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'lIhk 7' 3‘2??? , .. £11.”? 231‘] ‘ I ‘ g A” .3, ., I ‘- _ ' .2 . . .I ml?!» , {IIIII ”If? $I'u‘q.">‘t I .1‘! =1 i ‘. u ' If“; IJ ififl.’iL‘1;I'f§I,;.I ,4 I”; u “3W§112";‘g’\“ - ' .3 an: ~ 3f$ '0‘“ V'. ' I -|I"I'II I II-IIIIlIIIJ‘ IIIIII‘ I ”HM ' I‘- It'nnL“ .'.r|\.‘n"|llm All'l‘l’. l' 'I . . ad‘rfii 7:253: 5'“: I; 5 I. . ~!) #1 I I’|_. - THEE" This is to certify that the thesis entitled RESIDUES (E PENDIMETHALIN [N-(l-ETHYLPROPYL)-3,4-DIMETHYL-2,6- DINITROBEBLENAMINE], TRIFLURALIN (a,a,a-TRIELUORo-2,6-DINITR0- N,N-DIPROPYL-P-TOLUIDINE), AND ORYZALIN (3,5—DINITRo-N“,N“, DIPROPYLSULFANILAMIDE) IN SOIL ORGANIC MATTER presented by James Emery Nelson has been accepted towards fulfillment of the requirements for Ph.D. Crop 6 Soil Sciences degree in I 33 WMW Major professor [ QQ Date 5.44" 76' 0-7 639 OVERDUE FINES: 25¢ per day per item W: Place in book return to remove ‘ charge from circulation records T will]... 3\\\\‘ L \E‘ ~°“"W 1]. W RESIDUES OF PENDIMETHALIN [N—(l-ETHYLPROPYL)-3,4-DIMETHYL-2,6- DINITROBENZENAMINE], TRIFLURALIN (a,a,a-TRIFLUORO-2,6-DINITRO- N,N-DIPROPYL-P-TOLUIDINE), AND ORYZALIN (3,5-DINITROaN4,N4, DIPROPYLSULFANILAMIDE) IN SOIL ORGANIC MATTER BY James Emery Nelson A DISSERTATION Smeitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1979 ABSTRACT RESIDUES OF PENDIMETHALIN [N-(l-ETHYLPROPYL)-3,4-DIMETHYL-2,6- DINITROBENZENAMINE], TRIFLURALIN (a,a,a-TRIFLUORO—2,6-DINITRO- N,N-DIPROPYL-P-TOLUIDINE), AND ORYZALIN (3,5-DINITRO-N4,N4, DIPRDPYLSULFANILAMIDE) IN SOIL ORGANIC MATTER BY James Emery Nelson A "sequential" extraction procedure was developed to study the fate of pendimethalin [N-(l-ethylpropyl)-3,4-dimethyl-2,6-dinitrobenzenamine], trifluralin (a,a,a-trifluoro-2,6-dinitro—N,N-dipropyl-p-toluidine), and oryzalin (3,5—dinitro-N4,N4,dipropylsulfanilamide) in soil over a 6 month laboratory incubation period. The procedure includes an acidic methanol extraction followed by an extraction with 95:4:1 (v/v/v) ace- tone-HZO-HCI solution (95-4-1) and then 0.5 N_Na0H with further frac- tionization of the NaOH extract by partial precipitation. Soil bound radioactive residues of pendimethalin, trifluralin, and oryzalin con- tained 15%, 14%, and 23%, respectively, of the initial radioactivity in non-sterilized soil after 6 months. The 95-4-1 extraction solvent removed 45% of the bound radioactivity from all three herbicides. Thir- teen percent, 12%, and 30% of the bound radioactivity of each herbicide was associated with the fulvic acid, humic acid, and humin organic James Emery Nelson fractions, respectively. Bound residues of oryzalin increased most rapidly and were highest after 2 months in all organic matter fractions. Soil sterilization reduced parent herbicide degradation and meta- bolism by 47% during the 6 month experimental period. The 6 month analysis time yielded metabolites unique to non-sterile trifluralin and oryzalin treatments in nonpolar soil extracts containing nonbound herbicide materials. Radioactivity in the fulvic acid, humic acid, and humin organic fractions was reduced 37%, 37%, and 50%, respectively, by soil sterilization. Radioactivity in the 95-4-1 soluble fraction was reduced less than 10% by soil sterilization indicating that the primary mode of residue association with the 95-4-1 soluble fraction is by chemical or physical mechanisms rather than by biological processes. More than 90% of the radioactivity from each herbicide in the 95-4-1 solUble fraction was in the form of polar or soil organic matter associ- ated degradation products. No trends were observed over time in the proportion of radioactivity in the 95-4-1 soluble fraction associated with nondialyzable organic molecules larger than 12,000 molecular weight. After 6 months, nondialyzable residues accounted for 41%, 50%, and 32% of the 95-4-1 soluble radioactivity in pendimethalin, trifluralin, and oryzalin treatments, respectively. The 95-4-1 extraction solvent removed 80% of the radioactivity bound after the first month of incu- bation, indicating that the solvent removes herbicide residues in the early stages of complexing with soil organic matter. The 95-4-1 soluble fraction appeared to be implicated with early bound intermediates and polar degradation products in a dynamic state of transition. For Kimberly, whom I love, I promise to do the best I know how. For my mom and dad, to whom I owe everything. ii ACKNOWLEDGMENTS The author would like to express his sincere appreciation to his major professor, Dr. William F. Meggitt, fer his guidance, encouragement, and constructive criticism throughout this project; but particularly for the opportunity of involvement with weed control at Michigan State University. "I learned more than education could ever teach me." Special acknowledgments are extended to Dr. Donald Penner fOr his enthusiasm and assistance in the project as well as critical review of manuscripts, and to Elanco Products Company and American Cyanamid Company for the financial and technical support. The assistance given by Drs. Al Putnam, Matt Zabic, and Jim Tiedje for serving on my guidance com- mittee is also gratefully acknowledged. The author extends special thanks to Gary Leek and Dale Aaberg fbr their effbrts in critical review of manuscripts. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . vi LIST OF APPENDIX . . . . . . . . . . . . . . . . . . . . . . . . .viii INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . 3 MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . 11 Soil Treatment . . . . . . . . . . . . . . . . . . . . . . . . 11 Soil Extraction and Analysis . . . . . . . . . . . . . . . . . 13 Classical Extraction . . . . . . . . . . . . . . . . . . . . . 15 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . 16 CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 APPENDICES . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 109 iv Table 10. LIST OF TABLES Physical properties of the Sharpsburg silty clay loam and the Kawkawlin sandy loam soils used . . . . . . . . . . . . . Total 1"C-recovery from ll‘C-pendimethalin, 1“C-trifluralin, and 1"C-oryzalin treated Kawkawlin sandy loam soil . . . . . The effect of soil sterilization on evolution of 1"C02 from treated Kawkawlin sandy loam soil . . . . . . . . . . . . . . The influence of soil texture on the amount of organic matter removed with the 95-4-1 extraction solvent . . . . . . The influence of soil texture on the amount of radio- activity removed with the 95-4-1 extraction solvent . . . . . The proportion of partitionable, nonpolar herbicide residues removed with the 95-4-1 extraction solvent from Kawkawlin sandy loam $011 0 O O O O O I O O O O O O O O O O O O O O O I The effect of soil sterilization on the proportion of non- dialyzable residues removed with the 95-4-1 extraction solvent from Kawkawlin sandy loam soil . . . . . . . . . . . The effect of soil sterilization and extraction procedure on the proportion of radioactivity in fulvic acid, humic acid, and humin organic fractions in Kawkawlin sandy loam 8011 O O O I O O O 0 O O O O O O O O O O O O O O O O O O O O The amount of radioactivity in fulvic acid, humic acid, and humin organic fractions from Kawkawlin sandy loam soil treated with ll'C-pendimethalin, ll'C-trifluralin, or 1"C- oryzalin . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of extraction procedure on the recovery of radio- activity from Kawkawlin sandy loam soil . . . . . . . . . . . Page 12 24 39 45 47 48 51 66 67 68 Figure 10. 11. LIST OF FIGURES The distribution of radioactivity from 1"C-pendimethalin among parent compound, extractable transfbrmation products, and soil bound reisdues in Kawkawlin sandy loam soil . . . The distribution of radioactivity from 1"C-trifluralin among parent compound, extractable transfbrmation products, and soil bound residues in Kawkawlin sandy loam soil . . . The distribution of radioactivity from ll'C-oryzalin among parent compound, extractable transformation products, and soil bound residues in Kawkawlin sandy loam soil . . . . . The effect of soil sterilization on the dissipation of parent herbicide in Kawkawlin sandy loam soil . . . . . . . The effect of soil sterilization on the accumulation of extractable transformation products in Kawkawlin sandy loam sail O O O O I O O O O O O O O O O O O O O O O O O O O The effect of soil sterilization on the accumulation of soil bound residues in Kawkawlin sandy loam soil . . . . . The effect of soil sterilization on the formation of non- polar transfbrmation products in Kawkawlin sandy loam soil The effect of soil sterilization on the dissipation of total extractable radioactivity in Kawkawlin sandy loam $011 0 O O O C O O C O O O O O O O O O I O I O O O O O C 0 Evolution of 1"C02 from 1"C-pcndimethalin, ll’C-trifluralin, and 1"C-oryzalin treated Kawkawlin sandy loam soil over time D O O O O O O O O O O O O O O O O O O O O O I O O O O The effect of soil sterilization on the accumulation of radioactivity in the 95-4-1 soluble organic fraction in Kawkawlin sandy loam soil . . . . . . . . . . . . . . . . . The accumulation of radioactivity in the 95-4-1 soluble organic fraction in Kawkawlin sandy loam soil treated with 1"C-pendimethalin, ll‘C-trifluralin, and 1"C-oryzalin . . . vi Page 18 20 22 27 29 31 33 36 38 42 44 Figure ' 12. 13. 14. 15. 16. 17. 18. The proportion of non-dialyzable residues in the 95-4-1 soluble organic fraction in 1"C-pendimethalin, ll'C- trifluralin, or ll'C-oryzalin treated Kawkawlin sandy loam soil . . . . . . . . . . . . . . . . . . . . . . . . . The accumulation of radioactivity in the fulvic acid frac- tion in Kawkawlin sandy loam soil treated with 1"C- pendimethalin, 1"C-trifluralin, or 1|’C-oryzalin . . . . . . The effect of soil sterilization on the accumulation of radioactivity in the humic acid fraction in Kawkawlin sandy loam soil treated with ll‘C-pendimethalin, 1"C-trifluralin, or 1"C-oryzalin . . . . . . . . . . . . . . . . . . . . . . The accumulation of radioactivity in the humin fraction in Kawkawlin sandy loam soil treated with 1"C-pendimethalin, Ike'trifluralin, 01‘ ll'C-Oryzalin o o o o o o o o o o o o o The effect of soil sterilization on the accumulation of radioactivity in the fulvic acid fraction in Kawkawlin sandy loam soil . . . . . . . . . . . . . . . . . . . . . . The effect of soil sterilization on the accumulation of radioactivity in the humin fraction in Kawkawlin sandy loam SOiI O O O O O O O O I O O O O O O O O I O I O O O O O The dynamic relationship between the 95-4-1 soluble frac- tion, and the fulvic acid, humic acid, and humin organic fractions in Kawkawlin sandy loam soil . . . . . . . . . . vii Page 50 53 55 S7 60 62 64 LIST OF APPENDIX APPENDIX A-l. B’lo C‘lo D-l. E-l. 1.10 The distribution of radioactivity from 1"C-pendimethalin among parent compound, extractable transformation products, and soil bound residues in Sharpsburg silty clay loam sail O O O O O O O I O O O O O O O O O O O O O O O C I O O The distribution of radioactivity from 1"C-trifluralin among parent compound, extractable transformation products, and soil bound residues in Sharpsburg silty clay loam 5011 O O O O O O O O O O O I O O O O O O O 0 O C O O O O O The distribution of radioactivity from 1|'C-oryzalin among parent compound, extractable transfermation products, and soil bound residues in Sharpsburg silty clay loam soil . . Total 1"C-recovery from 1"C-pendimethalin, 1"C-trifluralin, and 1"C-oryzalin treated Sharpsburg silty clay loam soil . The effect of soil sterilization on the accumulation of extractable transformation products in Sharpsburg silty Clay loam soil '0 O O I O O O O O O O O O O O O O O O I O O The effect of soil sterilization on the accumulation of soil bound residues in Sharpsburg silty clay loam soil . . The effect of soil sterilization on the formation of non- polar transfbrmation products in Sharpsburg silty clay loam 5011 O O O O O O O O I O O O O O O O O I O C O O O O The effect of soil sterilization on the dissipation of total extractable radioactivity from the Sharpsburg silty clay loam soil . . . . . . . . . . . . . . . . . . . . . . Evolution of 1"C02 from 1"C-pendimethalin, 1"C-trifluralin, and 1"C-oryzalin treated Sharpsburg silty clay loam soil over time 0 O O O I O O O I O C O O O O O O O I O O O O O The effect of soil sterilization on evolution of 1‘'C02 from treated Sharpsburg silty clay loam soil . . . . . . . . . The effect of soil sterilization on the accumulation of radioactivity in the 95-4-1 soluble organic fraction in Sharpsburg silty clay loam soil . . . . . . . . . . . . . viii Page 73 75 77 78 80 82 84 85 87 88 90 Appendix L—l. M-l. N-l. P-l. T-l. ".10 The accumulation of radioactivity in the 95-4-1 soluble organic fraction in Sharpsburg silty clay loam soil treated with 1"C-pendimethalin, I’C-trifluralin, or1“C-oryzalin . . The proportion of nondialyzable residues in the 95-4-1 solu- ble organic fraction from uC-pendimethalin, 1"C-triflura- lin, or 1"C-oryzalin treated Sharpsburg silty clay loam $011 I O O O O O O O O O O O O O O O O O I O O O O O C O O O The effect of soil sterilization on the proportion of non- dialyzable residues removed with the 95-4-1 extraction sol- vent from Sharpsburg silty clay loam soil . . . . . . . . . The effect of soil sterilization on the accumulation of radioactivity in the fu1vic acid fraction in Sharpsburg silty clay loam soil treated with 1"C-pendimethalin, 1“C- trifluralin, or 1"C-oryzalin . . . . . . . . . . . . . . . . The effect of soil sterilization on the accumulation of radioactivity in the humic acid fraction in Sharpsburg silty clay loam soil treated with 1"C-pendimethalin, 1“C- trifluralin, or 1"C-oryzalin . . . . . . . . . . . . . . . . The accumulation of radioactivity in the humin fraction in Sharpsburg silty clay loam soil treated with 1"C-pendimetha- lin, 1"C-trifluralin, or 1"C-oryzalin . . . . . . . . . . . The effect of soil sterilization on the accumulation of radioactivity in the humin fraction in Sharpsburg silty clay loam soil . . . . . . . . . . . . . . . . . . . . . . . The dynamic relationship between the 95-4-1 soluble frac- tion, and the fulvic acid, humic acid, and humin fractions in Sharpsburg silty clay loam soil . . . . . . . . . . . . . The effect of soil sterilization and extraction procedure on the proportion of radioactivity in fulvic acid, humic acid, and humin organic fractions in Sharpsburg silty clay loam soil . . . . . . . . . . . . . . . . . . . . . . . . . The amount of radioactivity in fulvic acid, humic acid, and humin organic fractions from Sha sburg silty clay loam soil treated with 1"C-pendimethalin, 1 C-trifluralin, or 1"C- oryzalin . . . . . . . . . . . . . . . . . . . . . . . . . . The effect of extraction procedure on the recovery of radioactivity from Sharpsburg silty clay loam soil . . . . . ix Page 92 94 95 97 99 101 103 105 106 107 108 INTRODUCTION Radiotracer studies on the fate of dinitroaniline herbicides in the environment have shown that after one year the majority of radioactivity remains in soil as nonextractable or soil bound residues (10, ll, 12, 13, 28). Complete conversion of these materials to mineral constituents has not been indicated. Degradation pathways of several dinitroaniline herbicides leading to bound residues are well documented (12, 13, 28). Yet little is understood about the nature of the herbicide residues and the mechanism by which they become part of, or bound to, soil organic matter. Consequently, there is concern that herbicide residues may be liberated, allowing fer uptake by plants, and also that residues may accumulate in soil influencing soil properties and natural soil processes. The classical method of extracting soil organic matter with 0.5 N NaOH has been criticized for causing severe alteration of extracted or- ganic matter and associated herbicide residues (4, 6, 9, 24, 31). Other less destructive methods of organic matter extraction have proved unsati- factory because of much lower extraction efficiencies (6, 7, 13, 24, 31). Any determination of "bound" residues in soil based on a defined solvent system is arbitrary for that herbicide and soil. Artifacts due to the extraction procedure present themselves in the relative proportions of bound and nonbound residues and of parent compound and metabolites. Admittedly, the "ideal" solvent for extraction has not yet been found and a need for standardization of methods still exists. 1 2 A 95:4:1 (v/v/v) acetone-H O-HCI (95-4-1) solution has been reported 2 capable of extracting 10 to 23% of the organic matter from soil with little alteration of extracted organic matter (25). A "sequential" ex- traction procedure was developed for this study that first extracted soil with acidic methanol followed by the 95:4:1 (v/v/v) acetone-HZO-HCI solu- tion and then 0.5 N_NaOH with further fractionation of the NaOH extract by partial precipitation. The purposes of this study were to 1) develop a standardized procedure fer the determination of soil bound residues involving nonpolar pesticides, 2) determine the identity and quantity of 14C-pendimethalin [N-(l-ethylpropyl)-3,4-dimethyl-2,6-dinitrobenzenamine], l4C-trifluralin (a,a,a-trif1uoro-2,6-dinitro-N,N-dipropyl-p-toluidine), and 14C-oryzalin (3,5-dinitro-N4,N4-dipropylsulfanilamide) residues bound and associated with each organic matter fraction over time, and 3) deter- mine the influence of soil type and microbial activity on the fate of these herbicides in soil organic matter. LITERATURE REVIEW Russell (1952) defined soil organic matter (0.M.) as "... a whole series of products which range from undecayed plant and animal tissues through ephemeral products of decomposition to fairly stable amorphous brown to black material bearing no trace of anatomical structure of the material from which it was derived; and it is the latter material that is normally defined as soil humus." In addition to plant and animal residues which have been altered by microbial decomposition, soil O.M. also contains products of microbial synthesis not fbund in higher plants (24). The majority of soil O.M. is composed of materials of high mole- cular weight, although some organic compounds exist in free monomeric form (24). Several theories suggest that humus formation involves the synthesis of polymeric materials (8, 9, 20, 30, 34). Hydrogen-bonding, van der Waal's forces, free radical reactions, polymeric condensation, and autocatalytic reactions are examples of mechanisms involved in soil O.M. formation. Insoluble complexes form with certain metallic cations, such as calcium, iron and aluminum by chelation and salt-bridging (24). The complexity of these large heterogenous polymers and their diverse composition (i.e. resins, polysaccharides, proteins, pigments, etc.) prevent complete solubility in a single extracting solvent. In addition to solubility problems, adsorption of O.M. on the in- organic matrix makes extraction difficult. O.M. may become entrapped in interlayer spaces between expanding clays or actually enter into the 3 5 crystal lattice of clay minerals (6). Organic matter may also become adsorbed onto the surface of inorganic particles by cation/anion ex- change, hydrogen-bonding, van der Waal's forces, and ion-dipole inter- actions. Calcium, iron, and aluminum are also involved in clay-O.M. com- plexes through chelation and polyvalent cation bridging (6, 24). There is a growing volume of information on the fate of organic chemicals, mainly pesticides, in the soil environment (1. 14, 25, 35). Protonation of strong Lewis bases, cation exchange, hemisalt formation, anion exchange, salt bridging, and hydrogen and covalent bonding are examples of clay-organic and organic-organic interactions that influence pesticide fate in soil. Other possible physicochemical interactions are ligand exchange, charge transfer complexes, ion dipole-interactions, van der Waal‘s forces, entropy effects, hydrophillic-lipOphillic interactions, physical entrapment, and occlusion (36). In addition to physicochemical associations, the soil microflora and fauna play an integral role in pesticide transformations in soil. Complexed and often immobilized residues result from reactions between humic substances and introduced compounds. The mechanisms and rate at which a pesticide becomes bound to or part of soil O.M. are not understood. Once intimately associated with soil O.M., complete mineralization becomes a slow, lengthy process paralleling the rate of mineralization of soil O.M. (24). A critical question in studying bound residues is the adequacy of current analysis methods. EPA "Guidelines for Registering Pesticides in the 0.5." (Federal Register 40[123]:26802-26928, June 25. 1975) involves extraction of O.M. from soils with caustic alkali with further subdivi- sion of extracted material by partial precipitation with mineral acids. Although the classical alkali extraction of O.M. still remains 6 quantitatively the most efficient available, evidence indicates that the chemical structure of the organic matter is altered by the strong extrac- tion agent (4, 6, 9, 24, 31). Studies show that auto-oxidation of humic constituents occurs under alkaline conditions when in contact with oxygen. This results in carbon dioxide evolution, hydrolysis, and decarboxyla- tion. The bound pesticide moiety may also be altered by the alkali extraction so that it no longer resembles what was initially bound to the organic matrix. The search for suitable solvents for extraction has been, and will continue to be, a matter of high priority. Neutral salts of mineral and organic acids that are good polyvalent- metal solvents have been used for extraction of humic substances, how- ever, the yields are usually low (6, 24). Sodium pyrophosphate is the most satisfactory reagent in this group (6, 24, 31). The efficiency of extraction is considerably less than sodium hydroxide, especially from mineral soils, however, the humic polymers probably undergo less altera- tion during extraction (24, 31). Soil O.M. is not very soluble in dilute mineral acids with the ex- ception of a hydrofluoric-hydrochloric acid mixture, although some hy- drolysis occurs with hydrochloric acid alone and sulfuric acid (24). Hydrofluoric acid dissolves silicates and other compounds and couples aluminum and iron releasing O.M. from inorganic soil particles. Hydro- fluoric and hydrochloric acids have been used alone and in combination as a pretreatment to improve the efficiency of O.M. extraction with other reagents (6, 24, 31). Exchange resins offer a less destructive, but less efficient method of O.M. extraction (13, 31). Meilke et 31. (21) reported a reversal in the radiocarbon content of humic and fulvic acid fractions when ditalimbos 7 (0,0-diethyl phthalimido-phosphono-thioate) treated soil was extracted with a clelating resin or with hot NaOH. They concluded that hot alkali causes greater destruction of the humic acid polymers than does the resin which resulted in a greater proportion of low molecular weight polymers that fractionate as fulvic acid. The extractive power of resins is related to their selectivity for polyvalent cations (5). Organic reagents have been used to extract components of soil O.M. based on solubility. Organic compounds capable of chelating metals such as cupferron, 8-hydroxyquinoline, and acetylacetone are not as effective as neutral sodium pyrophosphate in extracting O.M. (6). Nonpolar pesti- cides present a problem because they are insoluble in the aqueous, high ionic strength extracting solvents commonly used. Difficulties also arise in the use of aqueous reagents when samples must be concentrated within limits of analytical detection. Theoretically, an extraction system based on solubility would be nondestructive to the O.M. and pesticide residues it removes. Porter (27) extracted about 10 to 23% of the total carbon from five soils with a 95:4:1 (v/v/v) acetone-HZO-HCI mixture. The aqueous acidic acetone combination is believed to extract the newer, more recently formed O.M. Stevenson (personal communication) experimented with several aqueous acidic organic solvent combinations including the acetone-H O-HCl mix- 2 ture. He found the 95:4:1 (v/v/v) acetone-H O-HCl combination to be 2 most effective in extraction of O.M. More recently, dispersion of organic colloids by ultrasonic vibration of soil suspensions has been used with much success (7). This method shows much promise for nondestructive removal of O.M. and pesticide resi- dues from soils. 8 Organic matter content is the soil property most often correlated with dinitroaniline herbicide activity (2, 10, 17, 23, 26, 29, 33). Bardsley §t_§1, (2) investigated the toxicity and persistance of surface applied trifluralin as related to organic matter content in soils. They incorporated either alkali-extractions of humic and fulvic acids or acti- vated charcoal with mineral soils of low organic matter content to obtain O.M. levels of 1.5, 3.0, 4.5, and 6%. The addition of O.M. to soils appeared to trap the trifluralin and resulted in increased toxicity to barley and cucumber seedlings 41 to 81 days after addition of trifluralin. The increased trifluralin toxicity with increasing O.M. content was attri- buted to the greater adsorption of the vapor phase of the herbicide. On the other hand, several researchers (6, 33) have found that increasing the O.M. content in the soil will reduce the phytotoxic effectiveness of several dinitroaniline herbicides. Segraves g£_§l, (32) studied the relationship of the soil properties of 24 soils to the herbicide activity of trifluralin and profluralin [N- (cyclopropylmethyl)-a,a,a-trif1uoro-2,6-dinitro-N-propy1-p-toluidine]. Herbicide activity in each soil was assessed by determining the rate of herbicide required for 50% sorghum root growth inhibition. The multiple correlation coefficient squared indicated that 91% and 84% of the vari- ability in the trifluralin and profluralin additions could be accounted fer by variability in the total soil carbon content. Talbert and Kennedy (33) studied effects of activated charcoal on the toxicity of trifluralin and nitralin [4-(methylsulfonyl)-2,6-dinitro-N,N-dipropyl- aniline] to redroot pigweed. Both trifluralin and nitralin were inacti- vated when applied to charcoal at 1.8 mg/g of charcoal. 9 Golab 35 31. (13) investigated the degradation of 14C-trifluralin in field soil over a 3-year period. Twenty-eight transformation products were isolated and identified. After 3 years, 38% of the applied radio- activity remained after extraction as soil-bound residues. An adsorption- desorption study was performed with selected degradation products using three adsorbants: sand, soil, and humic acid (12.5%) mixed with sand. Trifluralin and many of its derivatives were recoverable from the humic acid-sand mixture. However, the compounds that had both nitro groups reduced were not recoverable. The nonsubstituted triamine degradation product, a,a,a-trifluorotolune-3,4,5-triamine, appeared to be a key com- pound in the formation of soil bound residues. Golab et_al, (12) studied the degradation of 14C-oryzalin in a field experiment. After one year, 30% of the applied radioactivity was soil bound. Hot neutral and acidic hydrolysis yielded only a small amount of radioactivity. Hydrolysis with aqueous 20% ammonia in methanol released approximately one-fourth of the bound radioactivity. Aqueous alkaline hydrolysis with 0.5 N KOH released 90% of the soil bound radio- activity. A portion of the alkaline hydrolysis was chromatographed and was feund to contain many components ranging from relatively nonpolar to very polar. They concluded that the soil bound components were most probably altered by the alkaline hydrolysis. A second portion of the alkaline hydrolysate was separated into humic and fulvic acids. The majority of the radioactivity remained in the soluble fulvic acid frac- tion. Helling (15) found that the distribution of bound 14C between or- ganic fractions was similar among six dinitroaniline herbicides. The Gascho-Stevenson procedure employing NaZPzP7 or 0.3 N NaOH followed by 10 dialysis was used. Generally, 50+5% was in fulvic acid, 15 to 20% in humic acid, and 25 to 30% in humin. In another study, butralin [4-(1,l- dimethylethyl)-N-(l-methylpropyl)-2,6-dinitrobenzenamine] found residues were extracted from soil using the more rigorous classical extraction procedure with 0.5 N_NaOH. The change in procedure resulted in 61% of the bound butralin residues associated with fulvic acids and only 6% with humic acids. In a third study using the Gascho-Stevenson procedure, radioactive butralin residues were found to be concentrated in the humic acid fraction, humin, and in undecomposed organic matter. Little radio- activity was found in dialysates. No differences in relative 14C content among five humin fractions differing in molecular size were found. Bound butralin was principally localized in the more "highly humified" fraction and < 5% was associated with O.M. whose molecular weight was < 12,000 to 14,000. Booth g£_§l, (3) could extract only 14.73% of the total radioactivity from fluchloralin [N-(Z-chloroethyl)-2,6-dinitro-N-pr0py1-4-(trifluoro— methyl)aniline] treated soil 18 months fellowing application with an exhaustive extraction procedure that employed three extractions with methanol followed by hexane. Fluchloralin accounted for 76.3% of the extracted residues. Release of the bound radioactivity material into water was monitored over a 128 day period. Analysis of water with time showed that low concentrations of bound residue were released into water with values remaining constant after the 16th day. Fluchloralin accounted for approximately 85% of the total residue released into water. MATERIALS AND METHODS Soil Treatment Two soils were selected from different geographic regions of the cornbelt, chosen to represent extremes in clay and sand content with similar pH and organic matter levels (Table 1). Field moist soil was screened through a 9-mesh (0.2 mm) sieve. Twenty-five grams of soil (dry weight basis) and 0.0125 g of finely ground corn stocks (5 ton/ acre furrow slice) were placed in 500 m1 erlenmeyer flasks and thorough- ly mixed on a Burrell wrist-action shaker. Flasks were wrapped with aluminum foil to exclude light. Uniformly ring labelled 14C-pendimethalin (7.22 uCi/umole), 14C- trifluralin (2.52 uCi/umole) and 14C-oryzalin (2.45 uCi/umole) were amended with respective technical materials to give a 0.025 mg/ml solu- tion in methanol. Analyses indicated the purity of all 14C-herbicides to be greater than 99%. One m1 (1 ppmw soil) of the appropriate herbi- cide standard was pipetted evenly over the soil in each flask. The flasks were immediately stoppered and shaken for 2 minutes. Carbon dioxide traps were prepared suspending shell vials (12 x 30 mm) inside each flask with wire and pipetting 1.6 ml INLKOH into each. Soil moisture was then adjusted to 50% field capacity with distilled water. One half ml of propylene oxide was added to sterilize appropriate flasks, and all flasks were closed with aluminum fbil covered rubber stop- pers until analysis. Flasks were stored in closed metal cabinets between 11 12 Table 1. Physical properties of the Sharpsburg silty clay loam and the Kawkawlin sandy loam soils used. Soil Organic series Texture matter Clay Silt Sand pH Sharpsburg Silty clay loam 3.10 38 40 22 7.2 Kawkawlin Sandy loam 3.86 19 9 72 7.8 13 22 and 27°C. The flasks were arranged in a randomized complete block design and rerandomized periodically. The experiment contained 3 repli- cations and was repeated once. Soil Extraction and Analysis The carbon dioxide traps were first removed and the 14CO2 absorbant radioassayed by liquid scintillation spectroscopy using 15 ml ACS® aque- ous scintillation solution. The distinction of nonbound residues was made based on an acidic methanol extraction developed by American Cyanamid Company.1 The procedure was modified to accommodate thin-layer chroma- tography, autoradiography, and liquid scintillation spectrosc0py. Acidic methyl alcohol extracts from trifluralin and pendimethalin treated soils were liquid partitioned against hexane for purification. Methylene chloride was used for the liquid partition of extracts from soils treated with oryzalin. Aliquots (1 ml) of the acidic methanol extract were radioassayed following the partition using ACS(:> aqueous liquid scintil- lation solution. The hexane and methylene chloride fractions were concentrated en vagug_to 1 ml. Samples (100 pl) of the hexane or methylene chloride- soluble fractions were radioassayed using 4 g PPO (2,5-diphenyloxazole) plus 50 mg dimethyl POPOP [1,4-bis(2-(4-methyl-5-phenyloxazoly))-benzene] in Triton X-100 plus toluene (2:1) brought to a volume of 1 liter. A second (20 ml) was spotted on a 250 mm Silica Gel GF thin-layer chroma- tography plate. Thin-layer plates were developed twice in the same direction. Carbon tetrachloride was used to develop extracts of 1(CL 92, 553: Determination of N-(l-EthylprOpyl)-2,6-dinitro-3, 4-xylidine residues in soil.) l4 pendimethalin and trifluralin treated soils and a 60:40 (v/v) benzene- isopropyl ether mixture was used to develop extracts from oryzalin treated soils. Thin-layer plates were autoradiographed for 2 weeks and developed. -Locations of radioactivity concentration were scraped and radioassayed using the PPO plus POPOP scintillation solution. The remaining acidic methanol was removed by suction filtering through Whatman #1 filter paper and discarded. Soil samples were then extracted with two 250 ml portions of 95:4:1 (v/v/v) acetone-HZO-HCI using the first portion to quantitatively return all solids trapped by the filtering procedures. After 16 to 24 hours of shaking the aqueous, acidic acetone was removed by siphon. A 200 ml sample of the combined extracts was reduced to dryness en vagug_and freeze-dried. The residue was combusted in a resistance furnace combustion unit using oxygen as the carrier gas. 14002 was ab- sorbed in 15 m1 2:1 (v/v) Perma fluor V® liquid scintillation counting solution - CarboSorb II® CO2 absorbant and radioassayed by liquid scin- tillation spectrometry. All samples were combusted using this technique. The same procedure used to partition the acidic methanol fraction was applied to partition a 50 m1 sample of the aqueous acidic acetone extract. However, modifications were made due to solubility of acetone in the methylene chloride used in the partition cleanup of oryzalin treated soil extracts. The methylene chloride used in this partition was washed twice after the first partition and once after the second partition with 250 ml of distilled water. Sample preparation, radio- assay methods, and thin-layer chromatography techniques were identical to those used to assay the acidic methanol fraction. 15 A second 50 m1 sample was evaporated under nitrogen gas at room temperature to 7.5 m1. Samples were transferred to 1 cm diameter, cellu- lose dialysis tubing (12,000 molecular weight exclusion) and dialyzed against water for 24 hours. The precipitate formed from dialysis was transferred in water to a 50 ml, screw-cap polypropylene centrifuge tube and quickly frozen. After thawing, the dialysates were centrifuged at 2706 x G for 15 minutes and the supernatant fluid discarded. The dialysates were freeze-dried and combusted to determine radioactivity. The remaining residue was titrated using 2.5 N NaOH to pH 2. The residue was shaken with 250 ml 0.5 N_NaOH under N2 for 16 to 24 hours. The flask contents were quantitatively transferred to a polypropylene centrifuge bottle and centrifuged at 2706 x G for 20 minutes. The resi- due, classified as humin, was freeze-dried and combusted to determine radioactivity. The supernatant NaOH extract was titrated to pH 1 with concentrated hydrochloric acid. The precipitate fermed, classified as humic acid, was isolated by centrifugation at 2706 x G for 20 minutes, freeze-dried, and combusted. A 75 m1 sample of the supernatant, classi- fied as fulvic acid, was reduced to dryness en vacuo, freeze-dried, and combusted to determine radioactivity. Classical Extraction The classical analysis procedure was identical to the previously outlined procedure, however, the 95:4:1 extraction step was omitted. The soil residue was shaken with 250 ml of 0.5 N_Na0H under N following 2 removal of remaining acidic methanol by suction filtering. The fulvic acid, humic acid, and humin fractions were isolated using the previously described procedures and combusted to determine radioactivity. RESULTS AND DISCUSSION The fate of 14C-pendimethalin, l4C-trifluralin, and 14C-oryzalin were similar in the Kawkawlin sandy loam and Sharpsburg silty clay loam soils. Only the results obtained from the sandy loam soil will be pre— sented for clarity and ease of discussion. Any dissimilarity in re- sponse because of soil type will also be discussed. Tables and figures in the appendix contain results from the silty clay loam soil study. 1 14C The distribution of 14C-pendimethalin, 4C-trifluralin, and oryzalin among parent compound, extractable transformation products, and nonextractable soil bound residues from sterilized and nonsterile soil over a 6 month period is shown in Figures 1, 2, and 3. After 6 months, 60%, 49%, and 58% of the applied radioactivity was present as pendimethalin, trifluralin, and oryzalin, respectively, in nonsterile soil treatments. A decline in the percent parent compound resulted in increases in extractable degradation products and bound residues over time. The majority of parent pendimethalin dissipation had occurred by 2 months. However, declines in parent trifluralin and oryzalin occurred over a 4 to 6 month period. Decreases in parent oxyzalin were not detected until after the first month. The pattern of parent herbicide dissipation was similar in both soils. However, parent trifluralin dissipated to a lower level in steri- lized silty clay loam than sandy loam soil (Appendix Figure B—1) and less parent oryzalin was extracted from nonsterilized silty clay loam 16 17 Figure l. The distribution of radioactivity from 14C-pendimethalin among parent compound, extractable transformation products, and soil bound residues in Kawkawlin sandy loam soil. 18 70 PENDIMETHALIN, NON-STERILE SOIL \0/0 0 O X of INITIAL RADIOACTIVITY “o“ a ‘BOIND, NON-STERILE SOIL er, GRADATION PRODUCTS, ,) 0"", NON-STERILE son. I ”4* DEGRADATION. (mowers. STERILE SOL .......... ........ ‘0'"“2... ...."000.o'.’ *“‘ *0-0---- BOUND, STERILE SOIL 4 6 MONTHS 19 . . . . . . . l . . Figure 2. The distribution of radioactiVity from 4C-trifluralin among parent compound, extractable transformation products, and soil bound residues in Kawkawlin sandy loam soil. 20 100 90 80 ‘s \ $ O o “ 70 o TRIFLIRALIN, STERILE SOI TRIFLLRALIN, WN-S'TERILE SOIL \ X of INITIAL RADIOACTIVI'I’Y MGRADATION PRODIgTS STERILESO SOIL flDEGRADATION PRODUCTS, NON- .. STERILE SOIL '0... D/ “0.... o o o o ‘st Euatuvt, “KDNF' ...... A'n'ai}‘alll’n'a‘a‘lll'llllll'hh .0 STERILE sou ,1,” ”I, '. ‘3'” .quooooooooooooooooou. ’11----0‘-.. “’1'--. «'2... O ------. A: tBOUND, STERILE sou. * ° 1 2 4 e mourns 21 Figure 3. The distribution of radioactivity from 14C-oryzalin among parent compound, extractable transformation products, and soil bound residues in Kawkawlin sandy loam soil. 22 % of INITIAL RADIOACTIVITY 100 90 80 ORYZALIN, STERILE SOIL 70 .-..--------I---"° 60 ORYZALIN. NON-STERILE song. 50 40 3° pBOUND, NON-STERILE SOIL 1*» [1,1,1 '0”"""* I [I pBOUND STERILE son. . . 1,1?” '-- ,o /.-*---' Mou- sremtewo crs ’*' GRADATION mow 10 I,“ 00/ I goEGRAomou PRODUCTS $15ng sou. /0 ...........0000IoooooooooooooooooooooI oooooooooo- .0000... 0 1 A 2 4 5 MONTHS *-------- ---* 23 than sandy loam soil (Appendix C-l). Differences in soil type may have resulted from soil induced extraction artifacts or directly from the participation of clay surfaces in herbicide alteration and binding. Direct comparisons between parent trifluralin and parent oryzalin and pendimethalin cannot be made because of a 6 to 7% lower total recovery of trifluralin from soil (Table 2). Losses in parent herbicide and ex- tractable degradation products resulting from volatility and adsorption to glassware account for the lower recovery. Figures 1, 2, and 3 show that, after 6 months, 17%, 21%, and 15% of the applied radioactivity was present in nonsterile treatments as extract- 14 14 14C- able degradation products of C-pendimethalin, C-trifluralin, and oryzalin. The rate of appearance of extractable degradation products and bound residues coincides with the disappearance of parent herbicides. Extractable transfbrmation products of pendimethalin and trifluralin appeared rapidly, reaching a maximum level after 1 to 2 months. This level was maintained or declined slightly through 6 months. The decline from 2 to 6 months was most significant with transformation products of trifluralin. Extractable transformation products of oryzalin accumulated steadily throughout the 6 month period, but did not become significant until after the first month, which coincided with the disappearance of parent compound. Figures 1, 2, and 3 also show that, after 6 months, 15%, 14%, and 23% of the applied 14C-pendimethalin, 14C-trifluralin, and 14C-oryzalin, respectively, were present as soil "bound" radioactive residues in non- sterile treatments. Bound residues of oryzalin and pendimethalin appeared rapidly and were more abundant than residues of trifluralin from 0 to 2 months. However, bound residues of trifluralin were equal to or only 24 l4 l4 . . 14 . . Table 2. Total C-recovery from C-pendlmethalin, C-trifluralin, and 14C-oryzalin treated Kawkawlin sandy loam soil. Herbicide Recoveryfllh/ --% of initial radioactivity-- Pendimethalin 96.4 Trifluralin 89.3 Oryzalin 97.0 a/ .. --LSD.01 - 4.5. b/ —-Resu1ts are averaged over analysis time and soil sterility treatment. 25 slightly less prevalent than residues of pendimethalin after 4 months. Residues of oryzalin were present in greatest concentration after 1 month. The dynamic nature of introduced chemicals in the soil environment is exemplified by the balance between parent herbicide, extractable de- gradation products, and bound residues shown in Figures 1, 2, and 3. The relatively fast degradation rate of parent trifluralin and the slow rate at which trifluralin becomes bound to soil resulted in a larger extractable transformation product pool compared to the other two herbi- cides. Conversely, the rapid rate of soil bound residue formation of oxyzalin resulted in a smaller extractable transformation product pool. Six months after treatment, 47% more of the parent herbicide has been degraded in nonsterilized soil than in sterile treatments (Figure 4). Lower levels of parent herbicide in nonsterilized soil resulted in a 62% increase in extractable degradation products (Figure 5) and a 35% increase in bound radioactivity (Figure 6). Nonpolar, hexane/methylene chloride-soluble degradation products in nonsterilized soil were increased three or fbur times over the amount of these degradation products in sterilized treatments (Figure 7). All degradation products were located at the origin of thin-layer chroma- tography plates with two exceptions. In addition to origin localized metabolites, a single metabolite band appeared at 6 months in nonsterile trifluralin and oryzalin treated soils. The Rf values for the parent herbicide and respective metabolites are 0.775, 0.500, and 0.875 for trifluralin and oryzalin, respectively. Probst gt g1. (28) concluded from his work that biological degrada- tion could not be considered the major pathway of trifluralin degradation, 26 Figure 4. The effect of soil sterilization on the dissipation of parent herbicide in Kawkawlin sandy loam soil. a) Results are averaged over the three herbicides. 27 1.00 LSD... H O O ,.- ST ERILE SOIL ...-------.------I---'. NON-STERILE SOIL o—O % of INITIAL RADIOACTIVITY q 0 28 Figure 5. The effect of soil sterilization on the accumulation of extractable transformation products in Kawkawlin sandy loam soil. 3) Results are averaged over the three herbicides. % of INITIAL RADIOACTIVITY 29 20 Lsom NON-STERILE son MONTHS 30 Figure 6. The effect of soil sterilization on the accumulation of soil bound residues in Kawkawlin sandy loam soil. a) Results are averaged over the three herbicides. % of INITIAL RADIOACTIVITY 20 15 10 2 . 31 MONTHS NON-STERILE SOIL .\O STERILE SOIL 32 Figure 7. The effect of soil sterlization on the formation of non- polar transformation products in Kawkawlin sandy loam soil. % of INITIAL RADIOACTIVITY 10- TRIFLURALIN PENDIMETHALIN e e o e t e '9 ORYZALIN '? 33 NON-STERILE SOIL _ STERILE SOIL o-----..o s. I...- I. -—----.‘ /0 O 34 but that micro-organisms may contribute to eventual mineralization of applied herbicide. However, our results indicate that soil micro- organisms actively participate in the degradation and binding of these dinitroaniline herbicides in soil. Differences between sterilized and nonsterilized treatments did not develop until after the first month indicating that either an incubation period is required before biologi- cal degradation becomes apparent or that the additional corn stocks to the flask caused a catabolic repression that limited degradation during the first month (Figures 1, 2, and 3). The difference between total extractable radioactivity (parent plus extractable transformation products) from sterilized and nonsterilized treatments did not become significant until after 6 months (Figure 8). It appears that the major role of soil microorganisms lies in the forma- tion of metabolites from parent herbicide and secondly in the formation of soil bound residues. Less than 1% of the applied radioactivity was trapped as 14CO2 after 6 months (Figure 9). Mineralization of the organically complexed soil bound residues probably occurs concurrent with mineralization of soil O.M. Mineralization of 14C-pendimethalin and 14C-trifluralin followed 14CO from 14 2 never accumulated to detectable levels. Soil sterilization did not in- f 14 d 14 similar patterns over the 6 month period while C-oryzalin fluence mineralization o C-pendimethalin an C-oryzalin, however, 14 more 14CO was trapped in sterilized than nonsterilized C-trifluralin 2 treated soil (Table 3). The soil sterilant, propylene oxide, is a strong oxidant and possibly reacted with vapor 14C-trifluralin resulting in d 14 oxidative dealkylation an CO2 production. 35 Figure 8. The effect of soil sterilization on the dissipation of total extractable radioactivity in Kawkawlin sandy loam soil. a) Results are averaged over the three herbicides. 36 L50.“ NON-STERILE SOIL O o/ x of INITIAL RADIOACTIVI‘I'Y 3 MONTHS 37 Figure 9. Evolution of 14CO2 from 14C-pendimethalin, 14C-trifluralin, and 14C-oryzalin treated Kawkawlin sandy loam soil over time. 0 a) Results are averaged over soil sterilization treatment. 38 o_7 TRIFLURALIN q. PENDIMETHALIN * X of INITIAL RADIOACTIVITY LSD 0.3 _ For I l 0.2 -L ORYZALIN gag-0.000000000000000 no..." 4 6 MONTHS 39 Table 3. The effect of soil sterilization on evolution of 14CO2 from treated Kawkawlin sandy loam soil. Soil Treatmentéiy/ Herbicide Sterile Nonsterile --% of initial radioactivity-- Pendimethalin 0.40 0.48 Trifluralin 0.71 0.43 Oryzalin 0.00 0.08 a/ _ --LSD.01 — 0.12. b/ — Results are averaged over analysis time. 40 Soil O.M. was divided into feur fractions, 1) 95:4:1 (v/v/v) acetone- H O-HCl soluble (95-4-1), 2) fulvic acid, 3) humic acid, and 4) humin. 2 Soil sterilization had little effect on the amount of herbicide residues contained in the 95-4-1 soluble fraction. The majority of residues be- came associated with this fraction during the first month before biologi- cal degradation had become evident (Figure 10). Herbicide residues appear to become associated with the 95-4-1 fraction primarily by chemical or physical rather than by biological processes. The accumulation of 14C-herbicide residues in the 95-4-1 fraction is shown averaged over sterilized and nonsterilized treatments (Figure 11). After 6 months, 7.3%, 6.1%, and 10.1% of the applied 14C-pendimethalin, 14C-trifluralin, and 14C-oryzalin, respectively, was associated with the 95-4-1 fraction in nonsterilized treatments. The largest increase in residue incorporation occurred during the first month. Slight increase followed through 4 months in trifluralin and oryzalin treated soils with a small decrease between 4 and 6 months. Residues of trifluralin were about one-half those of oryzalin. Residues of pendimethalin reached a maximum at 1 month, followed by a decline between 1 and 6 months. Losses in radioactivity suggest movement of residues from the 95-4-1 fraction into the more humified O.M. fractions. The 95-4-1 extraction solvent removed 19.5% by weight, of the O.M. from the sandy loam soil and 45% of the soil bound radioactivity. Therefore, the proportion of radio- activity extracted with the 95-4-1 solvent is not due to the amount of soil O.M. removal, but rather results from the nature of the herbicide residues and the nature of the soil O.M. with which they are associated. A larger amount of O.M. was soluble in the 95-4-1 extraction solvent from the silty clay loam than the sandy loam soil (Table 4). This soil 41 Figure 10. The effect of soil sterilization on the accumulation of radioactivity in the 95-4-1 soluble organic fraction in Kawkawlin sandy loam soil. 3) Results are averaged over the three herbicides. p % of INITIAL RADIOACTIVITY Q Q h N . 42 NON-STERILE SOIL O /rERILE SOIL o a ' Q " vfl‘ - O Q ‘C Q Q C Q - C“‘ .-~ ‘~ ~o LS'ID.“ 1 2 4 6 MONTHS 43 Figure 11. The accumulation of radioactivity in the 95-4-1 soluble organic fraction in Kawkawlin sandy loam soil treated . l4 . . l4 . . l4 . with C-pendimethalin, C-trifluralin, and C-oryzalin. a) Results are averaged over soil sterilization treatment. 44 u N O I? O O D .< N > E Z p O .0. TRIFLURALIN p’ “ ...~Q “‘ ~... .0 % of INITIAL RADIOACTIVITY MONTHS 45 Table 4. The influence of soil texture on the amount of organic matter removed with the 95-4-1 extraction solvent. Soil Texturegih/ Analysis time Sandy loam Silty clay loam Months ---------- mg dry wt ........... l 179 350 2 174 308 4 228 363 6 172 350 a/ _ «LSD.01 — 35.48. b/ — Results are averaged over the three herbicides and soil sterilization treatment. 46 related extraction artifact resulted in the removal of a greater pro- portion of herbicide residues from the silty clay loam soil (Table 5). Less than 10% of the total radioactivity in the 95-4-1 fraction or less than 1% of the applied radioactivity could be partitioned into a nonpolar solvent (Table 6). Only parent herbicide and nonpolar trans- formation products formed during the early stages of degradation parti- tion into a suitable nonpolar solvent. Nonpolar materials were not separated using thin-layer chromatography because of the low quantities involved. Dialysis of the 95-4-1 soluble extract yielded a precipitate with the loss of acetone from the dialysis bag. Freezing of the aqueous sus— pension following dialysis flocculated the precipitate improving the effectiveness of the centrifugation used to isolate the material. The pattern of nondialyzable bound herbicide residues is unique for each herbicide in each soil (Figure 12). The dynamic, transient nature of the 95-4-1 soluble fraction is shown by the unpredictable changes that occur over time in the proportion of nondialyzable residues. The pro- portion of dialyzable radioactivity would be expected to decrease with time as a result of soil humification processes if the 95-4-1 soluble fraction were a static, nonintermediary residue pool. Soil sterilization did not have a significant effect on the complexing of herbicide degrada- tion products with 95-4-1 solvent extractable nondialyzable O.M. (Table 7). The accumulation of 14C-herbicide residues in the fulvic acid, humic acid, and humin organic fractions is shown in Figures 13, 14, and 15. After 6 months, 1.7%, 1.7%, and 2.8% of the applied 14C-pendimethalin, l4C-trifluralin, and 14C-oryzalin, respectively, had become associated with fulvic acid; 1.7%, 1.9%, and 2.6% was complexed with humic acid; and 47 Table 5. The influence of soil texture on the amount of radioactivity removed with the 95-4-1 extraction solvent. Soil texture 95-4-1 Soluble Residueséih/ --% of initial radioactivity-- Sandy loam 7.9 Silty clay loam 9.7 a/ = —-LSD.01 0.6. b/ —-Results are averaged over the three herbicides, soil sterilization treatment, and analysis times. 48 Table 6. The proportion of partitionable, nonpolar herbicide residues removed with the 95-4-1 extraction solvent from Kawkawlin sandy loam soil. Herbicide Nonpolar Residuesflih/ ——% of initial radioactivity-- Pendimethalin 0.8 Trifluralin 0.6 Oryzalin 0.7 a/ _ —-LSD.01 - 0.08. b/ —-Resu1ts are averaged over analysis time and soil sterilization treat- ment. 49 Figure 12. The proportion of non-dialyzable residues in the 95-4-1 soluble organic fraction in 14C-pendimethalin, 14C- trifluralin, or 14C-oryzalin treated Kawkawlin sandy loam soil. a) Results are averaged over soil sterilization treatment. 50 a L. m e» m. u § R . ~ U 0 Q l. 4 w .mL 4% m A O m N n O m m e. n o n .. N m z m. .. . m 0 coo C D .— ... a m . h .. .... a . a . 5 4 3 2 1 >h.>..—.U- I- E N 60 4“ IRELURAUN, STERILE SOIL II-Dl- -----------.-~ s so 1 TRIFLURALIN NON-STERILE SOIL ° E 40 E '8 3‘ 30 DEGRADATION PRODUCTS, 3 BOUND. 20 NON-STERILE SOIL Lemma sou fi" 11"”00” .‘_,..- 1 My ”".... U/: ..... fistazzzzzzzzla DEGRADATION PRODUCTS, STERILE SOIL STERILE SOIL 2 4 6 MONTHS 76 APPENDIX C Figure C-l. The distribution of radioactivity from 14C-oryzalin among parent compound, extractable transformation pro- ducts, and soil bound residues in Sharpsburg silty clay loam soil. 77 100 90 SO ... .‘-. ORYZALN, STERILE SOIL 70 ..~. .I. . .----------- 65 O ZALIN, NON-STERILE SOIL O\o % of INITIAL RADIOACTIVITY S 3 30 ,BOUND, NON-STERILE SOIL 'III ”fly/1”,” ”””lle 20 ”If: DEGRADATION PRODUCTS, NON-STERILE SOI ’ o------ * 1° ._. //' EBOLND. STERILE SOIL I I, ...oO'ooooo ooorooooooOOO°”......... / ”...“...u DEGRADATION PRODUCTS (man-m- STERILE SOIL O 1 2 4 6 MONTHS 78 APPENDIX D 14 14 . . 14 . . Table D-l. Total C-recovery from C-pendimethalin, C-trifluralin, and 14C-oryzalin treated Sharpsburg silty clay loam soil. Herbicide Recoverygih/ --% of initial radioactivity-- Pendimethalin 94.8 Trifluralin 35,4 Oryzalin 97.0 a/ .. --LSD.01 - 3.0. b/ -Results are averaged over analysis time and soil sterility treatment. 79 APPENDIX E Figure E-l. The effect of soil sterilization on the accumulation of extractable transformation products in Sharpsburg silty clay loam soil. a) Results are averaged over the three herbicides. N O 3.. 0| 3 X of INITIAL RADIOACTIVITY UI LSDm 80 WN-STERILE SOIL o_o 81 APPENDIX F Figure F-l. The effect of soil sterilization on the accumulation of soil bound residues in Sharpsburg silty clay loam soil. a) Results are averaged over the three herbicides. X of INITIAL RADIOACTIVITY 25 20 15 LSDm 82 NON-STERILE SOIL STERILE SOIL 83 APPENDIX G Figure G-l. The effect of soil sterilization on the formation of nonpolar transformation products in Sharpsburg silty clay loam soil. 96 of INITIAL HADIOACTIVITY u 0 a O PENDIMETHALIN TRIFLURALIN an 12 ORYZALIN CI 84 NON-STEIILE SOIL O—o “NILE SOIL .I-I-I-I. ¢"" ~— 0’... _ O O O o "““ I” Q- —-¢"’ " o :— I L50»: /0 0 4. o o" o" ,o" '0 'O O o I o ““. 1 2 4 5 MONTHS 85 APPENDIX H Table H-l. The effect of soil sterilization on the dissipation of total extractable radioactivity from the Sharpsburg silty clay loam soil. Soil Treatmentgih/ Herbicide Sterile Nonsterile --% of initial radioactivity-- Pendimethalin 81.1 79.4 Trifluralin 74.3 71.7 Oryzalin 82.7 73.2 a/ _ —-LSD.01 - 4.4. b/ -Resu1ts are averaged over analysis time. 86 APPENDIX I Figure I-l. Evolution of 14CO2 from 14C-pendimethalin, 14C- trifluralin, and 14C-oryzalin treated Sharpsburg silty clay loam soil over time. a) Results are averaged over soil sterilization treatment. 87 X of INITIAL RADIOACTIVITY ORYZALIN oooomtboooooooooooooooofl 4 6 MONTHS 88 APPENDIX J Table J-l. The effect of soil sterilization on evolution of 14CO2 from treated Sharpsburg silty clay loam soil. Soil Treatmentglk/ Herbicide Sterile Nonsterile --% of initial radioactivity-- Pendimethalin 0.55 0.64 Trifluralin 0.72 0.60 Oryzalin 0.00 0.08 a/ — LSD.01 0.12. E/Results are averaged over analysis time. 89 APPENDIX K Figure K-l. The effect of soil sterilization on the accumulation of radioactivity in the 95-4-1 soluble organic fraction in Sharpsburg silty clay loam soil. a) Results are averaged over the three herbicides. X of INITIAL RADIOACTIVITY 12 10 90 NON-STERILE SOIL I - STERILE SOIL I. ‘~. —.-———--——"-----------0 l .‘ l l l I . LSQ .. 01 I l l l l MONTHS Figure L-l. 91 APPENDIX L The accumulation of radioactivity in the 95-4-1 soluble organic fraction in Sharpsburg silty clay loam soil treated with 14C-pendimethalin, 14C-trifluralin, or 14C- oryzalin. a) Results are averaged over soil sterilization treatment. 92 ‘3 o" O n 3. I. PENDIMETHALIN O O O >- . s . " § -‘ ' 7 ,o' o" g :: .OO"‘.’ 3 I .3. 5: Z :‘I S " .' N o 1 3 ‘ MONTHS ORYZALIN ”00.000000000000000“: 93 APPENDIX M Figure M-l. The proportion of nondialyzable residues in the 95-4-1 soluble organic fraction from 14C-pendimethalin, 14 trifluralin, or 14C-oryzalin treated Sharpsburg silty C- clay loam soil. a) Results are averaged over soil sterilization treatment. 94 PENDIMETHALIN 6 5 4 3 LSDJln TRIFLURALIN ~ ~§ .. ORYZALIN >._._>_._.0