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UBC Theses and Dissertations

Mode of action of monuron (3- (4-chlorophenyl) -1, 1- dimethylurea. Baldwin, Richard William Ward 1960

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1 THE MODE OF ACTION OF MONURON (3- (4-chlorophenyl) -1,1- dimethylurea) by RICHARD W.W. BALDWIN B.S.A., University of Br i t i s h Columbia, 1956 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN AGRICULTURE in the Department of PLANT SCIENCE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1960 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree th a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood tha t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia, Vancouver S, Canada. ABSTRACT The herbicidal action of monuron, 3-(4-chlorophenyl)-1, 1-dimethylurea, cannot be completely realized until the mode of action is f u l l y understood. It is believed that the primary site of action is in the photosynthetic complex. The present investigations included: The study of the effects of monuron on some enzyme systems, greenhouse studies on the effects of monuron on the morphology of potato plants, f i e l d studies on the yield of barley treated with the substituted urea herbicides, the residual effects of the substituted urea herbicides applied to s o i l , the effects of monuron and the interaction of monuron and vitamin K on the rate of the photolysis of water by isolated chloro-plasts. The photolysis of water was followed by observing changes in potential of a potasium ferricyanide solution containing isolated chloroplasts. Monuron inhibited the protease enzyme system and stimulated the lipase enzyme system. This herbicide reduced the total weight of potato tubers per plant, the root to top ratio and the total weight of barley grain per acre. The top growth of potato plants and the bushel weight of barley were increased with monuron applications. 1 X lO"^ moles of monuron reduced the rate of the H i l l reaction by more than 507.. The data presented did not confirm vitamin K as a cofactor in the H i l l reaction. TABLE OF CONTENTS Introduction Page 1 Review of the Literature Page I Methods and Materials Page 12 Results Page 19 Discussion Page 36 Summary Page 40 Bibliography Page 41 Appendix Page 44 LIST OF FIGURES Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Page 20 Page 21 Page 24 Page 25 Page 28 Page 29 V Table I Table II Table III Table IV Table V Table VI LIST OF TABLES Page 31 Page 32 Page 32 Page 33 Page 34 Page 34 ACKNOWLEDGEMENTS The author wishes to express his appreciation for assistance received from the following during the preparation of this thesis. Dr. A.J. Renney, Associate Professor, Department of Plant Science, University of B.C., who showed endless patience and who provided guidance, encouragement and understanding throughout the preparation of this thesis. Dr. J.C. Campbell, Professor of Dairying, University of B.C. who graciously placed the f a c i l i t i e s of his laboratory at the disposal of the author and who also provided assistance with d i f f i c u l t i e s encountered with procedures and guidance in the preparation of the manuscript. Dr. V.C. Brink and Dr. CA. Hornby, Department of Plant Science, University of B.C., who provided assistance in the analysis of data and the preparation of the manuscript. The Presidents Committee on Research, who provided funds, without which this work would not have been possible. The graduate committee and members of the Faculty of Agriculture who have provided guidance throughout my graduate programme The many friends and relatives who donated the Ice which was required for the latter portion of the experimental work. 1 INTRODUCTION Monuron has become a widely used herbicide. The mode of action of this herbicide i s , at present, only partly understood. Until this is completely understood the potential uses of monuron w i l l not be f u l l y exploited. An attempt has been made in this essay to c l a r i f y some of the aspects of monuron's phototoxicity, particularly the inhibition of photosynthesis caused by low concentrations of monuron. REVIEW OF THE LITERATURE Monuron, 3- (4-chlorophenyl) -1,1- dimethylurea, f i r s t described in 1951 by Bucha and Todd (8) is one member of the substituted urea herbicides. Other commonly marketed substituted urea herbicides include diuron, fenuron and neburon. These herbicides are products of the E.I. du Pont de Nemours Co. Monuron is a s o i l sterilant at high rates of application (10-40 lbs. per acre) and i t is used as a selective herbicide in certain crops at lower rates (1.1/2 - 2 lbs. per acre). The selective herbicidal use of monuron is now largely being replaced by diuron, 3-(3,4- dichlorophenyl)-1,1- dimethylurea. The s o i l pH has a marked effect on the phototoxicity of the substituted urea herbicides as i t effects adsorption on the s o i l colloids-(10). In water solutions the substituted urea herbicides have a net positive charge (10). Monuron is lost from the s o i l by leaching, breakdown by s o i l microorganisms and by photodecomposition. Photo-decomposition is especially prevalent in areas of limited r a i n f a l l . (15,29,33,37). 2. Monuron causes one or more of the following symptoms to appear on leaves of susceptible plants: water soak blotch, wilt, petiole and/or stem collapse, indeterminated grey blotch, abscission, rapid yellowing and partial chlorosis. (25). The margins of the older leaves are the f i r s t to show chlorosis,- the chlorosis then proceeds towards the centre un t i l the entire leaf is chlorotic. Barley treated with monuron at 2 lbs. per acre had a deeper green colour than did the controls. (9). Maturation of oats was delayed by several days by the application of monuron at the 2 lb. rate. The same result was noted although less pronounced with barley and wheat. (32). Monuron appears to have some effect on t i l l e r i n g in some species. This has been found in sugar cane and to a small degree i n barley. (32). The 1000 kernel weight of winter barley was Increased and the production of anthocyanins was reduced following applications of monuron. (32). Excised roots may be grown in nutrient solutions containing lethal quantities of monuron. (26). This suggests that this herbicide is phytotoxic primarily to the aerial portions of the plant. Monuron enters the plant system most rapidly through the roots, although i t appears possible to k i l l plants by leaf applications and in some cases through stem applications. (12,19,26). Fang and his associates, (12) have found that when monuron, labelled with is applied to a leaf 10% of the Cj^ to be translocated throughout the plant very rapidly. The rate of monuron entry into the foliage depends on the thickness of the cuticle. (26). Following the application of Cj^ labelled monuron to the foliage, two compounds containing Cj^ occur. One of these compounds is monuron the second has not yet been identified. It is believed that the unidentified compound containing Cj^ i s the result of the plant incor-porating monuron into i t s metabolism. This belief is supported by the fact that the concentration of the unidentified labelled product increases with time, while the concentration of the labelled monuron decreases. (12). Translocation of monuron takes place largely through the xylem tissue, although as indicated above some translocation may occur in the phloem. (26). Plants grown i n monuron treated s o i l are more subject to injury i f post treatment conditions favour rapid transpiration. (23,26). This indeed would be expected as the main site of monuron entry i s through the roots and monuron is translocated primarily via the xylem. Monuron causes no gross morphological changes but does cause some abnormalities on the cellular level. (17). There is no c e l l proliferation (26), but there i s a collapse of the cambium, a disorganiza-tion of the phloem, mesophyll and pallisade cells and a decrease i n the number of xylem c e l l s . (9). The last mentioned phenomena may be the result of the disorganized cambium or a result of some change i n the biochemical constituents of the c e l l . It has been suggested that the pH within the plant may be altered by the presence of monuron. (32). Many workers believe that the pH determines the production of xylem and phloem from the cambium layer. It i s interesting to note that anthocyanin production within some species i s altered i n the presence of monuron, and that anthocyanin production is also believed to be related to the internal pH of the plant. Mitosis i s retarded by monuron i n barley and onion meristerns (9) and is arrested at the resting stage i n the onion root t i p . (16). A break-4. down of the nucleus has been observed in onion roots following treatment. (9). Minshall and McLarty (21) found that low concentrations of monuron stimulated root growth in several species, while higher concen-trations retarded root growth. It was also noted by these workers that the surface c e l l s i n the region of elongation were disrupted. The metabolic pathways within monuron treated plants are some-what altered. The nitrogen and protein balance is disturbed. The nitrate and the ammonia nitrogen content is lower and the protein content higher in treated plants as compared to non treated plants, (13,30) while the carbohydrate content is reduced. (13). The phosphorous content has been observed to be increased in some species (36) and the percentage ash appears to be increased i n barley following monuron treatments. (32). At the time the f i r s t monuron symptoms become vi s i b l e , growth has completely stopped. (27). In treated velvet beans, the dry weight of plants was at a maximum four days following treatment; thereafter the dry weight diminished until the eighteenth day when the plants were dead. (28). During this experiment i t was found that the monuron content of the leaves increased to a maximum at seven days after treatment and there-after decreased. The decrease may be due to the breakdown of the herbicide by the plant as suggested earlier. Using excised bean leaves, Minshall found that from fifteen to twenty micrograms per gram of leaf material reduced the dry weight production by 907.. (24) . Less monuron was required to inhibit the dry weight production in young leaves as compared to the mature leaves. Earlier Minshall found that 17, 30, 48 & 95 micrograms of monuron per gram of leaf material inhibited the production of dry matter by 89, 100, 100 & 1007. respectively and that transpiration was inhibited by 38, 57, 67 & 70% respectively. (22). From this work he suggested that monuron may act as a photosynthetic poison. In the same year Cooke (11) found that low concentrations (1 x 1 0 m o l e s ) of monuron inhibited the H i l l reaction, ie. the photolysis of water, and, Wessels and van der Veen (41) likewise found that this low concentration of monuron inhibited photosynthesis. The latter workers also reported that the inhibition of photosynthesis was localized, indicating that there was l i t t l e movement of the herbicide within the leaf once the site of action was reached. This does not agree with the results of other workers discussed earlier. Wessels and van der Veen also found that the photosynthetic activity could be retained by washing away the monuron and they assume that the monuron is adsorbed to the cyclopentanone ring of the chlorophyll molecule. The cyclopentanone ring is referred to as the active site. Abel (1) as well as Coggins and Crafts (10) refer to the work of Wessels and van der Veen and support the hypothesis that monuron is adsorbed to the active site of the chlorophyll molecule. It is believed that a l l of the substituted urea herbicides behave i n this manner - diuron being the most effective, followed by monuron and fenuron. Wessels and van der Veen assign a relative activity for these herbicides of diuron 2500, monuron 125, and fenuron 12.5. (41). The comparative effective-ness of neburon is not known. Neburon is adsorbed most strongly to cellulose particles, followed by diuron, monuron and fenuron in that order. (10). Wessels and van der Veen have postulated that vitamin K is the 6 energy acceptor of the light excited chlorophyll molecules. These workers have found that diuron, in a concentration just sufficient to inhibit photosynthesis and vitamin K occur in nearly the same concen-tration - that i s , about one hundredth of the concentration of chlorophyll molecules. They postulate further that the remaining chlorophyll molecules are either enolized or chelated and thus prevented from being biologically active. As the major part of the experimental work which follows involves photosynthetic responses i t was thought advisable to review br i e f l y the present theory concerning photosynthesis. Photosynthesis is the conversion of radiant energy to chemical energy. No l i f e on earth would be possible without this energy conver-sion group of reactions. Photosynthesis, until quite recently, was thought of as a simple, yet unresolved reaction or series of reactions that were represented by the following equation: 6 C 0 2 + 6H2Q ^ C6H 1 2 O5 + 60 2. It i s assumed that by some mechanism six atoms of carbon from atmospheric carbon dioxide were attached to six molecules of water. At the same time the six molecules of the newly formed carbohydrate fused to form a hexose sugar. It was sometime later that the workers queried the presence of the 3, 4 , 5 and 7 carbon sugars that were found i n plant material. It was then thought that photosynthesis may be best represented by the equation: n C 0 2 + 2 n H 2 0 + light energy (CH 20)n + n(>2 + ttf^O This reaction, referred to as the H i l l reaction, or the photo-lysis of water, may be followed by using an a r t i f i c i a l hydrogen acceptor in place of carbon dioxide. Frequently used hydrogen acceptors are ferricyanide and benzoquinone. These reactions are: + light + (1) 2 H 0 0 + 4 K - , Fe(CN), + 4 K _ > 4 K A Fe(CN), + 4 H T + 0 2 2 3 6 chlorophyll 4 6 * potassium ferricyanide potassium ferricyanide It is evident that the K ions replace the H ions leaving the H free in solution. / v u S h t A ^ ( 2 ) 2 0 » f / - 0 + 2 H 0 0 ^ 2 H 0 > O H + 0 / u " r > <nu " \ _ - z ' u 2 chlorophyll Benzoquinone Hydroquinone In may species the photolysis of water does not occur. These species have a different donor. Such is the case of the green sulfur bacteria. The H i l l reaction of the green sulfur bacteria is represented: light C 0 O + 2 H 0 S => CH„0 + H O 0 + 2 S . It is apparent that 2 2 chlorophyll 2 2 V V the same general scheme takes place as in the photolysis of water, thus a general equation for the H i l l reaction may be represented as (38): C 0 0 2H,A l l 8 h t „., > CH 9 0 + H O 0 + 2 A . It has been recognized 1 ' chlorophyll 4 for some time that photosynthesis consists of a light and a dark reaction. The photolysis of water is a light reaction and the reduction of carbon dioxide is a dark, or enzymic reaction. (3). If photosynthesis is considered to terminate at the synthesis of the hexose sugars, then other dark reactions include the incorporation of the reduced carbon dioxide into ribulose 1,5- diphosphate to form two molecules of phosphoglyceric acid, as well as a l l the other enzymic reactions which take place in the synthesis of hexose sugars. The light reaction consists of many more reactions than the photo-lysi s of water - a number of which have not yet been resolved. Fhotosynthetic phosphorylation is the synthesis of energy rich adenosine triphosphate (ATP) u t i l i z i n g light energy and adenosine diphosphate (ADP). This may be contrasted to mitochondrial phosphorylation in which the formation of ATP is at the expense of energy r i c h compounds within the c e l l and ADP. The ATP required for the metabolic processes within the plant may be supplied, during daylight, by photosynthetic phosphorylation, even though and/or water are not available. (2,5). It has been postulated that early forms of l i f e were more dependent on photosyn-thetic phosphorylation than on carbon assimilation. There are two types of photosynthetic phosphorylation: cyclic phosphorylation and terminal phosphorylation. (4,39,40). Cyclic phosphorylation i s that which does not require water or carbon dioxide. This may be represented by the following scheme. (39). light 2H20 chlorophyll TPN FMNH 20H -7* 2H20 ^ 2H ADP + Pi ATP Modified from Whatley et a l . (39). FMN i s the abbreviation for flavomononucleotide and TPN the abbreviation for triphosphopyridine nucleotide. These workers suggest that TPN and FMN are the energy transfer agents. Terminal transpiration requires the presence of water and follows a different scheme. (40). light 2H20 + 2TPN + 2ADF + 2Pi 2TPNH.H + 2ATP + 0-chlorophyll The reduced TPN and ATP are util i z e d i n the reduction of carbon dioxide to carbohydrate. The H i l l reaction and phosphorylation are linked reactions. The rate of the H i l l reaction may be increased by three and one half times by having conditions present which allow the formation of ATP. (6). Arnon (4) has prepared the following two systems of cyclic phosphorylation; one scheme requires vitamin K as an energy transfer, the other system requires FMN. The vitamin K pathway may be represented by the following equations. (4): light + 2 chlorophyll molecules > 2 chlorophyll + 2e vitamin K + 2e reduced vitamin K reduced vitamin K + 2 F e + + + cytochrome I >. vitamin K + 2 F e 4 4 cytochrome I 2 F e 4 4 cytochrome 1 + 2 chlorophyll"*" + ADP + Pi v 2 Fe 4 4" cytochrome 1 + 2 chlorophyll + ATP. The FMN pathway is represented by the following equations. (4): 2 chlorophyll — 1 : L 8 h t 5> 2 chlorophyll + 2e TPN + 2e > reduced TPN reduced TPN + FMN * TPN + reduced FMN I j | 11 reduced FMN + 2Fe cytochrome II .^ 2 FMN + 2 Fe + cytochrome II 2 Fe cytochrome II + 2 Fe cytochrome I > 2 Fe' '' cytochrome II + 2 Fe 4 4" cytochrome I 2 Fe 4 4" cytochrome 1 + 2 chlorophyll 4 + ADP + P i 2 cytochrome 1 + 2 chlorophyll + ATP. The complete system is represented on page 10. There is good evidence that vitamin K type compounds play a role in the H i l l reaction ( 7 ) . Chloroplasts which have been extracted with cold petroleum ether lose the faculty to carry on the H i l l reaction. This faculty may be returned by the addition of the petroleum ether extract, TERMINAL PHOSPHORYLATION CO, ASS IMILAT- | IOW CYCLIC PHOSPHORYLATION menadione (vitamin K^) or to a lesser degree by the addition of carotenoids. (7,20). Other evidence now indicates that the carotenoids do not play a role i n the H i l l reaction (31) but may have a function i n phototropisms, as i n some of the light stimuli experienced in the animal kingdom. (38). It must be pointed out that vitamin K type compounds occur associated with the chloroplasts of a l l green plants. Thus the role of the various plant pigments associated with photochemistry is not yet f u l l y understood. Chlorophyll a is associated with the photolysis of water and with the evolution of oxygen. Chlorophyll b is said to transfer the excitation energy of the fucoxanthins and the phycobilins is transferred to chlorophyll a with an efficiency of 100%. The carotenoids transfer the excitation energy with an efficiency of 20-40%. (35). These, as mentioned above, are also believed to play a role i n phototropisms. (38). MATERIALS AND METHODS A. The effect of monuron on various enzyme systems within the plant. (i) Catalase System The procedure followed for this and the other enzyme systems tested are essentially those described i n Loomis and Shull. (18). The material used for the catalase system was fresh leaves and buds of Spiraea. The time required to liberate 5.0 ml. of oxygen was recorded. 20.0 ml. of monuron in concentrations of 100, 10, 1.0, 0.1, 0.01, 0.001 and 0.000 ppm. were used to make up the total volume as described. ( i i ) Oxidase System The plant material used was freshly picked Forsythia flowers. 1.0 gram of the material was ground with mortar and pestle in 10 ml. of a monuron solution, i n one of the following concentrations: 100, 10, 1, 0.1, 0.01 and 0.00 ppm., then 5 drops of 17. guaiacum solution were added. In the case of one run 1.0 gram of the material was ground, 10.0 ml. of d i s t i l l e d water were added, the suspension was boiled, allowed to cool and 5 drops of 57. guaiacum added. ( i i i ) Peroxidase System The procedure employed here was essentially the same as that employed to follow the oxidase system, however, the material used was sweet potato. 1.0 gram portions of the material were ground in a mortar and 10.0 ml. of monuron added i n the same concentrations as used i n the oxidase system. 5 drops of 17. guaiacum solution were then added followed by 5 drops of 47. hydrogen peroxide. (iv) Lipase System A solution of monuron in varying concentrations, replaced the 10.0 ml. of water described in the procedure. The concentrations of monuron used were: 10, 1.0, 0.1, 0.01 and 0.00 ppm. of monuron. The control contained no monuron and boiled chloroplasts. These were used on husked castor beans. (v) Protease System The plant material used was Austrian Winter peas. B. Some effects of Monuron on potatoes. Whole seed potatoes were sown in six inch pots containing washed sand. Monuron was applied to the various pots at the following rates: 41b. per acre, 21b. per acre, l i b . per acre, 1/2lb. per acre and 0 lb. per acre. There were 10 pots per treatment. The pots were set in a randomized block pattern in a green house, and watered regularly every six days with 200 ml. of Hoagland1s complete solution. After a growing period of 108 days, observations were made. C. Some effects of the Substituted Urea Herbicides i n Barley. Monuron, diuron, fenuron and neburon were applied to 30 sq. foot plots which had been sown to barley. The herbicides were applied at two rates, 2 and 1 lbs. per acre and were applied either as an emergence or post-emergence treatments. There were four replicates of each treatment. The plots were randomized. Fenuron was ground to fine powder and the required weight was mixed with s o i l and applied to the required plots. A polyethylene screen, enclosing 30 square feet, was used to prevent d r i f t during application. A hand sprayer was used for a l l treatments, except the fenuron treatments described above. Observations were taken five times during the growing season. D. The residual effect of four Substituted Urea Herbicides. Treatment plots of thirty-six square feet la i d out i n the Agronomy Field, University of Bri t i s h Columbia, were each replicated four times. The herbicides used were monuron, fenuron, diuron and neburon at 4 and 2 lbs. per acre. The herbicides were applied to fallow ground. The residual effects of the herbicides were assessed 7 times by counting quadrats of seedling weeds on the treated plots as compared to the non-treated plots. Fenuron was applied by mixing the required weight of powdered herbicide with sand and applying i t to the plot. Monuron was applied with a small, wheeled pressure sprayer with a six foot boom. This apparatus was not satisfactory for herbicides formulated as wettable powders, and for diuron and neburon a small hand sprayer was used. E. Effect of monuron on the H i l l reaction and the interaction of vitamin K type compounds. The chloroplasts were prepared by taking 100 grams of fresh, washed mature market spinach without the midrib, and blending for thirty seconds in a Waring blender with cold 0.5 M. sucrose. The homogenate was strained through several layers of cheesecloth into a 250 ml. erlenmeyer flask in an ice bath. The erlenmeyer flask was covered with towelling to exclude as much light as possible. The crude chloroplast suspension was then placed in two 50 ml. tubes and centrifuged in a Servall refrigerated centrifuge, at 1000 X gravity for five minutes. The supernatant was discarded and the "pellet" in each tube was resuspended in 10 ml. of 0.5 M sucrose. The chloroplasts were washed three times in this manner. Following the washing a l l the chloroplasts were suspended in 80 ml. of 0.5 M. sucrose and placed i n an ice water bath. An ether-dry ice bath was prepared and 36 - 15 ml. plastic test tubes were placed in the ether-dry ice bath to cool, then 2.0 ml. of the chloroplast suspension was added to each of the 36 tubes. After 5 minutes i n the ether-dry ice bath the frozen chloroplast suspensions were stored under dry ice in a deep freeze. The temperature of storage was approximately - 25°C. This procedure was modified from Gorham and Clendenning (14). The chloroplasts as required were thawed at room temperature by s t i r r i n g with a glass st i r r i n g rod. As soon as i t was evident that there were no frozen particles i n the suspension, they were added to the reaction mixture. This was done at time zero. The electron acceptor, or H i l l solution, used was 0.001 M. K 3Fe(GN) 6 in 0.1 M phosphate buffer at the pH of 6.85. The above procedure was modified from the method of Spikes et a l . (34). Alkaline pyrogallol purified commercial nitrogen was bubbled into the H i l l solution i n order to prevent the chloroplast material from settling. It was found that the H i l l solution, with purified nitrogen being bubbled through i t , and without chloroplasts, maintained a constant potential reading for one hour with the lamp on. However, i t was necessary to maintain a constant temperature. The apparatus used to measure the potential consisted of a Beckman model H-2 pH meter equipped with a standard calomel electrode, a platinum electrode, and a glass bottomed water bath standing on an open ended box in which a 1000 watt light bulb was situated. The water bath was f i l l e d with cold water and enough ice to cover the water surface. A small s t i r r i n g propeller was emmersed in i n the ice water mix to keep the temperature constant throughout. A stand with a "snap" clamp was placed in the water bath in order to keep the 50 ml. beaker containing the reaction mixture a constant distance from the bottom of the water bath, thus the light in each experiment was f i l t e r e d through the same amount of water, which was three inches. The H i l l solution was placed i n a 50 ml. beaker which had previously been scrupulously cleaned. The beaker was then placed in the "snap" clamp i n the ice water bath. The ice water reached to within 3/8 of an inch from the top of the beaker. When the thermometer placed in the beaker registered 1.5°C. the nozzle attached to the nitrogen supply, and the electrodes were emersed in the H i l l solution. The electrodes were l e f t in the H i l l solution for at least five minutes to equilibrate the temperature. At this time, a frozen chloroplast sample was taken from the deep freeze and thawed. When the chloroplast suspension reached the slurrey state the pH meter was standardized and the lamp turned on. As soon as the chloroplast sus-pension was i n the f l u i d state i t was poured into the beaker, the stop watch started and the zero time potential reading taken. Potential read-ings were taken at one minute intervals for five minutes. The system used to calculate the m i l l i v o l t readings was that suggested in the book of instructions that is supplied with the pH meter. The formula i s : (7.0 - pH reading) X 60 = mi l l i v o l t s . The net m i l l i v o l t change was recorded. It must be noted that the i n i t i a l m i l l i v o l t readings for different samples varied greatly. This is thought to be due to deposits on the platinum electrode. Between readings the platinum electrode was washed in a detergent, scrubbed with a scouring powder, washed in an acid bath (20 - 40% HC1) and then rinsed in d i s t i l l e d water and dried. The net change in potential appeared to be a satisfactory method of measuring the rate of photolysis. Monuron (Dupont 80% wettable) was made in solutions which -3 -12 contained concentrations of 1 X 10 to 1 X 10 moles of monuron per ml. The required concentration of monuron was added by placing 1.0 ml. of the solution of the correct molarity in the reaction mixture. To remove the l i p i d material the thawed chloroplast sus-pension was placed in a 250 ml. separatory funnel with 25 ml. of pure petroleum ether and shaken in an ice bath for fifteen minutes. The chloroplast material was drawn off and added to the reaction and the H i l l reaction was followed as described above. Vitamin (menadione) was added to the chloroplast material in pure petroleum ether. One ml. aliquots were pipetted into a sep-aratory funnel containing the chloroplast suspension. The separatory funnel was kept in an ice water bath. Purified commercial nitrogen was passed through the stem of the separatory funnel at a rate which was just sufficient to prevent the chloroplast material from f a l l i n g down the stem. At this rate of nitrogen flow i t required five minutes to evaporate a l l of the petroleum ether. The nitrogen flow was l e f t on for six minutes to ensure that a l l the petroleum ether had been evaporated. Vitamin K,. (synkamin) was added directly to the reaction mixture as i t is a water soluble analogue of vitamin K. The literature referring to this analogue states that each ampule contains 4.0 mg. of active material. This was assumed to be correct, as the percentage of active material was not known. In any case, the same solution of vitamin K 3 was used for any one group of experiments so that any error introduced would be constant. During the spring of 1960 chloroplasts were used that were prepared from spinach grown In California. In May, 1960 spinach was no longer imported from California and locally produced spinach was on the market. It was found that chloroplasts prepared from the locally grown spinach were inactive during May and the f i r s t two weeks of June. It is believed that the inactive state of the chloroplasts i s a result of the unusually cold and wet weather experienced i n the Vancouver area at that time. RESULTS A. The Effect of Monuron on Various Enzyme Systems (i) Catalase System The evidence of this exploratory experiment (Appendix Table I) indicates that i f there is any influence shown on the catalase enzyme system by monuron i t is masked by time. It appears that there may be a natural inhibitor to the catalase system which breaks down after the leaves and buds have been severed from the plant. ( i i ) Oxidase System The results indicate that no influence i s exerted by monuron on the oxidase system (Appendix Table II). It must be pointed out that these tests were not quantitative. ( i i i ) Peroxidase System The results obtained i n these tests indicate that there i s l i t t l e or no effect on the peroxidase system shown by monuron (Appendix Table III). As i n the tests with the oxidase system, the tests with the peroxidase system were not quantitative. (iv) Lipase System Some activation of this system is apparent in this preliminary t r i a l (Appendix Table IV). Before any firm conclusions can be drawn more investigation w i l l be required. (v) Protease System The evidence gathered i n these experiments suggest that monuron inhibits this enzyme system (Appendix Table V). B. Some Effects of Monuron on Potatoes Monuron shows l i t t l e effect on the length of the root systems of Fig. 1 curve # 1; Weight of the root system, curve # 2; Weight of tubers, curve # 3; Weight of tops. 20 f i g . 2-potato plants, however the length of the tops was increased with a l l applications of monuron. There was an increase in the weight of the tops found in the plants treated up to two lbs. per acre. The weight of the root system and the total weight of tubers were reduced with increased rates of monuron applied. The total weight of tubers was reduced by 547. with the application of one lb. per acre and by 937. with the application of four lbs. per acre of monuron. ( f i g . 1, Appendix Table VI). The number of tubers per plant was reduced as the rate of monuron increased. There was a 64% reduction in the number of tubers per plant with the application of four lbs. per acre of monuron. The average weight per tuber was greatly reduced with monuron applications. For example there was a 52% reduction of tuber weight with the applica-tion of one lb. of monuron, and a reduction of 77% with the four lb. rate. The evidence does not suggest that monuron has any effect on the number of stems per plant. The root to top ratio was greatly reduced by monuron treatments. There is a 64% reduction in this ratio with one lb. per acre applied, a 747. reduction with four lbs. per acre applied, ( f i g . 2). There appears to be a tendency for monuron to cause swelling at the nodes. However, this tendency does not appear to increase uniformly with increased rates of monuron. The maximum swelling was observed at the one lb. rate. The size of the f i r s t leaf was reduced with monuron at rates which exceed 1/2 lb. per acre. The greatest reduction in leaf size was noticed between those treated at the 1/2 lb. rate and those treated at the one lb. rate. The degree of development of the potato plants appeared to be arrested with monuron applications. There is an increase in the leaf colour intensity of plants treated with monuron at one half and one lb. rates. At rates exceeding one lb. per acre, there is a decrease in green colour intensity and the leaves become yellow. Plants treated with monuron at rates exceeding one half lb. per acre tended to have chlorotic margins. Chlorosis of leaf margins is a recognized symptom of monuron damage. In a l l treatments there were some plants with dead leaves present. The two and four lbs. per acre treatments had a higher frequency of plants with dead leaves present (50 and 70% respectively). A l l the treatments had plants with dead spots on some of the leaves. This may have been due to a nutrient deficiency and thus may not be attributable to monuron damage directly. Many of the plants treated with monuron at the four lb. rate tended to have the main stem dying back. In some cases the lateral branches became dominant. Similar, but less obvious dying at the stem apices was observed in the plants treated at the two lb. rate. C. Some Effects of the Substituted Urea Herbicides on Barley A l l data may be seen on Table VII, VIII & IX of the Appendix. The four substituted urea herbicides used i n this experiment were most effective in controlling weed when applied as pre emergence herbicides rather than post emergence herbicides. In f i e l d observations the better crop growth was generally observed i n the post emergence treatments. Neburon was the exception here, there being l i t t l e difference between crops in the pre and post emergence treated plots. Fig. 3 #1 Control. #2 monuron at 2 lbs./A., as a pre emergent. #3 monuron at 2 lbs./A., as a post emergent. #4 monuron at 1 lb./A., as a pre emergent. #5 monuron at 1 lb./A., as a post emergent. #6 diuron at 21b./A., as a pre emergent. #7 diuron at 2 lbs./A., as a post emergent. #8 diuron at 1 lb./A., as a pre emergent. #9 diuron at 1 lb./A., as a post emergent. #10 neburon at 2 lbs. /A. , as a pre emergent. #11 neburon at 2 lbs./A., as a post emergent. #12 neburon at 1 lb./A., as a pre emergent. #13 neburon at 1 lb./A., as a post emergent. #14 fenuron at 2 lbs./A., as a pre emergent. #15 fenuron at 2 lbs./A., as a post emergent. #16 fenuron at 1 lb./A., as a pre emergent. #17 fenuron at 1 lb./A., as a post emergent. B U S H E L S P E R A C R E . O © 0> 69 H m to H _ Z O m OB 0 > f i g . 3. Fig. 4 #1 Control. #2 monuron at 2 lbs./A. , as a pre emergent. #3 monuron at 2 lbs./A. , as a post emergent. #4 monuron at 1 lb./A., as a pre emergent. #5 monuron at 1 lb./A., as a post emergent. #6 diuron at 2 lb&/A., as a pre emergent. #7 diuron at 2 lbs./A., as a post emergent. #8 diuron at 1 lb./A., as a pre emergent. #9 diuron at 1 lb./A., as a post emergent. #10 neburon at 2 lbs./A. , as a pre emergent. #11 neburon at 2 lbs./A. , as a post emergent. #12 neburon at 1 lb./A., as a pre emergent. #13 neburon at 1 lb./A., as a post emergent. #14 fenuron at 2 lbs./A. , as a pre emergent. #15 fenuron at 2 lbs./A. , as a post emergent. #16 fenuron at 1 lb./A., as a pre emergent. #17 fenuron at 1 lb./A., as a post emergent. B U S H E L WEiGHT f l IN L B S . , 2 •4-as <0 ro CM fig. 4. 26. Monuron and diuron at both the one and two lb. per acre treat-ments, applied both as pre and post emergence herbicides were the most effective weed control agents. Neburon at the two lb. per acre, applied as a pre emergent, gave only f a i r weed control. In general, the yield as measured by weight of grain was reduced by applications of the substituted urea herbicides. Diuron at the two lb. per acre, as a pre emergence herbicide, did not reduce the total weight of grain per acre, nor did neburon at one lb. per acre, fenuron at one lb., or monuron at one lb., as a post emergent. The number of bushels per acre was generally reduced by the application of these substituted urea herbicides. The noticeable exception was diuron at two lbs. per acre as a pre emergence herbicide, ( f i g , 3.). The bushel weight was found to be increased by a l l appli-cations of these herbicides except for diuron at two lbs. per acre, as a pre emergent, ( f i g . 4). Diuron, and possibly neburon, may offer promise as effective herbicides in cereal crops. From the evidence gathered in this experi-ment, diuron appears to hold the greatest promise. D. The Residual Effect of Four Substituted Urea Herbicides A l l data may be seen in Table IX of the Appendix. (i) Fenuron: Fenuron is the most water soluble of these four herbicides, 3,500 ppm. at room temperature. It would be expected that this herbicide would leach from the s o i l most readily. The degree of weed control of this herbicide increased until the twentieth day, thereafter the herbicidal action appeared to d i s s i -pate. After the twentieth day there was a r a i n f a l l of more than 0.7 inches. This rain probably leached a large percentage of the fenuron from the rhizophere. At the four lb. rate, the degree of weed control at 89 days was 3. (The weed control was measured on an arbitrary scale of 0 to 5, with 0 representing no weed control in the plots.) At the two lb. rate, the degree of control decreased after twenty days, and at the end of 89 days the degree of control in the plots was less than two. ( i i ) Monuron: Monuron is water soluble to the extent of 230 ppm. This herbicide exhibited i t s maximum degree of control within ten days after application, under the conditions present for this experiment. The degree of control decreased for the next twenty days, thereafter remaining f a i r l y constant. At the four lb. rate, monuron gave a maximum degree of control of 4.8, and at the end of 89 days the degree of control was four. The two lb. rate gave slightly less control than did the four lb. rate. The two rates appear to dissipate at nearly the same rate. ( i i i ) Diuron: Diuron is water soluble to the extent of 42 ppm. and appeared to be the most residual of the herbicides tested here. It controlled a l l vegetation for ten days at the four lb. rate and for twenty days at the two lb. rate. (The greater control shown at the two lb. rate is undoubtedly due to the weed populations within the plots.) The degree of control at the end of 89 days was 4.5 at the four lb. rate, and 4.0 at the two lb. rate. (iv) Neburon: Neburon, with a solubility of 4.8 ppm.,is the least soluble of Fig. 5 Curve #1 No monuron added. Curve #2 No monuron added, chloroplasts boiled. Curve #3 I X 10"3 moles of monuron added. Curve #4 I X 10""* moles of monuron added. Curve #5 I X 10"^  moles of monuron added. Curve #6 1 X 10 moles of monuron added. Curve #7 I X 1 0 m o l e s of monuron added. M I L L I V O L T D E C R E A S E . i Fig. 6 Curve #1 Curve #2 Curve #3 Curve #4 No monuron added. 1 X 1 0 m o l e s of monuron added. 1 X IO"** moles of monuron added. 1 X 1 0 m o l e s of monuron added. M I L L I V O L T D E C R E A S E the four herbicides studied here. It would be expected that the maximum degree of control would be reached at a later time than that of the other three herbicides. This, however, was not observed. The maximum degree of control at the four lb. rate was found to be at ten days after treatment, thereafter decreasing rapidly un t i l thirty days, then increasing for the next ten days and remaining f a i r l y constant until the end of the test period. The degree of control at 89 days was 3.0. The maximum degree of control at the two lb. rate was also reached within ten days. Thereafter the degree of control decreased until the end of the test period, 89 days, when the degree of control was 2.2. The most common weeds found on the majority of plots were: Lady's thumb Polygonum persicaria L., rib grass Plantago, lanceolata L,, broadleaf plantain Plantago major L., meadow foxtail Alopecurus  pratensis L., and orchard grass Dactylis glomerata L E. The Effect of Monuron on the Photolysis of Water and the Interaction of Vitamin K Type Compounds on the Photolysis Reaction Monuron in low concentrations inhibits the H i l l reaction of the photolysis of water. It was found that 1 X 10"^ moles of monuron in the reaction mixture w i l l inhibit the H i l l reaction by more than 50% (see Table I and fig.5). There appears, from this data, that there may be a slight stimulus given to this reaction by very low concentrations of monuron. (see Table II and f i g . 6). Table I THE EFFECT OF VARIOUS CONCENTRATIONS OF MONURON ON THE RATE OF THE HILL REACTION T ^ m e Change in potential, measured in m i l l i v o l t s . In Control control, 1 X 10"%. I X 10_5M. 1 X 10'7M. minutes boiled monuron monuron monuron 1 43.75 4.00 5.75 11.50 27.00 2 57.50 6.75 8.25 14.50 31.50 3 66.50 7.00 10.00 15.75 34.75 4 70.25 7.50 10.50 17.25 36.50 5 74.00 8.25 12.00 19.00 38.00 Table I (con't.) Time in minutes 1 X 10"9M. monuron 1 X 10"10M. monuron 1 39.50 45.25 2 51.25 59.50 3 58.00 66.75 4 62.25 71.75 5 66.00 75.75 32 Table II THE EFFECT OF EXTREMELY LOW CONCENTRATIONS OF MONURON ON THE RATE OF THE HILL REACTIONS in ' control 1 X 10"10M. 1 X 10" UM. 1 X 10"12M. minutes monuron monuron monuron 1 41.50 48.00 43.00 40.75 2 53.75 62.50 56.00 55.75 3 61.50 70.50 63.75 64.75 4 67.00 74.50 69.00 71.25 5 70.75 78.00 73.75 73.00 The addition of vitamin K type compounds resulted in a slight inhibition of the H i l l reaction, (see Tables III & IV). Table III THE EFFECT OF THE ADDITION OF VITAMIN K3 (Menadione) TO THE RATE OF THE HILL REACTION Time, Potential change, measured i n millivolts in control 1 ml. of 1 mg. of 0.1 mg. of minutes pet. ether v i t . v i t . K3 added, no v i t . K 3 1 48.75 38.75 33.75 37.00 2 64.25 55.25 47.75 52.00 3 70.50 62.25 56.00 59.00 4 76.25 67.75 61.25 63.75 5 80.25 71.50 65.50 70.00 Table IV THE EFFECT OF THE ADDITION OF VITAMIN Kc TO THE RATE OF THE HILL REACTION Time, in Control 0.1 mg. of minutes Vitamin Kc 1 44.75 30.75 2 59.25 43.25 3 66.50 51.75 4 72.50 57.00 5 76.50 61.50 The relative inhibition of the H i l l reaction was greater with the addition of vitamin K^  than i t was with the addition of vitamin Kj, indeed there was very l i t t l e inhibition shown by 0.1 mg. of vitamin K3. Much of the inhibition shown with the addition of vitamin K3 i s due to denaturation of the chloroplasts by light and warmth. This is evident when compared to the tests where petroleum ether was added without vitamin Kj. (see Tables III & IV). Vitamin K 5 is formulated as the hydrochloride of 4- amino - 2 -methyl - 1- napthol. It i s possible that the chloride ions are responsible for the inhibition. Removal of the petroleum ether soluble fraction resulted in a slight stimulation of the H i l l reaction (Table V). Table V THE REMOVAL OF THE PETROLEUM ETHER SOLUBLE FRACTION Time, Shaken, Shaken in Control with no with minutes petroleum petroleum ether ether 1 44.75 31.00 35.25 2 59.25 44.00 48.25 3 66.50 51.25 56.25 4 72.50 56.75 61.50 5 76.50 60.25 64.75 The addition of 0.1 mg. of vitamin to the reaction mixture, containing 1 X 10"7 moles of monuron almost completely inhibited the H i l l reaction, whereas the addition of vitamin K3 showed no inhibition, other than that due to the denaturation of the chloroplasts by light and warmth. (Table VI). Table VI THE INTERACTION OF MONURON AND ANALOGUES OF VITAMIN K. Potential change, measured in m i l l i v o l t s . 1 X 10"7M. 1 X 10"7M. 1 X 10"7M. 1 X 10"7M. monuron monuron monuron monuron plus 0.1 mg. plus 0.1 mg. with 1 ml. of Vit. K 5 of Vit. K 3 of pet.ether & no V i t . K. 1 56.75 34.50 4.50 24.25 24.00 2 70.75 43.00 7.00 27.75 28.75 3 79.25 45.50 7.00 30.75 31.25 4 85.75 48.50 9.00 31.75 33.75 5 89.00 49.50 10.00 34.00 34.75 Time, in Control minutes Each figure i n Tables I to VI is the average of four t r i a l s . DISCUSSION The mode of action of the substituted urea herbicides in a l l probability involves alterations i n the biochemistry of the plant. It has been shown in the literature that many deviations from the normal take place in plants subjected to the urea type herbicides. Many of these deviations are d i f f i c u l t to discern, such as the onion root tip growth being arrested at the resting stage of mitosis. (9). Other deviations are more pronounced, such as the recognized leaf symptoms of monuron injury. The present work confirms that monuron may alter the protein balance within the plant system, e.g. the protease system of enzymes may be inhibited. There also seems to be a stimulation to the lipase system of enzymes. Monuron reduced the root growth of potato plants. This may be due to a shortage of photosynthetic products. In the literature i t has been reported that excised roots may be grown in nutrient solutions containing lethal quantities of monuron. (26). However, i f there was a shortage of photosynthetic products one would expect a similar reduction in top growth, unless food transport is being restricted. The evidence gathered here shows that there is an increase i n top growth in plants that have been treated with up to two lbs. per acre of monuron. It is apparent that the inhibition of photosynthesis is not the complete answer here. It is possible for example that an imbalance with one of the growth hormones is created by monuron. The substituted urea herbicides enter the plant mainly through the root system. It i s not surprising therefore, that the best weed control was found i n the plots where the herbicides were applied as pre emergents. When applied as a post emergent much of the herbicide would remain on the foliage and would not be as free to enter the plant system. In pre emergence applications a l l of the herbicide would reach the s o i l surface and, following precipitation, would be leached to a rhizophere which would be much shallower than would be the case in later applications. Diuron, applied as a pre emergence herbicide, at two lbs. per acre, did not reduce the yield in barley, and at the same time provided good weed control As mentioned earlier diuron may offer good weed control in cereal crops. If this i s the case some of the disadvantages of using hormone type herbicides would be overcome. It is f e l t that this is an avenue of research that should be investigated thoroughly. Coggins and Crafts (10) have reported that the substituted urea herbicides have a not positive charge in aqueous solutions. It is reasonable to assume that the herbicide molecule retains this positive charge within the plant. The positively charged herbicide molecule would then be i n the position to change the configuration of many enzyme molecules by the disruption of hydrogen bonds, or other electrostatic forces, which are required to maintain enzyme configuration. The specificity of enzymes is believed to be largely dependent upon the configuration of the enzyme molecule, as well as the con-figuration of the substrate molecule. The photolysis of water appears to be stimulated by very dilute concentrations of monuron ( 1 X 1 0 " 1 0 moles added to the reaction mixture)-this stimulation is lost at lower and higher concentrations. It is not known i f the overall rate of photosynthesis is increased with these low concentrations of monuron. However i t was apparent that the potato plants treated with monuron up to one lb. per acre had a more intense green colour, and presumably these plants had a greater concentration of chlorophyll. It may be that the whole photosynthetic mechanism becomes more active with low concentrations of monuron. Whether the net assimilation is increased is s t i l l lacking proof. Monuron at higher concentrations ( I X 10'^moles added to the reaction mixture) inhibits the photolysis of water by more than 50%. If the photolysis of water is inhibited the whole photosynthetic series of reactions w i l l be inhibited. These reactions are dependent on the photolysis of water to provide hydrogen ions to reduce the carbon dioxide to carbohydrate and possibly to reduce TPN, which may be required in the synthesis of ATP. It is not known whether or not monuron w i l l inhibit the reduction of carbon dioxide or the synthesis of ATP per se, however as pointed out above these reactions w i l l be inhibited, even though the influence is indirect. It has been reported in the literature that vitamin K type compounds are required in the photolysis of water reactions. (7). The evidence gathered here does not support this report. Natural occurring vitamin K type compounds are petroleum ether soluble, but after shaking chloroplasts in cold petroleum ether, and separating,the rate of the H i l l reaction was found to increase. The procedure in the literature (7) for extracting the petroleum ether fraction from chloroplasts could not be repeated, as i t was not possible to recover the chloroplasts intact from the f r i t t e d glass. However shaking the chloroplasts i n 25 ml. of cold petroleum ether should have given similar results. One possible explanation for the contrary results would be that the petroleum ether was not able to penetrate the lipo-protein matrix which surrounds the chloroplasts, and thus was not reaching the vitamin K. The addition of vitamin K«j, and to a smaller degree vitamin K^, retarded the rate of the H i l l reaction. An excess of vitamin K type compounds following the addition of the synthetic annalogues of vitamin K might cause the pathway to become blocked. It is f e l t however, in the light of the evidence gathered here that the vitamin K type compounds do not play a role i n this phase of photosynthesis. These compounds probably do take part in either the c y l i c phosphory-lation or terminal phosphorylation, or both. Vitamin K3, which is prepared as the hydrochloride of 4 -amino-2-methyl-l-napthol, acted synergistically with monuron to inhibit the H i l l reaction. This synergistic relation with monuron may have been caused by the chlorine ion combining with the monuron at the meta position thereby forming diuron. In support of this explanation the vitamin shows no effect, (Table VI), either stimulatory or inhibitory, when in the presence of 1 x IO - 7 moles of monuron. Vitamin K3 and have the same physiological properties, at least i n animals. The apparatus used to measure the progress of the H i l l reaction was modified from that described in the literature. It was found that by having ice in the water bath the temperature of the reaction mixture, even with the lamp on, could be kept constant for the length of the run. When running water was used i t was not possible to keep the temperature from ri s i n g at least two degrees C. i n the five minute test period. The measurement of potential is very sensitive to temperature fluctuations. S U M M A R Y (1) Monuron has no evident effects on the catalase, oxidase or peroxidase systems of the test plants. An inhibition of the protease system and a stimulation of the lipase system was evident. (2) Monuron modifies growth of the potato plant by decreasing the weight of roots, the number and weight of tubers, and by increasing the weight of the tops. Low rates of application tend to increase the intensity of leaf colour, while applications greater than one lb. per acre appear to decrease the green colour intensity. (3) The weight of barley grain per acre, and the number of bushels per acre tend to be reduced with applications of the substituted urea herbicides. The weight per bushel, however, tends to be increased. (4) Diuron appears to be the most residual of the four herbicides tested. Neburon, which is almost ten times less soluble in water than diuron did not give as satisfactory weed control. (5) Monuron inhibits the photolysis of water by 507. at concentrations as low as 1 X 10"'' moles in the reaction mixture. A concentration of 1 X 10 _ 1 , 0 moles in the reaction mixture increased the rate of the H i l l reaction. These experiments did not show any evidence that vitamin K type compounds play a role in the photolysis of water. The removal of the petroleum ether soluble fraction resulted in an increase in the rate of the H i l l reaction. BIBLIOGRAPHY Able, A.S., The substituted urea herbicides, Chemistry and Industry, 33,1106 - 1112, 1957. Arnon, D.I., F.R. Whatley and M.B. Allen, Assimilatory power in photosynthesis, Science, 127,1026 - 1034, 1958. , Photosynthesis by isolated chloroplasts VIII. Photosynthetic phosphorylation and generation of assimilatory power, Biochimica et Biophysica Acta, 32, 47-57, 1959. 4 Arnon, D.I., Conversion of light into chemical energy in photosyn-thesis, Nature, 184, 10 - 21, 1959. 5 Arnoff, S., Photosynthesis, Botanical Review, 23, 65 - 107, 1957. 6 Avron, M., and A.T. Jagendorf, Evidence concerning the mechanism of adenosine triphosphate formation of spinach chloroplasts, The Journal of Biological Chemistry, 234, 967 - 972, 1959. 7 Bishop, N.I., Vitamin K, an essential factor for photochemical activity of isolated chloroplasts, Proceedings of the National Academy of Sciences of the U.S.A., 44, 501 - 504, 1958. 8 Bucha, H.C. and C.W. Todd, 3-(p-chlorophenyl)-1,1-dimethylurea a new herbicide, Science, 114, 493 - 494, 1951. 9 Christoph, R.J. and E.L. Fisk, Responses of plants to the herbicide 3-(p-chlorophenyl)-1,1-dimethylurea (CMU). Botany Gazette, 116, 1 - 4 , 1954. 10 Coggins, C.W.(Jr.), and A.S. Crafts, Substituted urea herbicides; their electrophoretic behaviour and the influence of clay colloid in nutrient solution on their phytotoxicity, Weeds, 7, 349 - 358, 1959. 11 Cook, A.R., A possible mechanism of the urea type herbicides, Weeds, 4, 397 - 398, 1956. 12 Fang, S.C., V.H. Freed, R.H. Johnson and D.R. Coffee, Absorption, translocation and metabolism of radioactive 3-(p-chlorophenyl) -1,1-dimethylurea (CMU) by bean plants, Journal of Agriculture and Food Chemistry, 3, 400 - 402, 1955. 13 Freed, V.H., Herbicide mechanism mode of action other than aryl oxyalkyl acids, Journal of Agriculture and Food Chemistry, 1, 47 - 51, 1953. 42 14 Gorham, K.A. and P.R. Clendenning, Storage of isolated chloroplasts without loss of photochemical activity, Canadian Journal of Research, C28, 177 - 187, 1954. 15 H i l l , G.D, J.W. McGahen, H.M. Baker, D.W. Finnerty and C.W. Bingeham, Fate of the substituted urea herbicides in agriculture s o i l s , Agronomy Journal, 47, 93 - 104, 1955. 16 Jasmin, J.J. and W. Ferguson, CMU for weed control in horticultural crops, Proceedings of the National Weed Committee, Eastern Section, 6, 61 - 66, 1953. 17 Levi, E., Some aspects of the toxicity of 3-(p-chlorophenyl)-1, 1-dimethylurea (CMU) to plants, Australian Journal of Agricul-ture Research, 6, 27 - 32, 1955. 18 Loomis, W.E. and CA. Shull, Experiments in Plant Physiology, McGraw-Hill, 1939. 19 Loustalot, A.J., a private communication to H.J. Cruzado. 20 Lynch, V.H. and C.S. French, B Carotene, an active component of chloroplasts, Archieves of Biochemistry and Biophysics, 70, 382 - 391, 1957. 21 Minshall, W.H. and D.A. McLarty, Preliminary investigations on the effects of some urea compounds on the morphology and physiology of plant roots, Proceedings of the Canadian National Weed Committee, Eastern Section, 6, 95 - 97, 1953. 22 Minshall, W.H., Effects of 3-(p-chlorophenyl)-1,1-dimethylurea (CMU) on the dry matter production and transpiration in excised primary leaves of bean, Research Report, National Weed Committee, Eastern Section, 9, 43, 1956. 23 , Influence of light on the effect of 3-(p-chloro-phenyl)-1,1-dimethylurea on plants, Weeds, 5, 29 - 33, 1957. 24 , Effect of 3-(4-chlorophenyl)-1,1-dimethylurea (monuron) on dry matter production and transpiration, Plant Physiology, 32, Supplement v i i , 1957. 25 , Primary place of action and symptons induced in plants by 3-(4-chlorophenyl) -1,1-dimethylurea, Canadian Journal of Plant Science, 37, 157 - 166, 1957. 26 Muzik, T.J., H.J. Cruzado and A.J. Loustalot, Studies on the absorp-tion, transpiration and action of CMU, Botany Gazette, 116, 65 - 73, 1954. 27 Muzik, T.J., M.P. Morris and H.J. Cruzado, Translocation and metabo-lism of 3-(p-chlorophenyl)-1,1-dimethylurea in velvet beans, Plant Physiology, 31, Supplement v i i i , 1956. 4 28 Muzik, T.J., H.J. Cruzado and M.P. Morris, a note on the transloca-tion and metabolism of monuron in velvet beans, Weeds, 5, 133 -134, 1957. 29 Newman, A.S. and CR. Downing, Herbicides and the s o i l , Journal of Agriculture and Food Chemistry, 6, 352 - 353, 1958. 30 Piper, K.C and V.H. Freed, Some effects of CMU and 2,4-D upon the nitrogen uptake and reserve sugars of bean and sunflower plants, Western Weed Control Conference, Research Report, 6, 92, 1953. 31 Piatt, J.R., Carotene donor acceptor complexes in photosynthesis, Science, 129, 372 - 374, 1959. 32 Renney, A.J., A PhD. Thesis Submitted to Oregon State College, 1956. 33 Sheets, T.J. and A.S. Crafts, The phytotoxicity of four phenyl-urea herbicides i n s o i l , Weeds, 5, 93 - 101, 1957. 34 Spikes, J.D., R. Lumry, H. Eyring and R.E. Wogrymen, Potential changes in suspensions of chloroplasts on illumination, Archieves of Biochemistry, 28, 48 -67, 1950. 35 Thomas, J.B., Chloroplast structure and function, Endeavour, 67, 156 - 161, 1958. 36 Tomizawa, C , Effects of 2,4-D and CMU on phosphorous metabolism, Nogyo Gijutsu Kentyujo Hokoku, Service Circular No. 6, 103 -109, 1956. (Abstracted in the Chemistry Abstracts,7513h, 1957.) 37 Upchurch, R.P. and W.R. Pierce, The leaching of monuron from Lakeland sand s o i l , Part I, The effect of amount, intensity and fre-quency of simulated r a i n f a l l , Weeds, 5, 321 -330, 1957. 38 Wald, C , Life and light, Scientific American, 201, No.4, 92-108,1959. 39 Whatley, F.R., M.B. Allen, and D.I. Arnon, Photosynthesis by isolated chloroplasts VII. Vitamin K and riboflavin as cofactors of cyclic phosphorylation, Biochimica et Biophsica Acta, 32, 32 - 46, 1959. 40 Whatley, F.R., M.B. Allen, A.V. Trebst and D.I. Arnon, Photosynthesis by isolated chloroplasts IX. Photosynthetic phosphorylation and C0 2 assimilation in different species, Plant Physiology, 35, 188 - 193, 1960. 41 Wessels, J.S.C and R. van der Veen, The action of some derivatives of phenylurethan and of 3-phenyl-l,l-dimethylurea on the H i l l reaction, Biochimica et Biophysica Acta, 19, 548 - 549, 1956. APPENDIX Table I The Effect of Monuron on the Catalase System Concentration of Time, i n seconds, monuron, in ppm. required to liberate 5.0 ml. of oxygen 0.000 100.0 10.0 1.00 0.10 0.01 0.001 100.0 0.000 72.0 69.5 93.2 87.8 41.6 36.4 34.4 22.9 21.8 (The length of time between each test was approximately ten minutes. The time lapse between the last test of the 1.00 ] series and the f i r s t of the 0.10 ppm. series was 90 minutes.) Table II The Effect of Monuron on the Oxidase System Concentration of monuron in ppm. Immediate colour change with 1% guaiacum 100.0 10.0 1.0 0.1 0.01 0.000 boiled 0.000 Yes Yes Yes Yes Yes Yes No Table III The Effect of Monuron on the Peroxidase System Concentration of monuron in ppm. Immediate colour change with 17. guaiacum. Immediate colour change with 47. H 20 2. 100.0 10.0 1.0 0.1 0.01 0.000 boiled 0.000 No No No No No No No Yes Yes Yes Yes Yes Yes No Table IV The Effect of Monuron on the Lipase System Concentration of Mis. of 0.1 N monuron in ppm. fatty acids present 10.0 0.644 1.0 0.568 0.1 0.533 0.01 0.546 0.000 0.430 boiled 0.000 0.333 Table V The Effect of Monuron on the Protease System Concentration of % Protein monuron, in ppm. 100.0 20.7 10.0 26.0 1.0 30.5 0.1 29.7 0.01 31.2 0.000 32.5 boiled 0.000 26.5 4 Table VI Some Effects of Monuron on Potato Plants (Each figure is an average of 10 pots.) Rate of Length in cm. Weight in gms. Weight Root to monuron, lbs./A tops roots tops roots of tubers in gms. top ratio 4.0 44.7 31.3 59.1 32.1 6.3 0.563 2.0 44.5 33.5 72.3 52.7 27.4 0.715 1.0 48.1 32.4 81.4 70.9 44.8 0.803 0.5 42.3 28.3 74.8 110.4 74.4 1.575 0.0 38.3 33.0 65.4 134.3 98.6 2.198 Rate of monuron, lbs./A Number tubers of stems Swelling at nodes Relative size of f i r s t leaf. Degree of Develop-ment. Intensity of colour 4.0 1.5 2.7 2.4 1.6 0.7 2.6 2.0 4.1 2.3 2.1 2.5 1.0 3.2 1.0 5.3 1.8 2.5 2.9 1.5 4.1 0.5 5.1 2.1 2.2 3.8 1.0 3.5 0.0 5.5 2.5 1.2 3.8 1.7 3.4 Rate of monuron in lbs./A % having bleached margins 7. having dead leaves 7. having dead spots in leaves 4.0 50 70 90 2.0 60 50 100 1.0 60 20 90 0.5 10 30 60 0.0 0 20 70 Swelling at the nodes, 0 indicates no swelling and 5 indicates maximum swelling. Table VI CROP GROWTH The Effects of Monuron on Barley Herbicide Rate, Time observations, days after applic. in lbs./ applied 27 32 42 52 73 A. monuron 2.0 pre e. 2.0 2.0 2.0 2.0 1.25 monuron 2.0 post e. 3.25 3.5 3.75 3.75 4.0 # monuron 1.0 pre e. 2.5 2.75 3.0 3.75 2.6 # monuron 1.0 post e. 3.25 3.75 3.75 4.25 3.0 # diuron 2.0 pre e. 2.25 2.75 3.0 3.75 2.6 # diuron 2.0 post e. 3.25 3.75 4.25 3.75 4.0 diuron 1.0 pre e. 2.75 2.75 3.25 3.75 3.25 diuron 1.0 post e. 4.0 4.0 4.0 4.25 4.25 neburon 2.0 pre e. 3.25 3.75 3.75 4.25 4.25 neburon 2.0 post e. 3.25 3.5 3.25 3.75 4.0 # neburon 1.0 pre e. 3.5 4.25 4.25 4.5 4.25 neburon 1.0 post e. 3.25 4.0 4.75 4.0 4.3 # fenuron 2.0 pre e. 2.0 2.0 2.0 2.75 2.3 # fenuron 2.0 post e. 4.0 3.75 3.75 4.0 4.0 M fenuron 1.0 pre e. 2.5 2.25 2.5 3.5 3.75 fenuron 1.0 post e. 3.5 4.25 3.0 4.0 4.0 # control n/a n/a 3.5 3.5 3.5 4.0 4.25 control n/a n/a 3.25 4.0 4.25 4.25 4.3 # control n/a n/a 3.5 4.25 3.75 4.25 3.5 ## control n/a n/a 3.5 3.5 3.5 3.75 4.3 # An arbitrary scale was used to measure the vegetation on the plots. The scale was from 1 to 5, with 1 representing no plants alive and 5 representing the most active plant growth. 4 # one plot was lost by trampling etc. before the last observation was made. ## two plots were lost by trampling etc. before the last observation was made. Relative size of the first leaf, 0 indicates a small first leaf and 5 indicates a large first leaf. Degree of Development, 0 indicates the least and 5 indicates the greatest development in the plant. Intensity of colour, 0 indicates the least intense colour and 5 indicates the most intense colour. 5i Table VII Weed Infestation Herbicide Rate,in lbs./A. Time applied Observation, 27 32 days 42 after application. 52 73 monuron 2.0 pre e. 1.0 1.0 1.0 1.0 1.25 monuron 2.0 post e. 3.0 4.25 4.75 4.5 3.0 # monuron 1.0 pre e. 1.0 1.25 1.25 2.0 1.6 # monuron 1.0 post e. 3.25 4.25 3.75 3.75 3.0 # diuron 2.0 pre e. 1.0 1.0 1.25 1.75 1.3 # diuron 2.0 post e. 3.25 4.0 4.75 4.5 2.0 diuron 1.0 pre e. 1.0 1.0 1.25 1.5 1.25 diuron 1.0 post e. 4.25 4.25 4.75 4.5 2.5 neburon 2.0 pre e. 1.5 1.25 1.25 1.75 1.75 neburon 2.0 post e. 3.25 3.75 4.0 4.0 2.6 # neburon 1.0 pre e. 1.5 1.75 1.75 1.75 2.25 neburon 1.0 post e. 4.0 4.25 4.25 4.5 3.3 # fenuron 2.0 pre e. 1.0 1.5 1.25 1.75 2.0 # fenuron 2.0 post e. 3.25 4.0 4.0 4.0 2.0 ## fenuron 1.0 pre e. 1.25 2.5 2.25 3.0 2.75 fenuron 1.0 post e. 4.0 3.75 4.25 4.5 3.0 # control n/a n/a 3.75 4.75 4.75 4.75 4.0 control n/a n/a 4.5 4.25 4.5 5.0 3.0 control n/a / n/a 3.75 4.5 4.75 5.0 3.5 ## control 'n/a n/a 3.0 4.0 4.25 5.0 3.0 # # one plot was lost by trampling etc. before the last observation was made. ## two plots were lost before the last observation was made. 5 An arbitrary scale was used to measure the weed cover in the plots. The scale was from 1 to 5, with 1 representing no weeds present and 5 representing a heavy weed cover. Table VII The Effect of the Substituted Urea Herbicides on Barley Yields Herbicide Rate, in Time Ave. Weight Ave. Yield, lbs./A applied Weight per A. bushel bu./A. per plot weight in lbs. monuron 2.0 pre e. 0.399 4345 46.75 9.29 monuron 2.0 post e. 0.567 6175 45.25 13.65 monuron 1.0 pre e. 0.577 6284 46.25 13.59 monuron 1.0 post e. 0.678 9540 45.0 21.20 diuron 2.0 pre e. 0.679 10629 45.72 23.25 diuron.# 2.0 post e. 0.571 6218 42.7 14.56 diuron 1.0 pre e. 0.811 8832 45.5 19.42 diuron 1.0 post e. 0.743 8091 45.75 17.69 neburon 2.0 pre i e. 0.820 8930 42.5 21.01 neburon 2.0 post e. 0.792 8625 44.0 19.6 neburon 1.0 pre e. 0.901 9812 44.25 22.17 neburon # 1.0 post e. 0.890 9692 47.96 20.21 fenuron 2.0 pre e. 0.497 5412 45.25 11.96 fenuron 2.0 post e. 0.811 8832 45.25 19.52 fenuron # 1.0 pre e. 0.926 10084 45.25 22.29 fenuron # 1.0 post e. 0.866 9431 43.67 21.60 control n/a n/a 0.786 8560 43.25 19.79 control # n/a n/a 0.986 10738 44.0 24.40 control# n/a n/a 0.819 8920 43.3 20.6 control # n/a n/a 0.845 9202 43.3 21.25 # one plot was lost and not harvested. 5 Table IX The Residual Effects of the Four Substituted Urea Herbicides Herbicide Rate, in Observations, days after treatment. lbs./A 4 7 11 20 30 51 89 monuron 4.0 4.0 4.8 4.2 4.7 4.0 4.2 4.0 monuron 2.0 2.8 4.0 4.7 4.2 3.7 4.0 3.7 diuron 4.0 3.2 4.2 5.0 4.7 4.7 4.2 4.5 diuron 2.0 3.0 4.2 5.0 5.0 4.2 4.0 4.0 neburon 4.0 2.5 2.8 4.7 4.0 3.2 3.7 3.5 neburon 2.0 1.7 3.0 3.5 3.5 3.2 2.7 2.2 fenuron 4.0 1.0 2.8 3.2 3.5 2.7 3.0 3.0 fenuron 2.0 0.75 1.7 2.5 2.7 2.7 2.5 1.7 control n/a 0.5 0.75 0.25 0.5 0.0 0.0 0.0 control n/a 0.0 0.25 0.5 0.0 0.0 0.0 0.0 The vegetation cover was measured on a scale of 0 to 5, where 0 represented a maximum vegetation cover and 5 represented no plant l i f e on the plot, i.e. maximum residual effect. During the time of the experiment a total of 4.19 inches of rain was recorded at the University of B.C.meteorological station. A p p a r a t u s u s e d t o m e a s u r e p o t e n t i a l c h a n g e 

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