5‘”; 31.; :5 . 434;} fig}? g» _ . fig; ' €31 1.51;} $3.96 ,- ‘ HQS‘ {fig-“i u; ‘W $63551) #11313 1: (’9?- 3 ;. » . w .. - _ ”(39%?" "£5.21: ‘ «an ".0 w k '5 £- ' 53": 4...]: _ my. I-I: w : 4‘35. Perri} *‘f‘fié ‘ J? . {fa '='$:«tm%‘§r"?th=~:fi:%% “ ;, '3 r”. Huey: ‘fiyfi’g" - '- .a-tr'n: ' .-. a& v. . h ' \(1‘1 givgfi' "k": 4 _ Qfiv‘“ . V. 32'" ~. '0. 131% . ‘ .4 . \M'. r 3W4 .‘f‘ H a: ‘ “2%,,“ "Ry-‘QE‘T '1‘. a . 3 . m» u; , “ mu—i‘?’ m ,r - ”1.91,..- " u. “‘w‘um _V m. .513 , . . . f- m . . 92f - fi’t.b;..«.z'.w.umvm .. . -4 - m. - ~ THES§ This is to certify that the dissertation entitled CHARACTERIZING HERBICIDE INTERACTIONS WITH NUCLEAR MAGNETIC RESONANCE SPECTROMETRY presented by KURT DAVID THELEN has been accepted towards fulfillment of the requirements for DOCTOR 0F Wdegreeinwl. SCIENCES Major professor Date APRH ; Jggg MS U is an Affirmative Action/Equal Opportunity Institution 0— 12771 MICHIGAN STATE UNIVERSITY LIBRARIES . |\\\\\\\\\\ \\ \\\ \\\\\\\\\\\\\\\\\\\ \\\\\\\\\\\\\\\\\\\\\l 3 ]293 01025 9061 LIBRARY Michigan State University PLACE II RETURN BOX to roman this checkoutm your record. TO AVOID FINES return on or baton date duo. DATE DUE DATE DUE DATE DUE 4n- 4- ’“ 3' E _. -T, I ’ 1“ A_____. ‘__ 7 !_ 7' q ——1 l'l.'“fi :‘ . l I .\ . ‘ _ ‘ ““ 4.“-..i‘ o CHARACTERIZING HERBICIDE INTERACTIONS WITH NUCLEAR MAGNETIC RESONANCE SPECTROMETRY BY Kurt David Theien 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 Sciences 1994 ABSTRACT CHARACTERIZING HERBICIDE INTERACTIONS WITH NUCLEAR MAGNETIC RESONANCE SPECTROMETRY BY Kurt David Thelen Nuclear Magnetic Resonance (NMR) was used to evaluate herbicide interactions with carrier solution salts (glyphosate/Ca“, glyphosate/Mg“), other herbicides (Na-bentazon/paraquat), herbicide formulation products (sethoxydim/Na-bentazon, imazethapyr/Na-bentazon), and adjuvants (glyphosate/diammonium sulfate (AMS), gIyphosate/organosilicones). NMR was found to be an effective technique for determining chemical interactions in the biologically active compounds. “C-glyphosate absorption by sunflower was reduced in the presence of Ca”. The addition of ammonium sulfate overcame the observed loss of “C- glyphosate absorption. NMFi Spectral analysis of the glyphosate/Ca”. glyphosate/Mg“, and glyphosate/AMS pairs indicated a direct chemical interaction. No spectral changes were observed for the glyphosate/organosilicone pair. Data indicated an association of Na‘ from Na—bentazon, NaHCOa, and NaCI with the sethoxydim molecule. NH; from AMS appeared to associate spatially with sethoxydim but did not exert the same electronic effect on the sethoxydim ring protons as observed with Na’ or Li’. The addition of AMS to sethoxydim plus Na- bentazon or NaHCO3 prevented the complexation of Na’ with sethoxydim. No spectral changes were observed from the Na—bentazon/paraquat, and imazethapyr/Na—bentazon pairs. Analytical techniques employed included ‘H, 31P, and 13C NMR, and (‘H, 1H) homonuclear double quantum filter correlated spectrometry. Nomenclature: Glyphosate, N- (phosphonomethyl)gchine; bentazon, 3-(1-methylethyl)-(1H)-2,1 ,3- benzothiadiazin-4(3H)-one 2,2-dioxide; paraquat, 1,1'-dimethyl- 4,4'bipyridinium ion; sethoxydim, 2-[1-(ethoxyimino)butyl]-5-[2- (ethylthio)propyI]-3-hydroxy-2-cyclohexen-1-one; imazethapyr, 2-[4,5- dihydro-4-methyI-4-(1-methylethyl)-5-oxo-1 H-imidazol-z-yn-s-ethyI-a- pyridinecarboxylic acid; AMS, diammonium sulfate (NH.)2SO.; sunflower, Helianthus annuus L. ACKNOWLEDGEMENTS I sincerely thank the members of my graduate committee, Dr. Don Penner, Dr. James Kells, Dr. Karen Renner and Dr. Matthew Zabik. Special thanks to Dr. Penner for serving as my major adviser and Frank Fioggenbuck for helping with the experimental procedures. I also thank Dr. Evelyn Jackson, Kermit, Long, and Dennis at the NMR Center. My sincerest gratitude goes to my wife Carol and my children Renee, Derek, Taylor, and Kelsey for their love and encouragement which made this work possible. TABLE OF CONTENTS PAGE LIST OF TABLES ............................................................... vii LIST OF FIGURES ............................................................. viii CHAPTER 1: REVIEW OF LITERATURE NUCLEAR MAGNETIC RESONANCE ............................ 1 Introduction ................................................................ 1 Determining Chemical Structure .............................. 2 Herbicide Metabolism Studies in Plants .................. 4 Herbicide Metabolism in Animals ............................. 7 Microbial Metabolism ................................................ 8 Herbicide Environmental Fate Studies .................... 10 Determining Molecular Complexation ...................... 13 GLYPHOSATE ANTAGONISM ........................................ 15 Introduction ................................................................ 15 Antagonism ................................................................ 15 Overcoming the Antagonism .................................... 18 Na-BENTAZON ANTAGONISM OF SET HOXYDIM ...... 21 Introduction ................................................................ 21 Antagonism ................................................................ 21 Proposed Mechanisms ............................................. 23 Overcoming the Antagonism .................................... 28 LITERATURE CITED ....................................................... 32 CHAPTER 2: THE BASIS FOR THE CALCIUM ANTAGONISM OF GLYPHOSATE ACTIVITY ABSTRACT ...................................................................... 48 INTRODUCTION .............................................................. 50 TABLE OF CONTENTS CONTINUED: PAGE MATERIALS AND METHODS ........................................ 53 Absorption Studies .................................................... 53 Nuclear Magnetic Resonance .................................. 54 RESULTS AND DISCUSSION ........................................ 56 Absorption Studies .................................................... 56 Nuclear Magnetic Resonance .................................. 57 ACKNOWLEDGEMENTS ................................................. 63 LITERATURE CITED ....................................................... 80 CHAPTER 3: CHARACTERIZING THE SETHOXYDIM- BENTAZON INTERACTION WITH PROTON NUCLEAR MAGNETIC RESONANCE ABSTRACT ...................................................................... 83 INTRODUCTION .............................................................. 85 MATERIALS AND METHODS ........................................ 88 RESULTS AND DISCUSSION ........................................ 90 Sethoxydim ............................................................... 90 Sethoxydim plus Na—Bentazon ................................. 92 Effect of Diammonium Sulfate ................................. 94 ACKNOWLEDGEMENTS ................................................. 96 LITERATURE CITED ....................................................... 105 CHAPTER 4: CHARACTERIZING HERBICIDE INTERACTIONS WITH NUCLEAR MAGNETIC RESONANCE SPECTROMETRY ABSTRACT ...................................................................... 107 INTRODUCTION .............................................................. 109 MATERIALS AND METHODS ........................................ 112 Glyphosate, Imazethapyr, and Paraquat ................. 112 Sethoxydim Treatments... ........................................ 113 V TABLE OF CONTENTS CONTINUED: PAGE RESULTS AND DISCUSSION ........................................ 114 Herbicide plus Herbicide .......................................... 114 Herbicide plus Herbicide Formulation Products ...... 114 Herbicide plus Adjuvants .......................................... 115 ACKNOWLEDGEMENTS ................................................. 1 17 LITERATURE CITED ....................................................... 123 CHAPTER 5: SUMMARY AND CONCLUSIONS INTRODUCTION .............................................................. 129 Addition of Organic Acids ........................................ 129 Diammonium Sulfate ................................................ 130 Nonionic Surfactants ................................................ 130 Low Volume Rates ................................................... 131 vi CHAPTER 2: CHAPTER 2: CHAPTER 4: LIST OF TABLES TABLE 1: 2: 1: The effect of the conjugate salts: IPA (is0propyiamine), Ca (calcium), AMS (ammonium sulfate), and Ca + AMS calcium + ammonium sulfate) on “C-glyphosate absorption by sunflower. Absorption is presented as % of total applied .......................................... The effect of the conjugate salts: IPA (isopropylamine), Ca (calcium), AMS (ammonium sulfate), Ca + AMS calcium + ammonium sulfate) on 1‘C-glyphosate absorption by tall morningglory. Absorption is presented as % of total applied ............................ ‘H-NMR chemical shift data for paraquat and imazethapyr as affected by Na-bentazon and Na-salts .............. vii PAGE 64 65 118 LIST OF FIGURES FIGURE PAGE CHAPTER 2: 1. ‘H-NMR spectrum of a) technical grade glyphosate plus calcium acetate plus ammonium sulfate; b) technical grade glyphosate plus AMS; c) technical grade glyphosate plus calcium acetate 1:1 molar ratio; d) 3‘P decoupled ‘H-NMR spectrum of technical grade glyphosate; and, e) technical grade glyphosate. 020 was the carrier solvent in all treatments ............................................. 67 2. “C-NMR spectrum of a) technical grade glyphosate plus calcium acetate 1:1 molar ratio; and, b) technical grade glyphosate. 020 was the carrier solvent in both treatments ............................................. 69 - 3. 31P-NMR spectrum of a) technical grade glyphosate plus calcium acetate plus AMS 1:1 :3 molar ratio; b) technical grade glyphosate plus AMS; 0) technical grade glyphosate plus calcium acetate 1:1 molar ratio; and, d) technical grade glyphosate. 020 was the carrier solvent in all treatments ............................................. 71 4. ‘H-NMR spectrum of technical grade glyphosate plus calcium acetate 1:1 molar ratio in 020. Spectrum obtained from 40 minutes (bottom) to 820 minutes (top) after mixing ................... 73 viii LIST OF FIGURES CONTINUED: Chapter 3: FIGURE 5. ‘H-NMR spectrum of a) commercially formulated isopropylamine glyphosate plus calcium acetate 1:4 molar ratio; b) commercially formulated isopropylamine glyphosate plus calcium acetate 1:1 molar ratio; and, c) commercially formulated isopropylamine glyphosate. 020 was the carrier solvent in all treatments... 31P-NMR spectrum of a) commercially formulated isopropylamine glyphosate plus calcium acetate 1:4 molar ratio; b) commercially formulated iSOpropyIamine glyphosate plus calcium acetate 1:1 molar ratio; and, c) commercially formulated isopropylamine glyphosate. 020 was the carrier solvent in all treatments... Scheme of hard-water cation association with glyphosate in solution: step 1) proposed association of Ca“ with carboxyl and phosphonate functional groups of glyphosate molecule based on Figures 1 through 3; step 2) progression towards tridentate and tetradentate ligand as supported by Figure 4; step 3) proposed formation of NH,-glyphosate and CaSO, based on Figures 1 and 3 ..................................... 1H-NMR spectrum and peak assignments of technical grade sethoxydim in CDCI3 ............................ PAGE 75 77 79 98 LIST OF FIGURES CONTINUED: Chapter 4: FIGURE 2. 1H-NMR spectrum of a) commercially formulated sethoxydim plus NaCI; b) commercially formulated sethoxydim plus NaHCO,; c) commercially formulated sethoxydim plus commercially formulated Na-bentazon; (1) technical grade Li-sethoxydim; e) commercially formulated sethoxydim. CDCI3 was the carrier solvent for all treatments ............................................. 1H, ‘H homonuclear double quantum filter correlated spectrometry (COSY NMR) spectrum for a) commercially formulated sethoxydim plus commercially formulated Na-bentazon; and, b) commercially formulated sethoxydim. CDCI3 was the carrier solvent for both treatments .................. ‘H-NMR spectrum of a) commercially formulated sethoxydim plus NaHCO, plus AMS; b) commercially formulated sethoxydim plus commercially formulated Na-bentazon plus AMS; 0) commercially formulated sethoxydim plus AMS; d) commercially formulated sethoxydim plus commercially formulated Na-bentazon; e) commercially formulated sethoxydim. CDCI3 was the carrier solution for all treatments .................... Ultra violet spectra for a) paraquat plus Na-bentazon; b) Na-bentazon; and, c) paraquat ................................... PAGE 100 102 104 120 LIST OF FIGURES CONTINUED: FIGURE 2. PAGE ‘H-NMR spectrum of a) technical grade glyphosate plus calcium acetate 1:4 molar ratio plus Sylgard 309; b) technical grade glyphosate plus Sylgard 309; c) technical grade glyphosate plus calcium acetate 1:1 molar ratio; and, d) technical grade glyphosate. 020 was the carrier solvent in all treatments ...................... 122 xi (grit-nth: u‘ .- Chapter 1 REVIEW OF LITERATURE Chemical Based Herbicide Antagonism and the Use of NMR In the Wed Science Discipline Nuclear Magnetic Resonance. Nuclear magnetic resonance, NMR, is an effective tool for deriving structural information on molecules. The theory of NMR is based on the premise that some nuclei behave as if they are spinning about an axis. Since they are positively charged, these spinning nuclei behave like tiny magnets and can interact with an externallyapplied magnetic field. Nuclei exhibiting spin properties include nuclei with odd numbered masses: 1H, 1‘B, 13C, ”F, 31P, and nuclei with even masses and odd atomic numbers: 2H, and “N. Of these, H-NMR is perhaps the most common form of NMR used in making structural determinations. In the presence of an applied magnetic field, the nuclear spins align parallel (low energy) or anti-parallel (high energy) to the applied magnetic field. When the oriented nuclei are irradiated with radio waves of a 1 certain frequency, energy absorption occurs and the low energy state spin-flips to the higher energy state, thus gaining resonance with the applied radiation. The energy required to spin flip nuclei is proportional to the applied magnetic field as denoted by the following equation (83): v = 8Ho/2rr where: v = frequency (MHz) Ho 8 applied magnetic field (gauss) magnetogyric ratio, proportionality constant between the magnetic moment and the spin number Nuclei in molecules reside in slightly different electronic environments due to magnetic shielding from circulating electron clouds. Thus, each unique proton experiences a slightly different magnetic field and absorbs energy at a correspondingly different radio frequency. This slight difference in energy absorption gives rise to the NMR spectra. Use of NMR In determining herbicide chemical structure. A primary role of NMR technology in weed science is in determining the chemical structure of new herbicide products. Hayashi and Kouji (31) synthesized two geometrical isomers of methyl 1-5-2-chlore-4-(trifluoromethyl)phenoxy- 2-nitrophenyI-2-methoxyethylIdene, aminooxyacetate and used two- dimensional NMR spectroscopy to confirm molecular confirmations. 2 Mixtures of the two isomers in various proportions exhibited practically the same biological effects as those of each isomer alone. Lynch et al. (44) prepared a variety of 1-aIkyI-5-cyano-1H-pyrazole-4carboxamides by directly alkylating pyrazoles under basic conditions. Regioisomers produced using this method were separated by'chromatography and identified using NMR techniques. These products were found to have activity against Amaranthus species, lgomoea species, velvetleaf (Abutilon thegghrasti Medic.) and jimsonweed (Datura stramonium L.). NMR has also been used extensively to identify microbially produced herbicidal compounds. Nakajima et al. (33) extracted a compound identified as hydantocidin from Streptomyces hygroscopicus. The molecular formula and structure was determined utilizing NMR and mass spectrometry. The compound when tested against 17 annual weeds was found to be as efficacious as glyphosate [N-(phosphonomethyl)glycine. Babczinski et al. (6) isolated the antibiotic vulgamycin from a new strain of Streptomyces sp. (WS 2611). The antibiotic which was characterized by mass spectrometry and NMR was found to exhibit postemergence control of several common weeds including Amaranthug retroflexus L., Chenggodium album L., Igomoea sp., and Setaria viridis (L.) Beauv., whereas cotton (Gossygigm sp.), barley (Hordeum vulgare L.), and maize (Zea mays L.) were only minimally sensitive. Biggens et al. (9) used NMR to determine that cyanobacterin, a secondary metabolite produced by the cyanobacterium Scytonema hofmanni, does not act as an uncoupler of photophosphorylation. Rather, the authors proposed that cyanobacterin acts by binding to a thylakoid membrane protein which facilitates proton transport. In addition to identifying herbicidally active ingredients, NMR technology has also been adapted for identifying inert ingredients in herbicide formulations. Nishizawa et al. (57) in 1989 developed a method for identifying inert ingredients using NMR analysis. More recently, in 1992, Nishizawa (58) described a method to detect inert ingredients in emulsifiable concentrates using NMR. The method utilizes both 1H and ”C NMR, and compares peaks to standards for surfactants and stabilizers. Herbicide metabolism studies In plants. A common adaptation of NMR technology is in the field of herbicide metabolite identification (89). During the early 1970’s, Still and Mansager (90) used NMR, infra-red, and mass spectral analysis to examine isopropyl carbanilate metabolism in soybean plants. The authors identified an aglycone derivative of isopropyl 2-hydroxycarbanilate which was in part conjugated as a glycoside. Also during this time period, Shimabukuro et al. (79) used NMR to identify a glutathione conjugate as a primary metabolite of 4 fluorodifen (2,-4’-dinitro-4-trifluoromethyl-diphenyl ether) metabolism in peanuts (Arachis hymgaea L.). In the mid 1980’s, Kelly and Smith (36) determined an aspartate conjugate as the major polar metabolite of benazolin-ethyl (4-chloro-2-oxobenzothiazolin-3-ylacetic acid) metabolism in soybeans (Glycine max (L.) Merr.). More recently, Lamoureux et al. (41) used proton NMR in conjunction with electron impact mass spectrometry, radio labels, protein liquid chromatography, and hydrolysis with beta- glucosidase to identify chlorimuron ethyl (2-[[[[(4-chIoro-6-methoxy-2- pyriidinyl)amino]carbonyl]amino]sulfonyljbenzoic acid) metabolites in corn. Tanaka et al. (94) determined that the major metabolic pathway for the detoxification of diclofop-methyl ((i)-2-[4-(2,4- dichlorophenoxy)phenoxy]propanoic acid) in wheat (Triticum sp.)_is by ring hydroxylation followed by glucoside conjugation. The workers utilized GLC, MS and NMR to identify three isomeric hydroxylated metabolites of diclofop-methyl after acid hydrolysis of the glucoside conjugates. Frear et al. (20) used NMR to characterize flumetsulam (N-(2,6-difluorophenyl)-5- methyl(1,2,4)triazolo(1,5-a)pyrimidine-2-sulfonamide) metabolism in wheat, corn, and barley. Similar hydroxylation and glucose conjugation metabolic pathways were identified in the tolerant species seedlings. In work with an herbicide safener, Nadeau et al. (50) used proton NMR to determine that “C label from phenyI-"C’e-z-(diphenylmethoxy)acetic acid methyl ester (DME) safener in rice was incorporated into alpha and 5 beta isomers of D-glucose pentaacetate, natural glucose units of starch. Still et al. (91) used 13C NMR to study the structure of synthetic chloroaniline-lignin copolymers. When rice (Oryza sativa L.) plants were grown hydroponically and treated with 1‘Ce’-3—chloroaniline or 1‘Ce’-3,4- dichloroaniline, more than 40% of the radio label was found in the roots, in isolated lignin fractions. A coniferyl alcohol polymerization system was developed for the preparation of a model copolymerization to yield synthetic chloroaniline-Iignin cepolymers. Using 13C NMR, the chloranilines were determined to be bound covalently to lignin via 1, 6 addition to a quinone methide intermediate during lignin synthesis. Law and Arnold (42) used NMR to evaluate the effect of EPTC (S-ethyl dipropyl carbamothioate) and acifluorfen (5-[2-chloro-4— (trifluoromethyl)phenoxy]-2-nitrobenzoic acid) on the chemical structure of sunflower (Helianthus annuus L.) epicuticular wax. The NMR data indicated no change in the chemical structure of the wax, however, SEM scans revealed a definite alteration in the surface leaf wax. Rollins et al. (71) conducted experiments to evaluate the possibility of using NMR imaging and spectroscopy to study the movement and distribution of xenobiotics non-invasively in stems and leaves of tomatoes (Lycopersicon esculentum Mill. cv. Ailsa Craig). They found the technique to be suitable for translocation studies in whole plants. However, the authors concluded that it was unlikely that NMR will, in the near future, be of use in detecting the very low concentration of herbicides used in field applications. Herbicide metabolism In animals. NMR technology has also been used extensively in identifying pesticide metabolites in animal physiological pathways. In the early 1970’s Paulson et al. (61) characterized radiolabelled urinary metabolites of propham (1-methylethyl phenylcarbamate) in rat (Rattus sp.) and goat (Capra sp.). The animals were given single oral doses (100 mg/kg body wt) of propham. After purification, the derivatives were characterized by infra red, NMR, and mass spectrometry. In the late 1970’s Sate et al. (74) identified urinary metabolites of dymrone (1-(alpha, alpha,dimethylbenzyl)-3-p-tolyl urea) in male Wistar rats. After intraperitoneal administration of carbonyl-“C- dymrone, 77% of the dose was eliminated in the urine. Thin layer chromatography, infra red, NMR, and mass spectra were employed to identify the major metabolites. In 1978, a technique for extraction of the chlorophenoxy alkyl acid herbicides was developed for human urine samples by Beer at al. (8). Conversion of the chlorophenoxy alkyl acids to the corresponding pentafluorobenzyl esters facilitated comparison with recorded MS, NMR, UV, and IR spectra. The use of NMR in identifying animal metabolites of pesticides has continued into the current decade. Leung et al. (43) used NMR to characterize the metabolic fate and distribution of the triazolone herbicide 1-2,4-dichloro-5-N-(methylsuIfonyl)aminophenyl-I ,4-dihydro-3-methyl-4- (difluoromethyl)-5H-triazol-5-one in the rat, goat and hen @Hgs domesticus). Hackett et al. (27) used rat liver enzyme preparations to investigate the pathways of triallate ((S-(2,3,3-trichloro-2-propenyl) bis(1- methylethyl)carbamothioate) metabolism. Metabolite identification was accomplished by mass spectrometry and chemical synthesis, and by heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. Yoshioka et al. (103) used "P NMR to demonstrate that thiamine pyrophosphate was absorbed by boar (Sus scrofa) spermatozoan cells. The thiamine pyrophosphate enhanced the formation of N- arylacetohydroxamic acids from nitroso derivatives of chlorinated 4- nitrodiphenyl ether herbicides. The results indicate that the activities of the nitroso compounds in the formation of N-arylacetohydroxamic acids increase with a decreasing number of chlorine substituents through a dechlorinative degradation pathway. Microbial metabolism. In addition to plant and mammalian metabolism, NMR is also utilized in characterizing and identifying microbial metabolic pathways and herbicide degradation products. Wallnofer et al. (95,96) used NMR to evaluate the herbicide metabolites produced by the fungus Rhizgpus jaggnicus. The authors (96) used NMR and mass spectrometric analysis to identify N-(3,4-dichIorophenyl)-2-methyl-2,3- dihydroxypropicnamide as the hydroxylation product produced by the fungus Rhizogus iamnicus cultured with the herbicide dicryl [N—(3,4— dichlorOphenyl)methacrylamide]. In other studies, Wallnofer et al. (95) identified metabolites of the phenylurea herbicides monuron (N'-(4- chlorophenyl)-N, N-dimethylurea), fluometuron (N,N-dimethyI-N’-[3- (trifluoromethyl)phenyl]urea), monolinuron (N-(4—chloro-phenyl)-N' methoxy- N methylurea), and buturon (N’-(4—chlorophenyl-N-methyl-N-(1-methyl-2— propynil)-urea). Schocken et al. (75) identified six metabolites of the tetrazolinone herbicide F5231 cultured with the filamentous fungus Afiigia pseuocylindospora Hesseltine et Ellis (ATCC 24169). Pothuluri et al. (65) also reported on the degradation of acetanilides by fungi. They found that the fungus Cunninghamella elegarls transformed alachlor (2-chloro- N-(2,6-diethylphenyl)-N-methoxymethyl)acetamide) into four metabolites. Using NMR, the authors found that metabolism occurred primarily by benzylic hydroxylation of one of the arylethyl side chains. In work with an anaerobic methanogenic consortium, Suflita et al. (93) used NMR to identify a dechlorinated metabolite of 2,4,5—T [(2,4,5— trichlorophenoxy)acetic acid]. The NMR spectra indicated that the 2,4,5- T was dechlorinated at the para position to form 2,5- dichlorophenoxyacetic acid. From this work the authors concluded that anaerobes may possess capacities to degrade some xenobiotic compounds that are considered recalcitrant under aerobic conditions. Krause et al. (40) evaluated the transformation of metolachlor (2-chloro- N-(2-ethyI-6-methylphenyI-N-(Z-methoxy-1-methylethyl)acetamide) by a soil actinomycete strain isolated from a metolachlor contaminated soil. Eight metabolites were obtained and identified by NMR and mass spectral analyses. Benzylic hydroxylation of the aralkyl side chains and/or demethylation at the N-alkyl substituent appeared to be the only transformations involved. The authors did not observe any dehalogenation of the chloroacetyl moiety. Use of NMR In herbicide environmental fate studies. NMR has been used successfully as a tool in examining herbicide interaction with soil organic matter. Senesi and Testini (76) used NMR to characterize interactions between substituted urea herbicides and soil humic acids. The workers found that in addition to H-bonding, a prominent role was played by electron donor-acceptor processes involving organic free radicals. Stearman et al. (88) evaluated soil humus fractions from no-till and conventionally tilled cotton plots utilizing 1°C-NMR. Herbicide activity was found to be inversely related to soil carbon, extractable carbon, carboxyl groups of humic acid, and fulvlc-acid carbon. Decreased 10 herbicide activity in tilled soil was largely attributed to the carbon in tilled soil being in a more reactive state than carbon in no-till soil. Piccolo et al. (64) compared atrazine (6-chloro-N-ethyI-N'-(1-methylethyl)-1,3,5- triazine-2,4odiamine) interactions with three humic acid fractions extracted from a volcanic soil, a North Dakota Ieonardite, and an oxidized coal. Characterization with “C-NMR indicated that humic acid aromaticity decreased and aliphaticity increased in the order oxidized coal followed by the Ieonardite, followed by the volcanic soil. The authors concluded that atrazine is mainly adsorbed through a charge-transfer mechanism between electron peer groups of humic acids and electron rich atoms in atrazine. In addition, the higher the aromaticity, the polycondensatlon, and the molecular size of humic acids, the more atrazine was adsorbed. Rueppel et al. (72) used 1H, 3‘P, and “C, NMR to identify both bound and unbound glyphosate metabolites in soil. Aminomethylphosphonic acid was found to be the primary soil metabolite, however, it underwent further rapid degradation in the soil. The soil persistence and metabolism of dibutalin (N-sec-butyl-4-tert-butyl-26dinitroaniline), was characterized by Kearney et al. (35). The major product isolated from soil was identified using NMR as the dealkylated derivative 2,6dinitm-4-t-butylaniline. 11 The photodegradation products of atrazine and ametryne (N-ethyI-N’-(1- methylethyl)—6-(methylthio)-1,3,5-triazine-2,4-diamine) in water were characterized with NMR by Rejto et al. (67). The primary degradation products were found to be deethylated s-triazines and 6-acetamido-s- triazines. These In turn were further decomposed to 4,6-diamino-s- triazine derivatives. Kissel et al. (37) used NMR and other analytical methods to assay acrolein (2-propenal) degradation in aqueous media. This data was then correlated to bioassay data. Analytical methods measuring acrolein directly, such as NMR, correlated well with bioassay data over the range of 1 to 4 acrolein half-lives. However, methods involving the conversion of acrolein to a derivative such as DNPH (2,4- dinitrophenylhydrazone) followed by analysis, did not correlate well with bioassay data. Osredkar and Kadaba (59) developed a procedure for using NMR techniques for the detection of pesticides in water at low concentrations. Although the sensitivity was found to be inferior to that of electron capture gas chromatography, one advantage to NMR was that it could be used to detect heat-labile compounds, since measurements are made at below room temperature. In addition, with NMR, clean-up and separation procedures did not need to be elaborate. Thus, the authors proposed the NMR techniques could be used for the analysis of triazine pesticides in wastewater at concentrations 10 to 100 ug/L. 12 Deterrnlnlng molecular complexation. In working with a light-induced pyrazole phenyl ether, Garbow and Gaede (21) utilized a form of solid state 13C-NMR. After failing to conclusively demonstrate the formation of an inclusion complex between the pyrazole phenyl ether and B- cyclodextrin using elemental analysis, x-ray powder diffraction and solution NMR, the authors used a form of solid state 13C-NMR, known as cross-polarization magic-angle spinning (CPMAS). Analyses of the resulting spectra of the solid complex demonstrated that it was a true I inclusion complex. The authors hypothesized that the formation of an inclusion complex would significantly alter the optical properties of the pyrazole phenyl ether sufficient to modify the biological activity. However, no significant difference in biological activity was observed using a multispecies whole plant assay. Chrystal et al. (15) used NMR in an attempt to show that herbicides derived from 4-[(benzyloxy)methyl}-1,3—dioxolanes and benzyl methyl ethers of poly(ethylene glycols) exerted their mode of action by chelating biologically important metal Ions such as Na“, K”, Mg”, and Ca”. The authors demonstrated that the herbicides did indeed complex with the metal ions. However, this association was not correlated with herbicidal activity. In fact, one of the poorer herbicides showed a greater affinity for the metal ions than the most active compound. In summary, they found 13 no evidence that herbicidal activity in this class of compounds is associated with their general complexing ability towards alkali or alkaline- earth metal ions. NMR has been used extensively in evaluating the complexation properties of paraquat (1,1’-dimethyl-4,4’-bipyridinium ion). In 1969, Haque et al. (28) reported a strong tendency for ion association of paraquat with electron-donating anions. Iodide and ferrocyanide ions were found to form charge transfer type complexes with paraquat, while ferricyanide ions formed outer-sphere type ion pairs. In 1987, Allwood et al. (2) characterized the complexation of paraquat and diquat (6,7- dihydrodipyrido1,2-2’,1’-c]pyrazinedium ion) by a bismethaphenylene—32- crown-10 derivative. That same year, Ashton et al. (4), also working out of the Sheffield University, London, reported on the complex formation between paraquat and bisparaphenylene-(an + 4)-crown-n ethers. More recently, Ashton et al. (5) used NMR to show the complexation of cyclobis(paraquat-phenylene) and 1,5-dinaphtho-38-crown10 in solution to form a highly ordered [2]catenane. The workers used x-ray crystallography to verify the complex formation in the solid crystal state. Philp et al. (63) also demonstrated complexation of the tetracationic cyclobis(paraquat-p-phenylene). In Philp’s work, NMR was used to characterize the complexation of cyclobis(paraquat-p-phenylene) with the powerful electron donor, tetrathiafulvalene in solution. As in the work 14 noted above by Ashton et al. (5), x-ray crystallography was also used to verify complex formation in the solid state. Glyphosate Antagonism Hatzios and Penner (30) have classified herbicide antagonism into four possible mechanisms. These include: biochemical antagonism, competitive antagonism, physiological antagonism, and chemical antagonism. Chemical antagonism is defined as a chemical reaction which occurs between a herbicide and another chemical in the spray mixture. Antagonism. The structure of glyphosate has been evaluated with NMR (14, 39). The molecule has ionizable moieties at the phosphonate, carboxyl, and amine locations (82). Acid dissociation constants of glyphosate are reported to range from 2.2 to 2.6 (pk,), 5.5 to 5.9 (pkz), and 10.1 to 10.9 (pig) (45, 46, 49, 86, 100,). These multiple negative charges on the glyphosate molecule make it reactive with positively charged sites in mineral and organic soil fractions (85) and with cations in aqueous solution (22). In examining the inactivation of glyphosate in soils, Sprankle et al. (86) 15 found that glyphosate was rapidly inactivated in Fe’” and Al’“ saturated clays and organic matter but not in washed quartz sand. This was supported by work done by Hensley et al. (32). They found a significant reduction in the activity of glyphosate on sorghum (Sorghum vunare Pers. var. RS. 610) root length when Fe“, Fe“, and Al’“ salts were added to glyphosate treated soils. The chelating properties of glyphosate in solution have been documented (22, 80). Motekaitis and Martell (49) investigated the possibility of a 2:1 glyphosate to Ca“ ligand. However, using potentiometric equilibrium data, they found that even a fourfold excess of glyphosate would not form exclusively 2:1 complexes. Therefore, the authors proposed a 1:1 bidentate planer arrangement with the metal chelated by two oxygens on the phosphonate functional group. Madsen et al. (45) and Appleton et al. (3) proposed that glyphosate formed a trident ligand. More recently, Subramaniam and Hoggard (92) used infrared data to show that glyphosate acts as a tetradentate ligand with coordination through the amine nitrogen, the carboxylate oxygen and the phosphonate oxygens. Recently, there has been renewed interest (51) in the previously reported (73) glyphosate antagonism by cations present in hard water. Shea and Tupy (78) reported a significant reduction in glyphosate activity on wheat (Triticum aestivum L. ’Centurk’) in the presence of 50 ppm calcium. 16 Nalewaja (54) has reported reduced control of wheat, sunflower, kochia (Kochia scogaria L. Shrad. #3) and soybean with glyphosate in the presence of calcium. In addition, sodium salt, butoxyethyl ester, and diethanolamine formulations of 2,4-D antagonized glyphosate phytotoxicity to wheat (52). From this work, it was suggested that 2.40 formulation cations may exchange with the glyphosate isopropylamine formulation cation (IPA) resulting in a less absorptive compound than the glyphosate IPA. Several researchers have documented an increase in glyphosate activity by decreasing carrier volume (10, 11, 73, 87). This can be attributed to fewer cations in the spray solution to associate with and deactivate the glyphosate molecule. Stahlman and Phillips (87) showed that as spray solutions became more alkaline, glyphosate activity tended to decrease. Buhler and Burnside (11) documented an increase in glyphosate activity by adding various acids, HCI, H280” and acetic acid. H280, was found to be the most effective acid at overcoming the antagonism and it was proposed that $0," ions may compete for Ca“ forming CaSO,. In addition to forming conjugate salts and removing cations from solution, the addition of acid to the spray solution may result in the protonation of the negatively charged moieties on the glyphosate molecule. The resultant nondissociated free 17 acid glyphosate molecule would be expected to more readily pass through the cuticle as compared to the conjugate cation-glyphosate salt. Nalewaja et al. (56) evaluated scanning electron micrographs of glyphosate spray droplets. They observed that glyphosate applied with antagonistic calcium chloride salts formed spray deposits that were amorphous, thick, and without crystals. From this work, the researchers interpreted the micrographs as indicating that the antagonism of glyphosate phytotoxicity by salts may be in part from physical entrapment of glyphosate in the spray deposit. Overcoming the antagonism. Calcium antagonism of glyphosate has been overcome with ammonium sulfate (51,53). This effect has been found to vary with plant species (54) and spray carrier volume (60). Nalewaja and Matysiak (55) proposed an equation for determining the amount of ammonium sulfate needed to overcome cation antagonism of glyphosate. The equation is based on the assumption that the antagonistic effect of individual cation salts on glyphosate is additive: Diammonium sulfate (grams per 100 L) = 0.6(sodium mg/L) + 0.2(potassium mg/L) + 1.0(calcium mg/L) + 1.7 (magnesium mg/L). 18 Sulfate lens may compete with glyphosate for the antagonistic Ca” cations thus deterring the formation of glyphosate Ca“ complexes. In their work with scanning electron micrographs of glyphosate spray droplets, Nalewaja et al. (56) observed crystal formation when ammonium sulfate was applied with or without calcium. In the presence of calcium the crystals were attributed to CaSO, forming in solution. In the absence of calcium in the carrier solution, the researchers postulated that calcium from the leaf cuticle may combine with sulfate to produce the observed crystals. Another possible mechanism for the reversal of the observed antagonism is a direct interaction between the NH,’ cation and the glyphosate molecule resulting in NH,-glyphosate, which may be more readily absorbed than the conjugate salt of Ca-glyphosate (52). Non-ionic surfactants are commonly used with glyphosate to improve efficacy. Knoche and Bukovac (38) evaluated the effect of oxyethylene chain length of three homologous series of nonionic surfactants on glyphosate uptake. Their results showed that the enhancement of glyphosate uptake and wetting characteristics varied markedly between species evaluated. This observed specificity between species with nonionic surfactants is consistent with the findings of Nalewaja & Matysiak (54) using AMS. 19 Another class of surfactants, the organosilicones, have also been used to improve rainfastness and efficacy of glyphosate (66, 70). In their work with the silicone adjuvant commercially known as Kinetic‘, Reddy and Singh (66) improved efficacy when the critical rain free period was reduced to 15 minutes for velvetleaf, sickle pod, and yellow foxtail. However, rainfastness was not improved on barnyardgrass thereby indicating a specificity for certain species. Roggenbuck et al. (70) evaluated a series of silicone adjuvants for increasing the efficacy and rainfastness of glyphosate on velvetleaf and common Iambsquarters. The researchers found that the different silicone adjuvants varied in response to different weed species and among other herbicides tested. The mechanism of action of the silicone adjuvants has not been established. However, Roggenbuck et al. (70) concluded that the increase in rainfastness and efficacy is not solely based on reducing surface tension sufficient to allow stomatal infiltration, which had been previously proposed as a possible mode of action for another silicone adjuvant by Field and Bishop (19). 1Helena Chemical Company, Memphis, TN 38119 20 Na-bentazon Antagonism of Sethoxydim Tank mixing POST applied herbicides for broad spectrum weed control is a commonly used management practice in soybean production. Bentazon plus a graminicide, such as sethoxydim, is a popular herbicide combination for control of many broadleaf and grass weeds. Antagonism. Hartzler and Fay (29) found that tank mixes of sethoxydim and bentazon gave reduced control of large crabgrass (Digitaria sanguinalis L.) as compared to sethoxydim applied alone. Rhodes and Coble (68) reported that tank-mixing sethoxydim and bentazon resulted in reduced control of broadleaf signalgrass (Brachiaria glatthylla Griseb.), fall panicum (Panicum dichotomiflorum Michx.), and large crabgrass as compared to sethoxydim applied alone. Jordan and York (34) found that bentazon antagonized sethoxydim activity on corn and large crabgrass. Grichar (25) observed reduced control of southern crabgrass (Digitaria ciliaris Retz.) and Texas panicum (Panicum texanum Buckl.) when bentazon was tank mixed with sethoxydim. Control of annual grasses (Texas panicum and southern crabgrass) with sethoxydim has also been found to be antagonized by other broadleaf herbicides (25) including acifluorfen, acifluorfen plus bentazon, pyridate and naptalam plus 2,4-D. Minton et al. (47, 48) reported antagonism of 21 sethoxydim with chlorimuron and imazaquin. Antagonism of barnyardgrass (Echinochloa crus;galli (L.) Beauv.) (47) and red rice (Oma sativa L.) (48) occurred when sethoxydim was tank mixed with imazaquin or chlorimuron. Bentazon has also been found to antagonize other grass herbicides when applied as a tank mix. Campbell and Penner (12) found that bentazon antagonized diclofop activity on annual grasses. Wilhm et al. (102) reported that bentazon inhibited absorption of quizalofop {2-[4-[(6.chloro- 2-quinoxalinyl)oxy]phenoxy]propionic acid} on quackgrass (Elytm' ia refins (L.)) leaves. Croon et al. (18) reported reduced absorption and I translocation of haloxyfop-methyl 2-{4-[[3-chloro-5-(trifluoro-methyI)-2- pyridinyljoxy]phenoxyj-propanoic acid, when applied with bentazon on sorghum (Soghum bicolor (L.)). When studying herbicide antagonism the treatment method may affect observed results. Westburg and Coble (101) reported reduced absorption and acropetal translocation of 1‘C-chlorimuron when acifluorfen was present. This contrasted with the findings of Shaw and Wesley (77) who found that acifluorfen increased absorption of “C-chlorimuron in common cocklebur. Shaw and Wesley (77) concluded that localized treatment rather than whole plant coverage with a spray solution was the basis for the differing results, indicating that with acetolactate synthase 22 (ALS) inhibiting herbicides, absorption and translocation are reduced when rapid, widespread membrane damage occurs, but that this does not occur when treatment IS confined to a IOCBIIZBO area. Hartzler and Foy (29) found that the antagonism could be eliminated by applying the bentazon and sethoxydim as separate applications. When bentazon was applied 1.5 hr or longer prior to the sethoxydim no antagonism of large crabgrass control was observed. A 0.2 hr interval between applications decreased control compared to sethoxydim applied alone, but the decrease was significantly less than occurred with a tank mix application of the two herbicides. Similar results were reported by Rhodes and Cable (68) who also demonstrated that sequential applications of the two herbicides did not result in antagonism. They found no trend to suggest that the order of the applications or the time between applications had any effect on sethoxydim efficacy on broadleaf signalgrass (Brachiaria glatyphylla (Griseb.) Nash. #3), fall panicum, and large crabgrass. Application intervals ranged from 1 second to 48 hours. The order of application had no effect on sethoxydim efficacy. Proposed Mechanisms. Rhodes and Coble (69) evaluated the effect of bentazon on goosegrass (Eleusine Indica (L.) Gaertn. #3) control with sethoxydim. The presence of bentazon decreased the foliar absorption of sethoxydim by about one-half. They also found that sethoxydim 23 translocation occurred in goosegrass whether or not bentazon was included in the spray mixture. When a bentazon formulation blank was substituted for bentazon, no antagonism occurred. Therefore, the authors concluded that the observed reduction in leaf penetration was due to the active ingredient (Na salt) of the bentazon formulation. Gerwick (23) reported that the bentazon antagonism of the methyl ester of haloxyfop could not be explained by direct chemical interactions or by contact injury from the bentazon. From his work he concluded that absorption was the primary locus of interaction between bentazon and the methyl ester of haloxyfop. However, chromatographic separation of the leaf rinsate showed greater than 90% of the label present as methyl ester both when the methyl ester of haloxyfop was applied alone or in the presence of bentazon. Furthermore, Gerwick’s (23) work did not support bentazon contact injury as a viable mechanism. In addition to bentazon, several analogs of bentazon with reduced activity were found to be effective antagonists of the methyl ester of haloxyfop. This data combined with the reported inhibitory action of bentazon on the absorption of other herbicides led Gemick to postulate that the crystallization of bentazon on the leaf surface may form a physical barrier that occludes the penetration of other xenobiotics. 24 In other studies with haloxyfop, Green et al. (18) examined the ester and free acid levels of haloxyfop in aqueous mixtures with bentazon, imazaquin, and chlorimuron. Using an n-hexane based extraction procedure combined with gas chromatography, they determined that the free acid and ester levels of haloxyfop were not reduced by the presence of the other herbicides. Aguiero-Alvarado et al. (I) examined the effect of bentazon and dicamba relative to preventing the haloxyfop induced inhibition of acetyl-CoA carboxylase. Based on their work, they concluded that interaction directly at the enzyme level was not a plausible explanation for the observed antagonism. Wanamarta et al. (98) observed that Na-bentazon inhibited the diffusion of “C-sethoxydim into and through isolated tomato fruit cuticles. This absorption inhibition was also evident on quackgrass leaves. In addition to Na-bentazon, other monovalent (Li, K, Cs) and divalent (Ca, Mg) cations produced the same inhibitory effect on sethoxydim absorption. The addition of organic acids to the solution overcame the absorption inhibition suggesting a direct molecular or ion association as the basis for the antagonism. They also found that Na-bentazon increased the partitioning of 1‘C-sethoxydim into dichloromethane and decreased partitioning into ethyl acetate. This bentazon induced change in partitioning further supports evidence of a direct interaction of the sethoxydim with part or all of the sodium salt of bentazon. Ultra violet 25 spectra of mixtures of the two herbicides gave no indication that complexes of the two compounds were formed. This suggests that the sodium salt of sethoxydim rather than a molecular complex is formed upon interaction with Na-bentazon. The Na+ from the Na-bentazon may be exchanged for a proton from the hydroxyl group on the sethoxydim ring structure, thus producing Na-sethoxydim (62). The formation of Na- sethoxydim or other alkaline or alkaline earth salts of sethoxydim results in a less preferred absorption form of sethoxydim (62). Couderchet and Retzlaff (16, 17) proposed that bentazon induced suppression of plasma membrane ATPase was responsible for the observed antagonism. By increasing ATP concentrations up to 2.0 mM in wheat leaf sections, uptake of sethoxydim was increased two fold. Addition of bentazon to the solution resulted in decreased sethoxydim absorption by the wheat leaf sections. In addition, bentazon solutions of 100 and 200 uM decreased ATPase activity 1 to 3% in a plasma membrane fraction isolated from wheat leaves. This difference was not statistically significant compared to untreated controls. However, when the bentazon concentration was increased to 500 uM, ATPase activity was decreased significantly. When the bentazon concentration was increased to 5mM, inhibition of ATPase activity reached 40%. At higher concentrations, bentazon precipitated from solution and inhibition was not much higher than that observed at 5mM. 26 The authors postulated that sethoxydim uptake is passive, however, decreased uptake of sethoxydim in the presence of bentazon is probably an indirect consequence of the inhibition of ATPases since it is unlikely that ATPases are directly responsible for sethoxydim uptake. This is based on the premise that ATPases are known to transfer small cations through membranes, and sethoxydim exits either as a nondissociated molecule or as an anion (pKa = 4.6). Inhibition of the ATPase would result in fewer protons pumped to the apoplast resulting in an increase in pH. The increased pH of the cell wall area would result in the dissociation of sethoxydim rendering it less lipophilic and less likely to pass through the plasmalemma. Conversely, an acidification of the apoplast outside the cell membrane in response to ATP activation of the ATPase would result in a greater concentration of nondissociated sethoxydim which would be the preferred form for uptake through the lipophilic plasma membrane. Therefore, the authors propose that bentazon inhibition of ATPase is responsible for the observed bentazon antagonism of sethoxydim. Shoaf and Carlson (81) reported that sethoxydim reacts spontaneously with water resulting in immediate structural changes. They found that light, moisture, oxygen, pH and soil duplicated this lability. Rhodes and Cable (69) observed the effect of bentazon on degradation of sethoxydim 27 using HPLC. Their data suggest that bentazon has no effect on the breakdown of sethoxydim, at least over the 6 hour duration of the study. Overcoming the Antagonism. The use of adjuvants to overcome the bentazon antagonism of sethoxydim and other graminicides has been examined. Gerwick et al. (24) studied the effect of urea ammonium nitrate (UAN) on overcoming this antagonism. They found that adding UAN to sethoxydim or haloxyfop in the presence of bentazon decreased the bentazon antagonism on grass activity. However, UAN increased this antagonism when used in conjunction with the bentazon and the methyl ester of haloxyfop. Gerwick speculated that the NH4 may interact directly with both the ionic herbicides as suggested by Wanamarta and with the plasmalemma as suggested by Carlson et al. (13) to produce complex differential effects on absorption. In identifying efficacious adjuvants, Wanamarta et al. (97) found no correlation between sethoxydim droplet spread and absorption on quackgrass and suggested that using 1‘C--herbicide absorption provided a more accurate basis for evaluating adjuvants than did droplet Spreadability. Wanamarta et al. (99) overcame the bentazon antagonism of “C-sethoxydim absorption in quackgrass with ammonium sulfate, ammonium phosphate or ammonium nitrate. In addition, by using an NH, salt of bentazon they did not observe the antagonism seen with Na- 28 bentazon. Wanamarta et al. (99) also evaluated the effect of the adjuvant BCH 815 00 on overcoming the bentazon antagonism of “C- sethoxydim. Based on their findings they concluded that the BCH 815 00 facilitated the penetration of sethoxydim through the quackgrass leaf cuticle. However, it probably did not prevent the formation of Na- sethoxydim, in sethoxydim bentazon tank mixes. Jordan and York (34) found that (NH,).,SO4 at 1.4 or 2.8 kg/ha was more effective at overcoming the antagonism than 5.6 kg/ha (NH,).,SO, at the 0.1 kg/ha rate of sethoxydim. The authors did not offer an explanation for the decrease in effectiveness at the higher rate of AMS. However, research on the lability of sethoxydim in solution (81) suggests that the Inability to overcome the antagonism may be a result of increased lability of the sethoxydim molecule under conditions of excessive AMS concentrations. Jordan and York (34) also partially alleviated the antagonism on corn and large crabgrass by substituting BCH 81508 for crop oil In sethoxydim bentazon tank mixes. Smith and Vanden Born (84) increased the absorption and translocation of sethoxydim on wild oats and barley with AMS plus crop oil concentrate. They found that AMS had little effect on sethoxydim absorption beyond 1 h after application. However, translocation of sethoxydim was increased 12 and 24 h after application. It was 29 speculated that the increased translocation was indirectly a result of the increased absorption. It appeared that the increase in sethoxydim absorption from AMS was dependent on the presence of COC. Beckett et al. (7) found a synergistic interaction when a nonionic surfactant or petroleum oil concentrate plus urea ammonium nitrate or ammonium polyphosphate were combined with sethoxydim to control giant foxtail. However, this reported synergism was inconsistent among differing environmental conditions and differing weed size at treatment. Gronwald et al. (26) reported that a surfactant was needed with AMS to promote imazethapyr (2-[4,5-dihydro-4-methyl-4-(1-methylethyl-5-oxo-1 H-imidazol-2- yI]-5-ethyI-3-pyridinecarboxylic acid) accumulation in quackgrass leaves but was not needed to promote imazethapyr accumulation in Black Mexican Sweet Maize cell cultures. The workers speculated that the surfactant plays an important role in the transcuticular penetration of both the herbicide and AMS. A surfactant is not always necessary to facilitate AMS reversal of cation antagonism of glyphosate (51,54,55). In the case of glyphosate, a water- soluble herbicide, it has been proposed that NH,+ may complex with glyphosate forming a readily absorbed compound (52). However, the absorption mechanism involved in the case of sethoxydim, a lipophilic molecule, is likely different from that of glyphosate a highly water-soluble molecule (84). 30 956 he DI ar Gronwald et al. (26) proposed an ion trapping mechanism for the increased absorption of imazethapyr in Black Mexican Sweet Maize cell cultures with AMS. Cellular uptake of NH,+ results in the generation of protons in the cytoplasm from the assimilation of NH,+ into organic nitrogen via glutamine synthetase and glutamate synthase. These protons would then be extruded via the plasma membrane ATPase. 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Food Chem. 40:2446-2452. 47 Chapter 2 The Basis for the Calcium Antagonism of Glyphosate Activity ABSTRACT Hard-water cations, such as Ca” and Mg”, present in the spray solution can greatly reduce the efficacy of glyphosate. These cations potentially compete with the isopropylamine in the formulation for association with the glyphosate anion. “C-glyphosate absorption by sunflower was reduced in the presence of Ca“. The addition of ammonium sulfate overcame the observed decrease in “C-glyphosate absorption. Nuclear Magnetic Resonance (NMR) was used to study the chemical effects of calcium and calcium plus ammonium sulfate (AMS) on the glyphosate molecule. Data indicate an association of calcium with both the carboxyl and phosphonate groups on the glyphosate molecule. Initially, a random association of the compounds occurred, however, the reaction progressed to yield a more structured, chelate type complex over time. NH,+ from AMS effectively competed with calcium for complexation sites on the glyphosate molecule. The data suggest that the observed calcium antagonism of glyphosate and AMS reversal of the antagonism are chemically based. Nomenclature: Glyphosate, N- 43 (phosponomethyl)glycine; AMS, diammonium sulfate (NH,)ZSO,; sunflower, Helianthus annuus L. 49 Introduction Recently, there has been renewed interest (10) in the previously reported (13) glyphosate antagonism by cations present in hard water. Shea and Tupy (14) reported a reduction in glyphosate activity on wheat (Triticum aestivum L. ’Centurk’) in the presence of 50 ppm calcium. Nalewaja (8) reported reduced control of wheat (Iriticum aestivum L.), sunflower (Helianthus annuus L.), kochia (Kochia scoparia (L.) Shrad. #3) and soybean (Glycine max (L.) Merr.) by glyphosate in the presence of calcium. In addition, the sodium salt, butoxyethyl ester, and diethanolamine formulations of 2,4-D [(2,4-dichlorophenoxy)acetic acid] antagonized glyphosate phytotoxicity to wheat (7). The authors suggested that 2,4-D formulation cations may exchange with the glyphosate is0propylamine formulation cation (IPA) resulting in a less absorptive compound than the IPA-glyphosate. Several researchers have documented an increase in glyphosate activity by decreasing carrier volume (2, 3, 13, 16). This was attributed to fewer cations such as Ca“ and Mg“ in the spray solution to associate with and deactivate the glyphosate molecule. 50 Stahlman and Phillips (16) showed that as spray solutions became more alkaline, glyphosate activity tended to decrease. Buhler and Burnside (3) documented an increase in glyphosate activity by adding various acids, HCI, H280“ and acetic acid. H280, was found to be the most effective acid at overcoming the antagonism and it was proposed that 80,4 lens may compete for Ca” forming CaSO,. In addition to forming conjugate salts and removing cations from solution, the addition of acid to the spray solution may result in the protonation of the negatively charged moieties on the glyphosate molecule. The resultant nonionic glyphosate molecule would be expected to more readily pass through the cuticle as compared to the conjugate cation-glyphosate salt. Calcium antagonism of glyphosate has been overcome with ammonium sulfate (7, 8, 10). This effect has been found to vary with plant species (8) and spray carrier volume (12). Sulfate ions may compete with glyphosate for the antagonistic Ca” cations thus deterring the formation of glyphosate Ca” complexes (11). Another possible mechanism for the reversal of the observed antagonism is a direct interaction between the NH,+ cation and the glyphosate molecule resulting in NH,-glyphosate, which may be more readily absorbed than the conjugate salt of Ca- glyphosate (7). 51 The chelating properties of glyphosate in solution have been documented (4, 15). Motekaitis and Martell (6) investigated the possibility of a 2:1 glyphosate to Ca” ligand. However, using potentiometric equilibrium data, they found that even a fourfold excess of glyphosate would not form exclusively 2:1 complexes. Therefore, the authors proposed a 1:1 bidentate planer arrangement with the metal chelated by two oxygens on the phosphonate functional group. Madsen et al. (5) proposed that glyphosate formed a trident ligand. More recently, Subramaniam and Hoggard (17) used infrared data to show that glyphosate acts as a tetradentate ligand with coordination through the amine nitrogen, the carboxylate oxygen and the phosphonate oxygens. The objective of this research was to apply the techniques of NMR to determine if glyphosate chemically interacts with Ca” in solution and, if so, to determine if NH: ions could effectively compete with Ca“ for active sites on the glyphosate molecule. 52 Materials and Methods Absorption studies. Sunflower (Interstate hybrid 7000, West Fargo, ND.) and tall morningglory (lgomoea purpurea (L.) Roth) seeds were planted in 945-ml plastic pots. After emergence, plants were thinned to two plants per pot. Plants were grown at (24 i 2 C) with supplemental lighting from high-pressure sodium lights which produced an irradiance of 600 uE rn'2 s‘1 PPFD to provide a total of 1200 uE m“2 s" for both supplemental and natural light. Day length was 16 h. Sunflower plants were at the four-leaf stage and tall morningglory plants were at the two- leaf stage at the time of treatment. Treatment consisted of one-2 ul. drop, containing formulated glyphosate (Roundup‘), spiked with 0.021 uCi of 1‘C-glyphosate (Spec. act. = 11.0 MBq mg" labeled at the methyl carbon of the N-phosphonomethyl glycine). Total glyphosate concentration was equivalent to 100 g/ha, at 160 Uha carrier. Ca-glyphosate was prepared by adding CaCl, at 12.5 mM (500 ppm Ca”) to the spotting solution (8). Diammonium sulfate was included alone or with calcium chloride at 0.5% w/v, equivalent to a 3:1 molar ratio with CaClz, which was reported as the ratio necessary to 1Roundup, from Monsanto company, 800 N. Linbergh Blvd., St. Louis, MO 63167 53 overcome Ca” antagonism (9). The drops were applied to the adaxial surface of the first true leaf pair. At 4, 24, and 48 hr after treatment the treated leaves were excised and rinsed with 4 to 5 ml of distilled water to remove unabsorbed 1‘C- glyphosate. Rinsate was radioassayed by liquid scintillation spectrometry. A completely randomized design was used with four replications per treatment. Experiments were conducted twice and the results were combined. Following an analysis of variance, treatment means were separated by the least significant difference test at the 0.05 probability level. Nuclear Magnetic Resonance. Analytical experiments were conducted to determine the effect of Ca“ on the free acid glyphosate zwitterion, and the isopropylamine formulation of glyphosate. In addition, the effect of AMS ((NH,)ZSO,) on glyphosate and glyphosate plus Ca” was examined. Technical grade glyphosate (87%) and isopropylamine formulations of glyphosate, sold as Roundup were obtained. Reagent grade calcium acetate (CH,COO),CaH.,O, calcium carbonate CaCO,, magnesium acetate (CH3COO)2MgHZO, and diammonium sulfate (NH,)ZSO, (AMS) were used in the indicated treatments. Deuterium oxide (020) was used as the carrier solvent for all NMR analytical observations. 54 NMR spectra were obtained on a Varian2 VXR 500. 1H spectra were obtained on a Varian 5 mm high resolution ‘H/"F probe. "P and 13C spectra were obtained on a Varian 5 mm broadband 15N/"‘P probe and decoupled spectra were obtained on a Varian 5 mm inverse detection broadband probe. The HOD peak was standardized to 4.65 ppm for all spectra. The glyphosate concentration was 0.026 molar which corresponds to a field application rate of 1.7 kg/ha at 370 Uha carrier (1.5 lb glyphosate per acre at 40 gpa). Calcium was added at either a 1:1 or 4:1 ratio with glyphosate and magnesium at a 1:1 ratio. This corresponds to a Ca‘+ concentrations of 1064 mg/L and 4258 mg/L and a Mg’+ concentration of 632 mg/L. AMS was used at concentrations of three times the Ca“ ion concentration as reported to be effective in overcoming Ca++ antagonism in wheat (9). 2Varian Associates Inc., Nuclear Magnetic Resonance Instruments, 3120 Hansen Way, Palo Alto, CA 94304-1030 55 Results and Discussion Absorption studies. The results for the 1‘C-glyphosate absorption greenhouse study on sunflower and tall morningglory are depicted In Tables 1 and 2. Significantly less “C from Ca-glyphosate was absorbed by sunflower than NH,-glyphosate at all subsequent harvest times (Table 1.). Ca-glyphosate also showed less “C absorption by sunflower than isopropyl amine (IPA) glyphosate at the 4 hr and 48 hr harvest times. The addition of AMS overcame the Ca” antagonism. These results are consistent with the findings of Nalewaja and Matysiak (8) who reported an increase in sunflower fresh weights when calcium was present in the spray carrier. In addition, Nalewaja and Matysiak reported that sunflower fresh weights decreased with the inclusion of AMS to the glyphosate and CaCl solution. This supports the hypothesis that calcium interacts chemically with glyphosate in solution to create a less readily absorbed Ca-glyphosate complex. Concomitantly, the reversal of the antagonism with AMS appears to be due to the formation of NH,-glyphosate. However, definitive proof on the formation of the proposed glyphosate conjugates is necessary to establish that the observed antagonism is chemically based. 56 Absorption of “C-glyphosate by tall morningglory was very poor for all treatments and statistical analyses indicated no differences between treatments (Table 2.). The tall morningglory plants were in the two-leaf stage and four-inches tall at the time of treatment which is taller than the current recommended height for control with glyphosate. Dissimilar responses to glyphosate treatments by different plant species has been reported previously by Nalewaja and Matysiak (8). Nuclear Magnetic Resonance. The results from the “C-glyphosate absorption study suggest the formation of a Ca-glyphosate conjugate in solution which Is less readily absorbed than IPA-glyphosate or NH,- glyphosate. Nuclear magnetic resonance spectrometry was used to evaluate various glyphosate spray carrier solutions to determine whether glyphosate conjugates are formed in the spray solution. The resultant ‘H NMR spectrum for the technical grade free acid glyphosate Is depicted in Figure 1-F. Area integration of the peaks present at 3.9 and 3.18 ppm show a 1:1 relationship. These peaks correspond to the two protons on each of the two methylene groups. In the "P decoupled 1H spectrum (Figure 1-E) the doublet at 3.18 ppm collapsed to yield a singlet. Therefore, it can be concluded that the doublet at 3.18 ppm represents the protons on the methylene carbon adjacent to the phosphonate group and the peak at 3.9 ppm corresponds 57 to the protons on the methylene carbon adjacent to the carboxyl group. This is consistent with the findings of Appleton et al. (1) who also determined the above peak assignments. With the introduction of Ca”, several changes were observed in the NMR spectra (Figure 1-D). The peak at 3.9 ppm shifted upfield indicating that the protons are in a more electronically shielded environment. Therefore, It appears that Ca” is associated with the carboxyl group, forming a conjugate salt. The doublet at 3.18 ppm also shifts upfield, to a somewhat lesser extent than the peak corresponding to the carboxyl methylene protons, suggesting that Ca” may also be binding to the phosphonate group. To verify the association of the Ca” with the carboxyl group, 1"'C NMR spectra (Figure 2) were obtained for the glyphosate free acid and glyphosate plus Ca” treatments. These spectra appear less defined with much more noise in the base line than do 1H NMR spectra, since 1“C accounts for only 1.1% of the total C population. Thus, we detected a significantly smaller segment of the total elemental pepulation with “C NMR. The top spectrum (2-A) which corresponds to the glyphosate plus Ca” treatment, shows a downfield shift for the carboxyl carbon peak when compared to the spectra for the free acid glyphosate (2-B). This Indicates that the carbon was in a more deshielded electronic 58 environment which would be expected since the ‘H NMR spectrum indicated that the methylene protons were electronically shielded in the presence of Ca“. Therefore, the "C NMR supports the hypothesis that the Ca” associates with the carboxyl group of glyphosate to form a Ca- glyphosate conjugate salt. 31P spectra were also obtained for the glyphosate free acid (Figure 3-D) and glyphosate plus Ca” (1:1 molar ratio) (Figure 3-C) treatments. One triplet peak is apparent representing the phosphonate P. The peak is split into a triplet by coupling with the two adjacent protons. The spectra show a downfield shift in the presence of Ca”. This verifies the contention based on the ‘H-NMR work that Ca” is also associated with the phosphonate group of the glyphosate molecule. Mg’+ elucidated a similar 1H-NMR peak shift response as Ca“ (Figure 1- C). The peak shifts were consistent with those of Ca“ indicating that Mg“ also associates with both the carboxyl and phosphonate groups of the glyphosate molecule. Based on these findings, a random association of Ca“ and Mg“ with glyphosate is proposed. In the presence of dilute concentrations of hard water cations, glyphosate would be expected to associate with the cations at both the carboxyl and phosphonate groups of the glyphosate molecule. A series of ‘H spectra were produced over time (Figure 4). These results indicate that the Ca-glyphosate complex formed a more structured chelate type orientation over time. The formation of a metal ligand is consistent with the findings of Madsen et al. (5) and Subramaniam and Hoggard (17). The shifting of the peaks corresponding to the methylene protons nearest the phosphonate group suggest the chelated, or more structured orientation occurred, under laboratory conditions, approximately 70 min after introduction of the Ca” to the spray solution. The ‘H NMR spectra for the IPA formulation of glyphosate and IPA formulated glyphosate in the presence of Ca“ at 1:1 and 1:4 molar ratios are shown in Figure 5. Although the components of the formulation complicate the spectra, the peaks corresponding to the methylene protons are visible. The ‘H-NMR spectra for IPA-glyphosate (Figure 5-C) did not indicate a relative shift of the singlet peak corresponding to the carboxylate methylene protons when Ca++ (Figure 5-B) was added. Because the position of the singlet peak for IPA-glyphosate (Figure 5-C) is similar to that of Ca-glyphosate (Figure 1-C), ‘H-NMR did not provide the resolution necessary to discern a substitution of Ca” for IPA on the carboxylate group of the glyphosate molecule. However, the ‘H-NMR spectra do show a relative upfield shift of the doublet peak Indicating the substitution of Ca” for IPA. on the phosphonate group of the glyphosate molecule. When the Ca” concentration was increased to a 1:4 molar 60 ratio with the IPA-glyphosate a correspondingly increased upfield shift in the phosphonate methylene proton doublet (Figure 5-A) was observed. Therefore, it appears that in the presence of excess Ca”, the Ca” associates at multiple sites on the phosphonate group. 31P-NMR was also used to examine the effect of Ca’+ on IPA glyphosate (Figure 6-B). The upfield shift apparent in the "P-NMR spectra more clearly indicates the replacement of the isopropylamine with Ca” on the phosphonate group of the glyphosate molecule. When the Ca++ concentration was increased to a 1:4 ratio (Figure 6-A) a corresponding increase in the upfield peak shift was observed. This supports the findings of the ‘H-NMR work that in the presence of excess Ca”, the Ca” associates at multiple oxygens on the phosponate group. Therefore, we may conclude that the Ca” associates with the IPA formulated glyphosate in the same manner as the technical grade free acid glyphosate. Figure 1-B shows the ‘H NMR spectra of glyphosate plus (NH,)ZSO,. In the presence of AMS, both peaks shift upfield, similar to that observed with Ca” plus glyphosate. Therefore, NH,‘ complexes directly with the glyphosate molecule similar to Ca”. In the ‘H NMR spectrum for the glyphosate + Ca“ + NH,’ three-way mixture (Figure 1-A), the potential substitution of NH; for Ca” on the phosphonate or carboxylate group is 61 not readily discernible by observation of shift changes at the methylene proton level. However, as seen with IPA-glyphosate plus Ca“. the shift change is apparent by observation of the "P spectra (Figure 3). This shift change indicates that NH,’ is effectively competing with Ca” and residing on the phosphonate group of the glyphosate molecule in solution. In conclusion, the results indicate that hard-water cations such as Ca” and Mg++ interact with both the phosphonate and carboxyl functional groups of the glyphosate molecule (Figure 7: step 1) to form less readily absorbed glyphosate salts. Over time, the association of the cations with glyphosate progresses to a more structured chelate orientation (Figure 7: step 2). This research did not support the proposed cation association at only the phosphonate group of the glyphosate molecule as proposed by Motekaitis and Martell (6). NH,‘ was found to compete with Ca++ for bonding sites on the glyphosate molecule (Figure 7: step 3). The substitution of NH; for Ca” on the glyphosate molecule results in a more readily absorbed form of glyphosate. The formation of NH,-glyphosate and the removal of Ca“ from solution by the conjugate sulfate ion appear to be the bases for the reversal of the Ca” antagonism of glyphosate absorption in the presence of AMS. 62 ACKNOWLEDGEMENTS The NMR data were obtained on instrumentation that was purchased in part with funds from HIH grant #1 -S10-RR04750, NSF grant #CHE-88- 00770, and NSF grant #CHE-92-13241. 63 lab_lei The effect of the conjugate salts: IPA (isopropylamine), Ca (calcium) AMS (ammonium sulfate) Ca + AMS (calcium + ammonium sulfate) on “C-Glyphosate absorption by sunflower. Absorption presented as % of total applied. Time after application (h) Glyphosate salt 0 4 24 48 ------ % absorbed - - - - - - IPA' 1.4 22.6 20.3 32.5 Ca 1.2 4.9 8.0 6.5 AMSb 1.5 28.8 30.0 30.0 Ca + AMS ' 0.8 21.9 28.5 25.3 LSD (0.05) NS 15.2 13.3 18.6 ‘IPA is isopropylamine I’AMS is diammonium sulfate Table 2: The effect of the conjugate salts: IPA (isopropylamine), Ca (calcium) AMS (ammonium sulfate) Ca + AMS (calcium + ammonium sulfate) on "C-Glyphosate absorption by tall morningglory. Absorption presented as % of total applied. Time after application (h) Glyphosate salt 0 4 24 48 ------ % absorbed - - - - - - IPA' 2.5 3.3 4.6 3.8 Ca 2.0 5.8 4.8 6.4 AMSb 1.5 3.1 3.3 4.0 Ca + AMS 2.1 4.1 3.9 3.2 LSD (0.05) NS NS NS NS aIPA is isopropylamine bAMS is diammonium sulfate 65 Figure 1. 1H-NMR spectrum of a) technical grade glyphosate plus calcium acetate plus ammonium sulfate; b) technical grade glyphosate plus AMS; c) technical grade glyphosate plus magnesium acetate 1:1 molar ratio; d) technical grade glyphosate plus calcium acetate 1:1 molar ratio; 9) "P decoupled ‘H-NMR spectrum of technical grade glyphosate; and, f) technical grade glyphosate. D20 was the carrier solvent in all treatments. 66 Figure 2. 13C-NMR spectrum of a) technical grade glyphosate plus calcium acetate 1:1 molar ratio; and, b) technical grade glyphosate. D,_O was the carrier solvent in both treatments. 68 d :.-1‘ J Rum . Hum nu» pwo pm 5 =35? _ E. 7 :22 255.5: EEEEPEEE pm“ ”mu Hmm 9mm nus Figure 3 "P-NMR spectrum of a) technical grade glyphosate plus calcium acetate plus AMS 1:1:3 molar ratio; b) technical grade glyphosate plus AMS; 0) technical grade glyphosate plus calcium acetate 1:1 molar ratio; and, d) technical grade glyphosate. 020 was the carrier solvent in all treatments. 70 I I uidd Figure 4. ‘H-NMR spectrum of technical grade glyphosate plus calcium acetate 1:1 molar ratio in 020. Spectrum obtained from 40 minutes (bottom) to 820 minutes (top) after mixing. 2;; PPPPPFPPPFFFFHHHHH iiiihlllll:ltfiiffiié§ Figure 5. 1H-NMR spectrum of a) commercially formulated isopropylamine glyphosate plus calcium acetate 1:4 molar ratio; b) commercially formulated isopropylamine glyphosate plus calcium acetate 1:1 molar ratio; and, c) commercially formulated isopropylamine glyphosate. D20 was the carrier solvent in all treatments. 74 E: :— — MAI—H wb _ Figure 6. "P-NMR spectrum of a) commercially formulated isopropylamine glyphosate plus calcium acetate 1:4 molar ratio; b) commercially formulated isopropylamine glyphosate plus calcium acetate 1:1 molar ratio; and, c) commercially formulated is0propylamine glyphosate. D20 was the carrier solvent in all treatments. 76 _.1~__—-—_—__u—_____—______-_—q__——-__—_u-W——-___—-_—__-d—_—_ua1—|—J—__-—_____.fi—d—q~jJ—u—d_—_—._udd_ 0.5 0.m 10.0 10.m 10.5. 10.0 10.0 IE0 [EN 003 Figure 7. Scheme of hard-water cation association with glyphosate in solution: step 1) proposed association of Ca” with carboxyl and phosphonate functional groups of glyphosate based on Figures 1 through 3; step 2) progression towards tridentate and tetradentate ligand as supported by Figure 4; step 3) proposed formation of NH,—glyphosate and formation of CaSO, based on Figures 1 and 3. 78 ‘ NH * HO HH 0 "o-HH—oo' ‘ ‘7 v ‘7 I + + 03804 NH +,_O'E\/N\/“o— HO’fiVNvgo 4 O x O NH,t Random association of NH41 with glyphosate and formation of CaSO4 NH O +03” ( 0:43 HO H Ho--,Caf*"'9‘ H OH o”|\/Nvf’."0‘ HO'fi'Vfix/go Ca‘+ O 0 4:1 GIyphosate:Ca“ HO ”H“ OH Motekaitis and Martell (6) I -o-P N” n 0 Ca“- 3"” H H HO H HO",Ca W0 o’JVNvg‘ ‘O" C 3..., 1:1 Glyphosate20a” Step 1 Motekaitis and Martell (a) I— ...cat: -9 O- HOI-P " 'ISVl-fN 0 Cart" oH‘g HO.-'Ca::-- (v:- H H O_‘~Ca"" OI/K’NV Voqo— Hos-PVvao — Random association of Ca“ with glyphosate '— Ca” 1" 0 HO" I; : ~ ' ‘ I 0— L0 I — .5. —* P I. f HohfiVN\/go O: \V‘N o ‘ H Tridentate ligand Tetradentate ligand Madsen er al. (5) Subramaniam and Haggard (17) Literature Cited Appleton, T. G., J. R. Hall, and I. J. McMahon. 1986. NMR Spectra of iminobis (methylenephosphonic acid), HN(CH2PO.,H2)2 and related ligands and of their complexes with platinum(ll). Inorg. Chem. 25:726-734. Buhler, D. D., and O. C. Burnside. 1983. Effect of spray components on glyphosate toxicity to annual grasses. Weed Sci. 31:124-130. Buhler, D. D., and O. C. Burnside. 1983. Effect of water quality, carrier volume, and acid on glyphosate phytotoxicity. Weed Sci. 31:163-169. Glass, R. L. 1984. Metal complex formation by glyphosate. J. Agric. Food Chem. 32:1249-1253 Madsen, H. E. L., H. H. Christensen, and C. Gottlieb-Petersen. 1978. Stability constants of copper (II), zinc, manganese (ll), calcium, and magnesium complexes of N-(phosphonomethyl)glycine (glyphosate). Acta Chem. Scand. A32:79-83. Motekaitis, R.J. and A. E. Martell. 1985. Metal chelate formation by N-phosphonomethylglycine and related ligands. J. Coord. Chem, 142139-149. 10. 11. 12. 13. Nalewaja, J. D., and R. Matysiak. 1992. 2,4-D and salt combinations affect glyphosate phytotoxicity. Weed Technol. 6:322- 327. Nalewaja, J. D., and R. Matysiak. 1992. Species differ in response to adjuvants with glyphosate. Weed Technol. 6:561-566. Nalewaja, J. D., and R. Matysiak. 1992. Diammonium sulfate effects on glyphosate phytotoxicity. Abstr., Adjuvants for Agrochemicals, Third International Symposium Organized by the SCI Pesticides Group. Nalewaja, J. D., and R. Matysiak. 1991. Salt antagonism of glyphosate. Weed Sci. 39:622-628. Nalewaja, J. D., R. Matysiak, and 1T. P. Freeman. 1992. Spray drOpIet residual of glyphosate in various carriers. Weed Sci. 40:576-589. O’Sullivan, P. A., J. T. O’Donovan, and WM. Hamman. 1981. Influence on nonionic surfactants, ammonium sulfate, water quality and spray volume on the phytotoxicity of glyphosate. Can. J. Plant Sci. 61 :391-400. Sandberg, C. L., W. F. Meggitt, and D. Penner. 1978. Effect of diluent volume and calcium on glyphosate phytotoxicity. Weed Sci. 26:476-479 81 14. 15. 16. 17. Shea, P. J. and D. R. Tupy. 1984. Reversal of cation-induced reduction in glyphosate activity with EDTA. Weed Sci. 32:802- 806. Shkol’nikova, L. M., M. A. Porai-Koshits, N. M. Dyatlova, G. F. Yaroshenko, M. V. Rudomino, and E.K. Kolova. 1982. X-ray structural study of organic ligands of the complexone type. III. Crystal and molecular structure of phosphonomethylglycine and iminodiacetic-monomethylphosphonic acid. J. Struc. Chem. 23:737- 746. Stahlman, P. W., and W. M. Phillips. 1979. Effects of water quality and spray volume on glyphosate toxicity. Weed Sci. 27:38- 41. Subramaniam, V. and P. E. Hoggard. 1988. Metal complexes of glyphosate. J. Agric. Food Chem. 336:1326-1329 Chapter 3 Characterizing the Sethoxydim-Bentazon Interaction with Proton Nuclear Magnetic Resonance Spectrometry. ABSTRACT The antagonistic effect of Na-bentazon on sethoxydim absorption and herbicidal activity has been documented. The addition of ammonium sulfate (AMS) with a surfactant overcomes the observed loss of sethoxydim activity. Nuclear Magnetic Resonance (NMR) was used to study the chemical effects of commercially formulated Na-bentazon, NaCI, NaHCO,, Na-bentazon plus AMS, and NaHCO3 plus AMS on commercially formulated sethoxydim. Technical grade Li-sethoxydim and sethoxydim were analyzed and ‘H-NMR spectra were used as comparative standards. Data indicate an association of Na‘ from Na- bentazon, Ncho,, and NaCl with the sethoxydim molecule. NH,” from AMS appears to associate spatially with sethoxydim but does not exert the same electronic effect on the sethoxydim ring protons as observed with Na+ or Li‘. The addition of AMS to sethoxydim plus Na-bentazon or NaHCO3 treatments prevents the complexation of Na” with the sethoxydim 83 molecule. The data support the hypothesis that the observed Na- bentazon antagonism and ammonium sulfate reversal of antagonism are chemically based. Nomenclature: Bentazon, 3-(1-methylethyI)-(1H)-2,1,3- benzothiadiazIn-4(3H)-one 2,2-dioxlde; sethoxydim, 2-[1- (ethoxyimino)butylj-5-[2-(ethylthio)propyI]-3-hydroxy-2-cyclohexen-1 -one; ‘ AMS, diammonium sulfate (NH,)ZSO,. Introduction Several mechanisms have been proposed to explain bentazon antagonism (4, 6, 8) of sethoxydim activity. Couderchet and Retzlaff (1, 2) proposed that the bentazon induced suppression of plasma membrane ATPase was responsible for the observed antagonism. The authors concluded that the increased pH of the cell wall area due to the inhibition of the plasma membrane ATPase results in the deprotonation of sethoxydim rendering it ionic, less lipophilic, and less likely to pass through the plasmalemma. Other researchers have identified the leaf cuticle as the point of antagonism. Rhodes and Coble (9) reported that bentazon reduced the foliar absorption of sethoxydim on goosegrass (Eleusine indica (L.) Gaertn.) by about one-half. Wanamarta et al. (11) observed that Na- bentazon inhibited the diffusion of 1‘C-sethoxydim into and through isolated tomato (Lycopersicon esculentum Mill.) fruit cuticles. In addition to Na-bentazon, other monovalent (Li, K, Cs) and divalent (Ca, Mg) cations produced the same inhibitory effect on sethoxydim absorption through the detached cuticles. Furthermore, NH,-bentazon did not produce an antagonistic effect on sethoxydim absorption suggesting that the Na’ from the Na-bentazon formulation was the active antagonist. This was supported by Rhodes and Coble (9) who demonstrated that the observed Na-bentazon antagonism of sethoxydim activity on goosegrass could be avoided when a formulation blank was substituted for Na- bentazon. Sethoxydim exists as a weak acid in solution with the pKa of the ring hydroxyl reported to be 4.6 (2). Therefore, in neutral solutions, the sethoxydim molecule would be primarily in the deprotonated state which would facilitate association with Na’ or other cations in the spray solution. The formation of Na-sethoxydim or other alkaline or alkaline earth salts of sethoxydim results in a less preferred absorption form of sethoxydim (7). The bentazon antagonism of sethoxydim absorption and activity has been overcome with the addition of NH; ions in the spray solution (3, 5). Smith and Vanden Born (10) reported a two-fold increase in sethoxydim uptake in wild oats (Avena fatua L.) and barley (Hordeum vulgare L. ’Klondike’) with the addition of AMS. Wanamarta et al. (12) reported inhibition of sethoxydim absorption with alkaline and alkaline earth acetate salts. However, NH,-acetate did not Inhibit sethoxydim absorption, indicating that NH,-sethoxydim could be as readily absorbed as the parent acid sethoxydim. Further work by Wanamarta et al. (12) indicated a possible effect from the NH,+ conjugate base. Ammonium 86 sulfate, ammonium phosphate, and ammonium nitrate were found to be effective at overcoming Na—bentazon antagonism. However, ammonium acetate, urea, and ammonium hydroxide did not overcome the antagonism on sethoxydim absorption. The objective of this research was to apply the techniques of NMR to determine if Na’ from Na-bentazon formulations interacted chemically with sethoxydim in solution, and to determine the chemical interactions that are the bases for the reported reversal of the antagonism with ammonium sulfate. 87 Materials and Methods Analytical experiments were conducted to determine the chemical effects of Na-bentazon, NaCI, NaHCO,, Na-bentazon plus AMS, and NaHCO3 plus AMS on sethoxydim. Technical grade sethoxydim (96%), and Li- sethoxydim (75%) were obtained and used as standards for comparison. Commercial formulations of sethoxydim and Na-bentazon were obtained as Poast‘ and Basagran2 respectively. Reagent grade NaCI, NaHCO._,, and (NH,),SO, (AMS) were also used. Deuterated chloroform (CDCla) was used as the carrier solvent for all NMR analytical observations. Treatments were mixed in distilled water and a 0.35 ml aliquot was removed and kept In the dark at room temperature for 24 h. The sethoxydim concentration was 4.6mM which is equivalent to 0.42 kg/ha in 280 Uha carrier. The bentazon concentration was 33.6 mM which is equivalent to 2.24 kg/ha in 280 Uha carrier. The NaCI and NaHCO3 sodium salts were used at 33.6 mM concentrations which are equal to that used for Na-bentazon. The AMS concentration was 75.6 mM to provide a final solution concentration of 0.5% w/v. 1BASF Corp., 100 Cherry Hill Road, Parsippany, NJ 07054 2BASE“ Corp., 100 Cherry Hill Road, Parsippany, NJ 07054 After reaction in the distilled water the 0.35 ml aliquots were put under forced N2 gas for approximately 2 h until the water had evaporated. The treatments were then redissolved in 0.7 ml of CDCla. Technical grade Li-sethoxydim and sethoxydim were also dissolved directly in 0001, for comparative standards. NMR spectra were obtained on a Varian3 VXR 500. A Varian 5 mm high resolution 1H/"’F probe was used for all spectra. The CDCI3 solvent peak was standardized to 7.24 ppm for all spectra. 3Varian Associates Inc., Nuclear Magnetic Resonance Instruments, 3120 Hansen Way, Palo Alto, CA 94304-1030 Results and Discussion Sethoxydim. The chemical structure of sethoxydim is depicted in Figure 1. The molecule has 13 different carbons with protons which produce the relatively complex 1H-NMR spectra. The solvent peak is apparent at 7.24 ppm. The peaks beginning with the peak for the two methylene protons on carbon 16 appear on the spectrum in order of increasing electronic shielding. The relatively downfield position of the carbon 16 proton peak is due to the electronic deshielding of the adjacent ethoxy imino group. Sethoxydim is a weak acid with the pK. of the ring hydroxyl proton reported to be 4.6 (2). Therefore, at neutral solution pH the majority of the molecule is in the deprotonated state and exists as an anion. Molecular association with a cation would be expected to occur at the negatively charged ring hydroxyl. Because of the conjugation between the two oxygens on the ring, the negative charge would likely resonate at some equilibrium between the two oxygens. Association with cations in solution would be discemable with ‘H-NMR by measuring the change in the electronic environment surrounding the adjacent ring protons (carbon 4 and 6). The spectral peaks corresponding to these protons occur between 2 and 3 ppm on the ‘H-NMR scale. Peak assignments were confirmed with personal communication from BASF Aktiengesellschaft (unpublished data). 90 The peaks for the equatorial protons of carbons #4 and #6 appear between 2.6 and 2.7 ppm (Figure 2-E). The corresponding peaks for the axial protons on carbons 4 and 6 appear in the region of 2.2 ppm. The remaining ring proton, located on carbon 5 defines the peak near 2.3 ppm. The other technical grade standard was Li-sethoxydim. The 1H- NMR spectrum for Li-sethoxydim (Figure 2-D) compared to the parent acid formulated sethoxydim (Figure 2-E) shows several differences. The peaks corresponding to the equatorial protons on carbon 4 and 6 have widened in the upfield direction with the most electronically shielded peak now appearing at 2.46 ppm. The peaks corresponding to the axial protons on carbons 4 and 6 have split with approximately one-half of the integrated peak area moving upfield to 2.1 ppm and the remainder moving downfield under the existing peak at 2.3 ppm assigned to the proton at carbon 5. This provides evidence that the Li+ associates with the hydroxyl oxygen on the sethoxydim ring. The splitting of the peaks suggests that two or more orientations, possibly axial and equatorial to the ring structure occur. The conjugation between the carbonyl and hydroxyl oxygen and the resultant resonating negative charge may contribute to the observed isomerism. 91 Sethoxydim + Ila-bentazon. Figure 20 shows the spectrum obtained for the sethoxydim + Na-bentazon treatment. Na-bentazon does not exhibit peaks on the 1H-NMR spectrum scale between 2 and 3 ppm where the sethoxydim ring proton peaks are located (data not presented). Therefore, the Na-bentazon will not obscure the ‘H-NMR sethoxydim peaks when analyzed together In solution. As seen with Li-sethoxydim, the protons located on carbons 4 and 6 show a shift indicating that Na+ associates directly with the hydroxyl oxygen on the sethoxydim ring. Peak area integration of the split axial ring proton peak areas of Li- sethoxydim and sethoxydim + Na-bentazon show an identical ratio, indicating that the Na‘ associates with sethoxydim in a manner very similar to Li+ and results in a similar isomeric ratio as seen in the ‘H- NMR spectra of the technical grade Li-sethoxydim standard. If Na’ from Na-bentazon is associating with the sethoxydim molecule in solution, Na’ from other sources would be expected to give the same molecular association. To determine this, NaHCO3 (Figure 2-B) and NaCl (Figure 2-A) were also evaluated for their effect on the sethoxydim molecule. These spectra show a similar shift pattern of the peaks corresponding to the sethoxydim axial and equatorial ring protons at carbons 4 and 6 in the presence of the indicated alternate Na’ sources. Therefore, it can be concluded that the Na‘ ion from these alternate Na’ sources associates with the sethoxydim molecule In the same manner as 92 Na+ from Na-bentazon. The effect of Na-bentazon on the sethoxydim molecule in solution was further studied with ‘H, 1H homonuclear double quantum filter correlated spectrometry (COSY NMR). This technology is most useful in determining peak assignments as cross reference to perpendicular peaks indicates spin coupling. The 2-D COSY spectrum can also confirm conventionally obtained peak shifts. The COSY spectra for sethoxydim is shown in Figure 3-8. The peaks appearing on the diagonal axis correspond to the one dimensional spectrum shown to the left of the vertical axis. The individual peaks also appear as mirror images on either side of the diagonal. The vertical and horizontal scale for each respective side can be used to locate the peaks. For example, the axial ring proton peak is apparent at 2.2 ppm on the horizontal and vertical scale. By drawing a perpendicular line from either peak to the corresponding scale, the line positions at approximately 2.6 ppm which is the position for the equatorial ring proton peaks, indicating spin coupling between the two. The COSY spectrum for sethoxydim plus Na-bentazon is shown in Figure 3-A. More spectral noise is apparent with the addition of Na-bentazon due to the higher concentration of Na-bentazon relative to sethoxydim. The vertical resolution was maintained at a relatively low altitude to detect 93 the low concentration of sethoxydim. At the low altitude, much noise from the higher concentration of Na-bentazon was apparent. However, despite the increased noise, the functional sethoxydim peaks are still visible on the spectra. The axial proton peak which is apparent at 2.2 ppm for straight sethoxydim is now split into two separate peaks occurring at 2.1 and 2.3 ppm. These new peaks are coupled to peaks at 2.5 and 2.6 ppm respectively which correspond to the now shifted equatorial ring proton peaks. Thus, the COSY spectra confirm the results obtained with the conventional one dimensional spectra indicating a direct molecular association of the Na’ with the sethoxydim hydroxyl oxygen. Effect of AMS. Figure 4-C depicts the spectrum obtained from the treatment of sethoxydim + AMS. There is a slight upfield shift of the peak corresponding to the axial ring protons on carbons 4 and 6, however, the characteristic shift pattern seen with Na’ and Li’ was not apparent. This indicates that the NH,+ ion associates with the sethoxydim molecule, however, this association is dissimilar to that observed with Na” and Li’. The absence of the splitting of the axial and equatorial proton peaks for carbons 4 and 6 indicates a homogenous orientation of the NH,—sethoxydim, unlike the isomeric orientation observed with Na- sethoxydim and Li-sethoxydim. This may be a function of size, whereby the relatively larger size of the NH,‘ ion may result in a more spatially distant molecular association. Figure 4-8 shows the spectrum obtained 94 for the sethoxydim plus Na-bentazon plus AMS treatment. The characteristic shift pattern observed with Na—sethoxydim was not observed. Rather, the spectrum very closely resembled the spectrum obtained for the sethoxydim plus AMS treatment, indicating that NH,’ effectively prohibits the formation of Na-sethoxydim. The AMS effect was also evaluated for alternate Na’ sources. Figure 4-A shows the spectrum obtained for NaHCO3 plus sethoxydim plus AMS. The spectrum is consistent with that obtained for Na-bentazon plus sethoxydim plus AMS, indicating that the NH,+ effectively prohibits the formation of Na- sethoxydim. In conclusion, Na’ from Na-bentazon formulations associated with sethoxydim in solution. This supports the hypothesis that the observed Na-bentazon antagonism of sethoxydim activity is chemically based. Furthermore, NH,+ from AMS effectively precludes the physical association of Na’ with sethoxydim. The substitution of NH: for Na‘ may explain the reported reversal of the antagonism with AMS. 95 ACKNOWLEDGEMENTS The authors wish to thank BASF Aktiengesellschaft for confirmation of the determined sethoxydim peak assignments. The NMR data were obtained on instrumentation that was purchased in part with funds from HIH grant #1 -S10-RR04750, NSF grant #CHE-88-00770, and NSF grant #CHE—92- 13241. 96 Figure 1. ‘H-NMR spectrum and peak assignments of technical grade sethoxydim in CDCI3. 97 3 E0 01... Figure 2. 1H-NMR spectrum of a) commercially formulated sethoxydim plus NaCI; b) commercially formulated sethoxydim plus NaHCO,,; 0) commercially formulated sethoxydim plus commercially formulated Na-bentazon; d) technical grade Li- sethoxydim; e) commercially formulated sethoxydim. CDCI3 was the carrier solvent for all treatments. II 7 p, i I '1‘ T 1.111I I it! 111 .113 11111 q—_-—A—uq_W~—-—-q-——u—~—q-q—-—q-—d—-q—-_-——-_-—._-J.A m.m m.m NQ m.0 m.m NE m.w m.m m.» m0 003 Figure 3. ‘H, ‘H homonuclear double quantum filter correlated spectrometry (COSY NMR) spectrum for a) commercially formulated sethoxydim plus commercially formulated Na- bentazon; and, b) commercially formulated sethoxydim. CDCI3 was the carrier solvent for both treatments. 101 lated Imelclalll aled Ni )xydim. sethoxydim + Na-bentazon ('H, ‘I-I) dqf cosy _._J I -5 % 3.0 ' .‘V 0 ‘ . 'c . ' no. . .. n . n ' ' .._ o. z to . ° , . .~. . p '. ? »..'.- -. . . g 1‘. '.' . - u - in...“ ., . . . - .- a ' O u . .-.. ‘. o a V ,. a '. 0 - to. ."' ..I 1' o ‘ '. . 0.. - o ‘. ' . . "' c ‘ -.‘ 3 I a in... 'm ~ - '..$ . 1.2 7...... I, --.-...- -- - .-.: v . r- 1.. ’0. .' do. . u. q. _‘ - - '9‘ 5.2. .u. ' 9.. .- is - ' - .. ‘-“ ., ° "' 1.4 1:11..“ . .- _ . ~.-,,~-. ‘ 50.3'0 '— I' 'M 0. r ' . x ‘ ~ 1L 1 e. O “ .' a u .‘I ‘. o J n ‘ 'J‘“ ‘.”. . a. . . . . ’ . O .. , Q - x... I 3'-” : ‘- ‘ - ~ . o o | . " u ‘- 0 - o '. : n- . ' . u ' " Q. I‘ o J ‘ .' . 3,9 . -fi. . ; - .' ._ , ._ .. '-s ' ° a a .. - Io. . J . .o . ‘- .. ~. Q I r o " o ' ' 0., ' f n 0 ~ . - o-,-_ ‘ .u. '- .5 2: o a.'--'-~..s-° spur. o o.. -H _ g . P..- ‘ . . r . .‘ .' ;v .. o “1. .- ._‘. .‘_. - 3,. :- ":3 .-'-i.'.‘- ‘:. ~ g - -... , W " ". .‘ J“-- . .- .- -' J‘oo.:. I ‘ It. - ‘- .‘ .. . , . 3. , d . -, ..' fl. . - .- .h . ‘ 7‘ . ”a." u ‘ r. a 0 . 3.0.. S f. - '. a .3 .0. .’ 0’ ' I r r I I r70 1.6 1.2 1.8 sethoxydim ('i-i, 'I-i) dqf easy .9 2.41 8.. l 3.0'! "S 3.93 . '. O. r I I 3.6 2.0 2.3 3.0 8.. l. n Ivy-I I I... 8.8 I... 0.3 Figure 4. 1H-NMR spectrum of a) commercially formulated sethoxydim plus NaHCOa plus AMS; b) commercially formulated sethoxydim plus commercially formulated Na-bentazon plus AMS; c) commercially formulated sethoxydim plus AMS; d) commercially formulated sethoxydim plus commercially formulated Na-bentazon; e) commercially formulated sethoxydim. CDCI3 was the carrier solution for all treatments. 103 Literature Cited Couderchet, M. and G. Retzlaff. 1990. Bentazone-sethoxydim antagonism: the role of ATP and plasma membrane ATPase. Pestic. Sci. 30:415-460. Couderchet, M. and G. Retzlaff. 1991. The role of the plasma membrane ATPase in bentazone-sethoxydim antagonism. Pestic. Sci. 32:295-306. Gerwick, B. C., L. D. Tanguay, and F. G. Burroughs. 1990. Differential effects of UAN on antagonism with bentazon. Weed Technol. 4:620-624. Grichar, W. J. 1991. Sethoxydim and broadleaf herbicide interaction effects on annual grass control in peanuts (Ms hyugggaea). Weed Technol. 5:321 -324. Jordan, 0. L., and A. C. York. 1989. Effects of ammonium fertilizers and BCH 81508 S on antagonism with sethoxydim plus bentazon mixtures. Weed Technol. 3:450-454. Minton, B. W., M. E. Kurtz and D. R. Shaw. 1989. Barnyardgrass (Echinochloa crus-galli) control with grass and broadleaf weed herbicide combinations. Weed Sci. 37:223-227. Penner, D. 1989. The impact of adjuvants on herbicide antagonism. Weed Technol. 32227—231. 105 10. 11. 12. Rhodes, N. G. Jr., and H. D. Coble. 1984. Influence of application variables on antagonism between sethoxydim and bentazon. Weed Sci. 32:436-441. Rhodes, N. G. , and H. D. Coble. 1984. Influence of bentazon on absorption and translocation of sethoxydim in goosegrass (Eleusine indica). Weed Sci. 32:595-597. Smith, A. M., and W. H. Vanden Born. 1992. Ammonium sulfate increases efficacy of sethoxydim through increased absorption and translocation. Weed Sci. 40:351-358. Wanamarta, G., D. Penner, and J. J. Kells. 1989. The basis of bentazon antagonism on sethoxydim absorption and activity. Weed Sci. 37:400-404. Wanamarta, G., J. J. Kells, and D. Penner. 1993. Overcoming antagonistic effects of Na-bentazon on sethoxydim absorption. Weed Technol. 7:322-325. 106 ; I" ‘g‘ .P' Jr. Chapter 4 UtIIlty of Nuclear Magnetic Resonance In Determining Herblclde Interactlons ABSTRACT In the discipline of Weed Science, nuclear magnetic resonance (NMR) has been used extensively for obtaining structural information on herbicide compounds in the areas of herbicide synthesis, metabolism, and environmental degradation. Comparatively less research has been published with regard to the utilization of NMR in determining herbicide interactions in the spray solution. Interactions between herbicides (Na-bentazon/paraquat), herbicides and herbicide formulation products (imazethapyr/Na-bentazon), and herbicides and adjuvants ’ (glyphosate/organosiIicones) were analyzed with NMR spectrometry. NMR was found to be an effective technique for characterizing chemical interactions in the biologically active compounds. No 1H-NMR spectral changes were observed for the Na-bentazon/paraquat, imazethapyr/Na- bentazon and gIyphosate/organosilicone pairs. Nomenclature: ' Glyphosate, N-(phosphonomethyl)gchine; Bentazon, 3-(1-methylethyl)- (1H)-2,1,3-benzothiadiazin-4(3H)-one 2,2-dioxide; Paraquat, 1,1’-dimethyl- 4,4’bipyridinium ion; Sethoxydim, 2-[1-(ethoxyimino)butyl]-5-[2- 107 (ethylthio)propyI]-3-hydroxy-2-cyclohexen-1-one; Imazethapyr, 2-[4,5- dihydro-4-methyl-4-(1-methylethyI)-5-oxo-1 H-imidazol-2-yl]-5-ethyI-3- pyridinecarboxylic acid. 108 INTRODUCTION NMR has been used extensively for obtaining structural information on herbicide compounds, primarily in the areas of herbicide synthesis and degradation. However, to date, this technology has not been widely utilized in determining chemical interactions between herbicides and other compounds in the spray solution. A primary adaptation of NMR technology is in the identification of the chemical structure of newly synthesized herbicide products (13, 21) and microbially produced herbicidal compounds (4, 14). In addition to identifying herbicidally active compounds, NMR technology has also been utilized for identifying inert ingredients in herbicide formulations (24). Another common adaptation of NMR technology is in the field of herbicide metabolite identification in herbaceous plants (9, 15, 18,), animals (20, 31), and microbes (17, 26). The ability to determine structural information on molecules has also proven NMR as an effective tool in environmental fate studies of herbicides in soil (30, 32), water (16), and light (27). In related work with plants, Law and Arnold (19) used NMFI to evaluate the chemical structure of sunflower (Helianthus annuus L.) epicuticular 109 wax and determined that EPTC (S-ethyl dipropyl carbamothioate) and acifluorfen (5-[2-chloro-4-(trifluoromethyl) phenoxy]-2-nitrobenzoic acid) had no effect on its chemical structure. Rollins et al. (28) conducted experiments to evaluate the possibility of using NMR imaging and spectroscopy to study the movement and distribution of xenobiotics non- . invasively in stems and leaves of tomatoes (Lycopersicon esculentum Mill. cv. Ailsa Craig). They found the technique to be suitable for translocation studies in whole plants. However, the authors concluded that it was unlikely that NMR will, in the near future, be of use in detecting the very low concentrations of field applied herbicides in plants. The use of NMR in evaluating complexation in herbicides has been limited to a few compounds. Paraquat (1, 3, 12, 25), and glyphosate (7. 11, 22, 33), have been evaluated with NMR for purposes of determining complexation products. Garbow and Gaede (10) utilized a form of solid state 13C-NMFI to demonstrate the formation of an inclusion complex between a pyrazole phenyl ether and B-cyclodextrin. Chrystal et al. (8) used NMR in an attempt to show that herbicides derived from 4- [(benzyloxy)methyl]—1,3-dioxolanes and benzyl methyl ethers of poly(ethylene glycols) exerted their mode of action by chelating biologically important metal ions such as Na‘, K’, Mg”, and Ca“. The authors demonstrated that the herbicides did indeed complex with the metal ions. However, this association was not correlated with herbicidal 110 activity. The ability of NMR to detect structural differences in molecules would be useful in characterizing antagonistic herbicide interactions and herbicide interactions with adjuvants. The objective of this research was to use the techniques of NMR, to characterize herbicide interactions in solution. 111 MATERIALS AND METHODS Analytical experiments were conducted to characterize herbicide interactions with the following pairs: other herbicides (Na- bentazon/paraquat), herbicide formulation products (imazethapyr/Na- bentazon), and adjuvants (glyphosate/organosiIicones). Glyphosate, Imazethapyr, and paraquat treatments. Technical grade glyphosate (87%) was used in the glyphosate pairs. Reagent grade calcium acetate (CH,COO)2CaH20 The organosilicone adjuvant utilized was Sylgard 309‘. Technical grade imazethapyr (98%) and paraquat (98%) were used in their respective pairs with formulated Na-bentazon which was obtained as commercially formulated Basagran’. Deuterium oxide was used as the carrier solvent for all NMR analytical observations. The HOD peak was standardized to 4.65 ppm. Glyphosate treatment concentrations were 0.026 molar which corresponds to field application rate of 1.7 kg/ha at 370 Uha carrier (1.5 lb glyphosate per acre at 40 gpa). Calcium was added at a 1:1 ratio with glyphosate. 1Sylgard 309 is a product of Dow Corning Corp, Midland, MI 48686 2BASF Corporation, 100 Cherry Hill Rd., Parsippany, NJ 07054 112 This corresponds to a Ca“ concentration of 1040 mg/L. In the organosilicone treatments Ca“ was added at a 1:1 or 4:1 ratio with glyphosate. All NMR spectra were obtained on a Varian" VXR 500. ‘H-NMR spectra were obtained on a Varian 5 mm high resolution ‘H/‘°F probe. Ultra-violet spectrophotometry. A UV spectrophotometry study was conducted to confirm NMR results from the paraquat/Na-bentazon pair. Technical grade paraquat and Na-bentazon were dissolved in distilled water individually or mixed and diluted to give a final concentration of 6.25 ppm for paraquat and 5.75 ppm for Na-bentazon (1:1 molar ratio). All three herbicide solutions were scanned from 220 to 340 nm using a UV spectrophotometer. ‘ 3Varian Associates Inc., Nuclear Magnetic Resonance Instruments, 3120 Hansen Way, Palo Alto, CA 94304-1030 113 RESULTS AND DISCUSSION Herblclde + herblclde (paraquat/Na-bentazon). The 1H NMR peak shifts of paraquat are depicted in Table 1. The peak at 8.92 ppm corresponds to the ring protons at the 2, 2’, 6, and 6’ position and the peak at 8.39 ppm corresponds to the ring protons at the 3, 3’, 5, and 5’ which is consistent with the findings of Ross and Krieger (29). The introduction of Na-bentazon did not elicit a peak shift in the resulting spectra (Table 1) indicating that the two molecules are not associating or complexing in solution. This is supported by the UV spectral analyses (Figure 1). The absorption of the mixture was additive with respect to the absorption of the individual herbicides and did not shift the peaks from those observed independently. Therefore, it appears that the reported Na-bentazon antagonism of paraquat activity (34) is due to a mechanism other than a direct chemical association of one herbicide with the other. Herblclde + herblclde formulatlon products (Imazethapyr/Na- bentazon). The ‘H NMR peak shifts for imazethapyr and imazethapyr plus Na-bentazon are presented in Table 1. No peak shift was observed suggesting that the observed antagonism of imazethapyr by Na-bentazon (5, 6) does not appear to be based on a chemical association of the 114 imazethapyr molecule with Na-bentazon or its formulation products. Additional sources of Na+ (Table 1) were also evaluated but were not found to associate with the imazethapyr in solution. This contrasts with the findings reported in chapter 3 where the Na-bentazon antagonism of sethoxydim was found to be chemically based. ‘H-NMR and (‘H, ‘H) double quantum filter correlated spectrometry were used to show that Na‘ from Na-bentazon associates with the ring hydroxyl oxygen of the sethoxydim molecule. Herblclde plus adjuvant (glyphosate! organoslllcone). The 1H-NMR spectrum for technical grade free acid glyphosate is shown in Figure 2- D. Peak area integration and 31P decoupled ‘H-NMR were used to conclude that the doublet at 3.18 ppm represents the protons on the methylene carbon adjacent to the phosphonate group and the peak at 3.9 ppm corresponds to the protons on the methylene carbon adjacent to the carboxyl group. This is consistent with the findings of Appleton et al. (2) who also determined the above peak assignments. With the introduction of Ca“, several changes were observed in the NMR spectra (Figure 2-C). The peak corresponding to the carboxyl methylene protons shifted upfield. The upfield shift indicates that the protons are in a more electronically shielded environment. Therefore, it appears that Ca” associated with the carboxyl group, forming a conjugate salt. The 115 doublet peak corresponding to the phosphonate methylene protons also shifted upfield, which suggests that Ca” also associated with the phosphonate group. This is consistent with the data reported in chapter 2 whereby 1"C NMR and 31P-NMR were used to confirm the association of Ca” with glyphosate to form Ca-glyphosate. Figure 2-B shows the ‘H NMR spectrum of glyphosate plus Sylgard 309. The organosilicone adjuvant did not produce a glyphosate peak shift indicating that the Sylgard 309 did not associate with the glyphosate molecule. Figure 2-A shows the spectrum for glyphosate plus Sylgard 309 in the presence of Ca”. The position of the glyphosate peaks was consistent with that observed for glyphosate plus Ca++ indicating that the Sylgard 309 did not preclude the Ca“ from associating with the glyphosate molecule. This is in direct contrast to the results reported in chapter 2 using diammonium sulfate (AMS) as the adjuvant. 31P-NMR was used to show that NH,+ associated directy with the glyphosate molecule when AMS and glyphosate were combined in solution. In addition, NH,+ was found to effectively compete with Ca“ for active sites on the phosphonate group of the glyphosate molecule. The substitution of NH,‘ for Ca” on the glyphosate molecule may explain the reported reversal (23) of the hard water cation antagonism of glyphosate. 116 The mechanism by which the organosilicone adjuvant increases glyphosate efficacy does not appear to be the result of a direct chemical interaction with the glyphosate molecule. This is in direct contrast to the observed glyphosate interaction with AMS, reported in chapter 2, which does appear to be chemically based. ACKNOWLEDGEMENTS The NMR data were obtained on instrumentation that was purchased in part with funds from HIH grant #1-S10-RR04750, NSF grant #CHE-88- 00770, and NSF grant #CHE-92-13241. 117 Tail; ‘H-NMR chemical shift data for paraquat and imazethapyr as affected by Na- bentazon and Na—salts. Treatment ring chain (PPM) imaz 8.40 7.85 2.68 1.95 1.31 1.16 0.95 0.76 imaz + Nabent 8.40 7.84 2.67 1.94 - 0.94 0.75 imaz + NaHCO, 8.40 7.84 2.67 1.94 1.30 1.16 0.94 0.75 imaz + Naacetate 8.39 7.84 2.67 1.94 1.30 1.16 0.94 0.75 imaz + NaCl 8.40 7.84 2.67 1.95 1.30 1.16 0.94 0.75 pquat 8.92 8.39 4.37 pquat + Nabent 8.92 8.39 4.37 imaz = imazethapyr, Nabent - Na-bentazon, pquat - paraquat 118 Figure 1. Ultra violet spectra for a) paraquat plus Na-bentazon; b) Na- bentazon; and, c) paraquat 119 220 231. 242. 253. 264. 275. 286. 297. 308. 319. 330 .00 00 00 00 00 00 00 00 00 00 .00 OOOO'O 0008'0 4. «lil- 0009'0 lib w- 0005'0 i 4)- 0008°I di- "l' OOOQ'I i .1— 0008'I L qu- 0001'? «l- qh 0007'? 4)- OOOL'B cli- '0- Figure 2. ‘H-NMR spectrum of a) technical grade glyphosate plus calcium acetate 1:4 molar ratio plus Sylgard 309; b) technical grade glyphosate plus Sylgard 309; c) technical grade glyphosate plus calcium acetate 1:1 molar ratio; and, d) technical grade glyphosate. D,_O was the carrier solvent in all treatments. 121 9 plus 5) ieCilrzéoal fade Ind, d) solvent in L 9 'e E 8 llllllLllllllllllllIllllllllllllllllllllllllgllIJIJLLIII I mdd Fir-— F" LITERATURE CITED Allwood, B. L., H. Shahriari-Zavareh, J. F. Stoddart, and D. J. Williams. 1987. Complexation of paraquat and diquat by a bismethaphenylene-32-crown-10 derivative. J. Chem. Soc. Chem. Commun. 1058-1061. Appleton, T. G., J. R. Hall, and I. J. McMahon. 1986. NMR Spectra of iminobis (methylenephosphonic acid), HN(CH,PO,H2)2 and related ligands and of their complexes with platinum(ll). Inorg. Chem. 25:726-734. Ashton, P. R., A. M. Z. Slawin, N. Spencer, J. F. Stoddart, and D. J. Williams. 1987. Complex formation between bisparaphenlyene- (3n + 4)-crown-n ethers and the paraquat and diquat dications. J. Chem. Soc. Chem. Commun. 1066-1069. Babczinski, P., M. Dorgerloh, A. Lobberding, J. Santel, R. R. Schmidt, P. Schmitt, and C. Wunsche. 1991. Herbicidal activity and mode of action of vulgamycin. Pestic. Sci. 33:439-446. Bauer, T. A., K. A. Renner, and D. Penner. 1994. 'Olathe" pinto bean (Phaseolus vulgaris) response to postemergence imazethapyr and bentazon. Weed Sci. 42: (In press). Bauer, T. A., K. A. Renner, and D. Penner. 1994. Response of selected weed species to postemergence imazethapyr and bentazon. Weed Technol. (submitted). 123 10. 11. 12. 13. Castellino, 8., G. C. Leo, D. Sammons, and J. A. Sikorski. 1989. 31P, ‘5, and 1"C NMR of glyphosate: comparison of pH titrations to the herbicidal dead-end complex with 5-enolpyruvoylshikimate-3- phosphate synthase. Biochemistry 28:3856-3868. Chrystal, E. J. T., A. H. Haines, and R. Patel. 1990. Studies into the mode of action of herbicides derived from 4- [(Benzyloxy)methyl}-1,3-dioxolanes and benzyl methyl ethers of poly(ethylene glycols). J. Agric. Food Chem. 38:870-874. Frear, D. S., H. R. Swanson, F. S. Tanaka. 1993. Metabolism of flumetsulam (DE-498) in wheat, corn, and barley. Pestic. Biochem. Physiol. 45:178-192. Garbow, J. R., and B. J. Gaede. 1992. Analysis of a phenyl ether herbicide-cyclodextrin inclusion complex by CPMAS 13C NMR. J. Agric. Food Chem. 40:156-159. Glass, R. L. 1984. Metal complex formation by glyphosate. J. Agric. Food Chem. 32:1249—1253. Haque, R., W. R. Coshow, and L. F. Johnson. 1969. Nuclear magnetic resonance studies of diquat, paraquat, and their charge- transfer complexes. J. Amer. Chem. Soc. 91:3822-3827. Hayashi, Y., and H. Kouji. 1990. Synthesis and herbicidal activity of geometrical isomers of methyl 1-5-2—chloro-4- (trifluoromethyl)phenoxy-2-nitrophenyl-z-methoxyethylidene, aminooxyacetate (AKH-7088). J. Agric. Food Chem. 38:845-850. 124 14. 15. 16. 17. 18. 19. Jakajima, M., K. Itoi, Y. Takamatsu, T. Kinoshita, T. Okazaki, K. Kawakubo, M. Shindo, T. Honma, M. Tohjigamori, and T. Haneishi. 1991. Hydantocidin: a new compound with herbicidal activity from Streptomyces hygroscopicus. Journal of Antibiotics 44:293-300. Kelly, l. D., and S. Smith. 1986. Chromatographic purification and identification of polar metabolites of benazolin-ethyl from soybean. International J. Environ. Analytical Chem. 25:135-149. Kissel C. L., J. L. Brady, A. M. Guerra, J. K. Pau, B. A. Rockie, and F. F. Caserio Jr. 1978. Analysis of acrolein in aged aqueous media. Comparison of various analytical methods with bioassays. J. Agric. Food Chem. 26:1338-1343. Krause, A., W. G. Hancock, R. D. Minard, A. J. Freyer, R. C. Honeycutt, H. M. LeBaron, D. L. Paulson, S. Y. Liu, and J. M. Bollag. 1985. Microbial transformation of the herbicide metolachlor by a soil actinomycete. J. Agric. Food Chem. 332584-589. Lamoureux, G. L., D. G. Rusness, and F. S. Tanaka. 1991. Chlorimuron ethyl metabolism in corn. Pesticide Biochemistry and Physiology. 41 :66-81. Law, M. E., W. E. Arnold. 1985. Effect of EPTC and acifluorfen on sunflower. Proc. North Cent. Weed Cont. Conf. 40:18. 125 20. 21. 22. 23. 24. 25. Leung, L. Y., J. W. Lyga, and R. A. Robinson. 1991. Metabolism and distribution of the experimental triazolone herbicide F6285 1- 2,4-dichIoro-5-N-(methylsulfonyl)aminophenyl-1 ,4-dihydro-3-methyI-4- (difluoromethyl)-5H-triazol-5-one in the rat goat and hen. J. Agric. Food Chem.. 39:1509-1514. Lynch, M. P., J. R. Beck, E. V. P. Tao, J. Aikins, G. E. Babbitt, J. R. Rizzo, and T. W. Waldrep. 1991. 1-alkyl-5-cyano-1H-pyrazole- 4-carboxamides. Synthesis and herbicidal activity. ACS Symposium Series. (No. 443):144-157. Motekaitis, R.J. and A. E. Martell. 1985. Metal chelate formation by N-phosphonomethylglycine and related ligands. J. Coord. Chem. 14:139-149. Nalewaja, J. D., and R. Matysiak. 1991. Salt antagonism of glyphosate. Weed Sci. 39:622-628. Nishizawa, Y., K. Ogura, and H. Momo. 1989. Establishment of quick multiple analytical method for inert ingredients of pesticide formulation (part 1). Bulletin of the Agricultural Chemicals Inspection Station, Tokyo. 29:17-22. Philp, D., A. M. Z. Slawin, N. Spencer, J. F. Stoddart, and D. J. Williams. 1991. The complexation of tetrathiafulvalene by cyclobis(paraquat-p-phenylene). J. Chem. Soc. Chem. Commun. 1584-1586. 126 26. 27. 28. 29. 30. 31. Pothuluri, J. V., J. P. Freeman, F. E. Evans, T. B. Moorman, and G. E. Cerniglia. 1993. Metabolism of alachlor by the fungus Cunninghamella elegans. J. Agric. Food Chem.. 41:483-488. Retjo, M., S. Saltzman, A. J. Archer, and L. Muszkat. 1983. Identification of sensitized photooxidation products of s-triazine herbicides in water. J. Agric. Food Chem. 31:138-142. Rollins, A., J. Barber, R. Elliott, and B. Wood. 1989. Xenobiotic monitoring in plants by 1“F and 'H nuclear magnetic resonance imaging and spectroscopy. Uptake of trifluoroacetic acid in Lycopersicon esculentum. Plant Physiol. 91:1243-1246. Ross, J. H., and R. l. Krieger. 1980. Synthesis and properties of paraquat (methyl viologen) and other herbicidal alkyl homologues. J. Agric. Food Chem. 28:1026-1031. Rueppel, M. l... B. B. Brightwell, J. Schaefer, and J. T. Marvel. 1977. Metabolism and degradation of glyphosate in soil and water. J. Agric. Food Chem. 25:517-528. Sato, K., Y. Kato, S. Maki, O. Matano, and S. Goto. 1979. Identification of urinary metabolites of 1-(alpha, alpha- dimethylbenzyl)-3-(p-tolyl)urea, dymrone in rats. J. Pestic. Sci., Japan 4:11-16. 127 32. 33. 34. Stearman, G. K., R. J. Lewis, L. J. Tortorelli, and D. D. Tyler. 1989. Herbicide reactivity of soil organic matter fractions in no- tilled and tilled cotton. Soil Sci. Soc. Amer. J. 1989. 53:1690- 1694. Subramaniam, V. and P. E. Hoggard. 1988. Metal complexes of glyphosate. J. Agric. Food Chem. 336:1326-1329. Wehtje, G., J. W. Wilcut, and J.A. McGuire. 1992. Influence of bentazon on the phytotoxicity of paraquat to peanuts (Arachfi hymgaea) and associated weeds. Weed Sci. 40:90-95. 128 Chapter 5 Summary and Conclusions The research detailed in the previous chapters has identified the formation of Ca“, Mg”, and other conjugate salts of glyphosate in hard- water solutions. Radio-labelled glyphosate absorption Studies showed these salts to be less readily absorbed by plants than commercially formulated isopropylamine glyphosate. These salts develop when alkaline or hard-water is used as a carrier for field applications of glyphosate and can result in reduced weed control. Several approaches can be utilized to adjust the spray tank chemistry to overcome the reduced control observed from the hard-water antagonism of glyphosate. Addltlon of Organic Acids. The addition of a weak acid such as food grade citric acid will effectively remove hard-water cations from solution. The conjugate base of the acid is the active agent in removing cations from solution and not the acid (H‘) itself. A weak acid, such as citric acid will provide a stronger conjugate base, and, therefore, will be more 129 effective than a strong acid such as nitric or hydrochloric acid. Addition of the organic acid to the point that the spray solution drops to pH 5 should provide a sufficient amount to overcome the hard-water antagonism of glyphosate. The organic acid should be added to the water carrier prior to the addition of the glyphosate. Acidifiers should not be used in conjunction with organo-silicone adjuvants as increased acidity may enhance chemical breakdown of the organo-silicone adjuvant. Ammonlum Sulfate (AMS). AMS has been used successfully in increasing glyphosate efficacy on a broad spectrum of weed species. The conjugate sulfate ion is very effective in forming conjugate salts with the hard-water cations. These salts are less active chemically due to their low ionization potential. In addition, research has demonstrated the formation of NH,-glyphosate which has been found to be a preferred absorption form on some weed species. The glyphosate label recommends the addition of 2% AMS by weight or 17 lb of dry AMS per 100 gallons of water for most applications. As with organic acids, the AMS should be added to the spray carrier solution prior to the glyphosate. Nonlonlc Surfactants. Nonionic surfactants will generally enhance glyphosate activity on most weed species. However, they will L191 overcome hard-water glyphosate antagonism. Therefore, under hard- 130 water conditions, AMS or organic acids should be used in conjunction with nonionic surfactants to maximize glyphosate absorption. Low Volume Rates. Decreasing the spray carrier volume has also been found to reduce hard-water antagonism of glyphosate. When the volume of carrier water is reduced the ratio of the antagonistic cations to glyphosate molecules is proportionally reduced. Reducing the spray carrier volume will help reduce glyphosate antagonism however, AMS or an organic acid, plus a nonionic surfactant is still recommended under hard-water conditions. Water treated with ion exchange water softeners contains monovalent Na” cations in place of hard-water divalent and trivalent cations. Although generally not as antagonistic as hard-water cations, Na’ has also been found to antagonize glyphosate herbicidal activity. Consequently, glyphosate activity may be increased with the addition of AMS or an organic acid plus a nonionic surfactant. Surface water sources such as streams or ponds generally have significant levels of dissolved solids and organic particulate matter. These particles decrease glyphosate activity and generally will not respond to organic acids or AMS. Surface water carrier sources should be avoided for glyphosate applications, especially if they are high in dissolved solids and particulate matter. 131 Adjusting the chemistry of spray carrier solutions can overcome hard- water antagonism and improve weed control with glyphosate. These recommendations are not intended to broaden glyphosate control to off label weed heights or weed species. 132 HIGRN STRTE UNIV. mlllgllii 2llllll3 lllllllllllllllllllliillillillllll