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Aminopyralid fate in plant tissues and soil

Permanent Link: http://ufdc.ufl.edu/UFE0041982/00001

Material Information

Title: Aminopyralid fate in plant tissues and soil
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Fast, Brandon
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: absorption, aminopyralid, carryover, metabolism, mineralization, picloram, sorption, translocation
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Aminopyralid fate in plant tissues and soil Aminopyralid is a synthetic auxin herbicide of the pyridinecarboxylic acid family that was introduced in 2005; thus, the amount of information available on this herbicide is limited. Greenhouse experiments were conducted to determine the absorption, translocation, and metabolism of 14C-aminopyralid in the southern forage bahiagrass (Paspalum notatum Fluegge acute). Absorption of 14C-aminopyralid was 99% at 4 DAT with 89% of the 14C recovered remaining in the treated leaves. Shoots of plants contained 9% of recovered 14C, and less than 1.0% of recovered 14C was translocated to the roots of bahiagrass plants. The portion of recovered 14C that remained in the form of aminopyralid was 94% or greater. Due to the environmental persistence of this herbicide, field experiments were conducted to determine the sensitivity of bell pepper, Capsicum annuum L.; eggplant, Solanum melongena L.; muskmelon, Cucumis melo L.; tomato, Lycopersicon esculentum Mill.; watermelon, Citrullus lanatus (Thunb.) Matsum. & Nakai to aminopyralid soil residiues. Aminopyralid was applied at six rates from 0.0014 kg ae ha 1 to 0.0448 kg ae ha 1, and vegetable crops were planted in the treated areas. At an aminopyralid soil concentration of 0.2 microg kg 1, the limit of quantitation (LOQ) for aminopyralid in this research, crop injury ratings 6 wk after planting were 48 (bell pepper), 67 (eggplant), 71 (tomato), 3 (muskmelon), and 3% (watermelon), and fruit yield losses (relative to the untreated control) at 0.2 microg kg 1 aminopyralid were 61, 64, 95, 8, and 14% in those respective crops. Laboratory experiments were conducted to determine the sorption of aminopyralid to soil and clay minerals. Freundlich distribution coefficients (Kf) for aminopyralid ranged from 0.35 in a Cecil sandy loam to 0.96 in an Arredondo fine sand. It was concluded that soil sorption of aminopyralid was greater than that of picloram and that the potential for off-target movement of aminopyralid is less than that of picloram. Mineralization of picloram and aminopyralid in Sharkey clay was 15.2 and 23.7%, respectively, and mineralization of those respective herbicides in Arredondo fine sand was 5.4 and 9.2%.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Brandon Fast.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Ferrell, Jason A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041982:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041982/00001

Material Information

Title: Aminopyralid fate in plant tissues and soil
Physical Description: 1 online resource (62 p.)
Language: english
Creator: Fast, Brandon
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: absorption, aminopyralid, carryover, metabolism, mineralization, picloram, sorption, translocation
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Aminopyralid fate in plant tissues and soil Aminopyralid is a synthetic auxin herbicide of the pyridinecarboxylic acid family that was introduced in 2005; thus, the amount of information available on this herbicide is limited. Greenhouse experiments were conducted to determine the absorption, translocation, and metabolism of 14C-aminopyralid in the southern forage bahiagrass (Paspalum notatum Fluegge acute). Absorption of 14C-aminopyralid was 99% at 4 DAT with 89% of the 14C recovered remaining in the treated leaves. Shoots of plants contained 9% of recovered 14C, and less than 1.0% of recovered 14C was translocated to the roots of bahiagrass plants. The portion of recovered 14C that remained in the form of aminopyralid was 94% or greater. Due to the environmental persistence of this herbicide, field experiments were conducted to determine the sensitivity of bell pepper, Capsicum annuum L.; eggplant, Solanum melongena L.; muskmelon, Cucumis melo L.; tomato, Lycopersicon esculentum Mill.; watermelon, Citrullus lanatus (Thunb.) Matsum. & Nakai to aminopyralid soil residiues. Aminopyralid was applied at six rates from 0.0014 kg ae ha 1 to 0.0448 kg ae ha 1, and vegetable crops were planted in the treated areas. At an aminopyralid soil concentration of 0.2 microg kg 1, the limit of quantitation (LOQ) for aminopyralid in this research, crop injury ratings 6 wk after planting were 48 (bell pepper), 67 (eggplant), 71 (tomato), 3 (muskmelon), and 3% (watermelon), and fruit yield losses (relative to the untreated control) at 0.2 microg kg 1 aminopyralid were 61, 64, 95, 8, and 14% in those respective crops. Laboratory experiments were conducted to determine the sorption of aminopyralid to soil and clay minerals. Freundlich distribution coefficients (Kf) for aminopyralid ranged from 0.35 in a Cecil sandy loam to 0.96 in an Arredondo fine sand. It was concluded that soil sorption of aminopyralid was greater than that of picloram and that the potential for off-target movement of aminopyralid is less than that of picloram. Mineralization of picloram and aminopyralid in Sharkey clay was 15.2 and 23.7%, respectively, and mineralization of those respective herbicides in Arredondo fine sand was 5.4 and 9.2%.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Brandon Fast.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Ferrell, Jason A.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041982:00001


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1 AMINOPYRALID FATE IN PLANT TISSUES AND SOIL By BRANDON JAMES FAST A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Brandon James Fast

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3 To my wife Sandra in appreciation for her love, s upport, help and encouragement

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4 ACKNOWLEDGMENTS I thank God for loving me unconditionally, for providing me with a loving wife and beautiful family, for His patience with me as I learn from my mistakes, and for the countless other ways in which He has truly blessed me I thank my wife, Sandra S. Fast, and stepchildren, Emilie N. Kosbab, Kacie L Kosbab, and Daniel B. Kosbab, Jr. for the immeasurable joy they bring to my life and for their continual love, support, and encouragement. I also thank my parents, Larry D. and Ruth Anne Fast, and my brother i n law, D. Jaye Rose, sister, Carmel A. Rose, and niece, L aney J. Rose for their love and support. I express sincere appreciation to my graduate advisor, Dr. Jason A. Ferrell, for the invaluable instruction, advice help, and friendship that he has provide d. I also thank the members of my graduate committee for the advice, instruction, and friendship that they have provided. Members of the committee include d Drs. Gregory E. MacDonald, Brent A. Sellers, Andrew W. MacRae, David L. Wright, and L. Jason Krutz

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5 TABLE OF CONTENTS ACKNOWLEDGMENTS ...................................................................................................... 4 page LIST OF TABLES ................................................................................................................. 7 LIST OF FIGURES ............................................................................................................... 8 ABSTRACT .......................................................................................................................... 9 CHAPTER 1 ABSORPTION, TRANSLOCATION, AND METABOLISM OF AMINOPYRALID AND PICLORAM IN BAHIAGRASS ..................................................................................... 11 Introduction ................................................................................................................................. 11 Materials and Methods ................................................................................................................ 14 Absorption and Translocation ............................................................................................. 14 Metabolism ........................................................................................................................... 15 Results and Discussion ............................................................................................................... 16 Absorption and Translocation ............................................................................................. 16 Metabolism ........................................................................................................................... 17 Sources of Materials .................................................................................................................... 18 2 AMINOPYRALID SOIL RESIDUES AFFECT VEGETABLE CROPS .............................. 20 Introduction ................................................................................................................................. 20 Materials and Methods ................................................................................................................ 22 Results and Discussion ............................................................................................................... 24 Sources of Materials .................................................................................................................... 27 3 PICLORAM AND AMINOPYRALID SORPTION TO SOIL AND CLAY MINERALS ................................................................................................................................. 32 Introduction ................................................................................................................................. 32 Materials and Methods ................................................................................................................ 34 Sorption Kinetics ................................................................................................................. 34 Sorption to Soil and Clay Minerals .................................................................................... 35 Molecular Modeling ............................................................................................................ 36 Statistical Analyses .............................................................................................................. 36 Results and Discussion ............................................................................................................... 37 Sorption K inetics ................................................................................................................. 3 7 Sorption to Soil and Clay Minerals .................................................................................... 37 Molecular Modeling ............................................................................................................ 40

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6 4 PICLORAM AND AMINOPYRALID MINERALIZATION IN SOIL ................................. 47 Introduction ................................................................................................................................. 47 Materials and Methods ................................................................................................................ 48 Results and Discussion ............................................................................................................... 49 APPENDIX: PICLORAM AND AMINOPYRALID DEGRADATION EXPERIMENT .......... 53 LIST OF REFERENCES ................................................................................................................... 55 BIOGRAPHICAL S KETCH ............................................................................................................. 62

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7 LIST OF TABLES Table page 1 1 Absorption and distribution of 14C aminopyralid and 14C -picloram in bahiagrass 1 and 4 d after treatment (DAT). .............................................................................................. 19 1 2 Percentage of recovered 14C remaining in the form of the parent herbicide (14Caminoppyralid or 14C -picloram) in bahiagrass 1 and 4 d after treatment (DAT). .............. 19 1 3 Tomato fresh weight reductions from plants grown in soil that received 0.12 kg ae ha1 aminopyralid or soil that contained 1 .2 g of bahiagrass that had been treated with 0.12 kg ae ha1 amino pyralid 0 or 2 d before harvest ........................................................... 19 2 1 Regression parameters and R2 values for graphs in Figures 21 and 22 ........................... 28 2 2 Values of crop injury, plant height reduction, bloom number reduction, and fruit yield loss that are predicted to occur at an aminopyralid soil concentration of 0.2 g kg1. .......................................................................................................................................... 28 2 3 Pearsons correlation coefficients for the correlation between fruit yield loss and crop injury, plant height reduction, and bloom number reduction. ............................................. 29 2 4 Aminopyralid soil concentrations predicted to cause 10% crop injury. ............................. 29 3 1 State of origin, taxonomy, and characteristics of the five soils included in this research. .................................................................................................................................. 43 3 2 Freundlich equation parameters ( Kf and n ) and R2 values for picloram and aminopyralid in the five soils and three clay minerals included in this research. .............. 43 3 3 Pearsons correlation coefficients between picloram and aminopyralid sorption ( Kf) and sand, silt, clay, pH, OM, and CEC of the five soils included in this research. ........... 44 4 1 State of origin, taxonomy, and characteristics of the five soils included in this research. .................................................................................................................................. 51 4 2 Regression parameters for the graph in Figure 4 1. ............................................................. 51

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8 LIST OF FIGURES Figure page 2 1 Crop injury, plant height reduction, bloom number reduction, and fruit yield loss (all expressed as a percentage of the untreated control) of bell pepper, eggplant, and tomato as a function of aminopyralid soil concentration. .................................................... 30 2 2 Crop injury, plant height reduction, bloom number reduction, and fruit yield loss (all expressed as a percentage of the untreated control) of muskmelon and watermelon as a function of aminopyralid soil concentration. ..................................................................... 31 3 1 Chemical structures of picloram and aminopyralid. ............................................................ 45 3 2 Picloram and aminopyralid sorption as a function of time. ................................................. 45 3 3 Molecular models of picloram in the undissociated-nonionic (A) and dissociatedanionic ( B) forms and aminopyralid in the undissociated-nonionic (C ), and dis sociated anionic (D) forms ............................................................................................... 46 4 1 14CO2 evolution from 14C -picloram and 14C aminopyralid as a function of time in Sharkey clay and Arredondo fine sand. ................................................................................ 52

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9 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy AMINOPYRALID FATE IN PLANT TISSUES AND SOIL By Brandon James Fast August 2010 Chair: Jason A. Ferrell Major: Agronomy Aminopyralid is a synthetic auxin herbicide of the pyridinecarboxylic acid family that was introduced in 2005; thus, the amount of inform ation available on this herbicide is limited G reenhouse experiments were conducted to determine the absorption, translocation, and metabolism of 14C aminopyralid in the southern forage bahiagrass ( Paspalum notatum Fluegg) Absorption of 14C aminopyralid was 99% at 4 DAT with 89% of the 14C recovered remaining in the treated leaves Shoots of plants contained 9 % of recovered 14C and l ess than 1.0% of recovered 14C was translocated to the roots of bahiagrass plants. The portion of recovered 14C that remained in the form of aminopyralid was 94% or greater. Due to the environmental persistence of this herbicide, f ield experiments w ere conducted to determine the sensitivity of bell pepper, Capsicum annuum L.; eggplant, Solanum melongena L.; muskmelon, Cucumis melo L.; tomato, Lycopersicon esculentum Mill.; watermelon, Citrullus lanatus (Thunb.) Matsum. & Nakai to a minopyralid soil re sidiues. Aminopyralid was applied at six rates from 0.0014 kg ae ha1 to 0.0448 kg ae ha1, and vegetable crops were planted in the treated areas. At an aminopyralid soil concentration of 0.2 g kg1, the limit of quantitation (LOQ) for aminopyralid in t his research, crop injury ratings 6 wk after planting were 48 (bell pepper), 67 (eggplant), 71 (tomato), 3 (muskmelon), and 3% (watermelon), and fruit yield losses (relative to the untreated

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10 control) at 0.2 g kg1 aminopyralid were 61, 64, 95, 8, and 14% in those respective crops. Laboratory experiments were conducted to determine the sorption of aminopyralid to soil and clay minerals. Freundlich distribution coefficients ( Kf) for aminopyralid ranged from 0.35 in a Cecil sandy loam to 0.96 in an Arredondo fine sand. It was concluded that soil sorption of aminopyralid was greater than that of picloram and that the potential for off -target movement of aminopyralid is less than that of picloram. Mineralization of picloram and aminopyralid in Sharkey clay w as 15.2 and 23.7%, respectively, and mineralization of those respective herbicides in Arredondo fine sand was 5.4 and 9.2%.

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11 CHAPTER 1 ABSORPTION, TR ANSLOCATION, AND MET ABOLISM OF A MINOPYRALID AND PICLORAM IN BAHIAGRASS Introduction Aminopyralid is a pyridinecarboxylic acid herbicide that was registered in 2005 for annual and perennial broadleaf weed control in rangeland, permanent grass pastures, an d non -cropland areas at rates of 0.05 to 0.12 kg ae ha1 (Anonymous 2008; USOPPEPTS 2005). It provides foliar and soil residual weed control, is moderately sorbed ( Kf = 0.35 persistent herbicide in the soil (half life = 34.5 d) (Fast et al. 2010a; Senseman 2007b ). While the residual activity of aminopyralid is desirable from a weed management standpoint, aminopyralid carryover can injure bro adleaf crops that are planted on a site where aminopyralid was previously applied. This issue is of extreme importance in Florida where aminopyralid is commonly used for weed control in bahiagrass ( Paspalum notatum Fluegg) pastures. V egetable crops are often grown in rotation with bahiagrass pasture (Hopkins and Elmstrom 1984), and several of Floridas most economically important vegetable crops, including bell pepper (Capsicum annuum L.), eggplant, ( Solanum melongena L.), and tomato (Lycopersicon escule ntum Mill.) are sensitive to aminopyralid at soil concentrations of 1 g kg 1 or less (Fast et al. 2010b). Tropical soda apple ( Solanum viarum Dunal.) is one of the most common and troublesome pasture weeds in Florida (Webster 2008) due to prolific seed production, high seed germination rates ( 70 to 90% ), and limited herbicide options (Bryson and Byrd 2007; Mullahey and Cornell 1994). P lants typically produce 150 to 400 fruits per plant w ith 400 to 500 seeds per fruit with rapid germination and seedling establishment (Bryson and Byrd 2007). T he herbicide t riclopyr is capable of providing 90% control of emerged tropical soda apple but control is ephemeral as seedlings quickly re infest the treated area in the months following herbicide application (Ferre ll

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12 et al. 2006; Mullahey et al. 1993). Conversely, aminopyralid provides excellent control of emerged plants and residual control of seedlings for up to one year after treatment (Ferrell et al. 2006). Most her bicides that are used for soil residual contr ol are applied directly to bare soil ; however, several of these types of herbicides are intercepted by cover -crop foliage and mulch residues in notill production systems and herbicide efficacy is reduced (Chokor et al. 2008; Ghadiri et al. 1984; Schmitz et al. 2001). Likewise, a minopyralid is commonly applied to dense and vigorously growing vege tation (Ferrell et al. 2006), and i t is likely that this vegetation intercepts the vast majority of the herbicid e, preventing direct soil sorption. The fact that aminopyralid has soil residual activity even when the herbicide is not applied directly to the soil suggests that some mecha nism may be present to allow the herbicide to move from vegetation to the soil. P icloram and clopyralid are two herbicides that are nearly structurally identical to aminopyralid. The m etabolism of picloram in pea ( Pisum sativum L.), barley ( Hordeum vulgare L.), soybean [ Glycine max (L.) Merr.], Canada thistle [Cirsium arvense (L.) Sco p.], horsenettle (Solanum carolinense L.), and leafy spurge ( Euphorbia esula L.) is negligible (Gorrell et al. 1988; Lym and Moxness 1989; Scott and Morris 1970; Sharma and Vanden Born 1973). Additionally, clopyralid i s not readily metabolized in hemp dog bane ( Apocynum cannabinum L.) and yellow starthistle ( Centaurea solstitialis L.) (Orfanedes et al. 1993; Valenzuela -Valenzuela et al. 2001). In many plants, these herbicides are conjugated in the parent form and sequestered in the cell vacuole. Thus, the herbicide remains in the active form within the plant tissues. The lack of metabolism of these herbicides is problematic, particularly when treated plants are used for mulch in sites where sensitive crop species will be grown. Because picloram and clopy ralid are not typically metabolized, mulch that is composed of plants that were treated with these

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13 herbicides may contain enough herbicide to injure susceptible broadleaf plants as it decomposes and the herbicide is released (Anonymous 2009a, 2009b). When spearmint ( Mentha cardiaca Baker) was treated with clopyralid and the spearmint residue was incorporated into soil, potato (Solanum tuberosum L.) grown in the treated soil sustained injury (Boydston 1994) Compost that contained 50 g kg1 clopyralid was applied on a site at 112 metric tons ha1, and tomato plants that were grown on the site sustained injury (Blewett et al. 2005). It has also been reported that clopyralid in mulch can injure susceptible plants at concentrations as low as 10 g kg1 (Rynk 2002). Clopyralid can remain in plant residue at concentrations high enough to injure sensitive plants for a long period of time; moreover, grass clippings that had an initial clopyralid concentration of 200 g kg1 showed a concentration of 20 g kg1 98 weeks after treatment (Miltner et al. 2003). Problems with clopyralid in mulch causing crop injury became so prevalent in California, Oregon, and Washington that restrictions were placed on clopyralid use in those states (Brank 2003; ODA 2003; WSDA 2003). Due to the chemical similarity of aminopyralid, picloram, and clopyralid, i t is questioned whether or not the behavior of aminopyralid in plants is similar to that of clopyralid and picloram. The aminopyralid product label states that aminopyralid -t reated plant residues should not be used in mulch that will be applied where susceptible broadleaf plants will be grown (Anonymous 2008). It has also been reported that aminopyralid was not metabolized in Canada thistle (Bukun et al. 2009). This informat ion suggests that aminopyralid, like picloram and clopyralid, is sequeste red in plants and released in the active form as the plant tissue decomposes H owever, no published data are available on aminopyralid absorption, translocation, and metabolism in ba hiagrass or the release of aminopyralid from treated plant residue. It is important to have a clear understanding of the anthropomorphic behavior of aminopyralid in plants and release into the soil to prevent

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14 unwanted crop injury from occurring as a resul t of aminopyralid carryover. Therefore, t he objective of this research was to quantify aminopyralid absorption, translocation, and metabolism in bahiagrass and compare to the known herbicide picloram Materials and Methods Bahiagrass rhizomes were collect ed from established pasture in North Central Florida, cut into 8 cm segments and placed in 12 cm diameter pots containing potting soil media. Pots were placed in a greenhouse and plants were treated with aminopyralid (0.12 kg ae ha1) or picloram (0.56 kg ae ha1) after reaching a height of 25 cm (approximately 4 weeks) Herbicides were applied with a track sprayer calibrated to deliver 140 L ha1 with an 8002E nozzle, and each spray solution contained 0.25% v/v non-ionic surfactant. Immediately after application, plants were spotted with 6.7 kBq of ringlabeled 14C aminopyralid (specifi c activity = 1.01 109 kBq mol1, radiochemical purity = 98%) or 14C -picloram (specifi c activity = 9.10 108 kBq mol 1, radiochemical purity = 99%) in a total volume o f 10 L. Spotting consisted of four 2.5 L droplets of 14C herbici de solution placed on the adaxial surface of four random leaves of each plant. Absorption a nd Translo cation Plants were harvested 1 and 4 days after treatment (DAT). Treated leaves were excised and washed with 5 mL of deionized water (five 1 -mL aliquots). The leaf rinse solutions were combined for each plant and 2 mL of the combined leaf wash was added to 15 mL of scintillation cocktail1. Soil was washed from roots, and plants were plac ed in a p lant press and oven dried at 40 C for 48 hr. Oven -dried plants were then sectioned into treated leaves, shoot, and roots, and sections were combusted in a biological oxidizer2. Evolved 14CO2 was trapped in 20 mL of scintillation cocktail3, and r adioactivity in leaf washes and oxidations was quantified using liquid scintillation spectrometry4. Mean 14C recovery was determined by summing the 14C present in

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15 the leaf washes and plant segments. Average 14C recovery was 97% of applied for aminopyrali d and 85% of applied for picloram. The experimental design was a completely randomized design with three replications, and the experiment was repeated. Metabolism Plants were grown and treated with 14C -he rbicide as described above and harvested for meta bolism analysis at 1 and 4 DAT. Leaf washes were performed, and plants were sectioned into roots and shoots, immediately frozen in liquid nitrogen and placed in -10 C storage until laboratory analysis was performed. Herbicide metabolism was determined u sing thin layer chromatography (TLC) as described by Lym and Moxness (1989). Tissue from treated leaves (0.2 g) was ground with a tissue homogenizer5 in 2 mL of 80:20 (v/v) ethanol/water. The suspension was then centrifuged at 5000 g fo r 8 min, and the supernatant decanted. An additional 1 mL of 80:20 (v/v) ethanol/water was added, and the pellet was rehomogenized. The suspension was again centrifuged for 8 min at 5000 g and the supernatant was added to the test tube. The supernatant was evaporated to a volume of 200 L in a nitrogen evaporator6 at 5 0 C. A two -way TLC analysis was then performed using 20 by 20 -cm silica gel plates7. The 200 L of plant extract was placed on the plates using a micropipette, and the plates were then developed using 80:40:10:5 (v/v/v/v) benzene/acetone/acetic acid/methanol. Plates were then air dried, rotated 90 degrees, and developed in 65:25:4 (v/v/v) chloroform/methanol/water. Plates were air dried and scanned using a radiochromatogram scanner8. For comparison, separate plates were also developed using this procedure that contained 10 L of stock radiolabeled solution. The experimental design was a completely randomized design with three replications, and the experiment was repeated. A bioassay experiment was c onducted with aminopyralid to provide additional metabolism data. Aminopyralid (0.12 kg ae ha1) was applied to Arredondo fine sand (Loamy, siliceous,

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16 semiactive, hyperthermic Groassarenic Paleudults ) in 8 cm square pots and was also applied to field -grow n bahiagrass. Aboveground bahiagrass biomass was harvested 0 DAT (immediately after application when herbicide solution had dried on the leaf) and 2 DAT (after 2.5 -cm of simulated rainfall had been applied to the treated area to wash unabsorbed herbicide from leaves ). Bahiagrass was dried at 40 C for 48 hr and ground to pass a 2 mm sieve. A 1.2 g sample of the bahiagrass was incorporated into 590 g of Arre dondo fine sand in 8 -cm square pots. Water was added to bring soil to 80% field capacity, and three tomato seeds were planted in the pots three weeks later. Tomato plants were thinned to one plant per pot one week after emergence. Aboveground tomato plant biomass was harvested 4 weeks after planting and percentage fresh weight reduction relative to th e untreated control was calculated. The experimental design was a completely randomized design with three replications and the experiment was repeated. Results and Discussion Absorption and Translocation 14C aminopyralid was readily absorbed by bahiagrass with approximately 93.0 and 98 .7 % absorbed at 1 and 4 DAT, respectively (Tab le 1 1 ). Of the 14C recovered, 82 .1 % (1 DAT) and 88.7 % (4 DAT) remained in the treated leaves, 10 .3 % (1 DAT) and 9 .5 % (4 DAT) was translocated to the shoot, and less than 1.0% ( 1 and 4 DAT) was translocated to the roots. Absorption of 14C -picloram was less than that of 14C aminopyralid. Moreover, 14C -picloram absorption was 6 3.6 % at 1 DAT and 92.1 % at 4 DAT. The distribution of 14C among plant segments was similar for plants treated with either aminopyralid or picloram. Of the 14C recovered from plants treated with 14C -picloram, 56 .0% (1 DAT) and 80.8% (4 DAT) remained in the treated leaves, 7 .0% (1 DAT) and 10.6 % (4 DAT) was tran slocated to the shoot, and less than 1 .0 % (1 and 4 DAT) was translocated to the roots. O ther researchers have likewise shown

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17 limited translocation from foliar applications on Can ada thistle and woolly loco ( Astragalus mollisimus Torr.) (Bukun et al. 2009; Sterling and Jochem 1995). Metabolism Metabolism of both aminopyralid and picloram was negligible in bahiagrass (Table 1 2). Approximately 94.0 and 96.2 % of 14C was found to be in the parent form of 14C aminopyralid at 1 and 4 DAT, respectively. Piclora m was found to be unmetabolized wi t h 99.0 and 96.6% of 14C remaining in the form of 14C -picloram 1 and 4 DAT, respectively. Plants were not harvested beyond 4 DAT because metabolism of other synthetic auxin herbicides typically occurs within 4 DAT; moreo ver, metabolism of triclopyr in rice (Oryza sativa L. ), 2,4 D common milkweed (Asclepias syriaca L. ) and honeyvine milkweed [Cyanchum lae ve (Michx. ) Pers.] and picloram in broom snakeweed [Gutierrezia sarothrae (Pursh) Britt. & Rusby] occurred within 4 DAT (Braverman 1995; Coble et al. 1970; Gibbs and Sterling 2004; Wyrill and Burnside 1976). In the bioassay experiment, a 93% reduction in tomato fresh weight was observed when plants were grown in soil treated with 0.12 kg ae ha1 aminopyralid (Table 1 3) When aminopyralid treated bahiagrass leaves were harvested 0 and 2 DAT and mixed with soil growth reductions in seedling tomato were 85 and 67%, respectively Clopyralid can also injure susceptible plant species when clopyralid treated plants are used for mulch (Anonymous 2009b; Blewett et al. 2005 ; Boydston 1994). These data indicate that aminopyralid is not metabolized in bahiagrass and that it can be released into the soil and affect susceptible p lant species as the bahiagrass decomposes. Based on the results of this research, it was concluded that aminopyralid and picloram translocation from foliar applications in bahiagrass is minimal and that these herbicides are not readily metabolized in bahiagrass. It was also concluded that aminopyr alid can be sequestered in bahiagrass and released into the soil as the plant tissue decomposes. This suggests that

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18 bahiagrass contributes to the soil residual activity of aminopyralid by absorbing the herbicide and slowly releasing it into the soil as th e plant tissue decomposes Sources of Materials 1 Scintisafe Plus Liquid Scintillation Cocktail, Fisher Scientific Company, Fairlawn, NJ 07410. 2 Biological Oxidizer, Model OX500, R. J. Harvey Instrument Corporation, Hillsdale, NJ 07642. 3 Carbon 14 Cocktail, R. J. Harvey Instrument Corporation, Hillsdale, NJ 07642. 4 Packard 1600 CA TRI CARB Liquid Scintillation Analyzer, Packard Instrument Company, Downers Grove, IL 60515. 5 Dremel Model 300 Tissue Homogenizer, Dremel Corporation, Racine, WI. 6 Zymark TurboVap LV Evaporator, SOTAX Corporation, Hopkinton, MA 01748. 7 Whatman K6F Silica Gel 60 Thin Layer Chromatography Plates, Whatman Incorporated, Clifton, NJ 07013. 8 Bioscan AR 2000 Imaging Scanner, Bioscan, Washington, DC 20007.

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19 Table 1 1 Absorption and distribution of 14C aminopyralid and 14C -picloram in bahiagrass 1 and 4 d after treatment (DAT). 14 C aminopyralid 14 C picloram 1 DAT 4 DAT 1 DAT 4 DAT % of recovered 14 C (SE) Treated leaf 82.1 (3.2) 88.7 (1.5) 56.0 (2.1) 80.8 (2.4) Shoot 10.3 (2.0) 9.5 (1.7) 7.0 (2.3) 10.6 (3.5) Root 0.7 (0.1) 0.5 (0.1) 0.6 (0.1) 0.7 (0.1) Unabsorbed 6.9 (1.5) 1.3 (0.2) 36.4 (5.1) 7.9 (1.0) Table 1 2. Percentage of recovered 14C remaining in the form of the parent herbicide (14C amino pyralid or 14C picloram) in bahiagrass 1 and 4 d after treatment (DAT). Harvest timing 14 C recovered as parent herbicide Herbicide DAT % of recovered (SE) 14 C aminopyralid Standard 1 99.6 (0.2) 14 C aminopyralid 1 94.0 (1.5) 14 C aminopyralid 4 96.2 (1.2) 14 C picloram Standard 99.3 (0.3) 14 C picloram 1 99.0 (0.5) 14 C picloram 4 96.6 (1.6) 1Standard = 333 Bq of 14C herbicide applied directly to TLC plate Table 1 3. Percentage reduction in t omato fresh weight from plants grown in soil that received 0.12 kg ae ha1 aminopyralid or soil that contained 1.2 g of bahiagrass that had been treated with 0.12 kg ae ha1 aminopyralid 0 or 2 d before harvest. Tomato plant fresh weight reduction Treatment % relative to untreated control (SE) Soil 93 (3) Bahiagrass harvested 0 DAT 85 (9) Bahiagrass harvested 2 DAT 67 (3)

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20 CHAPTER 2 AMINOPYRALID SOIL RE SIDUES AFFECT VEGETA BLE CROPS Introduction Florida is the second largest producer of fresh market vegetables in the US, with nearly 2 million metric tons va lued at 1.4 billion dollars produced in 2009 (USDA warm, humid climate and sandy soils in Florida are a favorable environment for soilborne pests, including damping-off ( Rhizoctonia solani Khn and Pythium spp.), Fusarium wilt ( Fusarium oxysporum Schltdl.), Phytophthora root rot ( Phytophthora spp.), Verticillium wilt ( Verticillium dahliae Kleb.), white mold [ Sclerotinia sclerotiorum (Lib.) de Bary], and sting (Belonolaimus longicaudatus Rau) and root knot ( Meloidogyne spp.) nematode s (Koike et al. 2003; Noling 2007; Sumner et al. 1999) These major soilborne pests of vegetable crops in Florida have been managed effectively since the 1960s with the use of methyl bromide as the primary method of soilborne pest control (Chellemi 2002). The production and import of methyl bromide in the US was banned by the EPA in 2005 (USEPA 2008a ) as a result of its negative effects on the stratospheric ozone layer. Methyl bromide is curre ntly being phased out with new production only possible for quarantine and preshipment uses and those crops that qualify for a critical use exemption (CUE) as decided by the Parties of the Montreal Protocol (UNEP 2009). This management tool will no longer be available when CUEs are no longer granted and the existing supplies of methyl bromide are depleted. The reduction in the availability of methyl bromide for soilborne pest control has forced producers to rely more heavily on other control methods. An effective and inexpensive way to control soilborne pests is to use crop rotations in which vegetable crops are rotated with crops that do not serve as host plants for these soilborne pests (Chellemi 2002; Momol et al. 2007; Noling 2007).

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21 Bahiagrass ( Paspal um notatum Fluegg) is the most commonly planted warm -season grass in Florida, and it occupies an estimated 1 million ha in the state (Chambliss et al. 1999). It tolerates a wide range of environmental conditions and fertility regimes while requiring less intensive management than many other forage grasses (Ball et al. 2002;). Since bahiagrass pasture is abundant in Florida and bahiagrass is not a suitable host plant for numerous soilborne pests, vegetable crops are commonly grown in rotation with bahiagr ass pasture (Hopki ns and Elmstrom 1984). Research has demonstrated that d amping off, fusarium wilt, yellow nutsedge (Cyperus esculentus L.), and root galls from root knot nematode decreased and crop yields increased when cucumber ( Cucumis sativus L.), sna p bean ( Phaseolus vulgaris L.), and watermelon were planted after three years of bahiagrass (Hopkins and Elmstrom 1984; Sumner et al. 1999). In addition to controlling pests, crop rotations with bahiagrass increase crop yields by improving the water holdi ng capacity, nitrogen content, organic matter content, and soil tilth (Beaty and Tan 1972; Breneman et al. 1995; Burton 1954; Hopkins and Elmstrom 1984). It should be noted that some weed species that grow in rotational crops can serve as alternative host s for soilborne pests (Rich et al. 2009). Tropical soda apple ( Solanum viarum Dunal), for example, is a common and troublesome weed of pastures in Florida (Mullahey and Cornell 1994), and it can serve as a host plant for root knot nematode (Church and Ros skopf 2005). Therefore, weed control is imperative in bahiagrass pastures that are employed in a vegetable crop rotation. Aminopyralid, a synthetic auxin herbicide registered in 2005, is labeled for annual and perennial broadleaf weed control in rangeland, permanent grass pastures, and non -cropland areas at rates of 0.05 to 0.12 kg ae ha1 (Anonymous 2008; USOPPEPTS 2005). Because it is relatively persistent in the soil (half life = 34.5 days), aminopyralid provides both foliar and soil

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22 residual weed cont rol; moreover, it provides excellent control of tropical soda apple for up to one year after application ( Senseman 2007b ; Ferrell et al. 2006). Because of its high efficacy and residual activity on several common and troublesome pasture weed species, amin opyralid is used for weed control in pastures throughout Florida. However, an aminopyralid product label states that pastures treated with aminopyralid are not to be rotated to any crop within one year after treatment Further label restrictions state th at a broadleaf crop should not be planted in a treated area until an adequately sensitive field bioassay reveals that the crop will not be adversely affected (Anonymous 2008). While the residual activity of aminopyralid is desirable from a weed control st andpoint, injury in subsequent broadleaf crops arising from aminopyralid carryover is a potential negative consequence. As a result of the heavy use of aminopyralid for pasture weed control and the fact that vegetable crops are commonly planted in rotation with bahiagrass pasture, there is a need for information on the effects of aminopyralid on vegetable crops. The objective of this research was to quantify the sensitivity of bell pepper ( Capsicum annuum L.), eggplant ( Solanum melongena L.) tomato ( Lycopersicon esculentum Mill.) muskmelon (Cucumis melo L.) and watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai.] to soil residues of aminopyralid. Materials and Methods Field experiments were conducted on a Lakeland fine sand (Thermic, coated Typi c Quartzipsamments) at the University of Florida North Florida Research and Education Center near Live Oak, FL and on an Arredondo fine sand (Loamy, siliceous, semiactive, hyperthermic Groassarenic Paleudults) at the University of Florida Plant Science Res earch and Education Unit near Citra, FL. Bell pepper, eggplant, and tomato were seeded on 7 March 2008 in a greenhouse in Gainesville, FL and were transplanted in the field on 18 April 2008 (Live Oak) and 23 April 2008 (Citra). Muskmelon and watermelon w ere direct -seeded in the field when the other species

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23 were transplanted. Plants were grown on polyethylene -mulched beds in plots that were five beds wide (one bed per crop species) by 4.6 m long. Beds at Live Oak were 94 cm wide with 1.5 m row spacing for bell pepper, eggplant, and tomato and 1.8 m row spacing for muskmelon and watermelon. Beds at Citra were 76 cm wide with 1.8 m row spacing for bell pepper, eggplant, and tomato and 2.4 m row spacing for muskmelon and watermelon. Eggplant, tomato, mus kmelon, and watermelon were planted in a single row in the center of the beds, while beds for bell pepper contained two rows of plants per bed. Inrow plant spacings were 30 cm (bell pepper), 45 cm (eggplant and tomato), 60 cm (muskmelon), and 91 cm (wate rmelon). Aminopyralid was applied to bare soil in plots three weeks before planting at rates of 0.0014, 0.0028, 0.0056, 0.0112, 0.0224, and 0.0448 kg ae ha1. The soil was tilled with a disk, beds were formed using a PTO -driven power bedder methyl bromi de was applied, and polyethylene mulch was set in place the day following aminopyralid application. A soil sample was collected from the center of the beds in each plot to a depth of 15 cm on the day that vegetables were transplanted. Samples were transpo rted to the laboratory on ice and ultimately stored at 10C. Aminopyralid soil concentration was then quantified via high performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS) at a commercial laboratory1 (limit of quantitation was 0.2 g kg1) At 6 weeks after planting (WAP) crop injury was visually estimated on a scale of 0 to 100 (0 = no injury, 100 = plant death), plant height or vine length of three random plants within each plot was measured, and open blooms on the three rand om plants in each plot were counted. For simplicity, vine length will be referred to as plant height herein. Marketable fruit was harvested four times at both locations ( 19 June, 26 June, 3 July, and 10 July 2008), with the culls being discarded.

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24 The experimental design was a randomized complete block, and treatments were replicated four times. Plant height, bloom number, and yield data were converted to percentage reduction relative to the untreated control. No location interactions were detected; therefore, data were pooled across experiment sites. Crop injury, plant height and bloom number reduction, and crop yield loss were regressed as a function of aminopyralid soil concentration. The regression model, parameters, and R2 values for the depend ent variables of each crop are provided in Table 2 1. Results and Discussion Aminopyralid caused severe crop injury and plant height reduction in bell pepper, eggplant, and tomato (Figure 2 1). Plant height was reduced 30 to 40% as aminopyralid soil concentration increased from 0 to 1 g kg1, with crop injury increasing by approximately twice the rate of plant height reduction. Conversely, little additional increase (<20%) in plant response was documented as aminopyralid concentration increased from 1 g kg1 up to the maximum of 14.8 g kg1. Maximum values of crop injury were 90 (bell pepper), 90 (eggplant), and 89% (tomato), and maximum plant height reductions were 69, 61, and 54%, respectively. These res ults were not surprising because syntheti c auxin herbicides such as picloram and clopyralid are typically highly efficacious on species in the Solanaceae family, which includes bell pepper, eggplant, and tomato (OSullivan et al. 1999; Rocha and Yamashita 2009). In addition to affecting the veg etative growth of bell pepper, eggplant, and tomato, aminopyralid also affected the reproductive capacity of those plants. Response curves of bloom number reduction and fruit yield loss for bell pepper, eggplant, and tomato were similar to those of crop i njury and plant height reduction. Interestingly, there was a dramatic initial reduction between 0 and 1 g kg1 aminopyralid with very little further reduction ( at concentrations above 1 g kg1. At the maximum concentration tested (14 g kg1), aminopyralid caused bloom

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25 number reductions of 100, 92, and 100% and yield losses of 100, 95, and 100% in bell pepper, eggplant, and tomato, respectively. The effects of aminopyralid on muskmelon and watermelon were less severe than those observed on be ll pepper, eggplant, and tomato. As aminopyralid soil concentration increased, muskme lon and watermelon crop injury, reductions in plant height and bloom number and fruit yield loss increased in a curvilinear manner (Figure 2 2 ). This contrasts the more drastic bi phasic increase that occurred in bell pepper, eggplant, and tomato. C rop injury, plant height reduction, bloom number reduction, and fruit yield loss were 51, 49, 41, and 68%, respectively, in muskmelon, and values of those respective variable s were 42, 28, 73, and 67% in watermelon. The limit of quantitation (LOQ) for aminopyralid in soil was 0.2 g kg1 in this research T he values of crop injury, plant height reduction, bloom number reduction, and fruit yield loss that were predict ed to occ ur in those crops evaluat ed at this concentration ar e provided in Table 2 2. Crop injury values for bell pepper, eggplant, tomato, muskmelon, and watermelon were 48, 67, 71, 3, and 3%, respectively, and yield losses for those respective crops were 61, 64, 95, 8, and 14% at 0.2 g kg1 aminopyralid. This demonstrates that aminopyralid can cause substantial injury and yield loss in bell pepper, eggplant, and tomato at soil concentrations below the LOQ; therefore, an alternative method of detection (e.g., a field bioassay) must be used to determine if the aminopyralid soil concentration is sufficient to cause unacceptable crop injury and yield loss. Pearsons correlation coefficients were calculated to determine the strength of the correlation between fruit yield loss and crop injury, plant height reduction, and bloom number reduction (Table 2 3). With the exception of muskmelon, correlation coefficients for crop injury were P -values were <0.0001 for all crops. Correlation coefficients for plant h eight reduction and bloom number reduction were P -values were

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26 except watermelon and tomato. The strong correlation between fruit yield loss and crop injury, plant height reduction, and bloom number reduction suggests that am inopyralid soil concentration and the resulting fruit yield loss can be reliably predicted using a field bioassay. Although the aminopyralid soil concentration can be predicted using a field bioass ay, it should be noted that these parameters were collecte d 6 WAP; therefore, a substantial amount of time is required to use this method. The regression model and parameters from Table 2 1 were used to predict the soil concentrations of aminopyralid that would result in 10% crop injury (the maximum acceptable le vel of injury in vegetable crops) in the five crops evaluated (Table 2 4). The estimated time (mo) required for aminopyralid (when applied at the maximum labeled rate of 0.12 kg ae ha1) to reach those soil concentrations were then calculated using aminopyralids reported half -life of 34.5 d (Senseman 2007b ) (Table 2 4). Our calculations suggest that bell pepper, eggplant, and tomato should not be planted within two yr after aminopyralid application, and muskmelon and watermelon should not be planted within one yr after aminopyralid application to avoid crop injury and yield loss from aminopyralid car ryover. These estimates are not meant to serve as a replacement for a field bioassay, but to provide a general guideline as to when it is appropriate to conduct a field bioassay. Only through this accurate confirm ation can a decision be made concerning w hen crops can be planted without sustaining unacceptable injury and yield loss from aminopyralid carryover. The rate of herbicide dissipation is influenced by a complex of dynamic factors (e.g., environmental conditions and soil chemical and physical properties) and is therefore highly variable (Nash 1988). Additionally, when clopyralid is applied in grass crops, the herbicide can be sequestered in the crop residue and then be re released into the soil in a biologically active form as the crop residue dec omposes (Branham and Lickfeldt 1997; Brinton

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27 et al. 2006). Based on aminopyralid label statements regarding plant residues that contain aminopyralid, it appears that aminopyralid behaves in a similar manner to clopyralid and picloram (Anonymous 2008). Th is introduces an additional factor that influences the rate of aminopyralid dissipation because environmental conditions and management practices affect the rate and timing at which the crop residue decomposes and aminopyralid is re released into the soil. Due to the extreme sensitivity of the crops included in this research to low aminopyralid soil concentrations (less than 1 g kg1) and the unpredictability of the rate of aminopyralid dissipation in the soil, it is imperative that a field bioassay be co nducted before planting a broadleaf crop on a site where aminopyralid has been applied. The data that we utilized was collected 6 WAP, which is unpractical for most producers; therefore, additional research needs to b e conducted to determine if the bioass ay period could be reduced to 1 or 2 WAP. Sources of Materials 1Carbon Dynamics Institute, LLC, 2835 Via Verde Drive, Springfield, IL 62703.

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28 Table 2 1. Regression parameters and R2 values for graphs in F igures 2 1 and 22 The hyperbolic regression mode l Y = AX / (B + X) was used, where Y = dependent variable, X = independent variable, A = vertical asymptote of the dependent variable, and B = equation constant. Crop Dependent variable A B R 2 Bell pepper Crop injury 91.108 0.178 0.95 Bell pepper Plant height reduction 71.325 0.593 0.94 Bell pepper Bloom number reduction 102.487 0.073 0.98 Bell pepper Fruit yield loss 103.011 0.136 0.97 Eggplant Crop injury 89.913 0.069 0.90 Eggplant Plant height reduction 62.796 0.343 0.57 Eggplant Bloom number reduction 91.919 0.082 0.90 Eggplant Fruit yield loss 94.476 0.095 0.96 Tomato Crop injury 89.163 0.052 0.92 Tomato Plant height reduction 54.498 0.273 0.78 Tomato Bloom number reduction 99.585 0.076 0.91 Tomato Fruit yield loss 98.878 0.008 0.96 Muskmelon Crop injury 63.436 3.608 0.58 Muskmelon Plant height reduction 56.568 2.262 0.77 Muskmelon Bloom number reduction 41.509 0.124 0.44 Muskmelon Fruit yield loss 75.679 1.764 0.73 Watermelon Crop injury 52.742 3.467 0.70 Watermelon Plant height reduction 30.654 1.464 0.55 Watermelon Bloom number reduction 92.089 3.875 0.66 Watermelon Fruit yield loss 70.791 0.781 0.85 Table 2 2. Values of crop injury, plant height reduction, bloom number reduction, and fruit yield loss that are predicted to occur at an aminopyralid soil concentration of 0.2 g kg1 (the limit of quantitation for aminopyralid in this research) using the regression model and parameters from Table 2 1. Dependent variable Crop injury Plant height reduction Bloom number reduction Fruit yield loss Crop % Bell pepper 48 18 75 61 Eggplant 67 23 65 64 Tomato 71 23 72 95 Muskmelon 3 5 26 8 Watermelon 3 4 5 14

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29 Table 2 3. Pearsons correlation coefficients for the correlation between fruit yield loss and crop injury, plant height reduction, and bloom number reduction. Crop Dependent variable Pearsons correlation coefficient P Bell pepper Crop injury 0.97 <0.0001 Bell pepper Plant height reduction 0.89 <0.0001 Bell pepper Bloom number reduction 0.96 <0.0001 Eggplant Crop injury 0.97 <0.0001 Eggplant Plant height reduction 0.81 0.0005 Eggplant Bloom number reduction 0.98 <0.0001 Tomato Crop injury 0.94 <0.0001 Tomato Plant height reduction 0.74 0.0025 Tomato Bloom number reduction 0.93 < 0.0001 Muskmelon Crop injury 0.83 0.0002 Muskmelon Plant height reduction 0.92 <0.0001 Muskmelon Bloom number reduction 0.80 0.0006 Watermelon Crop injury 0.91 <0.0001 Watermelon Plant height reduction 0.71 0.0048 Watermelon Bloom number reduction 0.83 0.0002 Table 2 4. Aminopyra lid soil concentrations predicted to cause 10% crop injury. Aminopyralid soil concentration pre dicted to cause 10% crop injury a Time required to reach aminopyralid soil concentration predicted to cause 10% crop injury b Crop g kg 1 (SE) mo Bell pepper 0.022 ( 0.040 ) 13 Eggplant 0.009 ( 0.003 ) 15 Tomato 0.007 ( 0.002 ) 19 Muskmelon 0.675 ( 0.205 ) 7 Watermelon 0.811 ( 0.447 ) 7 aConcentrations were calculated using the regression model and parameters in Table 2 1. bTimes were calculated using a half life of 3 4. 5 d (Senseman 2007b ) and assuming that aminopyralid was applied at the maximum labeled rate (0.12 kg ae ha1), which would achieve a soil concentration of 50 g kg1.

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30 Figure 2 1. Crop injury, plant height reduction, bloom number reduction, and fruit yield loss (all expressed as a percentage of the untreated control) of bell pepper, eggplant, and tomato as a function of amino pyralid soil concentration Crop injury, plant height reduction, and bloom number reduction data were collected 6 WAP.

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31 Figure 2 2 Crop injury, plant height reduction, bloom number reduction, and fruit yield loss (all expressed as a percentage of the untreated control) of muskmelon and watermelon as a function of amino pyralid soil concentration Crop injury, plant height reduction, and bloom number reduction data were collected 6 WAP.

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32 CHAPTE R 3 PICLORAM AND AMINOPYRALID SORPTION TO SO IL AND CLAY MINERALS Introduction Picloram and aminopyralid are auxin-ty pe herbicides of the pyridine carboxylic acid family Picloram ( Figure 3 1 ) was introduced in 1963 for the control of broadleaf weeds, including several deep rooted perennial herbaceous species and woody brush species ( Gantz and Laning 1963; Hamaker et al. 1963) and is currently labeled for use in pasture and rangeland, wheat, oats, barley, and non -crop areas at application rates of 0.07 to 1.12 kg ae ha1 (Anonymous 2009a ). Picloram is a moderately sorbed ( Kd = 0.5) we a k acid herbicide (p Ka = 2.3) with a water solubility of 430 mg L1 (Senseman 2007a ). Aminopyralid ( Figure 3 1 ), which is structurally similar to picloram, was first regis tered in 2005 (USOPPEPTS 2005) and is labeled for use in range and pastureland and non -crop areas at application rates of 0.05 to 0.12 kg ae ha1 for the control of several annual and perennial herbaceous broadleaf weed species (Anonymous 2008). Aminopyra lid is a weak acid herbicide (p Ka = 2.56) that has a water solubility of 2480 mg L1 and is moderately sorbed ( Kd = 0.72) (Senseman 2007b ). Picloram is relatively mobile in soil, and picloram sorption to soil and clay minerals has been the focus of numerous experiments over the past 40 years. Light textured soils with low organic matter (OM) content and high application rates cause picloram soil mobility to increase (Baur et al. 1972; Herr et al. 1966; Scifres et al. 1969), and the potassium salt fo rmulation of picloram is more mobile than the triisopropanolamine salt formulation (Hunter and Stobbe 1972). In addition to the experiments that have been conducted on picloram soil mobility, numerous experiments have been conducted where picloram sorptio n was measured and reported in the form of the soil sorption constant ( Kd) or the Freundlich distribution coefficient ( Kf) (Arnold and Farmer 1979; Biggar and Cheung 1973; Farmer and Aochi 1974; Grover 1971).

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33 Among those experiments, the lowest reported Kf of picloram was 0.07 on an Ephrata sandy loam ( 60% sand, 32% silt, 8% clay, 0.94% OM, pH = 7. 14) (Farmer and Aochi 1974), and the highest reported Kf was 1.74 on a Palouse silt (12% sand, 61% silt, 27% clay, 3% OM, pH = 5.9) (Biggar and Cheung 1973). Add itionally, picloram sorption is positively correlated to OM content (Farmer and Aochi 1974; Grover 1971; Hamaker et al. 1966) and negatively correlated to soil pH (Arnold and Farmer 1979; Biggar and Cheung 1973). Piclorams weak soil sorption, correlation between sorption and soil pH, and the resulting high degree of soil mobility can be attributed to its weak acid nature. More than half of the molecules of a weak acid compound are in the undissociated nonionic form at pH values that are below the p Ka of the compound, and the number of molecules in the dissociated anionic form increases as pH increases (Wauchope et al. 2002; Nicholls and Evans 1991). Because the pH of field soils is typically far above the p Ka of picloram (2.30) and aminopyralid (2.56) (S enseman 2007a, 2007b), most of those molecules are in the dissociated anionic form when present in field soil. The dissociation of weak acid herbicide molecules in soil affects sorption because dissociated anionic molecules are repelled by the net negativ e charge of soil surfaces (Green and Karickhoff 1990). Weak acid herbicides are often strongly sorbed to clay minerals because the pH at the surface of many clay minerals can be 3 to 4 pH units lower than that of the soil solution (Bailey et al. 1968; Noy an et al. 2006); therefore, molecules at clay surfaces can convert to the undissociated nonionic form and be adsorbed to the clay surfaces via coulombic interactions. Due t o its weak soil sorption and the resulting high degree of mobility in the soil, picl oram can potentially move off -target to ground and surface water in and adjacent to treated areas (Lym and Messersmith 1988; Smith et al. 1988; Wood and Anthony 1997). Despite the environmental concerns caused by off -target movement to water, picloram con tinues to be heavily used because

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34 of the high degree of efficacy and residual control that it provides on numerous broadleaf weed species. Aminopyralid, like picloram, is also highly efficacious on broadleaf weeds and provides residual weed control; howev er, aminopyralid has much lower use rates and a shorter half life than picloram (Senseman 2007a, 2007b), which suggests that the potential for negative environmental impacts is much lower with aminopyralid than with picloram. In addit ion to its lower use rates and shorter soil half life, the Kd of aminopyralid (0.72) is greater than that of picloram (0.50) (Senseman 2007a, 2007b), which suggests that aminopyralid soil sorption is greater than that of picloram. N o Kf values or additional published data on aminopyralid sorption relative to soil texture and OM content were found in the literature The objective of this research was to characterize picloram and aminopyralid sorption to five soils and three clay minerals and use that information to determine if the potential for off target movement of aminopyralid in the soil is less than that of picloram. Materials and Methods Sorption Kinetics Sorption kinetics of the potassium salt formulation of picloram and aminopyra lid were determined on an Arredondo fine sand ( see Table 3 1 for soil characteristics ) using the batch equilibrium method (Weber 1986). Soil was placed in 50 mL plastic centrifuge tubes (2 g tube1) and 10 mL of 0.01 M calcium chloride solution that conta ined either picloram or aminopyralid at concentrations of 0.1 and 1.0 g ae L1 were added to each tube. Herbicide solutions contained mixtures of either picloram plus 14C -picloram or aminopyralid plus 14C aminopyralid (supplied by Dow AgroSciences, Indian apolis, IN) The specific activity and radiochemical purity of picloram were 9.10 108 kBq mol1 and >99%, respectively, and the specific activity and radiochemical purity of aminopyralid were 1.01 109 kBq mol1 and 98%, respectively. Centrifuge tubes were placed on a wrist action shaker removed after 1, 6, 12, 24, or 48 h, and

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35 centrifuged at 3600 g for 4 min A 2 mL aliquot of the supernatant from each centrifuge tube was added to 15 mL of liquid scintillation cocktail, and the amount of radioactivity was quantified via liquid scintillation spectrometry (LSS). Herbicide not in solution was con sidered sorbed; hence, the amount of herbicide sorbed was calculated by subtracting the supernatant concentration from the concentration of herbici de initially added. Test concentrations did not exceed herbicide solubility, and a preliminary quality assurance experiment revealed that herbicide sorption to centrifuge tubes was negligible. Based on the relatively long half -lives o f these compounds (S enseman 2007a; 2007b) and the report of Biggar et al. (1978) that no degradation of picloram occurred during a sorption experiment, we assumed that picloram and aminopyralid degradation would not occur in our experiments. Sorption to Soil and Clay Mineral s S orption of aminopyralid and picloram to five soils and three clay minerals was conducted in the same manner as the sorption kinetics experiments (see Table 3 1 for soil characteristics) Centrifuge tubes contained 2 g of soil or 0.15 g of clay, and her bicide concentrations added to the tubes were 0.01, 0.05, 0.1, and 1.0 g ae L1. Tubes were placed on a wrist action shaker for 24 h, and all other procedures were identical to those used in the sorption kinetics experiment. Distribution of herbicide bet ween the sorbed and solution phases was calculated using the Freundlich equation (Equation 3 1) : x/m = KfC1/ n (3 1 ) Where: x/m is the amount of herbicide adsorbed per weight of dry soil (mg kg1), C is the concentration of the herbicide in soil solution at equilibrium (mg L1), and Kf and n are empirical constants.

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36 Molecular Modeling M olecular modeling software (ArgusLab 2004) was used to generate electrostatic potential -mapped electron density surface models of undissociated-nonionic and dissociated anionic picloram and aminopyralid molecules T he software generated three -dimensional models of the herbicide molecules in their most logical configurations based on electron density and a surface layer w as then added to each model that displayed electronegativity colorimetrically. As others have done ( Ferrell et al. 2005; Grey et al. 2000 ), we assumed that electronegative regions of the molecules would be repelled by negatively charged surfaces while positively charged regions would contribute to the adsorption of the molecules to negatively charged surfaces via coulombic forces. Statist ical Analyses Kinetics and sorption experiments were conducted twice using a completely randomized design, and each experiment contained three replications. Kinetics data were subject ed to analysis of variance (SAS 2007), which revealed that the two-way interaction between experiment and herbicide concentration, the two-way interaction between experiment and time, and the three -way interaction between experiment, herbi cide concentration, and time were not signi ficant ( P >0.05) for piclora m and aminopyralid; therefore, sorption data were pooled across experiments and across herbicide concentrations within each herbicide. Percentage herbicide sorption was plotted against time and a hyperbolic regression model (Eq uation 3 2) was fit to the data (SigmaPlot 2006): Y = aX / (b + X) (3 2 ) Where: Y = percentage of herbicide sorbed (dependent variable), a = vertical asymptote (percentage herbicide sorption as time approaches infinity), X = time (independent variable), and b = the initial increase in Y per unit increase in X. Sorption kinetics of the two herbicides were

PAGE 37

37 compared using the 95% confidence intervals of the regression model parameters. Results and Discussion Sorption Kinetics Picloram and aminopyralid sorption occurred in a short, initial phase of rapid sorption followed by a long phase during which sorption increased very little ( Figure 3 2 ). The vertical asymptotes (maximum theoretical percentages of herbicide sorption) of picloram and aminopyralid were 10.3 and 15.2%, respectively, which indicates that aminopyralid sorption to an Arredondo fine sand is greater than that of picloram. Moreo ver, 46% (picloram) and 59% (aminopyralid) of the maximum theoretical sorption had occurred within 1 h, and more than 80% of the maximum theoretical sorption had occurred for both herbicides within 6 h. These results are consistent with the report of Hama ker et al. (1966) that picloram equilibr ates rapidly in low OM soils. Sorption to Soil and Clay Minerals When herbicide sorption is described using the Freundlich equation, the empirical constant n is an indicator of the linearity of the sorption isotherm; moreover, h erbicide sorption is linearly proportional to the equilibrium solution concentration when n = 1. When herbicide sorption is a linear function of the equilibrium solution concentration, it is appropriate to use the soil sorption constant ( Kd) to describe herbicide sorption and make comparisons between treatments; however, it is more appropriate to use Kf when herbicide sorption is nonlinear (Seybold and Mersie 1996). Because our values of n ranged from 0.62 to 1.24, we used Kf to describe picl oram and aminopyralid sorption in the soils and clay minerals included in this research. The Kf values of picloram and aminopyralid were less than 1 in all five soils included in this research ( Table 3 2 ). The lowest Kf values for picloram (0.12) and aminopyralid (0.35) were

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38 observed in the Cecil sandy loam, and the highest Kf values for picloram (0.81) and aminopyralid (0.96) were observed in the Arredondo fine sand. The low Kf values (< 1) that we obtained for piclora m are similar to those reported by others in several soils ( Arnold and Farmer 1979; Biggar and Cheung 1973; Farmer and Aochi 1974). Kf values of aminopyralid were greater than those of picloram within each soil which suggests that soil sorption of aminop yralid is greater than that of picloram. Herbicide sorption varied significantly among the three clay minerals included in this research ( Table 3 2 ). Kf values for picloram were 0.25 (kaolinite), 1.17 (bentonite), and 1016.40 (montmorillonite) and Kf v alues for aminopyralid were 5.63 (kaolinite), 2.29 (bentonite), and 608.90 (montmorillonite). Kaolinite is a nonexpanding 1:1 clay (surface area = 29 m2 g1), bentonite is a 2:1 expanding clay (surface area = 171 m2 g1), and montmorillonite is a 2:1 expanding clay (surface area = 323 m2 g1) (Fushiwaki and Urano 2001). Differences in surface area and expansion between kaolinite and montmorillonite explain why sorption was several times greater in montmorillonite than kaolinite. The differences in he rbicide sorption among the three clay minerals included in this research suggest that clay minerals can significantly influence the amount of herbicide sorption that occurs in soil; moreover, the importance of clay surfaces to picloram soil sorption has be en previously reported (Biggar and Cheung 1973). A correlation analysis of our data revealed that sorption was not significantly correlated to soil texture, OM content, pH, or cation exchange capacity (CEC) (Table 3 3), which conflict s with previous report s that picloram sorption was positively correlated to OM content ( Biggar and Cheung 1973; Farmer and Aochi 1974; Grover 1971) and negatively correlated to pH ( Arnold and Farmer 1979; Biggar and Cheung 1973). In addition to the correlation analysis between sorption and OM, KO M values were also calculated for picloram and aminopyralid in the five

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39 soils by dividing Kf by percentage OM and multiplying by 100. If the contribution of OM to herbicide sorption was similar in the five soils, KO M should not vary su bstantially among soils. However, KO M values of the five soils included in this research were highly variable (data not shown), indicating that the contribution of OM to herbicide sorption differs among soils. The lack of correlation between herbicide so rption and soil texture, OM content, pH, and CEC indicates that additional factors or an interaction between factors influenced picloram and aminopyralid sorption. OM is not the only sorbent of herbicides in soil (Wauchope et al. 2002), and sorbents such as hydrated metal oxides and the mineral fraction of soil can contribute to picloram sorption (Hamaker et al. 1966; Cox et al. 1998). Clay minerals provide a suitable surface for weak acid herbicide sorption because their surface pH is often lower than th at of the soil solution. The lower pH on the clay surface allows dissociated anionic picloram molecules to enter the undissociated -nonionic form, which can be adsorbed to the clay surfaces by coulombic forces (Biggar and Cheung 1973; Bailey et al. 1968; N oyan et al. 2006). It has been reported that picloram sorption was positively correlated to OM content, that picloram sorption was negatively correlated to pH, and that picloram movement was greater in light textured soils with low OM content compared to h eavier soils with more OM (Arnold and Farmer 1979; Baur et al. 1972; Herr et al. 1966; Scifres et al. 1969; Biggar and Cheung 1973; Farmer and Aochi 1974; Hamaker et al. 1966). The Cecil sandy loam soil had 15% less sand, 0.47% more OM, and a pH that was 1.4 units below that of the Arredondo fine sand, which would lead one to predict that less picloram and aminopyralid sorption would occur in the Arredondo fine sand than in the Cecil sandy loam. However, Kf values of picloram and aminopyralid were higher in the Arredondo fine sand than in the Cecil sandy loam. The fact that the Cecil sandy loam is a kaolinitic soil could provide an explanation for this phenomenon.

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40 Sorption of picloram and aminopyralid to kaolinite was the lowest of the three clay mineral s included in this research. Even though the Cecil sandy loam contained a greater percentage of clay, picloram and aminopyralid sorption may have been greater on the clay minerals that were present in the Arredondo fine sand. Given the differences we obs erved in sorption to the different clay minerals, the effect of differing clay minerals in different soils could outweigh the effect of soil texture, OM content, and pH. Results similar to ours have been reported with other weak acid herbicides; moreover, sorption of imazapyr, imazethapyr, and sulfometuron was lowest in the soils with the highest clay content (Gan et al. 1994; Wehtje et al. 1987). It should also be noted that OM can obstruct clay mineral surfaces and thus make them less available for her bicide sorption (Cox et al. 1998; Hance 1969). Another possible explanation for our results is that additional factors such as herbicide sorption to hydrated metal oxides may have contributed to picloram and aminopyralid sorption. Molecular Modeling Herbicide molecules that contain highly electronegative regions are repelled by the net negative charge of soil surfaces. In the undissociated nonionic form, most regions of picloram and aminopyralid molecules are positively charged; however, small region s of high electronegativity exist on the nitrogen atom of the pyridine ring and on the oxygen atoms of the carboxyl group ( Figure 3 3 ). All regions of picloram and aminopyralid molecules are highly electronegative when present in the dissociated anionic form, except for the hydrogen atoms on the amino group. Because the pH of typical field soils is greater than the p Ka values of those herbicides, t he majority of picloram and aminopyralid molecules in soil solution are in the dissociated anionic form ; this helps explain why these herbicides are weakly sorbed to soil. Decreasing pH can cause picloram and amino pyralid molecules to change from the dissociated anionic to undissociated -nonionic form, which explains why the low surface pH of clay minerals

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41 can in crease the soil sorption of the se herbicides. P iclora m and aminopyralid are weakly sorbed to soils as a result of their weak acid nature and the potential for these herbicides to move off-target is greater than that of many other herbicides. Kf values of aminopyralid and picloram were less than 1 .00 in this research, and reported Kf values of atrazine and sulfentrazone were 11.08 and 3.35, respectively (DeSutter et al. 2003; Reddy and Locke 1998) Although there is potential for both piclor am and aminopyralid to move off -target in the soil, Kf values of aminopyralid were significantly greater than those of picloram for the five soils included in this research. This indicates that there is less potential for off target movement of aminopyralid in t he soil compared to that of picloram. Sorption to clay minerals varied by an order of magnitude among the three clay minerals included in this research, which suggests that the type of clay mineral present in soils can affect picloram and aminopyralid sor ption. Picloram and aminopyralid sorption to the five soils included in this research was not significantly correlated to soil texture, OM content, pH, or CEC; therefore, it was concluded that additional factors such as the type of clay minerals present i n the soil or herbicide sorption to hydrated metal oxides have a significant influence on picloram and aminopyralid sorption to soil. Models of picloram and aminopyralid molecules with a colorimetric display of electronegativity indicate that herbicide mo lecules in the dissociated anionic form are unlikely to be adsorbed because they would be repelled by the net negative charge of soil surfaces; moreover, most molecules are in the dissociated anionic form at the pH values of typical field soils. T he maxim um labeled application rate of aminopyralid is less than that of picloram (Anonymous 2008; Anonymous 2009a ), the avera ge half life of aminopyralid (34.5 d) is less than that of picloram (90 d) (Senseman 2007a, 2007b), and aminopyralid soil sorption was gre ater than that of picloram. The low Kf values of picloram and aminopyralid

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42 indicate that there is potential for off -target movement of these compounds in the soil. Additionally, the higher Kf values of aminopyralid compared to those of picloram indicate that the potential for aminopyralid to move off target is slightly less than that of picloram.

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43 Table 3 1. State of origin, taxonomy, and characteristics of the five soils included in this research. Mineral composition CEC OM Sand Silt Clay State Series Taxonomy pH cmol kg 1 % Florida Arredondo fine sand Loamy, siliceous, semiactive, hyperthermic Grossarenic Paleudults 6.6 3.94 0.84 96.4 1.0 2.6 Georgia Cecil sandy loam Fine, kaolinitic, thermic Typic Kanhapludults 5.2 3.35 1.31 81.4 10.0 8.6 Kentucky Maury silt loam Fine, mixed, semiactive, mesic Typic Paleudalfs 5.6 27.90 2.31 33.4 47.0 19.6 Mississippi Bosket fine sandy loam Fine loamy, mixed, active, thermic Mollic Hapludalfs 7.0 20.11 2.31 37.4 47.0 15.6 Mississippi Sharkey clay Very fine, smectitic, thermic Chromic Epiaquerts 6.0 11.16 1.11 24.2 23.7 52.1 Table 3 2. Freundlich equation parameters ( Kf and n ) for picloram and aminopyralid in the five soils and three clay minerals included in this research. All R2 values were 0.99, and n umbers in parentheses are 95% confidence intervals. Pyridinecarboxylic acid herbicide Picloram Aminopyralid Soil /clay K f n K f n Arredondo fine sand 0.81 (0.78 0.84) 0.88 (0.77 0.99) 0.96 (0.94 0.98) 0.99 (0.9 2 1.0 6 ) Cecil sandy loam 0.12 (0.1 1 0.13) 0.97 (0.88 1.06) 0.35 (0.32 0.38) 0.91 (0.58 1.2 4 ) Maury silt loam 0.3 6 (0.35 0.3 7 ) 1.21 (1.10 1.32) 0.95 (0.93 0.97) 1.00 (0.95 1.0 5 ) Bosket fine sandy loam 0.40 (0.39 0.41) 1.02 (0.9 2 1.1 2 ) 0.68 (0.66 0.70) 1.11 (1.01 1.21) Sharkey clay 0.52 (0.50 0.5 4 ) 1.24 (1.09 1.39) 0.79 (0.77 0.8 1 ) 1.04 (0.97 1.11) Bentonite 1.17 ( 1.05 1.28 ) 1. 08 ( 0.74 1.42 ) 2.29 (2.17 2.41) 0.99 (0.80 1.18) Kaolinite 0.25 (0.23 0.27) 0.62 (0.08 1.16) 5.63 (5.30 5.96) 1.05 (0.85 1.25) Montmorillonite 1016.40 (979.70 1053.10) 1.12 (1.10 1.1 4 ) 608.90 (564.30 653.6 0 ) 1.03 (1.00 1.06)

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44 Table 3 3. Pearson s correlation coefficients between picloram and aminopyralid sorption ( Kf) and sand, silt, clay, pH, OM, and CEC of the five soils included in this research. Soil property Picloram K f Aminopyralid K f Sand 0.21 ( P = 0.73) 0.19 ( P = 0.76) Silt 0.32 ( P = 0.59) 0.18 ( P = 0.77) Clay 0.001 ( P = 0.99) 0.13 ( P = 0.84) OM 0.45 ( P = 0.45) 0.04 ( P = 0.95) pH 0.64 ( P = 0.25) 0.41 ( P = 0.50) CEC 0.18 ( P = 0.78) 0.41 ( P = 0.49)

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45 Figure 3 1. Chemical structures of picloram and aminopyralid. Figure 3 2. Picloram and aminopyralid sorption as a function of time.

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46 Figure 3 3 Molecular models of picloram in the undissociated -nonionic (A) and dissociated anionic (B) forms and aminopyralid in the undissociated-nonionic (C ), and dissociated anionic (D ) forms. Ball and -stick models directly below the surface mapped molecules indicate the configuration of the atoms within the surface -mapped models.

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47 CHAPTER 4 PICLORAM AND AMINOPYRALID MINERALIZATION IN SO IL Introduction Herbicide dissipation occurs when herbicide residues are removed from an environmental compartment (e.g., soil) by microbial or chemical degradation or transferred to another environm ental compartment. T he microbial fraction of herbicide degradation is re ferred to as mineralization ( Stephenson et al. 2006). D egradation is an important component of herbicide dissipation, and mineralization makes a substantial contribution to degradation because of the abundant and diverse population of microorganisms tha t is typically found in soils (Walker 1987). T he rate at which a n herbicide degrades is influenced by numerous factors, including the molecular structure of the herbicide the physical and chemical properties of the soil, and environmental conditions ; the refore, the rate of herbicide degradation can be highly variable over time and space ( Nash 1988; Walker 1987) Herbicide sorption to soil colloids is one factor that can affect herbicide mineralization; moreover, herbicides that are weakly sorbed can be m ineralized more quickly than herbicides that are strongly sorbed because they are more readily available to soil microorganisms. ( Wauchope et al. 2002). Picloram is a sy nthetic auxin herbicide of the pyridine carboxylic acid family that was introduced in the 1960s and provides foliar and soil residual control of many annual and perennial broadleaf weed s, including several woody brush species (Gantz and Laning 1963; Hamaker et al. 1963) Picloram has relatively high use rates (0.07 to 1.12 kg ae ha1), is persistent in the soil (half life = 90 d) and is weak ly sorbed to soil ( Kf = 0.12 0.17) ( Anonymous 2009a; Fast et al. 2010a; Senseman 2007a). A lthough these facts raise concern about the potential negative impacts of this herbici de on the environment, picloram is still commonly used because of unmatched efficacy and residual activity on common and troublesome weed species

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48 Aminopyralid, another pyridine carboxylic acid herbicide, was introduced in 2005 and provides foliar and soil residual control of numerous annual and perennial broadleaf weed species in rangeland, permanent grass pastures, and non-cropland areas at rates of 0.05 to 0.12 kg ae ha1 (Anonymous 2008). Aminopyralid is a weakly sorbed herbicide ( Kf = 0.35 0.96 ) with a soil half -life of 34.5 days ( Senseman 2007b; Fast et al. 2010a ). A minopyralid has lower use rates, greater soil sorption, and a short er soil half -life than picloram; therefore, it appears that aminopyralid would have less negative environmental impacts compared to picloram The persistence of aminopyralid and picloram in the soil can provide the benefit of residual weed control ; however, the persistence of these herbicides also increases the p robability of offtarget movement and environmental contamina t ion The reported half -lives of picloram and aminopyralid are 90 and 34. 5 d, respectively (S enseman 2007a; 2007b ), which implies that aminopyralid i s less persistent than picloram; h owever, it should be noted that those reported half life values were aver aged across several field experiments that were conducted in a wide range of soi ls and environmental conditions. In order to compare the persistence of aminopyralid and picloram, experiments should be conducted in the laboratory where environmental condit ions are controlled and the same soil type is used for both herbicides. The objective o f this research was to determine if aminopyralid mineralization is greater than that of picloram in two soils Materials and Methods Mineralization of p icloram and amin opyralid was quantified in Arredondo fine sand from Florida and Sharkey clay from Mississippi (see Table 4 1 for soil characteristics) using EPA testing guidelines (USEPA 2008b ) and the methods described by Pramer and Bartha (1965). Soil s were collected from field sites with no history of picloram or aminopyralid application, air dried, and passed through a 2 mm sieve. Fifty g of dry soil was then added to b iometer flasks and

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49 deionized wa ter was added to bring soil to 40% field capacity. Flask s were then incub ated in darkness for 1 wk at 25 C to allow soil microbial populations to establish. After the 1 wk incubation period solutions of picloram plus 14C -picloram or aminopyralid plus 14C aminopyralid were distributed to the soil surface to ac hieve a herbicide concentration in the soil of 1 g g1. The specific activity and radiochemical purity of picloram were 9.10 108 kBq mol1 and >99%, respectively, and the specific activity and radiochemical purity of aminopyralid were 1.01 109 kBq mo l1 and 98%, respectively. Flasks were sealed and 10 mL of 1 N KOH trapping solution was added to the sidearm of the biometer flasks to capture evolved 14CO2. Flasks were placed in dark incubation, and KOH trapping solution was removed at sampling times of 1, 2, 4, 8, 16, 32, 48, and 64 DAT. At each sampling time, the biometer flasks were rinsed with 5 mL of trapping solution, 10 mL of fresh trapping solution was added, and 10 mL of fresh air was injected into the flasks to maintain an ae robic environment Radioactivity in the trapping solution from each sampling time was quantified by adding a 2 mL aliquot of the trapping solution to 15 mL of scintillation cocktail and determining radioactivity via liquid scintillation counting. The exp eriment contained three replications and was conducted twice Data were subjected to analysis of variance, which revealed that the effect of soil type on mineralization was significant. Mean 14CO2 evolution was plotted against time for each soil type and each herbicide, and the two compartment biexponential regression model was fit to the data (Equation 4 1). Y = a (1 b X) + c (1 d X) (4 1 ) The regression parameters for the data sets are provided in Table 4 2. Results and Discussion Mineralization of 14C aminopyralid was greater than that of picloram within each soil type, and mineralization of both herbicides was greater in the Sharkey clay than it was in Arredondo

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50 fine sand (Figure 4 1). At 64 DAT, m iner alization of aminopyralid, as indicated by 14CO2 evolution was 23.7 and 15.2% in Sharkey clay and Arredondo fine sand, respectively, a n d picloram mineralization was 9 .2 and 5.4% in those respective soils. The greater mineralization of both herbicides in the Sharkey clay compared to the Arredondo fine sand could be a result of differences in the microbial populations in the soils. Also, picloram and aminopyralid sorption was slightly greater in Arredondo fine sand than it was in Sharkey clay, which indic ates that the herbicide would be less available to soil microbes for minerali zation in Arredondo fine sand. The only structural difference between picloram and aminopyral id is that picloram has a chlorine atom in the number 5 position of the pyridine ring that is not present in aminopyralid. This small change in chemical structure may contribute to the increased mineralization of aminopyralid compared to picloram within each soil. Mineralization of picloram and aminopyralid was greater than that of flumi oxazin (2.0 2.2%) and sulfentrazone (1.7 2.1% ); moreover, soil sorption of aminopyralid and picloram appears to be less than that of flumioxazin and sulfentrazone (Fast et al. 2010a; Ferrell et al. 2003; Reddy and Locke 1998) This indicates that the weak sorption of picloram and aminopyralid contributes to their mineralization by making them more readily available to soil microbes. Research will be conducted to determine picloram and aminopyralid degradation (including mineraliztion and chemical breakdow n) in these soils to provide further information on their persistence in the soil.

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51 Table 4 1 State of origin, taxonomy, and characteristics of the five soils included in this research. Mineral composition CEC OM Sand Silt Clay State Series Taxonomy pH cmol kg 1 % Florida Arredondo fine sand Loamy, siliceous, semiactive, hyperthermic Grossarenic Paleudults 6.6 3.94 0.84 96.4 1.0 2.6 Mississippi Sharkey clay Very fine, smectitic, thermic Chromic Epiaquerts 6.0 11.16 1.11 24.2 23.7 52.1 Table 4 2. Regression parameters for the graph in Figure 4 1. The model Y = a(1 eb X) + c(1 dX) (biexponential two compartment model) was fit to the data. R2 values for both soils and both herbicides were 0.99. Regression parameter Soil Herbicide a b c d Sharkey clay A minopyralid 10.126 1.493 43.932 0.006 Arredondo fine sand A minopyralid 9.062 2.272 7.410 0.027 Sharkey clay P icloram 2.895 1.350 119.728 0.001 Arredondo fine sand P icloram 2.263 1.784 5.958 0.012

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52 Figure 4 1. 14CO2 evolution from 14C picloram and 14C aminopyralid as a function of time in Sharkey clay and Arredondo fine sand. Data points are means of six replications with standard error bars.

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53 APPENDIX PICLORAM AND AMINOPYRALID DEGRADATION EXPERIME NT An additional experiment was conducted on the degradation of picloram and aminopyralid in soil. Herbicide extracted from soil samples is being quantified, and this research will be incorporated into Chapter 4 when the results of the research become available. Picloram and aminopyralid dissipation were evaluated in Arredondo fine sand from Florida and Sharkey clay from Mississippi (See Table 1 for soil characteristics) in 50 -mL polypropylene centrifuge tubes. Dry soil (15 g) was added to centrifuge tubes, deionized water was added to bring soil to the appropriate moisture content (40 or 70% field capacity), and tubes were sealed and incubated in darkness at the ap propriate temperature (10 or 20 C) for 1 wk to allow the soil microbial population to become established. After the 1 wk incubation period, soil was fortified with picloram or aminopyralid to achieve an initial herbicide concentration of 1 g g1. Tubes were then sealed and incubated for 1, 2, 4, 8, 16, 32, 48, and 64 DA T in darkness, and they were placed in freezer storage ( 10 C) at the end of the incubation period until herbicide extraction occurred. All tubes were opened once per week during incubation to ensure that aerobic conditions were maintained in the tubes. A minopyralid was extracted from soil by adding 25 mL 90:10 (v/v) acetonitrile /1N HCl to the centrifuge tubes. The suspension was agitated on a wrist action shaker for 1 h and centrifuged at 3600 g for 6 min; supernatant was then transferred to 50 -mL beak ers. The extraction procedure was repeated using 15 mL of the 90:10 (v/v) acetonitrile /1N HCl solution, and the tubes were agitated for 30 min. The supernatants from the two extractions were combined and 10 mL of the supernatant was transferred to 15-mL polypropylene centrifuge tubes and evaporated to dryness at 50C in a nitrogen evaporator. Samples were brought to 3 mL with 1N HCl, and 2 of the 3 mL was concentrated on a Strata X SPE column that was

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54 co nditioned with 1 mL each of methanol and distilled water. The column was washed with 0.75 mL 95:5 (v/v) water/methanol dried under negative pressure for 15 min, and eluted twice with 0.5 mL acetonitrile per elution. Samples were then evaporated to dryness at 50C in a nitrogen eva porator; 0.2 mL 22:2:1 (v/v) acetonitrile /pyridine/butanol, 0.1 mL butyl chloroformate, and 0.7 mL 50:50:0.1 (v/v/v ) methanol / water /acetic acid were then added to bring samples to a final volume of 1 mL. Samples were then transferred to HPLC vials and analyzed with high perform ance liquid chromatography/mass spectrometry Picloram was extracted from soil in the same manner as aminopyralid; however, extraction columns were conditioned with 1 mL methanol followed by 1 mL of 1N HCl. Additionally, columns we re eluted twice with 0.5 mL methanol for picloram extraction. Unlike the aminopyralid extraction, no reconstitution was required with picloram; the extract was added to HPLC vials for analysis immediately after column elution.

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55 LIST OF REFERENCES Anonymous. 2008. Milestone herbicide product label. Dow AgroSciences publication No. D02 879002. Indianapolis, IN: Dow AgroSciences. 9 p. Anonymous. 2009a. Tordon 22K herbicide product label. Dow AgroSciences publication No. D02 111014. Indianapolis, IN: Dow AgroSciences. 13 p Anonymous. 2009b. Stinger herbicide product label. Dow AgroSciences publication No. D02 111014. Indianapolis, IN: Dow AgroSciences. 13 p. ArgusLab. 2004. ArgusLab Version 4.0.1. Planaria Software LLC, Seattle, WA. Arnold, J.S. and W.J. Farmer. 1979. Exchangeable cations and picloram sorption by soil and model adsorbents. Weed Sci. 27:257 Bailey, G.W., J.L. White, and I. Rothberg. 1968. Adsor ption of organic herbicides by montmorillonite: Role of pH and chemical character of adsorbate. Soil Sci. S oc. Am. Proc. 32:222 Ball, D. M., C. S. Hoveland, and G. D. Lacefield. 2002. Southern Forages. 3rd ed. Norcross, GA: Potash and Phosphate Institute. p. 26. Baur, J.R., R.D. Baker, R.W. Bovey, and J.D. Smith. 1972. Concentration of picloram in the so il profile. Weed Sci. 20:305 Beaty, E. R. and K. H. Tan. 1972. Organic matter, N, and bas e accumulation under Pensacola bahiagrass. J. Range Manage. 25:38 Biggar, J.W. and M.W. Cheung. 1973. Adsorption of picloram (4 amino 3,5,6 trichloropicolinic acid) on Panoche, Ephrata, and Palouse soils: a thermodynami c approach to the adsorption mechanism. Soil Sci. Soc. Am. Proc. 37:863 Biggar, J. W., U. Mingelgrin, and M. W. Cheung. 1978. Equilibrium and kinetics of adso rption of picloram and parathion with soils. J. Agric. Food Chem. 26:1306 1312. Blewett, T. C., D. W. Roberts, and W. F. Brinton. 2005. Phytotoxcitity factors and herbicide contamination in relation to compost quality management practices. Renew. Agr. Food Syst. 20:67 Boydston, R. A. 1994. Clopyralid persistence in spearmint ( Mentha cardiac ) hay injures potato (Solanum tuberosum ). Weed Technol. 8:296 Branham, B. E. and D. W. Lickfeldt. 1997. Effect of pesticide t reated grass clippings used as mulch on ornamental plants. HortScience 32:1216 Brank, G. 2003. DPR announces restrictions to protect compost. California Department of Pesticide Regulation Release. 3 p.

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56 Braverman, M. P. 1995. Absorption, translo cation, and metabolism of triclopyr in rice ( Oryza sativa ). Weed Technol. 9:490 493. Brenneman, T. B., D. R. Summer, R. E. Baird, G. W. Burton, and N. A. Minton. 19 95. Suppression of foliar and soilborne peanut dise ases in bahiagrass rotations. Phytopa thology 85:948 Brinton, W. F., E. Evans, and T. C. Blewett. 2006. Reliability of bioassay tests to indicate herbicide residues in compost of varying salinity and herbi cide levels. Comp. Sci. Util. 14:244 Bryson, C. T. and J. D. Byrd, Jr. 2007. Bilogy, reproductive pot ential, and winter survival of tropical soda apple ( Solanum viarum ). Weed Technol. 21:791 Bukun, B., T. A. Gaines, S. J. Nissen, P. Westra, G. Brunk, D. L. S haner, B. B. Sleugh, and V. F. Peterson. 2009. Aminopyralid and clopyralid absorption and t ranslocation in Canada thistle (Cirsium arvense ). Weed Sci. 57:10 Burton, G. W., E. H. DeVane, and R. L. Carter. 1954. Root penetration, distribution and activity in southern grasses measured by yields, drought symptom s, and P32 uptake. Agron. J. 46:229 Chambliss, C. G. 1999. Bahiagrass. Florida Forage Handbook. University of Florida Publication No. SP 253. Pp. 17 Chellemi, D. O. 2002. Nonchemical management of soilborne pests in fresh market vegetable production systems. Phytopathology 92:1367 Chokor, J. U., C. E. Ikuenobe, and C. N. Odoh. 2008. Effect of tillage on the efficacy of CGA362622 on weed control in maize. Afr. J. Biotechnol. 7:4288 4290. Church, G. T. and E. N. Rosskopf. 2005. Fi rst report of the root knot nematode Meloidogyne arenaria on tropical soda apple ( Solanum viarum ) in Florida. Plant Disease 89:527. Coble, H. D., F. W. Slife, and H. S. Butler. 1970. Absorption, metaboli sm, and translocation of 2,4 D by honeyvine milkwe ed. Weed Sci. 18:653 656. Cox, L., W.C. Koskinen, R. Celis, P.Y. Yen, M.C. Hermosin, and J. Cornejo. 1998. Sorption of imidacloprid on soil clay mineral and organic components. Soil Sci. Am. J. 62:911 DeSutter, T. M., S. A. Clay, and D. E. Clay. 2003. Atrazine sorpt ion and desorption as affected by aggregate size, particle size, and soil type. Weed Sci. 51:456 462. Farmer, W.J. and Y. Aochi. 1974. Picloram sorption by soils. Soil Sci. Soc. Am Proc. 38:418 Fast, B. J., J. A. Ferrell, G. E. MacDonald, L. J. Krutz, and W. N. Kline. 2010a. Picloram and Aminopyralid sorption to soil and clay m inerals. Weed Sci. In review.

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57 Fast, B. J., J. A. Ferrell, G. E. MacDonald, B. A. Sellers, A. W. MacRae, and L. J. Krutz. 2010b. Aminopyralid soil r es idues affect vegetable c rops. Weed Tchnol. In review. Ferrell, J. A. and W. K. Vencill. 2003. Flumioxazin soil persistence and mineralization in laboratory experiments. J. Agric. Food Chem. 51:4719 4721. Ferrell, J. A., J. J. Mullahey, K. A. Langeland, and W. N. Kline. 2 006. Control of tropical soda apple ( Solanum viarum ) with aminopyralid. Weed Technol. 20:453 Ferrell, J.A., W.K. Vencill, K. Xia, and T.L. Grey. 2005. Sorption and desorption of flumioxazin to soil, clay minerals, and ion -exchang e resin. Pest Manag. Sci. 61:4046. Fushiwaki, Y. and K. Urano. 2001. Adsorption of pesticides and their biodegraded products on clay minerals and soils. J. Health Sci. 47:429 Gan, J., M. R. Weimer, W. C. Koskinen, D. D. Buhler, D. L. W yse, and R. L. Becker. 1994. Sorption and desorption of imazethapyr and 5 -hydroxyim azethapyr in Minnesota soils. Weed Sci. 42:92 97. Gantz, R.L. and E.R. Laning. 1963. Tordon for the control of woody rangeland species in the western United States. Down to Earth 19(3):10 Ghadiri, H., P. J. Shea, and G. A. Wicks. 1984. Interception and retention of atrazine by wheat (Triticum aestivum L.) stubble. Weed Sci. 32:24 27. Gibbs, L. A. and T. M. Sterling. 2004. Seasonal variation of picloram metabolism in broom (Gutierrezia sarothrae ) and threadleaf ( Gutierrezia microcephala ) snakeweed populations in a common garden. Weed Sci. 52:206 212. Gorrell, R. M., S. W. Bingham, and C. L. Foy. 1988. Translocation and fate of dicamba, piclo ram, and triclopyr in horsenettle, Solanum carolinense Weed Sci. 36:447 Green, R.E. and S.W. Karickhoff. 1990. Sorption estimates for modeling. Pp. 79 In : H.H. Cheng (ed.) Pesticides in the soil environment: processes, impacts, and modeling. Soil Science Society of America, Madison, WI. Grey, T.L., R.H. Walker, G.R. Wehtje, J. Adams, Jr., F.E. Da yan, J.D. Weete, H.G. Hancock, and O. Kwon. 2000. Behavior of sulfentrazone in ionic exchange resins, electrophoresis gels, and cation -saturated soils Weed Sci. 48:239 247. Grover, R. 1971. Adsorption of picloram by soil colloids and various other adsorbents. Weed Sci. 19:417 Hamaker, J.W., C.A. Goring, and C.R. Youngson. 1966. Sorption and leaching of 4 amino 3,5,6 trichloropcolinic acid in soils Adv. Chem. Ser. 60:23 Hamaker, J.W., H. Johnston, R.T. Martin, and Carl T. R edeman. 1963. A picolinic acid derivative: A plant growth regulator. Science 141:363.

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58 Hance, R.J. 1969. Influence of pH, exchangeable cation, and the presence of organic matter on the adsorption of some herbicides by montmorillonite. Can. J. Soil Sci. 49:357 Herr, D.E., E.W. Stroube, and Dale A. Ray. 1966. The movement and persistence of picloram in s oil. Weeds 14:248 Hopkins, D. L. and G.W. Elmstrom. 1984. Eff ect of nonhost crop plants on watermelon Fusarium wilt. Plant Disease 68:239 Hunter, J.H. and E.H. Stobbe. 1972. Movement and persistence of picloram in soil. Weed Sci. 20:486 Koike, S. T., K. V. Subbarao, R. M. Davis, and T. A. Turini. 2003. Ve getable Diseases Caused by Soilborne Pathogens. Davis, CA: University of California Publication 8099. 13 p. Lym, R.G. and C.G. Messersmith. 1988. Survey for picloram in North Dakota groundwater. Weed Technol. 2:217 222. Lym, R. G. and K. D. Moxness. 1989. Absorption, translocati on, and metabolism of picloram and 2,4 D in leafy spurge ( Euphorbia esula). Weed Sci. 37:498 Miltner, E., A. Bary, and C. Cogger. 2003. Clopyralid and c ompost: formulation and mowing effects on herbicide content of grass clippings. Comp. Sci. Util. 11:289 Momol, T., J. Marois, K. Pernezy, and S. Olson. 2007. Int egrated disease management for vegetable crops in Florida. Pages 89 nd E. Simmone, eds. Vegetab le Production Handbook for Florida 2007 Mullahey, J. J. and J. A. Cornell. 1994. Biology of tropical soda apple ( Solanum viarum ) an introduced weed in Florida. Weed Technol. 8:465 Mullahey, J. J., J. A. Cornell, and D. L. Colvin. 1993. Tropical soda apple ( Solanum viarum ) control. Weed Technol. 7:723 Nash, R. G. 1988. Dissipation from Soil. Pages 131 n R. Grover, ed. Environmental Chemistry of Herbicides, Volume 1. Boca Raton, Florida: CR C Press. Nicholls, P.H. and A.A. Evans. 1991. Sorption of ionisable organic compounds by field soils. Part 1: Acids. Pestic. Sci. 33:319 Noling, J. W. Nematodes and their management. 2007. Pages 73 Simmone, eds. Vegetable Production Handbook for Flori da 2007 University of Florida. Noyan, H., M. nal, and Y. Sarikaya. 2006. The effect of heating on the surface area, porosity and surface acidity of a bentonite. Clays Clay Miner. 54:375

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62 BIOGRAPHICAL SKETCH Brandon James Fast was born in Fai rview, Oklahoma in 1979. After graduating from Fairview High School in 1999, he attended Oklahoma State University in Stillwater, Oklahoma and obtained a bachelor of science degree in plant and soil s ciences in 2003 and a master of science degree in plant and soil s ciences in 2007. Upon completion of his master of science degree, Brandon attended the Univers ity of Florida an d obtained a doctor of philosophy degree in a gronomy in summer 2010. Upon comple tion of his doctor of philosophy degree Brandon began employment with Dow AgroSciences in Indianapolis, Indiana