanz m.noon3O on mannoooo Ho>oH enHHHnononq n mO. onn no OHnnoOHMHanm nomeo non oo nonnoH oEom onn en ooonHom nEnHoo o nH mnoos o * HO em 0* OO onHo> oonoonxm unoEnoonp ooom o Oe o Om o mm nonsmonnoo + z IOH .oeoH poo n mO n OO n HO 2 uOH .oooH pom n eO n eO n mOH nnnoEpoonn noon nonneonnoo AnnoHQ\Eo mHv Hpnon\OE NHO AnnoHQ\mEo :Hv n OOH . n OOH on OOH Honnnoo nanom nanoz HMO oono moon nnoEnoone Honnnoo eo nnoonom .eoHnon oHonmooqu mo nnzonw on» no nonnaonnoo ono opoHennn no nOHnoanEoo onn Ono nonsmonnoo .oponpsn mo poommo one .OH oHnoe A3 .Honnnoo mo pnoonoq o no oowmonaxo monHo> nOHnoanEoo oonooaxo ono oo>nomno one soosooo own ooeosaemo so so HosoH seaHaoooono me. one so oocononnao esooaeacon o .Ooow no nH OOH\No : mo onon onn no mo3 unoEnoonn ooom nonsmonnoo one .nmoe omnom oHQHanz m.noon:O on wnHonoooo Ho>oH OnHHHnononQ n mO. onn no eHnnoOHmHanm noOMHo non on nonnoH oEom on» On noSOHHom nEOHoo o nH mnooz o HmH OOH cs Oom osHo> ooeoodxo nnoEnoonn ooom n mOH o mHH n OOm nonsmonnoo + 2 IOH .onoH pom o OmH o OHH o mmH z IOH .onoH pom o OO o OO o OOH nnnoEnoonn Ooow nonsmonnoo m HmEo\nHE\ OO O: OOm.OV AmEo\nHE\mOO w: OOH.OV AEm\nHE\mOO m: HOHV o OOH o OOH oo OOH Honnnoo mHmonnnawononm mHmonnnewononm. nOHnonHQmom Honoe noz nnoEnoone Honnnoo mo nnoonom .onnon OHoazooazH mo mHmonnnzmonona Ono nOHnonHQmon no nonsmonnoo ono oponpnn mo nOHnoanEoo onn Ono nonsmonnoo .ononnzn mo uoommo one .HH oHnoe AA leaf area per plant and plant height. The shorter foliage was thicker as the dry weight per plant was not reduced. Respiration was synergistically increased by the combination treatment but net photosynthesis was unchanged. The large increase in respiration contributed to the significant total photosynthesis difference, however, it was only an additive reSponse. The data in Table 12 show that the oxygen uptake by barley leaf tips, as a measure of respiration, was synergistically enhanced by the butylate plus carbofuran treatment. Thus the respiration response measured with the oxygen polarograph supports the results obtained with the 002 analyzer. Previously thiocarbamate herbicides have not been considered to directly effect photosynthesis or respiration. Application of butylate to carbofuran treated corn seed significantly decreased leaf area per pot and dry weight per pot from the butylate treatment itself, however, at no time were any of the treatments significantly different from the control plants for any of the growth characteristics measured (Table 13). Neither photosynthesis nor respiration differed significantly from the controls (Table 1A). Butylate is rapidly absorbed by corn roots and translocated to the foliage (68). The results in Table 15 indicate a synergistic reduction of leaf area per plant and per pot, dry weight per pot, and plant height from the butylate and 10"5 M carbofuran combination treatment. However, at no time were these parameters significantly different from the Table 12. The effect of butylate, A5 carbofuran and the combination of butylate and carbofuran on respiration of excised barley leaf tips from lA-day-old barley plants. Treatment 02 Uptake (Percent of control) Control 100 a (5.A9 mu moles/min/mg) Carbofuran b . seed treatment 92 aa BUt late 3 10' M 89 a 81.113 18.139 3 10‘ M + carbofuran 150 b seed treatment Expected value 82 *0 a Means followed by the same letter do not differ significantly at the .05 probability level according to Duncan's Multiple Range Test. 9 The carbofuran seed treatment rate was at A oz/lOO lb seed. Significant difference at the .05 probability level by an estimated LSD between the observed and expected combination values expressed as a percent of control. A6 .ooom mo nH OOH\no : mo onon on» no mo: nnoEnoonn ooom nonsmonnoo one n .nmoe ownom oHQHanz m.noonnm on wnHOnoooo Ho>oH anHHnononQ mO. onn no OHnnoOHmHanm nommHo non oo nonnoH oeom on» an OoonHom nESHoo o nH mnooz m OO O» mm OOH OO osHo> ooeoooxo nnoEnoonn Ooom o HO o :O o NO o :O o eO nonsmonnoo + z IOH .oooH pom no eO o mOH n OHH o OO n OOH z |OH .oocH poo n HOH o eO o OO o HOH no HO npnoEnoonp ooom nonsmonnoo / AoooHO\Eo ONO AesoHoxoe OOO nood\os eeev AesOHd\mso mOO Aeodxm5o Hmev no OOH o OOH no OOH o OOH ono OOH Honpnoo emeon nnOHoz Ono oono moon nnoEnoone HOLUCOO MO UCmOhmm .nnoo OHonmooue mo nnzonw one no nonsmonnoo Ono onoHOnsn no nOHnoanEoo onn ono nonsmonnoo .onoHOnzn mo noommo one .OH oHnoe A7 .ooom nH OOH\No : mo onon onn no moz nnoEnoonn ooom nonsnonnoo one n .nmoe omnom oHQHanz m.noonnO on wnHonoooo Ho>oH OOHHHnonona mO. onn no OHnnoOHOHanm nommHo non oo nonnoH oEom on» On ooonHom nESHoo o nH mnooz o mOH NO OO osHo> oonoomxm nnoEnoonn ooom nonsmonnoo o OOH o eO o mO + z IOH .oeoH esm o OO o OO o :e z mnOH .onoH pom o OOH o MO o OO nnnoEnoonn Ooom nonnponnoo m Am5o\cae\ OO we :O0.00 “Nao\eas\moo we NO:.OO Aswxeasxmoo we Ommv o OOH o OOH oo OOH Honnnoo mHmonnnOwononQ mHmonpnmmononml nOHnoanoom . Honoe poz . unoEpoone Honnnoo mo nnoonom .nnoo oHouzooue mo mHmonunmmononQ ono nOHnonHomon no nonsmonnoo ono ouonpnn mo nOHnoanEoo on» ono nonnnonnoo .opoHOpnn mo poommo one .zH oHnoe A8 .Honnnoo mo nnoonoa o mo commonaxo wonHo> nOHpoanEoo Oonooaxo Ono oo>nomno one nooseoe OOH ooeoeHeao so as Ho>oH OOHHHoooond mO. one no oesononoHo osmoHanon o .nmoe ownom oHaHanz m.noon:O on mnHonoooo Ho>oH OnHHHnononO mO. onn no sznoOHMHanm nommHO non ow nonnoH oEom onn an ooonHom nESHoo o nH mnooz o * OOH smH * mmH * :mH o* mzH osHo> ooooodxo z OIOH o OO o OOH so eOH o OO o mOH eonsooenoo + 2 -OH .oooH ago a NO o NOH so OHH o OO o OOH z mIOH .oeoH usm o mHH o HmH o ONH o emH o mmH z muOH nonsmonnoo ApnoaneO ONO ApnoHn\Oe OOV Hpon\me eeev ApnoHd\m5o MOO Huon\m5o Hmev o OOH o OOH o OOH _ o OOH om OOH Honoeoo uanom nanoz Ono oono moon unoEnoone Honnnoo no pnoonoe .nnoo OHOIOoOIe mo nnzonw on» no nonsmonnoo ono Oponnzn no nOHnoanEoo on» ono nonsmonnoo .ononnon no poomuo one .mH oHnoe “9 control plants. These measurements appeared to be synergistic only because of the stimulation of growth by carbofuran applied in this manner. Photosynthesis and respiration did not significantly differ from the control in this combination treatment (Table 16). The chlorbromuron treatment did not reduce the leaf area per plant significantly from that of the control plants, but it did reduce the dry weight and height of barley plants (Table 17). The carbofuran seed treatment did not further influence the chlorbromuron response. The data in Table 18 indicate that respiration by barley was significantly increased by the combination treatment compared to the control while net photosynthesis was significantly reduced at this level of chlorbromuron treatment. The combination treatment of carbofuran and chlorbromuron did not significantly differ from the chlorbromuron treatment with respect to respiration and photosynthesis itself. Chlorbromuron, a substituted urea herbicide, might be expected to act similarly on susceptible plants as other substituted ureas. Thus, chlorbromuron should be a potent photosynthesis inhibitor. Geissbuhler (38) stated that the site of action of these herbicides can be located with reasonable certainty to that part of the photosynthetic mechanism which is connected with the process of oxygen evolution. It is reasonable to assume chlorbromuron has some influence at this point on photosynthesis. As shown in Table 19, chlorbromuron with or without carbofuran did 50 .nmoe ownom oHQHanz m noonsa on wnHonoooo Ho>oH OOHHHnononQ mO. on» no mHnnoOHmHanm noOmHo non oo nonnoH oEom onn On OoZOHHom nESHoO o nH mnooz o ON mO mm oSHo> oonooaxm z muOH o NO o HO o HN .nonnmonnoo + z IOH .oeoH oso o OO o OO o ON m .onoH n: o HO o HN o ON 2 OIOH nnonnmonnoo Honoste\mOO we OOO.OO Hmso\eHE\mOO ma OOH.OO AswstE\mOO o: OONO o OOH o OOH oo OOH Honnnoo mHmonnnNmononm mHmonnanononm. nOHponHmmom Honoe noz nnoEnoone Honnnoo mo nnoonom .nnoo oHOImoouN no mHmonnnOmononQ ono noHnonHOmon no nonsmonnoo ono ononpzn mo nOHpoaneOo on» Ono nonsmonnoo .ononnon mo uoommo one .OH oHnoe .ooom no nH OOH\No : mo onon onn no mo: nnoEnoonn ooom nonsmonnoo one n .nmoe ownom oHQHanz m.noon5O on wnHonoooo Ho>oH OnHHHnononQ OO. onn no OHpnoOHeHanm noMMHo non oo nonnoH oEom on» an noSOHHom nESHoo o nH mnooz o 51 :O OO NO osHo> oonoonxm nnoEnoonn ooom no mO o HN o mN nonsmonnoo + z IOH .nonOEonnn HnO o OO o ON om mO z OIOH .nonnEonnLOHnO n NO n NO 0 mOH nnnoEpoonn ooom nonsmonnoo HoeoHONEo mHO HoeoHoxme NHV HocoHO\NEo :HO n OOH o OOH, oon OOH Honnnoo nanon nanoz mum oono moon nnoEpoone HONnnoo mo unoonom .onnon oHOIOoOIOH mo nnsonw onn no nonsmonnoo ono nonneonnnOHno mo nOHnoanEoo on» Ono nonsmonnoo .nonOEonnnoHno mo noommo one .NH oHnoe 52 .ooom no nH OOH\No : no onon onn no mo: nnoEnoonn ooom nonsmonnoo one n .pmoe omnom oHQHanz m.noonnO on wnHonoooo Ho>oH OpHHfinononq mO. onn no annoOHmHanm nommHU non OO nopnoH oEom on» On OoonHom nEOHoo o nH mnooz o OO :O :Om osHo> Uonooaxm nnoEnoonn ooom o OOH o NO 0 OOH nonsmonnoo + z IOH .nOLSEonnn HnO o OO no ON on ONH z IOH .nonnEonnn Hno o OO on OO no OOH nnnosnoonn ooom nonnmonnoo m Honc\eHemeO ma OON.OO ANEo\cHs\ OO O: ONH.OO ANoncHe\mOO ma OHO o OOH o OOH oo OOH Honnnoo meonunamononm. mHmonpnNmononm. nOHponHmwom Honoe noz pnoEnoone Honnnoo mo unoonom .onnon OHOIOoOIOH mo mHmonnnOmononQ ono noHponHQmon no nonsmonnoo ono non380nnnOHno no nOHOoaneOo on» Ono nonsmonnoo .nonOEOnnnOHno mo noommo one .OH oHnoe 53 Table 19. The effect of chlorbromuron, carbofuran and the combination of chlorbromuron and carbofuran on respiration of excised barley leaf tips from lO-day-old barley plants. Treatment 02 Uptake (Percent of control) Control 100 aa (5.A9 mu moles/min/mg) Carbofuran seed treatmentb Chlorbromuron, 10-6 M Chlorbromuron, 10-6 M + carbofuran seed treatment Expected value ' 92 a 91 a 97a 8A a Means followed by the same letter do not differ significantly at the .05 probability level according to Duncan's Multiple Range Test. b The carbofuran seed treatment was at the rate of A oz/100 lb seed. 5:. not significantly change the 02 uptake from that of the controls. Chlorbromuron is promoted for use in corn and thus would not be expected to affect corn. The results presented in Table 20 show this to be true, except for plant height where a small but significant reduction was observed. The combination with carbofuran, on the other hand, synergistically reduced the leaf area and dry weight per pot, but did not further reduce plant height. These results are similar to those obtained in the greenhouse (Table 2). As noted in Table 2l, respiration was not significantly altered from the control by the carbofuran seed treatment, the chlorbromuron treatment, or the combination of the two, whereas net photosynthesis was significantly reduced only by chlorbromuron. The synergistic plant height and dry weight reduction may have been the result of the adverse effect on net photosynthesis. The data in Table 22 show that when carbofuran was applied to corn at 10‘5 M, the combination with chlorbromuron synergistically reduced leaf area per pot and plant height. The corn height, however, was not significantly different from that of the control, possibly because carbofuran significantly increased plant height. The leaf area per pot was reduced by the combination treatment containing carbofuran on a molar basis in the same manner as the carbofuran on a molar basis significantly increased the leaf area per pot. The dry weight reduction per pot by the combination treatment differed when carbofuran was applied as a seed treatment 55 .Honnnoo mo nnoonoa o no oommonaxo mosHo> nOHnoanEoo oonooaxo Ono oo>nomno one coozooo OOH ooeoeHeno so On Ho>oH OOHHHoooond OO. one so oosonOOOHo enooHOchHm o .Ooom mo nH OOH\No : mo onon on» no mo: unosnoonn ooom nonsmonnoo one n .nwoe omnom oHQHanz m.noon:O on mnHonoooo Ho>oH zuHHHnonona mO. onn no annoOHMHanw noMMHo non oo nonnoH oEow on» an ooonHom nESHoo o nH mnooz o HO HO * HO NOH cs OO osHo> ooocodxo .nnoEnoonn noon o OO o HO o :O o OOH o NN nonsmonnoo + 2 OIOH .nonnnonnLOHno no mm o :O on mm o mOH n mOH OH 2 I anonnEonnthno o HOH o NO n OO o HOH n HO nnnoEpoonp coon nonsmonnoo AesoHO\Eo ONO AncoHe\we OOO Hoooxwe NNOO HonoHaxm5o MOO Heooxm5o Hmsv o OOH o OOH o OOH o OOH on OOH _ , Hoepnoo nanon uanoz ONO oono moon nnosnoone Honnnoo no nnoonom .nnoo OHOIzoOIN mo nnzonw on» no nonsmonnoo ono non:EonnnoHno mo nOHnoanEoo onn Ono nonsmoneoo .nonnEonnnoHno mo poommo one .Om oHnoe 56 .ooom mo nH OOH\No : mo onon onn no mo: nnoEnoonp Ooom nonnOonnoo one n .nmoe ownom oHQHanz m.noon5O on wnHonoooo Ho>oH OnHHHnononQ. mO. onn no OHnnoOHmHanm nomnHo non 0o nonnoH oEom onn an ooSOHHoe nEnHoo o nH mnooz o OO O: HO onHo> Oopooaxm nnoEnoonn Ooom o :O o O: o NO _ nonsmonhoo + 2 IOH .nonnEonnn HnO o NO o O: o OO 2 -OH .nonnEonnannO n OOH n OO o OO nnnoEnoonp Ooom nonnaonnoo N N HNoneHE\NOO Os OO0.00 Aso\eHe\ OO O; NO0.00 Hem\sHs\ OO O; ONNO n OOH o OOH oo OOH Honpnoo mHmonnnzmononm meonnnszuonm. . nOHponHmmom Honoe noz nnoanoone Honnnoo mo unoonom .nnoo OHouzoonN no meonnnmmononQ ono nOHnonHamon no nonsmonnoo Ono nonOEonnnOHno mo nOHnoanEoo on» ono nonnmonnoo .nonOEonnLOHno mo poommo one .HN oHnoe 57 .Honnnoo mo nnoonoe o no oommonaxo mosHo> nOHnoanEoo Oonooaxo Ono oo>nomno on» nooznon OOH oonoEHnmo no On Ho>oH OnHHHnononQ OO. onn.uo oononommHo pnoOHmHanm n .nmoe ownom oHQHanz m.noon3O on wnHonoooo Ho>oH OpHHHnononQ OO. onn no OHnnoOHmHanm noOMHo non OO nonpoH oEom onn On ooonHom nEOHoo o nH mnooz o * OOH OHH OHH HOH ea HOH osHo> ooeooaxm z OIOH no :O o OO o OOH n OOH e NHH .consnoonoo + z OIOH .nOLSEonnLOHnO o OO o :O o OO o OOH o OOH z IOH .nonSEonnannO o NHH n HNH n ONH n NOH o OOH 2 OIOH .nonnmonnoo ApnoHQ\Eo ONO AnnoHO\wE OOV Anoa\we Nsz AunoHQ\NEo OOV Anoa\NEo HOzv n OOH o OOH o OOH o OOH oo OOH Honnnoo nanom nanoz OnO oono moon . pnoEpoone Honnnoo mo unoonom .nnoo OHOIOoOIN mo nn30nw on» no nonsmonnoo Ono nonsnonnnOHno mo nOHpoanEoo on» ono nonsmonnoo .nonnEonnnoHno mo uoommo one .NN oHnoe 58 versus application as a molar concentration, but again the reason is likely due to the significant dry weight increase by carbofuran itself. The data in Table 23 indicate that the chlorbromuron and carbofuran 10‘5 M treatments both reduced photosynthesis. The combination of the two pesticides did not cause a synergistic reduction of photosynthesis. Thus, while chlorbromuron is suggested for use on corn, these greenhouse and growth chamber studies .show that it can influence the early growth of the plant, through a reduced net photosynthetic rate. Uptake, translocation and metabolism of lLAC-alachlor Extraction with 80 percent acetone following the addition of lL‘c-lahelled herbicide to untreated plant material indicated none of the herbicides used in this study were bound to the ground plant tissue. Partitioning between 1 ml of hexane and 5 ml of water of the ethanol- soluble stock ltic-alachlor, lLAC-butylate and lAC_ chlorbromuron indicated 92, 85, and 97 percent, respectively, partitioned into the hexane fraction. Alachlor is absorbed mainly by the germinating plant shoot, secondarily by the roots, and then translocated ‘throughout the plant (68). This occurred in both barley and corn (Figures A and 5). The greatest accumulation zeppeared to be in the coleoptile of corn when treated with zalachlor alone, but in both cases movement throughout the plants did occur. The uptake was greater in the susceptible spnecies, barley, compared to the tolerant corn as shown in .nmoe omnom oHQHanz m.noon:O on mnHonoooo Ho>oH OnHHHnononQ OO. onn no OHnnoOHmHanm nommHo non on nonnoH oEom on» On ooonHom nEOHoO o nH mnooz o OO OO O: onHo> nonooaxm 2 OIOH n OO o OO o OO .nonnmonnoo + z IOH .nonOEonnn HnO mu O OO o O: o OO 2 OuOH .nonnEonnnOHnO o HO n HN o ON 2 OIOH .nonneonnoo N HNEo\cHex OO O: OOO.OV Ano\sHs\mOO Os OOO.OO HeO\sHs\NOO O; Ommv o OOH o OOH oo OOH Honpnoo mHmonnnmwononm mHmonnnOwononm. nOHnonHmmom unoEnoone. Honoe poz Honnnoo mo nnoonom .nnoo OHOIOoOIN mo meonnnOmonona ono nOHnonHOmon no nonnuonnoo ono non580nnnOHno mo nOHnoanEoo onp ono nonsmonnoo .nonnnonnnOHno mo pooemo one .ON oHnoe 6O Figure A. Barley plants (left) and radioautographs (right) of barley plants harvested 10 days after lAC~ alachlor was placed in the nutrient solution. Upper: lac-alachlor treated plants. Lower: luc- alachlor treated plants in the presence of carbofuran as a seed treatment. h—— -—. —-—-———-——- ‘-—— ‘_———. 61 62 Figure 5. Corn plants (left) and radioautographs (right) of corn plants harvested 5 days after l“C-alachlor was placed in the nutrient solution. Upper: lAC- alachlor treated plants. Lower: l“C-alachlor treated plants in the presence of carbofuran as a seed treatment. 63 6A Figures A and 5 and Table 2A. There was almost three times the amount of 1“O in the barley as in the corn although the barley was exposed for twice as long. There was only about one-third more 1”C label in the barley root than in the shoot while the corn root contained half again as much as the shoot. Jaworski (38) has reported that corn, a species resistant to chloroacetamides, invariably absorbed less of these chemicals than soybeans, another resistant species, which took up more than the susceptible plant species, cucumbers and oats. Therefore, it would appear that two factors may be contributing to the tolerance Of corn: less alachlor is absorbed and less is translocated throughout the shoot. The addition of carbofuran as a seed treatment appeared to increase the total alachlor uptake in both barley and corn. The amount present was greater for barley than corn and for the roots than the shoots in both species. The greater amount of 114C in the roots can be seen in Figures A and 5 (lower). Carbofuran not only increases the uptake of alachlor into the plant but may slightly 1AC change the uptake and translocation pattern by causing accumulation in the root. The partitioning of the 1”C in the roots of barley and corn is similar, with corn having a little more in the 80 percent acetone—insoluble fraction (Table 25). The amount of 1“C in the hexane-soluble fraction in corn root is higher than it is in the shoot, whereas for 65 .ooom No nH OOH\No : no nnoEnoonn Ooom o mo ooHHaQo nonneonnoo o nonsmonnoo OOOO OOOH ONO NOOO NOOO OOOH HO OeeN + z OuOH .noHnonHO & OIOH OHHO ONOH OON OONO NNOO OOOH OH OOOH .noHnooHO unnoo _ ononnmonnoo OOOOH ONOO NOO OOOO OOOHH NONO NON :OOO + 2 OIOH .noHeooHO OOONH OOOH Om: NOON OHOO OOH: OON OOOO z OuOH .toHnooHO "NoHnom bw\EQov OO\EQOV Aw\EQUO Aw\EQOObw\anvbw\EQOv Hw\EQOv Aw\sanv Honoe nOHnoonm nOHnoonm oHnnHomnH Honoe nOHpoonu nOHpoonm oHnsHomnH pnoEpoone oHnsHom oHnsHom Iononooo oHnnH0m oHnSHom IonOpooo Inonoz Ionoxom unoonom Om Inonoz Ionoxom unoonoe OO noon nH OOH noonm nH OOH .nOHnoOHHan nOHnooHouozH nopmo.OHo>Huooanon maoo O ono OH.nOHnooHo wanHopnoo nOHanom nnoansn nH nzonw nnoo OHOIOoo IN ono OoHnon oHonzooazH nH NOHnooHouozH mo nOHnnanano ono wanOHannoQ one .ON oHnoe 66 .ooom no nH OOH\No : no nnoEnooen Ooom o mo OoHHQQo nonsmonnoo o nonsmonnoo HO O :O OO N NO + S IOH . .noHe oHO HO O :O m: H NO 2 OnOH .LOHnooHO unnoo ononnmonnoo NO O OO O: O O: + z OuOH .noHeooH< OO : NO O: O NO 2 OIOH .LOHnooH< ”HoHnom AO\EQOV Aw\EQOV Aw\eanv AO\EQUV. Aw\EQUV Aw\EQOv nOHnoonm nOHuoonm oHnnHomnH nOHpoonm nOHuoonm oHnnHonnH pnoEpoone OHnSHom oHnnHom IonOOooo oHnsHom oHnnHOO IonOOooo Inonoz Ionoxom nnoonom OO Inonoz Ionoxom pnoonom OO noon nH OOH .nOHnoOHHQQo nOHnooHoIO: eooea sH o:H H nonmo .OHo>Hpoonon mmoo O Ono OH .nOHnooHo wanHonnoo nOHnnHom unoHnnnn nH nzonw nnoo OHOIOoOIN ono onnon OHOIOoo1:H Mo noon no noonm one nH pnomonm O:H on» no pnoonoq o no O :H no OcHsoHaHenoa one .ON oHnoe 67 barley it is relatively unchanged. The carbofuran treatment did not influence the fractionation with respect to species or within the barley plant or corn root, but it did increase the 80 percent acetone-insoluble fraction in the corn shoot at the expense of the water-soluble fraction. In both plant species, most of the 1“C was in the 80 percent acetone- insoluble fraction. The hexane-soluble portion made up only a small part of the soluble fraction. The hexane-soluble portion contained so little 114C in barley that it was not chromatographed on thin layer chromatography (TLC). The barley shoot appeared to contain twice the concentration Of parent lac-alachlor in the water- soluble fraction than did the barley root (Table 26). The 140 in the water-soluble fraction which co-chromatographed with the standard was considered parent herbicide in all cases. The majority of the 1“C was found in one metabolite in both the shoot and the root, with one other metabolite occurring in each. The carbofuran treatment appeared to decrease the proportion of parent l[AC-alachlor and increase the percent in the major metabolite in the shoot. The root was affected in the opposite manner with more 1”C as alachlor and less as the major metabolite. The minor metabolite in the barley shoots and roots appeared to be the same, following alachlor treatment, but different from either of the minor compounds found following the alachlor- carbofuran combination treatment. It is inferred in Table Elliu'i-JILIUI . . L y i. .l. . . . . . .. .I ........I.u. ..u.......r.c.r.: .14.. 1.1114... x L. 7... .q. .47.... .O.:... .flb). 0Lo'. .u....,. 68 .OnsoanoO nnonoa monoOHonH n .ooom no nH OOH\NO : no nnoEnoonn ooom o no ooHHQQo no: nonsmonnoo o nOH nNH nmm O.H 0.0 0.0 .O N.O 0.0 0.0 :.O m.O N.O ON :O :O ON H.O 0.0 km nonnomn Honon mo nnoonom Gmedhonemo + z O-OH .noHeooHO z O:OH .noHnooHO oanHomlonoxom Cmezmonemo + 2 O-OH .noHeooHO 2 OuOH .noHeooHO oHndHOOILonoz "noom nonnmonnoo + z OIOH .noHeooHO s OuOH .noHecoHO oHnnHomuonoxom o nonneonnoo + z OIOH .noHeoOH< zOuOH .noHnooHO oHnnHomnnopoz nnoEnoone ”eccem enom OcoHo OH OoHnon OHOIOoOI:H Eonm muoonnxo noon ono poonm no one no COHOOLOQOO OEE .nnoEnoonn nOHnooHouO:H nonmo mOoo .ON OHooe 69 26 that more alachlor remains in the root in the presence of carbofuran. The data in Table 27 indicate that this is in fact what occurs in barley. Although there is an increase in parent compound in the root, there is an even greater decrease in the shoot. Since alachlor inhibited initial seedling growth in the greenhouse and in germination studies it would seem likely that the increased susceptibility of barley to the pesticide combination is in part due to increased uptake, decreased movement and also to decreased metabolism of alachlor. The alachlor treatment by itself on barley showed more parent material to be present in the shoot than in the root. Since these plants showed less toxicity than those receiving the combination, this would indicate that alachlor once in the shoot has less influence. It also suggests that if a susceptible plant root can get past the initial germination stage, following alachlor treatment, that while it is not entirely unharmed it can continue to grow. 1 In corn treated with luC-alachlor most of the “C was found in a major metabolite staying near or at the origin On the TLC plates (Table 28). In the water—soluble fraction ffi‘om the combination treatment a different major metabolite nuiy have been detected (Table 28). The shoot had three nflgnor metabolites in the water-soluble fraction and the root haHnooamon mOoO O Ono OH .onoHOpnn wanHonnoo nOHnnHom unoHnunn nH nzonw nnoo OHOIOoOIN Odo NoHnoo oHouNoos:H OH oOaHNanuO:H no :oHeseHneaHo new OcHsoHOHenoo one .ON oHnoe 75 .ooow Mo nH OOH\No : no nnoEnoonn ooom o no ooHHaqo nonsmonnoo o nonsmonnoo NO :O :O OO O O: + 2 uOH .oOoH osO N: ON OO O: O OO 2 OtOH .ononndm unnoo ononnmonnoo Om : NO OO O N: + 2 -OH .oeoH Osm z MIOH HO : OO NO : OO .onoH pom ONoHnom Aw\EQOV Aw\eaov Aw\EQoV Aw\EQOv nm\EQUV Am\EQOO nOHpoonm nOHnoonN oHnnHOmnH nOHnoonm nOHnoonm oHnnHomnH nnoEnoone oHnnHom oHnsHom IonOOooo oHnnHom oHnnHom Iononooo Inonoz Ionoxom unoonom OO Inopoz Ionoxom pnoonom OO noon nH O :H poonm nH O:H .nOHpoOHHQQo onoHOnnnIO:H nonmo .OHo>Hnoonon mNoO O ono OH .onoHOnnn wanHonnoO nOHusHow pnoannn nH nzonw .nnoo OHonmoouN ono OoHnon OHOIOoOI:H mo noon no poonm onn nH unomone O:H on» no pnoonog o no O:H mo wanOHannoQ one .OO oHnoe 76 Li Figure 6. Barley plants (left) and radioautographs (right) of barley plants harvested 10 days after lAc- butylate was placed in the nutrient solution. Upper: ltic-butylate treated plants. Lower: lAc- butylate treated plants in the presence of carbofuran as a seed treatment. 77 K1 78 Figure 7. Corn plants (left) and radioautographs (right) of corn plants harvested 5 days after 1 C- butylate was placed in the nutrient solution. Upper: l“C-butylate treated plants. Lower: lAC— butylate treated plants in the presence of carbofuran as a seed treatment. .‘__—__.——_~_ - 79 80 80 percent acetone-insoluble fraction in the barley root is almost double that in the barley shoot when treated with butylate alone. The combination treatment of carbofuran 1A and butylate decreased the C in the 80 percent acetone- insoluble portion, but increased the 1“C level in the water— soluble portion as well as the total 1“C in the barley root. The percent of 1“C in the various fractions in the Shoot was altered very little by the carbofuran treatment (Table 30). The tolerant species (corn) reacts differently. The shoot contained a relatively low concentration of 1”C which is almost equally divided between the 80 percent acetone- insoluble and the water-soluble fraction with the hexane- soluble portion being very small. By contrast, the hexane- soluble portion in the corn root was much larger and when the seed was treated with carbofuran it was equal to the other two portions even though the total level of 1“C in the root was lower. The water-soluble fraction of the barley shoot contained a large amount of one metabolite, a small percent of a second metabolite, and about the same proportion of parent material as the second metabolite (Table 31). As might be expected, by the small influence of the carbofuran treatment on other measured parameters in the shoot, the metabolites were the same and in the same proportions. The data in Table 32 support these results in that the percent parent material was only slightly changed. In the root, the water-soluble fraction from the butylate treated plants .OnOOQEoo nnonoa monoOHonH n .ooom mo nH OOH\No : no unoEnoonn Ooom o no ooHHQQo mos nonsmonnoo o 81 HN N O OO nonsmonnoo + z O-OH .oooHNesO oHO OH :N OH 2 O-OH .oeoHNesO oHnnHoonnoxom NO O :O nonswonnoo + z O-OH .oeoHNesO OH O :O 2 O-OH .onoHOosm oHnnHomInonoB "noom nonsmonnoo + z O-OH .oonHNesm 2 O-OH .oooHNOOO oHnnHOOIonoxom nO O OO ononneonnoo + z O-OH .oeoHNOsO oO O OO 2 O-OH .oeoHNesm oHnnHomlnopoz "noonm O.H 0.0 0.0 N.O O.O O.O :.O O.O N.O H.O O.O Om peoanoone upon ecoHN Oonnomn Hopon mo pnoonom . .nnoEnoonn onoHOnnnIO H nonmo wNoO OH .NoHnon oHouzoou:H Eonm mnoonnxo noon ono noonm mo one nw nOHnonoaom one .HO oHnoe 82 Table 32. The percent extractable l“Cubutylate remaining in lA-day-Old barley and 7-day-old corn shoots and roots, 10 and 5 days respectively, after ll‘C-butylate treatment. 130 in shoot *Ific in root Hexane- Water- Hexane- Water- soluble soluble soluble soluble Treatment fraction fraction Total fraction fraction Total Barley: Butglate, 0 3 5 3 5 1.9 3 O A 9 Butglate, 0 3 l 3.1 0 8 12.2 13.0 carbofurana Corn: Butylate, 0 1.5 1.5 0 2.3 2.3 Butylate, 10-5 M + 0 0 0 6.8 7.8 lA.6 carbofuran a Carbofuran of seed. was applied as a seed treatment at A oz/lOO lb 83 contained metabolites similar to those in the shoot. The major metabolite was the same and the minor one was perhaps the same, but moved a little further on the TLC plate. The parent compound made up a greater proportion of the total 114C than the minor metabolite. The carbofuran seed treat- ment greatly altered lL‘C—butylate metabolism in the barley root. In this treatment there was half as much parent compound as there was major metabolite and the latter differed from the butylate only treatment. There was also one minor metabolite, and again it was not the same as the other minor metabolites obtained in the root or shoot. Fifty percent of the hexane-soluble fraction separated as parent material with one other metabolite being 2A percent and two others equally making up the final 26 percent. One Of the minor metabolites was the same as the major water- soluble compound obtained in the root with the carbofuran and butylate combination treatment. The herbicide-insecticide combination influenced the hexane-soluble fraction in a similar manner to the water-soluble fraction. The same metabolites were found with the reduction Of the 1”c- butylate parent fraction by the same amount that an additional metabolite increased. There appeared to be a substantial amount of 1“C remaining as 1“ C-butylate in the root in the hexane-soluble fraction. Since the hexane-soluble fraction contained only a small portion of the total luc this did not greatly affect the total amount of luC-butylate present (Table 32). The three-fold increase in the parent luc- 8A butylate remaining in the root due to the carbofuran seed treatment may, in part, explain the synergistic action on barley. The shoots from corn grown for 5 days in lLAC-butylate contained three metabolites and a very small portion Of parent compound in the water-soluble fraction. Shoots from corn treated with the carbofuran and butylate combination contained only two metabolites. These were the same as the major ones from the butylate treatment. No parent luc- butylate was found in corn shoots receiving the combination treatment (Tables 32 and 33). The water-soluble extracts from corn roots contained three metabolites from the butylate treatment (one major and two minor); similarly, the combination treatment with carbofuran contained one major and two minor metabolites, (Table 33). The proportion of parent compound was five times greater in corn roots receiving the combination treatment. In the hexane-soluble fraction from corn roots, no parent 1” C-butylate was found unless the plants received the carbofuran-butylate combination treatment (Table 33). Roots receiving l[AC-butylate contained a luC-labelled metabolite which contained 77 percent of the 1“ C. This metabolite was reduced to A0 percent of the total 1”C present if the plants received the carbofuran-butylate combination treatment. Two other metabolites were found in a hexane-soluble extract from corn roots (Table 33). One staying at or near the origin in the TLC procedure increased 85 .OnSOQEoo nnonoa monoOHOnH n .ooow mo nH OOH\No : no nnoEnoonp noon o no OoHHaao mo: nonsmonnoo o nON o: H OO nonsmonnoo + z O-OH .oOoHNOOO NN O NH 2 O-OH .oeoHNOsm oHnsHomIonoxom nON O : OO noesmoneoo + z O-OH .oonHNeso OO O OH NN z O-OH .oooHNesO oHnnHomInonox “noom nonsmonnoo + 2 OIOH .oOoHNeso z O-OH .oooHNOsO oHnnHomlonoxom HN ON ononnmonnoo + z O-OH .opoHOosm mm m :N ON : O-OH .OOOHOOOO oHnnHomInonoz ”poonm O.H 0.0 O.O N.O O.O 0.0 :.O 0.0 N.O H.O 0.0 mm Ososeoone anon . . peoHN nonnomm Honon mo nnoonom .nnoEnoonn onoHOunnIO: nopmo mOoO O nnoo oHonOoOIN Eonm mpoonnxo noon Ono noonm no one no nOHnonomom one .OO oHnoe 86 from 17 to 38 percent due to the carbofuran seed treatment. The butylate-carbofuran treatment substantially increased the amount of parent material in the corn root over the butylate alone (Table 32). A similar difference was used to explain the increased phytotoxicity of the combination treatment to barley. The results obtained with corn would appear to discount this hypothesis. The mode of action of butylate has been reported to be an inhibition of growth in the meristematic region of the leaves Of grass plants (68). The question remains as to why carbofuran-butylate synergistically decreased barley growth when the percent of parent luC-butylate in the roots of barley and corn was increased almost equally by the carbofuran seed treatment. Kaufman gt gt. (37) have found methylcarbamates to inhibit phenylcarbamate metabolism. They suggested that perhaps l-naphthyl-flemethylcarbamate (carbaryl) increased oat sensitivity to t—chlorocarbanilate (chlorpropham) rather than increased chlorpropham persistence in the soil. This may be the situation which exists between barley and corn where the sensitivity of barley respiration has been increased by the combination of carbofuran, a methylcarbamate and butylate a diisobutylthiocarbamate. Carbofuran combined with butylate to synergistically reduce barley but not corn root and shoot growth. The bases for these interactions appeared to be the greater accumulation of butylate in the roots of barley plants treated with carbofuran, due to both 87 increased absorption of butylate and decreased metabolism of butylate. This same metabolism trend was apparent in corn, however the butylate level was much lower as the absorption of butylate by corn was reduced by the carbofuran treatment. This latter effect may have been due to competition for uptake sites in the corn root. Uptake, translocation and metabolism of l[AC-chlorbromuron 1A About 80 percent of the C from lL‘C-chlorbromuron was translocated to the shoot in 6 days (Table 3A). The 1A radioautographs in Figure 8 shows the C translocation in barley to be different than that in corn (Figure 9). The amount of lLAC in the corn root and coleoptile appeared to be greater than in the barley. However, the data in Table 3A indicate that the accumulation of 1”C in the roots of corn plants was much less than it was for the barley. In 1“C was found in the shoot versus corn, two-thirds of the 85 percent for barley. Geissbuhler (38) pointed out that substituted urea herbicides are readily taken up from soil and nutrient solutions by root systems and are rapidly translocated to the leaves and stem by the transpiration stream. It is important to note that different substituted urea herbicides have exhibited different mobilities in plant systems (38). It might also be considered, as shown by the corn and barley species here, that different mobilities in different plants could exist for the same substituted urea herbicide. Frank gt gt. (25) suggested that the selectivity of 5-aminO-A-chlorOO2-pheny1-3(2H)- 88 .ooom mo nH OOH\No : no nnoEnoonO noom o no OoHHaao mo; nonsmonnoo o nonsmonnoo ONNO O:: OO: OOON :OOO HOOH OOO OOOO + z OIOH .nonnEonnLOHnO OO:: :OO NHO HNOO OOHN :OOH ONO NOO: : OnOH .nonnsonnnOHnO unnoo ononnmonnoo ONHN HOO NOO :OOO NH::O OOOOH OOOO OOOON + z OIOH .nOLOEonnAOHnO O:NO :OO :O: NOOO ONHOm HO:NH OOON O:OON z O:OH .nonnsonnnOHnO "NoHnom iNONEaaO HONeaoO aaNean waeaoc .waeaeN Na\eaea .meaaON HONEan Honoe noHnoonm nOHpoonm nOHpoonm Hopoe nOHuoonm nOHpoonm oHnnHownH nnoEnoone oHnnHom oHnsHom iononooo oHnsHom oHnnHom Iononooo Inonoz Ionoxom unoonom om Inonoz Ionoxom nnoonom Om noon nH OOH Oooeo OH O:H .nOHnoOHHQQo nonSEonnNOHnOIO:H nonmo .OHo>Hpoonon mOoo O ono O .nonnsonnnoHno wanHonnoo noHnnHom nnoHnnnn nH nzonm .nnooIOHOIOoOIN ono OoHnon OHOIOoOIOH nH nonSEonnnOHnono:H mo nOHnnanano ono wanoHpHpnoQ one .:O oHnoe 89 Figure 8. Barley plants (left) and radioautographs (right) of barley plants harvested 6 days after l“C- chlorbromuron was placed in the nutrient solution. Upper: ll‘C—chlorbromuron treated plants. Lower: 1 C—chlorbromuron treated plants in the presence of carbofuran as a seed treatment. 91 Figure 9. Corn plants (left) and radioautographs (right) of corn plants harvested 5 days after 1 - chlorbromuron was laced in the nutrient solution. Upper: “C-chlorbromuron treated plants. Lower: ltic-chlorbromuron treated plants in the presence of carbofuran as a seed treatment. 92 93 pyridozinone (DYrazon) for lambsquarters (ChenOpodium gttgg L.) in sugar beets is based in part on differential translocation within the two species. The proposed mechanism of action of the substituted urea herbicides is the inhibition of photosynthesis (68). Nashed gt gt. (50) found no aniline metabolites,which could be monitered as lLICOZ loss in corn until 8 days after treatment with lLAC—chlorbromuron. The combination of carbofuran with chlorbromuron very slightly lowered the lac concentration in the barley shoots and raised it in the root by a similar amount. In corn, lLAC concentration raised the situation was reversed with the in the shoot and lowered in the root. Due to the overall lower l“C concentration in the corn, the shift was proportionately higher in corn. The 80 percent acetone-insoluble portion of the root contained 60 percent of the 1A C label in the barley plant and the water-soluble fraction contained one-half as much (Table 35). The addition of carbofuran did not appreciably alter these values except to slightly increase the hexane- soluble fraction in the shoot. Partitioning values in the root were unaltered by the combination treatment (Table 35). In the barley root the acetone-insoluble portion contained 85 percent of the 14C and the hexane- and water—soluble portions contained the remaining 15 percent. In the corn shoot, 27 percent of the 1“C was found in the water-soluble fraction and 68 percent was found in the 80 percent acetone-insoluble fraction (Table 35). The 9A .Ooom no nH OOH\No : no nnoEnoonn ooom o mo noHano nonnnonnoo o nonnnonnoo NH NH ON OH O :N + z OuOH .nonsEonnnOHnO OH O OO NN O OO 5 OIOH nnonOEonnnOHnO unnoo ononsnonnoo O N :O OO HH OO + z OIOH . .nonSEonnnOHnO O N OO NO O OO 2 OIOH anonsnonnnOHnO "NoHnom nONaan 1a\eaec Ha\eaea HONean AONaaaO Am\aaec nOHnoonn nOHnoonn oHnnHomnH nOHnoonn nOHnoonn oHnsHomnH nnoEnoone oHnnHom oHnsHom Iononooo oHnsHom oHnnHom Iononooo Inono3 nonoxom nnoonom OO nono3 Ionoxom nnoonom om noon nH OOH noonm nH OOH .nOHnoOHHmao non:EonnnOHnOIO:H nonno.xHo>Hnooqwon mnon O ono O .nonsEonnnOHno wanHonnoo nOHnsHow nnoHnnnn nH nzonw .nnoo oHoumoUiN Ono OoHnon OHOIOoOIOH no noon no noonm onn nH nnomona O:H onn no nnoonoa o no O:H no wanOHannoQ one .OO oHnoe 95 carbofuran-chlorbromuron combination treatment decreased the luc in the acetone—insoluble fraction. The hexane- soluble portion in the shoot remained virtually unchanged by the carbofuran treatment. In the root, the hexane- soluble fraction went from 8 to 12 percent with the addition of carbofuran while the 80 percent acetone-insoluble fraction remained the same. Carbofuran appeared to increase the percent of 1&0 in the hexane-soluble fraction-in the barley shoot and in corn root at the expense of the water—soluble and 80 percent acetone-insoluble fractions respectively. In barley shoots 6 days after treatment, luc- chlorbromuron made up only 1.3 percent of the water— soluble fraction separated by TLC (Tables 36 and 37). Three luC—metaboli’ces (Table 36) were found in the shoot, one major and two others each containing about 20 percent of the lac. In the root the proportion of label in the major metabolite was greater and less in the other two metabolites and a small amount appeared in a fourth metabolite. Due to 1“ the low amount of water-soluble C extractable from the f lL‘C-chlorbromuron in the root the actual concentration 0 root was less than in the shoot (Table 37). The combination treatment of carbofuran and chlorbromuron did-not change the number of water-soluble metabolites found in the barley shoot, however the Rf values were different than when chlorbromuron alone was the treatment (Table 36). The major metabolite decreased and a minor one increased by a similar amount. The other minor metabolite decreased slightly, 96 oczooEoo ucopmo mmumofiocH .omom mo 9H OOH\No : um pcoepmopp ooom m mm omHHoom mm; nonsmoopmo m m 9mm mm mH nonsmoohmo + E wloa .COLSEopnpoago pom OH mH 0H mm 2 QIOH .:0t350tnp0Hno oanHomuocmxmm m O_oH Hm em H: catsoontmo + z mica .COLSEouomoago o m mH :H so 2 ouoH .c0t350npt0Hno o oHozaomlpopmz "poom m 9mm om om cmpSMOQLMo + z mica «COLSEomopOHno om 9mm NH NH em 2 ©IOH .con350tpn0Hno mHooHomumcmxmm ow mm mH a: mcmhsmoopmo + z oloa acopsfiopopoano a: mH mm am 2 $-0H .cotsEOtnpOHno manzaomlnmpmz upoozm O.H m.o m.o m.o m.o m.o :.o m.o N.O H.O 0.0 mm pcoEpmomB whom . pCMHm omppommlampOp mo pcoohom .zoaamo oHo:szIOH Eopm mpomppxm poop can poocm ho DAB :0 coapmpmoom one .pcospmonu copssoponoHnouoza gonna ammo m .mm mHDMB 97 Table 37. The percent extractable lL‘C-chlorbromuron remain- ing in lO-day-old barley and 7-day-old corn shoots and roots, 8 and 5 days respectively, after luc- chlorbromuron treatment. 1AL‘C in shoot 15C in root Hexane- Water- Hexane- Water- soluble soluble soluble soluble Treatment fraction fraction Total fraction fraction Total Barley: Chlorbromuron, 10-6 M 2.3 1.3 3.6 1.9 0.5 2.9 Chlorbromuron, 10-6 M + 5.6 2.u 8.0 9.1 0.8 u.9 carbofurana Corn: Chlorbromuron, 10-6 M 1.7 0 1.7 2.2 2.9 5.1 Chlorbromuron, 10-6 M + 1.9 0.9 2.3 1.3 3.1 9.9 carbofuran a Carbofuran was applied as a seed treatment at u oz/lOO lb of seed. 98 but enough to double the lL’C-chlorbromuron content (Table 36). The l"‘C-chlorbromuron in the water-soluble portion of the barley root, from plants receiving the combination treatment, did not run at the front. The data in Table 36 show that one major metabolite and two other metabolites, each approximately half the major one, were present in the f lL’C-chlorbromuron parent barley root. The amount 0 material present in the root was only slightly increased by the combination treatment (Table 37). The hexane-soluble fraction in the lL‘C-chlorbromuron treated barley shoot contained four metabolites of almost equal concentration and the parent lL‘C-chlorbromuron in a slightly higher proportion (Table 36). The carbofuran seed treatment resulted in a greater portion of 1“C present as the parent compound in the hexane-soluble fraction of the barley shoot. Furthermore, one metabolite was absent and another greatly decreased compared to the herbicide only treatment (Table 36). The fate of the 140 in the hexane-soluble barley root fraction for the herbicide treatment alone was the same as in the barley shoot except that none of the 1"‘C metabolites lac-chlorbromuron on the TLC plate. The ran ahead of the presence of carbofuran changed the l“Cuchlorbromuron metabolism in the root in a manner similar to that observed in the shoot (Table 36 and 37), again increasing the content of parent lac-chlorbromuron. 99 The data in Table 37 show that more parent 1“c— chlorbromuron was present in the hexane—soluble fraction than in the water-soluble fraction, in both shoots and roots of barley plants receiving the carbofuran—chlorbromuron treatment. These results contrast with those of Nashed gt al. (50) who found no metabolites formed in the susceptible cucumber, H days after chlorbromuron treatment. However, they found "binding" of a portion of the original chlorbromuron. It is possible that cucumber, a dicotyledonous plant, may be unable to metabolize chlorbromuron whereas barley, a monocotyledonous plant, does to a certain extent even though both are susceptible. t l”Cochlorbromuron remained in the water- No paren soluble fraction of the corn shoot 5 days after treatment (Table 38). One major and two minor metabolites were found. The carbofuran seed treatment altered chlorbromuron metabolism by causing the disappearance of one minor metabolite and the appearance of another as well as the presence of a low level of parent luC-chlorbromuron. Corn roots contained identical water-soluble metabolites to the corn shoots following lLlC-chlorbromuron treatment (Table 38). However, they contained less of the two minor metabolites and 23 percent of the 1“C was present as the parent luc- chlorbromuron. The carbofuran seed treatment decreased the major metabolite in favor of the other metabolites. The hexane-soluble fraction from the corn shoot treated lOO .ocsoasoo ucmpmo mopmoHocH n .ommm mo pH OOH\No : pm ucmEpmoLo boom m mm omHHaom was cmgomonpwo m oOH mm H mm cmLSNOQLMo + z mIOH .COLSEOLQLOHQQ pom :H mm . om z ouOH .consEOLonHeo oHnsHomlocmxom on m :m o: nonsmoppmo + z oIOH .comSEopohono 3mm 0 m mm 2 QIOH «COMSEOLQHOHQQ oHQSHomunmpmz "poom omm mH NH mm nonsmonhmo . + z mIoH .QOLSEOLQLOHQQ nHm NH mm mm s ouOH .e09350nen0Heo oHozHomlmcmxom m cm Hm mN mcwmsmoonmo + z mIOH .copsEopoHono Hm NH mm 2 oloH .COQSEOLQQOHEU O.H m.o w.o N.O m.o m.o :.O m.o N.O H.O 0.0 mm oopuomm Hmu0p mo ucoopom oHnoHOm;popm3upoozm pomEpmmpB whom pcmHm .pcoEumohp COQSEopnhOHnolo: popmm mzwo m .cmoo oHonzmouN Eopm muomhpxm poop ocm poonm mo DAB :0 :oHamnmwo m 089 .mm mHnme 101 with chlorbromuron contained three lL‘Cnmetabolites and parent luC—chlorbromuron (Table 38). The parent compound and a metabolite which occurred at the same Rf as the major one in the water-soluble fraction were present in almost equal proportions. The other two metabolites contained 23 and 17 percent of the 1”C. The carbofuran seed treatment increased the level of the major metabolites at the expense of the others. The number of metabolites found was not changed. The water-soluble luC-metabolites from the luc- chlorbromuron treatment found in corn roots were similar to those found in the shoot (Table 38). However, 23 percent l“C remained as the parent luC-chlorbromuron. The of the carbofuran seed treatment reduced the level of the major metabolite and increased by the same amount a metabolite t l”Cuchlorbromuron with an Rf of 0.3. The level of paren in the water-soluble fraction was unaffected, whereas in the hexane-soluble portion there was a threefold reduction. Furthermore the carbofuran seed treatment caused the accumulation of a new metabolite, the disappearance of two minor metabolites and an increase in the major metabolite. The amount of luC-chlorbromuron present in the corn shoot was slightly increased by the carbofuran seed treatment, while that in the root decreased by almost the same amount (Table 37). The greatest change in the root came from a reduction in the content of parent luC-chlorbromuron in the hexane-soluble fraction. 102 The data shown in Table 38 for the chlorbromuron treat- ment of corn support the results of Nashed gt_al. (50) with regards to the number of metabolites found by TLC. Due to the different treatment procedures, it can only be speculated that they may be the same compounds. Chlorbromuron markedly increased respiration and reduced photosynthesis and growth of barley. The carbofuran seed treatment more than doubled the amount of luc- chlorbromuron found in both the shoot and the root (Tables 36 and 37). .Perhaps chlorbromuron was already so toxic to barley at the chlorbromuron level used in the experiment that no additional toxic response could be obtained by the carbofuran seed treatment. Photosynthesis in corn was not further reduced by the addition of the carbofuran seed treatment to the chlorbromuron and yet synergistic reductions in leaf area and dry weight were obtained (Table 2 and 20). It might logically be prOposed that if the increased amount of parent compound in the corn leaf is not causing a further reduction in photosynthesis that it is merely prolonging the time over which it occurs, and thus giving the synergistic effect observed. It would appear then if the photosynthesis reduction were not too severe and the plant could metabolize the chemical that perhaps the plant might overcome the effect of chlorbromuron. Carbofuran interacted synergistically with chlorbromuron to reduce the height and weight of ul-day-old corn, the root length of 3-day-old barley and the leaf area 103 and dry weight of 7-day-old corn grown in sand culture. The basis for this interaction appeared to be related to the increased accumulation of chlorbromuron and its metabolites in the corn shoots. Furthermore, carbofuran reduced chlorbromuron metabolism in corn shoots causing an increase in the parent chlorbromuron level in the corn shoot. SUMMARY AND CONCLUSIONS In the greenhouse, carbofuran in combination with alachlor caused a synergistic reduction of barley dry weight. The combination effect of carbofuran with chlorbromuron on barley dry weight was antagonistic. A synergistic response was obtained with carbofuran in combination with butylate or chlorbromuron on corn dry weight. Corn height was also synergistically reduced by the chlorbromuron and carbofuran combination treatment. Barley height was similarly reduced by the combination of butylate and carbofuran. Alachlor, butylate and chlorbromuron when combined with carbofuran all synergistically reduced the radical length of germinating barley. The carbofuran combination with alachlor also reduced barley germination in a synergistic manner. The respiration of 10 and 14-day-old barley plants grown in the growth chamber was significantly increased by each of the herbicides studied in combination with carbofuran applied as a seed treatment. The butylate- carbofuran combination treatment also synergistically increased 02 uptake. Net photosynthesis of barley was significantly reduced only by the chlorbromuron—carbofuran combination. The results of these altered physiological processes were manifested in significantly reduced leaf area, dry weight and height. A synergistic reduction was evident on leaf area and plant height for butylate or IOU 105 alachlor in combination with carbofuran, as well as dry weight for the latter combination. The respiration of corn grown in a growth chamber was unaffected by the pesticide combinations, however, chlorbromuron with or without carbofuran significantly reduced net photosynthesis. This resulted in synergistically reduced leaf area and dry weight per pot and a significant reduction in plant height by the chlorbromuron—carbofuran combination treatment. Butylate and chlorbromuron were absorbed by barley roots and translocated to the shoot where they accumulated. Alachlor was present in about equal amounts in the barley shoot and roots. The addition of carbofuran to the herbicide treatments lowered the concentration of butylate and chlorbromuron in the shoot and slightly raised the content in the root. The alachlor concentration was increased in both barley shoots and roots by the herbicide—insecticide combination.‘ The corn shoots contained proportionately less butylate and alachlor but more chlorbromuron than the root. The carbofuran treatment lowered the butylate concentration in the shoot and raised it in the roots. This pattern was reversed for chlorbromuron while the alachlor concentration was raised in both shoots and roots. The 1"'0 contained in the barley and corn shoots and roots was divided between the 80 percent acetone-insoluble fraction, a water-soluble fraction, and a small portion found in the hexane-soluble fraction. The corn root treated .I Ill ('1': II." I I if j— 106 IM with C-butylate was the only treatment with a substantial amount of hexane-soluble 1”C. The hexane-soluble fraction from the lac-alachlor- treated corn contained four metabolites and no parent material. The presence of carbofuran changed the concentrations of the metabolites, but not their identity. The water-soluble extract from the lL‘C-alachloratreated barley shoots and roots contained one major metabolite with or without the carbofuran seed treatment. The minor metabolite in the shoot was different from the minor metabolite in the root for the alachlor treatment. The minor metabolite in the root and shoot were similar for the alachlor—carbofuran treatment. The minor metabolite from the alachlor-carbofuran treatment was not the same as the alachlor treatment alone. The corn shoot contained four l[JG-metabolites in the water-soluble fraction from the lL’C-alachlor treatment. One minor compound disappeared due to the carbofuran treatment and a minor and a major metabolite remained the same. The water-soluble extract luC—alachlor treatment contained from corn roots following the three metabolites with or without the carbofuran combination. Of these metabolites, only one minor one appeared to be the same following the combination treatment, the others tiu were different. The amount of paren C-alachlor remaining in barley and corn, 10 and 5 days respectively, after 107 treatment was small. The barley shoot contained a greater prOportion of l1"C-alachlor than the root. This proportion was reduced in the shoot and raised in the root by the carbofuran combination. In corn these results were completely reversed. Barley root extracts contained three metabolites in the hexane-soluble fraction from 1“ C-butylate treatment. The addition of carbofuran produced, of the three metabolites found following the lL‘C-butylate-carbofuran treatment, only one metabolite similar to that found for the butylate treatment alone. Two metabolites were present in the hexane- soluble extract from corn roots following butylate treatment. Following the luC-butylate-carbofuran treatment, three different metabolites were found. The water—soluble extract from roots and shoots of luc- luC—metabolites. In butylate-treated barley contained two the shoot they remained unchanged on the addition of carbofuran to the treatment. In the root the combination treatment resulted in the presence of two different metabolites. The water-soluble extract from the shoots of l”Cubutylateutreated corn had three metabolites present. The combination treatment of butylate and carbofuran reduced the number of lac-butylate metabolites to two. The corn root contained three water-soluble lL‘C-metabolites following the luc- butylate treatment whether or not the corn seeds had been treated with carbofuran. Only two of these metabolites may 108 have been the same. Barley contained more parent luc— butylate than corn and for both species the roots contained more parent l“Cubutylate than the shoots. The amount of luC-—butylate in the shoot was reduced for both species parent when the insecticide was combined with the herbicide while the amount in the root increased. Following lL‘C-chlorbromuron treatment four luc- metabolites were found in the barley shoots and roots in luC-metabolites were the hexane-soluble extracts. Three found in corn. When carbofuran was combined with chlorbromuron both shoots and roots of the two species contained one less metabolite in the hexane—soluble fraction. The water-soluble extract from the shoots of chlorbromuron treated barley contained three metabolites while the root had four. The number of metabolites remained the same in the roots and shoots following the carbofuran-chlorbromuron treatment but the metabolites were not all similar. The water—soluble fraction from the corn shoots had three metabolites, at least one of which was different following the combination treatment of carbofuran with chlorbromuron. This was not the case for the root which had the same three water-soluble 1“ C-metabolites, regardless of the presence of carbofuran. The amount of parent luC-chlorbromuron present in the barley shoot and root doubled on the addition of carbofuran to the chlorbromuron treatment. The amount of parent luc- 109 chlorbromuron in the corn shoot was also increased under these conditions, while the amount in the root decreased. Alachlor was absorbed to a greater extent by the susceptible barley plant than by the tolerant corn plant. The alachlor content increased in the root and metabolism decreased in the presence of carbofuran seed treatment These factors would appear to contribute to the decreased growth of plants receiving the combination treatment. INC Although the combination treatment also increased the content in the shoot, the increased respiration may be related to the less parent alachlor present. Butylate was taken up by barley to a much greater extent than by corn. The addition of the carbofuran seed treatment lMC present in the barley decreased the concentration of shoot, corn shoot and root. The corn root treated with the pesticide combination contained more parent lL‘C-butylate than barley although the barley root contained more total 1”C. Under the conditions of the experiment the level of luC-butylate found in the roots was apparently not critical as corn remained tolerant. The decreased l“C level in the barley and corn shoot coupled with a decrease in parent butylate content indicates little change in the metabolism within the shoot due to the carbofuran treatment. Thus the reduced growth of barley may be related to the observed increase in respiration. This response may be caused by an increase in susceptibility of barley to butylate, a carbamate herbicide and the carbamate insecticide, 110 carbofuran. The carbofuran seed treatment with l”Cuchlorbromuron increased the 1”C content of barley roots, whereas the 1”C content in the shoots was lowered. The combination treatment greatly increased the amount of parent luc- chlorbromuron. However, no synergistic growth reduction occurred. Respiration was significantly increased and photosynthesis significantly decreased by the chlorbromuron treatment alone resulting in decreased corn growth. The 1”C content as well as the parent luC-chlorbromuron content increased in corn shoots and decreased in corn roots if the seed was treated with carbofuran. Since net photosynthesis was not further reduced by the carbofuran seed treatment the slight increase in shoot content of chlorbromuron plus a decrease in ability to metabolize the herbicide, might account for a longer period of contact of chlorbromuron with the leaf tissue resulting in decreased leaf area and dry weight. LIST OF REFERENCES 10. 11. 12. LIST‘OF REFERENCES Agbakoba, C. S. O. and J. R. 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Conf. p. 36. - APPENDICES APPENDIX A Modified Hoaglands Solution 1. 1 M KH2POu KNO3 Ca(NO3)2.UH20 g/l MnCl3 g/l H380“ g/l ZHC12 .05 g/l CUCl2-2H2O .05 g/l MoO3 M M u. l M MgSOM'7H2O 5 '“H2O 5 OOONH .4 .05 M ETDA .05 M F830“ 0\ 0CD or 26.3 g/l Sequestrene® pH 6.5-6.8 with 1 M NaOH 117 ml/l ml/l ml/l ml/l ml/l ml/l ml/l I'llllllllll1l'll‘"| I'l'l APPENDIX B Method of recorder chart paper conversion to ug/CO2/min. Compressed air flow rate 500 cc/min CO2 content of compressed air 330 ppm or .033 percent Molecular weight of CO2 MA I Standard volume 22.4 l/mole 22.“ 1 contains 1 mole of gas .5 l/min contains .0223 M (g/l) of gas/min 1 mole of gas contains .033 percent CO2 therefore .022 M/min contains 7.359 x 10"6 M CO2/min or 7.359 x 10- x uu = 3.23 x 10-3 g COz/min or 3.23 mg CO /min = 23 ug CO2/min therefore on ARGENT catalog no. S-72l66 recorder paper 1 unit change = 3.23 pg COz/min 118 119 O NO OO O NOH OOH O.H OOHOHHOOHO N EOOOOOOOOHOO O OOH OOH O NOH OOH O.H OOOOOHOOHO O.H EOOOOHOOOHOO .HmHHHcmeHO mocmmHmEompm O NO OO O OO HO O OumOON OOOO O OHOHNOOO O OO OO O OO OO O OumOON OOOO O.H OOHOEOOOHOHOO O OO NN O NO NO O mumOON OOOO N.O OOHOEOHOOOHOO O ON OO O NOH OO O.N OumOON OOOO O OOOHNOOO O ON OO O OO NO O.N mumOON OOOO N OOOHOHOO O NN OH O ON OO N OHOOH OO N :OOOEOOOEOHOO O HO OO O OO OO H OHOOH Om O.H OctagothtoHOO O OH OH O OO OO O OHOOH Om ON.O OctagothtoHOO O OO OO O OO NO H OHOOH Om ON.O OOHOEOOOOOHOO O NN NO O HOH HOH O emchotosHm H OHHOOOHOHtB O NO NHH O ON OO. 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