MOVEMENT AND PERSESTENCE OF A HERBECIDE. S-AMlNOo 4-CHLOkO-2-PHENYL-3 (Em-PYRIQAZINONE (F‘YRAZGN), EN SOIL Thesis an 15:: Degree 0? pk. D. MICHIGAN STATE UNIVERSlTY Dudiey "E. Smith 1968 LIBRARY i“ ‘ Michigan State University l--:L'. ath- This is to certify that the thesis entitled Mevement and Persistence of a Herbicide, S-Amino-h- chloro-Q-Phenyl-3(2H) Pyridazinone (Pyrazon), in Soil presented by Dudley T. Smith has been accepted towards fulfillment of the requirements for Ph.D degree in Crgg Science (i3 {cum—— m “\mfi' E Major professoxflvl Date Feb. 15, 1968 0-169 ABSTRACT MOVEMENT AND PERSISTENCE or A HERBICIDE, S-AMINO— U—CHLORO—2-PHENYL-3(2H)-PYRIDAZINONE (PYRAZON), IN SOIL by Dudley T. Smith Pyrazon (S—amino-u-chloro-2-phenyl—3(2H)—pyridazinone) was applied alone and in combination with TCA (trichloro— acetic acid) in commercial sugar beet fields to determine pyrazon movement and persistence in soil under natural environmental conditions. Soils were sampled through the growing season and bioassayed with mustard (Brassica Juncea L). In laboratory studies, pyrazon treated soils were in- cubated at n.5, 12.5, 21.0, and 29.5 C for 20 weeks to determine bacteria and actinomycete populations in response to pyrazon. Radioactive labelled pyrazon was incubated in warm, moist soil to determine herbicide disappearance and possible breakdown products. Pyrazon adsorption was deter- mined in nine soils ranging from 2.5 to ll.U% organic matter and 11 to 37% clay. Pyrazon adsorption was correlated with soil organic matter (r=O.92) but not with clay content (r=0.09) or soil pH (r=—0.08) between 6.3 and 7.9. In the field 70 to 90% of the pyrazon residue was retained in the upper 2 inches of soil. Movement in the profile was retarded by a high Dudley T. Smith organic matter content and was not related to rainfall intensity in these soils. Pyrazon did not accumulate in field soils at lower depths as a result of movement. Residues and phytotoxicity were low in soils containing 20 to 30% clay and 2.5 to U.3% organic matter where rainfall was well distributed after herbicide application. Pyrazon was more persistent in soils containing organic matter in excess of 10% and where rainfall was low or poorly distri- buted. The persistence of phytotoxicity was related to soil organic matter; however, the level or intensity of phytotoxicity appeared to be associated with the clay con— tent. Following a 6 lb/acre application, pyrazon decreased from 2.5 to 0.25 ug/g of soil between 2 and 4 months after applications on a sandy loam containing 10% clay and 2.9% organic matter. Under the same environmental conditions pyrazon decreased from 2.75 to 2.25 ug/g in a sandy clay loam containing 26% clay and 12.9% organic matter. In three soils bacterial response to pyrazon was maxi- mal after 5 weeks of incubation at 21.0 C. With lower incu- bation temperatures more time was required for bacteria to respond to pyrazon. Bacteria increased gradually with time in amended soil incubated at u.5 C. In a laboratory incubation study pyrazon decreased from 11.6 to 9.6 mu moles/g between 3 and 7“ days of incu- bation. Part of the decrease in pyrazon was accounted for by the formation of a breakdown product that increased Dudley T. Smith from 0.0“ to 0.94 mu moles/g over the same period. The breakdown product did not contain the phenyl ring of pyrazon but retained the primary amino group. The de— gradation product was identified as 5—amino-u—chloro-3- pyridazinone and was subject to additional degradation in the soil. MOVEMENT AND PERSISTENCE OF A HERBICIDE, S-AMINO- M—CHLORO—2—PHENYL—3(2H)—PYRIDAZINONE (PYRAZON), IN SOIL By Dudley T. Smith A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crop Science 1968 "7‘ -D m?) 9!- U \ OLJ‘ ACKNOWLEDGMENTS The objective attitude of Dr. William F. Meggitt in this and other related research is sincerely appreciated. His encouragement and confidence in individual initiative provided the impetus for this study and other research endeavors. The assistance of Guidance Committee members, Drs. Max M. Mortland, Clifford J. Pollard, Stanley K. Ries, and F. Woodrow Synder in technical aspects and interpretation of data is gratefully acknowledged. The technical assistance, constructive comments and cooperation of all the members in the week control project in the Department of Crop Science, and Mr. Gerald Stephen- son, Department of Horticulture is appreciated. The interest and financial support of Amchem Products, Inc., Ambler, Pennsylvania; Farmers and Manufacturers Beet Sugar Association, Saginaw, Michigan; Northern Ohio Sugar Com- pany, Fremont, Ohio; and Great Western Sugar Company, Longmont, Colorado deserve special mention. The author is most grateful to his wife, Angela, for her assistance, encouragement, and many helpful comments in conducting this study and preparing the original manuscript. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . iv LIST OF FIGURES . . . . . . . . . . . v LIST OF APPENDIX TABLES . . . . . . . . vi INTRODUCTION . . . . . . . . . . . . 1 REVIEW OF LITERATURE 3 Introduction 3 Factors Affecting Soil Microorganism Degradation of Herbicides. . . U Retention of Herbicides in Soil. . . 7 Climatic Factors and Field Studies on Herbicide Persistence . . . . . . . 10 Pyrazon in Plants and Soil . . . . . . 12 MATERIALS AND METHODS . . . . . . . . . 18 RESULTS AND DISCUSSION. . . . . . . . . 31 Mustard as a Bioassay for Pyrazon . . . . 31 Pyrazon Distribution and Movement . . . . 3A The Persistence and Disappearance of Pyrazon from the Upper 2 Inches of Soil . U0 Adsorption of Pyrazon in Soil . . . . 47 Response of Soil Microorganisms to Pyrazon . A9 Disappearance of Pyrazon in Soil . . . . 53 SUMMARY AND CONCLUSIONS . . . . . . . . 60 LITERATURE CITED. . . . . . . . . . . 67 APPENDIX . . . . . . . . . . . . . 73 iii LIST OF TABLES Table Page 1. Characteristics of Soils in Field Studies . l9 2. Characteristics of Soils in the Pyrazon Incubation Study . . . . . . . . 23 3. Characteristics of Soils in the Pyrazon Adsorption Study . . . . . . . . 29 A. Fresh Weight of Mustard from Soil Treated with Pyrazon, TCA, and Combinations of Both Herbicides. . . . . . . . . 32 5. Enhancement of Mustard Growth by Pyrazon Added to Four Soils . . . . . . . 33 6. Distribution of Pyrazon in Two Clay Loam Soils in 1965 as Accessed by Bioassay Injury Ratings . . . . . . . . . 36 7. Pyrazon Residues at Three Depths in Three Soils as Related to Time of Sampling as Accessed by Bioassay Injury Ratings . . 37 8. Simple Correlation Coefficients (r) Be- tween Pyrazon Adsorption and Soil Properties . . . . . . . . . . A7 9. Relationship Between Soil Organic Matter and Herbicide Adsorption from Two Con— centrations of Pyrazon . . . . . . M9 10. Recovery of Radioactivit from Soils Amended with 3H and l c Labelled Pyrazon . . . . . . . . . . . 54 11. Distribution of Radioactivity on Chromato- grams of Extracts from Soil Incubated for 30 to 7“ Days with lac-Pyrazon . . 56 iv LIST OF FIGURES Figure Page 1. Pyrazon Movement in Two Clay Loam Soils with Varying Clay and Organic Matter Contents, 6 Pounds Per Acre Applied . . 39 2. Pyrazon Persistence in the Upper Two Inches of Seven Soils with Varying Clay and Organic Matter Contents, 0, A, and 6 Pounds Per Acre Applied . . . . . . M1 3. Changes in Bacteria POpulations in Three Soils Amended with Pyrazon and Incubated at 21 C . . . . . . . . . . . 50 A. Changes in Bacteria Populations in a Sandy Clay Loam Amended with Pyrazon and Incubated at Four Temperatures . . 50 5. Correspondence of Colored Bands and lLAC Radioactivity on a Thin Layer Chromato— gram After Incubation of lAC-Pyrazon in a Sandy Loam Soil for 74 Days . . . . 58 6. Recovery of Pyrazon and a Meta olite from a Sandy Loam Amended with l c Labelled Pyrazon . . . . . . . . . . . 58 Table II. III. IV. VI. VII. VIII. IX. LIST OF APPENDIX TABLES Page Summary of Significance in Analyses of Vari- ance and Interactions in Pyrazon Bio- assays in 1965 Field Samples . . . . . 7“ Summary of Significance in Analyses of Vari- ance and Interactions in Pyrazon Bio- assays in 1966 Field Samples . . . . . 7H Bioassay Injury Ratings in Seven Bioassay Standards . . . . . . . . . . . 75 Pyrazon Residues in Three Soils During 1965 as Related to Rate of Application, as Accessed by Bioassay Injury Ratings. . . 75 Fresh Weight of Mustard with Several Rates of Pyrazon Added to Three Soils in 1966 . 76 Pyrazon Concentrations and Disappearance in Seven Soils as Related to Soil Properties and Rainfall, 6 Pounds Per Acre Applied . 76 Pyrazon Residues in Two Clay Loam Soils as Related to Rate of Pyrazon Bioassay Application and Time and Depth of Sampl- ing in 1965, as Accessed by Injury Ratings . . . . . . . . . Pyrazon Concentrations in the Upper Two Inches of Six Soils in 1966 . . . . . 78 Bacteria and Actinomycete Populations in Three Soils Amended with Pyrazon, Incu— bated at 21.0 C . . . . . . . . . 79 Bacteria and Actinomycete Populations in a Sandy Clay Loam Amended with Pyrazon, Incubated at Four Temperatures . . . . 80 vi INTRODUCTION The accumulation and persistence of organic residues is a common feature of soils (17), particularly since soils are generally regarded as a dumping ground for waste mater- ials (2). Usually residues arise from plant and animal sources but in recent years man has added increasing amounts of organic residues to soils, primarily in the form of pesticides. Chemical week control is an essential agricultural practice today, extending into all areas of crop and plant production. Efficient growth of desirable plants is still a simple basic requirement for man's existence. Man must be intelligent enough to observe his natural environment and manipulate it prOperly in order to survive. There has been an increasing concern regarding the use of biologically active materials in our environment. Herbicide behavior in soils has been investigated in numerous laboratory, greenhouse, and field studies; however, there are few studies where laboratory and greenhouse find— ings have been directly correlated with field observations. This study was conducted to investigate the movement, per- sistence, and possible accumulation of pyrazon in sugar beet soils under field conditions. Adsorption, degradation, and microbial response to pyrazon in soil were investi- gated in a laboratory to investigate and explain pheno- mena observed in the field. .W‘i 'a‘l .a. REVIEW OF LITERATURE Introduction In 1907 Schreiner and Reed (5“) found that phyto- toxic materials such as terpenes, courmarin, and vanillin, resulting from microbial degradation of plant material, 'F. L..._L. {LO—LE“ contributed to low productivity in soil. These investi— . gators showed that toxic substances could be removed by carbon black and recognized soil organic matter as a complex adsorbing material. In 19U6 (19), during the initial development of chemical weed control, the leach— ing, disappearance, and phytotoxicity of herbicides in soil was investigated at Camp Detrick. After observing the disappearance of two phenoxyacetic acids and a phenyl— carbamate in soil, De Rose (19) stated, "At the present time [1946] no information is available to explain this difference in persistence." Now, less than 25 years later, there are extensive reviews concerning the behavior and disappearance of herbicides in soil. Aldrich (l), in an early review, discussed herbi— cide persistence in relationship to microorganisms, re- tention by soil colloids, leaching, and decomposition in soil. More recent reviews concern the nature of the herbicide adsorption phenomena in soil (6, l7), relation— ships with soil microorganisms (2, 8, H0), effects of 3 climatic and environmental factors and agricultural practices on herbicides in soil (55, 58, 60) and the practical significance of pesticides in the soil en- vironment (22, “3, A8). Factors Affecting Soil Microorganism Degradation of Herbicides A portion of all herbicide applications eventually reach the soil, either directly or indirectly from crop residues (57). Alexander (2) emphasized that despite man's concept of microbial infallibility, soil organisms do have limitations in the materials degraded. Furthermore, microbes may generate metabolic products that are more toxic than the original substance. As explained by Kearney (H0),soil microbes are in reality complex enzyme systems capable of degrading a large number of organic pesticides. Frequently a lag period occurs between the time of pesti- cide application and the first observed loss while there is a buildup of adaptive organisms. Schlegel et_al. (53) re— gard the mixed microbial populations of soil and water as a community of competing species and metabolic types. Growth of one or a few species will be favored at the ex- pense of others as external conditions change. Aldrich (l) and Bollen (8) pointed out that herbi— cide persistance is mainly determined by conditions affect- ing microbial activity in soil. Techniques in isolating soil organisms were reviewed by Schegel et a1. (53). The .— mi In. importance of bioassays in herbicide persistance studies involving soil microorganisms were pointed out by Alexander (2). Structure Molecular structure is of major importance in deter- mining the susceptibility of herbicides to microbial at- tack. In chlorinated phenoxy compounds, resistance to degradation is governed more by the position of the halo- gen on the ring rather than the number (3, 42), whereas the number of halogens is more important in the case of benzoic acids (U2). Kearney et_al. (Al) indicated that chlorosubstitution, the addition of large electronegative atoms, influenced the disappearance of aliphatic acids as well as aromatic compounds. In a classical experiment Jenson (36) observed an 80% loss of mono- and dichloro- acetic acids in 2 to A and 8 to 1A days, respectively, in soil, whereas I“ to 28 days were required for a comparable loss of trichloroacetic acid (TCA). It was emphasized (36) that only a limited number of soil bacteria develop dechlorinating enzymes. Kaufman (39) showed that the presence of one molecular structure influenced the degradation of another. Amitrole apparently interfered with the proliferation of organisms capable of degrading dalapon, a chlorinated aliphatic acid, however, the converse was not observed with this combi- nation. Temperature and Moisture Effects of temperature on microorganisms were re- viewed by Farrell e£_al. (2U). Burnside (10) and Ercegovich gt_al. (23) showed relationships between soil temperature and herbicide disappearance. The breakdown of an s—triazine was clearly temperature dependent. Atrazine was not observed after 1, 2, and 9 months in soils incubated at 35, 25, and 15 C, respectively. Amitrole disappeared more rapidly as soil temperature increased from 8 to 100 C and also disappeared more rapidly in moist soil than in dry soil (23). Persistance of amiben (10) and atrazine (10, A2) in laboratory incubation studies was contrary to that usually observed under field conditions. Bollen (8) discussed herbicide losses in incubation studies in relation to losses observed under field conditions and indicated that some variables, such as unequal herbicide distribution in the field micro—environment, soil drainage, and rhizosphere conditions could not be duplicated in laboratory incu- bation studies. Soil pH and Fertility Persistance may be affected by soil pH and fertility. Corbin and Upchurch (l6) determined the optimum soil pH for detoxification of several herbicides. Marked differ- ences were observed. Cochrane (l3) pointed out that actinomycetes tend to be sensitive to phosphates in the soil; hence, soil pH is a factor in regulating the growth and activity of those organisms. Bacteria generally re- spond best in soil near a neutral pH while fungi are more predominant in acid soils. Retention of Herbicides in Soil Bailey and White (6) discussed the nature of soil colloids, pH, moisture, temperature, and herbicide formu- lation with regard to herbicide sorption phenomena. Dean (17) indicated that hydrogen bonding, polar molecules, and basic amino groups are all probably involved in retaining and stabilizing organic pesticides in soil. Although not all compounds are particularly resistant to decomposition, many exhibit some degree of stability in the soil colloid system. Herbicide Structure In a study with five s—triazines, Harris (31) indi— cated that there was little relationship between herbicide structure and adsorption in soil. HOwever, it was sug- gested that electron distribution in the s—triazine molecule, as affected by substitution on the ring, may account for some differences observed in the adsorption of those compounds. Ward and Upchurch (62) studied ad— sorption of 52 related N—phenylcarbamates, acetanilides, ‘and anilines on nylon, cellulose, and cellulose acetate. Adsorption occurred by hydrogen bonding between the amido hydrogen of the adsorbate and the carbonyl oxygen of the adsorbent. Soil Colloids There has been some controversy as to the relative importance of clay in comparison to organic matter in herbicide adsorption. In an objective study, Harris and Sheets (33) stated that there was no single factor from which herbicide adsorption could be predicted. Montmoril- lonite clay may be important in Midwestern areas while organic matter may dictate the extent of adsorption in Southern soils (3U). Doherty (20) showed that adsorption may vary with different types of organic matter, depend- ing on the degree or stage of decomposition of the ad— sorbent. The importance of the organic matter in herbi- cide adsorption is evident in many instances (20, 3A, 35, 56) but the importance of clays should not be under— estimated (61) since there is ample evidence of the role of clay in herbicide adsorption (9, 30, 34, A5, 46). Harris gt_al. (3“) point out that herbicide research is at a stage where broad generalizations regarding adsorption are not very useful. Soil pH The influence of pH on adsorption was shown by Harris et a1. (3M) where DNBP and an s-triazine were readily adsorbed by a bentonite clay at pH 2.3 but not at pH 8.“. Adsorption of the two herbicides by muck and a cation ex- change resin was intermediate between the two extreme values, depending on the pH of the adsorbent. Adsorption of three unrelated herbicides was not related to soil pH in 32 soils, ranging from pH A.3 to 7.7 (33). Nearpass (A6) observed that adsorption of an s-triazine was corre- lated with titratable acidity,which was a better indicator of adsorption than soil pH. In a later study (A7), ad- sorption was related to the degree of base saturation rather than to a predominance of any specific cation on clay. With increased base saturation apparently more sites were occupied by cations and less herbicide ad- sorption occurred. Competition for adsorption sites has been observed in other instances (18, 30) in which less adsorption occurred when water was present and occupied the sites. Leaching Movement of herbicides in soil has been related to the adsorption and water solubility of herbicides. In a study of s—triazines (31), mobility in soil was more re- lated to adsorption than to water solubility. The mobility of s-triazines has also been related to pH dependent ad- sorption (46). Hilton et_al. (35) reported that although a substituted urea was adsorbed by soil, the herbicide was readily leached by water. Bayer (7) in studying the hi? IO role of surfactants in leaching of herbicides concluded that differences in herbicide movement were due to the wetting ability of surfactants and not their ionic char- acter. Photodecomposition and Volatility Jordan gt_al. (37) showed that after exposure to ultra violet light the spectrophotometric adsorption of herbicides was altered, indicating that photodecomposition occurred. Photodecomposition may be more of a problem in warm sunny areas than in those of frequent rainfall and low sunlight. The phytotoxic properties of a specific herbicide may be modified after exposure to sunlight or specific artificial light but the relative importance of photodecomposition under field conditions has not been demonstrated (60). In regard to herbicide losses by volatility, Up— church (60) pointed out that volatile herbicides are generally lost more readily in moist soil, due to com- petitive adsorption with water, than in dry soil. Ef- fects of competitive adsorption of water on the loss of a volatile herbicide have been demonstrated (l8). Climatic Factors and Field Studies on Herbicide Persistence The research previously described in this review was performed primarily under laboratory and greenhouse 11 conditions. Upchurch (60) indicated that although in— dividual "component reactions" should be understood, weed researchers have been slow to conduct herbicide experiments considering all the diverse conditions en- countered in the field. Bailey and White (6) indicated that soil structure, moisture, and temperature affected the persistence of herbicides indirectly but were of as major importance, nevertheless. h 9 Sheets and Harris (58) indicated that favorable 5‘ weather both before and after application is important in the degradation of herbicides. The significance of herbi- cide residues was also discussed in relationship to soil properties and subsequent crop production. Rainfall, soil properties, and the total environment must be con- sidered when evaluating herbicide disappearance in soil (55). In one investigation (61) phytotoxicity of five herbicides was studied over a three year period at 17 field sites. Approximately 60% of the variability in plant response was accounted for on the basis of soil and climatic factors. Disappearance of a substituted benzoic acid herbicide was accounted for by geographical differ— ences in soil type and rainfall (11). Wide variation in the loss of pichloram, a highly persistent herbicide, was observed (29) but rates of disappearance were correlated with soil and environmental factors. 12 The persistence and mobility of endothall, a herbi- cide used in sugar beets, was investigated in an arid region (1A). Movement depended on irrigation methods and soil clay content and was not affected by the amount of soil moisture at application. Phytotoxic residues disappeared rapidly in soils with low retention capacity and were not found at the end of the growing season. Pyrazon in Plants and Soil Development and Use In 1962 Fischer (25) reported that a group of phenylpyridazones were effective herbicides in sugar beets. Pyrazon (l—phenyl-A-amino-5-chloro-6-pyridazone) exhibited the best herbicidal properties of 500 derivah tives investigated. Accepted nomenclature for pyrazon in the United States is 5-amino-U-chloro-2-phenyl-3(2H)- pyridazinone or PCA. In Michigan (MA) in 1963 pyrazon applied preemergence in sugar beet fields was most ef- fective at 3 and A lbs/acre in combination with TCA, in- dicating that low rates of this herbicide were effective in humid regions in the United States. Comparable per- formance was observed in Ontario, Canada (27). Use, application methods, and performance of pyrazon and other herbicides used in sugar beets were reviewed by Alley (A). Soil Microorganisms Jung (38) reported that CO2 evolution from soils amended with glucose, starch, or cellulose was not ad- versely affected by 60 and 660 ppm of pyrazon in the soil. In soils amended with glucose or wheat straw, CO2 evolu— tion was stimulated slightly by the addition of pyrazon, indicating that microbial activity was enhanced by the presence of the herbicide. Nitrification in soil was not affected by pyrazon at rates up to 100 kg/ha, however, respiration and nitrification were reduced by rates equi- valent to 1/2 and l ton/acre. Wolcott (63) observed that 250 ppm of pyrazon had no effect on nitrification in combined pure cultures of Nitrosomonas europaea and Nitrobacter agilis. In fungal cultures Pommer (50) observed that the growth and repro- duction of Aspergillus, Penicillium, Trichoderma, and Rhizopus were not affected by 10 ppm of pyrazon. Develop— ment and reproduction of naturally occurring fungi in soils at pH 5.3 and 7.5 were not affected by 100 ppm of pyIazon. Fate in Plants Stephenson et_al, (59) showed that roots of sus— Cexotible species absorbed and accumulated pyrazon more Peéadily than roots of a tolerant species, red beet. Trainslocation to the shoot and foliar absorption of pyr’azon was also greater in two susceptible species 14 than in red beet. A metabolite was extracted from red beet tissue but was not found in susceptible species. This compound was later conclusively identified as N-glucosyl pyrazon (52). It appears that in addition to differential uptake of the herbicide there is an addi— tional mode of selectivity, in which the herbicide may be detoxified in tolerant plants. The importance of the presence and the position of the amino group on the pyridazine ring for herbicidal activity has been shown (26). In contrast to work in the United States (52, 59), other researchers (21, 26) have observed a different meta- bolite in sugar beets. Dephenylated pyrazon or 5—amino- U-chloro—3-pyridazinone (metabolite A), was found in beet extracts five days after the herbicide was applied (26). Apparently the metabolite was not phytotoxic although no evidence was offered. Egte in Soils Drescher (21) stated that the breakdown of pyrazon wads slower in soils than in plants. In laboratory trials 4C) weeks were required for the dissipation of 12 ppm of thrazon in incubated loamy sand. No residues from a 20 Dprn application were detected in potted soil after 12 Weeeks of exposure outdoors. In field experiments pyrazon was; applied at 3.2 kg/ha and immediately after appli- cat ion a residue of 4 ppm was observed in the upper 4 inches 15 of soil. This residue decreased to 0.2 ppm within 11 weeks. Fischer (26) reported that the residual phyto— toxicity of pyrazon in loamy sand was shorter than that of monuron or simazine and that corn or small grain was not injured when planted 8 to 10 weeks after pyrazon was applied. Metabolite A has been observed in soil shortly after pyrazon application (26). Cooke (15) found that pyrazon adsorption in five pure clays was highest with hectorite, intermediate with illite and attapulite, and lowest with halloysite and montmorillonite. When pyrazon was added at 500 ug/g in 0.01 M CaCl2 hectorite adsorbed 100 ug/g while montmoril- lonite adsorbed A ug/g; however, both of these are a type of montmorillonite clay. Doherty (20) studied pyrazon adsorption by organic matter and bentonite by mixing 1% of the adsorbent with quartz sand. Adsorption was deter— mined by a bioassay and was in the order of fibrous peat > muck soil >> Sphagnum moss > bentonite clay. The fibrous peat was highest in organic matter while the Sphagnum was lheast decomposed. Indications were that adsorption was ITélated to the stage of decomposition as well as the per- Celntage of organic matter. Adsorption of pyrazon was not dinrectly related to surface area or cation exchange capa- Ciizy of the adsorbents. In Frank's work (28) soil type acczounted for differences in phytotoxicity from pyrazon. Phytoxicity in bioassays was greater in a sandy loam than in two clay soils, indicating that less adsorption occurred in the lighter soil. Movement Although pyrazon was adsorbed by soil colloids (15, 20) extensive leaching has been observed. In one study (15) pyrazon was applied at 6 lbs/acre to a moist sandy loam soil in a column. After A inches of water were applied over a A hour period the column was split longi- tudinally and bioassayed. Pyrazon trailed from the sur- face to 5 1/2 inches below the surface. TCA, which is frequently applied with pyrazon, exhibits an ionic character in water, is highly soluble, and adsorption is extremely limited (Al). The mobility of this herbicide was demonstrated by Ogle e£_al. (49), in which TCA moved U to 6 inches in fine sand, silt loam, and a muck soil after an application of 2 inches of water. This high degree of mobility has been confirmed by others (M1), but there was no report of the effect of this herbi— cide on the movement of another less mobile compound. The rapid loss of TCA in field soils is associated with its mobility. Photodecomposition and Volatility Zabik (6A) subjected a pyrazon solution to irradi- ation from a mercury arc lamp. After exposure, 17 approximately six different fragments or breakdown pro— ducts were observed with about 80% existing as one product. Although fragments were not positively identi- fied, pyrazon was not recovered. This work indicated that photodecomposition of pyrazon may occur if exposed to sunlight under field conditions. Fischer (26) negated the possibility of any significant loss of pyrazon by volatilization since the vapor pressure of the compound was low at temperatures usually encountered. At 40 C the vapor pressure was 0.074 mm of Hg. MATERIALS AND METHODS Field Experiments Pyrazon residues were evaluated in several soils during 1965 and 1966 under natural environmental con- ditions. In 1965 pyrazon was applied at three field I‘ locations at 0, 2, 3, U, and 6 lbs/acre and, except for the 2— and 6;pound rates, at the same rates in combi- nation with TCA at 8 lbs/acre. Pyrazon was also applied at 10 lbs/acre at location 2. In 1966 treatments were ap- plied at five locations at 2, 3, A, and 6 lbs/acre of pyrazon in combination with TCA at 8 lbs/acre and at 0, A, and 6 lbs/acre of pyrazone alone. Herbicide treat— ments were applied between May 1 and 13 in 1965 and April 15 and May 10 in 1966, in three replications in commercial sugar beet fields in Michigan. All herbicides were ap— plied in 23 gal/acre with a tractor mounted plot sprayer. Plots were A rows wide (approximately 10 ft.) by 50 to 100 ft. long. Soil composition was determined from a composite sample at the respective locations (Table 1). Soil samples were obtained three times during 1965 in a two to five month period after application to deter- mine residual concentrations of pyrazon at three soil 18 l9 depths. Samples were obtained from 0 to 2, 2 to 4, and 4 to 8 inches below the surface by dividing 15 to 20 soil core probes from each plot into three segments. During 1966 soil samples were obtained two to three times be- tween 2 and 6 months following application from the upper 2 inches of soil. One pound samples were sealed in wax cups and stored at 4.5 C or lower for 3 to 7 months until bioassayed. Daily distribution and intensity of rainfall TABLE l.--Characteristics of soils in field studies. Soil Characteristics Soil Soil No. Texture Organic Sand Silt Clay Matter pH % % % % 1 Clay loam 44.0 24.0 32.0 3.8 6.8 2 Clay loam 32.0 29.0 39.0 6.8 6.7 3 Sandy loam 67.0 24.0 9.0 13.7 7.0 4 Sandy clay loam 54.0 21.6 24.4 4.2 7.3 5 Sandy clay loam 48.2 23.8 28.0 4.3 7.5 6 Sandy clay loam 52.8 19.0 28.2 4.2 6.2 7 Sandy clay loam 46.6 26.8 26.6 12.9 7.6 8 Sandy loam 79.1 10.9 10.0 2.9 7.3 20 during the season was determined from data compiled by regional weather stations (5). Soil Analysis All soils were crushed and sieved through a 10 mesh screen prior to laboratory analyses. Soil mechanical analyses were determined using the Bouyoucos hydrometer method from which soil texture was determined. Organic matter was determined from 15 to 20 g of oven dried soil (105 C) by weight difference after combusion at 400 C. Soil pH was determined from a soil—water suspension (1:1) with a glass-calomel electrode system, one hour after initial mixing. Water holding capacity was determined by the water retained by 100 g of dry soil after six hours of free drainage. All analyses and determinations were made in duplicate. Cation exchange capacity was deter- mined from the amount of cations extracted from soil with 1.0 N ammonium acetate, pH 7.0 and from buffer suppression by hydrogen ions, in nine soils in an adsorption study. Biological Assay for Pyrazon Since many field samples may have contained TCA as well as pyrazon, a preliminary experiment was conducted to determine the selectivity and sensitivity of brown mustard (Brassica juncea (L) Cosson, var. Florida Broadleaf) for pyrazon. Pyrazon and TCA were added alone and in combi- nation up to 5 ug/g of soil in four replications. Plant 21 growth was observed and fresh weight of mustard plants was determined 3 weeks after planting. Pyrazon was determined in soil samples with a mustard bioassay. Samples from field plots were crushed and sieved and 250 g of air—dried soil were placed in a wax cup with holes punched to allow drainage. Mustard seed was covered with coarse washed sand and seedlings were thinned to ten plants per cup at an early cotyledon stage. Fresh weight and plant injury were determined 30 to 40 days after planting. Visual injury from pyrazon was evaluated on a scale of 0 to 10 where 0 indicated no ap— parent injury while 10 indicated that all plants were killed. Injury ratings between 0 and 10 were based on the intensity of damage and area of chlorosis on the foliage. Soil containing known amounts of pyrazon was included in the bioassay of all samples, except those from location 1 in 1965, to access residual concentration of pyrazon in field samples. Pyrazon was added to untreated soil from each location at rates ranging from 0.0 to 5.0 pg/g. Twenty—five m1 of prepared pyrazon standards were pipetted on 250 g of soil spread out on paper. Mixing was accom- plished by lifting alternate corners and sides of the paper, tumbling the soil back and forth across the sur- face. Clumps of moist soil were broken and redistributed during mixing. Excessive soil wetting, loss of herbicide 22 and contamination were reduced or avoided by this method while soil structure was maintained. Standards were replicated three times and were randomized with samples from the same soil during the bioassay. Pyrazon con- centrations in field samples were determined by compari- son of bioassay injury ratings to a standard curve from the same soil. Response of Soil Micro- organisms to Pyrazon The influence of temperature and time of incubation on the response of microorganisms to pyrazon was investi- gated in three soils. A sandy clay loam (Table 2) was ob- tained from a sugar beet field after recent tillage and was crushed, sieved, and thoroughly mixed prior to treat- ment. Treatments consisted of two soil treatments; an un- treated control, and soil amended with pyrazon, four in- cubation temperatures; 4.5, 12.5, 21.0, and 29.5 C, and four times of incubation; 5, 10, 15, and 20 weeks. In addition, soil at 21.0 ngas removed after 2 weeks of incubation. Samples incubated at 4.5 C were not obtained at the 15 week interval. Soil was amended with pyrazon at the rate of 4 ug/g. The amount of herbicide necessary for soil treatment was first mixed with washed quartz Sand and then sprinkled over the soil with intermittent SOil mixing. Mixing was accomplished as previously de- scribed in connection with the bioassay standards. Two 23 hundred g of soil were weighed into incubation containers. Containers were prepared by punching holes in the side of low-form wax cups near the lip and plugging holes with sterile cotton. Sterilized water was added to soil, equi- valent to 60% of the water-holding capacity, and containers were covered with tight fitting lids during incubation. TABLE 2.—-Characteristics of soils in the pyrazon incu- bation study. Soil Characteristics Texture Clay Mzgigic WHCl % % g/lOOg Sandy clay loam 23 4.8 35 Sandy loam (soil 3) 25 13.7 41 Clay loam (soil 1) 32 3.8 30 1 Soil water-holding capacity. Two additional soils, a sandy loam and a clay loam, were prepared and treated in the same manner described above, with some exceptions. These soils had been in a dry, dormant condition in a greenhouse for 7 months prior to treatment. In this study these soils were amended with DYrazon at 3 ug/g, incubated at 21 C, and samples were removed after 2, 5, 10, and 15 weeks of incubation. Time and soil treatments were randomized within each temperature treatment. All cups were weighed 24 periodically and soil moisture was maintained at 40 to 60% of water-holding capacity by the addition of steri— lized water. Only soils incubated at the highest tempera- ture longer than 10 weeks required appreciable amounts of water. Bacteria and actinomycete populations were deter— mined from soil as samples were removed from incubation. Microbe populations were determined by plating 2 m1 u, 10‘5, and lo‘6 dilutions of moist soil in tripli— of 10- cate in a soil extract agar (52). Plates were incubated in the dark at room temperature for 7 to 10 days before colonies were counted. Colony characteristics were re- 6 corded. Bacteria colonies were counted from a 10- dilution while actinomycetes were determined from a 10—5 dilution. Counts were corrected to a dry soil basis. Data were statistically analyzed on the basis of counts per g of soil but bacteria counts were expressed graphi- cally as a percentage of the untreated control from the same incubation time and temperature treatment to evaluate response to pyrazon. This percentage expression removed DOpulation fluctuations in amended soils occurring as a result of natural microbial responses to incubation en- Vironments. Soil pH was determined in untreated soils for each time and temperature treatment. Eggradation of Pyrazon in Soil. Radioactive labelled pyrazon was incorporated in a Sandy loam and incubated in a warm moist environment to 25 observe herbicide degradation in the soil. This soil con— tained 18.4% clay, 3.5% organic matter, and had a holding capacity of 29 g/100g of soil. One mg of pyrazon, uniformly tritiated in the phenyl ring, was applied in 20 m1 of water to 200 g of soil by the mixing process previously described. In preliminary isotope work, differences between pyrazon concentrations in soil were reduced from 41 to 4% after 10 and 20 minutes of mixing by the soil rolling process. In this study soil was mixed for 20 minutes and uniform herbicide distribution was con- firmed. Pyrazon, labelled with 1“C in positions 4 and 5 of the pyridazine ring, was added to a second batch of soil in a similar manner. Hence, two soils were amended with 5 pg of pyrazon/g (22.6 mu moles/g) with a radio- isotope label in each ring of the compound. Specific 3H-pyrazon and activity after dilution was 14.6 uc/mg of 19.6 uc/mg of lac-pyrazon. Both compounds were synthesized by BASF Corporation. Amended soils and an untreated control were incubated in closed glass containers at approximately 20 C at 60% of the water-holding capacity. Ten g samples of soil were removed after 3, 10, 15, 30, and 74 days of incubation. 3 Duplicate soil samples containing H-pyrazon were obtained after 30 days of incubation while duplicates of soil amended with lLAC—pyrazon were obtained after 30 and 74 days. Sample moisture was calculated from changes in 26 soil weight during the experiment. Soils were frozen until all samples were obtained. Samples were extracted with 10 ml of absolute ethanol for 25 minutes on a horizonal shaker and centri— fuged at 2000 x g for 10 minutes. Radioactivity in the supernatant was determined by counting 100 p1 dissolved in 2 m1 of absolute ethanol in 15 m1 of a BBOT (2,5,bis (2-tert—buyt1benzoxazolyl) thiophene)—to1uene) counting system (4 gm/L). A11 counting was done in a Packard 3003 Liquid Scintillation Spectrometer. There was no evidence of quenching by materials extracted from soil. One hundred pl of each supernatant solution were also chromato— grammed to determine the percentage of the source(s) of radioactivity in the soil extracts. Chromatographic techniques described by Stephenson et_al. (60) were em— ployed, utilizing Eastman prepared silica gel Chromagrams 3 for all chromatograms. Both H—pyrazon and lLAC—pyrazon were added to aliquots of supernatant solutions from un— treated controls and were co-chromatogrammed as standards. Chromatograms were developed in a benzene—ethanol (3:1, V/v) solvent system (solvent system I). Extracts from soils incubated for 74 days with 3H— Ixyrazon and lLAC—pyrazon (250 and 350 pl, respectively) weere co—chromatogrammed with a standard of lLAC—pyrazon arid non-labelled 5—amino-4-chloro-3—pyridazlnone; “metabolite A). After development in a benzene-ethanol (855:15, v/v) solvent (solvent system 11) chromatograms A“ d( were visualized by diazotizing and coupling the primary amine on the pyridazine ring to produce an azo compound. Developed chromatOgrams were exposed to nitrous acid fumes for 5 minutes by adding NaNO2 to concentrated HCl. Chromatograms were sprayed with a beta—napthol solution (1 g napthol, 10 g urea, and 50 ml each of water and ethanol) and eXposed to NHuOH fumes to observe color. If Colored bands on chromatograms were outlined, and chroma- tograms were segmented and counted to establish the position(s) of radioactivity in relation to the bands observed. Supernatants from soils incubated for 74 days with 1 lLAC—pyrazon were combined and concentrated approximately ten—fold, at 35 C, to confirm the presence of a 140 labelled compound occurring in that extract. Five hundred ul of the concentrated greenish liquid were chromatogrammed in solvent system I and visualized by the color method procedure previously described. Colored bands and intermediate areas were scraped off and counted for radioactivity. The concentrated extract and a standard containing pyrazon and metabolite A were also co-chromato— gyraphed in benzine—chloroform—methanol (3:1:1, v/v/v) (solvent system III). fifigrazon Adsorption lfl Soils The relationship between soil composition and pyra— 20:1 adsorption was determined in nine soils with different m LL properties (Table 3). Clay content ranged from 11 to 36% while organic matter ranged from 2.5 to 11.3%. Soils were obtained from untreated areas in sugar beet fields and were air-dried, crushed, and sieved through a 40 mesh screen. One g amounts were weighed into 15 m1 poly— carbonate centrifuge tubes. Tritium labelled pyrazon (specific activity after dilution, 1.91 uc/mg) was added at rates of 50 and 500 ug/g of soil in 10 m1 of 0.01 M 03012. In a preliminary experiment there was no differ- ence in pyrazon adsorption after 5 and 15 hours of equilibration time. In this experiment soils were placed on a horizonal shaker at a high speed for 6 hours of equilibration at 21 i 1 C and then centrifuged at 2000 x g. Radioactivity was determined by counting 50 ul of the supernatant solution, dissolved in 1 m1 of absolute ethanol. Herbicide adsorption in the soil was assumed to be the difference between the initial concentration added and that found in the supernatant after equilibration. Pyrazon was not absorbed by polycarbonate tubes or caps. Adsorption was expressed as pg of pyrazon/g of dry soil. The supernatant solution from the soil with the flighest organic matter content was chromatogrammed on Silica gel in solvent systems I and II to confirm pyrazon Els the source of radioactivity in the solution. Labelled DSWrazon, in 0.01 M CaC12, was co—chromatographed as 51 standard. A multiple regression analysis was employed 29 to evaluate the relationship between various soil proper— ties and adsorption from two concentrations of pyrazon. TABLE 3.-—Characteristics of soils in the pyrazon ad— sorption study. Texture Clay Sigigic pH C.E C % % me/lOOg Clay loam 32.0 3.21 7.34 13.2 Sandy clay loam 24.4 2.54 7.84 11.3 Sandy clay loam 28.4 3.91 7.95 15.0 Sandy loam 18.4 3.50 7.81 13.5 Clay loam 36.8 5.08 6.35 13.6 Sandy loam 11.2 3.06 6.57 6.6 Sandy loam 16.2 4.94 6.73 11.6 Sandy clay loam 26.6 11.38 7.92 23.7 Sandy loam 16.7 8.44 6.76 24.5 §tatistica1 Analyses Computer programs written by the Department of Agxdcultural Economics, Michigan State University, were Iitilized for analyses of variance and regression analysis fo experimental data. Significant differences in analy— Seas of variance were determined at the 5% level while iiignificance in correlation coefficients was determined 3 O at the 1% level. Treatment means were separated using the Duncan multiple range test. RESULTS AND DISCUSSION Mustard as a Bioassay for Pyrazon Brown mustard was an excellent bioassay species for pyrazon and exhibited many desirable characteristics. Plants were easy to establish, uniform in growth, and specific and sensitive to pyrazon. Pyrazon added at 0.5 and 1.5 ug/g reduced mustard gncowth significantly whereas TCA showed little or no ef- fkect on plant growth (Table 4). When pyrazon and TCA tvexwe applied in combination, the resultant fresh weight :in. each treatment was comparable to that observed from thee same rate of pyrazon applied alone. Plant injury was not observed from TCA treatments; lknveaver, injury from pyrazon was evident and could be evaHliiated Visually. The size of chlorotic areas on leaf tHIDS and the peripheral distance of chlorosis around the leaf‘ margin increased proportionally with higher pyrazon ratens. Consequently the intensity of plant injury could be eValuated on an empirical scale of 0 to 10. In St5U1d£irds from soil 2 there was a significant correlation betweuen herbicide concentrations and injury ratings (P = 0.85) and fresh weight (r = —0.93). Approximately 31 .Amo.o v av peoLoQMHo mapchHMchflm poc ohm popped osmm on» new: memozfi oa.o o.m + m.a ma.H o.m sa.o m.a ”x sa.a m.a + m.o sm.m s.m sm.a m.o sm.m m.a sH.m mm.o sm.m o d:o\w w\mn Q3o\m m\mn Q:o\m m\ms d:o\w m\wn sewed: spam panama seem sewed; some names; spam <09 + commszm <09 commszd Hohpcoo H.mmofiowoho£ neon do mQOHpmcfloEoo new .doe “commgxd new: oopmmep Hfiom Eonm pudenda mo pcwfloz cmopmll.a mqmde 33 70 to 85% of the differences in injury ratings and fresh weights could be attributed to differences in pyrazon in the soil. In some instances mustard vigor and fresh weight production in bioassay standards increased when low rates of pyrazon were added to soil. This enhancement was most apparent with applications of 0.2 to 0.5 ug/g in four .- soils (Table 5), but was evident at higher concentrations in some instances. Fresh weight of mustard increased 10 to {ET-M ‘43. -_.. _ C O A 4 (CABLE 5.-—Enhancement of mustard growth by pyrazon added to four soils. Pjurazon Scll Number Atided 2 3 6 7 ug/Qg soil g/cup %2 g/cup % g/cup % g/cup % 0.0 1.0b 1.4ab 1.6ab 1.0ab 0.253 1 3a 30 1.7ab 6 1 7a 70 0.53 1.2ab 2o 1.5ab 7 2 4a 53 1 7a 70 1.03 l.or 2 2a 57 1.50 1.3ab 3o 1—.53 0 (c 1 7ab 21 1 4b 0 9b 2.0 04d 1 8a 28 1 lb 0 7b 2.5 0.3d 0.9b 0.8b 0.7b \ 1 Means with the same letter in the same column are not ESignificantly different (P < 0.05). t 2Percentage increase in fresh weight compared to un l”salted control. and 31m $0115 2 and 3 pyrazon added at 0.2, 0.4, 0.8 :1° 2 ug/g, respectively. 70% as a result of some pyrazon treatments; however, the presence of small amounts of pyrazon could be detected by observing foliar injury. Pyrazon Distribution and Movement Distinct patterns in pyrazon distribution and move- ment were evident in two of the three soils during 1965. In two clay loam soils, pyrazon residues were signifi- cantly different due to rate of application, depth, and time of sampllng (Tables IV and VII), as well as soil composition. Interactions between application rates and concentrations at various depths, and between time of sampling and soil depth were also meaningful. These trends were more discernible when pyrazon was applied at 4 and 6 lbs/acre since bioassay responses were greater, particularly in samples below the surface layer of soil. Since rainfall intensity and distribution were comparable at field locations 1 and 2, it was assumed that differ— ences in herb cide residues were due primarily to treat— ments and soil properties and Jere not due to local en— vironmental conditions. In a sandy loam (soil 3) pyrazon residues were not affected by herbicide treatments (Table I) and herbicide distribution and movement patterns were not observed in this soil (Table 7). LA.) Kg Distribution Approximately 70 to 90% of the residual pyrazon observed in the clay loam soils remained in the upper 2 inches of soil (Table 6). The amount of pyrazon in both the surface layer and soil 2 to 4 inches below the sur- face tended to increase With higher rates of application. Consequently the percentage of total residue at each soil level remained constant for a given soil and was not af— fected by the rate of application. While 70% of the total residue in soil 1 resided in the upper 2 inches of soil, approximately 30% was located in the adjacent 2- to 4—inch zone. In soil 2, with a higher clay and organic matter content, nearly all of the residue was located in the top layer of sOll and only 10% or less occurred below 2 inches. Pyrazon was not found in either soil between 4 and 8 inches below the surface, even where the herbicide was applied at 6 or 10 lbs/acre. There was no indication that TCA applied in combination with pyrazon affected the distribution of the latter herbicide in the soil profile. Movement HerbiClde concentrations decreased with increasing soil depth and in soil 1 were different in each layer from those in adjacent zones (Table 7). Between the first and second sampling dates in soil 1, pyrazon concentrations approximately doubled 2 to 4 inches below the surface. No aq‘ .czoom mucoEpmmgp o» cowpaoom CH “<08 pooopfls pom spas meow cod mocsoq m com .m .0 pm omwaodm oommsmo moosaoch .Amo.o v m0 pcoememfio manom0H0chfim poo ohm mmmo oemm one mo coppoa meow who boas aHOm m cficpflz memo: .poflawx who: mpcmao Ham poop popmo Iflocfl 0a pom mesmcH o: oopmoaoofi o memos .csocm AmHV mmowpms anomsw ammmmOHm H mm m .o 0.0 m.o 0.0 0.0 H.o mus mm m um 0.0 w.o H.o m.o m.o sum aoa smuoa w.m 0.0 a.s m.m m.o muo Am HasmV smog mafia em a -o H.o a.o o.o 0.0 ml: mm mmm omuam m.m m.a s.a o 0 sum ams Halos m.m m.m m.m m.o muo AH aaom0 Essa Mafia a a ma mH ma ma ca cam: Abmcme OH <09 + 0 <09 + z s o comma m coHpSQHEpmHQ «ocom\moav oowado< commemm mo comm Meow a.mwcfipbs zpzncfi mmmmmOfih an commooom mm mmma CH mHfiOm Emoa mode 03» CH commend 00 cownooaepmflmnu.0 mqmetween 2.“ and 3.“ months after application. U6 Soil 2.--Pyrazon disappeared rapidly when applied at u lbs/acre, however, disappearance was slow following a 6-pound application during the first sampling period compared to that observed during the last period or at other locations. Disappearance was more rapid during the last sampling period with increased rainfall. In this soil herbicide disappearance appeared to be associated with rainfall intensity and distribution. This soil con- tained 6.8% organic matter and the highest amount of clay of any soil in the study. H. Consistent Loss of Pyrazon Soil 5.--Pyrazon disappeared linearly with time and after 6 months following application residues were negligi- ble. This soil contained intermediate amounts of clay and organic matter and rainfall was well distributed through- out the growing season. Soil 6.--Two months after application pyrazon ex- ceeded 3 ug/g of soil but disappeared rapidly during the next 3 months. High rainfall was well distributed through- out the season. Clay and organic matter contents were intermediate compared to other soils. Soil 8.-—Initial pyrazon concentrations were 0.5 51nd 2.5 ug/g following applications of u and 6 lbs/acre, Inespectively. Disappearance was rapid following the (S-pound application and residues from both application rates were negligible A months after application. Rain— fall was well distributed throughout the season. Soil was exceedingly low in clay (10%) and contained less than 3% organic matter. Adsorption of Pyrazon in Soil Adsorption ranged from 1.7 to 103.2 ug/g of soil and was related to soil properties. Pyrazon adsorption IF-- _‘ fl _. was correlated with organic matter and cation exchange ‘2‘. Av I capacity to the same extent; however, cation exchange capacity appeared to be dependent on soil organic matter (Table 8). Adsorption was not related to clay content TABLE 8.——Simple correlation coefficients (r) between pyrazon adsorption and soil properties. Soil Properties pH cgii‘int C-E-C- £22532? Adsorption from 50 ug/g soil —0.08 0.09 0.87* 0.88* 500 ug/g soil -o.01 0.02 0.92* 0.97* Exchange Capacity (C.E.C.) 0.22 0.15 1.00 0.87* *Indicates significant correlation coefficients (P < 0.01). tnetween 11 and 37%. Organic matter content was the only Esignificant independent variable in a multiple regression analysis. A “8 There was close agreement between the relative in- crease in soil organic matter and pyrazon adsorption when organic matter was 3.2% or higher (Table 9). Adsorption was greater when more pyrazon was added in the equili- brating solution in soils that contained 3.5% or more organic matter. The interaction for adsorption, between organic matter and the concentration of pyrazon added to ' ‘3. soil was significant since proportionally more adsorption occurred from the higher concentration as organic matter increased. Since pyrazon was equilibrated with soil in a solu- tion containing Ca++, differences in pH between soils may have been negated if soils were saturated with calcium. However, in two soils with the same organic matter con— tent (“.9“ and 5.08%), adsorption was lower in the soil with the lower pH and higher clay content (pH 6.3 and 37% clay). Radioactivity in an equilibrated water extract co— chromatogrammed with a pyrazon standard which indicated that activity in the equilibrated solutions was from 3H-pyrazon. “9 TABLE 9.--Re1ationship between soil organic matter and herbicide adsorption from two concentrations of pyrazon. Pyrazon Adsorbed (pg/g) Organic Matger 50 ug/g 500 ug/g Added Added 2.5“G 3.9“ “.17 3.06F 3.85 1.7“ . 3.21EF 7.8“ 11.37 ? 3.50E 8.50 13.72 i 3.91D 9.66 27.67 ‘ “.9“C 1“.08 “0.3“ 5.080 ll.““ 37.72 8.““B 19.20 90.90 11.38A 18.98 103.21 Mean 10.80a 36.80b 1 Means followed by the same letter of the same case are not significantly different (P < 0.05). Response of Soil Microorganisms to Pyrazon In three soils the response of bacteria to pyrazon was maximal after approximately 5 weeks of incubation at 21 C, when compared to untreated controls (Figure 3). The highest plate counts in treated soils also occurred at that time except in the sandy clay loam. In this soil bacteria counts in treated soil were highest after 10 weeks of incubation but when compared to the population 50 FIGURE 3. Changes in bacteria populations in three soils amended With pyrazon and incubated at 21 C. 10001 soo— — sanov cuv LOAM Zor ‘7 —-—- sauov LOAM UNTREATED \ '—-°—0 CLAY LOAN 800- CONTROL (Bacteria counts) .. 100— l T l J 1 2 s 10 15 20 INCUBATION (Weeks) FIGURE 4.Changes in bacteria populations in a sandy clay loam amended with pyrazon and incubated at four temperatures. ‘ 10001 00.0.0000000029-5 c 800- —— 21.0 c 7 —— - —12.5 c OF . 9 °——o—-4.5 C UNTREATEO 500 CONTROL ‘- 1 II (Bacteria 30 0.: counts) I 200- 100- “--—---'*-K ..... 'seeeeoeeo.’.. I) I I I I I 2 5 10 15 20 INCUIATION (Weeks) 51 in the control the greatest enhancement from pyrazon occurred prior to that time. In the amended clay loam soil bacterial counts were significantly higher in treated soil after 5 weeks of incubation than at other dates or in untreated soil. In the sandy loam the re- sponse to pyrazon after 5 weeks of incubation was con- sidered as being meaningful although plate counts were not statistically different. Bacteria populations in amended soils ranged from 10 to 38“ X 106/g while counts in untreated controls ranged from 10 to 163 x 106/g. In both soil treatments bacteria developed as convex circular colonies predominantly on the surface of the agar. Colon- ies were generally 2 to 6 mm in diameter and were whitish, cream, or light yellow. These bacteria were considered to be aerobic, based on colony growth characteristics and location. Only a limited number of colonies were em- bedded in the agar below the surface. Bacteria populations in each soil were not considered to be a result of minor pH fluctuations. Bacteria in amended sandy loam also responded to different incubation temperatures. With higher incubation temperatures less time was required for the organisms to respond to pyrazon (Figure “). Bacteria in amended soil at 29.5 C decreased from 280 to 65% of the untreated con— trol between 5 and 10 weeks of incubation and continued to decrease after that. This trend was assumed to be a 5,7 decline of a maximum population achieved sometime prior to the first sampling at 5 weeks. Soil incubated at this temperature dried out after 15 weeks and the in- creased population observed at 20 weeks was believed to be a cyclic response from an addition of water at week 18. When amended soil was incubated at 21 C a maximum response occurred at 5 weeks, as previously explained, and occurred after the peak that probably occurred in soil incubated at 29.5 C. In amended soil incubated at 12.5 C 15 weeks were required for organisms to show a response to pyrazon in the soil media. There was a general decline in the number of bacteria between 5 and 10 weeks of incubation at “.5 C. In the following 10 week period at this temperature the population in the untreated control remained the same; however, there was a two and one-half fold increase in bacteria in the treated soil. This response in the amended soil indicated that the species surviving and functioning at the lowest incubation temperature eventually responded to pyrazon in the soil media. In soils incubated at 29.5 and 12.5 C bacteria colonies were similar to those described previously in soils incubated at 21 C and were considered to be aerobic. Colonies from soil incubated at “.5 C were distinctly different from those in soil at higher temperatures. Colonies developed both in and on the agar and were minute, usually one mm in diameter, white or yellow in color, with occasional colonies displaying a pink pigmen— tation. The difference in the actual number, response, and type of colonies observed at this temperature com- pared to those at higher temperatures was undoubtly due to the difference in species that survived and responded in each environment. Actinomycete populations ranged from 0 to 70 X 105/g; however, most counts were between 3 and 15/g at a 10—5 di— lution. Colonies were crusty, formed only on the surface and usually developed a tan or brownish pigment with limited mycelium. Actinomycete development and popu- lations were not related to any particular soil treatment but populations were higher in soil incubated at 29.5 C than at other temperatures. There was no evidence that actinomycetes responded to or were inhibited by the addi- tion of pyrazon to soils. Disappearance of Ryrazon in Soil Total radioactivity recovered from soils decreased between 3 and 7“ days of soil incubation. Total activity decreased more consistently in soils containing 1“C_ pyrazon than in soils amended with 3H—pyrazon. Approxi- mately 50% of the pyrazon added initially was recovered. 0n thin layer chromatograms, 89 and 97% of the activity 3 from H-pyrazon and 1uC—pyrazon standards, respectively, was located at R 0.73 to 0.78 in solvent system I f 5“ (Table 10). Activity from soil extracts at this location was assumed to be pyrazon on the basis of these standard controls. Differences in recovered identifiable pyrazon ranged from 5.7 to 11.0% between duplicate soil extracts. TABLE 10.--Recovery of radioactivity from soils amended with 3H and 190 labelled pyrazon.l 1“ 3 C-Pyridazine Labelled Time of LH—Phenyl Compounds . abelled Incubation P razon y Rf 0.“7 Pyrazon days % mu % mu % mu moles/g moles/g moles/g 3 88.5 10.u5 0.3 0.0“ 95.6 11.65 10 76.9 8.28 0.9 0.11 96.“ 11.65 15 91.2 10.25 1.7 0.19 95.9 10.90 30 96.1 10.85 2.9 0.32 95.1 10.“5 30 91.“ 10.25 2.9 0.30 9“.0 9.85 7“ 92.8 9.“8 8.7 0.99 88.6 10.15 7“ 8.2 0.8“ 89.0 9.10 Standard 89.6 0 97.2 1 Based on the distribution of radioactivity on thin layer chromatograms. The percentage of activity as 3 H-pyrazon in soil extracts varied irregularly and was particularly low after 10 days of incubation. Other than pyrazon, there was no indication that any other tritium labelled compound was present in the soil. Radioactivity as 1uC-pyrazon de- creased consistently with longer periods of soil 55 incubation and was accompanied by an increase in radio- activity in a compound at R 0.“7. f During the 71 day incubation period 3 H—pyrazon de- creased from 10.“5 to 9.“8 mu moles/g, a difference of 0.97. During the same period luC—pyrazon decreased from 11.6 to 9.6 mu moles/g, a difference of 2.08. Part of this decrease was accounted for by the formation of a I 1“C labelled compound which increased from 0.0“ to 0.9“ mu moles/g. The concentration of this new compound after 7“ days of incubation amounted to less than 10% of the pyrazon extracted while pyrazon decreased 18% during the same period. Initial evidence for the formation and presence of a new compound from luC-pyrazon was obtained from the distribution of radioactivity on chromatograms. In sol- vent system I this compound chromatogrammed at R 's of f 0.“ and 0.5, with a distinct decrease in activity at Rf 0.6 prior to the pyrazon peak at R 0.7 and 0.8. The f distribution of radioactivity on chromatograms was distinct in extracts after 30 and 7“ days of soil incu- bation (Table 11). The low activity between two peaks indicated that the unidentified compound was, in fact, different from pyrazon and was not the result of trailing activity from pyrazon. 56 TABLE ll.--Distribution of radioactivity on chromatograms of extracts from soil incubated for 30 to 7“ days with lL‘C-pyrazonJ- Position on Days of Incubation Chromatogram 30 7“ Rf % % 0 1 1.5 1.“ 0 2 0.0 0 1 0 3 0.1 0.2 0 “ 0.8 2 0 0 5 2.0 6 “ 0.6 0.7 0.8 0.7 6“.9 7“.l 0.8 29.6 1“.7 0.9 0.0 0.0 1.0 0 0 0.0 1 Percentage of total activity on chromatograms from duplicate samples. Two distinct colored bands were observed when a standard containing lLlC—pyrazon and non—labelled 5-amino- “-chloro-3-pYridazinone was co—chromatogrammed in solvent system II. Nearly 90% of the total activity corresponded to an intense dark maroon band occurring at Rf 0.65. A less intensely colored region was observed at R 0.“3 f but contained no activity and was considered to be the dephenylated pyrazon (metabolite A). Both bands tended 57 to trail due to the high concentration applied. 0n the chromatogram of ll‘C—pyrazon (incubated in soil 7“ days), 81% of the total activity occurred in an extremely faint pink band at R 0.62 while 7.3% of the activity occurred f at R 0.“2. Although no color was observed in the f second band the positions of activity on this chromato- gram corresponded to the activity and the color observed in the standard. Pyrazon was the only source of radio- activity on the chromatogram of the 3 H—pyrazon extract but was not colored since less material was spotted initially. 0n the chromatogram containing 500 pl of the concen- trated lac-pyrazon extract three irregularly shaped bands were observed (Figure 5). Band 1 appeared as the same yellow-greenish color observed in the concentrated extract at Rf 0.78 but contained no radioactivity. Band 2 was immediately behind and along the sides of band 1 and ap- peared as an intense dark maroon color at approximately Rf 0.73. Some trailing of the maroon color was observed. This maroon band contained 8“% of the total activity and was considered to be pyrazon based on the Rf and color observed. Between bands 2 and 3 there was a distinct break and change of color. Band 3 was light pink, well defined, and occurred at R 0.“5, the same position where activity f had been observed on other chromatograms of luC—pyrazon extracts. Chromatogram sections “ and 5, corresponding 58 FIGURE 5. Correspondence of colored bands and “C radioactlvlty on a thin layer chromatogram after incubation , o1 14 - r n in a sandy loam soil for 74 days. c W m IAN D N UM IER '-"""1 L___--..-c ‘234507 001011 SECTION "UM-IR ”q .. 1’1 or 7 70 3.- TOTAL acrrvurv "" 104 .- I T T I 7 1 2 3 4 5 6 7 8 910 11 secrlou Hansen FIGURE 6. Recovery of pyrazon and a metabolite from a sandy loam soil amended with 14.; labelled pyrazo’n. ‘2! \ 10d ~ ~—% qI-fi —© PYRAZON In a moles 3' per cm sell .1- ‘- ol o usrasoun " __ q._-fl:==?:f'; 1 10 us so 7° INCUIATION (Days) 59 to band 3, contained 3.7 and 3.8% of the total activity, respectively, or a total of 7.5% of the activity; the same percentage of activity observed in this region on previous chromatograms. Radioactivity between bands 2 and 3 amounted to 1.98% of the total. Total activity on the chromatogram exceeded 6.5 X 10“ dpm, which may have resulted in the slight activity observed between the two peaks on the chromatogram. When the concentrated luC-pyrazon extract and a standard were co-chromatographed in solvent system III, pyrazon in the standard migrated in a sharp band to Rf 0.63. Ninety per cent of the activity from the extract occurred in the same position; however, the remaining activity, observed previously as a separate peak in other solvent systems, tended to trail from the pyrazon peak and separation was not achieved. Although the presence of the primary amine on the pyridazine ring was confirmed in the product formed from pyrazon and the product corresponded to 5-amino-“—chloro- 3—pyridazinone on chromatograms; the other substituents were not positively identified (Figure 6). However, breakdown product probably contained the chloro and carbonyl groups since the product co-chromatogrammed identically with a standard that contained these substi- tuents, in two solvent systems. SUMMARY AND CONCLUSIONS Pyrazon residues were evaluated by both fresh weight and plant injury; however, greater reliance was placed on injury ratings. Low concentrations of the herbicide were evaluated more accurately in all soils by the injury rating system since fresh weight of the bioassay was enhanced 10 to 70% in some soils by small amounts of pyrazon. There was little or no TCA present in soil at the time samples were obtained. TCA is not persistent in soil at the rates applied in this study and leaches readily (“1). However, if any TCA was present in samples the bio- assay species would have responded only to pyrazon. Differ- ences in the movement or persistence of pyrazone due to TCA could have been detected by the method of evaluation. There was no evidence that pyrazon movement or residues were af- fected by TCA. In the pyrazon adsorption study herbicide retention was associated with the organic matter content of soil and was not related to clay content. The influence of organic matter in pyrazon movement and persistence was evident in the field studies. The importance of organic matter in the adsorption of pyrazon has been noted previously (20). 60 61 In observations on the distribution and movement of pyrazon in two clay loam soils, the herbicide was re- tained in the upper 2 inches of the soil with highest organic matter and clay content. Less pyrazon was re- tained in the upper 2 inches of the soil with less organic matter and clay, and significant movement occurred during the season. It was concluded that the difference in pyra- zon movement between soils was due to differences in soil organic matter. The small percentage change in clay con— tent between the two soils would not account for the vast differences in the movement observed. Since the herbicide was retained near the soil sur— face, primarily in the upper 2 inches of the profile, de- gradation of pyrazon would occur in the plow-depth layer of soil. Under most field conditions pyrazon is available for biological use and degradation in the soil and is not lost as a result of leaching or movement in soil. However, only a portion of the total amount of pyrazon applied is available for biological systems, due to adsorption by organic matter. Although there was no direct evidence that bacteria were involved in the degradation of pyrazon, it is highly probable that soil bacteria are responsible for the de- gradation of pyrazon. In three soils these organisms responded to pyrazon, indicating that bacteria were capable of utilizing the herbicide. Significant losses 62 of labelled pyrazon did not occur until after 10 days of incubation but, corresponded to the increased prolifer— ation of bacteria observed previously. Different bacteria species at four soil temperatures also responded to the herbicide in the soil media. No doubt pyrazon is exposed to a similar variety of organisms in the natural environment as a result of increasing soil temperature after early spring applications of the herbi- cide. There was no indication that actinomycetes re— sponded to or utilized pyrazon. Fungi would not be highly functional at the pH levels usually encountered in sugar beet soils due to competition from soil bacteria. It was concluded that the high initial loss of labelled pyrazon in incubated soils, between the time of application and the first sampling, was due to adsorption. After the initial loss of pyrazon due to adsorption the herbicide disappeared slowly in incubated soil. One would expect to observe a slow rate of disappearance con- sidering the length of persistence and weed control ob- served in the field and the general reluctance of soil organisms to utilize chlorinated compounds. Persistence and disappearance of pyrazon in field soils may be attributed to factors that enhance micro- bial activity and contribute to herbicide adsorption. In all instances, the rate of disappearance was highest in soils that contained 2.9 to “.3% organic matter where "9%. __I 33%;.- 63 rainfall was well distributed throughout the season. Pyrazon concentrations remained high or did not disappear as rapidly in soils that received low rainfall or con— tained high amounts of organic matter. Uniform distribution of rainfall is essential in maintaining favorable soil moisture for microbial activity while water movement in the soil would tend to remove . soluble toxic by-products. The complete loss of phyto- 1 toxicity would be prolonged in soils with high organic . matter contents since the herbicide would be replenished in the soil constantly by desorption. Microbial de- gradation of organic matter may also release the herbi- cide. Increased clay content may affect herbicide persis- tence by maintaining adequate soil moisture for microbial development; however, in this study it appeared that clay may have aided in maintaining phytotoxicity. In comparing residues in two soils that contained organic matter in ex- cess of 10%, phytotoxicity did not change during the grow— ing season; however, there was a marked difference in the level of phytotoxicity. In the soil with a low but per- sistent level of phytotoxicity the clay content was less than 10%. In contrast, the second soil contained two and one-half times more clay, and phytotoxicity was higher. The persistence of phytotoxicity was related to soil organic matter content. However, the level or intensity 6“ of phytotoxicity in the soil appeared to be associated with the clay content. Although there is no clear ex- planation for this phenomena, clay may tend to stabilize the herbicide in the soil after desorption from organic matter. A breakdown product of pyrazon was observed in in- cubated soil. The product was first observed after 10 days of incubation and continued to increase. Loss of a radioactive labelled phenyl ring and retention of the labelled pyridazine ring indicated that the compound lacked the phenyl substituent of pyrazon. The primary amine on the pyridazine ring was confirmed and since the breakdown products co-chromatogrammed with a known metabo— lite of pyrazon, it was concluded that the product was 5-amino-“-chloro-3—pyridazinone. Since the single break— down product did not completely account for the total loss of radioactivity from pyrazon, it was assumed that the identified product undergoes additional degradation in the soil. The slow loss of pyrazon after 30 days of incubation may be due to an accumulation of the breakdown product. The breakdown product(s) may be toxic to some soil micro- organisms but may be removed from the biological environ— ment in the field by rainfall. In summary, the movement and persistence of pyrazon, herbicide was investigated in field environments. In 65 laboratory studies adsorption of pyrazon was correlated with soil pr0perties and the degradation of pyrazon in soil and response of soil microorganisms to pyrazon was determined in long-term incubation studies. Many of the changes in pyrazon concentrations in field soils could be explained after consideration of soil properties and rainfall in relation to laboratory findings. Signifi- cant findings of this study were: f 1. Adsorption of pyrazon is correlated with soil 1 organic matter (r = 0.9) and is not dependent on clay content. 2. Pyrazon is immobile in soils and is retained primarily in the upper 2 inches of soil. Movement is dependent on the organic matter content of soils. 3. Disappearance of pyrazon in the field is en- hanced by evenly distributed rainfall follow- ing herbicide application. The rate of dis- appearance is dependent on rainfall intensity and distribution. Pyrazon is persistent in soils with organic matter contents in excess of 10%. “. Soil bacteria functioning between “.5 and 29.5 C are capable of utilizing pyrazon. Five to 10 weeks are required for maximum proliferation of bacteria in soils at 21.0 C. Antinomycete popu— lations are not affected by pyrazon. 66 The loss of pyrazon in soil probably occurs by bacterial degradation. Degradation of pyrazon in soil is initiated by the loss of the phenyl substituent. The breakdown product was con- firmed as 5-amino-“-chloro—3-pyridazinone. After 10 weeks of incubation the amount of the breakdown product in the soil is low (less than 10%) compared to the amount of pyrazon present. The initial breakdown pro- duct is subject to additional degradation. Plant growth may be stimulated or enhanced by low concentrations of pyrazon. Fresh weight of mustard increased 10 to 70% by the addition of 0.2 to 1.0 ug/g of soil. 10. 11. LITERATURE CITED Aldrich, R. J. 1953. Residues in soil. J. Agri. Food Chem. 1:257—260. Alexander, M. 1965. Persistence and biological reactions of pesticides in soils. Proc. Soil Sci. Soc. Amer. 29:1-7. , and M. I. H. Aleem. 1961. Effect of chemical structure on microbial decomposition of aromatic herbicides. J. Agri. Food Chem. 9:““-“7. Alley, H. P. 1965. An evaluation of herbicide mixtures for annual weed control in sugar beets. Ph.D. Thesis. Colorado State University. 173p. Anon. 1965, 1966. Climatological Data. U. S. Department of Commerce Environmental Science Services. Michigan 80, 81. Bailey, G. W., and J. L. White. 196“. Review of adsorption and desorption of organic pesticides by soil colloids with implications concerning bio— activity. 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Movement and persistence of endothal (3,6-endoxo- hexahydrophthalic acid) as influenced by soil tex- ture, temperature, and moisture levels. J. Amer. Soc. Sugar Beet Technol. 11:287-293. Cooke, A. R. 1966. Unpublished data. Amchem Pro- ducts, lnc., Ambler, Penn. Corbin, T. T., and R. P. Upchurch. 1967. Influence of pH on detoxication of herbicides in soil. Weeds. 15:370—377. Dean, L. A. 1960. Chemistry of pesticides in soils, pp. 63—69. In The nature and fate of chemicals applied to soils, plants, and animals. USDA ARS Pub. 20-9. Deming, J. M. 1963. Determination of volatility losses of Clu—CDAA from soil surfaces. Weeds. DeRose, H. R. l9“6. Persistence of some plant growth—regulators when applied to soil in herbicidal treatments. Bot. Gaz. 107:583—589. Doherty, P. J. 1967. The adsorption of four herbi- cides by different types of organic matter and a bentonite clay. M.S. Thesis. Purdue University, Lafayette, Indiana. Drescher, N. 196“. Bestimmung der rfickstande von Pyramin in pflanze und boden. [Determination of PCA residue in plants and soils] pp. 78—8“. In Vortrage amlasslick der Wissenschaftlichen Aussprache fiber chemische Unkrautbekampfung in Zuckerruben mit Pyramin. Badische Anilinand Soda-Fabrik. Ludwigshafen am Rhein, Germany. Eno, C. F. 1958. What pesticides do in the soil. II. Insecticides and the soil. J. Agri. Food Chem. 6:3“8-51. Ercegovich, C. D., and D. E. H. Frear. 196“. The fate of 3—amino—l,2,“-triazole in soils. J. Agri. Food Chem. 12:26-29. 2“. 25. 26. 27. 28. 29. 30. 31. 32. 33. 3“. 35. 36. 69 Farrell, J., and A. Rose. 1967. Temperature effects on microorganisms. Ann. Rev. Microbiol. 21:101-120. Fischer, A. 1962. l-phenyl-“-amino—5-chloro-pyridazon— 6 (PCA) als ein neues rebenherbizid. Weed Res. 2:177-18“. . 196“. Die wirkungsweise von Pyramin. IThe mode of action of Pyramin] pp. 19—2“. In Vortrage anlasslich der Wissenschaftlichen Aussprache fiber chemische Unkrautbekampfung in Zuckerruben mit Pyramin. Badische Anilin-and Soda-Fabrik. Lud— wigshafen am Rhein, Germany. 3 Frank, R. 1967. Pyrazon, a selective herbicide for sugar beets. Weeds. 15:197-201. fl ..——____ _1_. “ a -1...‘ . 1967. Studies on pyrazon under controlled environmental conditions. Weeds. 15:355—358. Hamaker, J. W., C. R. Youngson, and C. A. I. Goring. 1967. Prediction of the persistence and activity of Tordon herbicide in soils under field conditions. Down to Earth. 23:30-36.| Hance, R. J. 1965. Observations on the relationship between the adsorption of diuron and the nature of the adsorbent. Weed Res. 5:108—11“. Harris, C. I. 1966. Adsorption, movement, and phytotoxicity of monuron and s-triazine herbicides in soil. Weeds. l“:6-10. . 1967. Movement of herbicides in soil. Weeds. 15:2l“—2l6. , and T. J. Sheets. 1965. Influence of soil properties on adsorption and phytotoxicity of CIPC, diuron, and simazine. Weeds. 13:215-219. , and G. F. Warren. 196“. Adsorption and desorption of herbicides by soil. Weeds. 12:120—126. ('Hilton, H. W., and Q. H. Yuen. 1966. Adsorption and leaching of herbicides in Hawaiian sugar cane soils. J. Agri. Food Chem. 1“:86-90. Jensen, H. L. 1953. Decomposition of chloro— substituted aliphatic acids by soil bacteria. Can. J. Microbiol. 3:151-16“. 37. 38. 39. “0. “l. “2. “3. ““. “5. “6. “7. “8. 70 Jordan, L. S., C. W. Coggins, Jr., B. E. Day, and W. A. Clerx. 196“. Photodecomposition of substi- tuted phenylureas. Weeds. l2:l-“. Jung, J. 196“. Der einfluss von Pyramin duf bodenatmung und nitrifikation. [The influence of Pyramin on the soil respirations and nitrification] pp. 85-91. In Vortrage amlasslick der Wissenschaft- lichen Aussprache uber chemische Unkrautbekampfung in Zuckerruben mit Pyramin. Badische Anilin-and Soda—Fabrik. Ludwigshafen am Rhein, Germany. Kaufman, D. D. 1966. Microbial degradations of ‘ herbicide combinations: amitrol and dalapon. Weeds. , 1“:130-l3“. Kearney, P. C. 1966. Metabolism of herbicides in the soil. Adv. Chem. Series. 60:250-262. , C. 1. Harris, D. D. Kaufman, and T. J. Sheets. 1965. Behavior and fate of chlorinated aliphatic acids in soils. Adv. Pest Control Res. 6:1—30. MacRae, I. C., and M. Alexander. 1965. Microbial degradation of selected herbicides in soil. J. Agri. Food Chem. 13:72-76. Martin, J. P., and P. F. Pratt. 1958. What pesti— cides do in the soil. 1. Fumigants, fungicides and the soil. J. Agri. Food Chem. 6:3““—3“8. Meggitt, W. F. 1966. Environmental variables and weed control with pyrazon in sugar beets. Abstracts, Weed Soc. Amer. p. 11. Mortland, M. M., and W. F. Meggitt. 1966. Inter— action of ethyl N,N—di-n—propylthiolcarbamate (EPTC) with montmorillonite. J. Agri. Food Chem. 1“; 126-129. Nearpass, D. C. 1965. Effect of soil acidity on the adsorption, penetration, and persistence of simazine. Weeds. 13:3“1-3“6. 1967. Effect of the predominating cation on the adsorption of simazine and atrazine by Bayboro clay soil. Soil Sci. 103:177-182. Newman, A. S., and C. R. Downing. 1958. What pesticides do in the soil. III. Herbicides and the soil. J. Agri. Food Chem. 6:352—353. "'1- “9. 50. 51. 52. 53. 5“. 55. 56. 57. 58. 59. 60. 71 Ogle, R. E., and G. F. Warren. 195“. Fate and activity of herbicides in soils. Weeds. 3:257—273. Pommer, E. H. 196“. Der Einfluss von Pyramin auf die Bodenpilze. [Influence of Pyramin on soil fungi] pp. 92-9“. In_Vortrage amlassick der Wissenchafllichen Aussprache fiber chemische Unkrautbekampfung in Zucker— rfiben mit Pyramin. Badische Anilin— and Soda—Fabrik. Ludwigshafen am Rhein, Germany. Pramer, D., and E. L. Schmidt. 1965. Experimental Soil Microbiology. .Burgress Publishing, Minneapolis, Minnesota. pp. 35, 102. Ries, S. K., M. J. Zabik, G. R. Stephenson, and T. Chen. 1968. N-glucosyl metabolite of pyrazon in red beet. Weed Sci. l6:(In press). Schlegel, H. G., and H. W. Jannasch. 1967. Enrich— ment cultures. Ann. Rev. Microbiol. 21:“9-70. Schreiner, 0., and H. S. Reed. 1907. Certain organic constituents of soils in relation to soil fertility. U.S.D.A. Bur. Soils Bul. “7. 52p. Sheets, T. J. 196“. Review of disappearance of substituted urea herbicides from soils. J. Agri. Food Chem. 12:30-33. , A. S. Crafts, and H. R. Drever. 1962. Soil effects on herbicides. Influences of soil properties on the phytotoxicity of the s—triazine herbicides. J. Agri. Food Chem. 10:“58-“62. , and L. L. Danielson. 1960. Herbicides in soil, pp. 170-181. In The nature and fate of chemi- cals applied to soils, plants, and animals. USDA ARS Pub. 20-9. , and C. 1. Harris. 1965. Herbicide resi— dues in soils and their phytoxicities to crops grown in rotation. Residue Rev. ll:1l9—l“0. Stephenson, G. R., and S. K. Ries. 1967. The movement and metabolism of pyrazon in tolerant and susceptible species. Weed Res. 7:51-60. Upchurch, R. P. 1966. 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APPENDIX 73 TABLE I.-—Summary of significance in analys I‘- v es of variance and interactions in pyrazon bioassays in 1365 field sanples.l Soil 1 fail 2 Soil 3 Source Injury ”eig‘t -njzr' Jeight Injury Weight In- 1;: Rate of application 03 .25 33 -90 ~08 -99 “A Time of sampling 00 .03 10 .00 .10 .02 .fi "1 R X T .10 .19 06 .31 .67 .80 ‘ Depth of sampling 00 00 .00 00 .00 .19 R X D 00 :6 00 10 .17 .96 T X D .0f .00 .33 01 .02 .52 H X T X D 009 '53 005 009 077 083 1Determined by visual injury ratings and fresh weight of mustard. TABLE II.--Summary of significance in analyses of variance and interactions in pyrazon bioassays in 1966 field samples.l Q r ( <1 :3 Sr: 7 S Source Soil “ soil 3 .011 u 1 oil 8 Injury Weight Injury ieight Iniur 'eigrt rju: height Injury Weight Rate .00 .00 .02 .00 00 17 1; 23 .01 .00 Time .87 .92 .00 .07 30 .00 7 11 .01 .67 R x T .62 .80 .01 .01 .06 :0 i: .18 .57 .16 1Determined by visual injury ratings and fresh weight of mustard. TABLE III.--Bioassay injury ratings in seven bioassay standards.l Pyrazon 1965 Soils 1966 Soils Applied 2 3 u 5 6 7 8 ug/g . 0.0 0.3a 0.2a 0.3e 0.0cd 0.0e 1.00 (1.0c 0.252 1.0a 1.3cd 0.3e 1.00 3.3c 0.52 1.0a 0.5a 1.0de 1.6cd 1.0de 1.00 9.3ab 1.02 1.3a 1.2a 2.6d 3.3c 1.0de 1.0c 5.6bc 1.52 3.30 1.5ab 5.3c 7.0b 1.6de 2.6c 7.6ab 2.0 6.0c 2.5abc 9.3a 8.3ab 2 3cde 3.3c 9.0ab 2.5 6.3c 3.7bcd 7.0b 9.3ab “ 3bcd 3.0c 9.3ab 3.0 9.0d “.SCd 9.0a 9.6a 5.6bc 7.0ab 10.0a 3.5 9.0d “.5cd 9.3a 7.6ab 6.0b 6.00 10.0a “.0 9.6d “.2cd 9.6a 9.6a 7.3ab 7.3ab 10.0a “.5 5.5d 9.6a 9.3ab 9.6a 9.3a 10.0a lInjury ratings (IR), where 0 indicated no injury and 10 indicated that all plants were killed. Means in the same column followed by the same letter are not significantly different (P < 0.05). 2In soils 2 and 3 PYrazon applied at 0.25, 0.“, 0.8, and 1.2 ug/g, re— spectively. TABLE IV.--Pyrazon residues in three soils during 1965 as related to rate of application, as accessed by bioassay injury ratings.l Herbicide Treatment2 Soil 1 Soil 2 Soil 3 PCA TCA Injury Injury Injury lbs/A lbs/A IR IR IR 0 0 0.11c 0.“lc 0.08c 0 8 0.060 0.07d 0.1“bc 2 8 1.1“b 0.590 0.“2a 3 0 1.38b 0.89bc 0.25ab 3 8 1.110 0.85bc 0.36ab “ 0 1.6“b 1.330 0.“7a “ 8 1.50b 1.“0b 0.28ab 6 '0 2.56a 6 8 2.53a 3.33a 0.39a lO 0 3.56a 1Injury ratings (IR), where 0 indicated no injury and 10 indicated that all plants were killed. Means in the same column followed by the same letter are not significantly different (P < 0.05). TABLE V.—-Fresh weight of mustard with several rates of pyrazon added to three soils in 1966.1 3583328 Soil “ Soil 5 Soil 8 us/s s/cup a/CUp S/Cup 0.0 1.60a 2.02a 0.53a 0.25 1.“0b 0.81a 0.5 1.18b 1.“1b 0.09a 1.0 0.770 0.630 0.37a 1.5 0.33d 0.600 0.2“a 2.0 0.09d 0.1“d 0.13a 2.5 0.16d 0.27d 0.10a 3.0 0.05d 0.09d 0.01a 3.5 0.06d 0.0“d 0.00a “.0 0.02d 0.09d 0.00a “.5 0.05d 0.00d 0.00a 1 Means with the same letter in the same column are not significantly different (P < 0.05). TABLE VI.—-Pyrazon concentrations and disappearance in seven soils as related to soil prOperties and rainfall, 6 pounds per acre applied.l Rainfall/Month Pyrazon Residues Rate of Soil Cla Organic Prior Bet. 1st 2 to 2.5 After Disap— No. y Matter to 1st and Last Mo. After “ pearance Sample Sample Appl. Mo. % % in/mo in/mo ug/g ug/g ug/g/mo 2 39 6.8 1.8 “.8 3.3 2.2 0.70 3 9 13.7 1.5 3.5 0.“ 0.“ 0.00 “ 2“ “.2 1.6 2.0 2.1 1.3 0.13 5 28 “.3 2.3 2.7 2.“ l.“ 0.59 6 28 “.2 2.9 3.“ “.0 1.8 0.97 7 26 12.9 1.8 2.8 2.8 2.3 0.23 8 10 2.9 1.8 2.8 2.5 0.2 1.0“ lConcentrations determined by comparison of bioassay injury ratings to standards in the same soil. '77 TABLE VII.-—Pyrazon residues in two clay loam soils a related to rate of pyrazon bioassay 5 application and time and depth of sampling in 1965, as accessed by injury ratings.“ Soil Soil 1 (months after application) Soil 2 (months after application) Depth 2.u 3.“ “.6 2.0 3.0 “.2 in IR IR IR IR IR IR “ lbs/A applied “ lbs/A applied 0-2 3.75 3.50 3.25 5.66C 5.008 0.668 2-“ 1.25 1.75 1.25 0.308 0.668 0.008 “-8 0.00 0.00 0.00 0.008 0.008 0.008 “ lbs/A + TCA applied “ lbs/A + TCA applied 0-2 3.50 3.25 2.75 5.000 “.668 2.66d 2-“ 0.7 1.75 1.50 0.008 0.338 0.008 “-8 0.00 0.00 0.30 0.008 0.008 0. 08 6 lbs/A applied 0-2 8.66ah 8.00ab “.330 2-“ 0.008 1.008 0.668 “-8 0.008 0.008 0.338 6 lbs/A + TCA applied 6 lbs/A + TCA applied 0-2 7.25 5.53 3.“? l_.;3a 9.33a 7.66t 2—“ 2.00 3.25 1 E: 1 .3d 1.33d 0.338 “-8 0.00 0.25 0 LC C._ue 0.338 0.338 3 lbs/A applied 10 lbs/A applied 0-2 3.50 3.50 2.75 9.66a 10.00a 9.66s 2-“ 0.50 1.00 1.00 0.668 1.33d8 0.668 “—8 0.25 0.00 0.00 0.008 0.008 0.008 Mean of All Treatmentsd Mean of All Treatments2 0-2 3.03A 2.81A 1.9“8 “.33A “.76A 3.008 2.14 0.650 1.280 0.780 0.160 0.660 0.260 “-8 0.03E 0.15E 0.00E 0.00C 0.13C 0.16C 1Injury ratings (IR), where 0 indicated no injury and 10 indicated that all plants were killed. Means with a soil followed by the same letter of the same case are not significantly different (P < 0.05). 2Includes pyrazon applied at 0, 2, and 3 pounds per acre with and without TCA, in addition to treatments shown. TABLE VIII.--Pyrazon concentrations in the upper two inches of six soils in 1966.1 Rate of Pyrazon Applied (lbs/A) 2 Rainfall Time After _ 2 Mean Prior to3 Application 0 4 6 6 + TCA Sampling (8 lbs/A) months ug/g ug/g ug/g ug/g ug/g ~ in Sandy Clay Loam (Soil u) 2.5 0.0 1.2 2.1 l.“ 1.2A “.1 “.6 0.2 1.8 1.2 2.1 1.2A “.8 5.8 0.2 1.8 1.7 1.7 1.2A 1.8 Mean 0.13 1.63 1.73 1.72 Sandy Clay Loam (Soil 5) 2.5 0.10 1.5a 2.Ua 1.9a 1.DA 5.9 “.6 ' 0.10 1.0a 1.0a 2.3a l.UA 6.6 6.2 0.00 0.uc 0.20 0.00 0.28 3.6 Mean 0.13 1.03 1.23 1.U3 Sandy Clay Loam (Soil 6) 1.7 0.08 3.5ab U.2a 2.8b 2.5A 5.1 “.9 0.08 0.0d8 0 Ede 1.7Cd 0.88 10.7 Mean 0.03 1.93 2 53 2 23 Sandy Clay Loam (Soil 7) 1.8 0.6 0.8 2 8 1.1 1.101 3.5 4.0 0.1 1.3 2 3 2.7 1.4A 6.1 Mean 0.33 1.03 2.53 1.93 Sandy Clay Loam (Soil 8) 1.8 0.0 0.5 2.5 1.7 0.8A 3.5 “.0 0.0 0.1 0.2 1.2 0.08 6.1 Mean 0.03 0.33 1.33 ~1.113 1 Means within a soil followed by the same letter of the same case or design- ation or no letter are not significantly different (P < 0.05). 2Includes pyrazon applied alone at 3 lbs/acre and at 2 and 3 lbs/acre in combination with TCA, in addition to treatments shown. 3Between time of application and first sampling and between sampling periods. 79 TABLE lX.-—Bacteria and actinomycete populations in three soils amended with pyrazon, incubated at 21.0 C.1 Bacteria Actinomycetes Incubation Soil Time Amended Amended pH Control 80112 Control 80112 weeks 106 106 105 105 bact/g bact/g actino/b actino/g inn-v ”l Clay Loam 2 31b “70 U 3 6.8 5 163b 38Ua 30 7 7.0 10 85b 99b 5 7 6.8 15 64b 13b 6 6 6.7 Treatment Mean 850 1350 ll 7 Sandy Loam 2 10 12 u 3 6.7 5 18 50 6 13 7.2 10 2O 29 2 6 7.0 15 6M M6 u 33 7.1 Treatment Mean 28 3M M 1“ Sandy Clay Loam 2 M3 10 7 8 7.4 5 17 16M 4 2 7.u 10 138 282 ll 9 7.5 15 H5 60 7 N 7.5 20 105 “3 l O 7.3 Treatment Mean 83 112 6 H 1 Means followed by no letter or the same letter are not significantly different (P < 0.05). 23 ug/g added to clay loam and sandy loam, u ug/g added to sandy clay loam. Q r) w I “\ TABLE X.-—Bact8ria and actinomycete populations in a sandy clay loam amended with pyrazon incubated at four tempera— tures.i Bacteria Actinomycetes Incubation Time Amended Amended Control Soil Control Soil weeks 105 106 105 105 bact/gm bact/gm actino/gm actino/gm 4.5 C 5 218 267 6 1 10 36 68 3 9 2O 31 169 l 2 Treatment Mean 75 165 3 A 12.0 C 5 28 22 A 2 10 152 101 7 1A 15 U6 lUO 3 A 20 10 15 1 0 Treatment 3 Mean 59 7“ A 5 21.0 C (see Table IX) 29.5 C 5 150 419 70 60 10 279 177 6 5 15 151 79 U8 29 20 33 65 O 2 Treatment Mean 153 185 31 2A 1“ pg/gm added, means are not significantly different (P < 0.05).