EFFECT {3F 33H. PH 0N METREBUZW ' ' [4- -AMIE€G 8 ER? BU'IYL- 3 x {HWYLTHIG} -1‘.,2'4- TRfiAZW 5(é-H) 9H5) ACTMTY AND FAVE EH SGEL . Disseriatim‘s fer the flame of) Ph. D. . ‘ MICHEGAN STATE UHWER 8327‘! JAMES STEPHEN LADLIE E975 ' ' Jlllll H ll) 3 1293 0109 lllllll )lmllll ‘_ l12048 LIB R A ‘13- if Michigan S . -. University A 4 J 1’: This is to certify that the thesis entitled EFFECT OF SOIL PH ON METRIBUZIN [4-AMINO—6- TERT-BUTYL—3— (METHYLTHIO) -l, 2 , 4-TRIAZIN- 5(4H) ONE] ACTIVITY AND FATE IN SOIL presented by James Stephen Ladlie has been accepted towards fulfillment of the requirements for Ph.D. degepin Crop & Soil Sciences @599 i'mi‘é’tj < Major professor Date Januag 29I 1975 0-7639 m (L ABSTRACT EFFECT OF SOIL PH ON METRIBUZIN [4-AMIN0- 6-TERT-BUTYL-3-(METHYLTHIO)-l,2,4- TRIAZIN-5(4H)ONE] ACTIVITY AND FATE IN SOIL BY James Stephen Ladlie Metribuzin [4-amino-6-tert-butyl-3-(methylthio)- l,2,4-triazin-5(4H)one] activity in the field increased with increasing soil pH. Fall panicum (Panicum dichototomiflorum Michx.) control, number of dead corn (Zea mays L.) and soybean (Glycine max (L.) Merr.) plants, ari crop injury ratings increased, whereas, plant height and grain yield decreased as soil pH increased. Metri- buzin residue analysis of soil samples showed greater amounts of residue extractable at soil pH 6.7 than 4.6. Metribuzin leaching increased with increasing soil pH in the field. Metribuzin disappearance from soil followed pseudo first-order kinetics. Rate constants increased and the half life of metribuzin decreased as soil pH increased. Rate constants decreased and the half life of metribuzin increased at all soil pH levels as depth of sampling decreased. James Stephen Ladlie Metribuzin phytotoxicity to corn and soybeans grown in the greenhouse increased with increasing soil pH. Microbial degradation decreased as the soil became more acid. Metribuzin and atrazine [2-chloro-4-(ethyl- amino)-6-(isopropylamino)-§ftriazine] mobility increased 14C-metribuzin with increasing concentration and soil pH. had greater mobility on soil thin-layer plates than 14C- atrazine because of differences in water solubility and basicity. The pKa of metribuzin was determined to be 0.99 i 0.08. Adsorption increased gradually as the soil pH decreased toward the pKa of metribuzin. Protonation of the amine group with subsequent adsorption to soil colloids was postulated to be the reason for the reduction in phyto- toxicity, microbial degradation and mobility of metribuzin in acid soils. EFFECT OF SOIL PH ON METRIBUZIN [4-AMINO-6-TERT- BUTYL-B-(METHYLTHIO)-l,2,4-TRIAZIN-5(4H) ONE] ACTIVITY AND FATE IN SOIL BY James Stephen Ladlie A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Science 1975 ACKNOWLEDGMENT The author wishes to express his sincere appreci- ation to his major professor, Dr. William F. Meggitt, for his guidance throughout this study. The experience and opportunities received through involvement in the weed control project were very rewarding and appreciated. To Dr. Donald Penner is extended a special acknowledgment for his friendship, laboratory guidance and constructive criti- cism during the investigation and preparation of this manu- script. The assistance of Dr. A. R. Putnam and Dr. A. R. Wolcott as Guidance Committee members is gratefully acknowledged. A special thanks to Mr. Robert Bond for his technical assistance in the field aspects of this study. To the one very special to me, Mrs. Fran E. Ladlie, my thanks for her patience, support, and encouragement throughout the course of study. ii TABLE OF CONTENTS Page LIST OF TABLES. . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . viii INTRODUCTION. . . . . . . . . . . . . . . . . . . . . 1 CHAPTER 1. LITERATURE REVIEW . . . . . . . . . . . . 3 Fate and Behavior of Herbicides in the Soil Environment. . . . . . . . . . . . . . . . . 5 Factors Influencing Adsorption of Herbicides. . . 6 A. Physico-Chemical Character of Adsorbate . . . . . . . . . . . . . . . . 6 ’ Chemical Character, Shape, and Con- figuration of the Adsorbate . . . . . . . 7 Dissociation Constant . . . . . . . . . . 8 Water Solubility. . . . . . . . . . . . . 11 B. Physico-Chemical Character of Adsorbent . . . . . . . . . . . . . . . . 12 Nature and Properties of Soil Colloids. . . . . . . . . . . . . . . . . 12 Soil pH . . . . . . . . . . . . . . . . . 13 Surface Acidity . . . . . . . . . . . . . 17 Soil Adsorption of Herbicides . . . . . . . . . . l9 Mechanisms of Adsorption. . . . . . . . . . . 19 Chemical Adsorption . . . . . . . . . . . 19 Protonation . . . . . . . . . . . . . . . 20 iii Page Occurrence of Adsorption . . . . . . . 22 Influence of Soil pH on Herbicides . . . . . 25 Phytotoxicity. . . . . . . . . . . 25 Leaching, Movement and Solubility . . . . 26 Degradation and Biodegradation . . . . . 31 List of References . . . . . . . . . . 36 CHAPTER 2. EFFECT OF SOIL pH ON METRIBUZIN ACTIVITY AND DISSIPATION IN FIELD SOILS . . . . . . . . . . . . 45 Abstract. . . . . . . . . . . . . . 45 Introduction . . . . . . . . . . . . 46 Materials and Methods . . . . . . . . . 49 Field Experiments . . . . . . . . . 49 Field Residue Study. . . . . . . . . 50 Results and Discussion . . . . . . . . . 53 Effect of Soil pH on Metribuzin Activity in the Field . . . . . . . . . . . 53 Effect of Soil pH on Metribuzin Dissipation in the Field . . . . . . . 71 Acknowledgment. . . . . . . . . . . . 81 Literature Cited . . . . . . . . . . . 82 CHAPTER 3. EFFECT OF SOIL pH ON METRIBUZIN PHYTO- TOXICITY, MICROBIAL DEGRADATION, ADSORPTION, AND MOBILITY. . . . . . 85 Abstract. . . . . . . . . . . . . . 85 Introduction . . . . . . . . . . . . 86 Materials and Methods . . . . . . . . . 89 iv Page Phytotoxicity . . . . . . . . . . . 89 Microbial Degradation and 14CO2 Release . . 91 Determination of the Dissociation Constant (pKa). . . . . . . . . . . 92 Adsorption Study . . . . . . . . . . 93 Mobility on Soil Thin-Layer Plates . . . . 95 Results and Discussion . . . . . . . . . 96 Effect of Soil pH on Metribuzin PhytOtOXiCity O O O O O O O O O O O 96 Effect of Soil pH on Microbial Degradation and 14CO2 Release. . . . . . . . . . 97 Determination of the Dissociation Constant (pKa). O O O O O O O O O O O O O 109 Effect of Soil pH on Metribuzin Adsorption . 114 Effect of Soil pH on Mobility of Metribuzin and Atrazine . . . . . . . . . . . 118 Summary . . . . . . . . . . . . . 126 Acknowledgment . . . . . . . . . . . . 129 Literature Cited . . . . . . . . . . . 130 CHAPTER 4. SUMMARY AND CONCLUSIONS . . . . . . 133 APPENDICES. O O O O O O O O O O O O O O 137 LIST OF TABLES CHAPTER 2 1. 2. Rainfall data for 1972, 1973, and 1974 Soils Farm, East Lansing, Michigan. . . The effect of soil pH on metribuzin toxicity to fall panicum . . . . . . . . The effect of soil pH on metribuzin phytotoxicity to corn in the field, measured by visual injury ratings . . . . . The effect of soil pH on metribuzin phytotoxicity to corn in the field, measured in grain yield (kg/ha) . . . . . . The effect of soil pH on metribuzin phytotoxocity to soybean in the field, measured by visual injury ratings . . . . . The effect of soil pH on metribuzin phytotoxicity to soybean in the field, measured in grain yield (kg/ha) . . . . . . Effect of soil pH on metribuzin residue in soil, following application of metribuzin (1.12 kg/ha) in the field . . . . . . . . . . . . . . . . . . . Correlation values (r) of the log of concentration remaining in the soil with pH, first-order rate constant (k), half lives (t-1/2) of metribuzin in soil at three pH levels and at three depths, following appli- cation of 1.12 kg/ha in the field, 1973 - - vi Page 50 54 SS 56 57 58 72 73 CHAPTER 3 1. 2. 10. Physical and chemical properties of Hillsdale soil, East Lansing, Michigan. Effect of soil pH on metribuzin phytotoxicity to corn as measured by reduction in dry weight of 1-month-old corn plants. . Effect of pH on metribuzin phyto- toxicity to corn grown in a nutrient- sand culture, measured as reduction in dry weight of l-month-old corn plants . Effect of soil pH on metribuzin phyto- toxicity to soybeans as measured by reduction in dry weight of l-month-old soybean . . . . . . . . . . . Effect of pH on metribuzin phyto- toxicity to soybeans grown in a nutrient— sand culture, measured as reduction in dry weight Of l-month-old soybean . Metribuzin degradation to CO weeks, as affected by soil .pH azide . . . . . . . . . 14 in 12 and sodium C-metribuzin adsorption by Hillsdale sandy clay loam at various pH levels. Extraction efficiency of metribuzin from Hillsdale sandy clay loam at several pH levels . . . . . . . . . . Effect of soil pH on the mobility of 14C atrazine on soil TLC plates . Effect of soil pH on the mobility of 14c metribuzin on soil TLC plates vii Page 90 98 98 99 99 102 117 117 119 119 LIST OF FIGURES CHAPTER 2 1. Weed control with metribuzin at two soil pH levels. Treatment (A) check (B) metribuzin 1.12 kg/ha at pH range of 6.5 to 6.9 and (C) metribuzin 1.12 kg/ha at pH range of 4.5 to 4.9. . . . . . . . . . Metribuzin phytotoxicity to corn and soy- bean over several soil pH levels. Treat- ment (A) metribuzin 1.12 kg/ha at pH range of 6.5 to 6.9 (B) check pH range 6.5 to 6.9 (C) metribuzin 1.12 kg/ha at pH range of 4.5 to 4.9 and (D) check at pH range 4.5 to 4.9. . . . . . . . . . . . . . . . Metribuzin phytotoxicity to corn and soy- bean at several soil pH levels. Treat- ment (A) check at pH range 6.5 to 6.9 (B), (C), (D), (E), and (F) metribuzin 1.12 kg/ha with pH ranges of 6.5 to 6.9, 6.0 to 6.5, 5.5 to 5.9, 5.0 to 5.4, and 4.5 to 4.9, respectively. . . . . . . . . Corn injury (rating 0 = no injury; 10 = complete kill) minus control at given pH range. Metribuzin applied at 1.12 kg/ha in 1972 . . . . . . . . . . . . . . . . . Corn yield as percent of control. Metri- buzin applied at 1.12 kg/ha in 1972 . . Metribuzin degradation rate as the log of concentration in soil at pH 4.6 at three soil depths following application of 1.12 kg/ha in the field in 1973. . . . . . . . Metribuzin degradation rate as the log of concentration in soil at pH 5.6 at three soil depths following application of 1.12 kg/ha in the field in 1973. . . . . . . . viii Page 60 62 64 66 68 75 77 Metribuzin degradation rate as the log of concentration in soil at pH 6.7 at three soil depths following application of 1.12 kg/ha in the field in 1973. . . . . . . CHAPTER 3 1. Soil pH effect on tolerance of soybeans metribuzin (0.00 and 0.56 kg/ha). Soil pH from left to right (A) 4.6 (B) 5.1 (C) 5.6 (D) 6.1 and (E) 6.7 . . . . . . Effect of soil pH and sodium azide on the degradation of l4C-5-labeled metri- buzin to 14C02 at pH 4.6 and 6.7 1 sodium azide. . . . . . . . . . . . . . Effect of soil pH on the degradation of 14c-5-1abe1ed metribuzin to 14c02 at pH 4.6, 5.6 and 6.7. . . . . . . . . . . . Positive correlation with soil pH of percent 14C evolved as 14CO2 in 12 weeks. . . . . . . . . . . . . . . . The spectrophotometric determination of the pKa of metribuzin at 264 nm . . . . Chemical structure of propazine and the proposed ionization equation. . . . . . Chemical structure of metribuzin and the proposed ionization equation. . . . . . Effect of concentration on movement of atrazine at pH 6.7, using soil thin- layer chromatography. Radioactivity per (0.5-cm) segment (% total recovery) versus cm . . . . . . . . . . . . . . . Effect of concentration on movement of metribuzin at pH 6.7, using soil thin- layer chromatography. Radioactivity per (0.5-cm) segment (% total recovery) versus cm . . . . . . . . . . . . . . . ix Page 79 101 104 106 108 111 113 116 121 123 10. 11. Page Effect of soil pH on movement of 14C- atrazine, using soil thin-layer chromatography. Radioactivity per (0.5 cm) segment (% total recovery) versus cm. . . . . . . . . . . . . . . . . . . 125 Effect of soil pH on movement of 14C— metribuzin, using soil thin-layer chromatography. Radioactivity per (0.5 cm) segment (% total recovery) versus cm. . . . . . . . . . . . . . . . . . . 128 INTRODUCTION Metribuzin is a new selective herbicide, which con- trols weeds by contact and systemic action, and has provided excellent control of a wide range of broadleaved weeds in field, forage, and vegetable crops. It has been less effective on annual grasses. Because of the wide spectrum of broadleaved weeds controlled by this compound, especially hard-to-control weeds such as cocklebur (7,17,37,100) (Xanthium pensylvanicum Wallr.), jimsonwood (17,37,59) (Datura stramonium L.), velvetleaf (17,37,95) (Abutilon theophrasti Medic.), and common ragweed (l7,37,42,43) (Ambrosia artemisiifolia L.), metribuzin has the potential of helping producers increase profits by reducing losses due to weed competition. Therefore, the combination of metribuzin and an effective grass herbicide could provide the farmer with broad spectrum weed control, not previously available for weed control in soybeans (l6,36,37,40,42,95), in both seeded and transplant tomatoes (12,13,24,57), and potatoes (41,83). Metribuzin is the first registered herbicide with 1,2,4-triazinone chemistry (96) and there is little information available on its persistence and behavior in soil. Knowledge of its behavior in the soil environment is essential in evaluating its safety and full potential as an agricultural tool. The objectives of these studies were to determine the effect of soil pH on (1) metribuzin activity in rela- tion to crop tolerance and weed control, (2) degradation of metribuzin in soil, (3) adsorption to soil, and (4) rela- tive movement in the soil. CHAPTER 1 LITERATURE REVIEW Metribuzin is the first commercially available asymmetrical triazine (ggftriazine) herbicide. This com- pound, though structurally different from the symmetrical triazines (gftriazines), has a similar mode of action and wide spectrum broadleaf weed control. The mode of metri- buzin action is considered to be the inhibition of photo- synthesis in light reaction II (12). Metribuzin has been used primarily for weed control in soybeans (Glycine max (L.) Merr.), seeded and transplanted tomatoes (Lycopersicon esculentum Mill.) and potatoes (Solanum tuberosum L.). Preemergence and early post- emergence applications have given the best results. Pre- plant incorporated applications have also shown promise on tomatoes and soybeans. Effective rates are 0.28 to 1.12 kg/ha for soybeans and tomatoes (preemergence or preplant incorporated), and 0.56 to 1.12 kg/ha for potatoes (pre- emergence or postemergence) (96). Combinations of metri- buzin and an effective grass herbicide, either as a tank mix application or overlay treatment, provides the farmer with broad spectrum weed control. Increased grass control was obtained when metribuzin was used with alachlor (2-chloro-2',6'-diethyl N—(methoxymethyl) acetanilide), or trifluralin (a,a,a-trifluoro-2,6-dinitro-N,N—dipropyl-p— toluidine) on soybeans (l4,l6,36,37,40,42,95). Weed control obtained with metribuzin has been acceptable, but soybean (7,27,28,71), potato (41), and tomato (72) tolerance has been variable. This variability is due in part to differences in varietal susceptibility. Hargroder and Rogers (29) reported that 14C- metribuzin was readily taken up by soybeans and sesbania (Sesbania punicea (Cav. DC.)) grown in nutrient solution. The herbicide was slowly transported from the roots and concentrated in the foliage. Schumacher (64,66,67) showed that 14 C-labeled metribuzin supplied to the roots of soy- bean seedlings was readily absorbed and transported to the shoots. The major non-polar metabolite detected in the roots and shoots was the deaminated parent 6-(l,l- dimethylethyl)-3-(methylthio)-l,2,4-triazin-5-(4Hl-one (DA). Two additional compounds, the diketo parent 4- amino-6-(1,1-demethylethyl)-l,2,4-triazin-3,5-(2H,4H)- dione (DK) and the deaminated diketo parent 6-(l,l- dimethylethyl)-1,2,4-triazine-3,5 (2H,4H)-dione (DADK), were also detected. The primary metabolic pathway of metribuzin detoxication in soybeans appeared to be the conversion of metribuzin to water soluble metabolites. The amount of 14C in the polar fraction of plant extracts increased with time. Fate and Behavior of Herbicides in the Soil Environment Many factors influence the behavior of herbicides in agricultural soils. What happens to a soil applied herbicide depends both on the physical and chemical prop- erties of the herbicide involved and on certain properties of the soil and water. Both herbicide degradation pro- cesses and herbicide transfer processes play a part in determining the ultimate fate of the chemicals. Three specific degradation processes serve to break down the herbicides and change their chemical com- position (1) biological decomposition - degradation by a living organism, (2) chemical decomposition - breakdown by a chemical process in the absence of a living organism, (3) photodecomposition - degradation by processes involving activation of the molecule by sunlight (87,91). There are a number of transfer processes that influence the degradation rate of soil applied herbicides: (1) absorption and exudation by plants and animals, (2) retention in vegetation, (3) adsorption by soil par- ticles, a process by which herbicides transfer from solu- tion or vapor to a solid surface, (4) volatilization, a process by which the herbicide is changed from the solid or liquid state into a gas or vapor form and discharged into the atmosphere, (5) surface runoff; the herbicide may be either dissolved or suspended in water or be adsorbed to eroding soil particles from treated areas, (6) leaching, and (7) capillary action (91). All processes involved in herbicide degradation are intimately associated. Adsorption, which directly influences all others, is probably the key process. Living organisms, their metabolic products and residues, and humified organic matter, along with inorganic materials such as clays, and the hydrous oxides of iron and aluminum are soil particulate matter responsible for adsorption and inactivation of herbicides. The adsorption process greatly affects the absorp- tion rate of chemicals by plants and soil organisms. Thus, adsorption helps determine herbicide performance. Adsorption also influences leaching, capillary action and volatility of a herbicide through attachment of the chemical to soil particles. The discussion in this review will be restricted to soil pH and its effect on herbicide activity, adsorp- tion, leaching, and degradation of certain groups of herbicides. Factors Influencing Adsorption of Herbicides A. Physico-Chemical Character of Adsorbate The following properties determine the role of the adsorbate in adsorption of herbicides by soil colloids (6): (1) chemical character, shape, and configuration, (2) acidity or basicity of the molecule (pKa or pr), (3) water solubility, (4) charge distribution on the organic cation, (5) polarity, (6) molecular size, and (7) polariza- bility. There are many ways in which each of these prop- erties of the adsorbate may be manifested in the overall adsorption reaction. It should be realized that more than one of these factors may be, and probably are, operating simultaneously. Chemical Character, Shape, and Configuration of the Adsorbate.--Four factors determine the role of the chemical character of the adsorbate in the overall adsorp- tion reaction according to Bailey and White (S): (1) nature of the functional group, (2) nature of the substituting groups, (3) position of the substituting group with respect to the functional group, and (4) presence and magnitude of unsaturation in the molecule. Molecular con- figuration and shape are important in determining if there can be rotation about a double bond which, in turn, may influence the orientation of molecules at the col- loidal surface. The type of molecular orientation may, in turn, determine the contribution of each type of bond- ing mechanism and thus the overall adsorption potential. Bailey st 31. (6) in a study relating the effect of functional group, nature, and position of ring substitu- tion to the magnitude of adsorption by a wide variety of compounds, concluded that the chemical character of the 64 (I) V “v, molecule affects retention by colloidal systems in three different ways as it: (1) determines if the molecule is fundamentally acidic or basic in character and its rela- tive strength, (2) affects the water solubility of the molecule, and (3) determines the relative importance to (1) and (2) of Van der Waals type forces. Dissociation Constant.--The pKa values represent the pH level at which one-half of the species in solution is present in the cationic form and one-half is in the molecular form. Since ionic species are more water soluble than molecular species, basic herbicides have higher solubilities at low pH levels than they do at neutral pH levels (82). The pKa of a compound indicates the degree of acidity or basicity that a compound will exhibit and therefore should be very important in determining both the extent of adsorption and the ease of desorption by colloidal systems (5). The effect of pKa on the adsorption of a basic compound of varying chemical character by montmorillonite has been studied by Bailey gt El- (6). Through the use of adsorption isotherms, the adsorption of such basic com- pounds as g—triazines, substituted ureas, phenylcarbamates, anilides, and anilines on montmorillonite was investigated. They concluded that the major factor governing the mag- nitude of adsorption by different chemical families basic in character is the dissociation constant of the adsorbate. The higher the so-called basicity of the pesticide the greater is its adsorption by acid soil particles (94). Talbert and Fletchall (75) studied the adsorption of five gftriazines by 25 different soil types, the extent of adsorption being expressed by means of distribution coef- ficients, Kd (amount absorbed at equilibrium concentration remaining in equilibrium solution). The degree of adsorption based on the averaged Kd values was propazine [2-chloro-4,6-bis(isopropylamino)-§7triazine] (2.0) < atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-§- triazine] (2.7) < simazine [2-chloro-4,6-bis(ethylamino)- gftriazine] (3.7) < prometone [2,4-bis(isopropylamino)-6- methoxy-gétriazine] (7.8) < prometryne [2,4-bis(isopropyl- amino)-6-(methy1thio)-§-triazine] (9.1). The methylmercapto- and methoxy-derivatives are much more basic in chemical character (pKa 4.2 to 4.8) than are the chloro derivatives (pKa 1.5 to 2.0) which probably accounts for the observed differences in adsorption. Similar results were reported by Harris (30) for the same series of g- triazines. From a consideration of the pKa of the g—triazines, the basicity of the sftriazines disregarding their groups on the 4 and 6 positions are in the order hydroxy > methoxy > methylmercapto > chloro (4,84). Weber (84) nOted for the methoxy-analogues that basicity increased ‘With.increasing number of carbons on the amino alkyl be (1‘ 10 groups. In both cases increasing basicity can be explained in terms of the presence of more nucleophilic groups on the molecule. Carringer and Weber (15) studied the adsorp- tion of five gétriazines and one as-triazine (metribuzin) from aqueous solutions by organic soil colloids and Na+- saturated montmorillonite clay at pH levels from 1.0 to 6.5. Maximum adsorption occurred at pH levels in the vicinity of the pKa values of the respective compounds. The amounts adsorbed by montmorillonite clay were depen- dent upon the molecular structure of the compounds and pH of the suspension. Comparisons of shtriazines substituted in the 4 and 6 positions with monoalkylamino groups indi- cated decreased adsorption by montmorillonite in the order: 4,6-bis(nfpropylamino) > 4-nfpropylamino-6- ethylamino > 4,6-bis(isopropylamino). Adsorption of all five gftriazines was much greater than the adsorption of the ag-triazine at all pH levels. Differences in adsorp- tion were attributed to differences in basicity, water solubility, and steric effects. The basicity of the desorbing agent appears to be important in the overall adsorption-desorption reaction, especially if the adsorbed material is basic in nature. Raman and Mortland (56) have studied charge transfer reactions at clay mineral surfaces by infrared spectro- scopy. They concluded the proton transfer from the pro- tonated species adsorbed on the clay surface to an 11 uncharged molecule at the clay surface was dependent upon the relative basicities of the two interacting compounds as well as the relative concentrations or activities of the reactants and the products. Water Solubility.--Metribuzin has a water solu- bility of 1220 ppm at 20°C (96). The water solubility of metribuzin is high compared to the s—triazines. Atrazine for example, has a water solubility of only 70 ppm (96). There appears to be a relationship between water solubility and extent of adsorption, but only within family of compounds. Bailey gt 31. (6) compared the extent of adsorption of a wide variety of basic herbicides onto sodium and hydrogen montmorillonite. On the basis of these findings, Bailey gt 31. (6) concluded that within a chemical family or within an analogue series basic in chemical character, the magnitude of adsorption is related to and governed by the degree of water solubility. Talbert and Fletchall (75) reported that there was greater adsorption of the methoxy- and methylmercapto- derivatives than there was of the chloro-grtriazine deriva- tives, indicating there was a direct relationship between ‘water solubility and adsorbability. However, it should be irecalled that not only does the water solubility increase among the chloro-, the methoxy-, and the methylmercapto- deliivatives, but also that the methoxy- and methylmercapto- derivatives are more basic than are the chloro-derivatives. 12 Hance (26) studied the adsorption by soils of ureas and their derivatives and found no relationship between adsorption and water solubility. For a particular family of adsorbates, several factors may be interacting to determine whether there is a direct relationship between water solubility and adsorb~ ability. These may include such factors as surface acidity and relative polarity of the adsorbent (5). B. Physico-Chemical Character of Adsorbent Nature and PrOperties of Soil Colloids.--The chemical and physical properties of the soil are controlled by constituents with highly reactive surfaces. Since high specific surface is associated with small particle size, the colloidal fraction of the soil will be the dominant factor in influencing interaction between herbicide mole- cules and the soil. The colloidal fraction, having the most reactive surface, can be divided into the organic matter (humin, humic acid, and fulvic acids) and clay fraction (5). A major factor that needs to be considered in evaluating the .relative importance of organic matter and clay in herbicide (adsorption is that, in mineral soils, the two are intimately bound together, probably as a clay-metal-organic complex. Thlzs, two major types of adsorbing surfaces are normally ava«inlable to the herbicide, namely, clay-humus and clay alone (73) . 13 Humic substances, which represent the most active fraction of the organic matter in most soils, can best be described as a series of highly acidic, yellow to black colored, high-molecular weight polyelettrolytes whose ability to combine with herbicides is due primarily to their high content of oxygen containing functionalgroups, including COOH, phenolic—, aliphatic- and enolic-, and heterocyclic-OH, and C=O structures of various types. Amino, hetercyclic amino, imino, and sulfhydryl groups may also be present. The low-molecular-weight fulvic acid have lower carbon contents but higher oxygen contents than the humic acids. Fulvic acid contain substantially more COOH groups than humic acid. The total acidities of fulvic acids (usual range of 890 to 1,400 meg/100g) are considerably higher than humic acid (usual range of 485 to 870 meg/1009). Both COOH and acidic OH groups contribute to the acidic nature of these substances, with COOH being the most important (5,73,99). The crystalline clays can be divided into two main structural groups on the basis of the ratio of silicate to aluminum layers (5,86). Montmorillonite, a 2:1 type structure, is characterized by having a large surface area and a high exchange capacity. Illite, a non-eXpanding 2:1 ‘tYPe structure, has a lower cation exchange capacity. iKa<>linite, a 1:1 structure, has the lowest surface area and cation exchange capacity. The causes of the cation- exchange capacity of the clay minerals originates from 14 broken bonds, isormorphous substitution, and dissociation of hydrogen ions of exposed hydroxyls. Broken bonds around the edges of the silica-alumina units would give rise to unsatisfied charges, which would be balanced by adsorbed cations. Substitutions within the lattice structure of trivalent aluminum for quadrivalent silicon in the tetrahedral sheet and of ions of lower valence, particularly magnesium, for trivalent aluminum in the octahderal sheet result in unbalanced charges in the structural units of some clay minerals. The hydrogen of exposed hydroxyls may be replaced by a cation which might be exchangeable. However, it seems probable that such hydrogen would be relatively tightly held as compared with those associated with broken bonds and hence, in the main, not replaceable (5,76). The total charge on soil colloidal materials can be separated into two categories, one of which is called the permanent charge, and the other the pH-dependent charge. Permanent charge results from isomorphous sub- stitution and gives to these clay minerals properties of strong acids. About 80% of the CEC at pH 7.0 of Inontmorillonite and vermiculite is due to isomorphous .substitution. The balance derives mainly from broken bonds in the silica layer and is pH—dependent (5,76). Weak acid groups in soil organic matter include carboxyls and quinone hydroxyls which buffer over a range front pH 3.0 to pH 10.0 and phenolic hydroxyls, dienols and a.» vu-u In v s- n“ 15 other groups which buffer only in the alkaline range. In soil mineral fractions, pH-dependent negative change is associated with broken bonds at the edges of the silica layer of crystalline alumino-silicate clays and with silica complexed with hydrated iron and aluminum oxides in amorphous clay colloids. The acid groups associated with silicon buffer over a slightly acid to alkaline pH range (5,73,76, 99). Sites of positive change are also present in soils and are pH-dependent. They appear in the acid range through protonation of broken edge alumina in crystalline layer silicates and protonation of hydrated iron and aluminum oxides in amorphous clay fractions. Protonation of amine groups also gives rise to sites of positive change in the organic fraction of soils. There is a wide range in basiCity of amine groups in soil organic matter, and some of the more basic groups may be protonated in the alkaline range (5,73,76,99). The pH-dependency of weak acid groups and of protonation reactions involving basic groups in mineral and organic colloids contribute importantly to the pH-dependency (Df binding and activity of both cationic and anionic herbicides in soils (5,99). Soil pH.--The acidity of a soil is uaually mea- sured in an aqueous suspension. The composition of the 16 soil solution affects the acid-alkaline character of the soil. The water and ions organized by the mineral surface also affect acidity. Because many reactions are pH sen- sitive, the nature of soil acidity is an important con- sideration in the soil environment (63,70). Soil pH is normally measured with a glass elec- trode in soilzwater suspensions of from 1:1 to 1:2.5 for convenience. However, dilution can change the relative ion dissociation from the clay surfaces and a junction potential at the reference electrodes can introduce error; therefore, the environmental pH of the organic molecule lying next to an air dry clay surface may be entirely different than that measured by the glass elec- trode in a suspension (63,70). The characteristic of acid solutions is based on the activity of the hydrogen ion (H+). An acid when mixed with water dissociates or ionizes into hydrogen ions and the accompanying anions, as represented by the clay- hydrogen complex: + - clay - H H2O H + clay F The H+ ions to the right indicate "active" acidity, and the more the reaction tends toward that direction, the greater the activity of the H+ and the stronger the acid is said to be. The clay-hydrogen complex on the left side of the equation is the "potential" acidity (76). 17 According to generally held views, acidity in soils has several sources: humus or organic matter, alumino- silicate clays, hydrous oxides of iron and aluminum, soluble salts, carbon dioxide, nitrification, and the biological oxidation of reduced sulfure compounds (76). The pH dependency of acidic functional groups in soil organic matter is an important factor influencing the behavior of cationic and weakly basic herbicides (99). Surface Acidity.--It has been recognized for some time that the activity of protons in the bulk suspension and the activity of protons at or in close proximity to the colloidal surface may differ drastically. The term "surface acidity" as applied to soil systems is the acidity at or in close proximity to the colloidal surface and reflects the ability of the system to act both as a Bronsted acid and a Lewis acid. This is a composite term which reflects both the total number of acid sites and their relative degree of acidity (5). Bailey and White (5) state that surface acidity is probably the most important property of the soil or col- loidal system in determining the extent and nature of adsorption and desorption of basic organic compounds as well as determining if acid-catalyzed chemical degrada- tion occurs. The nature and properties of water in the inter- facial region differ from those of bulk water (5) . Hecht ES 91_ (33) have shown that water adsorbed by uh bl [u 18 montmorillonite has a higher degree of dissociation than that of normal water. The NMR studies of Ducros and DuPont (21) showed that the degree of dissociation of adsorbed water in montmorillonite is at least 1,000 times higher than that in liquid water. The clay surface increases in acidity or proton donating properties as the water content is decreased (98). There is overwhelming evidence, mainly from infrared studies, that there is protonation of compounds basic in chemical character both by clays where hydrogen and aluminum are the predominant exchangable cation and also those saturated with transition, alkaline, and alkali metal cations (5). The kind of metal cation occupying exchange sites will largely determine the acidity of the clay surface. Thus, molecules like urea may be protonated on H+ or Al+++ montmorillonite, but not when the clay is ++ or Na+. saturated with Ca Bailey e£_al. (6) concluded from an adsorption study of adsorbates of widely differing chemical character and basicity that the surface acidity of montmorillonite is three to four pH units lower than the pH of the bulk solution. A summary of recent investigations clearly indi- cates that the protonation of organics in the interfacial Jregion of clays is a function of basicity of the molecule, the nature of the exchangable cation, water content of 19 the system, and origin of negative charge in the alumina- silicate (5). Soil Adsorption of Herbicides Mechanisms of Adsorption Adsorption of organic pesticides in soils can be defined as the accumulation of the chemical at the colloid surface which is in contact with the solution. Adsorbed molecules are in equilibrium with molecules in solution, and a number of factors affect this equilibrium. Several mechanisms or combination of mechanisms can be postulated for adsorption of organic compounds by alumino-silicates and soil organic matter. Some of these are as follows: (1) physical adsorption--adsorption due to Van der Walls forces (a summation of dipole-dipole inter- action, dipole induced dipole interactions, and induced dipole-induced dipole interactions, ion dipole interactions), (2) hydrogen bonding, (3) coordination complexes, (4) chemi- cal adsorption, and (5) hydrOphobic bonding (5,98,99). It should be realized that not all mechanisms occur simultaneously; however, two or more may occur simultaneously depending on the nature of the functional groups and acidity of the system. Chemical Adsorption.--Chemica1 adsorption by soils and soil constituents can occur by at least four different Imechanisms (5,98): (1) ion exchange, (2) protonation at 20 the silicate surface or colloidal surface by reaction of the base with the hydronium ion on the exchange site, (3) protonation in the solution phase with subsequent adsorption of the organic molecule via ion exchange, and (4) in systems having water of hydration, protonation by reaction with dissociated protons from residual water present on the surface or in coordination with the exchangable cation. Protonation. As indicated earlier under the topic of surface acidity, adsorption may occur due to protonation at or near the colloidal surface. This process involves the uptake of a proton from the soil-water system by a neutral molecule to become a positively charged ion. This is a very important adsorption mechanism, especially in regard to the adsorption of pesticides basic in chemical character, such as the 3-triazines, 33etriazines, and triazoles. Russell 31 31. (60) found that the amitrole (3-amino-37triazole) molecule became protonated upon adsorption on the surface of montmorillonite to produce a 3-amino—triazolium cation. The infrared spectrum of a Mg++ saturated montmorillonite exposed to a solution of the neutral 3-aminotriazole was remarkably similar to that magnesium and > calcium. He attributed this to the decreasing polarizing power of the cation. Another example of protonation of pesticides at\ colloidal surfaces is the interaction of 3-triazines with montmorillonite (60,20). Infrared studies showed that the 3ftriazines such as propazine and atrazine were pro- tonated when adsorbed on the surface of montmorillonite. This protonation and subsequent hydrolysis was attributed to the surface acidity of the mineral surfaces. Proton- ation and hydrolysis of several of the 3etriazines occur in aqueous systems only at pH values of three or lower. Yet propazine and atrazine, when adsorbed on montmoril- lonite saturated with basic cations such as K+, Mg++p NH4+, Na+, Li+, and Ca++, were found to be protonated and hydrolyzed as a result of the interaction with the cation- water-clay system. McGlamery and Slife (48) found that the amount of atrazine adsorbed on humic acid was pH-dependent; large quantities of atrazine were adsorbed at low pH values. 22 They also reported very little atrazine desorption at acid pH values from the humic complex. Adsorption also increased with increasing temperature. The opposite effect usually is noted in intact soil systems, and it is normally concluded that physical bonding accounts for retention which is reversible to increasing temperature. The data of McGlamery and Slife (48) suggest that a chemical bond was formed (22). Occurrence of Adsorption The properties of the clay-water system influence the properties of both the adsorbent and adsorbate. The pH of the soil solution will determine the degree of dissoci- ation or association of adsorbate, the exact extent being a function of the actual value of the pKa of the adsorbate (5). Therefore, whether a compound is present in the molecular, the cationic or anionic form can affect the extent and magnitude of adsorption and the strength by which the adsorbed species is held, since the energy of adsorption may be vastly different between the dissociated and the associated form (5). Bailey 35.31. (6) found that regard- less of the chemical character of the adsorbate, adsorption occurred to the greatest extent on the highly acid hydrogen- montmorillonite (pH 3.35) compared to the near neutral sodium-montmorillonite (pH 6.8). The same authors con- cluded that the magnitude of adsorption of organic com- pounds with widely different chemical character was governed by three factors: (1) pH of the clay system, 23 (2) water solubility, and (3) the dissociation constant of the adsorbate. These studies reported that the adsorption of acidic-type compounds was dependent upon the pH of the suspension, while the adsorption of a basic compound was dependent upon the surface acidity. Bailey 33 31. (6) studied the effect of pH on the adsorption of a wide variety of chemically basic and acidic compounds by montmorillonite and concluded that: (1) maxi- mum retention of basic compounds would be expected to occur when the surface acidity was at least one to two pH units lower than the lowest dissociation constant of the molecule, (2) adsorption of basic compounds will be prin- cipally due to Van der Waals forces where the surface acidity is more than two pH units lower than the dis- sociation constant, (3) the surface acidity of montmoril- lonite appears to be three to four pH units lower than the pH of the bulk solution. Weber (84,86) demonstrated for a series of 33 triazine compounds that the maximum adsorption on mont- morillonite occurred at a suspension pH in the vicinity of the pKa value of each compound. A further lowering of the pH resulted in some desorption of the gftriazine compounds which was attributed to competition of the protonated species with H+. An alternate explanation would be that the low pH released A1+++ from the clay lattice which would be a greater competitor than would H+ to displace 24 the protonated organic cation from the exchange complex (51). This result is directly Opposite that found by Frissel and Bolt (24) for the adsorption of chloro-3- triazines at three to four pH units above the dissociation constant, adsorption started to increase as pH decreased down to pH 1.0; the amount of adsorption was essentially exponential in nature. In an adsorption study of simazine with 18 soils from the southeastern United States, Nearpass (52) found that the extent of adsorption was not significantly corre- lated with soil pH, but was highly significantly correlated with organic matter content and titratable acidity. Near- pass (53), studying the effect of the predominating cation on the adsorption of simazine and atrazine found that adsorption was governed largely by the hydrogen ion activity relationship which occurred between the solution and the solid phase of the soil. At equal concentrations, atrazine was less strongly adsorbed than simazine. The adsorption of purines, pyrimidines, and their nucleosides by Na+-, Li+-, Ca++-, and Mn++-montmorillonite over a pH range of 2 to 12 was found to be pH dependent, greater adsorption occurring as the pH decreased (44). Adsorption was postulated to occur primarily as a cation exchange reaction under acid conditions. 25 Influence of Soilng on Herbicides Phytotoxicity The phytotoxicity of various soil-applied herbi- cides are affected by many factors, some of which are climate, microbial decomposition, plant species in the application area, and adsorption by soil (69). Hydrogen ions in the soil associate with basic herbicides to form positively charged substances that are readily adsorbed by soil particles. Since it takes free hydrogen ions in the soil to form these cationic compounds, and since free hydrogen ions are present only in acid con- ditions, it means these compounds are strongly adsorbed in highly acid soils. In neutral or alkaline soils these compounds remain primarily in a non-ionized (molecular) form and are held much more weakly by soil particles. This means more of the chemical is available in non-acidic soil. Hartwig (31), Best 33 31. (9) reported that liming of acid soils will improve the effectiveness of the triazine herbicides. Liming to near neutral or alkaline pH will also have an effect on the rate of biological or chemical degradation of these chemicals, since the chemicals are more readily available for plants, micro-organisms, and chemical reactions when they are in the soil solution. The pH dependent adsorption and hydrolysis of basic pesticides in soil systems has been found to affect the biological availability of these compounds. Leefe 26 (46) measured greater damage to strawberries from simazine on soils which were limed as compared to unlimed soils. He attributed the lower activity on the acid soils to increased hydrolysis of simazine. Weber 33 31. (92), Weber and Best (88), and Best and Weber (8) showed that soil pH greatly affected the phytotoxicity and persistence of soil-applied prometryne. The much lower activity which occurred in systems at pH 4.5 as compared with sys- tems at pH 6.5 was attributed to increased adsorption by clay and organic soil colloids. Best 33 31. (9) have recently obtained analogous results in limed versus unlimed soils in field studies. Atrazine and prometryne were much more phytotoxic when applied to limed soils at pH 7.0 than when applied to unlimed soils at pH 5.0. Corbin 33 31. (19) studied the influence of soil pH (4.3 to 7.5) on the phytotoxicity of herbicides incorporated into high organic soils. Phytotoxicity increased as the soil pH increased and reached a maximum at pH 6.5 for the weak bases, prometone and amitrole. Leaching, Movement and Solubili3y The movement of a basic herbicide placed on or within the soil may influence both its effectiveness and its potential as a contaminant in adjacent soil, water or air. Movement may occur while in solution or adsorbed on migrating soil particles, or by transfer vapors and by 27 diffusion if an activity gradient exists (11,34). Upchurch and Pierce (77,78) indicated that at least two steps are involved in the movement of a herbicide down- ward in soil: (1) entrance of the herbicide into solution, and (2) adsorption of the herbicide to soil particles. These two processes, solution and adsorption, may be affected by several variables. A survey of 3&triazine mobility literature by Helling (34) suggested that tria- zines have low to moderate mobility, dependent on the soil adsorptive capacity and the triazine adsorptive strength. Helling (34) reviewed the parameters influencing movement of 3§triazine herbicides in soils: (1) adsorp- tion, (2) solubility, (3) flow rate and amount, (4) for- mulation, (5) rate of pesticide application, and (6) rate of degradation. Adsorption is probably the most significant factor affecting the overall behavior of 3ftriazine herbicides in soils. The rate and extent to which a compound is adsorbed and subsequently desorbed will govern its movement and leaching in soils and its susceptibility to degradation. Scott 33 31. (68) indicated that the magnitude of the (iiffusion coefficient of l4C-metribuzin in Dubbs soil depends primarily upon the amount in the adsorbed phase and «an the moisture content. The adsorbed phase diffusion coefficients were less than the apparent self-diffusion 28 coefficients and decreased with decreasing soil water con- tent. Since negative adsorbed phase diffusion coefficients were observed at the lower soil water contents, the data suggested that the amount of metribuzin in the adsorbed phase increased as soil water content decreased. Savage (62), studying the mobility of metribuzin over a wide range of soil texture, organic matter content, and chemi- cal properties, found a significant negative correlation between Rf values and clay content, while a significant positive correlation was observed between Rf value and sand content. The relative movement of metribuzin in soil was not correlated significantly with soil organic matter content. The order of decreasing mobility of seven 3§triazines (57) seems to coincide with increasing relative adsorption (6,52,75). Adsorption of simazine by 18 acid soils was highly correlated with organic matter and titratable acidity, and somewhat less correlated to clay content (52). A reduction in pH increases the adsorption of basic herbicides, as documented and discussed previously. The pH effect is predominantly on the herbicide itself. This increased adsorption of various 3-triazines by clay or organic matter is generally considered a result of {Irotonation of the somewhat basic molecule (3,5,6,24,30, 75) . McGlamery and Slife (48) showed atrazine adsorption CH1 Drummer clay loam soil increased markedly as the pH deGreased below six; desorption increased as pH and 29 temperature increased. Adsorption on humic acid (from leonardite) was ten-fold greater at pH 2.5 than at 7.0. Since the process was irreversible, atrazine would thus be rendered immobile to leaching in this model system. Best 33 31. (10), Best and Weber (8), studying the effect of soil pH on 3ftriazine dissipation using a balance-sheet approach, reported that plant uptake and leaching occurred to a greater extent in the more alkaline soil. Nearpass (52) found the mobility of simazine was greater in limed than unlimed soil, and suggested that decreased adsorption occurred at increased pH. The nature of the adsorbate and of specific adsorbents in soils also influence adsorption and movement of herbicides (3). Weber (84) compared adsorption of 13 3ftriazines on montmorillonite. Maximum adsorption occurred near the pKa of each compound. The solubilities of herbicides and salts of herbi- cides that may form in the soil are important properties affecting leaching (50,54). Correlation of solubility and adsorption with movement suggested that solubility may be important in initial movement from the point of applica- tion whereas adsorption may be the determining process later. Thus, adsorption in general will be a more accu- rate indicator of overall potential movement than will be SOlubility (11). In theory, however, low solubility may liJnit the ability of a chemical to diffuse into water moving grwavitationally or by capillarity (34). It appears that‘ inczreased solubility may correlate with decreased adsorption 30 (and therefore, increased mobility) for some individual classes of compounds (34). Bailey 33 _1. (6) concluded that the magnitude of adsorption of 3ftriazines on H- montmorillonite was directly related to and governed by the degree of water solubility, with the exception of atrazine. The order of adsorbability was simetone [2,4-bis(ethylamino)-6-methoxy-3-triazine] > atratone [2-(ethylamino)-4-(isopropylamino)-6-methoxy-3- triazine] > prometone > trietazine [2-chloro-4-(diethylamino)-6- (ethylamino)-3-triazine] > propazine > atrazine. Adsorp- tion on Na++—montmorillonite also decreased in this order. The leaching of seven triazines in sandy loam corresponded rather poorly with solubility; Rogers (57) concluded that measurement of relative adsorption was a better predictor. Ward and Weber (82) studied the aqueous solubility of alkylamino-3ftriazines as a function of pH and molec- ular structure. In general, solubilities of 2-OH, 2-OCH3, and ZS-CH3 triazines increased significantly only at pH 3.0 or lower; and of 2-Cl triazines, at pH 2.0 or lower. Results indicated that structural changes at either the 2-position or 4,6-position significantly affected solu- bility. Increased solubility was associated with increased basicity of the 2-substituent and decreased size and (Steric hindrance of the 4,6-N-alkyl substituents. Corre- liition of pKa values and solubilities at low pH suggest tJLat protonation occurs at an endocyclic nitrogen atom. 31 Degradation and Biodegradation Biodegradation is a significant factor affecting the residual life and toxicity of many pesticides in soils. There is considerable evidence indicating that soil microorganisms can utilize 3—triazine compounds as a source of energy (23,38,55). Numerous 3ftriazine degrad- ing microorganisms have been isolated and identified (38). Dealkylation appears to be the major mechanism involved in the microbial degradation of chloro-3ftriazines (39, 47). Evolution of 14 CO2 from microbial systems treated with 14C-ring-labeled 3etriazines has been reported (39, 47). These observations indicate that ring cleavage may occur, however limited it might be. In nearly all cases only low levels of 14 CO2 (zero to four percent) have been evolved from microbial systems treated with ring-labeled gftriazines. This would indicate that the 3ftriazine ring structure is somewhat resistant to microbial attack (38). Hawck and Stephenson (32) suggested that the sym- metrical resonating structure of 3—triazine molecules con- tributed to differences in degradation rates. Asymmetri- cal chlorotriazines were degraded more quickly than the corresponding symmetrically substituted chloro-triazines. They suggested that polymerization resulting in an increased symmetry would lead to stability, whereas labile products of biochemical transformations tend toward asymmetry. 32 The 33-triazines represent a new class of herbi- cides and there is little information available on their persistence and behavior in soil. Lay and Ilnicki (45) used an oat (Avena sativa L.) and cucumber (Cucumis sativa L.) bioassay and observed a 90% loss of metribuzin phyto- toxicity in 42 days from a Sassafras loam soil. Hyzak and Zimdahl (35) reported the half-life of metribuzin in sandy loam soil to be 46 days at 20°C and 16 days at 350C. The loss of metribuzin was linear at both temperatures. Rates of 1.12 and 2.24 kg/ha, applied in the field, remained in the upper 5 cm of the soil profile, and the pattern of herbicide degradation followed that found in the laboratory study at 20°C. First-order kinetics best described the rate of degradation. Waggoner 33 31. (79) studied the degradation of metribuzin in mineral and muck soils under laboratory conditions. Deamination and thiodealkylation resulted in three metabolites: DA, UK, 14 DADK. Schumacher (64,65) showed that C-metribuzin was degraded to 14CO2 in soil. The metabolism of metribuzin was independent of soil organic matter content and was not increased by amendment with glucose and yeast extract. Metribuzin metabolism was inhibited in autoclaved soils. CO evolution from 14C-3 and 14 2 soils indicated that the triazine ring was degraded in a C-S metribuzin applied to stepwise manner and C-3 was released more readily than was C-S. The primary soil metabolite was DADK. 33 The influence of metribuzin on soil microbial activity is not known. Lay and Ilnicki (45) reported that metribuzin at 0.29 to 0.56 kg/ha had no effect on the num- ber of soil microorganisms present. Avrik 33 31. (2) reported a 60% decrease in the number of soil algae in soil treated with 10 ppm of metribuzin. Chlorella sp. and Anabaena sp. appeared to be more resistant to the chemical than other species studied. Soil pH may directly or indirectly influence the detoxication of herbicides by affecting the ionic or molecular character of the chemical, the ionic character of soil colloids, the cation exchange capacity, and the inherent capacity of microbial populations to respond to a given chemical (18). Soil pH, moisture content, and aeration affect the activities of soil microorganisms. Soil bacteria and actinomycetes are most active in near neutral or alkaline, moist soils (80), whereas soil fungi are generally more active in soil under low moisture conditions (49) and lower pH levels (81). Since so many soil fungi appear to be involved in biodegradation of 3—triazines, one might expect decomposition to occur more rapidly in acid soils of low moisture content. However, this is not the case. Persistence of several 3ftriazines is less in moist soils than in dry soils (74). Adsorption of atrazine and simazine varies inversely with pH (30,75). 34 Obien and Green (55), examining soil characteristics such as pH, organic matter content, and atrazine adsorption, revealed that low pH appeared to be more closely related to the rapid degree of atrazine degradation than the other soil properties. Hydroxyatrazine was the main degradation product. It was concluded that chemical degradation (hydrolysis) was the major pathway of atrazine loss in the soils. A similar observation was made by Weber 33 31. (92) who found that the phytotoxicity of prometryne to wheat (Triticum aestivum L. em Thelli) was inversely related to soil acidity, i.e., lower phytotoxicity was obtained at pH 4.5 than at pH 6.5 in the presence of montmorillonite clay or organic matter. They attributed this result to increased adsorption of the protonated prometryne associated with a decrease in pH of the medium. Chemical hydrolysis of the triazines was catalyzed by adsorption on clay and organic matter (1,61). Corbin and Upchurch (19) found no influence of pH on the detoxication of prometone. Some of the possible factors given for the observed persistence are (1) inhibition of microbial growth at the chemical concentrations used, with subsequent failure of the microorganisms to produce detoxifying enzymes within the allocated time period, (2) low chemical snalubility with a resultant low uptake by the primary ndkcroflora, (3) strong adsorptive bonds between the Cheunicals and soil colloids, coupled with a low availability to 1:he microbial population. The pH of the soil environment 35 has a profound effect upon the inherent capacity of the microbial population to respond to chemical changes in the environment and exerts a vital role in the interactions which eventually result in the partial or total inactivation of the herbicide substrate (19). The dipyridylium herbicide diquat2+ [6,7-dihydrodipyrido(1,2-a',2',l'-c)-pyrazidinium dibromide] is strongly adsorbed by montmorillonite and not readily extractable (93,90). Weber and Coble (89) showed that the addition of montmorillonite clay in an amount calculated to adsorb one-half of the 14 14CO2 detected. C-diquat reduced the Best and Weber (8) found the pattern of atrazine degradation was characteristic of nonbiological pro- cesses, while prometryne degradation was by microbial action. The higher amounts of extractable prometryne in the acid soils suggested that the adsorption to soil colloids offered some protection from microbial degrada- tion. Unlike atrazine, prometryne was relatively stable in the acidic environment surrounding these colloids (97). In the pH 7.5 soil, more of the prometryne molecules were in the soil solution and consequently available to plants and microbes. List of References Armstrong, D. E., and G. Chesters. 1968. Adsorption catalyzed chemical hydrolysis of atrazine. Environ. Sci. and Tech. 2:683-689. Arvik, J. H., D. L. Hyzak, and R. L. Zimdalhl. 1973. Effect of metribuzin and two analogs on five species of algae. Weed Sci. 21:173-175. Bailey, G. W., and J. L. White. 1964. Review of adsorption and desorption of organic pesticides by soil colloids, with implications concerning pesticide bioactivity. J. Agr. Food Chem. 12: 324-332. Bailey, G. W., and J. L. White. 1965. Herbicides: a compilation of their physical, chemical, and bio— logical properties. Residue Reviews 10:97-122. Bailey, G. W., and J. L. White. 1970. Factors influencing the adsorption, desorption and move- ment of pesticides in soil. Residue Reviews 32: 29-92. Bailey, G. W., J. L. White, and T. Rothberg. 1968. Adsorption of organic herbicides by montmoril- lonite: Role of pH and chemical character of adsorbate. Soil Sci. Soc. Amer. Proc. 32:222-234. Barrentine, W. L., and C. G. McWorter. 1972. Cocklebur control with Bay 94337 and bentazon in soybeans. Proc. Southern Weed Contr. Conf. 25:104. Best, J. A., and J. B. Weber. 1974. Disappearance of 3—triazines as affected by soil pH using a balance-sheet approach. Weed Sci. 22:364-373. Best, J. A., J. B. Weber, and T. J. Monaco. 1971. Effect of liming on the activity of soil-applied triazines. Proc. Southern Weed Sci. Soc. 24:368. 36 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 2]” 22. 37 Best, J. A., J. B. Weber, and T. J. Monaco. 1974. The effect of soil pH on 3—triazine dissipation using a balance-sheet approach. Weed Sci. Soc. Amer. Abstr. P. 97. Biggar, J. W. 1970. Pesticide movement in soil water. Pesticides In the Soil: Ecology, Degrada- tion, and Movement. Pp. 107-119. Buchel, K. H. 1972. Mechanism of action and struc- ture activity relations of herbicides that inhibit photosynthesis. Pest. Sci. 3:89-110. Burgis, D. S. 1971. Herbicide test on tomato trans- plants and seeded tomatoes. Proc. Southern Weed Contr. Conf. 24:198-205. Carlson, W. C. 1973. Metribuzin and metribuzin com- binations for weed control in tomatoes. N. Cent. Weed Contr. Conf. 28:67. Carringer, R. D., and J. B. Weber. 1973. Adsorption of triazines by soil colloids as a function of pH and molecular structure. Proc. Southern Weed Sci. Soc. 26:397. Carter, M. T. 1974. Metribuzin--a versatile herbi- cide for weed control in soybeans. Weed Sci. Soc. Amer. Abstr. P. 124. Chemagro. 1971. Sencor (Bay 9337): Experimental herbicide. Technical Information Bulletin. Corbin, F. T., and R. P. Upchurch. 1967. Influence of pH on detoxication of herbicides in soil. Weeds 15:370-377. Corbin, F. T., R. P. Upchurch, and F. L. Selman. 1971. Influence of pH on the phytotoxicity of herbicides in soil. Weed Sci. 19:233-239. Cruz, M., J. L. White, and J. D. Russell. 1968. Montmorillonite-3etriazine interaction. Israel J. Chem. 6:315-323. Ducros, P., and M. Dupont. 1962. Etude par resonance magnetique nucleaire des protons dans les argiles. Compt. Rend. 254:1409. Dunigan, E. P., and T. H. McIntosh. 1971. Atrazine- soil organic matter interactions. Weed Sci. 19: 279-281. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 38 Ercegovich, C. D. 1964. What happens to triazines in soil. Paper presented at the Washington State Weed Control Conference, Yakima, Washington, November 3. Pp. 1-22. Frissel, M. J., and G. H. Bolt. 1962. Interaction between certain ionizable organic compounds (herbicides) and clay minerals. Soil Sci. 94: 284-291. Fortino, J., and W. E. Splittstoesser. 1973. Studies with metribuzin on tomatoes. Weed Sci. Soc. Amer. Abstr. P. 15. Hance, R. J. 1965. The adsorption of urea and some of its derivatives by a variety of soils. Weed Research 5:98-107. Hardcastle, W. S. 1973. Soybean varietal interaction with metribuzin. Weed Sci. Soc. Amer. Abstr. P. 2. Hardcastle, W. S. 1974. Soybean cultivar interaction with metribuzin. Weed Sci. Soc. Amer. Abstr. P. 3. Hardgroder, T. 3., and R. L. Rogers. 1972. The behavior of 4C-Bay-94337 in soybeans and sesbania. Proc. Southern Weed Contr. Conf. 25:422. Harris, C. I., and G. F. Warren. 1964. Adsorption and desorption of herbicides by soil. Weeds 12: 120-126. Hartwig, N. L. 1974. Soil and soil pH effects on the activity of some triazine herbicides. Ag. Lime Digest 6:3-4. Hauck, R. D., and H. F. Stephenson. 1964. Nitri- fication of triazine nitrogen. J. Agr. Food Chem. 12:147-150. Hecht, A. M., M. Dupont, and P. Ducros. 1966. Etude des phénoménes de transport de l'eau adsorbée dans certains mineraux argileux par la resonance magnetique nucléaire. Bull. Soc. Franc. Min. Crist. 89:6. Helling, C. S. 1970. Movement of 3—triazine herbi- cides in soil. Residue Review 32:175-210. Hyzak, D. L., and R. L. Zimdahl. 1974. Rate of degradation of metribuzin and two analogs in soil. Weed Sci. 22:75-79. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 39 Jennings, V. M., D. W. Staniforth, and W. G. Lovely. 1973. Soybean herbicide evaluations across Iowa in 1973. Proc. N. Cent. Weed Contr. Conf. 28: 42-45. Jones, M. L., J. L. Barrentine, J. B. Edmondson, J. A. Keaton, J. L. Pafford, and H. L. Webster. 1974. Trifluralin + metribuzin applied preplant soil incorporated for weed control of soybeans. Weed Sci. Soc. Amer. Abstr. P. 124. Kaufman, D. D., and P. C. Kearney. 1970. Microbial degradation of 3-triazines herbicides. Residue Review 32:235-265. Kaufman, D. D., P. C. Kearney, and T. J. Sheets. 1963. Simazine: Degradation by soil microorga- nisms. Science 142:405-406. Ladlie, J. S., and W. F. Meggitt. 1972. Herbicide and application combinations for weed control in soybeans. Proc. N. Cent. Weed Contr. Conf. 27:27. Ladlie, J. S., W. F. Meggitt, and R. C. Bond. 1972. Weed control results in field crops. Michigan State University (not for publication). Pp. 85-93. Ladlie, J. S., W. F. Meggitt, and R. C. Bond. 1973. Soybean weed control in Michigan. N. Cent. Weed Contr. Conf. Res. Rep. 30:112-115. Ladlie, J. S., W. F. Meggitt, and R. C. Bond. 1974. Weed control results in field crops. Michigan State University (not for publication). Pp. 10-18. Lailach, G. E., T. D. Thompson, and G. W. Brindley. 1968. Adsorption of pyridines, purines, and nucleosides by Li, Na-, Mg- and Ca-montmorillonite (clay organic studies XII). Clays and Clay Minerals 16:275. Lay, M., and R. P. Ilnicki. 1973. The dissipation of metribuzin in soil. Weed Sci. Soc. Amer. Abstr. P. 59. ‘ Leefe, J. S. 1968. Effect of soil pH on the phyto- toxicity of simazine to strawberries. Can. J. Plant Sci. 48:424-425. McCormick, L. L., and A. E. Hiltbold. 1966. Micro- biological decomposition of atrazine and divron in soil. Weeds 14:77-82. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 40 McGlamery, P. L., and F. W. Slife. 1966. The adsorption and desorption of atrazine in soil as affected by pH, temperature, and concentration. Weeds 14:237-239. Menon, S. R., and L. E. Williams. 1957. Effect of crop, crOp residues, temperature, and moisture on soil fungi. Phytopathol. 47:559. Minarik, C. E. 1951. Pre-emergence herbicides and their behavior. Proc. N. Cent. Weed Contr. Conf. 5:29-39. Mortland, M. M. 1970. Clay organic complexes and interaction. Advan. Agron. 22:75-117. Nearpass, D. C. 1965. Effects of soil acidity on the adsorption, penetration, and persistence of simazine. Weeds 13:341-346. Nearpass, D. C. 1967. Effect of the predominating cation on the adsorption of simazine and atrazine by Bayboro clay soil. Soil Sci. 103:177-182. Norman, A. G., and A. S. Newman. 1950. The persis- tence of herbicides in soils. Proc. N. Cent. Weed Contr. Conf. 4:7-12. Obien, S. R., and R. E. Green. 1969. Degradation of atrazine in four Hawaiian soils. Weed Sci. 17: 509-514. Raman, R. V., and M. M. Mortland. 1969. Proton transfer reactions at clay mineral surfaces. Soil Sci. Soc. Amer. Proc. 33:313. Rogers, E. G. 1968. Leaching of seven 3-triazines. Weed Sci. 16:177-120. Romanowski, R. R., and G. F. Warren. 1973. Research results with metribuzin in tomatoes. Proc. N. Cent. Weed Contr. Conf. 28:68. Ross, M. A., J. L. Williams, Jr., and T. T. Bauman. 1973. The jimsonweed problem in Indiana soybean production. Proc. N. Cent. Weed Contr. Conf. 28: 48. Russell, J. D., M. I. Cruz, and J. L. White. 1968. The adsorption of 3-aminotriazole by montmoril- lonite. J. Agr. Food Chem. 16:21-24. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 41 Russell, J. D., M. Cruz, J. L. White, G. W. Bailey, W. R. Payne, Jr., J. D. Pope, Jr., and J. I. Teasley. 1968. Mode of chemical degradation of 3-triazines by montmorillonite. Science 160: 1340-1342. Savage, K. E. 1974. Adsorption and mobility of metribuzin in soil. Weed Sci. Soc. Amer. Abstr. P. 120. Schofield, R. K., and A. W. Taylor. 1955. The measurement of soil pH. Soil Sci. Soc. Amer. Proc. 19:164-167. Schumacher, R. W. 1974. Metabolism of metribuzin in soybeans and soil. Ph.D. Thesis, University of Kentucky. Schumacher, R. W., C. E. Rieck, and L. P. Buch. 1973. Soil degradation of metribuzin. Proc. N. Cent. Weed Contr. Conf. 28:30. Schumacher, R. W., L. Thompson, Jr., and C. E. Rieck. 1973. Absorption, translocation, and metabolism of metribuzin in soybeans. Weed Sci. Soc. Amer. Abstr. P. 60. Schumacher, R. W., L. Thompson, Jr., and C. E. Rieck. 1974. Metribuzin metabolism in soybeans. Weed Sci. Soc. Amer. Abstr. P. 123. Scott, H. D., R. E. Phillips, and R. F. Paetzold. 1974. Diffusion of herbicides in the adsorbed phase. Soil Sci. Soc. Amer. Proc. 38:558-562. Scott, D. C., and J. B. Weber. 1967. Herbicide phytotoxicity as influenced by adsorption. Soil Sci. 104:151-158. Smiley, R. W., and R. J. Cook. 1972. Use and abuse of soil pH measurement. Phytopathology 62:193-194. Smith, A. E., and R. E. Wilkinson. 1974. Uptake and accumulation of metribuzin and metabolites by several soybean varieties. Weed Sci. Soc. Amer. Abstr. P. 123. Stephenson, G. R., M. McLeod, and S. C. Phatak. 1974. Varietal differences in the tolerance of tomatoes to metribuzin. Weed Sci. Soc. Amer. Abstr. P. 122- 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 42 Stevenson, F. J. 1972. Organic matter reactions involving herbicides in soil. J. Environ. Quality 1:333-342. Talbert, R. E., and O. H. Fletchall. 1964. Inactiva- tion of simazine and atrazine in the field. Weeds 12:33-37. Talbert, R. E., and O. H. Fletchall. 1965. The adsorption of some 3—triazines in soils. Weeds 13:46-52. Tisdale, S. L., and W. L. Nelson. 1968. Soil fer- tility and fertilizers. The Macmillan Company, London. Pp. 413-422. Upchurch, R. P. 1958. The influence of soil fac- tors on the phytotoxicity and plant selectivity of diuron. Weeds 6:161-171. Upchurch, R. P., and W. C. Pierce. 1957. The leaching of Monuron from Lakeland sand soil. Part I. The effect of amount, intensity, and frequency of simulated rainfall. Weeds 5:321-330. Waggoner, T. B., C. A. Anderson, J. A. Ciarletta, and T. J. Olson. 1974. Degradation of metribuzin in soil and effects on rotational crops. Weed Sci. Soc. Amer. Abstr. P. 121. Waksman, S. A. 1932. Principles of soil micro- biology. Baltimore: Williams and Wilkins. Warcup, J. H. 1951. The ecology of soil fungi. Brit. Mycol. Soc. Trans. 34:376. Ward, T. M., and J. B. Weber. 1968. Aqueous solu- bility of alkylamino-3-triazines as a function of pH and molecular structure. Agr. and Food Chemistry 6(6):959. Wascon, B. W., T. Hernandez, and R. Constantin. 1972. The effect of herbicides on the yield, grade and quality of sweet potatoes. Proc. South- ern Weed Contr. Conf. 25:202-205. Weber, J. B. 1966. Molecular structure and pH effects on the adsorption of 13 3-triazine com- pounds on montmorillonite clay. Amer. Mineral. 51:1657-1670. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 43 Weber, J. B. 1970. Adsorption of 3—triazines by montmorillonite as a function of pH and molecular structure. Soil Sci. Soc. Amer. Proc. 34:401-404. Weber, J. B. 1970. Mechanisms of adsorption of 3—triazines by clay colloids and factors affecting plant availability. Residue Rev. 32:93-127. Weber, J. B. 1970. Behavior of herbicides in soils. Beltwide Cotton Production Research Conf. Pp. 35-36. Weber, J. B., and J. A. Best. 1972. Activity and movement of 13 soil-applied herbicides as influ- enced by soil reaction. Proc. Southern Weed Sci. Soc. 25:403-413. Weber, J. B., and H. D. Coble. 1968. Microbial decomposition of diquat adsorbed on montmorillonite and kaolinite clays. J. Agric. Food Chem. 16: 475-478. Weber, J. B., R. C. Meek, and S. B. Ward. 1969. The effect of cation-exchang3 capacity on the retention of diquat2+ and paraquat + by three-layer type clay minerals. II. Plant availability of para- quat. Soil Sci. Soc. Amer. Proc. 33:382-385. Weber, J. B., T. J. Monaco, and A. D. Worsham. 1973. What happens to herbicides in the environment. Weeds Today 4(1):l6-l7,22. Weber, J. B., P. W. Perry, and K. Ibaraki. 1968. Effect of pH on the phytotoxicity of Prometryne applied to synthetic soil media. Weed Sci. 16: 134-136. Weber, J. B., P. W. Perry, and R. P. Upchurch. 1965. The influence of temperature and time on the adsorption of paraquat, diquat, 2,4-D and pro- metone by clays, charcoal, and an anion-exchange resin. Soil Sci. Soc. Amer. Proc. 29:678-688. Weber, J. B., S. B. Weed, and T. J. Sheets. 1972. Pesticides--how they move and react in the soil. Crops and Soil Mag. 25(1):l4-l7. Webster, H. L., J. B. Edmondson, R. William Gesink, M. L. Jones, and J. C. Walker. 1973. Trifluralin and metribuzin for broad spectrum weed control in soybeans. Proc. N. Cent. Weed Contr. Conf. 28:60-63. 96. 97. 98. 99. 100. 44 Weed Science Society of America. 1974. Herbicide Handbook. 3rd ed. W. F. Humphrey Press, Inc., Geneva, New York. Pp. 246-249. White, J. L., and M. Cruz. 1971. The role of soil colloids in the behavior and fate of pesticides in soils. Pest. Chem. 6:23-45. White, J. L., and M. M. Mortland. 1970. Pesticide retention by minerals. Pesticides in the Soil: Ecology, Degradation and Movement. Pp. 95-100. Wolcott, H. R. 1970. Retention of pesticides by organic materials in soils. Pesticides in the Soil: Ecology, Degradation and Movement. Pp. 128-138. Wright, T. H., and C. E. Rieck. 1973. Effect of Linuron, Metribuzin and Chlorbromuron on Cockle- bur control. Proc. N. Cent. Weed Contr. Conf. 28:28. CHAPTER 2 EFFECT OF SOIL PH ON METRIBUZIN ACTIVITY AND DISSIPATION IN FIELD SOILS Abstract Metribuzin [4-amino-6-tert-butyl-3-(methylthio)- l,2,5-triazin-5(4H)one] activity in the field increased with increasing soil pH. Fall panicum (Panicum dichotomiflorum Michx.) control, number of dead corn (Zea mays L.) and soybeans (Glycine max (L.) Merr.) plants, and crop injury ratings increased, whereas, plant height and grain yield decreased as the soil pH increased. Metribuzin residue analysis of soil samples showed greater amounts of residue extractable at soil pH 6.7 than 4.6. Metribuzin leaching increased with in- creasing soil pH. Metribuzin disappearance from soil followed pseudo first order-kinetics. Rate constants increased and the half life of metribuzin decreased as soil pH increased. Rate constants decreased and the half life of metribuzin increased at all soil pH levels as depth of sampling decreased. The decreased activity and decreased rate of metribuzin dissipation at lower soil pH is likely due to protonation and increased adsorption. 45 46 Introduction Adsorption of the 3-triazines by soil colloids is dependent on the pH of the system, and many plant studies (6,7,12,16,21) have shown that the 3-triazines are gene- rally less phytotoxic under acid conditions than under basic conditions. This increased adsorption of various 3-triazines by clay or organic matter is generally con- sidered a result of protonation of the somewhat basic molecule (3,4,5,10,21). It is apparent that the reduction in herbicide concentration resulting from adsorption by soil colloids results in less herbicide available for absorption by plant roots (3,24). Although the adsorption of the compounds by the soil colloids is in most cases reversible, the decrease in the rate of uptake as a result of dilution reduces herbicidal activity of the chemicals (3,24). The order of decreasing mobility of seven 3- triazines (18) seems to coincide with increasing relative adsorption (17,21). Savage (l9) studying the mobility of metribuzin over a wide range of soil texture, organic matter content, and chemical properties, found significant negative correlation between Rf values and clay content,. while a significant positive correlation was observed between Rf value and sand content. The relative movement of metribuzin in soil was not correlated significantly with soil organic matter content. Scott 33 31. (20) 47 indicated that the magnitude of the diffusion coefficient of metribuzin in Dubbs soil depended primarily upon the amount in the adsorbed phase and on the moisture content. Biodegradation is a significant factor affecting the residual life and toxicity of many pesticides in soils. There is considerable evidence indicating that soil microorganisms can utilize 3-triazines compounds as a source of energy (15). Hawck and Stephenson (13) found asymmetrical chloro-3-triazines were degraded more quickly than the corresponding symmetrical substituted chloro-3- triazines. Gysin (11) suggested that methylthio-3- triazines are more readily degraded than chloro-3- triazines. Best and Weber (6), using a balance-sheet approach, found atrazine [2-chloro-4-(ethylamino)-6- (isopropylamino)-3-triazine] degradation was character- istic of nonbiological process, while prometryne [2,4- bis(isopropylamino)-6-(methylthio-3-triazine] degradation was probably by microbial action. Extractable atrazine after 5 months amounted to 35% of the initial amount in the pH 7.5 and 11% in the pH 5.5 soil, while prometryne occurred as 10% of the original concentration in the pH 7.5 soil and 42% in the pH 5.5 soil. Waggoner 33 31. (22) studied the degradation of metribuzin in mineral and muck soils under laboratory and field conditions. Deamination and thio-dealkylation resulted in three metabolites: 6-(1,l-dimethylethyl)-3-(methylthio)-l,2,4-triazin-5-(4H)- 48 one (DA); 4-amino-6-(1,1-dimethylethyl)-l,2,4-triazin-3, 5-(2H,4H)-dione (DK); 6-(l,l-dimethylethyl)-l,2,4-triazin- 1 l4 3,5-(2H,4H)-dione (DADK). Schumacher showed that C- ring-labeled metribuzin was degraded to 14CO2 in soil. The metabolism of metribuzin was independent of soil organic matter and was not increased by amendment with glucose and yeast extract. The primary soil metabolite was DADK. Hyzak and Zimdahl (14) reported the half-life of metribuzin in sandy loam soil to be 46 days at 20°C and 16 days at 35°C. The loss of metribuzin was linear at both tempera- tures. Rates of 1.12 and 2.24 kg/ha, applied in the field, remained in the upper 5 cm of the soil profile, and the pattern of herbicide degradation followed that found in the laboratory study at 20°C. Soil pH may directly or indirectly influence activity and detoxication of herbicides by affecting the ionic or molecular character of the chemical, the ionic character of soil colloids, the cation exchange capacity, and the inherent capacity of microbial population to respond to a given chemical (8). The objectives of this study were to determine the effect of soil pH on metribuzin activity and dissipation in field soil. 1Schumacher, R. W. 1974. Metabolism of metribuzin in soybeans and soil. Ph.D. Thesis. University of Ken- tucky, Lexington, Kentucky. 176 pp. 49 Materials and Methods Field Experiments Field experiments were conducted in 1972, 1973, and 1974 at East Lansing, Michigan to study the effect of soil pH on metribuzin phytotoxicity to soybeans and corn. The experiments were conducted on Hillsdale sandy clay loam, East Lansing, Michigan, containing 1.5% organic matter with a pH adjusted over a lS-year period with ammonium sulfate, calcium nitrate or dolomitic limestone (26). The soil mechanical analyses were similar at all five pH levels; OM (1.5%), sand (53%), silt (22%), and clay (25%). Treat- ments were replicated four times in a completely randomized block design. There were variations within the pH ranges given, over the four replications. Metribuzin was applied preemergence at 0.00, 0.56, 0.84 and 1.12 kg/ha with a tractor-mounted sprayer that delivered 215 L/ha. Two rows of corn 'Michigan 400' and two rows of soybeans, 'Wirth' (1972), 'Hark' (1973), and 'Swift' (1974) were planted in each plot. Plot size was 3.0 m x 7.6 m with four rows in 0.76 m row widths. Planting dates were June 3, 1972, May 17, 1973, and May 22, 1974. Rainfall (Table 1) occurred within 7 days after application in all three years. All untreated and treated plots were hand weeded as needed early in the growing season. Population counts and injury ratings (0 = no injury; 10 = complete kill) were measured one month after planting. Plant height was measured six 50 Table l.--Rainfall data for 1972, 1973, and 1974 Soils Farm, East Lansing, Michigan. Year April May June July Aug. Sept. Oct. Total (cm) 1972 8.54 7.46 6.04 6.29 8.89 10.67 7.87 55.75 1973 10.00 12.64 9.30 2.21 5.69 8.33 5.59 53.77 1974 5.18 11.78 4.42 3.89 9.86 7.01 2.82 44.96 weeks after planting. At maturity grain yields were recorded. Due to the effect of pH on plant growth at the different pH levels, population counts and injury ratings were expressed after subtracting the control at the given pH range. Plant height and grain yield were reported as percent of the control at the given pH range. Weed control ratings were recorded on a 0 to 10 scale (0 = no control and 10 = complete control) for fall panicum late in the growing season. Field Residue Study Field residue analyses were conducted at the pre- viously described plots. Soil samples were taken from the 1.12 kg/ha metribuzin treated plots in 1973 and 1974. A split-plot design was used with soil pH as main plots and dates of sampling as subplots. Metribuzin had been used on the plots in 1972, therefore, samples were collected before application in 1973, and 1974 to establish residue 51 levels from application the previous year. Samples were collected in 1973 at 15, 36, 77, and 150 days and in 1974 at 3, 21, 84, and 142 days after herbicide application. Depths of sampling were 0.0-5.0, 5.0-10.0, and 10.0-20.0-cm. At each sampling date, subsamples were takenfrom each main plot, extracted and analyzed separately. Rainfall for 1973 and 1974 is given in Table 1. After each sampling date, the soil was air-dried, screened, thoroughly mixed, and frozen prior to analysis. The amount of herbicide present was determined by Soxhlet extraction and gas chromatograph analysis (14). Herbicide standards were prepared to check extraction efficiency by dissolving the analytical grade compound (99 to 100% purity) in distilled acetone. Fifty grams of soil, pH levels 4.6, 5.6, and 6.7, were placed in beakers and 5 m1 of acetone solution were uniformly pipetted over the soil surface. The herbicide was thoroughly mixed with the soil to yield a uniform concentration of 1.0 and 0.1 ppm, w/w basis. The soil and herbicide were allowed to equilibrate for 48 hours. The 50 grams of soil were extracted in a Soxhlet apparatus with 250 ml of 80% methanol for five hours with a recycling time of approxi- mately five minutes. After extraction, 25 m1 of distilled water was added to the extractant and the solvent was evaporated to 20 to 30 ml. The aqueous phase was extracted with one 50 ml and two 25 ml aliquots of glass distilled 52 benzene to bring the final volume to 100 ml. The aqueous- benzene mixture was vigorously shaken for 10 to 15 seconds and allowed to separate into its respective phases. Thirty grams of anhydrous sodium sulfate (NaZSO4) was added to each bottle. The aqueous phase was drained into a beaker and discarded. The samples were then placed in a refrig- erator until analysis. Samples from the field plots were handled as previously described. Herbicide concentrations in the benzene extracts were determined with a gas chromatograph equipped with a Beckman helium arc discharge electron-capture detector. A 183 cm by 2 mm (I.D.) glass column was packed with 80/100 mesh Chromosorb Gas-Chrom Q coated with 3% SE-30. Injector, column, and detector temperatures were 250, 190, and 310°C, respectively. Helium at a flow rate of 40 ml/min was used for the carrier gas. Under these conditions, a retention time of 1.85 min was obtained. The range of linearity was from 0.05 ng to 5 ng, and the detection limit of the method was 0.01 ppm in soil. Herbicide content was determined by comparing peak heights from sample to peak heights of known standards. A series of standards of different concentrations of herbicide was injected to determine the range of linearity and sensitivity before, during, and after each day's run of samples. Known soil pH standards were extracted and analyzed to determine extraction efficiency at the differ- ent soil pH levels. 53 Results and Discussion Effect of Soil pflygn Metribuzin Activity in the FieId Metribuzin activity in the field increased with increasing soil pH (Tables 2 to 6). Fall panicum control increased with increasing soil pH (Figure l). Metribuzin applied at 0.84'and 1.12 kg/ha gave similar results, however, there were greater differences between pH ranges at the 0.56 kg/ha rate than at the higher application rates (Table 2). It can be postulated that the reduction in herbicide concentration by soil colloids resulted in less absorption by the fall panicum roots at the lower appli- cation rate. Phytotoxicity to corn and soybean also increased as the soil pH increased (Figures 2 and 3). Corn was a more sensitive indicator of the metribuzin pH response than soybean. Because of the variation of soil pH within the four replications in a given pH range, pH levels were not always significantly different. This held true for all parameters measured, especially at the 0.56 kg/ha metribuzin rate. However, correlations, with soil pH obtained from field data were 0.971 and 0.977 for corn injury ratings and grain yield at 1.12 kg/ha metribuzin in 1972 (Figures 4 and 5). Injury ratings as an indication of vigor and stand reduction were taken one month after planting. Corn injury showed a positive correlation with increasing soil pH (Figure 4). Corn injury (Table 3) 54 . COmflmm measouc ms» ca wand cmouooou ouo3 Houucoo encased Adam now mmcwucu Houucoo 00030 .Houucoo mucamaoo u 0H «Houucoo on u on .umou moccu mamfluasa m.cmo:so an Ho>oa pm on» um uEOHONMAo SHDENOAMHEUHN no: one mumuuoa ucawewm Ends sumo m.uco> some no access m m.m no e.m e m.m o e.m e H.m e m.m o m.m co o.m e m.m m.e-m.e m m.m no A.e e e.m «so m.m e e.m e m.m m e.m no H.e e m.m e.euo.o m o.oA eo o.m e m.m moon e.m e m.m e e.m eon m.» no m.m eu o.m m.mum.m we o.m o e.m e m.m eon m.e one n.m eon 0.5 A m.m on e.m eon m.m «.m-o.m on m.e no m.e on m.e n 0.5 one m.m n m.e e e.m e m.e e e.e m.e-m.e onAHouucoo no as «has meme News «has meme apes seed meme News omens ~A.H em.o om.o mm Aunwmxv causnwuuoz e.saoaaom same on auaoexou eeusnsuuma no em Heom mo uomuuo was--.« manna 55 .mcflucmam umumm nucoe oco ocean can Homw> co moms ocwumu Suoflcuo .Hawx ouoamsoo u OH “hushed on u on .umou omen» oamwuasa m.ccocoo >n Ho>oH am on» us ucoummmao waucmowmwcmfim uoc mum mumuuoa umawsflm sues sumo m.umom come no memozm m h.m m m.h o m.v moo h.v moon m.v o m.v econ b.m 60nd v.m Ono N.N m.mlm.m mu o.m Moo b.m on b.m won o.m 000nm m.v on v.m con o.m Ono o.m Ono m.N v.olo.m woo m.v mm m.m one m.N non o.m 000nm o.v Ono N.N on m.N m o.m no h.H m.mlm.m Dad m.N moo o.m no b.H QM m.H wcoflm o.¢ Unm m.N QM m.H m o.N no m.H «.mlo.m m 0.0 and m.N m m.o 0&6 m.N Conn h.m Ono v.N am m.H n6 N.N no v.H m.vum.v OnAHm>mH mm co>wo um Houucoo mazes mcwumu musnch vema mead mea vbma Mbma tha whoa mnma mea macaw NH.H vm.o mm.o 3&9: 59323: as m.mmcwumu audflcw Hmsmw> an consumes .pamfim on» CA cuoo ou >uaowxouou>nm senonwuuoa so an Hwom mo uoommm one .m manna 56 Table 4.--The effect of soil pH on metribuzin phytotoxicity to corn in the field, measured in grain yield (kg/ha).a Metribuzin (kg/ha) pH Range 1972 1974 1972 1974 1972 1974 (% of control) 4.5-4.9 92 be 93 d 88 bc 88 cd 104 c 84 bed 5.0-5.4 86 be 88 cd 80 b 90 Cd 86 be 81 bcd 5.5-5.9 90 be 86 cd 85 bc 79 bed 57 a 77 bed 6.0-6.4 87 be 74 be 80 b 77 bed 57 a 69 b 6.5-6.9 81 b 81 bcd 57 a 69 b 46 a 54 a a . . . Means of each year's data with Similar letters are not significantly different at the 5% level by Duncan's multiple range test. 57 .0cuucsam nouns nucoe mco ocsum was Moow> so moss mcfiusu wasncHo .Haux sumamfiou n 0H «anoncw o: u on .umou moss» mamwuass m.csocso >n Ho>ma mm onu us ucoummuuc haucsoHuficmwm uoc mus muouuoa uusEAm nufl3 suso m.uso> cuss mo mesmzs m m.m £ 0.m o m.m mm m.N 30M 0.v o m.m mo 0.N wvon N.N ons m.H 0.0Im.0 mm e.~ some m.m on A.~ use o.m new o.e one m.H been m.H messes m.~ one e.H e.suo.e mmco m.H no N.¢ on 0.N moon m.H nmmmv m.m on 0.N moon m.H Uns h.H 03s v.H m.mlm.m moon v.H nmmmou m.m as 0.a Uns 0.0 SOHO m.m 09s m.H Gas 0.0 as H.H ons m.H v.mlo.m s 0.0 cons 0.N s m.0 ns v.0 mmvun m.N 09s m.H Dons 0.0 s m.0 ens m.H m.vnm.v onAmmcsu mm cs>w0 us Houucoo macwa onwusu SunnsHv «had mead tha vsma mhma whoa vhma mnma tha NH.H em.o em.o modem AeE\mxs euusnuuumz mm s.m0cuusu Susana HsSma> an omusmsoe .cHsum ecu cw csOQSOm ou huaoaxououhnm :uNanHuoE so an Hwom mo uoommm s:9nu.m mansa 58 onu us ucmuoumuo maucsouuACOAm uo: mus muouuoa usHfiEHm sues suso m.uso> nosm mo mcsmzs .umou omcsu mamuuasa m.csocso an Hm>ma wm e e.ee e ~.ee e ~.me be H.em e m.oe be 0.0» gm m.mm e m.me be H.Nm m.b-m.e be e.mm e m.se be e.~e be e.mm e o.Hm be m.me be m.om e e.Am be m.mm e.e-e.e be e.ee e 3.0m be m.oe be m.oe e m.Hm be e.ee one m.~m e e.mm be m.~m m.m-m.m be o.me e H.mm be e.mm on e.ee e m.me be e.me one e.em e m.em be N.em e.muo.m o e.eos e m.me n m.sos on m.em e e.em be m.sm on m.em e e.mm be e.mm m.e-m.e Aaouucoo no we sees meme News ends meme Need ends meme meme deems ~A.A em.o em.o Aeexmxv euusnuuumz mm e.1ee\mxs sums» ensue es omusmsoe .0HOAM mnu cw cswn>0m Ou wuuouxououxnm cuusnwuuoe :0 mm Hflom mo uommmo OEBII.0 passe 59 Fig. l.--Weed control with metribuzin at two soil pH levels. Treatment (A) check (B) metribuzin 1.12 kg/ha at pH range of 6.5 to 6.9 and (c) metribuzin 1.12 kg/ha at pH range of 4.5 to 4.9. 61 .m.e ou m.e seems we us semen and use m.e ou m.v mo omcsu mm us sn\mx NH.H cussnwuume A00 m.m ou m.m smcsu mm xomno Amy m.m ou m.o mo smash mm us sn\mx NH.H sunsnwuuoe Adv ucoausoua .mas>sa mm Hwom asum>mm um>o csOQMOm ccs :uoo ou wuuouxououwnm causnfluumzln.m .mum 63 .SHm>Auoedmeu .e.e o» m.e see .e.m 0» e.m .m.m ou m.m .m.m ou 0.0 .m.o ou m.w mo mmmcsu mm cues sn\0x NH.H Canonfluuse Amy was .Amv .on .AUV .Amv m.w ou m.m omcsu mm us xomno Adv ucmEussuB .mHo>mH mm Huom asum>om us csmnhom was cuoo ou muuouxououwnm awusnuuum2u1.m .mum m Fig. 65 Fig. 4.--Corn injury (rating 0 = no injury; 10 = complete kill) minus control at given pH range. Metri- buzin applied at 1.12 kg/ha in 1972. 66 10 Y=-l3.96+3.14x r = 0 . 9 7 1 "‘ * 8 z 6 x/ I: ll] . .5. - x" s 4 "I <3 1’;’ o ,I’ 2 , " II III M 4.5 5.5 6.5 S o i l p H Fig. 4 67 Fig. 5.--Corn yield as percent of control. Metribuzin applied at 1.12 kg/ha in 1972. Yield as 96 of control 100 80 60 4O 20 Fig. 5 68 \ . Y=250.0'32.2X \\ r=-o.977M \ \ \ \ \I \ \ ‘\ ‘\ \ \ \. \ \. \\. \ \ . \ \. \ \ \ I \ \ \\. \ 4.5 5.5 6.5 Soul pH 69 showed significant differences between pH ranges at 1.12 kg/ha. Corn yields were not taken in 1973 because of dry conditions during silking and pollination, which resulted in the lack of ear development. Corn yield for 1972 as percent of control gave a negative correlation with in- creasing soil pH (Figure 5). Significant differences occurred between the high and low pH ranges at 0.84 and 1.12 kg/ha (Table 4). Soybean injury (Table 5) was sig- nificantly different at 0.56 kg/ha in 1973 and 1974. Soy- bean yield showed significant differences from the high and low pH ranges in 1972 and 1974 at 1.12 kg/ha (Table 6). Number of dead corn and soybean plants increased and plant height as percent of control decreased with in- creasing soil pH (see Appendix A to F). Soybeans more readily overcame injury seen earlier in the growing season than corn as indicated by final grain yield. There appeared to be a dividing line at pH range 5.0 to 5.4, between slight injury and severe injury at the 1.12 kg/ha rate (Figure 3). This was apparently the effect of soil pH on the adsorptibn of metribuzin on soil colloids. It can be postulated that a reduction in herbi- cide concentration resulting from adsorption by soil colloids resulted in less absorption by plant roots below this pH range. Metribuzin activity continued to increase with increasing soil pH. Surface acidity is probably the most important property of the soil or colloidal system in determining the extent and nature of adsorption and 70 desorption of basic organic compounds (4). Bailey 33,31. (5) found the surface acidity of montmorillonite appears to be three to four pH units lower than the pH of the bulk solution. As the pH decreases, the adsorption of the protonated gftriazine molecule was attributed to cation exchange and to complexing of the 3-triazine molecules with hydrogen ions on the clay surface (24). Weber (23) found that maximum adsorption of 13 3-triazines occurred in the vicinity of the dissociation constant of each compound. Frissel and Bolt (10) found for the adsorption of chloro- 3-triazines, however, that about 3 to 4 pH units above the dissociation constant adsorption started to increase as pH decreased down to pH one. The pKa of metribuzin was found to be 0.99 i 0.08 (see Chapter 3). The same mechanism of protonation and adsorption by the soil col- loids appears to be taking place with metribuzin as seen with the 3-triazines. Greenhouse data on phytotoxicity to corn and soybean grown in a nutrient-sand culture versus soil at comparable pH levels supported the field data. Corn and soybeans grown in soil and treated with metribuzin showed a pH-sensitive response with a reduc- tion in dry weight per plant as pH increased. Whereas, in sand culture there was no difference in the reduction in dry weight as pH increased (see Chapter 3). me a: S ‘I éu he 71 Effect of Soil pH on Metribuzin DissipatiOhCin the Field Metribuzin residue analysis of soil samples from the field showed greater amounts of residue extractable at soil pH 6.7 than 4.6 (Tables 7 and 8). The increase in residue may be due to less adsorption by the soil. The data in Table 7 also shows that as the pH increased leaching also increased, most likely due to the higher concentration of unbound metribuzin at the highest soil pH value. Most of the metribuzin was found in the upper 5 cm of the soil profile. Extraction efficiency of metribuzin from soil standards at 0.1 ppmw were 95% at pH 6.7, 87% at pH 5.6, and 63% at pH 4.6. In 1973 and 1974 extraction efficiency was found to also increase as soil pH increased in the field plots. Leaching of metribuzin after 7.1 cm of rainfall in 1973, increased with increas- ing soil pH. This is an indication of increased adsorp- tion as pH decreased. Metribuzin dissipation followed pseudo first- order kinetics (Tables 7 and 8) which is an approximation of first-order kinetics (9). These results were similar to Hyzak and Zimdahl (14). Data was plotted as first-order plots relating the log of soil concentration (ppbw) of metri- buzin at soil pH levels 4.6, 5.6, and 6.7 at 0.0 to 5.0, 5.0 to 10.0, and 10.0 to 20.0-cm depths versus time (Figures 6, 7, and 8). Linear regression analysis indi- gated that the mean data points fit two lines, high 72 Table 7.--Effect of soil pH on metribuzin residue in soil, following application of metribuzin (1.12 kg/ha) in the field.a Soil concentration of metribuzin Days after app1ication PH Depth (cm) 1973 1974 15U 36C 150“, 3e 21‘ 1429 (PpbW) 4.6 0.0-5.0 191 e 48 be 17 a 671 b 44 a 21 a 5.0-10.0 67 c 23 ab 15 a 31 a 18 a 19 a 5.6 0.0-5.0 236 f 49 bc 16 a 986 c 37 a 23 a 5.0-10.0 116 d 33 ab 16 a 42 a 16 a 21 a 6.7 0.0-5.0 298 g 73 c 17 a 1143 d 66 a 21 a 5.0-10.0 192 e 30 ab 16 a 23 a 16 a 21 a aMeans of each year's data with similar letters are not significantly different at the 5% level by Duncan's multiple range test. Rainfall since application: b7.1-cm, Cll.9-cm, d34.0-em, e0.3-cm, 4.4-cm, g29.0-em. 73 00H OH x m0.m mmm.l hm 0a x 00.H Nam.l 0.0N00.0H MI NI NNH mnoa x 00.m Hmm.| ma mica x v0.m mmw.u 0.0Huo.m mm Nuoa x m~.H vvm.| 0H NIOH x mh.m mmm.u o.mno.o v.0 mma muoa x 00.m mom.n we muoa x mv.a mmh.u o.o~uo.o~ we mnoa x mo.m mmm.u 0m Nuoa x H>.~ 000.: 0.0Huo.m on m 0a x om.m 0mm.| om Nioa x mm.m nmm.| o.muo.0 0.m and muoa x vo.v 0mm.| m0 Nuoa x HH.H vam.u o.o~uo.oa 00H mnoa x em.m mmm.n vm muoH x Ho.~ mmm.| o.oHuo.m vs mnoa x mm.m ovm.| Hm NIoH x mm.m «mm.l o.muo.o 0.¢ Amasov Annmsov any mosHs> Ammsov Aalhscv any mosas> «\H u x cowusaouuou N\H u x coHusHsHuoo A800 numoo mm OmH ou on he ou ma coAusomems nouns muse .mnma .oasum snu cw sn\0x NH.H mo cowusouamms mausOAHom .msumsc sounu us was mHs>sH mm songs as Hues cu duusbuuuos no x~\u as mo>uu use: .13. assumeoo oueu nosuouuuuuu .mm nuu3 Huom ecu cu mcucusesu nodusuucsocoo mo 00H snu mo Ans mssHs> cowusasuuooln.m oansa 74 Fig. 6.--Metribuzin degradation rate as the log of con- centration in soil at pH 4.6 at three soil depths following application of 1.12kg/ha in the field in 1973. 1“ o 1.5 Soil concentration of metrlbuzln (ppbw) 1.0 Fig. 6 75 pH 4.6 Depth (can) —-a-—-—— 0.0 - 5.0 # 5.0 - 10.0 —"-.'- 10.0 - 20-0 Time (months) 76 Fig. 7.--Metribuzin degradation rate as the log of concentration in soil at pH 5.6 at three soil depths following application of 1.12 kg/ha in the field in 1973. P o 3.0 2.5 2.0 Soll concentration of metribuzin (ppbw) ." an .A 0 Fig. 7 77 Depth (cu) “"""-‘- 0.0- 5.0 —0—-o— 5.0-10.0 """""" 10.0-20.0 V \\~ \ s '\ \. \ \. '\ \ ‘\ \\~ 0 ‘\ \\\ 5 --- ~n“‘:§‘ - “‘“~ \\ “\‘ s ‘\ “\ . ~~~ \ \“ ~~~\£ \“‘ “s~~ v ~~~~~ Time (months) 78 Fig. 8.--Metribuzin degradation rate as the log of con- centration in soil at pH 6.7 at three soil depths following application of 1.12 kg/ha in the field in 1973. Fig. 3 4.0 a. .9 .E g 3.0 :2 f; 2.5 E '6 2.0 C .2 E g 1.5 0 C o 0 '3 m 1.0 8 O 79 pH 6.7 Depth (cm) c-ah-u-h— 0.0- 5.0 +‘— 5.0-10.0 -‘--¢-- 10.0-20.0 Time (months) 80 correlation was obtained when two lines were drawn to repre- sent the rate of degradation (Table 7). Rate constants increased and the half life of metribuzin decreased as soil pH increased. Rate constants decreased and the half life of metribuzin increased at all soil pH levels as depth of sampling decreased (Table 8). There was no indication of a lag period for metribuzin at any of the pH levels or depths of sampling. Hyzak and Zimdahl (14) found that the pattern of degradation of metribuzin and two analogs in the field fit one straight line. However, Figures 6 to 8 indicate that metribuzin dissipation may best be represented by Rate constants three or more straight line correlations. and half lives observed are dependent on several factors. The values obtained are dependent on rainfall, depth of Sampling, time and soil pH (Table 8) ., Caution should be used in extrapolating degradation patterns and predicting This value may not half lives and the kinetic rate law. be constant, and will change over time, as indicated \ above, The decreased rate of metribuzin degradation as soil pH decreased is very likely due to increased adsorp- tion - Hyzak and Zimdahl (14) reported that decreased 1:- ate of degradation of metribuzin and two analogs below Several o - . 1 Ppm at 35°C may have been due to adsorption. 3L I:7.”"estigators have shown that the decomposition of organic 81 compounds is greatly altered when adsorbed on soil colloids (1,2,24,25). A slow release of metribuzin into the soil solution may have resulted in a reduced rate of degradation by soil microorganisms. Schumacher1 reported the 33- triazine ring appeared to be broken down in a stepwise fashion and C-3 was released as 14CO2 at a faster rate than C-5 of the 33-triazine ring. This would suggest that ring cleavage and subsequent metabolism occurred second- arily to ring substituent metabolism. If substituent metabolism process was significant in soil and necessary for ring cleavage and subsequent metabolism, then it is likely that the rate of substituent metabolism and thus degradation may be reduced if metribuzin was protonated and adsorbed to soil colloids in acid soils. Acknowledgment The authors express their appreciation to Chemagro Corporation for supplying the radioisotope and technical herbicide used in this investigation. Ibid. 10. Literature Cited Alexander, M. 1964. Biochemical ecology of soil microorganisms. Ann. Rev. Microbial. 18:217-252. Alexander, M. 1965. Persistence and biological reactions of pesticides in soils. Soil Sci. Soc. Amer. Proc. 29:1-7. Bailey, G. W., and J. L. White. 1964. Review of adsorption and desorption of organic pesticides by soil colloids, with implications concerning pesticide bioactivity. J. Agr. Food Chem. 12: 324-332. Bailey, G. W., and J. L. White. 1970. Factors influencing the adsorption and movement of pesti- cides in soil. Residue Review 32:29-92. Bailey, G. W., J. L. White, and T. Rothberg. 1968. Adsorption of organic herbicides by montmoril- lonite: Role of pH and chemical character of adsorbate. Soil Sci. Soc. Amer. Proc. 32:222-234. Best, J. A., and J. B. Weber. 1974. Disappearance of 3-triazines as affected by soil pH using a balance-sheet approach. Weed Sci. 20:364-373. Best, J. A., J. B. Weber, and T. J. Monaco. 1971. Effect of liming on the activity of soil-applied triazines. Proc. Southern Weed Sci. Soc. 24:368. Corbin, F. T., and R. P. Upchurch. 1967. Influence of pH on detoxication of herbicides in soil. Weeds 15:370-377. Fleck, G. M. 1971. Chemical reaction mechanisms. Holt, Rinehart and Winston, Inc. New York. 235 pp. Frissel, M. J., and G. H. Bolt. 1962. Interaction between certain ionizable organic compounds (herbicides) and clay minerals. Soil Sci. 94: 284-291. 82 ll. 12. 13. 14. 15. l6. 17. 18. 19. 20. 21. 22. 23. 83 Gysin, H. 1962. Triazine herbicides, their chemis- try biological properties and mode of action. Chem. Ind. (London), 1393-1400 pp. Hartwig, N. L. 1974. Soil and soil pH effects on the activity of some triazine herbicides. Ag. Lime Digest 6:3-4. Hawck, R. D., and H. F. Stephenson. 1964. Nitrifi- cation of triazine nitrogen. J. Agr. Food Chem. 12:147-150. Hyzak, D. L., and R. L. Zimdahl. 1974. Rate of degradation of metribuzin and two analogs in soil. Weed Sci. 22:75-79. Kaufman, D. D., and P. C. Kearney. 1970. Microbial degradation of 3-triazine herbicides. Residue Review 32:235-265. Leefe, J. S. 1968. Effect of soil pH on the phyto- toxicity of simazine to strawberries. Can. J. Plant Sci. 48:424-425. Nearpass, D. C. 1965. Effect of soil acidity on the- adsorption, penetration, and persistence of simazine. Weeds 13:341-346. Rogers, E. G. 1968. Leaching of seven 3-triazines. Weed Sci. 16:177-120. Savage, K. E. 1974. Adsorption and mobility of metribuzin in soil. Weed Sci. Soc. Amer. Abstr. P. 120. Scott, H. D., R. E. Phillips, and R. F. Paetzold. 1974. Diffusion of herbicides in the adsorbed phase. Soil Sci. Soc. Amer. Proc. 38:558-562. Talbert, R. E., and O. H. Fletchall. 1965. The adsorption of some 3-triazines in soils. Weeds 13:46-52. Waggoner, T. B., C. A. Anderson, J. A. Ciarletta, and T. J. Olson. 1974. Degradation of metribuzin in soil and effects on rotational crops. Weed Sci. Soc. Amer. Abstr. P. 121. Weber, J. B. 1966. Molecular structure and pH effects on the adsorption of 13 3-triazine com- pounds on montmorillonite clay. Amer. Mineral 51:1657-1670. 84 24. Weber, J. B. 1970. Mechanisms of adsorption of s-triazines by clay colloids and factors affect- Ing plant availability. Residue Review 32:93-130. 25. Weber, J. B., and H. C. Coble. 1968. Microbial decomposition of diquat adsorbed on montmoril- lonite and kalotinite clays. J. Agric. Food Chem. 16:475-478. 26. Wolcott, A. R., H. D. Foth, J. F. Davis, and J. C. Shickluna. 1965. Nitrogen carriers: I. Soil effects. Soil Sci. Amer. Proc. 29:405-410. CHAPTER 3 EFFECT OF SOIL PH ON METRIBUZIN PHYTO- TOXICITY, MICROBIAL DEGRADATION, ABSORPTION, AND MOBILITY Abstract Metribuzin [4-amino-6-tert-butyl-3-(methylthio)- l,2,5-triazin-5(4H)one] phytotoxicity to corn (Zea mays L.) and soybeans (Glycine max (L.) Merr.) increased with increasing soil pH. Microbial degradation decreased as the soil became more acid. Metribuzin and atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-3-triazine] mobility increased with increasing concentration and soil pH. 14C-metribuzin had greater mobility on soil thin- layer plates than l4C-atrazine because of differences in water solubility and basicity. Adsorption increased with decreasing soil pH. The pKa was determined to be 0.99 i 0.08. Adsorption increased gradually as the soil pH decreased toward the pKa of metribuzin. Protonation of the amine group with subsequent adsorption to soil colloids was postulated to be the reason for the reduction in phytotoxicity, microbial degradation, and mobility of metribuzin at low pH levels. 85 86 Introduction Relatively little information is available on the behavior of the 33-triazines in soil as compared to the 3-triazines. The rate and extent to which a compound is adsorbed and subsequently desorbed may govern its biological tox- icity. Adsorption of the 3—triazines by soil colloids is dependent on the pH of the system, and many studies with plants have shown that the 3-triazines are less phytotoxic under acid soil conditions than under basic conditions (22). Talbert and Fletchall (19) found that an increase in pH resulted in the decreased adsorption of simazine [2-chloro-4,6-bis(ethylamino)-3-triazine] and atrazine by soils. McGlamery and Slife (16) found that atrazine adsorption onto soil humic acid isolated from leonardite increased as pH decreased. Surface acidity is probably the most important property of the soil or colloidal system in determining the extent and nature of adsorption and desorption of basic organic compounds (6). Bailey 33 31. (5) found the surface acidity of montmorillonite appears to be three to four pH units lower than the pH of the bulk solu- tion. As the pH decreases, the 3-triazine molecule becomes protonated. Adsorption at pH levels in which the compounds were in the cationic form was attributed to cation exchange and to complexing of the 3-triazines 87 molecules with hydrogen ions on the clay surface (22). Weber (23) also showed that the pH of the system and the molecular structure of the 3-triazines influenced their adsorption on organic soil colloids in a matter analogous to their adsorption on clay minerals. Adsorption of the compounds was attributed to the complexing of the 3- triazines molecules by ionizable H+ ions on functional groups of the organic colloids and adsorption of the pro- tonated species by cation exchange. Soil microorganisms are involved in the degradation of 3-triazine herbicides. Chemical hydrolysis is probably the major detoxification method of chloro-3-triazines in soil systems, whereas methylthio-3-triazine detoxication is believed to be dependent upon microbial action (13). In nearly all cases only low levels of 14C02 (0 to 4%) have been evolved from microbial systems treated with l4C-ring-labeled 3-triazines. This would indicate that the 3-triazine ring structure is fairly resistant to microbial attack (13). Hawck and Stephenson (10) sug- gested that the symmetrical resonating structure of the 3-triazine molecule contributed to differences in degra- dation rates. Asymmetrical chloro-triazines were degraded inore quickly than the corresponding symmetrically- substituted chloro-triazines. They suggested that poly- inerization resulting in an increased symmetry would lead to stability, whereas labile products of biochemical 88 transformation tend toward asymmetry. Schumacher1 14C-ring-labeled metribuzin was degraded to CO2 in soil. CO2 evolution from 14C-3 and 14C-5 reported 14 metribuzin applied soils indicated that the 33-triazine ring was degraded in a stepwise manner and C-3 was released more rapidly than was C-S. This would suggest that ring cleavage and subsequent metabolism occurred secondarily to ring substituent metabolism. Lay and Ilnicki (15) used an oat (Avena sativa L.) and cucumber (Cucumis sativus L.) bioassay and observed a 90% loss of metribuzin phytotoxicity in 42 days from a Sassafras loam soil. Hyzak and Zimdahl (12) reported the half-life of metribuzin in sandy loam soil to be 46 days at 20 C and 16 days at 35 C. The loss of metribuzin was linear at both temperatures. Soil pH may directly or indirectly influence the detoxication of herbicides by affecting the ionic or molecular character of the chemical, the ionic character of soil colloids, the cation exchange capacity, and the inherent capacity of the microbial population to respond to a given chemical (7) . The order of decreasing mobility of seven 3- tlriazines seems to coincide with increasing adsorption 1Schumacher, R. W. 1974. Metabolism of metri- buZLin in soybeans and soil. Ph.D. Thesis. University of Kentucky, Lexington, Kentucky. 76 pp.« 89 (5,18,19). Nearpass (17) found the mobility of simazine was greater in limed than unlimed soil and it was sug- gested that decreased adsorption occurred at increased pH. The objectives of this study were to determine the effect of soil pH on metribuzin (1) activity, (2) microbial degradation, (3) adsorption to soil, and (4) mobility in soil, and to determine the dissociation con- stant of metribuzin and show a relationship to adsorption. Materials and Methods Phytotoxicity Greenhouse studies were conducted to compare the activity of metribuzin on 'Wirth' soybean and 'Michigan 400' corn grown in soil and nutrient-sand culture at five pH levels. Soil was collected from a field site of Hillsdale sandy loam (Table 1), East Lansing, Michigan, containing 1.5% organic matter and with a pH which had been adjusted over a lS-year period with ammonium sulfate, calcium nitrate or calcium carbonate to 4.6, 5.1, 5.6, 65.1, and 6.7 (24). The pH levels in nutrient-sand culture “were adjusted to those of the field. The soil mechanical analysis were similar at all five pH levels. This soil was; also used for the study of the pH effect on microbial degradation, adsorption, and mobility of metribuzin. Seven soybean and corn seeds were planted in wastzead No. 7 graded quartz sand and soil in 295-ml paper 90 umsm .>uousuonsq .mao>oH mm m>ww Has us usawaum ms3 mumxascs aspecscooe Haom era a .csmflcouz .mcumcsq mcfiumme Huom >uumuo>uc0 musum cs0u£OHz onu >n cmeuouumm mammascs Hwoms ea ee me mm oee mos ems e.e m.H om ee em me oem mm was H.e o.H ma mm em oe ooe mm so e.m e.s m em mm owe em em mes H.m e.u 0H om mm on om mm mes o.e m.H mm mm mm esoH Seas useem mussmaaum Hom we sz m2 so s A mm so Esau uuum seem musuxme moss Huom 3mm nucwouom .cs0HLOHz .mcflmcsq umsm M .HAOm oascmaaum mo wouuuomoum Hsowsono was HsOHmwnmll.H manse Hi (1' I) 'f“ '2!" 91 cups These paper cups with drainage holes in the bottom were placed in plastic 295-m1 cups to allow for subirri- gation by daily additions of nutrient solution or dis- tilled water to promote optimum growth. The nutrient solu- tion was Hoagland's No. 1 solution, with pH levels adjusted to that of the soil. A preemergence application of metribuzin at 0.28, 0.56, 0.84, and 1.12 kg/hg at 935 L/ha and 2.11 kg/cm2 pressure was applied to the soil and nutrient-sand culture. Containers were then placed in the greenhouse at 20 to 30 C with natural illumination supplemented with fluorescent lighting. Plants were thinned to five in number after emergence. After 30 days, height, fresh and dry weight of the soybean and corn plants were measured. All treatments were arranged in a completely randomized design with four replications. The data presented are the means of two experiments, expressed as percent of control. Microbial Degradation and __392 Release Metribuzin degradation in the variable pH soils W113 investigated in the following way. Soils from the fireld test plots were collected in August and a 25 g Portion of soil was placed in a 300-ml flask. The soils 4 received 5 m1 of 6.3 x 10— M NH4NO while the sodium 3 aZine (NaN3) treated soils received 5 ml of 6.3 x 10-4 M NH4N03 plus 3.9 x 10"2 M NaN3 per flask. The sodium 92 azide treatment approximates sterilization without alter- ing the soil physical or chemical properties. A l-ml 14C-metribuzin (.0256 uCi; Sp. portion of radioactive. act. = 1.5 mCi/mM) was added to each flask. A 2-ml plastic beaker containing 1-ml of l M KOH was suspended in the flask to collect the 14C02. The flask was sealed with a rubber stopper and placed in a room at 25 i 2 C under low light intensity. The flasks were sampled at one week intervals for 12 weeks after treatments. At sampling time, the content of the 2-ml beaker were placed in a vial containing 15 ml of 1:1 (v/v) CAB-O-SILR solution:60 g naphthalene, 4 g PPO. 0.2 g POPOP, 100 ml methanol, 20 ml ethylene glycol filled to a liter with p-l,4-dioxane. The contents of the vials were shaken to assure even distribution and radio-assayed by scintil- lation spectrometry. The treatments were replicated two times in a completely randomized design. The data pre- sented are the means of two experiments. _23termination of the Dissoci- Ifltion Constant (pKa) The dissociation constant of metribuzin was determined spectrophotometrically (1,21) . The pH meter Vfiis standardized with buffers at pH 4, 7 and 10 at room temnperature. A 125-ml portion of 10'4 M solution of (953-100% purity) technical grade metribuzin was titrated frCJm.pH 5.5 to pH 0.0 with 6.0 N HCl and from pH 5.5 to pH for The of use ti< so ad 80 cc 24 re CE 36 5C 41 93 pH 12.0 with l N NaOH. The analytical wavelength used for determination of the isoelectric point was 264 nm. The isoelectric point was determined by finding the point of most rapid change in optical density and through the use of the equation for determining pKa (3). The titra- tion was repeated several times with similar results. Adsorption Study Adsorption of 14 C-metribuzin (sp. act. = 1.5 mCi/mM) was determined using slight modifications of the adsorption procedure developed by Talbert and Fletchall (19). With the soil previously air-dried and later screened through a 1 mm sieve, one gram was weighed into a 15-ml centrifuge tube and 5 ml of 0.0112 uCi/S ml solution of 14C-metribuzin was added. The solution added to the test tube had been adjusted to the pH of the soil. The tube was stoppered with a polyethylene- covered rubber stopper. Each sample was equilibrated for 24, 72, 144, and 288 hr at 25 C in a horizontal shaker reciprocating at 120 strokes per minute. The sample was (Zentrifuged at 12,000 x g for 20 minutes. A 0.5 ml Siiimple of the supernatant was removed and placed in 15 1W1 of a scintillation solution consisting of 0.1 g of 1r‘1-313_[2-(4-methyl-5-phenyloxazoyl)]-benzene (dimethyl-POPOP) , 5.0 g of 2,5-diphenyloxazole (PPO) , 50 'g of naphthalene, 380 ml of tolulene, 380 ml of 1,4- di<'>xane, and 240 ml of absolute ethanol. The samples 94 were counted for 10 minutes and metribuzin adsorption was determined by comparing the radioactivity in a sample with soil to a sample of solution without soil (79.4 nM 14C-metribuzin/test tube). The difference was considered to be the quantity of herbicide bound and was expressed as a percent of 14C-metribuzin adsorbed. The effect of soil pH on adsorption of metribuzin was also studied by Soxhlet extraction and gas chromato- graph analysis (12). The same soil used in the previous adsorption studies was used. Herbicide standards were prepared by dissolving the analytical grade metribuzin (99 to 100% purity) in distilled acetone. Fifty grams of soil, pH levels 4.6, 5.6, and 6.7, were placed in beakers and 5 ml of acetone solution were uniformly pipetted over the soil surface. The herbicide was thoroughly mixed with the soil to obtain a uniform con- centration of 1.0 and 0.1 ppm, w/w basis. The soil and herbicide were allowed to equilibrate for 48 hours. The 50 grams of soil were extracted in a Soxhlet apparatus ‘Vith 200 m1 of 80% methanol for five hours with a Iflacycling time of approximately five minutes. After BXtraction, 25 ml of distilled water was added to the eXtractant and the solvent was evaporated to 20 to 30 ml. The: aqueous phase was extracted with one 50 ml and two 25 rnl aliquots of glass distilled benzene to bring the finiil volume to 100 ml. The aqueous-benzene mixture 95 was vigorously shaken for 10 to 15 seconds and allowed to separate into its respective phases. Thirty grams of anhydrous sodium sulfate was added to each bottle. The aqueous phase was drained into a beaker and discarded. The samples were then placed in the refrigerator until analysis. Herbicide concentration in the benzene extracts was determined with a gas chromatograph equipped with a Beckman helium arc discharge electron-capture detector. A 183 cm by 2 mm (I.D.) glass column was packed with 80/100 mesh Chromosorb Gas-Chrom Q coated with 3% SE-30. Injector, column, and detector temperatures were 250, 190 and 310 C, respectively. Helium at a flow rate of 40 ml/min was used for the carrier gas. Retention time of 1.85 min was obtained. The range of linearity was from 0.05 ng to 5 ng, and the detection limit of the method was 0.01 ppm in soil. Herbicide content was determined by comparing heights of sample to peak heights of known standards. A series of standards of different concentrations of herbi- cide was injected to determine the range of linearity and sensitivity before, during, and after each day's run of samples. Egbility on Soil Thin- L-jlyer Plates The procedure of Helling (11) was used for making soil thin-layer plates using a standard thin-layer 96 chromatographic spreader. Soil collected from the field at variable pH levels was air dried and screened through a 500 um screen. Duplicate 20 by 20 cm plates were coated with a 500 um thick layer of soil at pH levels 4.6, 5.1, 5.6, 6.1, and 6.7. The plates were allowed to dry and then spotted 2 cm from the bottom with 0.005 and 0.10 l4C-metribuzin and 14C-atrazine in 10 ul of solu- UCi of tion. The plates were allowed to equilibrate for 30 minutes. The lower 5 mm of each plate was then submerged in water and allowed to chromatograph ascendingly to a distance of 15 cm above the origin. The plates were air- dried for 24 hr and exposed to Kodak no-screen medical x-ray film for 14 days. The frontal movement, or Rf values were calculated from the radioautographs. After radioautography, consecutive 0.5-cm zones were removed. Direct liquid scintillation radioassay of 14C-labeled herbicide in soil was used (14). The data presented are the means of two experiments replicated twice. Results and Discussion lgffect of Soilng on Metribuzin .Ehytotoxicity Metribuzin phytotoxicity on corn and soybeans :hicreased with increasing soil pH (Tables 2 and 4). Whereas, in a nutrient-sand culture adjusted to com- Palnable soil pH levels, there was no reduction in plant dry: weight as pH increased. The nutrient-sand culture 97 showed only a herbicide rate response (Tables 3 and 5). However, in soil with the same rate of metribuzin at soil pH levels 4.6, 5.1, 5.6, 6.1, and 6.7 phytotoxicity increased as soil pH increased (Tables 3 and 5). There was significant difference between the high and low pH levels. Figure 1 shows the effect of soil pH on metribuzin phytotoxicity to soybeans. The parameters of plant height and fresh weight also showed the sOil pH response. Similar results were observed in field studies (see Chapter 2). The lack of plant uptake of metribuzin at low soil pH levels could be due to rapid microbial degradation, adsorption to the soil, lack of plant absorp- tion, or several of these factors. Effect of Soil pHAon Microbial Degradation and "C0, Release 14C-S-ring-labeled metribuzin was readily degraded in the soil over a lZ-week period (Table 6). Treatment of the soil with sodium azide drastically reduced the evolution of 14CO2 release from the soil (Figure 2). Degradation of metribuzin increased with the increasing soil pH (Figure 3). All soil pH levels were significantly diqfferent from each other. Figure 4 shows a highly Significant positive correlation of percent 14C evolved as 14 14C evolved as 14C02 C02 on soil pH. The percent of at pH 4.6 and 6.7, 3 sodium azide, was 4.6 versus 0.4 and 17- 9 versus 1.1 respectively. The reduction in evolution 98 Table 2.--Effect of soil pH on metribuzin phytotoxicity to corn as measured by reduction in dry weight of l-month-old corn plants. Metribuzin (kg/ha) P“ 0.28 0.56 0.84 1.12 (% of control)a 4.6 93.0 j 78.9 ghi 82.0 ghij 61.3 cde 5.1 91.1 ij 76.3 fgh 66.0 def 56.9 bcd 5.6 86.1 hij 70.9 efg 59.3 bcde 53.1 bcd 6.1 81.5 ghij 64.4 def 54.9 bcd 47.0 ab 6.7 71.9 efg 60.4 cde 49.5 abc 39.9 a aMeans with common letters are not significantly different at the 5% level by Duncan's multiple range test. Table 3.--Effect of pH on metribuzin phytotoxicity to corn grown in a nutrient-sand culture, measured as reduction in dry weight of 1-month-old corn plants. H Metribuzin (kg/ha) p 0.28 0.56 0.84 1.12 (% of control)a 4.6 43.9 fg 34.5 cde 26.5 abc 23.9 ab 5.1 42.4 efg 30.0 bcd 21.8 ab 22.1 ab 5.6 46.1 g 24.5 ab 21.3 ab 20.8 a 6.1 34.5 cde 26.3 abc 24.0 ab 23.3 ab 6.7 36.1 def 28.5 abcd 22.4 ab 19.9 a , aMeans with common letters are not significantly dlffelreent at the 5% level by Duncan's multiple range test. 99 Table 4.--Effect of soil pH on metribuzin phytotoxicity to soybeans as measured by reduction in dry weight of 1-month-old soybean. H Metribuzin (kg/ha) P 0.28 0.56 0.84 1.12 (% of control)a 4.6 101.81 i 96.6 hi 87.4 gh 73.0 ef 5.1 95.5 hi 96.8 hi 86.8 fgh 64.3 cd 5.6 85.4 efgh 80.4 efg 71.3 de 48.0 b 6.1 95.5 hi 78.3 defg 57.0 bc 46.3 b 6.7 75.0 defg 71.6 de 53.5 be 24.3 a aMeans with common letters are not significantly different at the 5% level by Duncan's multiple range test. Table 5.--Effect of pH on metribuzin phytotoxicity to soy- beans grown in a nutrient-sand culture, measured as reduction in dry weight of l-month-old soy- bean. pH Metribuzin (kg/ha) 0.28 0.56 0.84 1.12 (% of control)a 4.6 54.5 c 20.4 b 19.9 b 14.6 ab 5.1 56.9 c 17.8 ab 17.6 ab 11.9 ab 5.6 48.8 c 16.8 ab 11.1 ab 8.8 a' 6.1 55.3 c 18.8 ab 13.4 ab 12.5 ab 6.7 54.4 c 17.4 ab 11.9 ab 11.3 ab aMeans with common letters are not significantly different at the 5% level by Duncan's multiple range test. 100 Fig. 1.--Soil pH effect on tolerance of soybeans to metri- buzin (0.00 and 0.56 kg/ha). Soil pH from left to right (A) 4.6 (B) 5.1 (C) 5.6 (D) 6.1 and (E) 6.7. lOl I ‘ V ~ ‘ V , l ' l D. E SE NCOR _ ; 0.0 KG/HA .‘s 4%“ I SENCOR (a 2 ' 0.56 KG/HA 102 Table 6.--Metribuzin degradation to CO in 12 weeks, as affected by soil pH and sodium azide. pH % 14C evolved as 14C02b 4.6 4.6 c 4.6 NaN3 0.4 a 5.1 V 8.6 d 5.6 10.2 e 6.1 13.5 f 6.7 17.9 g 6.7 NaN3 1.1 b aMeans within columns followed by similar letters are not significantly different at the 5% level by Duncan's multiple range test. The sodium azide treatment approxi- mates sterilization. b0.0256 uCi/flask. 103 UmamanlmIU «a .oownm EdwoOm.H h.m can w.¢ mm um moova ou Canonwuume mo cofiumomummo may no woman EdwoOm can mm HflOm mo poomwmun.m .mam 104 «8.02.: 0.5.— N .6: Nu «a Gd 0 C h C I Q Q N a 9 I I I l I IleII Illa,. i . . o "'I'||'|| "" \ as. s.- a- \ \ \ \ \ \ \ \ . \ . \ a v s x \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ \ ‘0 \ \ \ \ \ \ \\ \ \ \\ \‘ \\ \\ \\ \\ \\ \ Q: Cu in n so POA'OA. a"; 105 0» cflusnfluume nmamnmaumuu vH .5.6 6:6 .m.m .m.e am pm wooed mo codumomuomo on» so mm Haom mo uoowmmnu.m .mwm 106 Na «d o.— ..v a. 2 .. Ana—003v OE: h 0 n O u ‘03 I. .A 0A. 9! P I 39“ N a 0n 107 14 Fig. 4.--Positive correlation with soil of percent C evolved as 14C02 in 12 weeks. 00 f» . 25 3 N v- 5 20 801 $- 15 GD 0 '3 1o 2. O 3' ° 5 *0 ,. be Fig. 4 108 Y: 43.2 + 6.07 I' =+ 0593‘“ I)’ W 4.6 5 .1 5.6 6.1 6.7 3 0| L p H 109 of 14 C02 due to the addition of sodium azide, was signifi- cant at both pH levels. There are a number of reasons why metribuzin degradation may be reduced as the pH of the soil decreased: (1) metribuzin may have been bound more tightly to the soil and thus was less available to the organisms in acid soils, (2) variation in the micro- organism population over the pH range studied, and (3) a pH effeCt on microbial metabolism over the pH range studied. The most plausible explanation for the decreased rate of metribuzin degradation in acid soil, appears to be adsorption. Several investigators (2,3,12,22) have suggested that a substrate readily consumed by the soil microflora can be protected if it becomes adsorbed or deposited at microsites inaccessible to soil microorga- nisms. If the chemical hydrolysis process was signifi- cant insoil, then it is likely that the rate of hydrolysis and thus degradation may be reduced if Metribuzin is adsorbed to soil colloids (12). Determination of the Dissoci- ation Constant (pKa) Figure 5 shows the spectrophotometric determina- tion of the pKa of metribuzin at 264 nm and theresulting plot of absorbance versus pH. The pKa of metribuzin is 0.99 1.0.08 compared to atrazine with a pKa of 1.68 (22) and propazine with a pKa of 1.85 (22). Figure 6 shows the chemical structure of propazine and the proposed 110 .5: «mm on cHNoQHHqu mo mxm map mo coflumcHEumumo ofiuumfiouocmouuommm ch||.m .mE 0.7 PKo 09910.0! 111 "2 ‘1 “3 o O O OOUBQJOSQV 0.1 12 11 10 pH 5 Fig. 112 .Ammv coflumsvw coHumNHcoH Ummomoum map can mafinmmoum mo musuosuum HmoHEmnoul.m .98 113 m .35 ocfimoocd and “In 3... 114 ionization equation (22). Figure 7 shows the chemical structure of metribuzin and proposed ionization equation. The pKa of a compound indicates the degree of acidity or basicity that a compound will exhibit and therefore is important in determining both the extent of adsorption by and the ease of desorption from colloidal systems. There may be other possible ionization equations for metribuzin. Effect of Soilng on Metri- buzin Adsorption l4C-metribuzin increased with The adsorption of time and with a decrease in soil pH (Table 7). The dif- ferences between the five soil pH levels were signifi- cant. The pH of the soil had an influence on the extrac- tion efficiency of metribuzin from the soil (Table 8). There were significant differences between the three soil pH levels at both the 1.0 and 0.1 ppmw. A greater percentage of metribuzin was extracted at the higher con- centration (1.0 ppmw). As soil pH decreased, the percent- age of metribuzin extracted at both 1.0 and 0.1 ppmw decreased, indicating that more metribuzin was bound to the soil at the lower pH levels. The above findings of metribuzin activity in relation to soil pH are similar to those found with g: triazines. In an adsorption study of 13 related §7 triazines on montmorillonite, Weber (20) found that the 115 .coflumsvm cowumwacofl Ummoaoum may pom cHNanuumE mo musuoouum HmoHEmcunn.> .mE a .35 :oSoaoo co=o~_co_ oomoaoto ocm 52::me .o 0.32:? 3055.5 mOdH mad H at: 116 117 Table 7.--14C-metribuzin adsorption by Hillsdale sandy clay loam at various pH levels. Time after treatment (hr) pH 24 -172 144 288 Average I‘IC-metrlbuzin adsorgtiona (% adsorption) 4.6 15.6 f 17.8 21.1 27.1 20.4 5.1 12.4 Cd 13.0 18.5 21.9 16.4 5.6 10.4 b 11.8 16.8 20.8 14.9 6.1 9.9 ab 10.6 13.8 17.9 13.0 6.7 9.2 a 10.2 11.7 15.5 11.6 aMeans followed by similar letters are not signifi- cantly different at the 5% level by Duncan's multiple range test. - b79.4 nM/test tube. Table 8.--Extraction efficiency of metribuzin from Hills- dale sandy clay loam at several pH levels.a - b % extraction efficiency 8 011 pH 1.0 ppm 0.1 ppm 6.7 99.1 c 95.3 c 5.6 90.6 b 87.2 b 4.6 71.1 a 63.2 a aMeans within columns followed by similar letters are not significantly different at the 5% level by Duncan's multiple range test. bHillsdale sandy clay loam (Table 1). 118 maximum adsorption of all the compounds occurred at a pH in the vicinity of the dissociation constant of each compound. It has also been reported by Bailey (5) that the surface acidity of montmorillonite clay appears to be three to four pH units lower than the pH of the bulk solu- tion. The pKa of metribuzin has been reported here to be 0.99 i 0.08. Considering the surface acidity of clays and the pKa of metribuzin, one can postulate that similar mechanisms of adsorption are involved with metribuzin at low pH values as with the g-triazines. Frissel and Bolt (8) reported also, with the chloro-s-triazines, that at about three to four pH units above the dissociation constant adsorption started to increase as pH decreased. Effect of Soil pH on Mobility of Metribuzin and Atrazine Greater movement of metribuzin and atrazine, with the water front, was observed as pH and herbicide concentration increased on soil TLC plates. Table 9 shows the effect of concentration and pH on atrazine movement. There was a noticable increase in movement of metribuzin (Table 10) compared to atrazine (Table 9). Figures 8 and 9 show the quantitative movement of 14C-labeled herbicide with distance. Atrazine moved less and more remained at the origin compared to metribuzin. More atrazine was adsorbed at the origin at pH level 4.6 than at the higher pH levels (Figure 10) compared to 119 Table 9.--Effect of soil pH on the mobility of 14C atrazine on soil TLC plates. “w _.._ Rfa H . p 14C atra21ne uC1 ab 0.05 uCi 0.10 ' 4.6 .25 a .26 5.1 .27 b .27 5.6 .29 c .31 6.1 .31 d .32 6.7 .32 d .34 aMeans followed by similar letters are not signifi- cantly different at the 5% level by Duncan's multiple range test. Table 10.--Effect of soil pH on the mobility metribuzin on soil TLC plates. of 14C a pH c mefifiibuzin 0.05 uCi 0.10 uCi 4.6 .50 a .51 ab 5.1 .51 ab .52 be 5.6 .52 be .53 ca 6.1 .54 de .56 f 6.7 .58 g .60 h aMeans followed by similar letters are not signifi- cantly different at the 5% level by Duncan's multiple range test. 120 .EU momum> A>Hm>oomu Hmuou wv ucmEmmm AEonm.ov mom wufl>fluomoflnmm .>nmmumoumeouno mommaucflnu HHOm mcflms .n.m mm um mcflnmuum mo ucme>oe co coflumuucmocoo mo pommwmuu.m .mE 121 A53 cot—:3... ocmuuuu< (pualxoao: low; ,0 %) 536111143: 5.3611 0...." :3 - .9360 lo|o o... :a w nuewfias 19d Aunuoeogpeu 122 .EU m5mum> A>Hm>oomu Hmuou wv ucmammm ”Eonm.ov mom >ua>fluomoflpmm .mnmmumoumsouno umwmalcflnu HflOm momma .n.m mm um cflwsnfluuma mo ucmEm>OE co coflumuucmocoo mo uommmmun.m .6; 123 EU a: u3€> 0"! 66': | o .1: o. \ \ G} a 3 d .In (POJOAOOOJ logos §o %) zuawSas Jed Aunnoeogpeu Metribuzin (cm) migration Fig. 124 .EU mDmHm> A>u0>oomn Hmuou wo wv ucmfimmm AEo m.ov mom >ua>fiuomoflpmm .mnmmumouméouno mommaucflcu HHOm mcflm: .mcflumuumuo «a mo ucmEm>oE co mm HHOm mo pommmmul.oa .mE 125 .. -o.os Icl _o.10ICI 9H6] o 5 o s 2 1 1 Avoto>092 .22 .o 5 23:30» too 33:32.5: Atrazine migration (cm) 10 Fig. 126 metribuzin (Figure 11). The degree of adsorption at the origin at all three pH levels was less with metribuzin than with atrazine, whereas, mobility was greater. Dif- ferences in adsorption and movement may be attributed to differences in water solubility, basicity, and steric effects. All three herbicide properties would affect the entrance of the compound into solution and adsorption to soil particles. Metribuzin with a water solubility of 1200 ppm would enter into solution more quickly than atrazine. Atrazine is more basic in chemical character than is metribuzin which probably accounts for the observed differences in adsorption. Summary Studies with soils of varying pH indicate that metribuzin phytotoxicity increased with increasing soil pH. Whereas, in the nutrient-sand culture, there was no reduction in dry weight as pH increased. Microbial degradation increased as the soil pH increased. With 4.6% and 17.9 % 14CO2 being released at soil pH 4.6 and 6.7 respectively. Adsorption increased with decreasing soil pH. The dissociation constant was determined to be 0.99 + 0.08. The dissociation constant was shown to be important in determining the adsorption by colloidal sys- tems. Positive adsorption increased gradually as the soil pH decreased toward the pKa of metribuzin. Thus, adsorption and not microbial degradation, is the reason 127 .EU mSmH®> Axum>oomu Hmuou mo wv ucoEomm AEo m.ov mom >ua>auomoflomm .wcmmumoumEouno HmmeIcwcu HflOm anew: .cHNdnwnumEIU vH mo ucmEm>oE co mm HHom mo pomwmmuu.aa omflrm 128 2:3 coin-um:— 5:22.202 .9: .6 Till. no 3.. 55.61163. .2. 636.16.]. o... :a On mu .3 ([351voan logo; go %) iuswfias .Iad humoeogpsu .mE 129 for the decreased phytotoxicity observed at low pH levels on corn and soybeans. This is postulated to occur through protonation of the amine group on metribuzin; which . . . . l4 . . increases With a decrease in 5011 pH. C-metribuZin and l4C-atrazine mobility increased with increasing concentra- tion and soil pH. Metribuzin was shown to be more mobile on soil thin-layer plates than atrazine. A greater per- centage of atrazine was adsorbed at the origin of appli- cation and not allowed to migrate with the water front. Acknowledgment The authors express their appreciation to Chema- gro Corporation and CIBA-GEIGY Chemical Corporation for supplying the radioisotopes and technical herbicides used in this investigation. 10. Literature Cited Albert, A., and E. P. Serjeant. 1962. Ionization constants of acids and bases. John Wiley, New York. 68-92 pp. Alexander, M. 1965. Persistence and biological reactions of pesticides in soils. Soil Sci. Amer. Proc. 29:1-7. Alexander, M. 1964. Biochemical ecology of soil microorganisms. Ann. Rev. Microbiol. 18:217-252. Bailey, G. W., and J. L. White. 1964. Review of adsorption and desorption of organic pesticides by soil colloids, with implications concerning pesticide bioactivity. J. Agr. Food Chem. 12: 324-332. Bailey, G. W., J. L. White, and T. Rothberg. 1968. Adsorption of organic herbicides by montmoril- lonite: Role of pH and chemical character of adsorbate. Soil Sci. Soc. Amer. Proc. 32:222-234. Bailey, G. W., and J. L. White. 1970. Factors influencing the adsorption and movement of pesti- cides in soil. Residue Review 32:29-92. Corbin, F. T., and R. P. Upchurch. 1967. Influence of pH on detoxication of herbicides in soil. Weed Sci. 15:370-377. Frissel, M. J., and G. H. Bolt. 1962. Interaction between certain ionizable organic compounds (herbicides) and clay minerals. Soil Sci. 94: 284-291. Harris, C. I., and G. F. Warren. 1964. Adsorption and desorption of herbicides by soil. Weeds 12:120-126. Hauck, R. D., and H. F. Stephenson. 1964. Nitrifi- cation of triazine nitrogen. J. Agr. Food Chem. 12:147-150. 130 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 131 Helling, C. S. 1971. Pesticide mobility in soils. I. Parameters of soil thin-layer chromatography. Soil Sci. Soc. Amer. Proc. 35:732-737. Hyzak, D. L., and R. L. Zimdahl. 1974. Rate of degradation of metribuzin and two analogs in soil. Weed Sci. 22:75-79. Kaufman, D. D., and P. C. Kearney. 1970. Microbial degradation of s-triazine herbicides. Residue Review 32:235-265. Lavy, T. L., C. G. Messersmith, and H. W. Knoche. i 72. Direct liquid scintillation radioassay of C-labeled herbicide in soil. Weed Sci. 20: 215-219. Lay, M., and R. D. Ilnicki. 1973. The dissipa- tion of metribuzin in soil. Weed Sci. Soc. Amer. Abstr. P. 59. McGlamery, M. D., and F. W. Slife. 1966. The adsorption and desorption of atrazine as affected by pH, temperature and concentration. Weeds 14: 237-239. Nearpass, D. C. 1965. Effects of soil acidity on the adsorption, penetration, and persistence of simazine. Weeds 13:341-346. Rogers, E. G. 1968. Leaching of seven s—triazines. Weed Sci. 16:117-120. Talbert, R. E., and O. H. Fletchall. 1965. The adsorption of some s-triazines in soils. Weeds 13:46-52. Weber, J. B. 1966. Molecular structure and pH effects on the adsorption of 13 gftriazine com- pounds on montmorillonite clay. Amer. Mineral. 51:1657-1670. Weber, J. B. 1967. Spectrophotometrically deter- mined ionization constants of 13 alkyamino-g- triazines and the relationships of molecular structure and basicity. Spectrochimica Acta. 23A;458-461. Weber, J. B. 1970. Mechanisms of adsorption of s-triazines by clay colloids and factors affecting plant availability. Residue Review 32:93-130. 132 23. Weber, S. B., S. B. Weed, and T. M. Ward. 1969. Adsorption of s-triazines on soil organic matter. Weed Sci. 17:417-421. 24. Wolcott, A. R., H. D. Foth, J. F. Davis, and J. C. Shickluna. 1965. Nitrogen carriers: I. Soil effects. Soil Sci. Soc. Amer. Proc. 29:405-410. CHAPTER 4 SUMMARY AND CONCLUSIONS Field, greenhouse and laboratory studies were initiated to determine the effect of soil pH on metri- buzin activity, dissipation, microbial degradation, adsorption, mobility in soil, and to determine the dis- sociation constant and show how it relates to adsorption. Metribuzin activity in the field increased with increasing soil pH at 0.56, 0.84 and 1.12 kg/ha. Fall panicum control, number of dead corn and soybean plants, and crop injury ratings increased, whereas plant height and grain yield decreased as the soil pH increased. The lack of plant uptake of metribuzin at low soil pH levels could be due to rapid microbial degradation, adsorption to the soil colloids, lack of plant adsorption, or several of these factors. Greenhouse data on phytotoxicity to corn and soy- beans grown in a nutrient-sand culture versus soil at com- parable pH levels supported the field data. Corn and soy- beans grown in soil and treated with metribuzin showed a pH-sensitive response with a reduction in dry weight per plant as pH increased. Whereas, in nutrient-sand culture there was no difference in the reduction in dry weight as 133 134 pH increased. Reduction in herbicide concentration resulting from adsorption by soil colloids results in less absorption by plant roots at lower pH levels as observed in the field and greenhouse. Adsorption studies support these observations, with increased adsorption as soil pH decreases. Metribuzin residue analysis of soil samples from the field showed greater amounts of residue extractable at soil pH 6.7 than 4.6. Most of the metribuzin was found in the upper 5 cm of the soil profile. Metribuzin leach- ing increased with increasing soil pH. This is an indi- cation of increased adsorption as pH decreased. Metri- buzin disappearance from field soil followed pseudo first- order kinetics. Rate constants increased and the half life of metribuzin decreased as soil pH increased. Rate constants decreased and the half life of metribuzin in— creased at all soil pH levels as depth of sampling decreased. Laboratory studies on microbial degradation of l4C-5-labeled metribuzin showed decreased l4CO2 release as the soil became more acid. These results and those from the field indicate that metribuzin is being bound more tightly to the soil colloids and thus unavailable to the microorganisms in acid soils. If the substituent metabolism process is significant in soil and necessary for ring cleavage and subsequent metabolism, then it is likely that the rate of substituent metabolism and thus 135 degradation may be reduced if metribuzin was protonated and adsorbed to soil colloids in acid soils. Metribuzin and atrazine mobility increased with increasing concentration and soil pH. 14C-metribuzin had greater mobility on soil thin-layer plates than 14C- atrazine. A greater percentage of atrazine was adsorbed at the origin of application and did not migrate with the water front. Differences in adsorption and movement were attributed to differences in water solubility, basicity, and steric effects. All three herbicide properties would effect the entrance of the compounds into solution and adsorption to soil colloids. Adsorption increased with decreasing soil pH. The dissociation constant was determined to be 0.99 i 0.08. The dissociation constant was shown to be important in determining the adsorption by colloidal systems. Positive adsorption increased gradually as the soil pH decreased toward the pKa of metribuzin. This is postulated to occur through protonation of the amine group on metri- buzin, which increases with a decrease in soil pH. Adsorption at pH levels in which metribuzin is in the cationic form was attributed to cation exchange and to complexing with hydrogen ions on the soil colloids. Similar results are known to occur with the s—triazines. 136 Thus, protonation with subsequent adsorption to soil colloids was shown to be the reason for the reduction in activity, microbial degradation and mobility of metri— buzin as soil pH decreases. APPENDICES APPENDIX A Fig. A-1.--Number of dead corn plants per plot minus the control value at each given pH range. Metri- buzin applied at 1.12 kg/ha in 1972. 137 138 6.5 5.5 Soil pH Y = -37.8+9.58 X f= 0.979** / 4.5 «233.53 Eoo poop .oz .mnma as mean uon mom mucmHm camp mo Monaco commouocfl 0:» now omucoooom o>mc was coflumoflammm noumm xmc 0:0 50 m.H mo HHMNCHmmo .mcflucmHm umumm LDCOE moo coxmu mum3 mucmamusmmmzn .umou mmcmu mamfluaofi m.cmocoo >9 Hm>ma am on» on oomsmMMHb maucmoflmacmflm uoc mum mumuuma umHHEHm nuflz mump m.umo> some mo mommzm m o.NH U o.HN m N.mm mp b.0H U 0.0N mbo N.ma QM N.m m o.m no m.h m.®lm.o on o.oa o m.ma mp o.mm o 0.5 on m.@ o o.ma no o.m m o.m no m.n v.0no.o co o.m o m.mH to m.oH a o.v Q0 m.v no m.> mm o.m m o.m m m.H m.m|m.m n m.m on n.m n o.m am m.H no m.v no o.m m m.o m m.a mm m.m v.mno.m am o.m no m.m no h.> m m.o m m.m no m.h m m.o m m.o nm m.m m.v|m.v nammcmu mm co>Hm um Houucoo mocHEnImucmHm cuoo camp .ozv vnmfi mnma mama whoa mnma whoa gnma mbma mnma NH.H 5m.o om.o magma 16;\mxv causnfluumz mm II.‘ II - lull-I'll 1.1! 111 1|.11l- .. III om.uon mom mucmHm poop Hogan: mm UmMSmmmE .Uamfiw msu :H cuoo ou >uflofix0u0u>£m cflmsnflpuoe co mm HHow mo uomwwm m:9:u.~1m wands m xHozmmm< 139 APPENDIX D Fig. D-l.-—Corn height as percent of control. Metri- buzin applied at 1.12 kg/ha in 1972. 141 120 100 E E o 80 ‘5 x» 60 U) (0 E, 40 0 J: 20 Fig. D-l 4.5 142 Y=201.3 ' 20.1X r: -0.921* \\\ \ - \ x \ \\ \\ 5.5 Soil pH 6.5 .ucmHm onu co mama mcflmcmn mo» mo ucflom umwzmfln map 0» cmxmu mumz mucmEmHSmmoz .mcfiucmHm umumm mxmm3 me cwxmu mucmEmusmme unmflmmo Q .ummu omcmu mamfluaoe m.cmocsa >3 Hw>ma mm wnu um ucmumwmab waucmoHMHcmHm Doc mum mumuuma Hoafieflm Suez womb m.umw> comm mo mammzm 6 m6 6 mm 6 66 n6 66 66 mm 6 6e 666 mm 6666 m6 6 N6 6.6-6.6 666 56 n6 66 6 mm 66 mm 6666 66 6 66 666 66 666 66 6 66 6.6-6.6 n6 66 6 O6 6 66 666 66 6666 66 6 66 66 H6 6 66 66 6.66 6.6-6.6 666 66 on 66 66 mm 666 km 66 H6 6 66 66 am 66 mm 6 66 6.6-6.6 6 moH 666 M6 6 65H 666 66 666 m6 6 66 66 66 66 56 6 66 6.6-6.6 onAHouucoo mo wv 666a mema mead 666a mama mums 666a mums News NH.H 66.6 66.6 66666 mm .onHw ozu CH :uoo A6nxmxv 6666666662 .uanmS unwam :H cofluoopmu mm bmusmmms OD >uflonODoumnm sawsnfluuma co mm aeom mo uomwwm mnenl.aum manme m xHazmmm< 143 .mcflucmHm Houmm mxmwz xam coxmu mucoEmHSmmmE unmflomo .ucHom mcflzoum mnu mo mflu mzu ou :mxmu muwz mucmEousmmoZ n .ummp omcmu onHuHoE m.:mocso an Ho>mH wm 65» um “commMMHU >HucmoHMHcmflm Doc mum mumuuoa umHHEHm QDHB mump m.umo> comm mo mammZm m on m no QM Hm m am 6 no 6 Nb am om Guam mp am hm m.®Im.® 0 am 096 on Dam om 6 am U036 on nm em am mm mob hm onm mm v.0:o.© QM vm an on 36 mm no mm 009 om am vm QM mm mm «m on mm m.mlm.m mm mm mm bond we on mm QM mm moon mm on No no om H mm on Hm v.m|o.m o maa mbo mm o moa no am @036 an on am on HOH m hm QM hm m.vnm.v OQAHOHDCOU MO wv vhma muma mhma whoa mmma WWMH vwma mnma tha mound NH.H vm.o om.o mm i6n\mxv 6666666662 m .unvflmn ucmHm ca coauospmu mm pmHSmmmE .baoflm man be cmmn>om on xufloflxouou>nm cHNsnauuoE :0 mm HAOm mo pommmw 6591:.Hum magma m xHazmmm¢ 145 6660 66 sufloflxouousnd A6n\mx NH.HV U xHozmmm¢ .mcmwn>om pom cflmsnfluuma co pomwmm mm HHomuu.Huo .mflm 146 .I- ‘ ..m E :06 f . _ :Om <29. N... _ 4 (Val a _ .6. m Em In. :Cr. acid; . _ . APPENDIX H Table H-l.--l4C-metribuzin degradation to C02 in 12 weeks as affected by soil type.a Soil Sand Silt Clay OM % 14% evolved Texture % % % % 9“ as 1 €02 Sandy loam 65 18 17 2.5 6.2 16.6 a Clay loam 44 25 31 2.5 7.5 30.3 d Clay 10am 39 24 37 3.2 8.0 18.6 b Muck 7.6 23.1 c aValues are the means of two experiments two replications per experiment. Means in a followed by similar letters are not significantly different at the 5% level by Duncan's multiple range test. b0.0256 uCi/flask 147 many ummu\vmobm 2c «.mhn .ummu mocmu mamfluaoe m.cmocsa >n Hm>wa wm mnu D6 ucmumMMHb >HucmoHMAcmHm uoc mum mumuuma “MHHEHm >9 UmBOHHOM mammz .zomo mcofiumoflammu 03» zuHB mucmfifiummxo 03» mo momma on» mum mmsam>m m 0.6H mm m.mH m 6.6H p N.NH v.n m.m >.Hh EmoH >6Ho ovmaoe o o.oH o N.m Q >.> m H.m >.m >.m >.mm EmoH >6Ho >ccmm um>ocoo n.c0Humu0mpm w. a .m coeumuombm aflmsnfluumEuuwWJ‘ mm umuuma >6 mm>e aflom m6 6N NH 6. oflcmmuo 6H0 .unv ucoEummuu Houmm 0569 1“" II" "I III I'IUI Il'll.|llv m.ceusnfluumeluva mo coflumu0mbm co mm>u HHom mo uomwwm m£BII.HIH magma H xHozmmm< 148 APPENDIX J Fig. J-1.--Metribuzin degradation rate as the log of concentration in soil at three pH levels at a depth of 0.0-5.0-cm, following application of 1.12 kg/ha in the field in 1973. 149 150 .252: 533.205 .o cozmzcoocoo :o 0. 1 0 Time (months) lJ-l Fig. APPENDIX K Fig. K—l.--Metribuzin degradation rate as the log of concentration in soil at three pH levels at a depth of 0.0-5.0-cm, following application of 1.12 kg/ha in the field in 1974. 151 152 a x. H 6. .m 7 I p I. 5 6 \~~ . . \N . n I . . S. _ _ e... xx; \« \. \.\.~ \\ . \\~ . ~ \\\\\ ~ \\ x e \\ \ _ \.\\ \ s \x a \\ x e \ \ \ ~ \ \ x \ x . I. \ \ \ xx \ \ v \ x o. 6 s. 6 5 4 3 2 2 1. .259: £25.sz .o :oZmzcoocoo :om Time (months) 1.0 Fig. K-l APPENDIX L Modified No. l Hoagland's Solution 1 M KH2P04 2 m1/L l M KNO3 2 ml/L l M Ca(NO3)2'4H20 3 ml/L l M MgSO4°7H20 2 ml/L 1.5 g/L MnCl3°4H20 2.5 g/L H B04 3 0.1 g/L ZnClz l ml/L 0.05 g/L CuC12°2H20 0.05 g/L MoO3 26.3 g/L SequestreneR 1 ml/L pH 6.5 to 6.8 with l M NaOH 153