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Effect of Surfactants and Herbicide Combinations on Phytotoxicity of Diquat

Permanent Link: http://ufdc.ufl.edu/UFE0022102/00001

Material Information

Title: Effect of Surfactants and Herbicide Combinations on Phytotoxicity of Diquat
Physical Description: 1 online resource (165 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: 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: Diquat, a fast-acting contact herbicide, has been used for weed control in the US for over 45 years. It was widely recommended for control of hydrilla, often in combination with copper. Restriction of copper use in public waters in Florida in the 1980s and development of fluridone resistant hydrilla in the 1990s has resulted in renewed interest in using diquat for hydrilla control. Diquat is also being evaluated for aquatic weed control in irrigation canals due to its short half-life in water. Greenhouse studies indicated that rice seed germinated and maintained in diquat-treated DI water was very sensitive to diquat, with EC10 values of 0.016 and 0.004 mg L-1 for shoots and roots, respectively. Seeds of corn, cotton, squash, wheat and soybean germinated in sand and irrigated once with diquat-treated water at planting had EC10 values from 3 to 6 mg L-1. When treated at the seedling or more mature stages all crops exhibited EC10 values from 1 to 5 mg L-1, suggesting that diquat-treated water (0.370 mg L-1) may be used for irrigation with limited potential for phytotoxicity, except for freshly planted rice. Biomass and chlorophyll studies indicated that surfactants (CT-301, Cygnet Plus, SilEnergy and Timberland 90) applied alone were phytotoxic to hydrilla at concentrations greater than those recommended for aquatic weed control. Regression models yielded EC50 values from 26 to 592 mg L-1 for hydrilla dry weight and from 433 to 4,814 mg L-1 for chlorophyll reduction. Addition of surfactants to diquat at aquatic and terrestrial labeled rates was additive on hydrilla and landoltia based on Colby method. When applied on petunia, the aquatic and terrestrial surfactant rates plus diquat were additive and synergistic, respectively. The combination of copper, flumioxazin and endothall salts at different concentrations with diquat gave an additive effect based on dry weight. Acibenzolar added to diquat was synergistic based on hydrilla dry weight, as was carfentrazone based on hydrilla total length. These results indicate that some herbicides can be combined with diquat for improved hydrilla control, but the benefit of adding surfactants at aquatic labeled rates provides no more than additive effects.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Haller, William T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022102:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022102/00001

Material Information

Title: Effect of Surfactants and Herbicide Combinations on Phytotoxicity of Diquat
Physical Description: 1 online resource (165 p.)
Language: english
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: 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: Diquat, a fast-acting contact herbicide, has been used for weed control in the US for over 45 years. It was widely recommended for control of hydrilla, often in combination with copper. Restriction of copper use in public waters in Florida in the 1980s and development of fluridone resistant hydrilla in the 1990s has resulted in renewed interest in using diquat for hydrilla control. Diquat is also being evaluated for aquatic weed control in irrigation canals due to its short half-life in water. Greenhouse studies indicated that rice seed germinated and maintained in diquat-treated DI water was very sensitive to diquat, with EC10 values of 0.016 and 0.004 mg L-1 for shoots and roots, respectively. Seeds of corn, cotton, squash, wheat and soybean germinated in sand and irrigated once with diquat-treated water at planting had EC10 values from 3 to 6 mg L-1. When treated at the seedling or more mature stages all crops exhibited EC10 values from 1 to 5 mg L-1, suggesting that diquat-treated water (0.370 mg L-1) may be used for irrigation with limited potential for phytotoxicity, except for freshly planted rice. Biomass and chlorophyll studies indicated that surfactants (CT-301, Cygnet Plus, SilEnergy and Timberland 90) applied alone were phytotoxic to hydrilla at concentrations greater than those recommended for aquatic weed control. Regression models yielded EC50 values from 26 to 592 mg L-1 for hydrilla dry weight and from 433 to 4,814 mg L-1 for chlorophyll reduction. Addition of surfactants to diquat at aquatic and terrestrial labeled rates was additive on hydrilla and landoltia based on Colby method. When applied on petunia, the aquatic and terrestrial surfactant rates plus diquat were additive and synergistic, respectively. The combination of copper, flumioxazin and endothall salts at different concentrations with diquat gave an additive effect based on dry weight. Acibenzolar added to diquat was synergistic based on hydrilla dry weight, as was carfentrazone based on hydrilla total length. These results indicate that some herbicides can be combined with diquat for improved hydrilla control, but the benefit of adding surfactants at aquatic labeled rates provides no more than additive effects.
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.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Haller, William T.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-05-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022102:00001


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1 EFFECT OF SURFACTANTS AND HERBIC IDE COMBINATIONS ON PHYTOTOXICITY OF DIQUAT By TOMS F. CHICONELA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Toms F. Chiconela

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3 ACKNOWLEDGMENTS This study was m ade possible through the contri butions of several pe ople, and I would like to express my appreciation to each of them. Firs t, I would like to thank my advisor and mentor, Dr. William T. Haller, for his patience, encourag ement and guidance during the course of my studies at the University of Florida. I sincer ely appreciate his willingness to chair my DPM and PhD Supervisory Committees and for the aquatic weed control insights he gave me that, I am sure, will serve me for the rest of my caree r. I would also like to thank my supervisory committee members Dr. Gregory MacDonald, Dr Robert McGovern, Dr Michael Netherland and Dr. David Wofford for their inputs, as sistance and support th roughout my program. I extend my gratitude to Dr. Lyn Gettys, Dr. Ty ler Koschnick, Dr. Atul Puri, Dr. Christopher Mudge and Brett Bultemeier for their contributions and friendshi p. My appreciations also go to Margaret Glenn for her help. I would also like to thank David Mayo and William Jordan for their help in collecting data for this project. I extend my apprecia tion to the Ford Foundation for providing financial support. Sp ecial thanks go to Dr. Incio Maposse for his friendship and encouragement. Finally, I wish to thank my fa mily, paricularly my companion Veronica de Deus, for being supportive throughout my studies. My sincere gratitude al so goes to my sons Shelton, Arsnio, and Fernando for their friends hip and love. I extend my gratitude to my mother, Angelina Macie for her unfailing love, encourag ement and belief in me.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................3 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES.......................................................................................................................10 ABSTRACT...................................................................................................................................11 CHAP TER 1 INTRODUCTION..................................................................................................................13 Biology of Hydrilla.................................................................................................................13 Hydrilla Reproduction and Spread......................................................................................... 16 Importance of Hydrilla......................................................................................................... ..18 Hydrilla Management.............................................................................................................19 Diquat Use in Aquatic Weed Control..................................................................................... 23 Copper Use in Aquatic Weed Control.................................................................................... 25 Effects of Surfactants on Herbicides......................................................................................29 Herbicide Combinations......................................................................................................... 33 Herbicide Use in Irrigation Canals.........................................................................................35 Objectives...............................................................................................................................36 2 EFFECT OF DIQUAT IN IRRIGA TION WAT ER ON GE RMINATION AND VEGETATIVE GROWTH OF RICE.................................................................................... 38 Introduction................................................................................................................... ..........38 Materials and Methods...........................................................................................................39 Germination.................................................................................................................... .40 Irrigation..........................................................................................................................40 Statistical Analysis.......................................................................................................... 41 Results and Discussion......................................................................................................... ..41 Germination.................................................................................................................... .41 Irrigation..........................................................................................................................43 3 SENSITIVITY OF SELECTED AGRONOMIC PLANTS TO DIQUAT IN IRRIGATION WATER .......................................................................................................... 50 Introduction................................................................................................................... ..........50 Materials and Methods...........................................................................................................51 Results and Discussion......................................................................................................... ..52 4 PHYTOTOXICITY OF SURF ACTANTS TO HYDRILLA .................................................58

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5 Introduction................................................................................................................... ..........58 Materials and Methods...........................................................................................................59 Hydrilla Biomass.............................................................................................................60 Hydrilla Chlorophyll.......................................................................................................60 Results and Discussion......................................................................................................... ..61 Hydrilla Biomass.............................................................................................................61 Hydrilla Chlorophyll.......................................................................................................63 5 EFFECTS OF SURFACTANTS ON DIQU AT PHYTOTOXICITY TO SELECTED AQUATIC AND T ERRESTRIAL PLANTS......................................................................... 72 Introduction................................................................................................................... ..........72 Material and Methods.............................................................................................................75 Hydrilla Biomass.............................................................................................................75 Ornamental Biom ass....................................................................................................... 76 Hydrilla Chlorophyll.......................................................................................................76 Landoltia Chlorophyll.....................................................................................................77 Statistical Analysis.......................................................................................................... 78 Results and Discussion......................................................................................................... ..79 Hydrilla Biomass.............................................................................................................79 Experiment 1............................................................................................................ 79 Experiment 2............................................................................................................ 79 Ornamental Biom ass....................................................................................................... 81 Begonia.....................................................................................................................81 Petunia......................................................................................................................82 Hydrilla Chlorophyll.......................................................................................................83 Landoltia Chlorophyll.....................................................................................................84 Experiment 1............................................................................................................ 84 Experiment 2............................................................................................................ 85 6 HERBICIDE COMBINATIONS FOR THE E NHANCEMENT OF DIQUAT TOXICITY FOR HYDRILLA CONTROL........................................................................... 96 Introduction................................................................................................................... ..........96 Material and Methods.............................................................................................................98 Statistical Analysis........................................................................................................... .......99 Results and Discussion......................................................................................................... ..99 7 SUMMARY AND CONCLUSIONS...................................................................................110 APPENDIX A LABELS OF SURFACTANTS USED IN THIS STUDY ...................................................114 B COMPARISONS BETWEEN SURFACTANTS WHE N COMBINED WITH DIQUAT AT DIFFERENT CONCENTRATIONS.............................................................................120

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6 C COMPARISONS BETWEEN HERBICIDES WHEN COM BINED WITH DIQUAT AT DIFFERENT CONCENTRATIONS.............................................................................128 LIST OF REFERENCES.............................................................................................................135 BIOGRAPHICAL SKETCH.......................................................................................................165

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7 LIST OF TABLES Table page 2-1 Effect of diquat in irrigation water on rice germ ination and growth 14 DAT. Diquat applied once initially to s eeds and water level maintained with untreated tap water........ 47 2-2 Effect of diquat in irrigation water on rice at different stages of developm ent 14 DAT...................................................................................................................................47 3-1 Effect of diquat in irri gation water on corn, cotton, soybean, squash and wheat seed germ ination.................................................................................................................... ....57 3-2 Effect of diquat in irrigation water on corn, cotton, soybean, squash and wheat on initial seedling growth........................................................................................................ 57 4-1 Description of surfactants used for phytot oxicity studies on hydril la and in additional studies as described in this dissertation. Full labels are presented in Appendix A. ........... 67 4-2 Effect of surfactants on dry weight and total leng th of hydrilla Hydrilla tips were exposed to surfactants at various concentrations for 24 h................................................. 71 4-3 pH of surfactants in distilled water (p H 7.1.1) at various concentrations used in these studies. Each value presented as m ean standard error (n=3)................................. 71 4-4 Effect of surfactants on chlorophyll content of hydrilla. Hydrilla tips were exposed to surfactants at various concentrations fo r 48 h and chlorophyll content determ ined.......... 71 5-1 Effect of surfactants and diquat applied for 24 h alone or in combination on percent dry weight reduction of hydrilla. ....................................................................................... 88 5-2 Effect of surfactants and diquat applied for 24 h alone or in combination on percent dry weight reduction of hydrilla. ....................................................................................... 89 5-3 Effect of surfactants an d diquat applied for 24 h alone or in com bination on percent total length reduction of hydrilla........................................................................................ 90 5-4 Effect of surfactants and diquat applie d alone or in com bination on percent dry weight reduction of petunia............................................................................................... 91 5-5 Effect of surfactants and diquat applied for 48 h alone or in combination on percent total chlo rophyll reduction of hydrilla............................................................................... 92 5-6 Effect of surfactants and diquat applied for 48 h alone or in combination on percent total chlorophyll reduction of landoltia in Experim ent 1................................................... 93 5-7 Effect of surfactants and diquat applied for 48 h alone or in combination on percent total chlorophyll reduction of landoltia in Experim ent 2................................................... 94

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8 5-8 Interaction totals of the effects of surfactants ap plied alone and in combination with diquat at sublethal concentrations on hydrilla, landoltia and petunia................................ 95 6-1 Effect of diquat, acibenzolar and copper exposed for 24 h alone or in com bination on the dry weight (% reduction) of hydri lla. Hydrilla was harvested 14 DAT.................... 104 6-2 Effect of diquat, acibenzolar and copper exposure for 24 h alone or in combination on the total length (% reductio n) of hydrilla. Hydrilla was harvested 14 D AT...............105 6-3 Effect of diquat, carfentrazone and flum ioxazin exposure for 24 h alone or in combination on the dry weight (% reduction) of hydrilla............................................... 106 6-4 Effect of diquat, carfentrazone and fl um ioxazin applied for 24 h alone or in combination on the total lengt h (% reduction) of hydrilla...............................................107 6-5 Effect of diquat, dipotassium and al kylam ine salts of endothall exposure for 24 h alone or in combination on the dry weight (% reduction) of hydrilla............................. 108 6-6 Effect of diquat, dipotassium and al kylam ine salts of endothall exposure for 24 h alone or in combination on the tota l length (% reduction) of hydrilla............................. 109 B-1 Effect of surfactants and diquat applied in com bination for 24 h on percent dry weight reduction of hydrilla and p-va lues for combinations comparison........................ 121 B-2 Effect of surfactants and diquat applied in com bination for 24 h on percent dry weight reduction of hydrilla and pvalues for combinations comparison....................... 122 B-6 Effect of surfactants and diquat applied in com bination for 24 h on percent total chlorophyll reduction of landol tia and p-values for combinations comparison.............. 126 B-7 Effect of surfactants and diquat applied in com bination for 24 h on percent total chlorophyll reduction of landol tia and p-values for combinations comparison.............. 127 C-1 Effect of diquat on hydrilla dry weight when combined with acibenzolar and copper. Hydrilla was exposed to the com bination fo r 24 h and allowed to grow for 14 d after treatment...................................................................................................................... ....129 C-2 Effect of diquat on hydrilla total length when combined with acibenzolar and copper. Hydrilla was exposed to the com bination fo r 24 h and allowed to grow for 14 d after treatment...................................................................................................................... ....130 C-3 Effect of diquat on hydrilla dry weight when combined with carfentrazone and flum ioxazin.................................................................................................................... ..131 C-4 Effect of diquat on hydril la total length when combin ed with carfentrazone and flum ioxazin.................................................................................................................... ..132

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9 C-5 Effect of diquat on hydrilla dry weig ht when combined with dipotassium and alkylamine salts of endothall........................................................................................... 133 C-6 Effect of diquat on hydril la total length com b ined with dipotassium and alkylamine salts of endothall..............................................................................................................134

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10 LIST OF FIGURES Figure page 2-1 Relationship between dry weights of germ inating rice shoots and roots w hen exposed to various concentrations of diquat in water...................................................................... 48 2-2 Relationship between dry weight of rice seedling, tillering and m ature stages when exposed to various concentrations of diquat in irrigation water........................................ 49 3-1 Percent germination of corn, cotton, s oybean, squash and wheat when exposed to various concentrations of diquat in water.......................................................................... 55 3-2 Dry weights of corn, cotton, soybean, s quash and wheat shoots when exposed to various concentrations of diquat in irrigation water.......................................................... 56 4-1 Phytotoxicity of surfactants to hydrilla based on dry weight. H ydrilla was exposed for 24 h to surfactants at various concentrations............................................................... 68 4-2 Phytotoxicity of surfactants to hydrilla based on total length. Hydrilla was exposed for 24 h to surfactants at various concentrations. .............................................................. 69 4-3 Phytotoxicity of surfactants to hydrilla based on total chlorop hyll content. Hydrilla was exposed for 48 h to surfactants at various concentrations ..........................................70 A-1 Cygnet Plus label containi ng product ingredients, use ra te, handling and precautions for use...............................................................................................................................114 A-2 CT-301 label containing product ingredients, use rate, handling and precautions for use. ...................................................................................................................................115 A-3 SilEnergy label containi ng product ingredients, use ra te, handling and precautions for use...............................................................................................................................117 A-4 Timberland 90 label containing produc t ingredients, use rate, handling and precau tions for use........................................................................................................... 118 A-4 Timberland 90 label containing produc t ingredients, use rate, handling and precau tions for use........................................................................................................... 119

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy EFFECT OF SURFACTANTS AND HERBIC IDE COMBINATIONS ON PHYTOTOXICITY OF DIQUAT By Toms F. Chiconela May 2008 Chair: W. T. Haller Major: Agronomy Diquat, a fast-acting contact herbicide, has b een used for weed control in the US for over 45 years. It was widely recommended for contro l of hydrilla, often in combination with copper. Restriction of copper use in public waters in Fl orida in the 1980s and development of fluridone resistant hydrilla in the 1990s has resulted in renewed interest in us ing diquat for hydrilla control. Diquat is also being evaluated for aquatic weed control in irrigation canals due to its short halflife in water. Greenhouse studies indicated that rice seed germin ated and maintained in diquattreated DI water was very sensitive to diquat, with EC10 values of 0.016 and 0.004 mg L-1 for shoots and roots, respectively. S eeds of corn, cotton, squash, wheat and soybean germinated in sand and irrigated once with diquattreated water at planting had EC10 values from 3 to 6 mg L-1. When treated at the seedling or more mature stages all crops exhibited EC10 values from 1 to 5 mg L-1, suggesting that diquat-tr eated water (0.370 mg L-1) may be used for irrigation with limited potential for phytotoxicity, except fo r freshly planted rice. Biomass and chlorophyll studies indicated that surfact ants (CT-301, Cygnet Plus, SilEnergy and Timberland 90) applied alone were phytotoxic to hydrilla at concentrations greater th an those recommended for aquatic weed control. Regression models yielded EC50 values from 26 to 592 mg L-1 for hydrilla dry

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12 weight and from 433 to 4,814 mg L-1 for chlorophyll reduction. Add ition of surfactants to diquat at aquatic and terrestrial labe led rates was additive on hydril la and landoltia based on Colby method. When applied on pe tunia, the aquatic and terrestrial surf actant rates plus diquat were additive and synergistic, respectively. The co mbination of copper, flumioxazin and endothall salts at different concentrati ons with diquat gave an additi ve effect based on dry weight. Acibenzolar added to diquat was synergistic base d on hydrilla dry weight, as was carfentrazone based on hydrilla total le ngth. These results indicate that some herbicides can be combined with diquat for improved hydrilla control, but the benef it of adding surfactants at aquatic labeled rates provides no more than additive effects.

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13 CHAPTER I INTRODUCTION Biology of Hydrilla Hydrilla (Hy drilla verticillata (L.f.) Royle) is a perennial submersed freshwater herb native to tropical regions. Its exact cen ter of origin is unknown, but warmer regions of Asia, central Africa, and Australia have been suggested (C ook and Lnd, 1982; Mahler 1979; Tarver et al., 1986). It is a monocotyledonous a nd monotypic genus in the family Hydrocharitaceae (Fasset, 1969), which comprises 16 genera and 100 species. Hydrilla is considered a serious nuisance plant in several regions of the world (Steward et al., 1984; Verkleij et al., 1983; Lazor, 1975), except in Antarctica, where its occurrence has not been reported (Pieterse, 1981). According to Soerjani (1986), it is the second most important aquatic weed worldwid e after water hyacinth, Eichhornia crassipes (Mart.). Two distinct forms of hydrilla, in terms of vegetative growth habit, genotype and asexual propagule production, have been described in th e United States: the dioecious form, which produces either male or female flowers on sepa rate plants (although only female flowers have been reported in the US), and the monoecious form, which produces both male and female flowers on the same plant (Madeira et al., 1997 ; Steward and Van, 1987; Verkleij et al., 1983). Ryan et al. (1995) used random amplified polym orphic DNA (RAPD) to separate the two forms accurately based on the primer operon G17, which is present in the dioecious form, but is absent in the monoecious form. In terms of vegeta tive growth, monoecious hydrilla is generally distinguished by its smaller and diminutive appearance compared to the larger and more robust dioecious form (Nakamura and Kadono, 2000). However, DNA analysis is necessary to distinguish positively between the two forms.

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14 Dioecious hydrilla was first introduced in to Missouri in the 1950s from Sri Lanka (Blackburn et al., 1969), and monoecious hydrilla was introduced in the 1970s in Delaware and Virginia (Potomac River) from Korea (Madeira et al., 1997; Steward et al., 1984; Haller, 1982). In both cases, the introduction oc curred through the aquarium and ornamental trade (Schmitz et al., 1991). Florida was the first state where the di oecious form was described as naturalized in 1960 (Blackburn et al., 1969). Since then, it has spread throughout the southern US (Les et al., 1997). The monoecious form reportedly occurs in several north eastern states and in the western states of Washington, Oregon, California, and Ariz ona (Steward et al., 1984). Failure to remove hydrilla fragments from boats, trailers and fishin g gear before leaving bod ies of water, coupled with common misidentificati on of hydrilla as elodea ( Elodea canadensis Michx.) egeria ( Egeria densa Planch.) and Nuttalls elodea ( Elodea nuttallii Planch.), are considered to be the anthropogenic causes of interstate and intrastate spread of hydr illa from one body of water to another (Langeland, 1996; Steward et al., 1984; Yeo et al., 1984). In turn, apical stem fragments and axillary turion flotation are considered to be the natural mechanisms for intermediate and long-distance dispersal of hydrilla within a nd between waterbodies (Sutton, 1996; Sutton and Portier, 1985; Haller et al., 1976). Hydrilla is usually rooted on the bottom of wate rbodies and can grow in depths of 0-15 m depending upon water clarity, which limits its growth (Langeland, 1990). Under normal conditions, it may grow as much as 3 cm d-1 (Langeland, 1996) and produce one new ramet m-2 d-1 (Madsen and Smith, 1999). Thus, at the wa ter surface hydrilla can quickly form a dense canopy, which is capable of preventing light penetration by mo re than 95%, allowing hydrilla to dominate over lower growing native submersed species (Haller and Sutton, 1975).

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15 In the southeastern US, dioecious hydrilla growth occurs during warmer months (Madsen and Smith, 1999; Van et al., 1978), with vegetati ve propagule (tubers and turions) production occurring during winter months (Netherland, 1997; Haller et al., 1976). Hydrilla regrowth occurs from tubers, stolons and root crowns in re sponse to increasing temperatures, but asexual perennating structures are producced in response to short-day stimulus with a critical daylength of 13 h (Van et al., 1978). In northern states where the monoecious form is prevalent, tuber production occurs throughout the year inde pendent of photoperiod (Van, 1989), whereas vegetative growth primarily occurs in the warm er months (McFarland and Barko, 1999; Sutton et al., 1992). Hydrilla has a lower light compensation point and reduced photorespiration compared to native submersed species (Van et al., 1976) This is because hydrilla exhibits C3 or C4 photosynthetic characteristics depend ing on the concentrations of O2 and CO2, as well as temperature and irradiance (Bowes et al., 2002; Magnin et al., 1997). In addition, Salvucci and Bowes (1983) reported that hydrilla is able to use carbon dioxide when it is available and can utilize bicarbonate for photosynt hesis under high pH and high aque ous carbonate concentrations. The switch to bicarbonate use grea tly increases the pH of water, which also negatively affects native species. Haller et al. ( 1974) found that hydrilla grew in water containing 6.66 salinity and Steward and Van (1987) reported hydrilla gr owth in water with salinity of 13. These characteristics, coupled with the ability of hydrilla to grow in a wide range of nutrient conditions (Barko et al., 1991; Barko, 1982; Cook and Lnd, 1982) and pH ranges (Steward, 1991), allow it to grow and spread very rapidly. In 1988, th e Florida Department of Natural Resources estimated that more than 20,000 ha of water in Florida were infested with hydrilla and by 1995 hydrilla covered 40,000 ha (Schardt and Nall, 1988). In a previous study, Johnson and Manning

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16 (1974) reported hydrilla spread from 5 ha to 10 ha in 6 weeks, illustrating how problematic this species can become and emphasizes the need for active control measures to keep it in check. Hydrilla Reproduction and Spread Hydrilla reproduces by a variety of m eans includi ng: fragmentation of stems, growth from root crowns, tubers (ter minal subterranean turions), turions (axillary dormant buds that fall from the parent plant when mature), and seed (Madsen and Smith, 1999; Langeland, 1996; Sutton et al., 1992). However, viable seeds are produced only by monoecious hydrilla (Langeland and Smith, 1984; Sainty and Jacobs, 1981; Mitra, 1955) and seedlings have rarely been found in the wild, suggesting that th is means of reproduction is not an important factor in hydrilla multiplication and spread. Reproduction in both dioecious a nd monoecious hydrilla in the US is primarily dependent on stolon formation, stem fragmentation, tuber and turion production. Madsen and Owens (1998) found that stolon formation from root crowns acc ounted for essentially a ll radial expansion of hydrilla colonies in a si x-month period and was also responsible for spring regrowth after winter diebacks. In a separate study, Sutton et al. (1992) collected up to 6,046 tubers m-2 produced by monoecious hydrilla during the summer and 3,524 tubers m-2 produced by the dioecious form during winter. In both cases, the hydrilla colony wa s started from a single tuber and allowed to grow for one year. Tubers and turions produced in a season do not always sprout during the following growing season. Van and Steward (1990) found that some tubers and turions remained dormant in the sediment for five and one year, respectively, after the above ground part of the plant had been destroyed. New plants were rege nerated from remaining stems and stolons when conditions became favorable for meristematic regr owth. For this reason, tubers and turions are regarded as perennating structures in natural environments following adverse conditions, such as

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17 a severe winter, drought, herbicid e application, or activity of bi ological control agents (Sutton and Portier, 1985; Sculthorpe, 1967). Monoecious hydrilla is reportedly more prolif ic in terms of tuber production than the dioecious form. Sutton et al. (1992) found that m onoecious hydrilla produced an average of 56% more tubers than the dioecious form; however, tu bers of the latter superseded the former by 32% in average weight. Similar findings were prev iously reported by McFarland and Barko (1990) and Van (1989). Conflicting results have been repo rted in terms of biomass production. Steward and Van (1987) did not find differences in biom ass between monoecious and dioecious hydrilla in a temperature study, but Sutton et al. (1992) reported higher biomass production by the dioecious form during winter as well as a di fference in biomass production between the two forms in summer. Several authors have noted that turions are us ually smaller, are formed in fragmented or floating stems, and are outnumbe red by tubers by ten-fold (Mill er et al., 1993; Thullen, 1990; Mitra, 1955; Lakashmanan, 1951). However, turion production by races of hydrilla from Japan and Poland was up to four times that of tube r production (Steward, 2000), and turion production in these races was photoperiod dependent. Tu rion production under a 10 h photoperiod was highest and a shift to tuber production was observed at a 16 h photoperiod. Turion production under long days was greatly reduced compar ed to tuber production (Steward, 2000). Hydrilla has high genetic divers ity. Several biotypes have been differentiated in the US and elsewhere based on ecological adaptation and sex expression (monoecy or dioecy) (Cook and Lnd, 1982; Pieterse, 1981), ploidy (Lange land, 1989), random polymorphic DNA (Hofstra et al., 2000; Madeira et al., 1997) and isoe nzyme polymorphism (Nakamura and Kadono, 2000; Ryan et al., 1995). Based on these studies, the geographical distribution of hydrilla and the

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18 genetic relationship between strains from diff erent regions and within a region have been established. Cook and Lnd (1982) reported that dioecious hydrilla was more adapted to temperate regions, while monoecious hydrilla was more adapted to warmer, tropical climates. Distribution of hydrilla in the US is currently cont rary to this finding; however, the distribution and continuous spread (Madeira et al, 2000; Le s et al., 1997), including the coexistence of both forms of hydrilla in Lake Gaston in North Caro lina (Ryan et al., 1995) and elsewhere, suggests that the difference may be due to intr oduction rather than to plant adaptation. Madeira et al. (1997) conducted a DNA analysis of 44 accessions of hydrilla from different parts of the world and found that the US dioecious form, which is more prevalent in the southern US, was more closely related to accessions from Bangalore, I ndia, while the US monoecious forms were more similar to those collected in Se oul, South Korea. In a similar study, Pieterse et al. (1985) studied isoenzyme patterns, mor phology, and chromosome number of hydrilla accessions from Uganda, Rwanda and Burundi, and found no differences among the accessions. This is due to interbasin spread of aquatic plants (Howard, 2004), since most African drainage basins are shared by two to 13 countries (UN/Wa ter Africa, 2006). Verkleij and Pieterse (1991) compared isoenzyme patterns of hydrilla from Africa and Asia and found some relatedness among plants. However, a similar study of hydrilla from Asia revealed variability among plants within the same waterb ody and between populations from diff erent water systems (Verkleij and Pieterse, 1986). Cook and Lnd (1982) attributed the variability to possible origin and subsequent differentiation of hydr illa on that con tinent (Asia). Importance of Hydrilla Hydrilla infestations in a quatic system s usually produce environmental and economic problems. Detrimental impacts on water use as a re sult of hydrilla infestations include reduced flow in drainage and irrigation canals, changes in physical characteristics and nutrient cycles of

PAGE 19

19 lakes, oxygen depletion, fish kills, the formation of extensive mats capable of clogging intakes of pumps used for conveying water, and displacemen t of native plants and animals (Van et al., 1999; Bates and Smith, 1994; Pesacreta, 1988; Tarv er et al., 1986; van Dijk, 1985; Haller, 1978; Hogan, 1969). In addition, hydrilla in terferes with recreational use of lakes and rivers, affects public health by increasing the risk of drowning and harboring snails and mosquitoes (Pimentel et al., 2000; Hearnden and Kay, 1997; Dibble et al., 1996), and redu ces biodiversity in natural systems (Pieterse and Murphy, 1990; Haller, 1978). As a result, the economic impacts of hydrilla infestations on tourism, sport, and other wa ter uses are overwhelming. An economic study of Orange Lake in north central Florida indicated that the economic activity attributed to the 5000 ha lake was almost $11 million annually and that during severe hydrilla infestations these benefits were virtually lost (M ilon et al., 1986). A similar study in the Kissimmee chain of lakes and Lake Istokpoga in south Florida revealed lo st recreational revenues estimated at $10 million annually due to excessive hydrilla growth (S chardt, 1998). Approximately $158 million in state and federal funds were spent in Florida be tween 1980 and 2004 to manage hydrilla in public waters (FLDEP, 2004), and Center et al. (1997) stated that during this period management costs in certain years amounted to as much as $14.5 million/year. Hydrilla Management Hydrilla has been m anaged by chemical, mech anical, physical, and biological methods (Doyle et al., 2002; Doyle and Sm art, 2001; Shearer, 1998; Madsen, 1997; Cassani, 1996; Blackburn and Weldon, 1970). However, effectiveness is variable and dependent on the level of infestation, water uses, area and type of wa terbody (SE-SPPC, 2003; Haller, 2002; Netherland, 1999; Shireman et al., 1986). Several biological control agents have been evaluated for hydrilla management (Balciunas and Mino, 1984; Etheridge et al., 1983; Charuda ttan, 1973; Blackburn and Taylor, 1968). Snails

PAGE 20

20 were the first agents evaluated in the 1960s for biological control (Blackburn and Taylor, 1968). From the early 1970s to date, extensive surveys and evaluation of insect s (Balciunas et al., 2002, Balciunas et al., 1996; Buckingham, 1994) and pat hogens (Shabana et al., 2003; Shearer, 1998; Joye and Cofrancesco, 1991; Joye, 1990; Ch arudattan and McKinney, 1978; Charudattan and Linn, 1974; Charudattan, 1973) have been undert aken, and some are still being conducted worldwide. Based on these evaluations, four insects (hydri lla tuber weevil, Bagous affinis Hustache; hydrilla stem weevil, Bagous hydrillae OBrien; and two hydri lla leaf-mining flies, Hydrellia balciunasi Bock and Hydrellia pakistanae Deonier) have been released in the US as potential biological control agents (Julien and Gr iffiths, 1998; Center et al., 1991; Buckingham, 1990). Other herbivorous insects have also been fo und in natural areas in Florida. Del Fosse et al. (1976) reported an apparent acci dental introduction of a moth, Parapoynx diminutalis Snellen, which quickly spread to several hydrilla-infested areas in Florida, where it occasionally has caused severe damage to hydrilla. In addition, a midge, Cricotopus lebetis Sublette, was also found and evaluated as a potential biological control agent for hydri lla management (Cuda et al., 2002). However, none of these organisms is predicta ble or effective in th e natural environment, although under controlled conditions they have performed well (Langeland, 1996). According to Perkins (1978), fish predation, among other factor s, is likely the main reason for limited insect activity. As for pathogens, poor inoculum landing and attachment to the host are believed to result in the lack of eff ectiveness (Agrios, 2005). Unlike other biological control agents, sterile grass carp ( Ctenopharyngodon idella Val.) are effective in controlling hydrilla, even when not integrated with othe r methods. Shireman and Maciena (1981) reported eradication of hydrilla from Lake Baldwin (Florida) two years after stocking. Osborne and Sassic (1979) observed a 63% reduction in hydr illa biomass after stocking

PAGE 21

21 Lake Barton. In Santee Cooper Reservoir, Kirk et al. (2000) observed hy drilla reduction from 17,272 ha to a few ha within four years afte r stocking nearly 768,500 triploid grass carp. Management of aquatic vegetation through the use of grass carp has been investigated by other researchers as well (Van Dyke et al. 1984; Colle et al., 1978 ; Gasaway and Drda, 1978; Sutton, 1974). A fault of grass carp is the lack of host sp ecificity and extensive m ovement. This restricts their use in public lakes in several states wher e movement and total vegetation removal is not desirable (Sutton and Vandiver, 198 6). Another constraint related to grass carp use is removal from the water system when control is no longer n eeded (Bonar et al. 1993) However, if the lake or pond is privately owned, fairly small, without inlet or outlet, and total vegetation removal is acceptable, triploid grass carp use is legal by permit in many states (H amel, 2004; Cassani and Caton, 1986). Cultural and physical practices for hydrilla management, which include lowering the water level of a reservoir or a partic ular lake (drawdown) (Doyle and Smart, 2001; Halle r et al., 1976), benthic barriers or other methods of covering se diments to prevent light from reaching plants (Carter et al., 1994; Engel, 1990, 1984; Cooke, 1980), and application of natural or synthetic dyes (Anonymous, 1992), have been tested. Howeve r, these methods are effective for a very limited time period (Eichler et al., 1995; Ludlow 1995; Haller et al., 1976). In addition, methods such as benthic barriers or ot her bottom-coverings are costly for widespread use and affect the benthic community. Therefore, these methods are recommended only for small areas or used around docks, boat launch areas, and swimming ar eas (Madsen, 2000). Engel (1984) found that benthic barriers killed plants in one to two mont hs, but if barriers were removed the site was usually re-colonized quickly (Eichler et al., 1995). Also, where sediments are used, new plants

PAGE 22

22 can establish on top of the added layer (Engel an d Nichols, 1984), reduci ng the effectiveness of these methods. Doyle and Smart (2001) tested short and l ong-term drawdown and found that they had no effect on tuber viability and surv ival, despite more than 90% re duction in the number of tubers. Long-term viability and survival of tubers have been associated with dormancy and retention in undisturbed sediments (Sutton, 1996; Langeland, 1993; Van and Steward, 1990). Therefore, drawdown for hydrilla management is only effective for one to two years (Ludlow, 1995). Mechanical harvesting is another method wi dely recommended for hydrilla management, particularly near potable water in takes, flowing water sites where he rbicides are less effective, or for hydrilla control in certain sites where immediate clearance is required (H aller, 2002). However, the need for two to three cuttings per year due to rapid plant growth (Madsen et al., 1988; Nichols and Cottam, 1972), coupled with a cost of up to $1000 or more per hectare (SESPPC, 2003) and environmental concerns due to non-selective removal of turtles, fish and amphibians (Booms, 1999; Engel 1990; Haller et al ., 1980), limits the larg e scale use of this method. Dredging (the removal of sediments from la kes, reservoirs or other aquatic systems) is another mechanical method used for aquatic we ed management (Peterson, 1982). However, due to the high cost, environmental impacts, and the problem of sediment disposal, dredging is most often used for lake restoration rather than for aquatic plant management (Peterson, 1982). Chemical control is the mo st widely used method to control hydrilla due to its effectiveness and lower cost compared to mo st other methods (Langeland, 1996). It relies primarily on four compounds: fluridone, endot hall, diquat, and copper compounds (Gallagher and Haller, 1990). However, the dear th of registered compounds fo r control of hydrilla and other aquatic weeds has resulted in development of herbicide resistance. A biotype of hydrilla that

PAGE 23

23 developed resistance to fluridone was recently re ported in Florida (Arias et al., 2005; Pons, 2005; Michel et al., 2004; MacDonald et al., 2001), and a duckweed ( Landoltia punctata (G. Meyer) D.H. Les and D.J. Crawford) that developed resi stance to diquat was al so discovered in Lake County, Florida (Koschnick et al., 2006). These findings, in combination with restrictions on copper use in public waters and the establis hment of National Copper Standards in water (USEPA, 1985), have placed hydrilla control in jeopardy and primarily dependent upon the contact herbicide endothall. The integration of different methods has also been investigated and shown to be effective under laboratory conditions. Shear er and Nelson (2002) observed that combining an endemic pathogen, Mycoleptodiscus terretris (Gerd.) Ostazeski, with fluridone had a synergist effect compared to the application of either agent alone Similar findings have been previously reported (Nelson et al., 1998; Netherland and Shearer, 1996; Joye and Co francesco, 1991). Likewise, the integration of grass carp with lower herbicide us e rates was also found to be effective (Eggeman, 1994; Small et al., 1985; Tooby et al., 1980). In ad dition, Hestand and Carter (1978) noted that the carp/herbicide combination in small me socosms reduced phytoplankton blooms triggered by rapid plant control and nutrient release when he rbicides are used alone. Similarly, Shearer and Nelson (2002) reported that the combination of Mycoleptodiscus terretris with fluridone reduced herbicide rates for hydrilla management and, consequently, reduced damage to non-target species. Diquat Use in Aquatic Weed Control Diquat dibrom ide (6,7-dihydrodipyr ido [1,2-a:2,1-c] pyrazinediium dibromide salt) is a contact herbicide widely used since the 1960s fo r the control of floating and submersed aquatic weeds and as a desiccant in several crops, incl uding non-selective applicat ions in non-crop areas (Syngenta, 2004). It is a member of the bipyridylium family, along with paraquat and

PAGE 24

24 morfamquat (WSSA, 2002; Summers, 1980), and was fi rst registered for use in the US in 1961 (USEPA, 1986). Diquat is absorb ed rapidly by plants (usually within 5 minutes of its application) and acts quickly on gr een tissue (Brian et al., 1958), but root uptake is negligible due to inactivation by soil or sediment par ticles (Yeo, 1967; Funderburk and Lawrence, 1963). Phytotoxicity symptoms on sus ceptible plants are characterized by the occurrence of water soaked areas on treated leaves and subsequent desiccation and necrosis (Hess, 2000). Plant death usually occurs in 1 to 3 days, depending upon environmental conditi ons (WSSA, 2002). The bipyridylium herbicides as a group are re garded as membrane disruptors and electron diverters in photosystem I by competing with fe rredoxin as an electron acceptor (White, 1970). This competition results in inhibition of NADP reduction into NADPH in the plant chloroplasts (Zweig et al., 1965). Another char acteristic of these compounds is the need to be converted to their reduced state in order to exert their herb icidal action. The c onversion process is accomplished through a single electr on transfer, which depends on th e presence of light (Mees, 1960), temperature (Dodge et al., 1970), and oxygen (Homer et al., 1960). On ce in their reduced state, these compounds are re-oxidized in the pr esence of oxygen to their original cationic forms and a superoxide anion (O2) is produced (Monk, 1998). Continuous oxygen availability in the chloroplasts induces the cationic forms of the he rbicide to enter in a cyclic reduction-oxidation process (Kok et al., 1965), with subsequent forma tion of superoxide ani ons and accumulation of hydrogen peroxide (H2O2), hydroxyl radicals (OH), and molecular oxyge n, all of which are associated with polymerization a nd peroxidation of unsaturated lipid s. These reactions result in the malondialdehyde formation (Dodge et al., 1970) chlorophyll destruction (Dodge and Harris, 1970), tonoplast rupture (Calde rbank, 1966), and ultimately, membrane destruction (Farrington et al., 1978).

PAGE 25

25 Diquat is characterized by ha ving a half-life in water of usually less than 48 hours (FAO/WHO, 1995; Langeland and Warner, 1986; Coats et al., 1964). Half-life is influenced by hydrosoil/sediment type (Frank and Comes, 1967; Weber et al ., 1969), turbidity (Poovey and Getsinger, 2002; Hofstra et al., 2001), pH (D az et al., 2002), photolysis (Howard, 1989; Tucker, 1980), type of aquatic plants present (Langela nd et al., 1994; Summers, 1980), and levels of microorganisms (Simsiman and Chester, 1976). Losse s due to volatility are considered negligible (Coats et al., 1966). According to Yeo (1967), diquat disappears quickly from water due to adsorption by sediments, suspended particles, and aquatic plant uptake. Based on these residue characteristics, diquat must be rapidly absorb ed by plants if it is to be effective. Copper Use in Aquatic Weed Control Copper has been used in agricu lture operations for m ore than two centuries as a fungicide and bactericide to control several plant diseas es (Agrios, 2005). Copper use for aquatic weed control dates back to at least th e early 1900s, when it was first discovered to have algaecidal and bactericidal properties (Moore and Kellerman, 1905). Since then, copper has been extensively used for algae control in potab le water systems. Besides thes e properties, copper is also a required micronutrient for the activ ity of several essential enzyme s, is involved in oxidationreduction reactions, and is a cofactor in redox reactions in humans (Britton, 1996; Linder, 1991; Cordano et al., 1964), plants (Marschner, 1995; Fernandes and Henrigues, 1991), and animals (Underwood and Suttle, 1999; Hart et al., 1928). Copper was first used for aquatic plant manage ment in the 1950s (Chancellor et al., 1958; Hunkins, 1955). Its use further increased when se veral studies showed that when diquat and copper were applied together, the diquat upt ake by certain submerse d plants increased (Kammerer and Ledson, 2001; Pennington et al., 2001; Sutton et al., 1972; Sutton and Bingham, 1970; Mackenzie and Hall, 1967), but both compounds need to be above certain concentrations

PAGE 26

26 for increased uptake to occur (S utton et al., 1972). Copper-induced membrane loss of integrity and increased permeability to diquat is considered to be the cause of the enhanced effects of the combination (Demidchik et al., 2001; Gupta et al ., 1996; Sutton et al., 19 72; Gross et al., 1970). De Vos et al. (1992) attributed th e loss of membrane integrity to induced lipid peroxidation when copper binds to sulfidryl groups of membrane proteins. This results in alteration of antioxidant enzyme activity (Chen and Kao, 1999) and increased levels of free radical formation (Chen et al., 2000) in chloroplasts (Sandmann and Bger, 1980) roots (De Vos et al., 1993) and leaves (Chen and Kao, 1998; Weckx and Clijsters, 1996). Consequently, there is increa sed formation of non-specific ion pores in cell membranes, which increases ion influx and efflux (Demidchik et al. 2001). Several authors have reported that copper interferes with num erous other plant processes. Marschner (1995) noted that, desp ite coppers importance as an e ssential micronutrient, exposure to high levels of this metal had detrimental effects on plant growth, particularly on root development and morphology. Patsikka et al. (2002) found that copper reduced chlorophyll content of leaves, interfered w ith thylakoid alignment, and co mpeted with iron uptake. Copper has also been shown to be a potent photosynthe tic inhibitor in cyanoba cteria (Singh and Singh, 1987), green algae (Shioi et al., 1978a), and in thylakoids of higher plants (Samuelsson and Oquist, 1980; Shioi et al., 1978b). Although the exact site of action of copper has not been elucidated, Mohanty et al. (1989) suggested that the secondary quinone electron acceptor, QB, in photosystem II was the probable site of copper interf erence due, to inhibition of the Hill reaction. Coppers high affinity for organic matter and colloidal materials su spended within the water column has been reported in several studi es (Haughey et al., 2000; Sigg et al., 2000; Xue and Sigg, 1999; Hanson and Stefan, 1984). When non-chel ated copper is used with hard water, it

PAGE 27

27 tends to hydrolyze and form precipitates that are subsequently adsorbed by sediments (McKnight et al., 1983). Button et al. (1977) found that copper concentrations in the water column usually returned to pretreatment levels within two hours. As a result, copper conc entrations in sediments are usually high compared to the concentration in the water column (Van Hullebusch et al., 2003a). Leslie (1992) compiled data of copper content of sediments from several waterbodies in Florida and found that the highest average cumulative copper concentrations in the sediments was 464 mg kg-1 in potable water reservoirs, where coppe r was widely used for blue-green algae control. In other waterbodies where coppe r was being used for general macrophyte and filamentous algae management, the highest average cumulative copper concentration in the hydrosoil was 71 mg kg-1. In a similar study, Van Hullebusch et al. (2003b) reported copper concentrations of 27 to 58 g g-1 in lake sediments in France; however, copper concentrations in the water column were less than 3 g g-1. Several authors have reported th at the soluble fraction of copp er adsorbed in sediments can be available for uptake by aquatic and terrestrial organisms, including humans (Roberts et al., 2006; Olivares et al., 2002; Han et al., 2001; Mastin and Rodgers, 2000; Turnlund et al., 1989; Brown and Rattigan, 1979). Although multiple coppe r exposure pathways have been identified, direct exposures to dissolved copper where certain organisms dw ell (Morrisey et al., 1996) and consupmtionof copper-contaminated drinking water or dietary copper (Cl eawater et al., 2002; Morris et al., 2003; Tanner et al., 1979) are considered the most important. Hamdy (2000) found that macroalgae were good bioaccumulators of heavy metals, such as copper, lead, and zinc, and constituted a major exposure route to several organisms, including annelids, crustaceans, and mollusks, as well as fish which feed on them (Farag et al., 1999;

PAGE 28

28 Taylor, 1998). Copper bioaccumulation in bacter ia, insects, epiphytes, macrophytes (Lindqvist, 1992; Patrick and Loutit, 1977), and the herbivores that consume c ontaminated plants has also been reported (Danks, 1995; Hunter et al., 1987; O'Shea et al., 1984; Venuto and Trefry, 1983). Human disorders such as Indian Childhood dis ease (ICC) and Endemic Tyrolean Infantile Cirrhosis (ETIC) have been associated with copp er accumulation in the liver and other tissues in India and Austria, respectively, either from feeding infants with milk containing excess copper or from drinking water overly rich in this metal (Muller et al., 1996; Scheinberg and Sternlieb, 1996; Bhave et al., 1982). Despite the problems associated with copper us e elsewhere, it was the late 1970s before environmental problems associated with coppe r use were noticed in the US. Copper use was linked to the reduction of the Florida apple snail ( Pomacea paludosa ) (Kushlan, 1975), the primary food of the endangered Everglades kite ( Rostrhamus sociabilis ) (Emerson and Jacobson, 1976). There were other concerns with copper us e and the endangered manatee. As a result, copper use in public waters in Florida was restri cted in 1985 (Leslie, 1992), due to its persistence in the environment and toxicity to non-target organisms. Als o, fluridone was registered and became widely used for hydrilla control, furt her reducing the need for the diquat-copper combination. In 1991 and 1993, the Environmental Protection Agency and the World Health Organization, respectively, review ed their copper safety guidelin es for drinking-water and both institutions established enforceable copper conc entration action levels in drinking water of 1 mg L-1 and 2 mg L-1, respectively (WHO, 1993; USEPA, 1991). These events forced researchers to seek alternatives to replace c opper for use as algaecides and herbicides. Some algae control alternatives to copper have been studied, including barley straw (Brownlee et al., 2003; Barrett et al., 1999; Caffrey and Mo nahan, 1999), ultrasound (Zhang et

PAGE 29

29 al., 2006; Lee et al., 2002; Gior dano et al., 1976), sodium car bonate peroxyhydrate (Quimby et al., 1988), nutrient management (Paerl et al., 2004; USEPA, 2003; NRC, 2000), aeration (Sukias et al., 2003), mechanical mixing or mixing waters with waters of lower algae concentrations (Lewis et al., 2003; Toetz, 1977), and physical removal (Abbaszadegan et al., 2006); however, copper remains a very important algaecide, and the search for a diquat enhancer for aquatic macrophyte control is still being sought. Effects of Surfactants on Herbicides Surfactants or surface active agents, known collectively as ad j uvants, are chemicals that have pronounced effects on the interface forces of pesticide spray mixtures with the goal of providing more effective droplet deposition, sp read, and retention on treated plant surfaces (Monaco et al., 2002; Hartzler a nd Foy, 1983). The ability of surfact ants to modify interfacial forces and increase herbicide efficacy depends on the hydrophilic-li pophilic balance (HLB) (Norris, 1982), spray volume droplet size (Matysiak, 1995), concentration of surfactants, and structures present on the leaf surface which coul d alter the herbicidesurfactantplant surface interaction (Liu, 2004). When an appropriate selection is made, surf actants not only enhance herbicide selectivity (Hess and Foy, 2000), but may also reduce the amount of herbicide requir ed, reduce the overall cost of weed management, and improve the c onsistency of weed control (Zollinger, 2000). Sutton and Foy (1971) found that th e addition of several surfactant s at concentrations from 0.001 to 1.0% to diquat increased be tanin efflux from red beet ( Beta vulgaris L.) root discs. Menendez and Batisda (2004) reported 15 to 30% reduction of ED50 rates of diquat when combined with several surfactants at concentrations ranging from 0.05 to 1.0% compared to diquat alone on purslane (Portulaca oleracea L.) and ryegrass ( Lolium rigidum Gaudin).

PAGE 30

30 Similar findings have been reported on other plant species with other herbicides and surfactants. Smith and Foy (1967) noted that the addition of 8 surfactants at 1,000 and 10,000 mg L-1 to paraquat, a compound chemically related to diquat, increas ed its toxicity on corn; however, a specific concentration of each t ype of surfactant was required. Nalewaja et al. (1995) observed that nicosulfuron was able to reduce yellow foxtail [ Setaria glauca (L.) Beauv.] fresh weight from 10 to 92% depending on the ty pe of surfactant used in the spray solution Nalewaja et al. (1995) observed that nicosulfuron was able to reduce the fresh weight of yellow foxtail [ Setaria glauca (L.) Beauv.] from 10 to 92%, depending on the type of surfactant used in the spray solution Tonks and Eberlein (2001) used rimsulfuron in combination with crop oil concentrate, a nonionic surfactant, methylated seed oil and a s ilicone-polyether copolymer on common lambsquarters ( Chenopodium album L.), hairy nightshade ( Solanum sarrachoides Sendtner), kochia ( Kochia scoparia L.), redroot pigweed ( Amaranthus retroflexus L.), and volunteer oat ( Avena sativa L.) in potato. Based on this study, th ey concluded that effective weed control depended on herbicide appli cation rate; type of adjuvant selected, and target species. Research on surfactant-herbicide-water-pla nt relationships indicate that increased performance of herbicides when combined with surfactants on terrestrial and emergent aquatic weeds is due to more rapid fo liar uptake. Koger et al. (2007) found that the combination of organosilicone-based nonionic and methylated seed oil/organosilicone surfactants with urea ammonium nitrate reduced the rain-free period from 8 h to 1 or 4 h, respectively, and provided complete control of barnyardgrass [ Echinochloa crus-galli (L.) Beauv.] when applied with bispyribac. Reddy and Singh ( 1992) reported increased glyph osate efficacy and reduced critical rain-free period from 6 or 8 h to 15 min. when glyphosate was applied with Kinetic, an organosilicone-based adjuvant, on velvetleaf (Abutilon theophrasti Medik.), sicklepod (Cassia

PAGE 31

31 obtusifolia L.), and yellow foxtail [Setaria glauca (L.) Beauv.]. Molin and Hirase (2005) obtained 50% and 80% c ontrol of johnsongrass ( Sorghum halepense L.), with glyphosate in combination with surfactant even when simula ted rain was applied five and 15 min. after application. The reduction of the critical rain-free period with addition of adjuvants has been widely reported (Roggenbuck et al ., 1993, 1990; Kudsk, 1992; Fi eld and Bishop, 1988, 1987). These studies suggest that the faster uptake of some herbicides when combined with certain surfactants is due to cuticular and stomatal infiltration from the target surface. The addition of adjuvants has been shown to increase herbicide absorption (Hess and Foy, 2000), phytotoxicity (Ramsdale and Messersm ith, 2002) and control efficacy when the combination of herbicides plus adjuvants was foliarly applied in terrestrial (Devendra et al., 2004; Ivany, 2004; Smithet al., 1999; Coret and Ch amel, 1993), and emergent and floating plants (Syngenta, 2004; Fairchild et al., 2002; Shilling et al., 1990). However, no reports are available regarding improved efficacy when surfactants ar e added to herbicides for submersed aquatic weed control. Submersed aquatic plants (inclu ding hydrilla) lack or have a thin cuticle, and stomata are absent or non-functional (Verma and Verma, 2000; Pendland, 1979; Sculthorpe, 1967). The effect of adjuvants on herbicide efficacy in s ubmersed plants is unknown and the environments where these plants are found will influence not on ly herbicide performance but also that of surfactants due to extensive dilu tion (Cedergreen and Streibig, 2005). Aquatic applicators started to a dd surfactants to cont act herbicides applied directly to water in the 1980s, despite the fact that there was no scientific evidence suggesting surfactants increased herbicide efficacy in aquatic appl ications. The recommended surfactant rate (1 to 2 quarts/100 gallons of water) for mixing with foliar applied herbicides is 0.25 to 0.5% v/v.

PAGE 32

32 The concentration of surfactants in the herb icide mixture is therefore 2,500 to 5,000 mg L-1 in the tank mix. The recommended surfactant rate suggested by some surfactant labels for use with contact herbicides for submersed w eed control is 1 to 2 gallons acre-1 regardless of water depth. Assuming a water depth of 1 foot (1 acre-foot = 326,900 gallons), this ap plication rate would result in a surfactant co ncentration of 0.0003 to 0.0006% v/v or 3 to 6 mg L-1 with uniform dilution. Depending upon the ratios of the amount of surfactant use d, the aquatic concentrations of the surfactants per acre-foot is 833x times less than the concentrations used in foliar applications and also indicate that diluted surf actant concentrations (< 0.1%) are likely not to improve herbicidal activity. Green (1996) found that when surfactant was ap plied at 0.0008% in th e spray volume with rimsulfuron, less than 10% of green foxtail [ Setaria viridis (L.) Beauv.] wa s controlled, but control increased to 90% when the surfactant concentration was increased to 0.1%. Liu (2004) noted that glyphosate uptake by wheat ( Triticum aestivum L.) and bean (Vicia faba L.) foliage increased from 5% and 44% to 17% and 85%, resp ectively, when surfactant concentration was increased from 0.2 to 0.5% v/v in the spray mixtur e. Herbicide phytotoxicity also increased when the carrier volume was 23 to 47 L ha-1 compared to a more dilute 94 to 188 L ha-1, when several herbicides were applied on an area basis in a ta nk-mix with methylated vegetable oil (Ramsdale and Nalewaja, 2001; Nalewaja and Ahrens, 1998). Wh en surfactant concentrations of the spray mix increased, there was a consistent increase in herbicide efficacy. These findings show that surfactant concentration and carri er volume are critical for enha nced herbicide performance in foliar applications. Surfactants are widely used in agriculture. Th eir ability to reduce surface tension in water and other liquids allows them to be used for several purposes, such as such as penetration,

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33 wetting, emulsification, spreading, sticking and humectancy (Monaco et al., 2002; Matsumoto et al., 1992; Hartzler and Foy, 1983). There are more than 3,000 compounds registered in the US as emulsifiers and adjuvants, which include surf actants for agricultural purposes (USEPA, 2004). Underwood (2000) reviewed the worldwide pesticid e market and found that surfactants represent more than $400 million of the $8 billion sales of pesticides in the US. According to Allison (2003), 80%-90% of emulsifiers and adjuvants are used with herbicides, which represent more than 50% of total pesticide sales. Despite the exte nsive use of adjuvants, pa rticularly surfactants, few toxicity tests are conducte d on these products compared to the pesticide active ingredients they are usually mixed with (Cox and Surg an, 2006). Based on increasing environmental awareness and willingness to protect natural resour ces, surfactants are viewed by some as a tool to reduce the amount of herbicide required to co ntrol weeds. The common recommendation is to add surfactants to foliar applied herbicides for improved control of emergent and floating weeds. Now that these combinations are used on submersed weeds in aquatic systems as well, the amount of these products in aquatic areas is expected to rise. Some literature is available on the toxicity of surfactants to a quatic and terrestrial organisms (Hodges et al., 2006; Haller and Stocker, 2003; Ma et al ., 2003; Mann and Bidwell, 2001; Moreno-Garrido et al., 2001; Yoshimura, 1986; Wolfenbarger and Holscher, 19 67) and on humans (Richa rd et al., 2005), but little is known about the benef its of the use of these compounds for submersed aquatic weed control. Therefore, the effect of surfactants on herbicides used for submersed weed control needs to be evaluated. Herbicide Combinations Herbicid e combinations for weed control in aquatic and terrestrial settings have been utilized for decades. Increased efficacy of herbic ide combinations over the application of a single compound is a primary reason to mix different materials (Baldwin and Oliver 1985). Other

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34 benefits can also be accrued from herbicide mixtures. A greater spectrum of weed species can be targeted with a single application when herbicid es with different modes of action are combined (Norris et al., 2001). Combination of different herbicides can re duce the propensity of weeds to develop resistance (Marshall, 1998) and can also re duce crop damage (Webster et al., 2006; Brown et al., 2004; Omokawa et al., 1996; Hoffma n, 1953), application cost (Norris et al., 2001), and phytotoxicity of herbicides to non-targ et organisms (Follak and Hurle, 2003) and consequently, reduce environmental concerns (Brimner et al., 2005). One of the most common examples of increased herbicide efficacy in aquatic systems is the use of diquat-copper for hydrilla control when they are applie d in combination compared to the activity of each compound appl ied alone (Sutton et al., 1972; Sutton et al., 1971; Blackburn and Weldon, 1970; Sutton and Bingham, 1970; Mack enzie and Hall, 1967). Further studies involving the combination of diqua t with endothall in aquatic (P oovey et al., 2002; Nelson et al., 2001; Pennington et al., 2001) and te rrestrial (Ivany, 2004) settings also revealed that both compounds were more effective when applied to gether than singly. Improved performance of other herbicides when applied in combination with diquat for the control of terrestrial weeds has also been reported. Most recently, Ivany (2005) found that pyraflufen-ethyl efficacy on potato haulm desiccation was improved when applied after or in combina tion with diquat in a single application. Although increased herb icide efficacy has been observ ed in most studies involving herbicides combined with diquat, antagonistic eff ects have also been reported. Srensen et al. (2007) tested synergistic and antagonistic effects using a concentration addition model in binary mixture toxicity studies and re ported that acifluorfen was an tagonistic to diquat activity on duckweed ( Lemna minor L.). Cedergreen et al. (2007) examined the toxicity of six binary

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35 herbicide combinations on pigment content and pl ant population growth. They subjected the data to the concentration addition (CA) model and in dependent action (IA) reference models both of which showed that acifluorfen combined with diquat was antagonistic. In a previous study, Cedergreen et al. (2006) examined the synergistic effects of prochloraz, an imidazole fungicide, when applied in combination with diquat, azoxystrobin, acifluorfen, di methoate, chlorfenvinphos and pirimicarb on four aquatic species: bacteria ( Vibrio fischeri ), daphnia ( Daphnia magna Straus), algae [Pseudokirchneriella subcapitata (Korshikov) Hindak] and duckweed. They found that, although the mixtures between prochloraz-azoxystrobin a nd diquat-esfenvalerat had shown a synergistic effect on daphnia, and diquat-pr ochloraz on algae, all combinations were antagonistic on duckweed. These findings emphasi ze the need to test not only individual herbicides, but also mixtures of herbicides a nd herbicide/adjuvant combinations to identify possible adverse effects on non-target species and possible reductions in herbicide performance. Herbicide Use in Irrigation Canals Subm ersed aquatic weed control in irrigation ca nals, including waters used to irrigate rice in the US and elsewhere, is commonly accomplished with acrolein (acrylaldehyde, 2-propenal) (Bowmer and Smith, 1984; USEPA, 1980; Hill, 1960). This broad-spectrum herbicide is generally applied every 2-3 w eeks as a drip treatment in flowing canals at 75-150 L m-3 of water flow, depending upon the density and weed spec ies present (Hansen et al., 1983). Because acrolein is volatile, flammable, and toxic to fish, mammals, and othe r non-target organisms (WSSA, 2002; Anonymous, 2001; Ei sler, 1994; Albin, 1962) alternat ive herbicides are being sought to reduce or replace its use in irriga tion canals. Although diquat is a non-selective herbicide, it was chosen for evaluation for possi ble use in irrigation canals due to its lack of persistence in water (Langeland and Warner, 1986; Coats et al., 1964) and rapid uptake by submersed plants.

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36 Despite diquats short half-life in water (L angeland and Warner, 1986), its extensive use for aquatic weed control makes it imperative to understand more comp letely any potential phytotoxicity to non-target plants in order to minimize crop damage and environmental problems that can result from application of this herbicid e, particularly if used in irrigation canals. Moreover, herbicide applications might be made at certain growth stages to reduce possible adverse effects on crop development. If herbicide applications ar e made at the wrong stage of crop growth, reductions in seed germination, crop yield and quality may be observed (Baig et al., 2003; Bennett and Shaw, 2000; Ratnayake and Shaw, 1992). For example, Bond and Bollich (2007) reported that yield reductions in rice were influenced by cultivar, as well as time and rate of application of sodium chlorate and paraquat, when both he rbicides were used as desiccants three to seven days prior to harvest. Peterson et al. (1997) found that growth of duckweed, a species largely used for environmental risk assessment, and the cyanobacterium Anabaena inaequalis (Kutzing) Bornet and F is inhibited by 50% when exposed to diquat concentrations as low as 0.004 and 0.005 mg L-1, respectively. In another study, Fa irchild et al. (1997) found an EC50 (effective concentration 50 the concentration of diquat required to reduce plant dry weight by 50%) of 0.018 and 0.08 mg L-1 for duckweed and the algae Selenastrum capricornutum Printz, respectively. While duckweed and algae are very sensitive to diquat, these concentrations are much lower than the maximum aquatic labeled ra te, implying that crop plants irrigated with diquat-treated water may also be adversely affected. Objectives Diquat has been widely used for aquatic weed control for m ore than 45 years, and copper has usually been mixed with diquat and applied simultaneously for control of submersed weeds, particularly hydrilla. As copper use has become restricted by regulatory agencies, surfactant

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37 manufacturers have recommended th at aquatic applicators add surf actants or other adjuvants to diquat to improve submersed weed control. Some aquatic applic ators have reported improved submersed weed control when surfactants are added. However, there is no scientific evidence to support this hypothesis. In additi on, diquat is being considered for aquatic weed control in irrigation canals to reduce or re place acrolein, that is currently widely used. Thus the overall objectives of this research were to i) eval uate the phytotoxicity of diquat to different developmental stages of corn, cotton, rice, soyb ean, squash and wheat, ii) compare the effects of surfactants on diquat phytotoxicity on aquatic and terres trial plants, and iii) assess the effect of several herbicide and surfactant combinat ions on diquat toxi city to hydrilla.

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38 CHAPTER 2 EFFECT OF DIQUAT IN IRRIGATION WAT ER ON GERMI NATION AND VEGETATIVE GROWTH OF RICE Introduction Subm ersed aquatic weed control in irrigation ca nals, including waters us ed to irrigate rice in the US and elsewhere, is commonly accomplished with acrolein (acrylaldehyde or 2-propenal) (Bowmer and Smith, 1984; USEPA, 1980; Hill, 1960). This broad-spectrum herbicide is generally applied every 2-3 w eeks as a drip treatment in flowing canals at 75-150 L/m3 of water flow, depending upon weed density and weed species present (Hansen et al., 1983). Acrolein is volatile, flammable and toxic to fish, mammals and other non -target organisms (WSSA, 2002; Anonymous, 2001; Eisler, 1994; Albin, 1962). Prim arily because of a pplicator toxicity alternative herbicides are being sought to reduce or replace acrolein use in irrigation canals. Diquat dibromide (6,7-dihydrodipyr ido [1,2-a:2,1-c] pyrazinediium dibromide salt) is a contact herbicide used for the control of floati ng and submersed aquatic weeds in ponds, lakes, canals, and drainage ditches, as a desiccant in several crops, and for nonselective weed control in non-crop areas (Syngenta, 2004). Di quat is absorbed rapidly when applied to plants and acts quickly by interfering with electr on transport in photosystem I, ultimately leading to plasma membrane disruption (Hess, 2000). Susceptible plant tissue can exhibit symptoms within hours of treatment, and plant death usually occurs in 1 to 3 days, depending upon environmental conditions (WSSA, 2002). Diquat dissipation in water de pends on hydrosoil sediment t ype (Frank and Comes, 1967; Weber et al., 1969), turbidity (P oovey and Getsinger, 2002; Hofstra et al., 2001), pH (Daz et al., 2002), photolysis (Howard, 1989; Tuck er, 1980), type of aquatic plan ts present (Langeland et al., 1994; Summers, 1980), and presence of micr oorganisms (Simsiman and Chester, 1976). Volatility appears to be negligible (Coats et al., 1966). Although diquat has a short half-life in

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39 water (Langeland and Warner, 1986), and it is nontoxic to fish and other aquatic animals when applied at recommended rates for weed contro l in aquatic systems (Paul et al., 1994), its extensive use for aquatic weed control makes it imperative to understand more completely any potential phytotoxicity to non-target plants to, prevent environmenta l problems, especially if this herbicide is used in irrigation water. The water use restriction following diquat appl ication for aquatic w eed control at the highest concentration (0.37 mg L-1) is a 5-day interval between treatment and use of treated waters for irrigation purposes (Syngenta, 2004). However, Peterson et al. (1997) found that growth rates of duckweed ( Lemna minor L.), a species widely used for environmental risk assessment, and the cyanobacterium Anabaena inaequalis (Kutzing) Born et and F were inhibited by 50% when exposed to diquat c oncentrations as low as 0.004 and 0.005 mg L-1, respectively. In another study, Fair child et al. (1997) found an EC50 (effective concentration 50 the concentration of diquat re quired to reduce plant dry weight by 50%) of 0.018 and 0.08 mg L-1 for duckweed and the algae Selenastrum capricornutum Printz, respectively. These concentrations are much lower than the maximum aquatic labeled rate, implying that crop plants irrigated with diquat-containing water may be adversely affecte d. Therefore, this study was conducted to evaluate the phytotox icity of diquat on various grow th stages of rice. Rice was selected for this study due to its high sensistivity to pollutants in soil (Palazzo and Leggett, 1986) and water (Wang, 1992; Holst and Ellwanger, 1 982). Also, rice is a semi-aquatic plant and requires much more water for growth than other crop plants. Materials and Methods All experiments were conducted in 2005 in a tem perature-controlled greenhouse at the University of Florida, Center for Aquatic and In vasive Plants, Gainesville, Florida. Plants were grown under a 14 h photoperiod with maximum daytim e temperatures of 32 3 C and nighttime

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40 temperatures of 18 3 C. This study followed a completely randomized design with five replications and all expe riments were repeated. Germination Ten rice seeds were germ inated in 0.5 L plastic cups containing 200 ml of deionized (DI) water with 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.0, 1.5 and 2.0 mg L-1 a.i. of diquat. Germinating seedlings were observed daily and DI water was replenished to maintain the 200 ml initial volume. Percent germination, root and shoot length were determ ined 2 WAT (2 weeks after treatment) for each seedling in each replication. Roots and shoots were then separated and dried at 90 C for 72 h to determine dry weight. Irrigation Rice seeds were pre-g erminated in unchlorin ated well water for 10 to 14 d to allow easy handling and transplanting. Ten seedlings were pl anted in 3 L pots filled with builders sand amended with 2.6 g (1 g kg-1 dry sand) of 15-9-12 controlled release Osmocote 1. Pots were maintained under flooded conditions with unchlor inated well water until harvest. Subsequent applications of 0.5 g per pot of 46-0-0 (N-P-K) urea fertilizer and micronutrients were also administered to provide optimum plant growth. Three rice vegetative stages [seedling (3-4 leaf stage), tillering, and mature (before seed head setting)] were exposed to a single application of diquat in irrigation water. All pots were drained of excess water before treatment and then irrigated with 700 ml of tap water containing diquat, except for the control pl ants. Diquat-treated irrigation wate r was applied directly onto the sand to simulate flood irrigation. Seedling and tillering plants were irrigated with diquat (Reward 1 The Scotts Company, Marysville, OH. 43041.

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41 QIT) 2 concentrations of 0, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 75.0, and 100.0 mg L-1 a.i. (active ingredient), and mature plants were ir rigated with diquat con centrations of 0, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 75.0, 100.0, and 200 mg L-1 a.i. Four plants in each pot were randomly chosen 14 DAT and were measured from their base to the tip of the longest leaf. Plants were then clipped at the sand surface, dead tissue was removed, and live tissue was dried at 90 C for 72 h to determine dry weight. Statistical Analysis Data collected for germ ination, root lengt h, shoot height and dry weights for the germination experiment, and shoot height and dr y weight for the irriga tion experiments, were subjected to analysis of vari ance using SAS (SAS, 2002). Nonlinea r regression was performed to determine possible relationships between plant dr y weight and height as a function of diquat concentration applied in the simula ted irrigation water. Threshold EC10 (diquat rate that caused 10% reduction in dry weight compared to nontre ated plants) was calcu lated with regression models. Data from all studies were pooled since there was no significant difference between experiments. Means within each parameter were separated using Fishers LSD at P 0.05. Results and Discussion Germination Percent germ ination ranged from 89 to 95%, in dicating that the seed used for this study was highly viable. Percent germination did not differ across the rates tested, except for 1.0 mg L-1 a.i. of diquat, which was different from nontreated seed; however, germination did not differ from other diquat rates (data not shown). 2 Trademark of Syngenta Group Company. Syngenta Crop Protection. Professional Products. Greensboro, NC. 27419.

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42 Root and shoot length of nontreated seeds (control) were higher than those for seeds exposed to all diquat concentra tions tested, except for shoot lengt h at a diquat concentration of 0.05 mg L-1. Diquat concentrations as low as 0.05 mg L-1 a.i. reduced shoot and root length by 8 and 75%, respectively. While shoot length was re duced by 42% when diquat concentration was increased to 0.2 mg L-1, root length was reduced by 98% and root growth was completely inhibited at concentratio ns greater than 0.4 mg L-1. In contrast, although shoot length was reduced by 95% at diquat c oncentrations of 1.0 mg L-1 and higher, none of the tested rates completely inhibited shoot growth (data not shown). The calculated EC10 values for roots based on dry weight and length were similar (0.004 mg L-1). The EC10 for shoot dry weight and length were 0.016 and 0.035 mg L-1, respectively (Figure 2-1 and Table 2-1). There is no safety fact or when comparing the maximum diquat label rate (0.37 mg L-1) to the EC10 values for roots and shoots. The EC10 for roots and shoots were 93x and 23x, respectively, lower than the recommended maximum aquatic weed application rate. Based on these results, roots of germinating rice appear to be more sensitive than shoots to diquat in irriga tion water. Palazzo and Leggett ( 1986) also reported that root elongation was more sensitive to herbicides than seed germination. Li et al. (2005) found similar evidence of a sharp decline in rice growth in terms of number of roots, total length of roots, surface area of roots, and biomass wh en roots were exposed to 0.05 mg L-1 a.i. of metsulfuron-methyl as bound residues in the soil. Similar results have been reported in other species. Groninger and Bohanek (2000) found that applications of diquat at 0.48 and 0.96 g L-1 a.i. were able to cause a partial and complete suppression, respectively, of root growth of black willow ( Salix nigra Marsh.). Wright (1966) reported th at four lines of rye grass

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43 ( Lolium perenne L.) genotypes treated with diquat at rela tively low concentrations also exhibited rapid biomass decline. Although germination of rice seed was not imp acted in our study, adverse effects of diquat on seed germination for other species has been widely reported. Rahman et al. (2004) observed reduced germination of soybean ( Glycine max L.) seed when it was desiccated with diquat at seed moisture contents of >30%. Sund and No mura (1963) reported 50% inhibition of radish ( Raphanus sativus L.) and sweet Sudan grass ( Sorghum sudanense Stapf) germination when diquat was applied at 30 M. Roberts and Griffiths (1973) observed that treating perennial ryegrass with diquat a few days before the normal date for harvest resulted in subsequent low seed germination and abnormal seedling growth. Re sults from our studies indicate that rice establishment may also be severely affected by diquat at concentrations lower than 0.05 mg L-1 in irrigation water. Germinati on and subsequent growth of rice seedlings in our study was affected by diquat concentrations nearly 10 to 20 times lower than the maximum labeled rate for aquatic weed control. There was no soil in contact with these seeds and seedlings to adsorb or reduce diquat concentrations, so further stud ies with various soil types are warranted to determine if soils reduce the sens itivity of germinating rice to di quat in irrigation water. Irrigation The phytotoxic effects of diquat applied once in irrigation water to different developm ental stages of rice are presented in Table 2-2 and Figure 2-2. Rice plants that emerged above the irrigation water were more tolera nt of diquat than submersed germ inating seedlings; also, larger or more mature plants were less affected by diquat. Plants treated at the seedling and mature stages were not different from nontreated plants at diquat concentrations lower than 10 mg L-1. However, when the rate was increased from 10 to

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44 25 mg L-1, plant height of seedling rice decreased by 83% and was completely inhibited by higher diquat concentrations. Sim ilarly, the length of mature pl ants was reduced by 47% at 10 mg L-1. Despite continued decline in length as diquat concentrations increased, complete growth inhibition was only attained at diquat concentra tions of 200 mg L-1, compared to 50 mg L-1 for the seedlings (data not shown). Shoot elongation at the tiller ing stage was reduced by 22% at a diquat concentration of 5 mg L-1. There was no complete inhibition of growth even at 100 mg L-1, the highest diquat concentration evaluated for this growth stage (data not shown). The concentration of diquat in irrigation wate r that caused a 10% reduction in dry weight and height of rice was essentially similar due to overlapping 95% confidence intervals (Table 2-2). The EC10 for the mature stage, based upon dry we ight, was not different from that of rice at the seedling stage, but mature plants were more tolerant than those at the tillering stage. Similar results were found when EC10 was based upon plant height. In general, more mature plants were more tolerant of di quat in irrigation water, but due to variation, some overlap in the 95% confidence intervals occurred with the seedling stage. The sensitivity of rice to herbicides at the til lering stage has been previously reported with diquat, and also with other herbic ides. For example, Baylis et al (1991) found that tillering rice was more susceptible to several mono and di-substituted analogues of 2-phenoxypropionic acid than were wheat and barley. Nelson et al. (2003) examined diffe rent aquatic herbicides on wild rice and noted that the application of 2,4-D at the tillering stage was likely to affect subsequent seed production. Similar findings were reported by other authors. For example, Oelke and McClellan (1991) and Clay and Oelke (1990) found that application of 2,4-D at early or midtillering resulted in severe injury to wild rice.

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45 Rice at the tillering stage is sensitive not only to herbic ides, but also to adverse environmental (growth) conditi ons. Yang et al. (2007) reported the highest reduction of rice yield when water deficit was imposed at this stage, and Zheng and Shanon (2000) found that tillering was very sensitive to salinity stress. Ho wever, based on these data, the application of diquat in flood irrigation waters at the labeled rate of 0.37 mg L-1 should not have an effect on the growth (height or weight) of plants that have reached the 3-4 leaf st age of vegetative growth. Nevertheless, caution should be exercised to avoid exacerbating negative effects from any existing adverse environmental conditions. Mature plants showed the highest EC10 values (2.89 and 5.30 mg L-1) of all the growth stages. Nelson et al. (2003) also observed that wild rice treated w ith diquat when mature was not affected, regardless of rate or herbicide used; ho wever, diquat rates test ed in this study were much higher than those evaluate d in Nelsons work (1.03 mg L-1). Rice seedlings at the germination stage are more sensitive to diquat in irrigation water than subsequent stages of plant development. The di quat concentration capable of inhibiting plant growth by 10%, on average, for the ge rminating whole plant was 0.01 mg L-1. This theoretical value was obtained from the data in Table 2-1 by calculating the mean EC10 for the roots and shoots (dry weight) and is about 37 times less than the maximum concentration of diquat (0.37 mg L-1) that can be currently applied in water. The high susceptibility of germinating rice seed lings to diquat indicates that diquat-treated irrigation water should not be us ed to flood freshly planted fields However, restricting irrigation of rice with diquat treated water at later growth stages does not appear necessary. These studies were conducted with clean well water and pure sa nd, which would likely re sult in longer diquat half-lives and constitute worst case conditions.

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46 This study evaluated the effect of diquat on gr owth and dry weights of vegetative rice plants and did not evaluate a ny potential impacts on subsequent grain production; however, based on the known activity and physiological behavi or of diquat, grain yield should not be reduced as long as growth or height of mature pl ants is not affected. However, additional studies should be conducted to ascertain th at diquat in irrigation water does not affect seed production in rice.

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47 Table 2-1. Effect of diquat in irrigation wate r on rice germination and growth 14 DAT. Diquat applied once initially to seeds and water level maintained with untreated tap water Parameter EC10 1 (95% CI2) Regression equation r2 Safety factor3 Dry weight Roots 0.004 (0.004 0.005) y = 0.0479e-25.9548x 0.92 0.01 Shoots 0.016 (0.014 0.019) y = 0.1137e-6.6777x 0.91 0.04 Length Roots 0.004 (0.0036 0.004) y = 39.8694e-25.6295x 0.94 0.01 Shoots 0.035 (0.0300 0.040) y = 62.3133e-3.0043x 0.95 0.09 1 EC10 = Concentration (mg L-1) of diquat required to reduce plant dry weight by 10%. 2 95% CI = 95% Confidence Interval 3 Safety Factor = EC10/0.37 mg L-1 a.i. (Maximum recommended rate) Table 2-2 Effect of diquat in irrigation water on rice at different stages of development 14 DAT. Diquat applied once to plants at seedling, tillering, and mature stages. Water level was maintained with untreated ta pwater for 14 d when harvested Stage EC10 1 (95% CI2) Regression equation r2 Safety factor3 Dry weight Seedling 1.67 (1.28 2.38) y = 2.5571e-0.0631x 0.87 5 Tillering 1.11 (0.96 1.33) y = 6.0409e-0.0945x 0.96 3 Mature 2.89 (2.28 3.93) y = 5.9143e-0.0365x 0.90 8 Plant height Seedling 2.24 (1.60 4.00) y = 41.3535e-0.0470x 0.61 6 Tillering 2.40 (1.99 2.88) y = 45.3100e-0.0448x 0.88 7 Mature 5.30 (4.00 7.00) y = 44.9412e-0.0199x 0.72 19 1 EC10 = Concentration (mg L-1) of diquat required to reduce plant dry weight by 10%. 2 95% CI = 95% Confidence Interval 3 Safety Factor = EC10/0.37 mg L-1 a.i. (Maximum recommended rate)

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48 Diquat concentration (mg L-1) 0.0 0.5 1.0 1.5 2.0 Mean dry weight (g/pot) 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 Roots Shoots Figure 2-1. Relationship between dry weights of germinating rice shoots and roots when exposed to various concentrations of di quat in water. Data are pooled for two experiments. Each value is presented as the mean standard error (n=10). Diquat applied once initially to seeds and water level maintained with untreated tap water. Plants where harvested 14 DAT from treatment solutions.

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49 0 1 2 3 4 5 Seedling 020406080100120 Mean dry weight (g/pot) 0 2 4 6 8 Tillering Diquat concentration (mg L -1 ) 050100150200 0 2 4 6 8 10 Mature Figure 2-2. Relationship between dry weight of rice seedling, til lering and mature stages when exposed to various concentrations of diquat in irrigation water. Data are pooled for two experiments. Each value is presented as the mean standard error (n = 10). Diquat applied once to plants at seedling, ti llering and mature stages and water level was maintained with untreated tap water for 14 d when harvested.

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50 CHAPTER 3 SENSITIVITY OF SELECTED AGRONOMI C PLANT S TO DIQUAT IN IRRIGATION WATER Introduction Diquat is a n onselective herbicide that has been used for both submersed and floating macrophyte control, non-crop areas weed manageme nt, and as a crop desiccant in several crops (Ivany and Sanderson 2001; Groninger and Bohane k, 2000; Brian et al.; 1958). It is usually applied post-emergence, targeting plant green tissue, where it is rapidly absorbed and acts quickly (Brian et al., 1958). Root uptake is neg ligible due to adsorption and inactivation by soil or sediment particles (Yeo, 1967; Funderbur k and Lawrence, 1963). Rapid and differential adsorption of diquat by soil constituents is one of the most important characteristics behind its wide agricultural use. Coats et al. (1966) reported 0.3 mg g-1 diquat adsorption on a sand loam soil, and 2-2.5 and 80-100 mg g-1 on Georgia kaolinite and bentoni te clays, respectively. Similar results were also observed with paraquat on soil (0.3 mg g-1), kaolinite (2.5-3 mg g-1) and montmorillonite (75-85 mg g-1) (Coats et al., 1964). On the othe r hand, when diquat is applied in water it dissipates rapidly with a reported ha lf-life of 48 h (Langeland et al. 1994, Tucker, 1980; Gillett, 1970). There is a 5-day water use restriction between application of diquat for aquatic weed control at the maximum recommende d aquatic use rate (0.37 mg L-1) and the use of treated water for crop irrigation (Syngenta, 2004). However, phytotoxic effects on severa l species used for environmental risk assessment have been repo rted at concentrations much lower than the recommended aquatic use rate (Fairchild et al., 1997; Peterson et al., 1997) indicating that it is important to gather more information on nontarget effects of diquat, especially on crops, which at the moment is limited. Therefore, the objective of this study was to evaluate the sensitivity of varied agronomic crops to diquat in irrigation water.

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51 Materials and Methods Plants for this study were grown in a tem per ature and light regulated greenhouse in 2005 at the University of Florida, Center for Aquatic and Invasive Plants, Gain esville, Florida. The greenhouse was set to provide a 14 h photoperiod with maximum daytime temperatures of 32 3 C and nighttime temperatures of 18 3 C. The study followed a completely randomized design with five replications and was repeated. Tested crops consisted of sweet corn ( Zea mays L. Silver Queen), cotton (Gossypium hirsutus L. FiberMax), soybean ( Glycine max L. Merr. NG2328R), squash ( Cucurbita maxima Duchesne Crookneck Early), and wheat (Triticum aestivum L. Wakefield). Ten seeds of each crop were planted at a depth of 0.5-1 cm in 3 L pots filled with builders sand and amended with 1 g of Osmocote per kg of dry sand. Immediately after planting, all pots were irrigated with 100 ml of unchlorinated well water (pH 7.7) containing diquat at concentrations of 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 75.0, and 100.0 mg L-1 a.i. for cotton, soybean and wheat, and similar concentrations from 0 to 50 mg L-1 for sweet corn and squash. Irrigation water containing diquat was applied once directly onto the sand to simulate flood irrigation. Further irrigation used untreated well water and was applied as necessary for plant growth. Treated pots were observed daily and seed ge rmination was recorded. The heights of four plants in each pot were measured from their base to the tip of the longest leaf 2 WAT. Plants were then clipped at the sand surface, any dead tissue removed, and dried at 90 C for 72 h to determine dry weight. Data for pl ant germination, shoot height a nd dry weights were subjected to ANOVA using SAS (SAS, 2002). Nonlinear regr ession was also conducted to determine relationships between shoot height and dry weights to diquat concentrations. Threshold EC10 values were calculated using regression models. There was no significant difference between

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52 experiments; therefore, data were pooled for analysis. Mean s within each parameter were separated using Fishers LSD at P 0.05. Results and Discussion Germ ination of corn, cotton, soybean, s quash and wheat treated with various concentrations of diquat in irri gation water is presented in Figure 3-1. Germination of seeds in the control treatments was genera lly 90% or greater, as shown in Figure 3-1, with the exception of cotton which had a germination rate of 80-90%. Diquat concentrations of < 25 mg L-1 had little effect on germination of any of the seeds evaluated. Cotton, soybean and wheat were least sensitive to a diquat co ncentration of 25 mg L-1 in irrigation water, while corn was most sensitive. Diquat concentrations in th e initial irrigation water of 50 mg L-1 or greater reduced germination of all crops to less than 30-40%. Soybean seed was the most tolerant to diquat in irrigation water. Germination of soybean was greatly reduced but not completely inhi bited at the highest diquat concentration (100 mg L-1), while cotton and wheat we re completely inhibited. Table 3-1 presents the EC10 (diquat concentration require d to reduce germination by 10%) values for the plants studied. The EC10 varied between 3 and 6 mg L-1 among the 5 crops evaluated, which yielded a safety factor from 8 to 16 fold comparing the EC10 for germination to the maximum label application rate allowed fo r aquatic weed control (Syngenta, 2004). There was little difference among the EC10 values for germination among the crops evaluated (Table 3-1). High sensitivity of some grass sp ecies and tolerance of broadleaf plants to bipyridylium herbicides at the germinating st age has been previously reported. Roberts and Griffiths (1973) found that the application of diqu at to perennial ryegrass (Lolium perenne L.) reduced seed germination and induced the grow th of abnormal seedlings. On the other hand,

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53 Moyer et al. (1996) observed that ap plications of di quat to alfalfa ( Medicago sativa L.) had no effect on seed germination. Appleby and Brenchle y (1968) reported that di rect applications of paraquat, a chemically related compound to diquat, at 0.56 kg ha-1 on alfalfa and red clover ( Trifolium pratense L.) did not affect their germination, wh ile germination of seven grass species was greatly reduced. However, when a protectiv e layer of sandy loam soil (0.6 cm) was placed over the seed, the effect of paraquat was neutralized. The adsorption of diquat by soil particles has been widely reported (Coats et al., 1966; Harris and Warren, 1964). Reduced effects of diquat on corn and wheat germination in our study compared to the results reported by Appleby a nd Benchley (1968) may be the result of the sorbent effect of sand, similar to the protective effect not ed in their study. The dry weights of the 5 plant species 14 DAT are presented in Figure 3-2. Plant dry weight was reduced by diquat concentrations of 10 mg L-1 or greater, and concentrations of 50 mg L-1 were essentially lethal to sweet corn and squash. As shown in Table 3-2, the calculated EC10 values for seedlings based on dry weight varied from 2 to 5 mg L-1 and from 3 to 8 mg L-1 based on plant height. These values are very similar to the effects of diquat on seed germinat ion (Table 3-1). The estimated safety factors, derived by rationing the EC10 values to the maximum a pplication rate of 0.37 mg L-1, once again varied from 5 to 16 fold. These results suggest that fl ood irrigation of seeds planted in sand with diquat-treated water at concentrations of 0.37 mg L-1 would have no effect on seed germination and subsequent growth of these crops. Similar results on 5 orna mental plants (begonia, dianthus, impatiens, petunia and snapdragon) irrigate d once with diquat at the flow ering stage have been reported

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54 (Mudge et al., 2007). Conversely, seed s and seedlings in direct cont act with diquat-treated water were very sensitive to diquat in irrigation wa ter, as observed with rice in Chapter 2. These studies were conducted with dry, wash ed builders sand, where seeds were first irrigated with water containing various concentrati ons of diquat and subsequently irrigated with water containing no diquat. This is a likely scenario when diquat is applied once every few weeks to irrigation canals for aquatic weed cont rol. Washed builders sand, with a low cationic exchange capacity (CEC), would have little binding affinity for diquat compared to soils with higher organic matter or clay contents. These latt er soils would bind diquat and probably result in an even greater safety factor for diquat in irrigation water.

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55 0 20 40 60 80 100 120 Corn Percent germination 0 20 40 60 80 100 120 Cotton Diquat concentration (mg L-1) 0 20 40 60 80 100 120 Soybean 0 0 0 5 0 1 0 2 0 4 0 8 1 1 5 2 4 1 0 2 5 5 0 0 20 40 60 80 100 120 Squash Diquat concentration (mg L-1) 0 0 0 5 0 1 0 2 0 4 0 8 1 2 5 5 1 0 2 5 5 0 7 5 1 0 0 0 20 40 60 80 100 120 Wheat Figure 3-1. Percent germination of corn, cotton, soybean, squash and wheat when exposed to various concentrations of diquat in water. Seeds irrigated once at planting with diquat treated water. Thereafter plants were irriga ted with tap water as needed for 14 d when harvested. Data are pooled for two experiment s. Each value is presented as the mean standard error (n=10).

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56 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Corn Mean dry mass (g/pot) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Cotton Diquat concentration (mg L-1) 0.0 0.5 1.0 1.5 2.0 2.5 Soybean 0102030405060 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Squash Diquat concentration (mg L-1) 020406080100 0.0 0.1 0.2 0.3 0.4 Wheat Figure 3-2. Dry weights of co rn, cotton, soybean, squash and wheat shoots when exposed to various concentrations of diquat in irriga tion water. Diquat applied once initially to seeds and thereafter plants irrigated with untreated tap water. Plants where harvested 14 DAT. Data are pooled for two experiments and each value is presented as the mean standard error (n=10).

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57 Table 3-1. Effect of diquat in irrigation water on corn, cotton, soybean, squash and wheat seed germination. Diquat applied once immediat ely after planting and seed germination recorded 14 DAT when harvested. Percent germination EC10 1 (95% CI2) Regression equation r2 Safety factor3 Corn 3 (3 4) y = 96.4695e-0.0309x 0.78 8 Cotton 4 (3 5) y = 86.1386e-0.0272x 0.89 11 Soybean 6 (5 6) y = 99.9666e-0.0184x 0.96 16 Squash 4 (3 5) y = 96.7793e-0.0271x 0.88 11 Wheat 5 (4 5) y = 97.3297e-0.0226x 0.96 14 1 EC10 = Concentration (mg L-1) of diquat required to reduce plant dry weight by 10%. 2 95% CI = 95% Confidence Interval 3 Safety Factor = EC10/0.37 mg L-1 a.i. (Maximum recommended rate) Table 3-2. Effect of diquat in irrigation wate r on corn, cotton, soybean, squash and wheat on initial seedling growth. Diquat applied once immediately after planting. Plants were irrigated with untreated tap water as needed for 14 DAT when harvested. Dry weight EC10 1 (95% CI2) Regression equation r2 Safety factor3 Corn 2 (2 3) y = 0.4447e-0.0475x 0.89 5 Cotton 3 (3 4) y = 0.8810e-0.0307x 0.88 8 Squash 4 (3 6) y = 0.8719e-0.0240x 0.69 11 Soybean 5 (3 4) y = 1.8344e-0.0211x 0.92 14 Wheat 3 (3 4) y = 0.2730e-0.0330x 0.89 8 Plant height Corn 3 (2 3) y = 16.2617e-0.0426x 0.60 8 Cotton 4 (3 4) y = 10.6667e-0.0296x 0.93 11 Soybean 6 (5 6) y = 25.3851e-0.0190x 0.97 16 Squash 5 (3 8) y = 12.5570e-0.0331x 0.88 14 Wheat 4 (4 5) y = 14.3100e-0.0240x 0.97 11 1 EC10 = Concentration (mg L-1) of diquat required to reduce plant dry weight by 10%. 2 95% CI = 95% Confidence Interval 3 Safety Factor = EC10/0.37 mg L-1 a.i. (Maximum recommended rate)

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58 CHAPTER 4 PHYTOTOXICITY OF SURFACTANTS TO HYDRILLA Introduction Surfactants are widely u sed in agriculture. Their ability to reduce surface tension in water and other liquids makes them useful for several purposes, such as penetration, wetting, emulsification, spreading, sticking and humectancy (Monaco et al., 2002; Matsumoto et al., 1992; Hartzler and Foy, 1983). In the US, there are more than 3,000 co mpounds marketed as adjuvants, which include surfactants for agricultural use (USEPA, 2004). Underwood (2000) reviewed the worldwide pesticide market and found that adju vants in the US represent more than $400 million of the $8 billion total sales volume of pesticides. According to Allison (2003), 80%-90% of adjuvants are used with herbicides, and herbicides represent more than 50% of total pesticide sales. Despite the extensive use of adjuvants, particularly surfactants, few toxicity tests are conduc ted on these products compared to the active ingredients in pesticides that they are usually mixed with (Cox and Surgan, 2006). Based on the increasing environmental awaren ess and willingness to protect natural resources, surfactants are seen by some as tools for reducing both the amount of herbicide required to control weeds and the amount releas ed into the environm ent. Surfactants are typically added to foliar applied herbicides for improved control of emergent and floating weeds and recently have been recommended for use with herbicides to improve control of submersed weeds. The amount of adjuvant s and surfactants use in aquatic areas is expected to rise as invasive sp ecies spread throughout the US. Although some literature is available on the toxicity of surfactants to aquatic and terrestrial plants with func tional cuticle and stomata (Ramsdale and Messersmith, 2002;

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59 Caux et al., 1993, Caux and Weinberger, 1993; Sutton and Foy, 1971), to aquatic invertebrates, verteb rates and microalgae (Hodges et al ., 2006; Haller and Stocker, 2003; Ma et al., 2003; Mann and Bidwell, 2001; Moreno-Garrido et al., 2001; Yoshimura, 1986; Wolfenbarger and Holscher, 1967), and most recently to humans (Richard et al., 2005; Lin and Garry, 2000), litt le is known about the effect of these compounds on submersed aquatic plants, which have little or no cuticle and non-functional or absent stomata (Verma and Verma, 2000; Pendland, 1979; Sculthorpe, 1967). Some surfactant manufacturers are no w recommending adding surfactants to aquatic herbicides for improved submer sed weed control (See Appendix A). The recommended rate of application for use with herbicides for submer sed weed control is 9.3 to 18.7 L ha-1 (1-2 gallons acre-1), corresponding to a concentration of 3 to 6 mg L-1 of formulated product in water 30 cm (1 foot) deep. Because of lack of data on the effects of these surfactants, th is study was conducted to evaluate th e toxicity of surfactants to the submersed plant hydrilla. Materials and Methods Four different types of surfactants were chosen for study: a silicone-based product (SilEnergy), an acid-based product (CT-301) and a dlim onene and alcohol/glycol products (Cygnet Plus and Timberland 90), re spectively. General in formation on these four surfactants is provided in Table 4-1. The experiments on the phyt otoxicity of these surfact ants to hydrilla were conducted in greenhouse and laboratory studies at the Univers ity of Florida, Center for Aquatic and Invasive Plants, Gainesville, Flor ida in April 2007 and were repeated in May 2007. Hydrilla plants were coll ected from Rodman Reservoir (Florida) and maintained in culture in plastic tanks with well water. Ne w plants were collected every two weeks and

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60 the old ones discarded. The greenhouse was set to provide a 16 h photoperiod with maximum daytime temperatures of 30 3 C and nighttime temperatures of 18 3 C. Hydrilla Biomass Hydrilla tips m easuring 15 cm in length were exposed in 0.5 L plastic cups containing 400 ml of DI water (p H 7.1) and 0, 3, 10, 100, 1,000 and 5,000 mg L-1 of formulated Cygnet Plus, SilEnergy, CT-301 and Timberland 90 surfactants for 24 h to determine EC50 concentrations. All hydrilla tips were thoroughly rinsed after exposure and planted in 0.102 L (13.5 cm x 4 cm diam eter) plastic tubes filled with locally purchased topsoil (Earthgro Topsoil) amended with 0.2 g of 15-9-12 Osmocote controlled-release fertilizer. The tubes were topdressed with 3 cm of builders sand to cover the topsoil, placed in a rack and submersed in a tank (59 cm depth by 107.5 cm diameter) filled with well water (pH 7.7) and allowed to grow for 2 wk. Plants were then harvested 14 DAT and the total lengths (length of main stem plus length of axillary stems), and dry weights were determined. Pl ants were dried at 90 C for 72 h and then weighed. Hydrilla Chlorophyll To further define the effects of surfactants on hydrilla, 3 cm -long tips were excised and allowed to acclimate in dishpans contai ning DI water for 24 h in a growth chamber3 with a 14 h photoperiod and temperature of 27 3 C. Tips were transferred to 20-ml polypropylene scintillation vials4 with 18 mL of DI water and treated with the four previously described surfactants at 0, 3, 10, 100, 1000 and 5,000 mg L-1. Immediately after treatment, the surfactan t-water mixture was thoroughly mixed by covering each vial 3 Percival Scientific, Inc. Perry, IA. 50220. 4 Trademark of Penchiney Plastic Packaging. Menasha, WI. 54952.

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61 with Parafilm and inverting it four times. Tips were exposed to the surfactant solutions for 48 h. This study followed a completely ra ndomized design with three replications, and was repeated. Tips were rinsed immediately after the 48 h exposure and blotted with paper towels to remove excess water. A 100 mg subsample of hydrilla tissue was collected from each tip for chlorophyll extr action by dimethylsulfoxide (DMSO), for 6 h in a water bath at 65 C, according to the method of Hiscox and Israelstam (1979). The extractants were analyzed spectrophotometrically based on the method of Arnon (1949). Total extracted chlorophyll was calculated by using the formula: Total chlorophyll = [(0.0127*chlorophyll a 0.00269*chlorophyll b)*Vol ume of DMSO]/fresh weight. Results and Discussion Hydrilla Biomass The effects of surfactants on hydrilla dry weight after 24 h of exposure followed by a 14 d grow out period are presented in Figure 4-1. As surfactant c oncentration increased, dry weight o f hydrilla decreased. CT-301 wa s the most phytotoxic to hydrilla, followed by Cygnet Plus, Timberland 90 and SilEnergy (Table 4-2). Timberland 90 and SilEnergy had similar EC50 values of 569 and 592 mg L-1; however, there was no difference among Cygnet Plus, SilEnergy and Timberland 90. Silicone surfactants, such as SilEnergy are most effective at pH 6-8 (Knoche et al. 1991) because under acidic (pH 2 to 5) or alkaline (pH 9 to 10) conditions they rapidly degrade. In our study, hydrilla tips exposed for 24 h in plasti c cups increased the pH of DI water from 7.1 to 9.9. Large mats of hydrilla in natural environments have been reported to raise the pH of water to 10 (Spencer et al., 1994; Van et al., 1976). High pH values in plastic cups due to hydrilla photosynthesis in our study may have resulted in reduced phytotoxicity of SilEnergy. Mo reover, silicone-based surfactants and

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62 alcohol/glycol blends are classified as wettin g-spreader surfactants for the control of terrestrial weeds (Hazen, 2000). However, fo r the control of submersed weeds those properties are of no use, resul ting in their reduced effect under an aquatic situation as shown in these studies. In contrast, the acidification of the treat ment solution by CT-301 (Table 4-3) may have influenced its phytotoxicity, considering that although hydrilla tolerates a wide range of pH, its optimum growth occurs at pH 7 (Steward 1991). Furthermore, acids are also widely known for their ab ility to dissolve waxy materials and cell membranes that usually impair solute penetr ation through the cuticle, with subsequent loss of plant membrane integrity (Van Bilsen and Ho ekstra, 1993; Mukherj ee and Choudhuri, 1985). Hence CT-301 likely displayed pronounced phytot oxicity compared to other surfactants used in our study due to its acidic properties. The pH of herbicides and surfactants is widely ignored and can vary by many orders of magnitude, thus affecting compatib ility and chemical performance. The effects of high concentrations of CT-301 on hydrilla were obvious within moments of exposing tissue to this sulfuric acid-c ontaining surfactant as shown in Table 4-3. The pH of the concentrate of CT-301 was -1.6. The pH of th e other surfactant con centrates was much higher and ranged from 5.1 to 6.9, respectivel y, for Cygnet Plus and SilEnergy. The pH of CT-301 is lower than pH 0 because it is considered a super acid, and the pH of the other surfactant-water mixtures may be lower than either the water or concentrate alone due to the presence of H+ added in the water (Muwanga-Zaque, 2002). The acidic properties of CT-301 were evid ent 24 h after expo sure. Hydrilla tips exposed to Cygnet Plus and CT301 at concentrations >100 mg L-1 were bleached, and

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63 they had lost turgor and cell membrane inte grity. Loss of membrane integrity in hydrilla treated with Cygnet Plus require d a lag period of 24 h to occur, and the treatment solution remained milky white in appearance, while the treatment solution of CT-301 turned purple within 5 min. of hydrilla placement in the plastic cups. The color change induced by CT-301 might have been the result of memb rane dissolution and release of pigment contents, particularly anthocyanins, which at pH < 3 usually turn reddish-purple, while at pH 4-5 are colorless (Jackman and Yada, 1987) In contrast, hydrilla tips exposed to Timberland 90 and SilEnergy maintained me mbrane integrity, ev en at the highest surfactant concentra tions evaluated. The total length of hydrilla exposed to the four surfactants followed a pattern similar to the dry weight data (Figure 4-2 and Table 4-2). Timberland 90 and SilEnergy at concentrations < 100 mg L-1 did not differ from control pl ants. Conversely, Cygnet Plus and CT-301 at 100 mg L-1 significantly reduced hydril la total length, with CT-301 showing a greater effect at this concen tration than Cygnet Plus. At 1000 and 5000 mg L-1, all surfactants signific antly affected hydrilla growth, and Cygnet Plus and CT-301 were toxic to hydrilla at these rates. Hydrilla Chlorophyll The effect of CT-301, Cygnet Plus, S ilEnergy and Tim berland 90 on hydrilla chlorophyll content is presente d in Figure 4-3 and Table 4-4. In contrast to previously described effects of CT-301 on hydrilla dry weight and total lengt h, hydrilla chlorophyll content was reduced by Cygnet Plus and SilEnergy much more than CT-301 and Timberland 90. The SilEnergy and Timberland data show di fferent trends when comparing results for the amount of chlorophyll content vers us biomass in hydrilla. The effects of

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64 Timberland 90 on chlorophyll content were le ss pronounced compared to SilEnergy. As concentrations of both compounds increase d, the chlorophyll content decreased (Figure 4-3). SilEnergy concentrations as low as 10 mg L-1 were able to elicit a significant chlorophyll reduction compared to the nontreated plants and at 1000 mg L-1 severe symptoms of chlorosis (water soaking) appeared on the plant tissue. Similar trends were also observed with Cygnet Plus and Ti mberland 90, except that differences from the control plants began at c oncentrations of 100 mg L-1 for both compounds. The calculated EC50 for SilEnergy, Cygnet Plus and Timberland 90 were 408, 433 and 4,814 mg L-1, respectively (Table 4-2). The EC50 for Cygnet Plus is almost two times higher than that obtained in the biomass study, which may be due to differences in light and temperature that hydrilla was subjected to in each study. Plants in the greenhouse were exposed and grown unde r 70% sunlight (1050 1400 mol cm-2 s-1) and under temperatures ranging from 18 to 33 C. Hydrilla in the growth chamber was grown at a light concentration of 380 mol cm-2 s-1 and at a constant temperature of 27 C. Likewise, the adverse effects of SilE nergy and Timberland 90 on chlorophyll content were different from the results of the biomass study. Surfactants with high molecular mass or long hydrophilic ethylene oxide groups require more time to exert their dissolution and penetr ating effects on plant memb ranes (Bauer and Schnherr, 1992), and thus have lower phytotoxicity due to lower water solubility (Parr, 1982). Concentrations as low as 3 and 10 mg L-1 of CT-301 usually produced dark green color on hydrilla. However, at concentrations 100 mg L-1 hydrilla tips were bleached and lost membrane integrity, similar to the effects at these concentrations in the biomass study. Chlorophyll degradation or bleaching as a result of exposure to acidic compounds

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65 has been previously reported (Matile et al., 1989; Schanderl et al., 1962). Tett et al. (1975) noted that when hydrochloric acid (HCl ) is applied to benthic microalgae, the H+ rapidly reacts with carotenoids and the e poxidic groups, fucoxanthi n and diadinoxanthin, and causes increased absorption at 750 nm. Ther efore, they concluded that for increased accuracy of chlorophyll a determ inations, the final acid concentration must never be above 3 x 10-1 M. Otherwise, the aforementioned pigments will be converted to compounds absorbing in the 600-750 nm range and interfere with ch lorophyll readings (Riemann, 1978). Therefore, chlorophyll conten t of hydrilla was not a valid indicator of CT-301 phytotoxicity. The phytotoxicity and effects of surfactan ts on terrestrial and floating aquatic weeds have been reported. Haile et al. (2000) observed reduced photosynthesis for 2 d on lettuce (Lactuca sativa L.) seedlings caused by the sili cone surfactant Kinetic applied alone; however, photosynthetic rates of treated plants recovered 5 d after treatment and were not different from the control plants Uhlig and Wissemeier (2000) reported severe phytotoxic effects caused by Triton X100 and Genapol C-80 at 1000 mg L-1 on leaves and bracts of poinsettia ( Euphorbia pulcherrima Willd. ex. Klotzsch). Noga and Bukovac (1986) observed that the application of Citowett and Tween-20 on sour cherry ( Prunus cerasus L. 'Schattenmorelle') and apple trees ( Malus domestica Borkh. 'Golden Delicious') from full bloom to the final stages of fruit developmen t induced flower and fruit abscission, fruit russe ting and cracking, leaf necrosis and changes in plant photosynthesis and transpiration rates. Surfactant phytotoxicity to duckweed has also been reported (Caux and Weinberger, 1993; Caux et al., 1993). Based on these previous studies, cell membrane

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66 disruption resulted from surf actant induction of a desatu rase enzyme, with hydrophobic surfactants having more pronounced e ffects than hydrophilic surfactants. Previous studies have shown that surf actants are phytotoxic to terrestrial and floating aquatic plants. This study shows they ar e also toxic to a submersed aquatic plant, but at concentrations much higher (100 to 1,000 mg L-1) than suggested aquatic use rates (3 to 6 mg L-1).

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67Table 4-1. Description of surfactants used for phytotoxicity studies on hydrilla and in additional studies as described in this dissertation. Full labels are presented in Appendix A. Surfactant Manufacturer Class Ingredients Submersed aquatic use Use rate/379 L water Specific gravity @25 C CT-301 Cheltec, Inc. (Sarasota, FL) Inorganic acid Sulfuric acid 51% No Foliar (0.24 L) 1.35 Cygnet Plus Brewer International (Vero Beach, FL) Dllimonene Alkyl hydroxpoly oxyethylene 10%; dl-limonene 90% Yes Foliar (0.95 -1.89 L) Submersed (3.79-7.57 L) 0.87 SilEnergy Brewer International (Vero Beach, FL) Silicone Polalkyleneoxide-modified polydimethylsiloxane 99 % Yes Foliar (0.18-0.35 L) Submersed (0.24-0.41 L) 1.06 Timberland 90 Loveland Products, Inc. (Greeley, CO) Alcohol/glycol Alkyl Aryl Polyoxylkane Ether, Glycol and free fatty Acids 90%; Inert material 10% No Foliar (0.13 0.5 L) 1.025 .

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68 0.0 0.1 0.2 0.3 0.4 0.5 Cygnet Plus Concentration (mg L-1) 010002000300040005000 Mean dry weight (g/pot) 0.0 0.1 0.2 0.3 0.4 0.5 Sil Energy 0.0 0.1 0.2 0.3 0.4 0.5 CT-301 010002000300040005000 0.0 0.1 0.2 0.3 0.4 0.5 Timberland 90 Figure 4-1. Phytotoxicity of surf actants to hydrilla based on dry weight. Hydrilla was exposed for 24 h to surfactants at various concentra tions. Thereafter, it was rinsed, planted in tubes and grown in a tank with well water. Plants were harvested 14 DAT. Data were pooled for two experiments and each value is the mean standard error (n=10).

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69 Total length (cm) 0 20 40 60 80 100 120 140 Cygnet Plus CT-301 010002000300040005000 0 20 40 60 80 100 120 140 160 SilEnergy Surfactant concentration (mg L-1) 010002000300040005000 Timberland 90 Figure 4-2. Phytotoxicity of surf actants to hydrilla based on to tal length. Hydrilla was exposed for 24 h to surfactants at various concentra tions. Thereafter, it was rinsed, planted in tubes and grown in a tank with well water. Plants were harvested 14 DAT. Data were pooled for two experiments and each value is the mean standard error (n=10).

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70 Chlorophyll (mg/g fresh weight) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Cygnet Plus CT-301 Concentration (mg L-1) 010002000300040005000 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Sil Energy 010002000300040005000 Timberland 90 Figure 4-3. Phytotoxicity of surf actants to hydrilla based on tota l chlorophyll co ntent. Hydrilla was exposed for 48 h to surfactants at various concentrations and chlorophyll immediately extracted with DMSO for 6 h. Data were pooled for two experiments and each value is the mean standard error (n=6).

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71 Table 4-2. Effect of surfactants on dry weight and total length of hydr illa. Hydrilla tips were exposed to surfactants at various concentr ations for 24 h. Thereafter, it was rinsed, planted in tubes and grown in a tank with well water for 14 d and then harvested. Parameter EC50 1 (95% CI2) Regression equation r2 Dry weight CT-301 26 (17 57) y = 0.3216e-0.0265x 0.91 Cygnet Plus 277 (173 654) y = 0.282e-0.0025x 0.88 SilEnergy 592 (339 2,222) y = 0.2913e-0.00117x 0.51 Timberland 90 568 (341 1,639) y = 0.2986e-0.00122x 0.46 Total length CT-301 28 (18 70) y = 109.50e-0.0247x 0.80 Cygnet Plus 239 (127 1,843) y = 93.8633e-0.0029x 0.68 SilEnergy 770 (499 2,063) y = 95.1210e-0.0009x 0.56 Timberland 90 2,166 (498 7001) y = 96.0401e-0.00032x 0.43 1 EC50 = Concentration (mg L-1) of surfactant required to reduce plant dry weight by 50%. 2 95% CI = 95% Confidence Interval Table 4-3. pH of surfactants in distilled water (pH 7.1.1) at various concentrations used in these studies. Each value presented as mean standard error (n=3). Concentration CT-301 Cygnet Plus SilEnergy Timberland 90 3 4.2.0 6.5.1 6.8.5 7.2.2 6 3.9.0 6.3.1 6.8.5 6.6.0 10 3.8.0 6.2.1 6.8.5 6.4.0 12 3.7.0 6.2.1 6.7.5 6.3.0 24 3.4.0 6.2.2 6.6.4 6.1.1 100 2.7.1 5.2.1 6.4.4 5.8.0 1000 1.7.1 4.2.2 5.9.5 5.2.1 5000 1.0.0 5.3.1 6.1.4 4.8.0 Concentrate -1.6.1 6.6.3 6.9.5 5.1.2 Table 4-4. Effect of surfactants on chlorophyll content of hydrilla. Hydrilla tips were exposed to surfactants at various concentrations fo r 48 h and chlorophyll content determined. Surfactant EC50 1 (95% CI2) Regression equation r2 CT-3013 Cygnet Plus 433 (329 642) y = 0.9008e-0.0016x 0.95 SilEnergy 408 (273 825) y = 0.7496e-0.0017x 0.88 Timberland 90 4,814 (3,180 9,763) y = 0.8553e-0.0001x 0.59 1 EC50 = Concentration (mg L-1) of surfactant required to reduce chlorophyll content by 50%. 2 95% CI = 95% Confidence Interval 3 Not valid, see text.

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72 CHAPTER 5 EFFECTS OF SURFACTANTS ON DIQU AT PHYTOTOXICITY TO SELECTED AQUATIC AND T ERRESTRIAL PLANTS Introduction Surfactants or surface active agents, known collectively as ad j uvants, are chemicals that have pronounced effects on the interface forces between the spray mixture and plant surfaces with the objective of providi ng more effective droplet depo sition, spread, and retention (Monaco et al., 2002; Hartzler and Foy, 1983). When an appr opriate selection is made, surfactants not only enhance he rbicide selectivity (Hess and Foy, 2000), but may also reduce the amount of herbicide required, reduce the ove rall cost of weed management, and improve the consistency of weed control (Zollinger, 2000). Sutton and Foy (1971) found that the addition of several surfactants at concentr ations from 0.001 to 1.0% to diquat increased betanin efflux from red beet ( Beta vulgaris L.) root discs. Menendez and Batisda (2004) reported 15 to 30% reduction of ED50 rates of diquat when combin ed with several surfactants at concentrations ranging from 0.05 to 1.0% compared to diquat alone on purslane ( Portulaca oleracea L.) and ryegrass ( Lolium rigidum Gaudin). Similar findings have been reported on other plant species with other herbicides and surfactants. Smith and Foy (1967) noted that the addition of 8 surfactants at 1,000 and 10,000 mg L-1 to paraquat, a compound chemically related to diquat, increas ed its toxicity on corn; however, a specific concentration of each type of surfactant was required. Nalewaja et al. (1995) observed that nicosulfuron was able to reduce the fresh weight of yellow foxtail [ Setaria glauca (L.) Beauv.] from 10 to 92%, depending on the type of surfactant used in the spray solution Tonks and Eberlein (2001) used rimsul furon in combination with crop oil concentrate, a nonionic, methylated seed oil a nd a silicone-polyether copolymer surfactants on common lambsquarters ( Chenopodium album L.), hairy nightshade ( Solanum sarrachoides Sendtner), kochia ( Kochia scoparia L.), redroot pigweed ( Amaranthus retroflexus L.) and

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73 volunteer oat ( Avena sativa L.) in potato. They concluded that effective weed control depended on herbicide applicationrate; type of adjuvant select ed and target species to be controlled. The addition of adjuvants has been shown to increase herbicide absorption (Hess and Foy, 2000), phytotoxicity (Ramsdale and Messersm ith, 2002) and control efficacy when the combination of herbicides plus adjuvants was fo liarly applied in terrestrial (Devendra et al., 2004; Ivany, 2004; Smith et al., 1999; Coret an d Chamel, 1993), emergent and floating plants (Syngenta, 2004; Fairchild et al., 2002; Shilling et al., 1990). However, there is no scientific evidence that confirms improved efficacy when surfactants are added to herbicides for submersed aquatic weed control. Research on surfactant-herbicide-water-pla nt relationships indi cate that increased performance of herbicides when combined w ith surfactants on terrestrial and emergent aquatic weeds is due to more rapid foliar uptake of herbicide. Koger et al. (2007) found that the combination of organosilicone-based nonion ic and methylated seed oil/organosilicone surfactants with urea ammonium nitrate reduced the rain-free period from 8 h to 1 or 4 h, respectively, and provided comple te control of barnyardgrass [ Echinochloa crus-galli (L.) Beauv.] when applied with bispyribac. Reddy and Singh (1992) reported increased glyphosate efficacy, and reduced critical rain-free period from 6 or 8 h to 15 min., when glyphosate was applied with an organosi licone-based adjuvant on velvetleaf ( Abutilon theophrasti Medik.), sicklepod ( Cassia obtusifolia L.), and yellow foxtail. These studies suggest that the fa ster or greater uptake of he rbicides is due to cuticular and stomatal infiltration from the plant surf ace, but submersed aquatic plants (including hydrilla) have little or no cu ticle, and stomata are absent or non-functional (Verma and Verma, 2000; Pendland, 1979; Sculthorpe, 1967). Therefore, the effect of surfactants on herbicide efficacy in submersed plants s hould be evaluated. Moreover, the aquatic

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74 environment where these plants grow greatly influences not only herbicide performance, but would be expected to impact the efficacy of the surfactants due to high dilution (Cedergreen and Streibig, 2005). Surfactant manufacturers starte d to recommend that surfactan ts be added to herbicides applied directly to water in the 1990s despite the fact that there wa s no scientific evidence suggesting surfactants increased herbicide efficacy in submerse d aquatic weed applications. Recommended surfactant concentrations for foliar applications are usually 2,500,000 mg L-1 and recommended concentrations of surfactants for submersed applications are 3 to 6 mg L-1. This is equivalent to 1 to 2 gallons of surfactant in 1 acre-ft of water (Appendix A). The concentration of surfactants used in terrestrial spray mixes is at least 833x greater than the concen tration recommended for use on submersed weeds. Green (1996) found that when surfactant wa s applied at 0.0008% of the spray volume with rimsulfuron, green foxtail [ Setaria viridis (L.) Beauv.] biomass was reduced by 10% or less, but control increased to 90% when surfact ant concentration was increased to 0.1%. Liu (2004) reported that glyphosate uptake by wheat ( Triticum aestivum L.) and bean foliage ( Vicia faba L.) increased from 5% and 44% to 17 % and 85%, respectively, when surfactant concentration was increased from 0.2 to 0.5% v/v in the spray mixture. Herbicide phytotoxicity increased when the carrier volu me was more concentrated (23 to 47 L ha-1 compared to 94 to 188 L ha-1), when several herbicides were applied on an area basis in a tank-mix with methylated vegetable oil (R amsdale and Nalewaja, 2001; Nalewaja and Ahrens, 1998). On the other hand, when surfactants were applied based on increasing percentage of the spray volume, there was a consistent increase in herbicide efficacy. These findings clearly show that surfactant concentra tion and carrier volume are critical factors for enhanced herbicide performance by the additi on of surfactants and there are no studies on terrestrial or aquatic plants which show that 3 to 6 mg L-1 of surfactant (0.0003-0.0006% v/v)

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75 provides improved herbicidal activity on plants In contrast, studies have shown that surfactant concentrations are most effective when applied at concentrations > 0.1% (> 1000 mg L-1) on terrestrial plants (Liu, 2004; Gaskin et al., 1996). However, leaves of terrestrial plants have functiona l stomata and often distinctive cu ticular layers, in contrast to submersed aquatic plants. Therefore, this st udy was conducted to evaluate the effects of surfactants on diquat phytotoxic ity on selected aquatic (duckw eed and hydrilla) and terrestrial (begonia and petunia) plants. Material and Methods Hydrilla Biomass Greenhouse studies were conducted f ollowing methods described previously for the surfactant phytotoxicity study (Chapter 4) to determine the effects of surfactants on hydrilla when combined with diquat. Experiment 1 a nd its repeat were run concurrently with surfactant phytotoxicity studies in May-June 2007, while Experiment 2 and its repeat were conducted in July-August 2007. Hydrilla tips in Experiment 1 were exposed to four surfactants (Cygnet Plus, SilEnergy, CT-301 and Timberland 90) at 0, 3 and 5,000 mg L-1 alone and in combination with diquat at 10 g L-1. Concentrations of 3 and 5,000 mg L-1 are the lowest and highest labeled rates recommended for aquatic a nd terrestrial use, respectively. Experiment 2 evaluated the effect of the highest aquatic use rate (6 mg L-1) and included surfactant concentrations of 0, 3, 6, 12 and 24 mg L-1 alone and in combination with diquat at 10 g L-1. Diquat concentrations in bo th studies were selected ba sed on previous studies that determined 10 g L-1 as the concentration required to reduce hydril la biomass by 50% (EC50). Hydrilla tips in Experiment 1 and 2 were exposed for 24 h, rinsed and planted in plastic tubes, as previously described, and allowed to grow for 2 wk and then harvested.

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76 The exposure time used in this study was lim ited to 24 h to simulate field conditions where diquat phytotoxic effects are usually short due to rapid degradation, dilution and other environmental conditions (WSSA, 2002). The variable time exposure of submersed plants to herbicides is somewhat similar to the ra in-free period so widely used in studying surfactant/herbicide combinations on terrestria l plants. The experiment was arranged as a completely randomized design and treatments in both experiments were replicated five times. Ornamental Biomass The ornam ental plants petunias ( Petunia x hybrida Dream) and begonia ( Begonia x semperflorens-cultorum Vodka Cocktail) were purchas ed in March 2007 from local nurseries and maintained under a 14 h photoperi od with maximum daytime temperatures of 28 3 C and nighttime temperatures of 18 3 C. Foliage of begonia and petunia were trea ted with four surfactants (Cygnet Plus, SilEnergy, CT-301 and Timber land 90) at 3 and 5,000 mg L-1 alone and in combination with diquat at 200 mg L-1. Treatments of the foliage of these plants were applied with a CO2 powered sprayer with a single XR Teejet 8002 VS nozzle at 30 PSI. A diluent volume of 561 L ha-1 was used to deliver all solutions. Contro l plants were sprayed with plain water (i.e., without surfactant or herbicide). Plants were chosen at rando m and harvested 11 DAT. Height from each treatment (from their base to th e tip of the longest leaf) were recorded, then all above ground green tissue was harvested a nd dried at 90 C for 5 d to determine dry weights. The experiment was arranged in a completely randomi zed design with four replications and it was repeated. Hydrilla Chlorophyll The concentration of chlorophyll in treated hydrilla plants was also used to determ ine effects of surfactants in combination with di quat in two series of experiments conducted in June-July 2007.

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77 In the first experiment, hydrilla tips were exposed to all four surfactants at 0, 3 and 5,000 mg L-1 and in combination with diquat at 10 g L-1. In the second study tips were exposed to 0, 3, 6, 12 and 24 mg L-1 alone and in combination with diquat at 10 g L-1. These series of studies were conducted followi ng the same procedures described for the phytotoxicity study and chlorophyll analysis outlined in Chapter 4. Hydrilla tips were exposed to treatment solutions for 48 h, rinsed and 0.1 g of the hydrilla tips were subsampled for chlorophyll extraction by DMSO for 6 h in a wa ter bath at 65 C. The experiments were arranged in a completely randomized design wi th five replications and were repeated. Landoltia Chlorophyll The duckweed, landoltia [ Landoltia punctata (G. Meyer) D.H. Les and D.J. Crawford], was another aquatic plant that was used to evaluate the phytotoxicity of surfactants in com bination with diquat. Landoltia plants were collected from a pond in Gainesville, Alachua County, FL in April 2007. Plants were maintained at the University of Florida, Center for Aquatic and Invasive Plants in 9.5-L aquaria with standard growth medium (Wang, 1990). The cultures were cleaned and the growth room was set to provide temperature of 26 4 C and a 16 h photoperiod. Small aquarium air pumps were used to maintain constant agitation with forced air. The growth medium in each aquarium was changed every 3 to 6 wk. Algal growth was minimized by addition of the dye Aquashade5 and aquaria were maintained under light levels of 150 10 mol m-2 s-1. Surfactant concentrations and all other procedures were the same as those in the hydrilla chlorophyll study, except that th e diquat concentration (EC50) was 0.5 g L-1 as determined in preliminary studies. The fresh weight of landoltia was obtained by weighing all fronds contained in each vial and the chlorophyll extraction time was 3 h. 5 Registered trademark of Applied Bi ochemists. Germantown, WI. 53022.

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78 Statistical Analysis All data collected from biomass and chlor ophyll studies were conve rted to percent dry weight reduction or percent chlorophyll reduction according to the formula: DWR = 100 [(treated plant dry weight or chlorophyll/nontreated plant dry weight or chlorophyll) x 100]. Converted data were then standardized, i.e., values < 0, or treatments that outgrew the nontreated plants were replaced by 0. Interactions (synergy, addition or antagonistic effects) between surfactants and diquat were determined following the method described by Colby (1967). Several other methods for determining the interaction betw een mixtures of herbicides exist (Tammes, 1964; Crafts and Raynor, 1936; Bliss, 1934); however, due to their complexity in data analysis and interpretation of the resulting interactions, Colbys method was selected for comparisons in these studies. In this method of analysis, th e expected value of dr y mass reduction for the combination surfactant-diquat is determined us ing the formula: E = [(X + Y) (XY/100)], where E is the percent of expected dry ma ss reduction, X the observed percent of dry mass reduction with compound 1 at rate xi and Y the observed percent of dry mass reduction with compound 2 at rate yi. Expected and observed values were separated using Mixed Procedures (SAS, 2002) with experiment, replication (nested within expe riment), and all intera ctions considered as random effects. Experiments and their repeats we re also considered random effects, and each treatment was considered a fixed effect to allow inferences about treatment s at different levels (Hager et al., 2003; Carmer et al., 1989). Type I II statistics were used to test all possible effects of fixed factors. Least square m eans were used for mean separation at p 0.05. If the observed value of the surfactant-diquat combin ation was significantly less than the expected value, the interaction was cons idered antagonistic. In contrast, if the observed value was higher than the expected value, the combination was considered as havi ng a synergistic effect.

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79 When there was not a significant difference be tween the two values, the interaction was considered additive. Results and Discussion Hydrilla Biomass Experiment 1 Diquat applied alone at the EC50 concentration (10 g L-1) caused a 67% reduction in dry weight compared to the nontreated control (Table 5-1). Cygnet Plus at 3 mg L-1 caused a 20% reduction in dry weight and, wh en combined with diquat at 10 g L-1, caused a 67% reduction in dry weight. The Co lby analysis predicted an ex pected 71% reduction in dry weight from the combination, which was not attained, so the comb ined effects were considered additive. All surfactants evaluated alone at the lowest aquatic labeled rate (3 mg L-1) as well as diquat when applied alone at the pre-determined EC50 concentration of 10 g L-1 did not cause a change in percent dry weight. Dry weight reduction by diquat alone was 67% and the addition of CT-301, Cygnet Plus, SilEnergy and Timberland 90 applied at the aquatic rate did not differ from diquat alone. Surfactants at 3 mg L-1 did not increase diquat performance above the expected value predicted by the Colby method; therefore, the combination response was additive. The effects of the addition of CT-301 and Cygnet Plus to diquat on hydrilla dry weight were greater than the control at 5000 mg L-1 but were not different from those of SilEnergy. Despite the variability of the diquat-surfactant combinations, the interaction of their effects was additive due to high surfact ant phytotoxicity to hydrilla when applied alone. Total plant length analysis yielded simila r results (data not shown). Experiment 2 The effects of surfactants applied at 0, 3, 6, 12 and 24 m g L-1 alone and in combination with diquat at the EC50 of 10 g L-1 on hydrilla dry weight ar e presented in Table 5-2.

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80 Reduction of hydrilla dry weight when diquat wa s applied alone was 36%, and the reduction caused by surfactants alone ranged from 1 to 12%. All surfactants evaluated at 3, 6, 12 and 24 mg L-1 had no effect on hydrilla dry weight when applied in combination with diquat, except Cygnet Plus at 24 mg L-1 and Timberland 90 at 6 mg L-1. Cygnet Plus at 24 mg L-1, a rate 4 times higher than the maximum recomme nded aquatic use rate, was the only surfactant that was synergistic or induced a significant increase in diquat efficacy when both compounds were applied in combination. Al l other surfactants, regardless of rate, were additive to the effect of diquat alone. The reduction in hydrilla dry weight when treated at the previously determined EC50 value (10 g L-1) was 67% in Experiment 1 (17% more than 50%) and 36% in Experiment 2 (14% reduction from 50%). Experiment 1 was conducted twice in the greenhouse in MayJune 2007, and Experiment 2 was also conducte d twice in July-August 2007. Daylength and hydrilla growth were at maximum levels dur ing Experiment 1, but the latter study was conducted under conditions of progressively shorter days and perhaps reduced growth, which may have decreased the effects of diquat and surfactants on hydrilla. Not only did diquat efficacy decrease between Experiment 1 and Experi ment 2 (67% to 36%), but the toxicity of surfactants applied at 3 mg L-1 also decreased from a 10-20% re duction in Experiment 1 to a 2-6% reduction in Experiment 2. Van et al. (1978) reported that in North Florida (where these experiments were conducted) hydrilla growth is greater during the summer months from May to July. Reduced hydrilla growth, starting from July through fall and winter months, may have contributed to reduced chemical efficacy as reported in a previous study (Madsen and Owens, 1998). The results of this study on EC10 values of diquat applied al one on hydrilla varied by 15% simply as a result of the time of year when plants were treated.

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81 Percent total length reducti on of hydrilla in Experiment 2 when diquat was applied alone was 75%, and the reduction caused by surfact ants alone varied from 4 to 22% (Table 53). Combinations of diquat and surfactant s reduced length from 54 to 86%. Interaction analysis between expected and obser ved data for Cygnet Plus (3 mg L-1), SilEnergy (3 and 6 mg L-1) and Timberland 90 (6 and 12 mg L-1) were antagonistic, whereas other rates were additive. There appears to be no synergy or benef it of adding surfactants at the recommended aquatic labeled rates (3 to 6 mg L-1) to diquat when treati ng hydrilla under greenhouse conditions. Even if surfactants improved the e fficacy of diquat on hydrilla by 10 to 15%, the variance in susceptibility and growth of hydrilla would make it impossible to show this slight benefit statistically. There is also a large disparity between fiel d application rates and diquat concentrations used to produce an EC50 in the greenhouse. Field applicati on rates of diquat are usually in excess of 300 g L-1 for hydrilla control, and the EC50 in our greenhouse studies was at least 30x less (10 g L-1). Very young, immature and actively growing hydrilla tips, grown in clean well water, are much more susceptible to diqua t, and likely surfactants, when compared to mature plants growing under field conditions, wh ere water mixing is likely to occur and other factors that reduce the half-life of diquat are usually presen t (Daz et al., 2002; Poovey and Getsinger, 2002; WSSA, 2002; Langeland et al., 1994). Ornamental Biomass Begonia Begonia dry weight and heights of treated and nontreated plants were not different in either experiment. The interaction between surfactants and diquat when applied in combination were not significant and therefore all responses were additive (data not shown). Treatment of these plants, with fully develope d waxy cuticles at the onset of flowering, may

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82 have contributed to the reduced effect of surf actants in combination with diquat in this study. These results are similar to the effects of di quat on rice in Chapter 2, which showed that mature plants are somewhat less affected by diqu at. There were no antagonistic or synergistic effects of diquat-surfactant combina tions evaluated in these studies. Petunia The single and com bined effects of diquat a nd surfactants on pet unia dry weight are presented in Table 5-4. Diquat and surfactan ts applied alone provided 14% and 5-17% reductions in percent dry weight, respectivel y. Despite preliminary experimentation which suggested an EC50 to petunia at 200 mg L-1, the more mature plants at the time of treatment only decreased by 14% in dry weight. Comb inations of surfactants at 3 mg L-1 with diquat at 200 mg L-1 reduced dry weight from 8 to 24% and their interaction with diquat was additive except for Cygnet Plus, which was antagoni stic according to Colbys analysis. The addition of surfactants at 5000 mg L-1 to diquat at 200 mg L-1 further increased diquat performance from 36 to 52%, and all diqua t-surfactant combinations provided greater percent reduction in dry weight compared to diquat treatments alone. This information shows that the addition of surfactants at the terrestrial rate to diqua t had the intended consequence of increasing the efficacy of the treatment. When the interaction between the combinations was analyzed, CT-301, SilEnergy and Timberland 90 were synergistic, while Cygnet Plus at 5000 mg L-1 was additive when combined with diquat. The effect of addition of SilEnergy at 5000 mg L-1 to diquat at 200 mg L-1 was not different from those of CT-301 and Timberland 90, but was greater than that of Cygnet Plus. These results may be due to the dual ability of orga nosilicone to penetrate plants through the cuticle and stomata (Roggenbuck et al., 1990; Field and Bishop, 1988), hence their use to increase the uptake and efficacy of microbial bi ocontrol agents (Hoeft et al., 2001; Sheikh et al., 2001), uptake of foliar nutrients, growth re gulator effects (Greenbe rg and Goldschmidt,

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83 1990; Coker et al., 1987) and increase efficacy of herbicides on numerous species (Koger et al., 2007; Jansen, 1973). Hydrilla Chlorophyll The effect of diquat and diquat com binations with surfactants on chlorophyll content of hydrilla was studied under controlled conditions in growth chambers. Surfactants applied alone at 3 mg L-1 caused chlorophyll content reduction in hydrilla varying from 0 to 7%, similar to observations of hydrilla tips treate d with diquat alone (Table 5-5). Chlorophyll reduction caused by diquat applied alone at 10 g L-1 was 1%. Addition of surfactants at 3 mg L-1 to diquat at 10 g L-1 caused chlorophyll content re duction ranging from 0 to 8%, which was not different from diquat alone. Ther efore, the interactio ns of all diquat and surfactant combinations at 3 mg L-1 were additive. Surfactants alone applied at terrestrial rate s (5000 mg L-1) caused chlorophyll reduction from 17 to 89%. When surfactants at the same rate were combined with diquat, chlorophyll reduction varied from 8 to 83%, with the hi ghest values corresponding to SilEnergy (83%) and Cygnet Plus (76%), and the lowest to Timberland 90 (50%) and CT-301 (8%). Although CT-301 gave the lowest pe rcent chlorophyll reduction at 5000 mg L-1, treated plants were visually bleach ed and their tissue was completely disintegrated. High total chlorophy ll readings, despite plant visual bleaching, could have been caused by chlorophyll a destruction into phaeophytin a caused by sulfuric acid, resulting in a shift in the absorp tion spectrum to 667 m (Rao and LeBlanc, 1966) Therefore, chlorophyll content of hydrilla once again was not a valid indicator of CT-301 effects on enhancement of diquat phytotoxicity. SilEnergy and Timberland 90 provided simila r results as in the previous study (see Chapter 4), indicating that they require a longer interval to exert their membrane fluidizing

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84 effects compared to the faster acting CT301 and Cygnet Plus, which in 24 h produced noticeable results on hydrilla. The EC50 for Cygnet Plus, SilEnergy and Timberland 90 from Table 4-3 (Chapter 4) were 483, 408 and 4,814 mg L-1, respectively. In this study, 5000 mg L-1 concentrations caused 76, 83 and 50% re ductions in chlorophyll content. Surfactants concentrations at 0, 3, 6, 12 and 24 mg L-1 applied alone and in combination with diquat had no effects on hydrilla chlor ophyll content 48 HAE (d ata not shown). Landoltia Chlorophyll Experiment 1 Surfactant and diquat effects when applied alone and in combination on landoltia are presented in Table 5-6. D iquat applied alone at 0.5 g L-1 reduced chlorophyll content of landoltia by 17% and reduction by surfactants alone at 3 mg L-1 ranged from 4 to 15%. The combination of surfactants at 3 mg L-1 with diquat at the EC50 concentration (0.5 g L-1) reduced chlorophyll content in th e range of 7 to 24%; however, there was no increase in the effect of diquat, because when diquat was a pplied alone it reduced landoltia chlorophyll content by 17%. When surfactant concentrations were added to diqua t at the terrestrial recommended rates, CT-301 and Timberland 90 were antagonistic and reduced diquat efficacy. Although CT-301 gave an antagonistic response, expose d plants were completely bleached and disintegrated, indicating that chlorophyll a destruction and shifts in spectrophotometer readings might ha ve played a role in these results, as previously discussed (see Chapter 4). Cygnet Plus applied at 5000 mg L-1 with diquat was additi ve and reduced landoltia chlorophyll content (48%) compar ed to the control plants. Ne vertheless, the reduction was less than 50%, and it was not different from CT-301 and Timberland 90 at 3 mg L-1. Reduced effects of Timberland 90 and SilEnergy at the highest concentrations may be related to the lack of buoyancy of landoltia pl ants and subsequent sinking in the highly concentrated

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85 surfactant solutions. Floa ting plants exposed to low concentr ations of surfactants and higher oxygen content in the air had higher reductions in chlorophyll content compared to sunken plants exposed to greater surf actant concentrations. Kaurov et al. (1993) reported reduced rate of chlorophyll bleaching under anaerobic conditi ons. Results from our studies corroborate their findings, due to the sim ilar behavior shown by landoltia in water containing surfactants at terrestrial rates, which ha s also been observed by others6. Experiment 2 Surfactant treatments at aquatic use concentrations (3-6 mg L-1) once again had little effect on the chlorophyll content of landoltia. Colby s criteria determined that all had an additive response (Table 5-7) at these concentrations when combined with diquat. Chlorophyll content of landoltia decreased fr om 19% with diquat alone to 38% when surfactant was added. CT-301 applied at concentrations of 3 and 6 mg L-1 and Timberland 90 at 6 mg L-1 in combination with diquat provided greater chlorophyll reduction than diquat alone, and the interaction analysis deemed these were additive effects. Cygnet Plus applied at twoand four-fold the labele d aquatic rate reduced chlo rophyll content by 38 and 47%, respectively, and provided the only synergistic effects observe d in this experiment. Although CT-301 and Timberland 90 applied at the same rate gave 48 and 49% chlorophyll content reduction, respectively, their response was additiv e due to their increased phytotoxicity when applied alone. In conclusion, the Colby analysis allows the comparison of chemical combinations and categorizes the results depending upon antagonistic, synergistic or additive effects. It is well known that herbicide-surfactant interaction depends upon charac teristics of leaf surfaces, plant species and many environmental factors. Resu lts of the studies in th is chapter are varied, 6 Koschnick and Mudge, 2005 personal communication

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86 but over 80% of the herbicide-surfactant combin ations tested were classified as additive (Table 5-8). There were nearly as many anta gonistic responses as there were synergistic determinations in these studies. The lowest c oncentration that showed synergy on hydrilla was Cygnet Plus at 24 mg L-1 based upon hydrilla dry weight (Table 5-2), and the lowest concentration that was classifi ed as antagonistic was 3 mg L-1 of Cygnet Plus and SilEnergy based upon hydrilla plant le ngth (Table 5-3). Combined, these results suggest little more than an additive effect when surfactants are combin ed with diquat for submersed weed control at surfactant concentrations of 3 and 6 mg L-1. It should also be noted that these concentrations are based on application of surf actants in 30 cm (1 foot) of wa ter; greater water depths would further dilute the surfactants. The variance in the methods used in these st udies is of a magnitude in which it would be impossible to detect significant effects of <15% or even greater improved efficacy as a result of adding surfactants to herbicid es for submersed weed control. The EC50 of diquat on hydrilla (10 g L-1) was determined in earlier studies. In Experiment 1 this concentration produced a 67% reduction in biomass, while in Experiment 2 it caused only a 36% biomass reduction. Similarly, 3 mg L-1 Cygnet Plus caused biomass re duction of 20 and 2% in the same two experiments, respectively. The other surfactants also demonstrated differences (though not 10x) between the two expe riments. Obviously, a more se nsitive assay is needed to determine if these herbicide-surfactant combinations provide more than additive effects. The surfactant concentrations shown to improve or speed herbicide efficacy in terrestrial foliar applications are between 2500 and 5000 mg L-1. Submersed aquatic plants have little or no cuticle and few or no functional stomata. The addition of 3 to 6 mg L-1 of surfactant would be considered significant if improved control of submersed weeds equalled or exceeded the cost of additional herbicide. An additive effect of 20% is likely significant

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87 enough to make the addition of surfactant to herbicide tank mixes fiscally justified. The problem is, in some cases the benefits were only 2% or less. These studies showed no synerg istic benefits of using su rfactants with diquat at maximum recommended aquatic rates for hydrilla control. However, the question remains as to whether or not the additive effect is consistently sufficient to justify the additional cost. In addition, the recommended rates for floa ting species, particul arly duckweed, were not beneficial due to reduced buoyancy and reduced herbicide efficacy. However, when Cygnet Plus was applied at 12 and 24 mg L-1 in combination with diquat at 0.5 g L-1, the concentration was synergistic. Although the comb ination diquat-surfactant interaction of other surfactants evaluated was additive, there was an increasing trend in diquat efficacy as the concentrations increased from 3 to 24 mg L-1 (Table 5-7). This illustrates that optimal surfactant rates for use with herbicides for floating aquati c weed control are unknown since 2500-5000 mg L-1 rates reduced herbicide efficacy. Conversely, surfactants performed their intended use when applied on terrestrial plants at recommended rates. Although the diquat concentration used in this study was nearly 3-7 times below the rates recommended for terrestrial weed control, increased efficacy occurred in petunia when diquat was applied in combination with surfactants. The role of the leaf cuticle on the uptake of diquat may be illustrated by comparing the effects of diquat on petunia, landoltia and hydr illa. Though not measured, we assume that the cuticle is greater on petu nia than on hydrilla. The EC50 value for hydrilla was 10 g L-1, while 200 mg L-1, a 20000 higher diquat concentration, reduced petunia dry weight by 14%.

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88Table 5-1. Effect of surfactants and diquat applied for 24 h alone or in combination on percent dry weight reduction of hydrill a. Hydrilla was grown in a tank with well water for 14 DAT before harvesting. Data pooled for two expe riments. Each value is the mean of 10 replications. Diquat (10 g L-1) Surfactant Rate X1 Y2 Expected P value Response mg L-1 ---------------% dry wei ght reduction ---------------P > t 3 None 0 0 67 bcd 0 CT-301 3 8 65 cd 68 0.6803 Additive CT-301 5000 100 100 a 100 1.000 Additive Cygnet Plus 3 20 67 bcd 71 0.7057 Additive Cygnet Plus 5000 100 100 a 100 1.000 Additive SilEnergy 3 19 60 cd 71 0.2124 Additive SilEnergy 5000 84 88 ab 94 0.4791 Additive Timberland 90 3 11 53 d 69 0.0824 Additive Timberland 90 5000 55 75 bc 84 0.3239 Additive 1 observed percent dry weight reduction when compound applied alone 2 observed percent dry weight reduction when compound combined with diquat 3 P values used to compare the differences betw een the observed and the expected values at P 0.05

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89Table 5-2. Effect of surfactants and diquat applied for 24 h alone or in combination on percent dry weight reduction of hydrill a. Hydrilla was grown in a tank with well water for 14 DAT before harvesting. Data pooled for two expe riments. Each value is the mean of 10 replications. Diquat (10 g L-1) Surfactant Rate X1 Y2 Expected P value Response mg L-1 -------------------------% dry weight reduction ------------------------P > t 3 None 0 0 36 cd 0 CT-301 3 6 38 bcd 39 0.6779 Additive CT-301 6 8 52 abcd 40 0.4962 Additive CT-301 12 6 54 abc 40 0.4911 Additive CT-301 24 4 50 bcd 38 0.4453 Additive Cygnet Plus 3 2 36 cd 37 0.9274 Additive Cygnet Plus 6 1 47 bcd 36 0.1581 Additive Cygnet Plus 12 2 54 abc 37 0.2057 Additive Cygnet Plus 24 6 70 a 40 0.0002 Synergistic SilEnergy 3 6 36 cd 39 0.9357 Additive SilEnergy 6 7 35 d 39 0.1106 Additive SilEnergy 12 5 46 bcd 39 0.522 Additive SilEnergy 24 12 44 bcd 43 0.3995 Additive Timberland 90 3 2 42 bcd 37 0.4886 Additive Timberland 90 6 5 57 ab 39 0.1703 Additive Timberland 90 12 4 41 bcd 38 0.7158 Additive Timberland 90 24 10 48 bcd 39 0.2584 Additive 1 observed percent dry weight reduction when compound applied alone 2 observed percent dry weight reduction when compound combined with diquat 3 P values used to compare the differences betw een the observed and the expected values at P 0.05

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90Table 5-3. Effect of surfactants and diquat applied for 24 h alone or in combination on percent total length reduction of hydrilla. Hydrilla was grown in a tank with well water for 14 DAT before harvesting. Data pooled for two expe riments. Each value is the mean of 10 replications. Diquat (10 g L-1) Surfactant Rate X1 Y2 Expected P value Response mg L-1 -------------------------% total length reduction ------------------------P > t 3 None 0 0 75 abcd 0 0 CT-301 3 11 70 bcde 78 0.2030 Additive CT-301 6 6 72 bcde 76 0.4742 Additive CT-301 12 19 74 bcd 80 0.3570 Additive CT-301 24 22 77 abc 80 0.6159 Additive Cygnet Plus 3 8 57 fg 77 0.0017 Antagonistic Cygnet Plus 6 4 66 cdef 76 0.1162 Additive Cygnet Plus 12 12 79 ab 78 0.9151 Additive Cygnet Plus 24 17 86 a 79 0.2681 Additive SilEnergy 3 6 54 g 76 0.0006 Antagonistic SilEnergy 6 14 62 efg 78 0.0125 Antagonistic SilEnergy 12 12 69 bcde 77 0.1988 Additive SilEnergy 24 14 71 bcde 78 0.2841 Additive Timberland 90 3 8 68 b-f 77 0.1775 Additive Timberland 90 6 16 65 defg 80 0.0209 Antagonistic Timberland 90 12 9 64 defg 77 0.0414 Antagonistic Timberland 90 24 17 73 bcde 79 0.3865 Additive 1 observed percent dry weight reduction when compound applied alone 2 observed percent dry weight reduction when compound combined with diquat 3 P values used to compare the differences betw een the observed and the expected values at P 0.05

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91 Table 5-4. Effect of surfactants and diquat applied alone or in combination on percent dry weight reduction of petunia. Petunia was grown in a greenhouse for 11 DAT before harvesting. Data pooled for two e xperiments. Each value is the mean of 8 replications. Diquat (200 mg L-1) Surfactant Rate X1 Y2 Expected P value Response mg L-1 ---------------% dry wei ght reduction ---------------P > t 3 None 0 0 14 de 0 CT-301 3 5 18 de 18 0.3556 Additive CT-301 5000 9 44 ab 22 <0.0001 Synergistic Cygnet Plus 3 9 8 e 22 0.0305 Antagonistic Cygnet Plus 5000 17 36 bc 28 0.2089 Additive SilEnergy 3 8 24 cd 21 0.6829 Additive SilEnergy 5000 12 52 a 24 0.0014 Synergistic Timberland 90 3 10 24 cd 23 0.8716 Additive Timberland 90 5000 12 43 ab 25 0.0025 Synergistic 1 observed percent dry weight reduction when compound applied alone 2 observed percent dry weight reduction when compound combined with diquat 3 P values used to compare the differences betw een the observed and the expected values at P 0.05

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92Table 5-5. Effect of surfactants and diqua t applied for 48 h alone or in combinati on on percent total ch lorophyll reduction of hydrilla. Hydrilla exposed in a growth chamber room and chlorophyll extracted w ith DMSO. Data pooled for two experiments. Each value is the mean of 6 replications. Diquat (10 g L-1) Surfactant Rate X1 Y2 Expected P value Response mg L-1 -----------------% total chlorophyll reduc tion --------------P > t 3 None 0 0 1 c 0 CT-301 3 1 0 c 2 0.7752 Additive CT-301 5000 17 8 c 18 0.2054 Additive Cygnet Plus 3 0 0 c 1 0.9423 Additive Cygnet Plus 5000 89 76 a 89 0.0956 Additive SilEnergy 3 7 0 c 8 0.3003 Additive SilEnergy 5000 85 83 a 85 0.7571 Additive Timberland 90 3 0 8 c 1 0.3375 Additive Timberland 90 5000 44 50 b 45 0.5140 Additive 1 observed percent dry weight reduction when compound applied alone 2 observed percent dry weight reduction when compound combined with diquat 3 P values used to compare the differences betw een the observed and the expected values at P 0.05

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93Table 5-6. Effect of surfactants and diqua t applied for 48 h alone or in combinati on on percent total ch lorophyll reduction of landoltia in Experiment 1. Landoltia exposed in a growth chamber room and chlorophyll extracted with DMSO. Data pooled for two experiments. Each value is the mean of 6 replications. Diquat (0.5 g L-1) Surfactant Rate X1 Y2 Expected P value Response mg L-1 --------------------% total chlorophyll redu ction ---------------P > t 3 None 0 0 17 bc 0 CT-301 3 15 13 bc 26 0.2960 Additive CT-301 5000 70 39 ab 73 0.0084 Antagonistic Cygnet Plus 3 7 17 bc 23 0.5961 Additive Cygnet Plus 5000 33 48 a 38 0.4382 Additive SilEnergy 3 0.3 7 c 17 0.4561 Additive SilEnergy 5000 6 8 c 20 0.3304 Additive Timberland 90 3 4 24 abc 21 0.7986 Additive Timberland 90 5000 9 0 c 26 0.0422 Antagonistic 1 observed percent dry weight reduction when compound applied alone 2 observed percent dry weight reduction when compound combined with diquat 3 P values used to compare the differences betw een the observed and the expected values at P 0.05

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94Table 5-7. Effect of surfactants and diqua t applied for 48 h alone or in combinati on on percent total ch lorophyll reduction of landoltia in Experiment 2. Landoltia exposed in a growth chamber room and chlorophyll extracted with DMSO. Data pooled for two experiments. Each value is the mean of 6 replications. Diquat (0.5 g L-1) Surfactant Rate X1 Y2 Expected P value Response mg L-1 -------------------% total chlorophyll reduc tion ---------------P > t 3 None 0 0 19 e 0 0 CT-301 3 14 36 abcd 31 0.5392 Additive CT-301 6 17 37 abc 33 0.5917 Additive CT-301 12 18 40 ab 33 0.3838 Additive CT-301 24 20 48 a 36 0.1409 Additive Cygnet Plus 3 1 20 e 20 0.9666 Additive Cygnet Plus 6 2 23 cde 20 0.7123 Additive Cygnet Plus 12 2 38 ab 21 0.0391 Synergistic Cygnet Plus 24 5 47 ab 23 0.0043 Synergistic SilEnergy 3 3 19 e 29 0.2492 Additive SilEnergy 6 6 22 de 32 0.2473 Additive SilEnergy 12 7 23 cde 31 0.3276 Additive SilEnergy 24 10 33 bcde 33 0.9416 Additive Timberland 90 3 7 33 bcde 32 0.9767 Additive Timberland 90 6 9 35 abcd 32 0.7150 Additive Timberland 90 12 18 48 a 38 0.1944 Additive Timberland 90 24 17 49 a 36 0.1153 Additive 1 observed percent dry weight reduction when compound applied alone 2 observed percent dry weight reduction when compound combined with diquat 3 P values used to compare the differences betw een the observed and the expected values at P 0.05

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95 Table 5-8. Interaction totals of the effects of surfactants applie d alone and in combination with diquat at sublethal concentrations on hydrilla, landoltia and petunia. Surfactant Antagonistic Additive Synergistic CT-301 1 18 1 Cygnet Plus 2 15 3 SilEnergy 2 17 1 Timberland 90 3 16 1

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96 CHAPTER 6 HERBICIDE COMBINATIONS FOR THE E NHANCEM ENT OF DIQUAT TOXICITY FOR HYDRILLA CONTROL Introduction Herbicide com binations for improved weed c ontrol in aquatic and terrestrial programs have been utilized for decades. Increased efficac y of combinations over the application of a compound alone is the primary reason to mix di fferent materials (Bal dwin and Oliver 1985); however, other benefits can also be accrued from herbicide mixtures. A wi der spectrum of weed species can be controlled when herbicides with different modes of action are combined in a single application (Norris et al., 2001). Combinations of different herbicides can also reduce the propensity of weeds to develop resistance (Mar shall, 1998), and can also reduce crop damage (Webster et al., 2006; Brown et al., 2004; Om okawa et al., 1996; Hoffman, 1953), application cost (Norris et al., 2001) and re duce the phytotoxicity of herbic ides to non-target organisms (Follak and Hurle, 2003), and consequently reduc e environmental concerns (Brimner et al., 2005). One of the most common examples of increased herbicide efficacy used in aquatic weed control is the use of diquat-copp er for hydrilla control when they are applied together, compared to the activity of each compound applied alone (S utton et al., 1972; Sutton et al., 1971; Sutton and Bingham, 1970; Blackburn and Weldon, 1970; M ackenzie and Hall, 1967). Further studies involving the combination of diqua t with endothall in aquatic (P oovey et al., 2002; Nelson et al., 2001; Pennington et al., 2001) and te rrestrial (Ivany, 2004) settings also revealed that both compounds were more effective when applied to gether than singly. Improved performance of other herbicides when applied in combination with diquat for the control of terrestrial weeds has also been reported. Most recently, Ivany (2005) found that pyraflufen-ethyl efficacy on potato

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97 leaf and vine desiccation was improved wh en diquat was applied after it in a follow up application or in combination as a single application. Although increased herbicide effi cacy has been observed in ma ny studies involving diquat, antagonistic effects have also been reported. Srensen et al. (2007) tested synergistic and antagonistic effects using a concen tration addition model in binary mixture toxicity studies and reported that acifluorfen was antagonis tic to diquat on duckweed (Lemna minor L.). Cedergreen et al. (2007) examined the toxic ity of six binary herbicide comb inations on pigment content and plant growth. They subjected the data to concentration addition (CA) and independent action (IA) reference models, both of which showed that acifluorfen combined with diquat was antagonistic. In a previous study, Cedergreen et al. (2006) examined the synergistic effects of prochloraz, an imidazole fungicide, when app lied in combination with diquat, azoxystrobin, acifluorfen, dimethoate, chlorfenvinphos and pi rimicarb on four aquatic species [bacteria ( Vibrio fischeri ), daphnia (Daphnia magna Straus), algae [ Pseudokirchneriella subcapitata (Korshikov) Hindak)] and duckweed. They found that, a lthough the mixtures between prochlorazazoxystrobin and diquat-esfenvalerat had shown a synergistic effect on daphnia, and diquatprochloraz were synergistic on algae, all combinations were antagonistic on duckweed. These results emphasize the need to test not only individual he rbicides, but also mixt ures of herbicides to identify possible adverse effects on non-targ et species and possible reduction in herbicide performance on target species. Furthermore, despite the knowledge of increased efficacy as the result of certain combinations, the type of interaction (synergy, additivity or antagonism) involved in most cases is not known. Therefore, this study was conducted to determine the extent of any interactions involved when diquat is mixed with acibenzolar, carfentrazone, copper, flumioxazin, and the alkylamine and dipotassium salts of endothall.

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98 Material and Methods Three series of greenhouse studies were c onducted to determine the effects of five herbicides and acibenzolar-s-methyl ( 1,2,3-benzothiadiazole-7-thiocarboxylic acid-s-methyl-ester), a plant elic itor or systemic acquired resist ance inducer in vegetables and tobacco (Syngenta, 2006), on the enhancement of diquat phytotoxicity on hydrilla. Experimental procedures followed methods previously describe d for the phytotoxicity studies. All experiments were conducted in July-August 2007 w ith hydrilla tips collected from the Rainbow River, FL. In the first series of experiments, hydrilla tips were exposed for 24 h in 0.5 L plastic cups containing DI water plus acibenzolar-s-methyl (hereafter refe rred as acibenzolar) and copper at different concentrations. Acibenzolar was a pplied at concentrations of 125, 250, 500, 1000 and 2000 g L-1, and copper at concentrations of 31.3, 62.5, 125, 250 and 500 g L-1. The second series of experiments included carfentrazone-e thyl (hereafter referred as carfentrazone) and flumioxazin. Carfentrazone was applied at 10, 25, 50, 100 and 200 g L-1 and flumioxazin at 12.5, 25, 50, 100 and 200 g L-1. The last series of phytotoxi city experiments on hydrilla tips compared the dipotassium and alkylamine salts of endothall at 5, 10, 25, 50, 100, 250 and 500 g L-1. All compounds were applied alone and in combination with diquat at the previously determined diquat EC50 concentration of 10 g L-1. Treatments in each series of studies were assigned in a completely randomized design, rep eated and replicated five times. Two weeks following a 24 h exposure to the compounds, hydrilla was harvested and tota l length (main stem plus any lateral shoots) was meas ured as previously described. Pl ants were then dried for dry weight determination.

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99 Statistical Analysis The interaction between each compound and di quat was determined following the method described by Colby (1967). Data were converted to percent dry weight reduction for evaluation in this analysis by the formula: DWR = 100 [( treated plant dry weight/nontreated plant dry weight) x 100]. Converted data were then standardized, i.e., values < 0 or treatments that outgrew the nontreated plants were replaced by 0. Based on this method, the expected value of dry mass reduction for th e herbicide-diquat combination is determined using the formula: E = [(X + Y) (XY/100)], where E is the percent of expected dry mass reduction, X the observed percent of dry mass re duction with compound 1 at rate x and Y the observed percent of dry mass reduction with compound 2 at rate y. Expected and observed values were separated using Mixed Procedures (SAS Institute, 2002) with experiment, replication (nested within experiment), and all interactions considered as random effects. Experiments and their repeats we re also considered random effects, and each treatment was considered a fixed effect to allow inferences about treatment s at different levels (Hager et al., 2003; Carmer et al., 1989). Type III st atistics were used to te st all possible effects of fixed factors. Least square means were used for mean separation at p 0.05. If the observed value of the combination of a compound with di quat was significantly less than the expected value, the interaction was consid ered antagonistic. In contrast, if the value was significantly higher than the expected value, the combination was considered synergistic. The interaction was considered additive when there was no significant difference between the two values. Data from all repeated sets of experiment s were not different; therefore, they were pooled for analysis. Results and Discussion The height o f hydrilla plants following the 14 d grow-out period was highly variable due to the random sprouting of adven titious buds in the leaf axils, which often occurred if the

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100 growing tip was damaged or destroyed by the 24 h herbicide exposure peri od. The total length of the main stem and all lateral shoots was a more accurate measure than plant height in response to these herbicide treatments. Diquat applied alone at the pre-determined EC50 concentration (10 g L-1) in the first experimental series resulted in a 44% reduction in hydrilla dry weight compared to nontreated control plants (Table 6-1). The reduction of dry weight with the addition of acibenzolar ranged from 50 to 69%, and the copper addition ra nged from 64 to 96%. Copper applied at concentrations of 125 g L-1 and greater produced greater dry weight reductions compared to acibenzolar, which at its highest concentration (2000 g L-1) was not different from copper at 31.3 g L-1. Although copper produced the highest dry mass reduction, its response was additive, whereas acibenzolar had two syne rgistic responses at 1000 and 2000 g L-1. Synergistic effects between two or more compounds occur whenever th eir combined effects are greater than the sum of the effects of each compound applied alone (Kim et al., 2002). High copper toxicity to hydrilla when applied alone (21 to 89%) compar ed to acibenzolar (4 to 17%) and average efficacy increases of 21% and 49% when combined with diquat, respectively, contributed to the toxicity of the combination, but Colbys analysis determined this to be an additive response. The effects of these treatments on the total length of hydrilla are presented in Table 6-2. The response to all acibenzolar treatments with diquat was additive. Copper at 62.6 g L-1 antagonized diquat efficacy; howev er, at other concentrations, it was considered additive. In the second series of studies, diqu at applied alone at the expected EC50 concentration reduced dry weight by 46% (Table 6-3). Carfen trazone applied alone reduced dry weight by 12 to 27%, while flumioxazin reductions similarl y ranged from 7 to 28%. Combinations of carfentrazone with diquat furthe r increased hydrilla dry weight reduction from 49 to 66%, while

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101 flumioxazin addition reduced dr y weight similarly by 49 to 58%. Both carfentrazone and flumioxazin had very similar rate responses above 25 mg L-1, and there were few differences from the application of diquat alone. This similarity is likely due to their common mechanism of action (PPO inhibitors) (Iver son and Vandiver, 2005; WSSA 2002) and rapid hydrolytic degradation in high pH water (Elmarakby et al., 2001). The pH of the deionized water used in these studies was 7.1. Conversely, when interaction responses were analyzed based on total length of hydrilla, carfentrazone growth inhibition varied from 5 to 24% and was synergistic with diquat at all concentrations. Flumioxazin addition to diquat antagonized diquat activity at 12.5 g L-1 and reduced growth by 18 to 26%. At flumioxazin concentrations >12.5 g L-1 all responses were additive (Table 6-4). Based on the results of both compounds, the synergistic effect exhibited by carfentrazone may have resulted from the standa rdization of the data and correction of outliers, when it was applied alone, which lowered th e corresponding means and consequently the calculated expected valu e due to variability in length of hydrilla, particularly at 10 g L-1. This resulted in a synergistic effect, even though there was no difference between the herbicide mixture and diquat applied alone, in contrast to the synergistic e ffect observed in the Cygnet Plus and acibenzolar studies. Therefor e, whenever a mixture of two or more compounds results in a synergistic or additive interac tion, a second analysis must be made between the mixture and individual compounds alone to av oid misleading results. Cauti on in interpreting interaction results when using Colbys method has been called upon previously to avoid misjudgments (Gruzdev and Kalinin, 1967; Colby et al., 1965). While there are other methods or models that can be used to determine the type of interaction between herbicide mixt ures, all of these models are likely to be affected by the large

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102 variance in hydrilla susceptibility to diquat. Moreove r, other methods are considered more liberal than Colbys; therefore results analyzed using Colbys method are more robust and conservative. The dipotassium and alkylamine salts of e ndothall applied for 24 h alone followed by a 14 d growth period reduced hydrilla dry weight by 4 to 14% (Table 6-5) and 8 to 31%, respectively. Addition of diquat at the EC50 concentration to these tw o endothall salts increased dry weight reductions from 45 to 71% and 47 to 78%, respectively. However, the observed response for both compounds at all concentrations was additive. Total length of hydrilla yielded results very similar to the dr y weight data (Table 6-6). Higher dry weight reductions a nd complete control of hydrilla when endothall salts or copper were combined with diquat have been re ported (Pennington et al., 2001; Netherland et al., 1991; Blackburn and Weldon, 1970), and increased efficacy of these combinations on other aquatic weeds has also been observed in other studies (Nelson et al., 2001; Netherland et al., 2000). However, the concentrations of copper, endothall and diquat have always been higher than those evaluated in this study. The type of response from these combinations, including others involving diquat, has ne ver been determined nor modeled based upon exposure to sublethal doses. These results, with the exception of the carfentrazone an d diquat results on total hydrilla length, indicate that these combinations are all additive. The EC50 of a 24 h exposure to diquat, followed by a 14 d grow out period, was predetermined in several studies conducted prior to these experiments. The EC50 of diquat (10 g L-1) applied alone had dry weight reductions of 44, 46 and 53%, and reduced total length by 62, 69 and 70% compared to the growth of nont reated plants. The evaluation of herbicide combinations to determine additive or synergis tic responses can only be conducted at sublethal herbicide concentrations or exposure times; otherwise valid comparisons are impossible.

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103 The variability of the growth reduction at 10 g L-1 of diquat by the response in dry weight was 9% (44-53%), and for total plant length was 8% (62-70%), which suggests that this method was very effective at providing meaningful result s on the interaction (addi tive, antagonistic or synergistic) effects of these ch emical combinations. These results also suggest that this method would not likely determine if the addition of a he rbicide, surfactant, or other additive to diquat provides an improvement in efficacy of as lit tle as 5 to 10%, as claimed by some field applicators. Moreover, the inte rpretation of the results shoul d not be limited only to the comparison of the expected and mixture results but also to the mixture and each compound applied alone, since in some cases, an a dditive effect may also mean no effect. Reports by applicators which suggest improved efficacy in the field appear to be due largely to additive effects, with the exception of the synergistic diquat/acibenzolar effects on dry weight at 1000 and 2000 g L-1. Field or large scale studies w ith acibenzolar and carfentrazone are warranted to confirm these greenhouse studies, but variance in field applications will also increase. Carfentrazone will be subjected to furthe r studies due to the significant additive effect on dry weight and the synergistic effect on total length that exhibi ted in combination with diquat. Potentially, a shorter exposure period, e. g. 12 h, might be used with high er diquat and additive concentrations to determine the effects of c oncentrations more typical of field use.

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104Table 6-1. Effect of diquat, acibenzolar and copper exposed for 24 h alone or in combination on the dry weight (% reduction) of hydrilla. Hydrilla was harvested 14 DAT. Diquat (10 g L-1) Compound Rate X1 Y2,3 Expected P value Response g L-1 -----------------% dry weight reduc tion ----------------P > t 4 None 0 0 44 d 0 Acibenzolar 125 4 50 cd 46 0.5753 Additive Acibenzolar 250 10 56 bcd 50 0.4194 Additive Acibenzolar 500 17 64 bc 54 0.1630 Additive Acibenzolar 1000 14 69 b 46 0.0028 Synergistic Acibenzolar 2000 16 65 bc 48 0.0188 Synergistic Copper 31.3 21 65 bc 55 0.2086 Additive Copper 62.5 54 69 b 75 0.4743 Additive Copper 125 66 89 a 83 0.4128 Additive Copper 250 78 96 a 88 0.3185 Additive Copper 500 89 95 a 94 0.7974 Additive 1 Observed percent dry weight reduction when compound applied alone. 2 Observed percent dry weight reduction when compound combined with diquat. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05. 4 P values used to compare the differences betw een the observed and the expected values at P 0.05.

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105Table 6-2. Effect of diquat, acibenzolar and copper exposure for 24 h alone or in combination on the total length (% reduction) of hydrilla. Hydrilla was harvested 14 DAT. Diquat (10 g L-1) Compound Rate X1 Y2,3 Expected P value Response g L-1 ----------------% total length reduc tion ----------------P > t 4 None 10 0 69 cd 0 0 Acibenzolar 125 11 71 bcd 72 0.8628 Additive Acibenzolar 250 18 64 d 73 0.3012 Additive Acibenzolar 500 10 86 abc 71 0.1070 Additive Acibenzolar 1000 20 74 abcd 75 0.9291 Additive Acibenzolar 2000 22 76 abcd 75 0.9255 Additive Copper 31.3 16 88 ab 74 0.1183 Additive Copper 65.5 50 67 d 85 0.0101 Antagonistic Copper 125 44 80 abcd 83 0.7386 Additive Copper 250 56 92 a 86 0.5118 Additive Copper 500 76 91 a 92 0.9517 Additive 1 Observed percent dry weight reduction when compound applied alone. 2 Observed percent dry weight reduction when compound combined with diquat. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05. 4 P values used to compare the differences betw een the observed and the expected values at P 0.05.

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106Table 6-3. Effect of diquat, carfentrazone and flumioxazin exposure for 24 h alone or in combination on the dry weight (% reduction) of hydrilla. H ydrilla was harvested 14 DAT. Diquat (10 g L-1) Compound Rate X1 Y2,3 Expected P value Response g L-1 -----------------% dry weight reduc tion ----------------P > t 4 None 0 0 46 c 0 Carfentrazone 10 12 49 abc 52 0.7028 Additive Carfentrazone 25 16 66 a 55 0.1455 Additive Carfentrazone 50 17 61 abc 55 0.4618 Additive Carfentrazone 100 18 58 abc 56 0.7443 Additive Carfentrazone 200 27 66 a 60 0.4415 Additive Flumioxazin 12.5 7 49 abc 50 0.8539 Additive Flumioxazin 25 14 54 abc 54 0.9828 Additive Flumioxazin 50 17 50 abc 56 0.3890 Additive Flumioxazin 100 16 54 abc 54 0.9926 Additive Flumioxazin 200 28 58 abc 59 0.9290 Additive 1 Observed percent dry weight reduction when compound applied alone. 2 Observed percent dry weight reduction when compound combined with diquat. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05. 4 P values used to compare the differences betw een the observed and the expected values at P 0.05.

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107Table 6-4. Effect of diquat, carfentrazone and flumioxazin applied for 24 h alone or in combination on the total length (% redu ction) of hydrilla. Hydrilla was harvested 14 DAT. Diquat (10 g L-1) Compound Rate X1 Y2,3 Expected P value Response g L-1 ----------------% total length reduc tion ----------------P > t 4 None 10 0 70 abc 0 0 Carfentrazone 10 5 62 bc 34 0.0003 Synergistic Carfentrazone 25 16 82 a 51 0.0008 Synergistic Carfentrazone 50 14 77 abc 39 <0.0001 Synergistic Carfentrazone 100 11 71 abc 39 0.0006 Synergistic Carfentrazone 200 24 79 ab 48 0.0012 Synergistic Flumioxazin 12.5 26 60 c 79 0.0456 Antagonistic Flumioxazin 25 22 74 abc 78 0.7193 Additive Flumioxazin 50 21 62 bc 77 0.3588 Additive Flumioxazin 100 18 77 abc 76 0.4826 Additive Flumioxazin 200 24 76 abc 77 0.4103 Additive 1 Observed percent dry weight reduction when compound applied alone. 2 Observed percent dry weight reduction when compound combined with diquat. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05. 4 P values used to compare the differences betw een the observed and the expected values at P 0.05.

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108Table 6-5. Effect of diquat, dipotassium and alkylamine salts of endothall exposure fo r 24 h alone or in combination on the dry weight (% reduction) of hydrilla. H ydrilla was harvested 14 DAT. Diquat (10 g L-1) Compound Rate X1 Y2,3 Expected P value Response g L-1 -----------------% dry weight reduction -----------------P > t 4 None 0 0 53 bcde 0 Dipotassium 5 4 49 cde 55 0.5727 Additive Dipotassium 10 4 47 e 55 0.3615 Additive Dipotassium 25 8 52 bcde 58 0.5517 Additive Dipotassium 50 5 45 e 56 0.2361 Additive Dipotassium 100 13 49 cde 60 0.2576 Additive Dipotassium 250 11 60 abcde 57 0.6728 Additive Dipotassium 500 14 71 ab 56 0.0934 Additive Alkylamine 5 8 48 de 56 0.3622 Additive Alkylamine 10 11 47 e 60 0.1330 Additive Alkylamine 25 11 53 bcde 60 0.4690 Additive Alkylamine 50 14 70 abc 60 0.2573 Additive Alkylamine 100 9 69 abcd 58 0.2355 Additive Alkylamine 250 14 59 abcde 57 0.8133 Additive Alkylamine 500 31 78 a 67 0.2222 Additive 1 Observed percent dry weight reduction when compound applied alone. 2 Observed percent dry weight reduction when compound combined with diquat. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05. 4 P values used to compare the differences betw een the observed and the expected values at P 0.05.

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109Table 6-6. Effect of diquat, dipotassium and alkylamine salts of endothall exposure fo r 24 h alone or in combination on the tot al length (% reduction) of hydrilla. H ydrilla was harvested 14 DAT. Diquat (10 g L-1) Compound Rate X1 Y2,3 Expected P value Response g L-1 ----------------% total length reduc tion ----------------P > t 4 None 0 0 62 cd 0 Dipotassium 5 14 67 bcd 68 0.9228 Additive Dipotassium 10 14 59 d 70 0.2513 Additive Dipotassium 25 17 82 ab 70 0.2472 Additive Dipotassium 50 14 66 bcd 67 0.9435 Additive Dipotassium 100 23 75 abc 74 0.9229 Additive Dipotassium 250 16 73 abc 69 0.7001 Additive Dipotassium 500 16 85 a 67 0.0600 Additive Alkylamine 5 11 69 bcd 66 0.7641 Additive Alkylamine 10 23 63 cd 72 0.3554 Additive Alkylamine 25 16 65 cd 71 0.5162 Additive Alkylamine 50 13 86 a 67 0.0565 Additive Alkylamine 100 13 86 a 68 0.0638 Additive Alkylamine 250 27 76 abc 70 0.5183 Additive Alkylamine 500 46 85 a 81 0.7036 Additive 1 Observed percent dry weight reduction when compound applied alone. 2 Observed percent dry weight reduction when compound combined with diquat. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05. 4 P values used to compare the differences betw een the observed and the expected values at P 0.05.

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110 CHAPTER 7 SUMMARY AND CONCLUSIONS Irrigation studies were conducted to determ ine if diquat, with a high affinity for adsorption to soils and short half-life in wa ter, would be a candidate for aquatic weed control in irrigation canals. Six agronomic crops (corn, cotton, rice, soybean, squash and wheat) were selected and subjected once to various diquat concentrations ranging from 0.05 to 200 mg L-1 in irrigation water at the germination, seedli ng and vegetative growth stages. Rice seed germinated and maintained in treated water for 14 d was more sensitive to diquat than any of the other pl ants. Diquat concentra tions required to reduce dry weight of germinating rice seeds by 10% (EC10) were 0.016 mg L-1 and 0.004 mg L-1 for shoots and roots, respectively. Diquat concentrati ons required to reduce dry weight s of all crops at the seedling stage, including rice at the tillering and mature stages, varied from 1.11 to 5 mg L-1. In contrast, seeds of corn, cotton, soybean, squash, and wheat germinated in sand and treated once with diquat at planting were more tolerant to diquat. The EC10 values ranged from 3 to 6 mg L-1. These results suggest that irri gation of freshly planted rice with diquat-treated water at the aquatic labeled rate (0.37 mg L-1) needs to be restricted to avoid reduc tion of crop stand; how ever, restrictions on irrigation of mature plants or seeds planted in sand or other types of soil with diquat-treated water seems unnecessary, confirming previous reports by Appleby and Brenchley (1968) and Mudge et al. (2007). Biomass and chlorophyll studies indicated that CT-301, Cygnet Plus, SilEnergy and Timberland 90 surfactants applied alone were phytotoxic to hydrilla at concentrations much greater than the recommended c oncentrations for aquatic weed control. Regression models

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111 yielded EC50 values ranging from 26 to 592 mg L-1 for hydrilla dry weight and from 433 to 4,814 mg L-1 for chlorophyll reduction. Addition of surfactants and herbicides to the EC50 diquat concentration (10 g L-1) to determine synergy between chemical combinatio ns was analyzed using Colbys method (Colby, 1967). The combination of surfactan ts at the aquatic (3 mg L-1) and terrestrial (5000 mg L-1) labeled rates to diquat at pre-determined EC50 values of 0.5 and 10 g L-1 for landoltia and hydrilla, respectively, were additive based on the reduction of dry weight. However, Cygnet Plus applied at a concentration 4x hi gher than the recommended aquatic use rate was synergistic. Based upon these results, surfactant enhancemen t of diquat phytotoxic ity on hydrilla when applied at the maximum aquatic labeled rate (6 mg L-1) was -1, 11, 16 and 21% for SilEnergy, Cygnet Plus, CT-301 and Timberland 90, respectively. These results suggest th at there is little benefit of adding surfactants to diquat for hydri lla control. Also, SilEnergy reduced diquat performance (antagonistic), indicatin g that there is also a need to evaluate surfactants for their compatibility with herbicides to avoid adverse effects. Surfactants are often found to be synergists wh en added to foliarly applied herbicides at concentrations > 2,500 mg L-1, presumably increasing or speed ing the uptake of herbicides across the leaf cuticle. The combination of diqua t and surfactants at a quatic recommended rates was additive on petunia; however, the terrestrial rate was gene rally synergistic. This shows that surfactants perform their intended use on terrestrial species, but only at concentrations much higher than those recommended for aquatic use. Surfactant addition at 3 to 6 mg L-1 to diquat was also deemed additive on chlorophyll content of landoltia. These results are similar to those reported for hydrilla, indicating once again

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112 that the benefit of surf actant addition at aquatic labeled rates to diquat is minimal. However, Cygnet Plus applied at 12 and 24 mg L-1 was synergistic. The combination of diquat with the herbicides carfentrazone copper, flumioxazin, and the alkylamine and dipotassium salts of endothall al so provided an additive effect based on dry weight at all concentrations evaluated. Acibenzolar, based on dry weight, and carfentrazone, based on hydrilla total le ngth, were synergistic, warranting furt her field studies to confirm these greenhouse results. All herbicide and diquat combin ations tested in these studies yielded non-synergistic effects. They did increase diquat performance by 12 to 25%, except for copper, which increased diquat efficacy by 52% based on dry weight. These results suggest that higher hydrilla dry weight reductions can be expected if these herbicides are combined with diquat at labeled rates. Additionally, synergistic effects from labeled compounds for hydrilla control are unlikely or would require thorough titrations, assuming th at the effects of each compound under normal conditions can be predicted. Previous studies that compared the effect of two chemicals in combination using the Colby method of analysis depended upon a reliable estimate of the EC50 of at least one of the compounds. The contribution of each chemical in a combination is impossible to determine if all doses were lethal to the plants. Several rate titrations were conducted to determine the EC50 of diquat on hydrilla dry weight and reduction of chlorophyll in landoltia. In our combination studies, the EC50 diquat concentrations (10 g L-1) gave hydrilla dry weig ht reductions of 36, 44, 46, 53 and 67%. The EC50 for dry weight reduction thus varied by 14 to 17%, which was similar to variation in the initial studies conducted to determine the EC50 values for diquat.

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113 Though all these values were close to the EC50, this level of variance makes it impossible to detect improvements in efficacy of < 10 to 15%. Commercial aqua tic applicators have reported improved efficacy when using surfactants with contact herbicides for submersed weed control. This may incl ude more complete or faster cont rol, a broader sp ectrum of weeds controlled, or control of a difficult to manage species. This improved efficacy has never been quantified in replicated st udies. Interestingly, even in very tightly controlled experiments such as conducted in this study, plant response to diquat alone varied with time of year, source of plants, age of plants, light intensity, dayl ength and other factors. Thus, it is possible that the surfactants evaluated in this study do improve efficacy by as much as 10%. These improvements are likely to be additive only, not synergistic. Moreover, thes e studies, as designed, will not be able to detect those slight improveme nts if they do occur.

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114 APPENDIX A LABELS OF SURFACTANTS USED IN THIS STUDY Figure A-1. Cygnet Plus label c ontaining product ingredients, use rate, handling and precautions for use.

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115 Figure A-2. CT-301 label containi ng product ingredients, use rate, handling and precautions for use.

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116 Figure A-2. Continued.

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117 Figure A-3. SilEnergy label cont aining product ingredients, use rate, handling and precautions for use.

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118 Figure A-4. Timberland 90 label containing product ingredient s, use rate, handling and precautions for use.

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119 Figure A-4. Timberland 90 label containing product ingredient s, use rate, handling and precautions for use.

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120 APPENDIX B COMPARISONS BETWEEN SURFACTANTS WHE N COMBINED WITH DIQUAT AT DIFFERENT CONCENTRATIONS

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121Table B-1. Effect of surfactants and diqua t applied in combination for 24 h on percent dry weight reduction of hydrilla and p-v alues for combinations comparison. Diquat (10 g L-1) + Cygnet Plus CT-301 SilEnergy Timberland 90 Compound Rate 3 5000 3 5000 3 5000 3 5000 DRW2,3 mg L-1 P > t 1 % Diquat (10 g L-1) + 0.9475 0.0003 0.8182 0.0003 0.4123 0.0192 0.1219 0.3771 67 bcd Cygnet Plus 3 0.0003 0.7675 0.0003 0.3759 0.0228 0.1069 0.4136 67 bcd Cygnet Plus 5000 0.0001 1.000 <0.0001 0.1778 <0.0001 0.0052 100 a CT-301 3 0.0001 0.5550 0.0103 0.1873 0.2660 65 cd CT-301 5000 <0.0001 0.1778 <0.0001 0.0052 100 a SilEnergy 3 0.0017 0.4652 0.0894 60 cd SilEnergy 5000 0.0001 0.1419 88 ab Timberland 90 3 0.0156 53 d Timberland 90 5000 75 bc 1P values used to compare the differences betw een the observed (combinations) values at P 0.05. 2 DWR = Percent dry weight reduction 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05.

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122Table B-2. Effect of surfactants and diqua t applied in combination for 24 h on percent dry weight reduction of hydrilla and p-values for combinations comparison. Diquat (10 g L-1) + Cygnet Plus CT-301 SilEnergy Timberland 90 Compound Rate 3 6 12 24 3 6 12 24 3 6 12 24 3 6 12 24 DWR3,4 mg L-1 P > t 2 % Diquat 1 + 0.9236 0.1449 0.1631 <0.0001 0.7606 0.0398 0.0169 0.0697 0.9692 0.9236 0.2067 0.2844 0.4040 0.0634 0.5333 0.1150 36 cd Cygnet Plus 3 0.1736 0.1944 <0.0001 0.8356 0.0500 0.0219 0.0859 0.9533 0.8469 0.2538 0.3303 0.4608 0.7840 0.5979 0.1391 36 cd Cygnet Plus 6 0.9495 0.0041 0.2485 0.5474 0.3492 0.7205 0.1558 0.1204 0.8448 0.6978 0.5323 0.6886 0.4040 0.9055 47 bcd Cygnet Plus 12 0.0034 0.2754 0.5060 0.3146 0.6737 0.1751 0.1362 0.8947 0.7452 0.5747 0.6426 0.4406 0.8555 54 abc Cygnet Plus 24 <0.0001 0.0230 0.0521 0.0130 <0.0001 <0.0001 0.0022 0.0011 0.0005 0.0134 0.0002 0.0059 70 a CT-301 3 0.0795 0.0370 0.1309 0.7902 0.6888 0.0370 0.1309 0.5960 0.1203 0.7490 0.2032 38 bcd CT-301 6 0.7379 0.8072 0.0436 0.0314 0.4253 0.3224 0.2204 0.8406 0.1513 0.6291 52 abcd CT-301 12 0.5629 0.0188 0.0130 0.2579 0.1857 0.1190 0.5921 0.0770 0.4136 54 abc CT-301 24 0.0758 0.0562 0.5791 0.4557 0.3262 0.9658 0.2334 0.8110 50 bcd SilEnergy 3 0.8930 0.2209 0.3021 0.4261 0.0691 0.5579 0.1242 36 cd SilEnergy 6 0.1744 0.2435 0.3522 0.0510 0.4713 0.0946 35 d SilEnergy 12 0.8473 0.6681 0.5509 0.5229 0.7532 46 bcd SilEnergy 24 0.8132 0.4303 0.6552 0.6122 44 bcd Timberland 90 3 0.3055 0.8335 0.4574 42 bcd Timberland 90 6 0.2170 0.7780 57 ab Timberland 90 12 0.3406 41 bcd Timberland 90 24 48 bcd 1 Diquat applied at 10 g L-1 2 P values used to compare the differences between the observed (combinations) values at P 0.05. 3 DWR = Percent dry weight reduction 4 Means within a column followed by the same letter are not significantly different according to the t-test on difference of leas t square means at P 0.05.

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123Table B-3. Effect of surfactants and diqua t applied in combination for 24 h on perc ent total length reduction of hydrilla and p-values for combinations comparison. Diquat (10 g L-1) + Cygnet Plus CT-301 SilEnergy Timberland 90 Compound Rate 3 6 12 24 3 6 12 24 3 6 12 24 3 6 12 24 DWR3,4 mg L-1 P > t 2 % Diquat 1 + 0.0045 0.1709 0.5675 0.0717 0.4494 0.6157 0.8802 0.7720 0.0014 0.0456 0.3611 0.5742 0.2787 0.1212 0.0884 0.7999 75 abcd Cygnet Plus 3 0.1390 0.0007 <0.0001 0.0366 0.0191 0.0071 0.0018 0.7157 0.03961 0.0531 0.0224 0.0775 0.1939 0.2524 0.0096 57 fg Cygnet Plus 6 0.0526 0.0016 0.5392 0.3852 0.2228 0.0973 0.0655 0.5272 0.6475 0.4187 0.7741 0.8565 0.7369 0.2642 66 cdef Cygnet Plus 12 0.2181 0.1845 0.2832 0.4701 0.7780 0.0002 0.0103 0.1379 0.2572 0.0983 0.0342 0.0231 0.4094 79 ab Cygnet Plus 24 0.0107 0.0215 0.0511 0.1304 <0.0001 0.0002 0.0068 0.0183 0.0040 0.0009 0.0005 0.0401 86 a CT-301 3 0.7990 0.5446 0.2956 0.0142 0.2129 0.8753 0.8456 0.7436 0.4267 0.3423 0.6149 70 bcde CT-301 6 0.7253 0.4286 0.0069 0.1338 0.6807 0.9522 0.5608 0.2942 0.2287 0.8037 72 bcde CT-301 12 0.6596 0.0023 0.0645 0.4456 0.6809 0.3510 0.1617 0.1202 0.9181 74 bcd CT-301 24 0.0005 0.0222 0.2292 0.3945 0.1700 0.0661 0.0464 0.5870 77 abc SilEnergy 3 0.2254 0.0214 0.1500 0.0334 0.0965 0.1316 0.0032 54 g SilEnergy 6 0.2762 0.0123 0.3581 0.6516 0.7669 0.0807 62 efg SilEnergy 12 0.7250 0.8648 0.5235 0.4278 0.5093 69 bcde SilEnergy 24 0.6018 0.3225 0.2526 0.7577 71 bcde Timberland 90 3 0.3581 0.5334 0.4065 68 b-f Timberland 90 6 0.8768 0.1946 65 defg Timberland 90 12 0.1466 64 defg Timberland 90 24 73 bcde 1 Diquat applied at 10 g L-1 2 P values used to compare the differences between the observed (combinations) values at P 0.05. 3 DWR = Percent dry weight reduction. 4 Means within a column followed by the same letter are not significantly different according to the t-test on difference of leas t square means at P 0.05.

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124Table B-4. Effect of surfactants and diqua t applied in combination for 24 h on perc ent dry weight reduction of petunia and p-values for combinations comparison. Diquat (200 mg L-1) + Cygnet Plus CT-301 SilEnergy Timberland 90 Compound Rate 3 5000 3 5000 3 5000 3 5000 DWR2,3 mg L-1 P > t 1 % Diquat (200 mg L-1) + 0.3674 0.0004 0.4964 <0.0001 0.1070 <0.0001 0.1121 <0.000114 de Cygnet Plus 3 <0.0001 0.1147 <0.0001 0.0125 <0.0001 0.0133 <0.00018 e Cygnet Plus 5000 0.0042 0.1708 0.0512 0.0085 0.0485 0.2089 36 bc CT-301 3 <0.0001 0.3491 <0.0001 0.3612 <0.000118 de CT-301 5000 0.0010 0.2002 0.0010 0.9093 44 ab SilEnergy 3 <0.0001 0.9814 0.0015 24 cd SilEnergy 5000 <0.0001 0.1634 52 a Timberland 90 3 0.0014 24 cd Timberland 90 5000 43 ab 1 P values used to compare the differences betw een the observed (combinations) values at P 0.05. 2 DRW = Percent dry weight reduction. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05.

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125Table B-5. Effect of surfactants and diqua t applied in combination for 24 h on perc ent reduction of total chlorophyll of hydril la and pvalues for combinations comparison. Diquat (10 g L-1) + Cygnet Plus CT-301 SilEnergy Timberland 90 Compound Rate 3 5000 3 5000 3 5000 3 5000 DWR2,3 mg L-1 P > t 1 % Diquat (10 g L-1) + 0.9588 <0.0001 0.9177 0.3534 0. 9177 <0.0001 0.3240 <0.0001 1 c Cygnet Plus 3 <0.0001 0.9588 0. 3274 0.9588 <0.0001 0.2994 <0.0001 0.4 c Cygnet Plus 5000 <0.0001 <0.0001 <0.0001 0.3567 <0.0001 <0.0001 76 a CT-301 3 0.3027 1.000 <0.0001 0.2762 <0.0001 0 c CT-301 5000 0.3027 <0.0001 0.9532 <0.0001 8 c SilEnergy 3 <0.0001 0.2762 <0.0001 0 c SilEnergy 5000 <0.0001 <0.0001 83 a Timberland 90 3 <0.0001 8 c Timberland 90 5000 50 b 1 P values used to compare the differences betw een the observed (combinations) values at P 0.05. 2 DWR = Percent dry weight reduction. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05.

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126 Table B-6. Effect of surfactants and diqua t applied in combination for 24 h on percent total chlorophyll reduction of landoltia and pvalues for combinations comparison. Diquat (0.5 g L-1) + Cygnet Plus CT-301 SilEnergy Timberland 90 Compound Rate 3 5000 3 5000 3 5000 3 5000 DWR2,3 mg L-1 P > t 1 % Diquat (0.5 g L-1) + 0.4174 0.1978 0.8872 <0.00010.1932 0.3742 0.3232 0.1847 17 bc Cygnet Plus 3 0.1940 0.8958 <0.00010.1970 0.3802 0.3285 0.5472 17 bc Cygnet Plus 5000 0.0095 0.0829 0.0002 0.0010 0.0007 0.0024 48 a CT-301 3 <0.00010.3112 0.5494 0.4850 0.7472 13 bc CT-301 5000 0.0023 0.0079 0.0059 0.0168 39 ab SilEnergy 3 0.8756 0.7976 0.9044 7 c SilEnergy 5000 0.7662 0.9369 8 c Timberland 90 3 0.2363 24 abc Timberland 90 5000 0 c 1 P values used to compare the differences betw een the observed (combinations) values at P 0.05. 2 DWR = Percent dry weight reduction. 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05.

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127 Table B-7. Effect of surfactants and diqua t applied in combination for 24 h on percent total chlorophyll reduction of landoltia and pvalues for combinations comparison. Diquat (0.5 g L-1) + Cygnet Plus CT-301 SilEnergy Timberland 90 Compound Rate 3 6 12 24 3 6 12 24 3 6 12 24 3 6 12 24 DWR3 mg L-1 P > t 2 %4 Diquat 1 + 0.6815 0.9560 0.0316 0.0009 0.0744 0.0428 0.0151 0.0004 0.5964 0.8990 0.9705 0.1382 0.1711 0.0835 0.0004 0.0002 19 e Cygnet Plus 3 0.6868 0.0267 0.0013 0.0575 0.0350 0.0140 0.0007 0.9178 0.8060 0.7464 0.1014 0.1236 0.0639 0.0006 0.0004 20 e Cygnet Plus 6 0.0695 0.0047 0.1340 0.0876 0.0395 0.0026 0.6125 0.8746 0.9364 0.2162 0.2551 0.1465 0.0023 0.0017 23 cde Cygnet Plus 12 0.3065 0.7499 0.9142 0.8058 0.2258 0.0205 0.0486 0.0581 0.5608 0.4962 0.7147 0.2142 0.1812 38 ab Cygnet Plus 24 0.1801 0.2584 0.4370 0.8500 0.0009 0.0029 0.0037 0.1091 0.0890 0.1654 0.8258 0.7525 47 ab CT-301 3 0.8329 0.5725 0.1263 0.0453 0.0978 0.1145 0.7925 0.7175 0.9627 0.1189 0.0981 36 abcd CT-301 6 0.7237 0.1873 0.0271 0.0622 0.0738 0.6355 0.5668 0.7965 0.1772 0.1486 37 abc CT-301 12 0.3340 0.0105 0.0267 0.0325 0.4081 0.3545 0.5410 0.3187 0.2748 40 ab CT-301 24 0.0005 0.0015 0.0020 0.0734 0.0590 0.1152 0.9753 0.8996 48 a SilEnergy 3 0.7273 0.6696 0.0817 0.1005 0.0505 0.0004 0.0003 19 e SilEnergy 6 0.9379 0.1634 0.1952 0.1076 0.0014 0.0010 22 de SilEnergy 12 0.1881 0.2233 0.1256 0.0018 0.0013 23 cde SilEnergy 24 0.9213 0.8288 0.0686 0.0554 33 bcde Timberland 90 3 0.7527 0.0550 0.0441 33 bcde Timberland 90 6 0.1082 0.0890 35 abcd Timberland 90 12 0.9241 48 a Timberland 90 24 49 a 1 Diquat applied at 0.5 g L-1 2 P values used to compare the differences between the observed (combinations) values at P 0.05. 3 DWR = Dry weight reduction 4 Means within a column followed by the same letter are not significantly different according to the t-test on difference of leas t square means at P 0.05.

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128 APPENDIX C COMPARISONS BETWEEN HERBICIDES WH EN COM BINED WITH DIQUAT AT DIFFERENT CONCENTRATIONS

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129Table C-1. Effect of diquat on hydrilla dr y weight when combined with acibenzola r and copper. Hydrilla was exposed to the combination for 24 h and allowed to grow for 14 d after treatment. Diquat (10 g L-1) + Acibenzolar Copper Compound Rate 125 250 500 1000 2000 31.3 62.5 125 250 500 DWR2,3 g L-1 ------------------------------------------------------------P > t 1 --------------------------------------------------------------------% Diquat + 10 0.3997 0.1170 0.0078 0.0010 0.0048 0.0060 0.0010 <0.0001 <0.0001 <0.0001 44 d Acibenzolar 125 0.4668 0.0676 0.0139 0.0464 0.0548 0.0133 <0.0001 <0.0001 <0.0001 50 cd Acibenzolar 250 0.2696 0.0817 0.2045 0.2312 0.0792 <0.0001 <0.0001 <0.0001 56 bcd Acibenzolar 500 0.5221 0.8687 0.9254 0.5125 0.0012 <0.0001 <0.0001 64 bc Acibenzolar 1000 0.6348 0.5846 0.9882 0.0089 0.0005 0.0005 69 b Acibenzolar 2000 0.9428 0.6243 0.0021 <0.0001 <0.0001 65 bc Copper 31.3 0.5745 0.0016 <0.0001 <0.0001 71 b Copper 62.5 0.0093 0.0005 0.0005 69 b Copper 125 0.3701 0.3780 89 a Copper 250 0.9883 96 a Copper 500 95 a 1 P values used to compare the differences between the observed and the expected values at P 0.05. 2 DWR = Percent dry weight reduction 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05.

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130Table C-2. Effect of diquat on hydrilla to tal length when combined with acibenzola r and copper. Hydrilla was exposed to the combination for 24 h and allowed to grow for 14 d after treatment. Diquat (10 g L-1) + Acibenzolar Copper Compound Rate 125 250 500 1000 2000 31.3 65.5 125 250 500 DWR2,3 g L-1 -------------------------------------------------------------P > t 1 ---------------------------------------------------------------------% Diquat + 10 0.8191 0.6141 0.0563 0.5587 0.3995 0.0276 0.4898 0.2142 0.0083 0.0110 69 cd Acibenzolar 125 0.4637 0.0925 0.7217 0.5391 0.0481 0.3581 0.3106 0.0157 0.0204 71 bcd Acibenzolar 250 0.0161 0.2765 0.1785 0.0070 0.8521 0.0813 0.0017 0.0024 64 d Acibenzolar 500 0.1841 0.2842 0.7663 0.0096 0.5020 0.4585 0.5197 86 abc Acibenzolar 1000 0.7963 0.1045 0.02027 0.5102 0.0390 0.0492 74 abcd Acibenzolar 2000 0.1716 0.1258 0.6888 0.0706 0.0870 76 abcd Copper 31.3 0.0040 0.3331 0.6568 0.7288 88 ab Copper 65.5 0.0539 0.0009 0.0003 67 d Copper 125 0.1583 0.1890 80 abcd Copper 250 0.9222 92 a Copper 500 91 a 1 P values used to compare the differences between the observed and the expected values at P 0.05. 2 DWR = Percent dry weight reduction 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05

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131Table C-3. Effect of diquat on hydrilla dry weight when combined with carfentrazone and flumioxazin. Hydrilla was exposed to th e combination for 24 h and allowed to grow for 14 d after treatment. Diquat (10 g L-1) + Carfentrazone Flumioxazin Compound Rate 10 25 50 100 200 12.5 25 50 100 200 DWR2,3 g L-1 ------------------------------------------------------------P > t 1 -------------------------------------------------------------------% Diquat + 10 0.6759 0.0087 0.0496 0.0998 0.0105 0.7259 0.2987 0.6079 0.2789 0.1141 46 c Carfentrazone 10 0.0269 0.1214 0.2187 0.0318 0.9462 0.5340 0.9242 0.5057 0.2444 49 abc Carfentrazone 25 0.5025 0.3218 0.9466 0.0226 0.1102 0.0341 0.1202 0.2907 66 a Carfentrazone 50 0.7483 0.5461 0.1061 0.1456 0.3526 0.3756 0.6989 61 abc Carfentrazone 100 0.3555 0.1946 0.5423 0.2563 0.5716 0.9473 58 abc Carfentrazone 200 0.0268 0.1258 0.0401 0.1369 0.3223 66 a Flumioxazin 12.5 0.4906 0.8708 0.4636 0.2182 49 abc Flumioxazin 25 0.5983 0.9652 0.5869 54 abc Flumioxazin 50 0.5684 0.2849 50 abc Flumioxazin 100 0.6173 54 abc Flumioxazin 200 58 abc 1 P values used to compare the differences between the observed and the expected values at P 0.05. 2 DWR = Percent dry weight reduction 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05

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132Table C-4. Effect of diquat on hydr illa total length when combined with carfentrazone and flumioxazin. Hydrilla was exposed to the combination for 24 h and allowed to grow for 14 d after treatment. Diquat (10 g L-1) + Carfentrazone Flumioxazin Compound Rate 10 25 50 100 200 12.5 25 50 100 200 DWR2,3 g L-1 ------------------------------------------------------------P > t 1 -------------------------------------------------------------------% Diquat + 10 0.4459 0.1729 0.4220 0.8696 0.3331 0.3318 0.6033 0.8886 0.9750 0.9419 70 abc Carfentrazone 10 0.0341 0.1181 0.4648 0.0842 0.8347 0.2003 0.5337 0.4648 0.4905 62 bc Carfentrazone 25 0.5744 0.1633 0.6918 0.0201 0.3982 0.1330 0.1633 0.1512 82 a Carfentrazone 50 0.4041 0.3812 0.0768 0.7767 0.3458 0.4041 0.3812 77 abc Carfentrazone 100 0.4213 0.2567 0.7221 0.7610 0.8450 0.8126 71 abc Carfentrazone 200 0.0532 0.6535 0.2681 0.3177 0.2981 79 ab Flumioxazin 12.5 0.1367 0.4061 0.3476 0.3693 60 c Flumioxazin 25 0.5095 0.5816 0.5535 74 abc Flumioxazin 50 0.9135 0.9465 62 bc Flumioxazin 100 0.9669 77 abc Flumioxazin 200 76 abc 1 P values used to compare the differences between the observed and the expected values at P 0.05. 2 DWR = Percent dry weight reduction 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05

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133Table C-5. Effect of diquat on hydrilla dry weight when combined with dipotassium a nd alkylamine salts of endothall. Hydrilla w as exposed to the combination for 24 h and allowed to grow for 14 d after treatment. Diquat (10 g L-1) + Dipotassium Alkylamine Compound Rate 5 10 25 50 100 250 500 5 10 25 50 100 250 500 DWR2,3 g L-1 --------------------------------------------------------------------P > t 1 ----------------------------------------------------------------------% Diquat + 10 0.7031 0.4866 0.9571 0.3741 0.7040 0.4047 0.0399 0.5904 0.4653 0.9757 0.0566 0.0818 0.4876 0.0058 53 bcde Dipotassium 5 0.7529 0.7435 0.6114 0.9990 0.2249 0.0150 0.8751 0.7269 0.6807 0.0224 0.0341 0.2825 0.0017 49 cde Dipotassium 10 0.5209 0.8467 0.7519 0.1267 0.0061 0.8747 0.9725 0.4677 0.0095 0.0151 0.1649 0.0006 47 e Dipotassium 25 0.4037 0.7444 0.3750 0.0351 0.6281 0.4988 0.9328 0.0501 0.0729 0.4545 0.0050 52 bcde Dipotassium 50 0.6105 0.0855 0.0034 0.7256 0.8737 0.3579 0.0053 0.0088 0.1139 0.0003 45 e Dipotassium 100 0.2254 0.0151 0.8741 0.7260 0.6815 0.0225 0.0342 0.02830 0.0017 49 cde Dipotassium 250 0.2205 0.1706 0.1184 0.4221 0.2819 0.3631 0.8893 0.0533 60 a-e Dipotassium 500 0. 0097 0.0055 0.0430 0.8812 0.7517 0.1726 0.4773 71 ab Alkylamine 5 0.8477 0.5695 0.0147 0.0230 0.2181 0.0010 48 de Alkylamine 10 0.4469 0.0086 0.0137 0.1548 0.0005 47 e Alkylamine 25 0.0607 0.0873 0.5069 0.0064 53 bcde Alkylamine 50 0.8674 0.2244 0.3898 70 abc Alkylamine 100 0.2944 0.3046 69 abcd Alkylamine 250 0.0384 59 a-e Alkylamine 500 78 a 1 P values used to compare the differences between the observed and the expected values at P 0.05. 2 DWR = Percent dry weight reduction 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05

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134Table C-6. Effect of diquat on hydrilla total length comb ined with dipotassium and alkylamin e salts of endothall. Hydrilla was exposed to the combination for 24 h and allowed to grow for 14 d after treatment. Diquat (10 g L-1) + Dipotassium Alkylamine Compound Rate 5 10 25 50 100 250 500 5 10 25 50 100 250 500 DWR2,3 g L-1 --------------------------------------------------------------------P > t 1 ---------------------------------------------------------------------% Diquat + 10 0.6448 0.6929 0.0502 0.7058 0.2036 0.3059 0.0197 0.5190 0.9398 0. 8292 0.0186 0.0166 0.1713 0.0227 62 cd Dipotassium 5 0.3922 0.1337 0.9334 0.4172 0.5731 0.0608 0.8540 0.6998 0.8062 0.0579 0.0525 0.3638 0.0685 67 bcd Dipotassium 10 0.0188 0.4400 0.0960 0.1563 0.0065 0.2986 0.6380 0.5415 0.0061 0.0053 0.0782 0.0076 59 d Dipotassium 25 0.1135 0.4902 0.3483 0.7038 0.1880 0.0597 0. 0812 0.6899 0.6584 0.5531 0.7463 82 ab Dipotassium 50 0.3709 0.5177 0.0502 0.7890 0.7627 0.8715 0.0478 0.0432 0.3214 0.0568 66 bcd Dipotassium 100 0.8041 0.2860 0.5303 0.2317 0.2909 0.2764 0.2578 0.9227 0.3110 75 abc Dipotassium 250 0.1887 0.7043 0.3428 0.4188 0.1816 0.1679 0.7300 0.2075 73 abc Dipotassium 500 0.0906 0.0240 0.0341 0.9828 0.9484 0.3320 0.9570 85 a Alkylamine 5 0.5691 0.6677 0.0866 0.0790 0.4688 0.1013 69 bcd Alkylamine 10 0.8885 0.0227 0.0203 0.1961 0.0275 63 cd Alkylamine 25 0.0324 0.0291 0.2489 0.0389 65 cd Alkylamine 50 0.9656 0.3214 0.9398 86 a Alkylamine 100 0.3008 0.9056 86 a Alkylamine 250 0.3595 76 abc Alkylamine 500 85 a 1 P values used to compare the differences between the observed and the expected values at P 0.05. 2 DWR = Percent dry weight reduction 3 Means within a column followed by the same letter are not signif icantly different according to the t-test on difference of leas t square means at P 0.05

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165 BIOGRAPHICAL SKETCH Tom s Fernando Chiconela was born on April 12, 1966, in Bilene-Macia (Gaza), Mozambique. He graduated from high school in 1986 and enrolled at th e Faculty of Agronomy and Forestry Engineering of Eduardo Mondlan e University (EMU) in Maputo. In 1993, he earned his B.S. in agronomy, concentrating on weed ecology and his thesis was entitled Contribuio para o Estudo Flors tico-ecolgico da Vegetao Infestante do Vale do Umbelzi Thereafter, he joined EMU as a researcher and lecturer of weed science. After three years of lecturing and heading th e Plant Protection Secti on of the Department of Plant Production and Protection, he enrolled for graduate studies in we ed science at Orange Free State University in Blomf ontein, South Africa. In 1999, he earned his masters degree and the title of his thesis was Effect of Spray Volume, Water Q uality, Adjuvants and Ammonium Salts on Sethoxydim Activity Following the receipt of his masters degree, he returned to Mozambique and continued lecturing Weed Science and Pes ticides Application. In 1999, he was appointed Chair of the Department of Plant Production and Protection. In 2003, he was awarded a Ford Foundation Scholarship for his doctoral degree at the University of Florida in the Plant Medicine Program (DPM), concentrating on pest mana gement and received his DPM degree in 2006. Under the same sponsor, he enrolled for his PhD in weed science at the same university, under the guidance of Dr. William T. Haller, focusi ng on the effect of adjuvants and herbicide combinations on phytotoxicity of diquat to hydrill a. He authored several publications on the ecology and management of pests. While a graduate student at the University of Florida, he was member of the Southern, Midwest and Florida Weed Science Societies an d the Aquatic Plant Management Society where he gave several presentations.