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Characterization of Flumioxazin as an Aquatic Herbicide

Permanent Link: http://ufdc.ufl.edu/UFE0021559/00001

Material Information

Title: Characterization of Flumioxazin as an Aquatic Herbicide
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Mudge, Christopher R
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aquatic, flumioxazin, herbicide, hydrilla, invasive, irrigation, light, native, non, ph, photosynthesis, plant
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: The aquatic weed, Hydrilla verticillata has spread throughout Florida?s waterways, clogging irrigation canals, reducing productivity of recreational fisheries, impeding navigation, and displacing native plants. The state of Florida spends $20 million per year to control hydrilla. There are only five registered aquatic herbicides that control hydrilla and in recent years, hydrilla has developed resistance to the most commonly used of these herbicides. The focus of this research was to characterize and develop flumioxazin as an aquatic herbicide for hydrilla control. New herbicides such as flumioxazin will aid in resistance management and hinder further resistance development. Flumioxazin is desirable because it has a relatively short half-life (minutes to days), low environmental toxicity, and is applied at low use rates. The registration of this herbicide will aid in controlling hydrilla and other invasive aquatic species, returning Florida's lakes to its native flora.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Christopher R Mudge.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Haller, William T.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021559:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021559/00001

Material Information

Title: Characterization of Flumioxazin as an Aquatic Herbicide
Physical Description: 1 online resource (120 p.)
Language: english
Creator: Mudge, Christopher R
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: aquatic, flumioxazin, herbicide, hydrilla, invasive, irrigation, light, native, non, ph, photosynthesis, plant
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: The aquatic weed, Hydrilla verticillata has spread throughout Florida?s waterways, clogging irrigation canals, reducing productivity of recreational fisheries, impeding navigation, and displacing native plants. The state of Florida spends $20 million per year to control hydrilla. There are only five registered aquatic herbicides that control hydrilla and in recent years, hydrilla has developed resistance to the most commonly used of these herbicides. The focus of this research was to characterize and develop flumioxazin as an aquatic herbicide for hydrilla control. New herbicides such as flumioxazin will aid in resistance management and hinder further resistance development. Flumioxazin is desirable because it has a relatively short half-life (minutes to days), low environmental toxicity, and is applied at low use rates. The registration of this herbicide will aid in controlling hydrilla and other invasive aquatic species, returning Florida's lakes to its native flora.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Christopher R Mudge.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Haller, William T.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021559:00001


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CHARACTERIZATION OF FLUMIOXAZIN AS AN AQUATIC HERBICIDE


By

CHRISTOPHER R. MUDGE












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

































O 2007 Christopher R. Mudge




































To my patient and loving wife, Erin, as well as my parents Alvin and Wanda whose love and
support has never ended, and to my departed grandparents Houston and Hazel Doucet; I miss
you both.









ACKNOWLEDGMENTS

First and foremost, I thank God for all he has blessed me with throughout my life. Without

him, little would be possible. A thank you goes to Dr. Bill Haller for the opportunity to study

under his tutelage and teach me so much about aquatic weed research. He provided me with an

opportunity to learn about aquatic research by starting with a new compound and taking it close

to registration. Appreciation is also extended to my committee members Drs. Jay Ferrell, Greg

MacDonald, Bill Stall, and Kevin Kenworthy for your advice and guidance. It has been a

pleasure collaborating with Dr. Mike Netherland of the U. S. Army Corps of Engineers on many

aspects of my research, and I thank him also for his guidance and perspectives on several issues.

Margaret Glenn is greatly appreciated for all her help from the moment I arrived as a student

until the day I finished my dissertation. I am lucky to have worked with and learned from

several outstanding weed science graduate students and post-docs including Brett Bultemeier,

Dr. Tyler Koschnick, Tomas Chiconela, Dr. Lyn Gettys, Dr. Atul Puri, and Eileen Ketterer.

Brett and Tyler are especially appreciated for helping me with research and class work. David

Mayo, Cole Hullon, William Jordan, and Michael Aldridge were all vital in data collection.

Without the unyielding and relentless support of Drs. Mike Riffle and Joe Chamberlin of Valent

U.S.A. Corporation, flumioxazin would not have had the opportunity to be evaluated for use in

aquatics. I appreciate their suggestions for studies and discussions over the last two years. The

use of time, boats, and willingness to learn about new herbicides is acknowledged by the crews

at the St. John's River Water Management District (Johnnie Drew, Tom Boyette, Shawn Moore,

Richard Krantz, and James Godfrey). Much gratitude is due to Mr. Sonny Phillips, Dr. Seigfred

Fagerberg, Osceola County, and the South Florida Water Management District for use of ponds

in research. The financial assistance provided by the Aquatic Ecosystem Restoration Foundation

(AERF) and the Florida Department of Environmental Protection (FDEP) is greatly appreciated.










Finally, a most grateful and heartfelt thank you is long overdue to my entire family for

their support during this special time in my life. I am so blessed to have a wife who loved and

encouraged me throughout the course of this experience. I thank her for being at my side and

always loving me no matter what the circumstance. Finally, my parents taught me to never settle

for less than what I could accomplish. I have learned from them how to work hard and have fun

with what I love the most.











TABLE OF CONTENTS


page

ACKNOWLEDGMENT S ................. ...............4.......... ......


LIST OF TABLES ................ ...............8............ ....

LI ST OF FIGURE S .............. ...............9.....


AB S TRAC T ............._. .......... ..............._ 12...


CHAPTER


1 INTRODUCTION ................. ...............14.......... ......

2 THE EFFECT OF FLUMIOXAZIN ON HYDRILLA CONTROL IN NATURAL
SYSTEMS AND THE INFLUENCE OF WATER PH ON HYDRILLA CONTROL IN
ME SOCO SMS ............... ...............2


Introducti on ................. ...............24.................
Materials and Methods .............. ...............24....
Effcacy in Ponds ................. ...............24........... ....
Effcacy in Mesocosms............... ...............2
Impact of pH on Effcacy ................ ........................ ......... ..............26
Results and Discussion .............. ...............28....
Effcacy in Ponds ................. ...............28...............
Effcacy in Mesocosms............... ...............3
Impact of pH on Effcacy ........._..... .....___ ....___ ....___.. ......_._.......33

3 THE EFFECT OF FLUMIOXAZIN ON HYDRILLA PHOTOSYNTHESIS AND
CHLOROPHYLL CONTENT .............. ...............41....


Introducti on ........._.__...... ..__ ...............41....
Materials and Methods .............. ...............42....
Photosynthesis and pH .............. ...............42....
Photosynthesis and Light.................... ........ .........4
Effect of Contact Herbicides on Photosynthesis .............. ...............45....
Chlorophyll Content ................. ...............45.......... .....
Results and Discussion .............. ...............46....
Photosynthesis and pH .............. ...............46....
Photosynthesis and Light.................... ..... ...........4
Effect of Contact Herbicides on Photosynthesis .............. ...............50....
Chlorophyll Content ................. ...............51.......... .....

4 THE EFFECT OF FLUMIOXAZIN ON SUBMERSED, EMERGENT, AND
FLOATING AQUATIC PLANT PLANTS .............. ...............59....












Introducti on ................. ...............59.................
Materials and Methods .............. ...............60....
Floating Aquatic Plants .............. ...............60....
Submersed Aquatic Plants ................. ...............62........... ....
Emergent Aquatic Plants .............. ...............64....
Results and Discussion .............. ...............65....
Floating Aquatic Plants .............. ...............65....
Submersed Aquatic Plants ................. ...............68........... ....
Emergent Aquatic Plants .............. ...............70....

5 ORNAMENTAL AND ROW CROP SUSCEPTIBILITY TO FLUMIOXAZIN INT
IRRIGATION WATER............... ...............82.


Introducti on ................. ...............82.................
Materials and Methods .............. ...............82....
Ornamental Susceptibility .............. ...............82....
Crop Susceptibility ................. ...............84.......... .....
Results and Discussion .............. ...............85....
Ornamental Susceptibility .............. ...............85....
Crop Susceptibility .............. ...............86....

6 THE EFFECT OF FLUMIOXAZIN AND DIQUAT ON MEMBRANE
PERMEABILIITY AND CHLOROPHYLL CONTENT OF LANDOLTIA ..........._..........92


Introducti on ........ ................. ...............92.......
Materials and Methods .............. ...............92....
lon Leakage .............. ...............93....
Chlorophyll ................. ...............94........ ......
Results and Discussion .............. ...............95....
Ion Leakage .............. ...............95....
Chlorophyll ................. ...............95........ ......


7 SUMMARY AND DRAFT AQUATIC USE DIRECTIONS............... ...............9


Sum m ary ........._...... .. ......... ...............99.....
Draft Aquatic Use Directions .............. ...............102....
General information............... ..............10
Mixing Guidelines ........._...... ...............103...__..........
Control of Submersed Weeds ................. ...............103.__._. .....
Subsurface Application .............. ...............104....
Surface/Foliar Application .............. ............... 104...
Plant Susceptibility ................. ...............104......... ......
Irrigation Restrictions ............ ............ ...............105...

LIST OF REFERENCES ............ ............ ...............108...


BIOGRAPHICAL SKETCH ............ ........... ...............120....










LIST OF TABLES


Table page

2-1 Hydrilla infested ponds in Florida treated with flumioxazin under an Experimental
Use Permit in 2006............... ...............37..

3-1 The effect of flumioxazin on photosynthesis of apical hydrilla tips at high (9.0) and
low (6.0) pHa. .......... ..... ._ __ ...............55....

3-2 The effect of flumioxazin (400 Clg L^1 a.i.) at pH 9.0 on photosynthesis of apical
hydrilla tips cultured in a growth chamber for 168 h at low, medium, and high light
quantities. ........._.__...... .__ ...............57....

3-3 The effect of select contact herbicides at pH 9.0 on photosynthesis of apical hydrilla
tips cultured in a growth chamber for 96 h at 380 Cpmol m-2 S-la light quantity................_57

4-1 The effect of a single submersed flumioxazin application on dry weight of
submersed aquatic plants 28 d after exposure. ............ ...............77.....

4-2 The effect of a single submersed flumioxazin application on dry weight of emergent
aquatic species 40 d after exposure. ............ ...............79.....

4-3 The effect of a single foliar flumioxazin application on dry weight and height of
emergent aquatic species 40 d after treatment. ............ ...............81.....

5-1 The effect of a single overhead irrigation with 1.27 cm water containing flumioxazin
on ornamental species dry weight and height 14 d after treatment.a ............ ..................89

5-2 The effect of single overhead irrigation with 1.27 cm water containing flumioxazin
on crop species dry weight and height 14 DAT. a............ ...............91.....

7-1 Aquatic plant and algae control with flumioxazin in water with a pH of 7.0 to 9.5b .....106

7-2 Proposed water use restrictions to overhead irrigated crop and ornamental species
following submersed and foliar flumioxazin applications ................. ......................107









LIST OF FIGURES


Figure page

2-1 The effect of flumioxazin concentration on hydrilla dry weight 21 d after exposure
under 70% sunlight. Flumioxazin applied as a single application to hydrilla cultured
in 95 L tubs (pH 9.0-9.5). Data are shown as dry weight means a standard error
(n=10). ECso = effective concentration 50, concentration of flumioxazin in water
required to reduce hydrilla biomass by 50%............... ...............38..

2-2 The effect of flumioxazin at 400 Clg L^1 on hydrilla dry weight as influenced by low
(6.0 to 6.2), medium (7.0 to 7.2), and high (>8.5) water pH under 70% sunlight.
Hydrilla plants were added to low, medium, and high pH water treated with
flumioxazin 0 to 4 d after initial treatment and allowed to grow for 21 d after
treatment until harvest. Data are shown as percent of nontreated control of each pH &
standard error (n=8). Treatment means within a particular day were separated using
least square means (p<0.05)............... ...............39

2-3 Dissipation of flumioxazin applied at 400 Clg L-1 to low (6.0 to 6.2), medium (7.0 to
7.2) and high pH (>8.5) tap water in 95 L tubs under 70% sunlight. The dissipation
of flumioxazin was calculated using non-linear regression (exponential decay) for
the low (y 0.0178e-o.017sx; r2 0.92; half-life 39.0 h), medium (y 0.3074e-0.0373x;
r2 0.93; half-life 18.6 h), and high (y 0.3209e-0.3991x; 2 0.94; half-life 1.7 h) pH
treatments. All residues are reported as the mean a standard error (n=6). ........................40

3-1 The effect of flumioxazin rate at high (9.0) and low pH (6.0) on photosynthesis of
apical hydrilla tips cultured in a growth chamber for 168 h at 380 Cpmol m-2 S-1 Of
light quantity. Data are normalized to the control at each respective pH and shown as
means a standard error (n=8). .............. ...............54....

3-2 The effect of flumioxazin (400Clg L^1) at pH 9.0 on photosynthesis of apical hydrilla
tips cultured in a growth chamber for 96 h at low (20 Clmol m2 Sl), medium (170
Clmol m2 Sl), and high light (400 Clmol m2 Sl) quantity levels. Data are normalized to
the control at each respective light quantity and shown as means a standard error
(n=10) .........____....... .__ ...............56....

3-3 The effect of flumioxazin concentration on hydrilla chlorophyll content (mg kg-l
fresh weight) 1 to 4 d after treatment (DAT) under 70% sunlight. Flumioxazin was
applied as a single application to hydrilla cultured in 18.9 L buckets filled with tap
water (pH 9.0-9.5). Data are shown as actual means a standard error (n=6). ...................58

4-1 The effect of a foliar flumioxazin application (g ha-l a.i.) on water lettuce and water
lettuce dry weight 34 d after treatment under 100% sunlight. Flumioxazin was
applied as a single application by a CO2-pOwered sprayer at an equivalent of 379 L
ha- diluent with a non-ionic surfactant (0.25% v/v) to water lettuce and water
hyacinth grown in 95 L tubs (pH 7.5 to 8.0). Data are shown as dry weight means &
standard error (n=10). ECso = effective concentration 50, concentration of










flumioxazin (g ha-l a.i.) that is required to reduce water lettuce and water lettuce
biomass by 50% ............. ...............74.....

4-2 Percent control (visual) of landoltia 21 d after a foliar diquat (g ha-l a.i.) and
submersed flumioxazin application (Clg L^1 a.i.). Diquat and flumioxazin each
applied as a single application to landoltia cultured in 1 L pots (water pH 8.0) under
70% sunlight. Diquat was applied by a CO2-pOwered sprayer at an equivalent of 379
L ha-l diluent with a non-ionic surfactant (0.25% v/v). Percent control & 95%
confidence interval (CI) (n=10). Overlapping CI bars indicate no significant
difference. ............. ...............75.....

4-3 The effect of flumioxazin concentration on the dry weight of submersed aquatic
plants 28 d after exposure. Flumioxazin applied as a single application to submersed
aquatic species cultured in low (7.0) and high (9.0) pH water in 95 L tubs under 70%
sunlight. Data are shown as actual dry weight means a standard error (n=10 for low
pH, except for naiad and vallisneria n=5; n=5 for high pH). Dry weight means &
standard error (n=10). ............. ...............76.....

4-4 The effect of flumioxazin concentration on dry weight of emergent aquatic plants 40
d after exposure. Flumioxazin applied as a single application to emergent aquatic
species cultured in 95 L tubs (pH 7.5) under 70% sunlight. Data are shown as actual
dry weight means a standard error (n=5)............... ...............78.

4-5 The effect of flumioxazin rate on dry weight of emergent aquatic species 40 d after
treatment. Flumioxazin applied as a single application by a CO2-powered sprayer at
an equivalent of 379 L ha- diluent with a non-ionic surfactant (0.25% v/v) to
emergent aquatic species cultured in 277 L tanks (pH 7.5) under 70% sunlight. Data
are shown as actual dry weight means a standard error (n=5). ............. ....................80

5-1 The effect of flumioxazin concentration in irrigation water (pH 7.5) on ornamental
species dry weight 14 d after treatment. Flumioxazin was applied once to the plants
as an overhead irrigation with a sprinkle can (equivalent to 1.27 cm of irrigation
water). Data are shown as actual dry weight means a standard error (n=10). Data for
snapdragon were not included as flumioxazin resulted in minimum effects at all
rates. .............. ...............88....

5-2 The effect of flumioxazin concentration in irrigation water (pH 7.5) on crop species
dry weight 14 d after treatment. Flumioxazin was applied once to the plants as an
overhead irrigation with a sprinkle can (equivalent to 1.27 cm of irrigation water).
Data are shown as actual dry weight means a standard error (n=10), except for corn
(n=5, for each experiment) ................. ...............90................

6-1 The effect of diquat and flumioxazin on ion leakage from landoltia cultured in DI
water (pH 8.5) in a growth chamber for 96 h. Values are presented as means -t 95%
confidence interval (CI) (n=10). Overlapping CI bars indicate no significant
difference at a given time ................. ...............97........... ...










6-2 The effect of flumioxazin concentration on landoltia chlorophyll content 96 h after
treatment. Landoltia was cultured in 20 mL vials contain DI water (pH 8.5) in a
growth chamber. Data are shown as actual means + standard error (n=6). ECso =
effective concentration 50, concentration of flumioxazin in water required to reduce
landoltia chlorophyll content by 50%. .............. ...............98....









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

CHARACTERIZATION OF FLUMIOXAZIN AS AN AQUATIC HERBICIDE

By

Christopher R. Mudge

December 2007

Chair: W. T. Haller
Major: Agronomy

The suitability of flumioxazin as an aquatic herbicide for control of hydrilla and other

invasive aquatic plant species was evaluated in Hield, greenhouse, and laboratories studies.

Flumioxazin is a protoporphyrinogen oxidase inhibitor in plants, which is a precursor to

production of chlorophyll. It is degraded by hydrolysis and has a half-life of 17.5 min in water at

pH 9.0 compared to a half-life of 16. 1 h and 4. 1 d at pH 7.0 and 5.0, respectively. Flumioxazin

efficacy was evaluated at various concentrations, pH, and light levels to determine the impact on

hydrilla biomass, net photosynthesis, and chlorophyll content. The effective concentration of

flumioxazin required to reduce hydrilla dry weight by 50% (EC5o) was 56 Clg L^1 in mesocosm

studies; however, regrowth was noted in concentrations as high as 1600 Clg L^1 under high pH

conditions. Apical hydrilla tips treated under high pH (>9.0) with flumioxazin at 100 Clg L^1

failed to reduce net photosynthesis by 20% of the nontreated control. All concentrations >100

Clg L^1 at high pH and >100 Clg L^1 at low pH reduced net photosynthesis by at least 60% 168

HAT. Under low light quantity (20 Clmol m-2 S-1), flumioxazin failed to reduce net

photosynthesis of apical hydrilla tips compared to medium (170 Clmol m-2 S-1), and high light

(400 Clmol m-2 S-1) quantities. Non-target emergent plants appeared to tolerate flumioxazin at

high pH (>9.0), whereas selectivity decreased in waters of lower pH (7.0). Phytotoxicity to non-










target ornamental and row crop plants irrigated with flumioxazin treated water was dependent on

maturity as immature, actively growing plants were highly susceptible. Additionally, water

lettuce and landoltia were more susceptible to submersed flumioxazin applications than foliar

applications. These data provide evidence that flumioxazin has the potential for use as an

herbicide with submersed and foliar applications to control hydrilla and other weeds and that

application methods, environmental conditions, and concentrations influence non-target plant

selectivity.









CHAPTER 1
INTTRODUCTION

Currently, there are ten herbicides labeled (FIFRA-Section 3) by the U.S. Environmental

Protection Agency (EPA) for aquatic use in the U.S. including 2,4-D [(2,4-dichlorophenoxy)

acetic acid], carfentrazone (X,2-dichloro-5-[4-(difluromethyl)-4, 5-dihydro-3 -methyl-5-oxo-1H-

1,2,4-triazol-1 -yl]-4-fluorobenzenepropanic acid), copper (copper sulfate or copper chelate),

diquat (1,1'-ethylene-2,2' -bipyridylium dibromide), endothall (7-oxabicyclo[2.2.1l]heptane-2,3-

dicarboxylic acid), fluridone (1-methyrl-3-phenyr __'_~~l-5-[3-(t~rluorme hy)phnyrl]-4(1H)-

pyridinone), glyphosate N-(phosphonomethyl)glycine, imazapyr t(()-2-[4,5-dihydro-4-methyl-

4-(1 -methylethyl)-5-oxo- 1H-imidazol-2-yl]-3 -pyridinecarboxylic acid), penoxsulam (2-(2,2-

difluoroethoxy)-6-(trifluoromethyl-N-(5,8-diehoy1,2,4]triazolo[1 ,5-c]pyrimidin-2-

yrl))benzenesullfnonamide) andl trrilopyrr ([(356-trichlom3n~rro-2-pridinyl~oxyraceti acid. Of

these, only copper, diquat, endothall, and fluridone have historically been used for hydrilla

(Hydrilla verticillata (L. f.) Royle) control (Vandiver 2002).

Hydrilla is a submersed aquatic fresh-water angiosperm in the family Hydrocharitaceae

native to Asia or Africa that has become a serious weed problem in the United States and many

other countries (Cook 1985; Haller and Sutton 1975; USDA 2007; Van and Vandiver 1992).

Once established, hydrilla readily dominates and replaces native submersed species by forming a

canopy and reducing light penetration (Haller and Sutton 1975).

There are two biotypes of hydrilla in the U. S., which are the dioecious (plants produce

only female flowers) and monoecious (male and female flowers on the same plants) (Cook and

Luoind 1982; Langeland 1996). The dioecious female biotype was introduced from Sri Lanka to

the Tampa Bay, Florida area (Schmitz et al. 1990) in the early 1950's and was first observed

growing outside of nursery conditions in a canal in Miami and in a spring near Crystal River, FL









in 1960 (Blackburn et al. 1969). The dioecious female plant has spread throughout the southern

U.S. and as far west as California (Yeo and McHenry 1977; Yeo et al. 1984). The first

population of the monoecious biotype of hydrilla was discovered in Delaware in 1976 with a

second discovery in 1980 in the Potomac River (Haller 1982; Steward et al. 1984).

Hydrilla has been described as "the perfect aquatic weed" due to its specialized growth

habit, physiological characteristics, and various means of asexual reproduction (Langeland

1996). The dioecious biotype is especially problematic since its response to management efforts

results in rapid regrowth from vegetative propagules such as tubers, turions, and plant fragments

(Van and Vandiver 1992). Turions are compact dormant buds produced in leaf axils which

detach from the plant upon maturation, while tubers (subterranean turions) are formed terminally

on subterranean rhizomes (Langeland 1996). The Florida Department of Natural Resources

(now the Florida Department of Environmental Protection) estimated over 20,000 ha of water in

Florida contained hydrilla in 1988 (Schardt and Nall 1988), with hydrilla spreading to 40,000 ha

of water in 43% of public lakes in Florida by 1995 (Langeland 1996). Rapid hydrilla growth and

expansion is favored by its low light and CO2 compensation points, reduced photorespiration

due to a C4 like photosynthetic mechanism and its prolific reproductive capacity (Van et al.

1976; Holaday et al. 1983; Magnin et al. 1997). Hydrilla is typically rooted in the hydrosol,

although fragments frequently break loose and survive in a free-floating state (Langeland 1996).

The stems may be quite long especially when the plant grows in deep clear water and branching

usually does not occur until the plant grows near the water surface (Langeland 1996). Upon

reaching the surface, stems begin to branch profusely forming a surface mat. This density of

stem biomass causes pH in the upper 0.3 m of water during the summer to increase >10.0 during

the day by utilization of CO2 and HCO3- and to fall below 7.0 in the evening (Spencer et al.









1994; Van et al. 1976). Surface matted hydrilla can also cause dissolved oxygen levels to fall

below the air-saturated level during the night, but by noon levels may reach 16 mg L^1,

equivalent to over 200% air saturation (Van et al. 1976).

Various forms of weed control have been evaluated for hydrilla control in Florida

including biological, cultural, chemical, mechanical, and preventative techniques. Biological

control agents such as grass carp are usually unpredictable forms of control (Martyn 1985).

Cultural management techniques such as drawdowns have had limited success, due to regrowth

from subterranean turions (Haller et al. 1976). Mechanical removal of hydrilla is not practical

for large lakes, costing as much as $2500 ha yrl (SE-EPPC 2005). Due to the high costs or

limited effectiveness of mechanical, biological, and cultural techniques, early successful control

of hydrilla in the 1960's and 1970's was through the use of herbicides. These included the

contact herbicides diquat, endothall, and diquat plus copper combinations (Brian et al. 1958;

Hiltibrand 1963; Sutton et al. 1972; Simsiman 1976; Vencill 2002). Due to the rapid activity of

these herbicides, it is recommended that no more than half of an infested water body be treated at

one time due to potential reduced oxygen supply and fish toxicity (Anonymous 2007a;

Anonymous 2003). In addition, these herbicides possess a relatively short half-life (Simsiman

and Chesters 1975; Langeland et al. 1994); this usually results in rapid control but also

encourages rapid regrowth, so season long weed control has not been possible with single

applications of these contact herbicides.

The first residual herbicide for hydrilla control, fluridone, received a Section 3 EPA

registration for aquatic use in 1986 (Dayan and Netherland 2005). Although the maximum

labeled rate is 150 Clg L^1 active ingredient (a.i.), fluridone is commonly applied at 8 to 12 Clg L1

with concentrations as low as 4 to 7 Clg L^1 providing hydrilla control if the dosage was









maintained for several weeks (Van and Steward 1985). Large areas of hydrilla in Florida were

being controlled at 6 to 10 Clg L^1 in the late 1980's at costs of $250 ha-l or less, (Haller et al.

1990). Several factors, including low use rates, favorable native plant selectivity, slow activity

(reduced oxygen depletion), and often more than one year of hydrilla control have resulted in the

reliance on fluridone for hydrilla control in large, shallow Florida lakes (Puri et al. 2006).

However, poor performance was observed in the late 1990's and the development of fluridone

resistance in hydrilla was characterized in 2004 (Puri et al. 2007). Since then, fluridone resistant

populations of hydrilla have expanded in the waterways of Florida. This is likely a result of

continuous use of fluridone, low application rates (<20 Clg L^1) and persistent fluridone residue

(MacDonald et al. 2001; Arias et al. 2005). The loss of fluridone from an already limited

number of aquatic herbicides has resulted in the search for new and effective herbicides that can

be applied to aquatic systems.

Beginning in 2004, herbicide efficacy studies have been conducted at the Center for

Aquatic and Invasive Plants at the University of Florida to evaluate and identify herbicides that

are relatively non toxic to aquatic organisms, possess a short half-life, and show native plant

selectivity which are characteristics necessary for aquatic registration. Of the herbicides

evaluated, flumioxazin {2-[7-fluom' 2aro-,4dhydro-'2nn_3-oxo-4-(2-propynyl)-H-1,-benzoxazin-6-l]

4,5,6,7-tetrahydro- 1H-isoindole-1 ,3(2H_-dione} met the dlesimred critria Hdrlldyeih


was reduced by 63% and 99% in static tests when treated with 50 and 400 Clg L^1 a.i.,

respectively, in 2005 (Mudge and Haller 2006). Based upon these initial evaluations, Valent

U.S.A. Corporation applied for an Experimental Use Permit (EUP) from the U.S. Environmental

Protection Agency (EPA) and the Florida Dept. of Agriculture and Consumer Services (FDACS)


SChiconela, T. F. and W. T. Haller. 2007. Personal Conununication.









in 2006 to evaluate control of the submersed aquatic weed hydrilla with flumioxazin (FDACS

2006; Fishel 2006).

Flumioxazin is an N-phenylphthalimide herbicide that is registered for preemergence weed

control in peanut (Arachis hypogaea L.) and soybean (Glycine max L.) and for post-direct weed

control in cotton (Gossypium hirsutum L.) (Anonymous 2005; Askew et al. 1999; Burke et al.

2002; Clewis et al. 2002; Grichar and Colburn 1996; Main et al. 2003). Direct foliar contact

(postemergence) with flumioxazin results in unacceptable crop injury regardless of plant species

(Yoshida et al. 1991). Flumioxazin is a protoporphyrinogen oxidase (PPO) (protoporphyrin

IX:0xygen oxidoreductase, EC 1.3.3.4) inhibiting herbicide with both soil and foliar activity

(Cranmer et al. 2000; Hartzler 2004; Price et al. 2002; Price et al. 2004) and is a strong inhibitor

of chlorophyll synthesis. It prevents the transformation of protoporphyrinogen IX into

protoporphyrin IX which is a precursor to heme and chlorophyll production (Aizawa and Brown

1999; Matringe et al. 1989; Cobb 1992). Protoporphyrinogen IX accumulates in plastids due to

inhibition of the PPO enzyme and then diffuses through plastid membrane into the cytosol,

where it is oxidized to protoporphyrin IX by a plasma membrane-bound protox (Dayan and Duke

1997; Duke et al. 1991). Protoporphyrin IX reacts with light to produce toxic singlet oxygen

radicals leading to lipid peroxidation and the destruction of cellular components (Duke et al.

1991; Gupta and Tripathy 2000). Irreversible damage to the plasmalemma and tonoplast

membrane lipids is followed by swelling of organelles, rupture of organelle membranes, and

eventually, rupture of the cellular membranes in susceptible plants (Duke et al. 1989). Entire cell

contents (both cytoplasmic and vacuolar) are released after extensive membrane destruction

resulting in cell desiccation and electrolyte leakage (Becerril and Duke 1989; Duke and Kenyon

1993).









Flumioxazin generally provides 4 to 6 wk of residual broadleaf control when applied to the

soil and there is low potential for soil carryover to subsequent rotational crops (Vencill 2002).

The potential for phytotoxicity increases with high soil moisture (Sakaki et al. 1991). The half-

life of flumioxazin in a sandy clay loam soil was 17.9 and 16.0 d compared to 13.6 and 12.9 d in

a loamy sand soil (Ferrell and Vencill 2003a). The flumioxazin molecule is non-ionic and has a

water solubility of 1.79 mg L^1 (Vencill 2002); due to its low water solubility, flumioxazin has a

greater affinity for organic matter than for silicate clay, which prevents it from binding tightly to

the soil matrix and allows it to be readily removed from soil adsorption sites by soil water

(Ferrell et al. 2004). Flumioxazin use in row crops is environmentally beneficial because of low

use rates (71 to 107 g ha-l a.i.) and rapid soil dissipation (Lovell et al. 2001). Flumioxazin

possesses favorable characteristics for use in aquatic systems since half of the herbicide is

degraded by hydrolysis in 4.1 d and 16.1 h at pH 5.0 and 7.0, respectively; however, at pH 9.0,

the half-life decreases to 17.5 min (Katagi 2003) which may limit aquatic weed control in water

with high pH. Heavy hydrilla infestations have been shown to raise water pH to >9.0 by

utilizing free CO2 and HCO 3 (Van et al. 1976) which has the potential to reduce the efficacy of

this herbicide by causing rapid breakdown. Kwon et al. (2004) found the photolytic degradation

rate of flumioxazin increased as a function of pH with a half-life of 41.5 and 4.9 h at pH 5.0 and

7.0, respectively, after correction for the effects of hydrolysis (dark conditions).

Other forms of degradation can influence flumioxazin activity in the aquatic environment.

Flumioxazin showed maximum absorbance at 217 and 286 nm but could absorb UV energy of

sunlight at wavelengths greater than 290 nm (Kwon et al. 2004). In aquatic systems, natural

sunlight would cause the direct breakdown by photolysis near the surface of the water, but

degradation is more likely a function of hydrolysis and water pH.









Carfentrazone received federal registration in 2004 as the only PPO inhibiting aquatic

herbicide (FMC 2005; Iverson and Vandiver 2005). Carfentrazone is similar to flumioxazin

because of its rapid degradation by hydrolysis at high pH (Elmarakby et al. 2001; Ngim and

Crosby 2001). It is effective in controlling the submersed aquatic plant Eurasian watermilfoil

(M~yriophyllum spicatum L.) and floating plants such as water lettuce (Pistia stratiotes L.), water

fern (Salvinia minima ) watermeal (Wolffia spp.), and water hyacinth (Eichhornia crassipes

(Mart.) Solms) (FMC 2004; Koschnick et al. 2004).

Species of duckweed in the Lemnaceae family are commonly used in biochemical and

toxicity tests because of their small size, high reproductive rate and ease of culture (Gensemer et

al., 1999; Geoffroy et al. 2004; Lewis 1995; Ma et al. 2002; Parr et al. 2002). Studies evaluating

pigment content (chlorophyll a, b, and carotenoids) and oxygen evolution are often reliable

indicators of herbicide toxicity (Wang and Freemark 1995). Previous research has shown that

flumioxazin at 1, 10, and 50 Clg L-1 decreased photosynthetic capacity of common duckweed

(Lemna minor L.) by 23, 62, and 64%, respectively (Frankart et al. 2002). In these studies

photosynthetic capacity of common duckweed was inhibited more when plants were exposed to

200 Clg L^1 of copper mixed with flumioxazin than all rates of flumioxazin applied alone. A very

slight synergistic or additive effect was observed between the two chemicals.

One of the primary goals of aquatic weed control in public and private waters is to

eliminate invasive species while maintaining a diversity of native submersed and emergent

species. Native aquatic plants have been shown to improve water clarity and quality, provide

valuable fish and wildlife habitat, reduce rate of sediment resuspension, and help prevent the

spread of invasive plants (Dibble et al. 1996b; Heitmeyer and Vohs 1984; Savino and Stein

1982; Smart 1995). Selective removal of invasive species is beneficial for continued existence









and diversity of native vegetation. Invasive submersed aquatic species often form dense

canopies that significantly increase surface water temperature, reduce dissolved oxygen, and

decrease light penetration for native species (Bowes et al. 1979; Honnell et al. 1993). Native

plant density and diversity has been shown to increase when canopy forming exotic plants are

removed (Getsinger et al. 1997) and diversity of invertebrate and fish habitats are maintained

(Dibble et al. 1996a).

Damage to non-target native plants species is a major consideration in herbicide selection,

with favorable aquatic herbicides being able to selectively remove unwanted plants while

minimizing damage to non-target native plants. Various mesocosm studies have been conducted

to evaluate the sensitivity of native plant species to registered aquatic herbicides. Netherland et

al. (1997) evaluated the effects of fluridone on the submersed species elodea (Elodea Cana~densis

Michaux), American pondweed (Potttttttttttttttttamgton nodosus Poiret), sago pondweed (Pottttttttttttttttttamgto

pectinatus L), and vallisneria (Vallisneria amnericana Michaux). Emergent natives such as soft-

stem bulrush (Scirpus validus Vahl.), Egyptian panicgrass (Paspalidium geminatum Forssk),

maidencane (Panicum hemitomon Schult.), pickerelweed (Pontederia cordata L.), and sagittaria

(Sagittaria lan2cifolia L.) have been evaluated for tolerance to the systemic acetolactate synthase
(ALS) inhibiting herbicides bispyribac-sodium sodiumm 2,6-bis[(4,6-di~methoxypyr;;rimidn-2-


yl)oxylbenzoate}, imazamox {2-[4,5-dihydro-4-methyl-4-(1 -methylethyl)-5-oxo-1H-imidazol-2-

yl]-5-(methoxymethyl)-3 -pyridinecarboxylic acid}, and penoxsulam (Koschnick et al. 2007).

These ALS herbicides (as well as fluridone) usually have relatively long half-lives, while contact

herbicides have a relatively short half-life. Emergent, submersed, and floating species have also

been evaluated for sensitivity to the contact herbicide dipotassium salt of endothall (Skogerboe

and Getsinger 2002). Those species evaluated included Eurasian watermilfoil, curlyleaf










pondweed (Potttttttttttttttttamgton crispus L.), Illinois pondweed (Potttttttttttttttttamgton illinoensis Morong.), sago

pondweed, coontail (Ceratophyllum demersum L), elodea, vallisneria, cattail (Typha latifolia

L), smartweed (Polygonum hydropiperoides Michx.), pickerelweed and spatterdock (Nuphar

advena Aiton).

Homeowners, commercial nurseries, and farmers in Florida often irrigate plants from

surface waters (canals, ponds, lakes, etc.) (Hassell et al. 20004; Hodges and Haydu 2006); non-

target plants may be affected if these waters are treated with herbicides for aquatic weed control.

The use of herbicide treated irrigation water before herbicide residues dissipate below phytotoxic

levels will result in injury or death of irrigated plants. Similarly, farmers may irrigate food crops

from treated water, so the aquatic herbicides must have established tolerances or acceptable

levels of residue on or in food crops. Tolerances of flumioxazin on certain food crops have been

established by the EPA by determining the maximum amount of pesticide residue that can

remain in or on a treated food commodity to ensure food safety (EPA 2003), but no such

tolerances are required for ornamental plants (non-food crops). Phytotoxicity is a major concern

when water with aquatic herbicide residues is used for irrigation of both food and non-food

crops. Previous research has been conducted to evaluate the phytotoxic effects of irrigation

water containing 2,4-D, copper, diquat, endothall, and fluridone on non-target turf and

ornamental species (Andrew et al. 2003; Hiltibran and Turgeon 1977; Koschnick et al. 2005a;

Koschnick et al. 2005b; Mudge et al. 2007; Riemer and Motto 1980), but similar studies have not

been reported for flumioxazin.

Therefore, the obj ectives of this research were to determine the effect of flumioxazin on

hydrilla with respect to efficacy, photosynthesis, and chlorophyll content as influenced by rates

of application, water pH, and light. Further research was conducted to determine the impact of










flumioxazin on other aquatic invasive plants, non-target aquatic plants, non-target row crops and

non-target ornamental plants. An additional objective of this research was to judge the

suitability of flumioxazin as an aquatic herbicide, establish herbicide use patterns, and determine

irrigation restriction parameters through development of possible aquatic use directions.









CHAPTER 2
THE EFFECT OF FLUMIOXAZIN ON HYDRILLA CONTROL IN NATURAL SYSTEMS
AND THE INFLUENCE OF WATER PH ON HYDRELLA CONTROL IN MESOCOSMS

Introduction

Flumioxazin is fast acting herbicide which inhibits chlorophyll production and results in

the production of toxic singlet oxygen radicals leading to lipid peroxidation and the destruction

of cellular components (Duke et al. 1991; Gupta and Tripathy 2000). Injury symptoms may

occur within 1 d after plants are treated with flumioxazin and other herbicides in the N-

phenylphthalimide family (Ferrell et al. 2007b). Although flumioxazin acts rapidly to inhibit

chlorophyll and destroy membranes, this herbicide is rapidly hydrolyzed with an average half-

life of 4.1 d, 16.1 h, and 17.5 min at pH 5.0, 7.0, and 9.0, respectively (Katagi 2003; Vencill

2002). Hydrilla infested waters may have pH in excess of 9.0 (Van et al. 1976) and will likely

influence the efficacy of flumioxazin since water with a pH >9.0 results in rapid breakdown of

this herbicide by hydrolysis with an average half-life of 17.5 min under laboratory conditions

(Katagi 2003). Most aquatic herbicides are degraded by either photolysis or microbial activity

(Vencill 2002) so efficacy of these herbicides is unaffected by time of day during application;

however, higher pH waters (due to pH cycling throughout the day) may limit when flumioxazin

can be applied to successfully control hydrilla. Flumioxazin treatments at rates less than 400 Clg

L^ in water with a pH >8.0 may not be adequate for successful hydrilla control; therefore, the

obj ectives of these studies were to determine flumioxazin use rates and the effect of water pH.

Materials and Methods

Efficacy in Ponds

Eight ponds infested with hydrilla in Florida were treated in 2006 with submersed

flumioxazin applications to determine herbicide efficacy under an Experimental Use Permit

(EUP). Flumioxazin was applied at 100, 200, or 400 Clg L-1 to each pond (0.10 to 10.12 ha)

24









having water pH ranging from 6.7 to 10.0. Two of the ponds were treated with flumioxazin (400

Clg L^1) plus copper (chelated copper, EarthTec@2, 200 Clg L )~. One or more herbicide

applications were made to each pond between February and October. All ponds were treated

with the appropriate amount of herbicide in an equivalent of 93 5.0 L water ha-l in a 378.5 L

spray tank with hydraulic agitation. Most herbicide treatments were applied using a boat

equipped with 3 weighted hoses mounted on the bow of the boat and connected to the spray tank.

The weighted hoses were 1.2 to 3.7 m in length on the left, center, and right side of the boat,

respectively. Some ponds were treated with hoses (0.3 to 0.6 m long) that trailed behind the

boat, which applied the herbicide treatment to the surface of the water, while other treatments

were applied using a handgun sprayed at the water surface. All data were based on visual

observations (including injury symptoms, time of hydrilla regrowth and recovery time) prior to

and after herbicide treatments. Efficacy of flumioxazin treatments was primarily based on how

quickly hydrilla produced new apical tips from treated tissue and the amount of time hydrilla

needed to reach the water surface following treatments.

Efficacy in Mesocosms

A mesocosm study was conducted and repeated at the Center for Aquatic and Invasive

Plants at the University of Florida to determine the effect of flumioxazin on hydrilla. Hydrilla

was collected from Rodman Reservoir near Interlachen, FL in June 2005 and February 2007.

Four sprigs of hydrilla (15 cm) were planted in each 10 x 10 x 12 cm (1 L) pot filled with

masonry sand amended with Osmocote@3,4 15-9-12 fertilizer at a rate of 1g kg-l soil. Five pots


2 Registered trademark of Earth Sciences Laboratories. Rogers, AZ. 72756.
3 The Scotts Company. Marysville, OH. 43041.

4 Mention of a trademark or a proprietary product throughout this document does not constitute a guarantee,
warranty, or endorsement of the product by the author or the University of Florida, and does not imply its approval
to the exclusion of other products that may also be suitable.









were placed in each 95 L HDPE (high-density polyethylene) tub filled with tap water (pH 7.5 at

planting). The experiment was a completely randomized design with five replications (tubs).

The initial study was conducted outside in a shade house (70% sunlight) in July 2005, while the

repeated study was conducted in a greenhouse (70% sunlight) in April 2007. Hydrilla was

allowed to acclimate for 2 wk in 2005 and 6 wk in 2007 before herbicide treatment. Hydrilla

was immature (actively growing) and had just began to branch at the water surface (pH 8.5 to

9.5) at the time of treatment. Flumioxazin was applied as a submersed treatment at 0, 50, 100,

200, 400, 800, and 1600 Clg L^1 a.i. and the dipotasium salt of endothall was applied to other tubs

at 3000 Clg L^1 a.i. as a comparison treatment.

All plants were harvested 21 d after treatment (DAT) by clipping biomass at the soil line;

shoots were placed in a drying oven at 90 C for ca. 1 wk and then weighed. Plant dry weight

data were analyzed using non-linear regression (PROC NLIN, SAS Institute 2002) and

regression models were used to determine the effective concentration 50 (ECso), which is the

concentration of flumioxazin in water that resulted in a 50% reduction in dry weight compared to

control plants. Data from both studies were pooled as there was no difference between the

slopes of regression lines for both studies at the 95% confidence interval.

Impact of pH on Efficacy

Hydrilla was collected from 900 L concrete mesocosm stock tanks and one 15 cm sprig

was planted in a 50 mL plastic tube (13.5-cm by 4-cm in diameter). Tubes were filled with

potting media' amended with Osmocote fertilizer at a rate of 1g kg-l soil and topped off with a 1

cm sand cap. A total of 144 tubes were planted and hydrilla was acclimated in 900 L concrete

tanks for 2 wk prior to herbicide treatment. The initial study was conducted in September 2006



5 Earthgro Topsoil is a registered trademark of The Scotts Company. Marysville, OH. 43041.









and repeated in May 2007 under a shadehouse (70% sunlight). Efficacy of flumioxazin was

evaluated in low (6.0 to 6.2), medium (7.0 to 7.2), and high (>8.5) pH water. Prior to herbicide

treatment, one tube planted with hydrilla was placed into each 95 L HDPE tub that had been

filled with 83 L of tap water (pH 8.0). Hydrilla was 45 & 3.6 cm long with an average dry weight

of 0.51 & 0.3 g at treatment. Muriatic acid was added at a rate of 10 mL or 25 mL per tub to

establish medium or low pH treatments as needed. The high pH treatment was achieved by

placing extra pots filled with hydrilla in tubs 24 h before herbicide application. These additional

plants were kept in high pH treatment tubs so that photosynthesis would maintain water pH at a

level above 8.5.

Flumioxazin was applied at a rate of 400 Clg L1 and mixed in the water as a submersed

treatment. The pH was monitored daily and muriatic acid was added as needed to maintain

desired pH levels in the medium and low pH treatments. Following initial treatment, an

additional tube of hydrilla was added every 24 h to the treated water at 1, 2, 3, 4, and 5 DAT.

Day 0 plants were in the tubs at the time of treatment (Day 0) and removed from the treated

solutions after 4 d of exposure and harvested 21 d later. Plants were placed in the tubs at 1, 2, 3,

4, and 5 days after initial treatment to assess if pH or a pH mediated degradation of flumioxazin

impacted efficacy. All treated plants were given a 96 h exposure and were then removed from

treatment and placed into 900 L mesocosms with flowing tap water (pH 7.5) for 3 wk. This

experiment was conducted as a completely randomized design with four replications (tubs).

Water samples were collected from the low and medium pH treatment at 0.5, 1, 24, 48, 72, 96,

and 120 h after treatment (HAT). Due to anticipated rapid breakdown under high pH, water

samples were collected at 0.25, 0.5, 1, 4, 7, 19, 24, 48, 72, 96, and 120 HAT. All water samples

were immediately acidified with 0.5 mL of muriatic acid at the time of collection to a pH <4.0 to










prevent further breakdown by reducing hydrolysis, frozen and shipped to the Valent U. S.A.

Corporation laboratory (Walnut Creek, CA) for flumioxazin analysis by gas

chromatography/mass spectrometry (GC/MS) using methods reported by Hirota et al. (1992).

All biomass above the soil line was harvested 21 DAT. Shoots were placed in a drying

oven at 90 C for ca. 1 wk and then weighed. Plant weight data were converted to percent of the

respective non-treated plants at each pH for each day and analyzed as a mixed model (PROC

MIXED, SAS Institute 2002) with experiment used as a random factor. The pH of the water was

considered a fixed effect, while experiment, replication (nested within experiment), and all

interactions containing either of these effects were considered random effects. Classification of

experiment (or the combination of experiment and location) as an environmental or random

effect, permits inferences about pH to be made over a range of environments (Carmer et al. 1989;

Hager et al. 2003). Type III statistics were used to test all possible effects of fixed factors. Least

square means were used for mean separation at p<0.05. Data from both studies were pooled.

Water residue data were analyzed using non-linear regression (PROC NLIN, SAS Institute 2002)

to calculate flumioxazin half-life at each pH.

Results and Discussion

Efficacy in Ponds

The maximum EUP rate of 400 Clg L^1 was chosen to treat all the ponds until results

suggested lower rates could successfully control hydrilla. Preliminary studies conducted at the

Center for Aquatic and Invasive Plants indicated flumioxazin controlled hydrilla at

concentrations as low as 50 Clg L- but higher rates may be required in the field due to the rapid

hydrolysis of flumioxazin, especially in high pH waters where hydrilla infestations commonly

occur.









The Micanopy pond treated in February 2006 was the most successful of all flumioxazin

treatments (Table 2-1). Hydrilla began to exhibit apical tip bleaching within 3 DAT followed by

tip abscission and decay within 5 to 7 DAT. The stem segment immediately below the bleached

apical tip became chlorotic/necrotic and the tissue eventually reddened before the stem lost

cellular integrity and buoyancy. Hydrilla height and dry weight were measured weekly for 2 mo

and periodically sampled for 6 mo in the Micanopy pond (data not shown). In addition, a non-

replicated residue study was conducted in the Micanopy Pond and data suggested flumioxazin

possessed a 25 to 35 h half-life at pH 6.7 (data not shown). Flumioxazin provided >95% control

for at least 6 mo after treatment, as few new apical tips could be found throughout the pond;

however, flumioxazin was not solely responsible for the extensive hydrilla control due to very

low water levels 7 mo after herbicide treatment. Factors that could have influenced this

particular treatment included low water pH (6.7), clear water, lower water temperature, time of

year, and actively growing plants that were 0.6 to 1.0 m from the surface (non-matted).

The Apopka pond possessed a higher water pH at treatment (7.4), but flumioxazin still

provided >80% reduction in hydrilla biomass for 8 wk when treated at 400 Clg L^1 (Table 2-1).

The first flumioxazin treatments in February and March 2006 controlled hydrilla with minimal

regrowth in the first two mo after treatment (MAT). Due to the success of these initial

treatments at the maximum EUP rate, it appeared that flumioxazin at lower application rates

could be as effective in controlling hydrilla as the higher application rates. Three ponds were

treated in Kissimmee, FL with three rates of flumioxazin (100, 200, and 400 Clg L^1) in May 2006

(Table2-1). These were the first EUP ponds treated that had water with a pH > 9.0, whereas the

Micanopy and Apopka ponds had <7.5 pH. Hydrilla in all Kissimmee ponds (A-C)

demonstrated similar injury symptoms as the Micanopy and Apopka ponds; however, hydrilla









began to sprout new shoots from treated tissue within 3 to 5 wk after treatment (WAT) and

began to reach the surface by 7 WAT. These particular ponds were treated with a hand gun at

the water surface, whereas the Micanopy and Apopka ponds were treated with weighted and

trailing hoses, respectively. The Kissimmee ponds averaged 2.5 to 4.0 m in depth and it is

possible that the majority of the herbicide treatment remained in the upper 1.0 m due to thermal

stratification. Thermal stratification can occur in the summer when less dense warmer waters

overly colder more dense waters (Wetzel 1975). Thermal stratification is common in surface-

matted submersed plants, especially on sunny days in hydrilla stands (Getsinger et al 1990).

This thermal layer can create a physical barrier, isolating layers in the water column and

preventing surface-applied herbicides from immediately reaching the target vegetation. The

upper layer of these infested water bodies may be in excess of 10 C more than the layers below

the hydrilla mat. Consequently, the hand gun application technique may have failed to evenly

distribute the herbicide into the water column. In addition, these three ponds were more turbid

and heavily infested with hydrilla, permitting less light to reach the bottom. Although hydrilla in

the Kissimmee ponds had not begun to form a surface mat, the hydrilla was beginning to branch

near the water surface at the time of herbicide treatment.

The Apopka pond was retreated in June with only 200 Clg L^1 flumioxazin (Table 2-1);

however, hydrilla began to form new apical tips within 1 WAT of herbicide application and was

surface matted 3 WAT (Table 2-1). At treatment, this pond was shallow (0.5-1.3 m deep)

compared to other ponds, so hydrilla did not require a long period of time to once again reach the

water surface. The failure of this treatment and the low efficacy in the Kissimmee ponds at 100

and 200 Clg L^1 suggested that flumioxazin may not control hydrilla when rates <400 Clg L^1 are









used in the warmer months when hydrilla may cause pH in the water to increase to >10.0 during

the day by utilization of CO2 and HCO3- (Spencer et al. 1994; Van et al. 1976).

Flumioxazin treatments in the latter portion of June and throughout the rest of the year

were applied only with weighted hoses to ensure thorough herbicide distribution in the water

column. When Kissimmee pond C was retreated with flumioxazin, the pH was measured

throughout the water column; the pH was 6.5 to 7.0 near the bottom (2.5 m) compared to >9.0 at

and near the water surface. Weighted hose applications placed approximately one-third of the

flumioxazin treatment in direct contact with the lower portion of the hydrilla stand where it

would mix with low pH water and consequently degrade at a slower rate than the flumioxazin

applied in the upper 1 m of the water.

Due to reduced flumioxazin efficacy in the summer, this herbicide has too short of a half-

life in high pH water and does not provide acceptable control of mature hydrilla. Therefore,

flumioxazin treatments should be restricted to early season for hydrilla control or should be used

in combination with other herbicides as a tank mixture. Combinations of herbicides can result in

increased efficacy when used for aquatic weed management (Gray et al. 2007; Nelson et al.

2001). Copper was chosen because of increased efficacy when mixed with diquat for hydrilla

and common duckweed control (Langeland et al. 2002; Sutton et al. 1972). This flumioxazin

plus copper combination was evaluated as a fall treatment (September and October) in the

Interlachen and Kissimmee C pond and provided ca. 6 mo of control. These treatments were

successful because of potential synergism between copper and flumioxazin, later season

applications (when hydrilla began tuber production and may have been more susceptible to

flumioxazin), or lower water pH (7.2 to 8.5) (Haller et al. 1976; Sutton et al. 1972; Van et al.

1978).









These field studies indicate flumioxazin efficacy in ponds is highly variable. Early season

applications (February and March) provided longer hydrilla control than treatments in warmer

months. The level of control observed with flumioxazin varied according to factors such as

water pH, water temperature, maturity/growth stage of hydrilla, time of year, and placement of

herbicide in water column. Hydrilla is exposed to higher concentrations of flumioxazin for a

longer period of time at low pH than at higher pH. Flumioxazin applied to hydrilla in May

through August failed to provide more than a few weeks to a couple of months of control as new

apical shoots sprouted from treated tissue on most plants and began to reach the surface within 3

to 7 WAT (Table 2-1). Consequently, several of these ponds were treated with flumioxazin

multiple times. Although hydrilla recovered within a few WAT, flumioxazin at 400 Clg L^1

usually provided an additional 2 to 4 wk of control compared to 100 and 200 Clg L-1 treatments.

Efficacy in Mesocosms

Flumioxazin applied at 50 to 1600 Clg L-1 to actively growing immature hydrilla in

mesocosms resulted in bleaching of the upper 5 cm of all apical tips within 3 DAT and stems

began to redden (probably due to anthocyanin production) from 3 cm below the bleached tip to

the soil surface. Bleached apical tips began to abscise and decay within 3 to 7 DAT, when the

plants began to lose cellular integrity. Despite rapid bleaching, reddening, and loss of integrity,

hydrilla in all treatments began to regrow from treated tissue and formed new apical shoots at the

internodes within 5 to 13 DAT depending on flumioxazin concentration. The calculated ECso of

flumioxazin was 56 Clg L^1, while the EC90 for flumioxazin was 186 Clg L^1 (Figure 2-1). Low

herbicide use rates are desirable in aquatic ecosystems and the proposed flumioxazin labeled rate

of 400 Clg L^1 reduced dry weight by 90% of the nontreated control plants. As a comparison,

3000 Clg L^1 endothall reduced hydrilla dry weight by 91% in the same time period.









Impact of pH on Efficacy

Dry weights of hydrilla treated at high pH differed from those treated at low and medium

pH on all days except for hydrilla added 4 and 5 DAT (Figure 2-2). Although statistically

different from the other treatments at 0 DAT, hydrilla placed in the high pH flumioxazin treated

water was still reduced by approximately 90% of the nontreated control. There was no

difference in dry weight at low and medium pH treatments except for hydrilla placed in

treatment solutions 3 or 4 DAT. Hydrilla biomass generally increased daily following exposure

by the plants from 1 to 5 d after exposure as percent of nontreated control. This increase in

biomass corresponded with a decrease in flumioxazin residue (Figure 2-3). Plants in tubs treated

with flumioxazin at medium and low pH levels were still exposed to herbicide through the 96 h

exposure period, while approximately 98% of flumioxazin in the high pH treatment was

hydrolyzed by 3 DAT (Figure 2-3). Although the residue analysis detected less than 10 Clg L^1 of

flumioxazin in the high pH solution, hydrilla biomass was still reduced by ca. 10 to 20% on the

last 2 d of exposure. These data indicate flumioxazin is active at low concentrations (<10 Clg L^1)

and/or secondary metabolites possess activity on hydrilla as well.

The half-life of flumioxazin in low, medium, and high pH water (6.0 to 6.2, 7.0 to 7.2 and

>8.5, respectively) was 39, 18.6, and 1.7 h, respectively (Figure 2-3). Katagi (2003) reported

that the half-life of flumioxazin at pH 5.0, 7.0, and 9.0 under controlled laboratory conditions,

was 98.4, 16.1, and 0.3 h, respectively. Environmental factors in our studies (e.g., higher light

levels, potting media, water quality, and plants which possibly absorbed and/or metabolized the

flumioxazin molecule) most likely influenced the half-life of flumioxazin. The half-life data in

these studies is not directly comparable to previous research by Katagi (2003) since the water pH

evaluated differed between studies. However, flumioxazin degradation in both studies followed

a similar trend, as pH increased, half-life decreased. Under field conditions, pH fluctuates and









can not be controlled, whereas previously reported data (Katagi 2003) was conducted under

stable conditions. Consequently flumioxazin may be exposed to different pHs within the same

water body; therefore, degrading at a faster rate within the same system. As a result, some plants

would be exposed to higher herbicide concentrations than other plants

These Hield EUP trials and mesocosm studies provided further evidence of the impact of

pH on flumioxazin efficacy. Water pH does not directly influence flumioxazin efficacy; pH

influences degradation, which in turn reduces flumioxazin concentration and exposure time to

hydrilla. The pH efficacy study data indicated high pH treatments were successful, possibly due

to ample mixing and hydrilla exposure to higher concentrations for an extended period of time.

Conversely, Hield treatments usually don't provide adequate mixing and adequate exposure at

higher concentrations.

Flumioxazin applied to hydrilla in ponds under high pH conditions were less effective

since flumioxazin was rapidly hydrolyzed before it could control the weed; however, application

of flumioxazin to water with pH levels less than 8.0 generally provided adequate to complete

control of hydrilla. These data provided evidence of rapid flumioxazin uptake in hydrilla as

exhibited by biomass reduction in high pH treatments. If plants treated at the high pH did not

absorb flumioxazin within the first few minutes or hours after treatment, these plants would not

have become chlorotic and would not have been reduced by 90% as in the 0 d treatments. Other

contact herbicides such as endothall require exposures of at least 48 h at 2 mg L1 acid equivalent

(a.e.) to provide greater than 85% reduction in biomass of hydrilla (Netherland et al. 1991).

Flumioxazin appears to require greatly reduced exposure times and has increased activity in

lower pH water. The degradation of flumioxazin within minutes of application in the high pH

treatment prevented a significant reduction in hydrilla biomass 2 to 5 DAT. Flumioxazin is not









highly water soluble (1.79 mg L^1) (Vencill 2002), so uptake into hydrilla is probably rapid due

to the lipophilic nature of this molecule.

Hydrilla treated with flumioxazin in EUP ponds in Florida showed similar bleaching of

apical stem segments regardless of stem length or water depth. Field treated plants followed the

same bleaching pattern and reddening of the stem below the tip as noted in the preliminary

mesocosm study that demonstrated flumioxazin activity on hydrilla. However, symptoms of

hydrilla treated at depths greater than 1.5 m were less pronounced and stems had only 5 to 10 cm

of reddened tissue below the apical tip. Submersed plants growing at these depths are often light

limited (Haller and Sutton 1975) and net photosynthesis is restricted to the apical portions of the

plant. Upon forming a dense mat on and just below the water surface, hydrilla can further limit

light penetration. Flumioxazin and other PPO inhibitors are more active in the presence of light

and may require full sunlight for optimal activity (Sherman et al. 1991; Wright et al. 1995).

Additionally, oxygen-derived free radicals have very short half-lives ranging from milliseconds

to microseconds (Kobayashi et al. 1989) and the short half-life of flumioxazin in higher pH water

potentially reduces radical formation. By the time sufficient light reaches the lower apical tips

and stem segments, flumioxazin has been degraded by hydrolysis and is no longer present to

inhibit the PPO (Aizawa and Brown 1999; Matringe et al. 1989; Cobb 1992). High light

conditions (>1000 Cpmol m-2 S-1) in all greenhouse experiments facilitated bleaching as a result of

inhibition of chlorophyll and membrane disruption through radical production (Duke et al. 1989).

Lower efficacy observed in EUP pond treatments was likely due to high pH, low light intensity

in these deeper waters, and growth stage. Most treatments resulted in >95% control of hydrilla

within 1.5 m of shore, where water was shallow, hydrilla was less than 0.5 m in length and plants

were not shaded by emergent or floating vegetation. This evidence further supports previous









studies (Sherman et al. 1991; Wright et al. 1995) that show higher light levels increase

flumioxazin activity.









































Abbreviations: WAT, weeks after treatment; MAT, months after treatment; WH = weighted hose, 1.2 to 3.7 m long; TH = trailing hose, 0.3 to 0.6 m long
injected at rear of boat; HG = hand gun, using pressure to inject herbicide 0.6-0.9 m into the water ahead of boat.


Table 2-1. Hydrilla infested ponds in Florida treated with flumioxazin under an Experimental Use Permit in 2006.
Location Date DH Comment/Results


6.7 WH application at 400 Clg L 1; no regrowth for 6 MAT; >95% hydrilla control for 6-8
MAT
7.4 TH application at 400 Clg L^1; >80% hydrilla control for 8 wk; 30% hydrilla surface
matted by 3 MAT
9.7 HG application at 100 Clg L^1; hydrilla regrowth 3 WAT and near surface 7 WAT

9.7 HG application at 200 Clg L^1; hydrilla regrowth 3 WAT and near surface 7 WAT

9.7 HG application at 400 Clg L^1 on approximately one third of pond; bleaching and control
in whole pond but better control in treated area; hydrilla regrowth 5 WAT and near
surface 7 WAT
7.2 TH application at 200 Clg L 1; hydrilla regrowth at 1 WAT was surface matted 3 WAT

9.5 TH application at 400 Clg L 1; minimum injury symptoms and hydrilla reached surface 1
MAT
7.2 HG application at 200 Clg L 1; hydrilla controlled for 1 month but began to regrow and
reached surface 12 WAT
9.4 WH application at 400 Clg L 1; hydrilla regrowth 3 WAT, had not reached surface by 10
WAT
10.0 WH application at 400 Clg L 1; minimum hydrilla injury and reached surface 1 MAT

7.2 WH application at 400 Clg L 1; hydrilla regrowth at 1MAT and surfaced 3 MAT

8.5 WH application at 400 Clg L1 plus 200 Clg L^1 copper; hydrilla regrowth 2 MAT, but had
not reached surface by March 2007
7.2 WH application at 400 Clg L^1 on whole pond, but half of pond received copper at 200 Clg
L^:; >6 months of control: hydrilla bean to reach surface in March 2007


Micanopy

Apopka

Kissimmee A

Kissimmee B

Kissimmee C


Apopka
Eustis

Interlachen

Kissimmee C

Gainesville

Interlachen

Kissimmee C

Interlachen


Feb 2006

March 2006

May 2006

May 2006

May 2006


June 2006

June 2006

June 2006

June 2006

June 2006

July 2006

Sept 2006

October 2006























o -1 T ,"5 ,Vf ,,









O 200 400 600 800 1000 1200 1400 1600

Flumioxazin Concentration (Cpg L1 a.i.)
Figure 2-1. The effect of flumioxazin concentration on hydrilla dry weight 21 d after exposure
under 70% sunlight. Flumioxazin applied as a single application to hydrilla cultured
in 95 L tubs (pH 9.0-9.5). Data are shown as dry weight means + standard error
(n=10). ECso = effective concentration 50, concentration of flumioxazin in water
required to reduce hydrilla biomass by 50%.














100 -1 m eatum pn
[M High pH
a
80-a a
U a -rab
aa
60- -iil b


b
"o 40 -b


b E~b
20-
a c

0 I b
0 1 2 3 4 5
pH Replacement, Days After Treatment (DAT)

Figure 2-2. The effect of flumioxazin at 400 Clg L1 on hydrilla dry weight as influenced by low
(6.0 to 6.2), medium (7.0 to 7.2), and high (>8.5) water pH under 70% sunlight.
Hydrilla plants were added to low, medium, and high pH water treated with
flumioxazin 0 to 4 d after initial treatment and allowed to grow for 21 d after
treatment until harvest. Data are shown as percent of nontreated control of each pH +
standard error (n=8). Treatment means within a particular day were separated using
least square means (p<0.05).











400


j I THigh pH Water
to- Medium pH Water
3- 300 ------ Low pH water















0 20 40 60 80 100 120

Hours After Treatment (HAT)
Figure 2-3. Dissipation of flumioxazin applied at 400 Clg L-1 to low (6.0 to 6.2), medium (7.0 to
7.2) and high pH (>8.5) tap water in 95 L tubs under 70% sunlight. The dissipation of
flumioxazin was calculated using non-linear regression (exponential decay) for,~ thze
low (y 0.0178e-o.017sx; r2 0.92; half-life 39.0 h), medium (y 0.3074e-0.073x
0.93; half-life 18.6 h), and high (y = 0.3209e-0.3991x; 2 0.94; half-life 1.7 h) pH
treatments. All residues are reported as the mean + standard error (n=6).









CHAPTER 3
THE EFFECT OF FLUMIOXAZIN ON HYDRELLA PHOTOSYNTHESIS AND
CHLOROPHYLL CONTENT

Introduction

Flumioxazin was evaluated for hydrilla control in EUP field trials throughout Florida in

2006 and 2007 in water bodies ranging in pH from 6.7 to 10.0 at rates of 100 to 400 Clg L1 (see

Chapter 2). Despite early season success, summer applications of flumioxazin failed to provide

adequate season-long control. Most summer EUP trials resulted in rapid injury to the hydrilla

canopy followed by re-infestation within 1 to 4 months.

Higher water pH (either due to hydrilla infestations or pH cycling throughout the day) may

limit when flumioxazin can be applied for successful hydrilla control. Flumioxazin applied to

water with a pH >9.0 is rapidly degraded by hydrolysis and submersed aquatic plants growing at

greater depths are often light limited (Haller and Sutton 1975). Flumioxazin and other PPO

inhibitors require full sunlight for optimal activity (Sherman et al. 1991; Wright et al. 1995), so

the limited quantity of light at greater water depths may reduce flumioxazin activity on hydrilla.

Measurement of the net photosynthetic rate of submersed aquatic plants has been utilized

to study the effects of aquatic herbicides on submersed plants (MacDonald et al. 2003;

Netherland and Getsinger 1995a; Netherland and Getsinger 1995b). Carbon dioxide is absorbed

by plants and fixed as one of the primary products of photosynthesis, while Oz is OVOlVed from

the splitting of water (Oj a et al. 2007). Submersed aquatic plants treated with herbicides that

interfere with photosynthesis or the production of light capturing pigments (chlorophyll and

carotenoids) typically evolve less 02 COmpared to nontreated plants (MacDonald et al. 2002;

Netherland and Getsinger 1995a). Measuring total chlorophyll content in aquatic plants provides

further evidence of the impact of these herbicides on plant status (Doong et al. 1993; Netherland

and Getsinger 1995a). Such studies provide information regarding the health of plants treated









with herbicides that may not show visual damage for several days. Therefore, the objective of

these studies was to determine the effect of water pH, light, and flumioxazin rate on net

photosynthesis and chlorophyll content of hydrilla.

Materials and Methods

Photosynthesis and pH

The effects of flumioxazin at low (6.0) and high (9.0) pH on the net photosynthetic rates of

hydrilla were determined by measuring oxygen evolution (dissolved oxygen, DO) over time.

Hydrilla was collected at Rodman Reservoir near Interlachen, FL in January 2007. All DO

techniques were similar to those reported by Netherland and Lembi (1992) and Netherland and

Getsinger (1995b). The plants evaluated in those previously cited studies remained continuously

in biological oxygen demand (BOD) bottles as static exposures, whereas plants in these studies

were removed every 24 h from herbicide treatments to prevent oxygen saturation. Apical

hydrilla tips (4 cm) were excised from freshly collected plants and placed in clear plastic cups

(473 mL) with 350 mL deionized water (DI) and allowed to acclimate for 24 h before treatment.

Tips were acclimated and treated in a growth chamber at a constant temperature of 27 C with a

14 h light/10 h dark photoperiod. The daytime light intensity was 380 Cpmol m-2 S-1

Preliminary studies were conducted to determine appropriate concentrations of nutrients

and buffer (data not shown) to attain active growth and hold pH. Nutrients were supplied by 1%

v/v Hoagland's solution and 4.7mM NaHCO3 WAS added as a carbon source. The experimental

design was a randomized design with 4 replications. The culture solution of plants treated at the

low pH also received 10 CIL of muriatic acid (diluted HC1) and MES buffer (5 mM) to maintain a

pH of 6.0. Hydrilla tips were treated with flumioxazin at 0, 100, 200, 400, and 800 Clg L^1 in the


6 Percival Scientific, Inc. Perly, IA. 50220.









same plastic culture cups and the same hydrilla tip was reused daily to measure DO over time.

DO was measured pre-treatment (0), 24, 48, 72, 96, 120, 144, and 168 h after treatment (HAT)

using a dissolved oxygen meter To eliminate possible oxygen saturation, treated and

nontreated tips were removed from the plastic cups, rinsed twice in DI water to ensure no

herbicide residue transfer and placed in 300 mL BOD bottles filled with fresh DI water and the

same nutrient solution as the plastic culture cups for DO daily measurements. Initial DO

measurements were recorded for each bottle prior to tip placement in the bottles, then tips were

placed in BOD bottles and allowed to incubate for 1 h. Final DO was measured in each bottle

and tips were removed, gently blotted with a dry paper towel, and weighed (fresh weight). Tips

were immediately returned to their original cups after fresh weights were obtained. BOD bottles

were emptied each day and rinsed after completion of DO measurements. The formula for

calculating the net photosynthetic rate was:

(Final DO-Initial DO)/weight(g)/time(min)* 1000 = Clg 02 g freSh weight- min'

Many of the treated hydrilla tips lost turgor and became defoliated during the course of the

experiments and disintegrated by the conclusion of the study. Decayed tips were discarded when

they occurred and a value of 0 for net photosynthesis was assigned to that treatment for the

remainder of the experiment.

All data were normalized to the control to account for differences in photosynthetic rates at

each pH. Percent data were analyzed using non-linear regression (exponential decay) (PROC

NLIN, SAS Institute 2002) and regression models were used to determine an ETso, which is the

amount of time hydrilla was exposed to flumioxazin before a 50% reduction in photosynthesis


SAccumet Excel XL40 Dissolved Oxygen/BOD/OUR/SOUR Temperature Meter. Fisher Scientific. Pittsburgh, PA
15275.

SWheaton Science Products. Milkville, NJ. 08332.









was reached for each rate and pH. The ETso values were calculated using the slope of the line

based on the following formula: In(0.5)/-bl. Additionally, SAS produced upper and lower 95%

confidence intervals (CI) for each line. When placing an upper and lower 95% confidence

interval on the slope of a non-linear equation, calculated ETso values & 95% CI will not be & a

single value. This is because a non-linear equation will not always fit exactly between the range

of data values. This can then require differing 95% CI values for the upper or lower limits,

depending on which direction the equation trends toward. For these experiments, data were

pooled because were no difference between the slopes of regression equations for both

experiments at the 95% level of confidence.

Photosynthesis and Light

The effects of light quantity on net photosynthetic rates of apical hydrilla tips treated with

flumioxazin were evaluated in May and June 2007. Hydrilla was collected from the

Withlacoochee River near Dunnellon, FL in May 2007. Methods used were as described above

for the DO pH experiment except plants were only cultured at pH 9.0. Apical tips were

acclimated for 24 h and treated at low (20), medium (170), and high (400 Cpmol m-2 S-1) light

quantities in a growth chamber. The low light quantity chosen in this study is near the upper

threshold of the light compensation point for hydrilla and is typical of the quantity of light found

near the bottom of a pond/lake, whereas the high light quantity is less than the light saturation

point of hydrilla and is near the light quantity found near the surface of matted hydrilla (Van et

al. 1976; Bowes et al. 1979; Steward 1991). Hydrilla DO was measured every 24 h and the

experiment was concluded 168 HAT. Light levels in the chambers were adjusted by removing

incandescent and fluorescent bulbs to obtain desired light levels. Each light quantity treatment

consisted of 10 hydrilla tips (cups); 5 control plants and 5 receiving flumioxazin at 400 Clg L^1 for









a total of 30 tips (5 reps/treatment). Cups were placed in a randomized design in each of the

growth chambers.

All data were normalized to the control to account for differences in photosynthetic rates at

each light level. Percent data were analyzed using non-linear regression (exponential decay)

(PROC NLIN, SAS Institute 2002) and regression models were used to calculate ETso values at

each light quantity.

Effect of Contact Herbicides on Photosynthesis

The effect of flumioxazin on hydrilla photosynthesis was compared to the effect of other

registered contact aquatic herbicides in April 2007. Most culture and treatment techniques used

in this experiment were the same as those described above in the DO pH study but this

experiment had 5 replications, plants were treated at pH 9.0, and this experiment was concluded

96 HAT. Hydrilla was cultured and treated with a light quantity of 380 Cpmol m-2 S-1. Herbicide

treatments applied to hydrilla apical tips in this experiment included flumioxazin at 400 Clg L^1

(all flumioxazin treatment combinations were applied at this rate), copper (copper-

ethylenediamine complex, Komeen@9, 50 and 200 Clg L^1 a.i., hereinafter referred to as K50 and

K200), flumioxazin plus K50, flumioxazin plus K200, and the dipotassium salt of endothall

(5000 Clg L^)~. All data were normalized to the control and analyzed using non-linear regression

(exponential decay) (PROC NLIN, SAS Institute 2002) and regression models were used to

calculate ETso values for each herbicide treatment.

Chlorophyll Content

The effect of flumioxazin on chlorophyll content of hydrilla was determined using hydrilla

collected from the Withlacoochee River near Dunnellon, FL in April and May 2007. Four 15 cm



9 Registered trademark of SePRO Corporation. Carmel, IN. 46032.









sprigs were planted in each 10 x 10 x 12 cm (1 L) pot filled with masonry sand amended with

Osmocote (15-9-12) fertilizer at a rate of 1g kg-l soil. Two pots of hydrilla were placed in each

18.9 L bucket (28 cm diameter by 31 cm deep) filled with tap water (pH 7.5 at planting) under

70% sunlight. Hydrilla was allowed to acclimate for 4 and 3 wk for the initial and repeated

study, respectively, at which time plants had reached the water surface and begun to branch. The

pH ranged from 9.0 to 9.5 when flumioxazin was applied at 0, 50, 100, 200, 400, 800, and 1600

Clg L^1. This experiment was a randomized design with 3 replicates. Apical tips (ca. 2.5 cm,

0.09-0.11 g fresh weight) were collected from each treatment 1, 2, 3, and 4 DAT for chlorophyll

analysis. Bleaching of hydrilla continued beyond 4 DAT, but no apical tips could be used for

chlorophyll analysis as they had abscised the stems and were disintegrating. Excess water was

removed by gentle blotting with paper towels, then tips were immediately weighed (fresh

weight), placed in 20 mL scintillation vials, and frozen until chlorophyll analysis. Total

chlorophyll was extracted by placing the apical tips in tubes containing dimethylsulfoxide

(DMSO) (Hiscox and Israelstam 1979) in a water bath at 60 C for 6 h. Chlorophyll content was

determined spectrophotometrically (Arnon 1949) and expressed as mg chlorophyll kg-l of fresh

weight. Non-linear regression (PROC NLIN, SAS Institute 2002) was used to determine the

effect of flumioxazin on chlorophyll content.

Results and Discussion

Photosynthesis and pH

All flumioxazin treatments of 100 to 800 Clg L^1 showed similar trends with respect to

reduction in photosynthesis except 100 Clg L^1 at high pH (Figure 3-1) which only reduced

photosynthesis by approximately 20% of the nontreated control plants 168 HAT. The amount of

time required to reduce photosynthesis by 50% (ETso) of the control plants was 737 h for

flumioxazin applied at 100 Clg L^1 in the high pH solution (Table 3-1). All other treatments









reduced photosynthesis by 50% of the nontreated control between 68 and 1 18 HAT (2 to 5

DAT). Flumioxazin applied at 800 Clg L^1 in low pH was different from 200 Clg L^1 in the high

pH treatment and from 100 Clg L^1 in low pH treatment based on ETso (f 95% confidence

interval) values. No other treatment differences were observed at low or high pH.

Photosynthesis was reduced more rapidly as a function of flumioxazin concentration at both high

and low pH. Net photosynthesis of nontreated control plants prior to herbicide treatment (0

HAT) was 174.6 & 16.9 (low pH) and 155.9 & 8.7 Clg 02 g freSh weight- min' (mean a standard

error) (high pH), respectively, compared to 61.2 & 10.9 (low pH) and 65.3 A 10.4 Clg 02 g freSh

weight- min' (high pH) 168 HAT. The low and high pH nontreated control plants decreased in

net photosynthesis by 64.9 and 58.1%, respectively. This gradual decline in net photosynthesis

over the course of the experiment was possibly attributed to decline in NaHCO3 and Hoagland's

solution in the plastic cups in addition to stress caused by blotting them dry to obtain fresh

weight.

These data indicate a minimal pH effect on photosynthesis except for flumioxazin applied

at 100 Clg L-1 to apical tips in high pH treatment solutions. Based upon these data, flumioxazin

should be applied at rates higher than 100 Clg L^1 in high pH waters to over come the effects of

rapid breakdown. Regardless of pH, flumioxazin at 400 and 800 Clg L-1 had similar ETso values

and DO measurements at 168 HAT. The light quantity in this study (380 Cpmol m-2 S-1) in

combination with low pH or flumioxazin at rates above 100 Clg L^1 appears to be sufficient to

reduce photosynthesis and kill apical tips exposed for 168 h. Apical hydrilla tips in this

particular study began to bleach at ca. 48 HAT followed by whole tip disintegration within 120

HAT in all treatments except high pH 100 and 200 Clg L-1 treatments (data not shown). These

data suggest that flumioxazin is rapidly absorbed into hydrilla despite the half-life being very









short in high pH water (9.0) and is similar to the pH efficacy data (see Chapter 2). However,

these studies provide evidence that flumioxazin concentration greater than 200 Clg L^1 may be

required to overcome the rapid degradation when applied in high pH water.

Flumioxazin does not halt hydrilla photosynthesis as quickly as other herbicides such as

diquat. MacDonald et al. (2002) demonstrated diquat at 344 Clg L-1 decreased net photosynthesis

in apical hydrilla tips by 44% 10 min after treatment and completely inhibited net photosynthesis

2 HAT. Although diquat quickly inhibits photosynthesis under controlled conditions, it is

typically applied with copper to improve efficacy, due to difficulty of controlling hydrilla when

applied alone (Sutton et al. 1970; Sutton et al. 1972; Anonymous 2003).

Photosynthesis and Light

The effect of 400 Clg L^1 flumioxazin on photosynthesis of apical hydrilla tips at low (20),

medium (170), and high (400 Cpmol m-2 S-1) light levels showed apical hydrilla tips treated at

medium and high light levels followed a similar trend (Figure 3-2). The photosynthetic rates of

hydrilla tips treated under high light conditions were different from those at the medium light by

the conclusion of the study. Low light treated tips were still photosynthesizing at approximately

73% of the nontreated control plants by the conclusion of the study. The amount of time

required by flumioxazin to reduce photosynthesis by 50% (ETso) at medium light was not

different from the low or high light quantities (Table 3-2). The high and low light quantities

differed with respect to the calculated ETso values. Low light treated hydrilla would require an

estimated 303 h to achieve a 50% reduction, while high light plants only required 99 h. Low

light treated tips did not visually appear to be different from the low light nontreated control

plants. There was no bleaching or chlorosis of the apical tips in either treatment. In addition,

low light treated apical tips elongated more than 5 cm during the course of the study, whereas

those treated at medium and high light quantities elongated less than 1 cm (data not shown).









Low light conditions such as those used in this study are similar to those found at the mid-depths

or bottom of water bodies infested with hydrilla.

Previous research demonstrated that the light compensation point of hydrilla ranged from 7

to 20 Cpmol m-2 S-1 (Van et al. 1976; Bowes et al. 1979; Steward 1991). The low light quantities

in these studies were ample for hydrilla to produce Oz, but were not sufficient enough for

flumioxazin to completely halt photosynthesis or bleach the apical tips. Conversely, the high

light levels reduced photosynthesis to about 30% of the nontreated control plants by the

conclusion of the study. Flumioxazin is a strong inhibitor of chlorophyll synthesis and prevents

the transformation of protoporphyrinogen IX into protoporphyrin IX. (Aizawa and Brown 1999;

Matringe et al. 1989; Cobb 1992). Protoporphyrinogen IX accumulates in plastids due to

inhibition of the PPO enzyme and then diffuses through the plastid membrane into the cytosol,

where it is oxidized to protoporphyrin IX by a plasma membrane-bound protox (Dayan and Duke

1997; Duke et al. 1991). Protoporphyrin IX reacts with light to produce toxic singlet oxygen

radicals leading to lipid peroxidation and the destruction of cellular components (Duke et al.

1991; Gupta and Tripathy 2000). The low light treated plants did not exhibit bleaching within

the course of these studies. However, chlorophyll turnover is continuously occurring as the

average half-life of chlorophyll is relatively short (minutes to days) (Grumbach et al. 1978;

Hendry and Stobart 1986); therefore, protoporphyrinogen IX should have been inhibited during

the course of the experiment and result in bleaching of the apical tip. Apparently, chlorophyll

turnover in hydrilla tips grown under low light was reduced, but not rapid enough to result in

bleaching.

Hydrilla infested lakes form dense canopies and restrict light penetration to the lower

portions of the plant (Haller and Sutton 1975). This study provides a possible explanation for the









observations noted at EUP treatment sites where the surface canopy of hydrilla was injured, the

lower stems received minimal injury, and rapid regrowth occurred from the lower stem segments

(see Chapter 2). These data provide evidence that low light levels hinder flumioxazin activity

and explains why rapid regrowth occurs from apical tips growing under the hydrilla canopy.

Effect of Contact Herbicides on Photosynthesis

Flumioxazin applied alone to apical hydrilla tips at 400 Clg L^1 required 156 h to reduce

photosynthesis by 50% (ETso) of the nontreated control plants (Table 3-3). Copper applied alone

at 50 Clg L-1 had minimal impact on photosynthesis of hydrilla, whereas copper at 200 Clg L^1

reduced DO by 50% within 35 HAT. Flumioxazin plus K200 reduced photosynthesis by 50%

more rapidly than any other treatments evaluated; however, this increase in activity was minimal

compared to K200 applied alone. The combination of flumioxazin and copper may be beneficial

for control of surface matted hydrilla. Flumioxazin activity has been variable in EUP field

research (see Chapter 2) and the combination of these products could aid in overcoming the rapid

breakdown in high pH water. Copper alone has activity on submersed and floating aquatic

weeds such as elodea (Elodea densis), coontail, giant salvinia (Salvinia molest D. S. Mitchell),

and duckweed (Angelo et al. 1998; Filbin and Hough 1979, Nelson et al. 2001; Ware 1966). In

addition, copper has been used successfully in combination with diquat for hydrilla control

(Sutton et al. 1972; Langeland et al. 2002) and overcomes diquat resistance in landoltia

[Landoltia punctat (G. Meyer) D.H. Les and D.J. Crawford] (Koschnick and Haller 2006).

Previous research has shown that flumioxazin at 1, 10, and 50 Clg L-1 decreased

photosynthetic capacity of common duckweed (Lemna minor L.) by 23, 62, and 64%,

respectively (Frankart et al. 2002). In these studies photosynthetic capacity of common

duckweed was inhibited more when plants were exposed to 200 Clg L^1 of copper mixed with

flumioxazin than all rates of flumioxazin applied alone.









Photosynthesis in this study was only reduced to 60% of the nontreated control with 5000

Clg L^1 of endothall after a 96 h exposure. MacDonald et al. (2002) reported technical grade

endothall acid applied at 372 mg L-1 stopped photosynthesis 120 min after treatment; however,

this rate of endothall is approximately 75x higher than the maximum label rate of the

dipotassium salt of endothall (Anonymous 2007a). Previous research has shown that this

formulation of endothall kills various terrestrial and aquatic plants by inhibiting respiration

(MacDonald et al. 1993), photosynthesis (Turgeon et al. 1972), lipid synthesis (Mann and Pu

1968), and protein synthesis (Mann et al. 1965) as well as causing cellular disruption (Keckemet

1968; Keckemet and Nelson 1968). According to the calculated photosynthesis ETso values,

flumioxazin alone is slower than the dipotassium salt of endothall at reducing DO by 50%.

Flumioxazin and endothall were applied at the maximum labeled rate compared to 5 and

20% of maximum labeled rate for copper at 50 and 200 Clg L^1, respectively. These data indicate

that copper at 200Clg L^1 reduced hydrilla photosynthesis faster than flumioxazin or the

dipotasium salt of endothall. The addition of 50 Clg L^1 copper to 400 Clg L^1 flumioxazin did not

decrease the calculated ETso value from 400 Clg L^1 flumioxazin alone. However, the addition of

200 Clg L^1 copper to the flumioxazin did provide a faster reduction in photosynthesis.

Chlorophyll Content

Flumioxazin applied to hydrilla in a mesocosm study at rates of 50 to 1600 Clg L^1 showed

that chlorophyll content was reduced as function of increased flumioxazin concentrations (Figure

3-3). Hydrilla chlorophyll content 3 and 4 d after the flumioxazin treatment reflected more of a

flumioxazin rate response than 1 and 2 DAT, as indicated by the trend of the regression lines.

These data demonstrate the ability of flumioxazin to rapidly bleach apical hydrilla tips due to the

inhibition of protoporphyrinogen oxidase which is a precursor to chlorophyll (Aizawa and

Brown 1999; Matringe et al. 1989; Cobb 1992). Hydrilla treated with flumioxazin in EUP trials









(see Chapter 2) showed similar apical tip bleaching in the field within 1 to 3 DAT followed by

reddening of the stem 5 cm below the apical tip. These results are similar to those observed in

these studies. Some of the red coloration in treated plants was likely due to anthocyanin

formation or destruction of other secondary chlorophyll protecting pigments which were visible

because of chlorophyll inhibition or were produced in response to the stress of the herbicide

treatment (Hrazdina 1982; Sandmann et al. 1991; Spencer and Ksander 1990). Susceptible

terrestrial crops and weeds exposed to other PPO inhibiting herbicides rapidly show injury

symptoms including chlorosis, leaf crinkling, and stunting followed by necrosis (Lovell et al.

2001; Wilcut et al. 2001; Vencill 2002). Hydrilla may grow as much as an inch per day

(Langeland 1996) and new tissue and chlorophyll is being produced in the tip much more

quickly than in the lower stem segments. PPO inhibitors would inhibit chlorophyll and

consequently produce more toxic radicals in the tip region than in the more mature and shaded

lower stem segments. Lower stem segments of plants treated with flumioxazin were not tested

for chlorophyll content in these studies since flumioxazin primarily causes bleaching in the upper

apical tip (1 to 3 cm) where chlorophyll turnover is occurring more rapidly. In these chlorophyll

studies, flumioxazin was applied to hydrilla cultured in high pH water. Further studies could be

conducted to evaluate the effects of flumioxazin on chlorophyll content in low pH water;

however, regardless of pH, only the apical portion of the plant will bleach since chlorophyll is

being produced more rapidly in this region and consequently flumioxazin toxicity symptoms are

more pronounced.

In conclusion, these data once again show flumioxazin is rate dependent up to 400 Clg L1

when applied to hydrilla in high pH solutions. Most of the treatments applied to apical hydrilla

tips reduced photosynthesis before the conclusion of the experiments. Approximately half of









hydrilla' s biomass occurs in the upper 0.5 m of the water column when the plant forms a dense

surface mat (Haller and Sutton 1975). Most of the apical tips can be found in the upper surface

of these mats; hence, flumioxazin treatments tend to work exceptionally well on the upper

canopy because of ample light. Treated apical tips growing >1.5 m from the surface under low

light conditions at and near the lake bottom are bleached, but the lower stem sections typically

don't become chlorotic and disintegrate in the same manner as the upper stem segments which

receive high light intensity. Stem segments treated with flumioxazin responded by reduced

photosynthesis but didn't disintegrate similar to the apical tips. The light study confirmed the

lack of flumioxazin efficacy at light levels near hydrilla's light compensation point which is

typically found below hydrilla mats in greater water depths. These studies provide further

evidence that pH is not the only factor influencing flumioxazin efficacy at 400 Clg L 1. Light

quantity and tissue type can reduce efficacy in field treatments. If flumioxazin received a

Section 3 label for aquatic use, treatments applied to high pH water (>9.0) may result in failure.

Based on these data and EUP research, applications should be made early in the morning before

the pH is cycled to above 9.0, early in the year, or before hydrilla forms dense canopies.

Flumioxazin is most effective in rapidly growing tissue where chlorophyll turnover is high.

Lower pH water results in a longer half-life and increases exposure of plants to higher

flumioxazin concentrations for a longer period of time. Non-matted hydrilla allows more light to

penetrate to the lower canopy, where flumioxazin will more injury to lower portions of the

hydrilla plant. Thus, optimum conditions for aquatic weed control with flumioxazin included

rapid growth, low pH, and high light intensity.





























0 20 40 60 80 100 120 140 160 180


0 20 40 60 80 100 120 140 160 180


high pH (9.0), 800 stg L
\ -- -low pH (6.0), 800 stg L'


120

100 -

80 -

60 -

40

20


S I I I I I I I I I I
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180
Flumioxazin Exposure Time (h) Flumioxazin Exposure Time (h)

Figure 3-1. The effect of flumioxazin rate at high (9.0) and low pH (6.0) on photosynthesis of
apical hydrilla tips cultured in a growth chamber for 168 h at 380 Cpmol m-2 S-1 of light
quantity. Data are normalized to the control at each respective pH and shown as
means + standard error (n=8).










Table 3-1. The effect of flumioxazin on photosynthesis of apical
hydrilla tips at high (9.0) and low (6.0) pHa.


r2

0.93

0.88

0.75

0.71



0.85

0.85

0.86

0.89


pH 9.0b ETSOC (95% CId)


Regression equation

y = 91.3356e-0.000940x

y = 87.9257e-0.00585x

y = 90.3356e-0.00801x

y = 92.0237e-0.00852x



y = 92.3011e-0.00564x

y =108.8e-0.00753x

y = 94.0648e-0.0101x
v =113.4e-0.0102x


100

200

400

800

pH 6.0e
100

200

400

800


737 (315-2166)

118 (90-175)

87 (61-148)

81 (57-144)



123 (89-197)

92 (70-134)

69 (55-91)

68 (56-87)


a Hydrilla cultured in a growth chamber for 168 h at 380 pLmol nf2 S-1 of light
quantity at high and low pHa. Data are normalized to the control at each respective
pH.
b High pH: 9.0.
" Effective time 50: ETso = time required by flumioxazin (h) to reduce hydrilla
photosynthesis by 50%. Each value is a mean of two experiments with a total of 8
replications.
d 95% CI = 95% Confidence Interval.
e Low pH: 6.0, pH reduced with muriatic acid.













100-







60


40 Low Light

-- -- Med Light
20~ High Light



0 20 40 60 80 100 120 140 160 180

Flumioxazin Exposure Time (h)
Figure 3-2. The effect of flumioxazin (400Clg L^1) at pH 9.0 on photosynthesis of apical hydrilla
tips cultured in a growth chamber for 96 h at low (20 Clmol m2 Sl), medium (170 Clmol
m2 Sl), and high light (400 Clmol m2 Sl) quantity levels. Data are normalized to the
control at each respective light quantity and shown as means + standard error (n=10).










Table 3-2. The effect of flumioxazin (400 Clg L^1 a.i.) at pH 9.0 on
photosynthesis of apical hydrilla tips cultured in a growth
chamber for 168 h at low, medium, and high light quantities.


Light Quantityb ETSOC (95% CId)

Low 303 (198-648)

Medium 140 (105-209)


Regression equation

y = 95.4156e-0.00229x

y = 95.9852e-0.00495x


0.92

0.88


High 99 (81-130) y =102.4e-0.00697x 0.90
a Data are normalized to the control at each respective light quantity.
b Low: 20pLmol m s : Medium 170pLmol m s : High 400pLmol m s .
" Effective time 50: ET50 = time required by flumioxazin (h) to reduce hydrilla
photosynthesis by 50%. Each value is a mean of two experiments with a total of 10
replications (sprigs).
d 95% CI = 95% Confidence Interval.


Table 3-3. The effect of select contact herbicides at pH 9.0 on photosynthesis of apical hydrilla tips
cultured in a growth chamber for 96 h at 380 ymol m-2 S-la light quantity.


Herbicideb

Flumi 400e


ETSOC (95% CId)


% Label rate


Regression equation


156 (117-233) y = 88.9311e-0.00444x 0.99


1568 (291-6931) y =103.8e-0.000442x


K50


0.98

0.92


K200


35 (26-53)


y = 93.2636e-0.0198x


Flumi 400 + K50

Flumi 400 + K200

Endothall 5000


100 + 5

100 + 20

100


126 (100-171) y = 93.0193e-0.00549x 0.99


20 (17-25)

71 (62-82)


y = 96.3408e-0.0340x

y =101.4e-0.00978x


0.97

0.98


a Data are normalized to the control.
bHerbicide rate: pLg L' a.i.
" Effective time 50: ET,, = time required by the herbicide (h) to reduce hydrilla photosynthesis by 50%. Each value is a
mean of two experiments with a total of 10 replications (sprigs).
d 95% CI = 95% Confidence Interval.
e Abbreviations: Flumi, flumioxazin; K, Komeen (copper chelate); endothall = dipotasium salt.





0.4 -




r 0.3-P




S0.2-P


-- 3DAT

0.1
0 200 400 600 800 1000 1200 1400 1600

Flumioxazin Concentration (pg L-1 a.i.)
Figure 3-3. The effect of flumioxazin concentration on hydrilla chlorophyll content (mg kgl
fresh weight) 1 to 4 d after treatment (DAT) under 70% sunlight. Flumioxazin was
applied as a single application to hydrilla cultured in 18.9 L buckets filled with tap
water (pH 9.0-9.5). Data are shown as actual means + standard error (n=6).









CHAPTER 4
THE EFFECT OF FLUMIOXAZIN ON SUBMERSED, EMERGENT, AND FLOATING
AQUATIC PLANT PLANTS

Introduction

One of the primary goals of aquatic weed control in public and private waters is to

eliminate invasive plants while maintaining a diversity of native submersed and emergent

species. Native aquatic plants may improve water clarity and quality, provide valuable fish and

wildlife habitat, reduce rates of sediment resuspension, and help prevent the spread of invasive

plants (Savino and Stein 1982; Heitmeyer and Vohs 1984; Smart 1995; Dibble et al. 1996b).

Hydrilla is a submersed aquatic fresh water angiosperm that is considered native to Asia or

Africa and has become a serious weed problem in the United States and many other countries

(Cook 1985; Haller and Sutton 1975; USDA 2007; Van and Vandiver 1992). Once established

in a body of water, hydrilla readily dominates and replaces native submersed species by forming

a canopy that reduces light penetration, increases surface water temperature, and reduces

dissolved oxygen (Bowes et al. 1979; Haller and Sutton 1975; Honnell et al. 1993).

Floating aquatic plants such as water hyacinth, water lettuce, and duckweed form dense,

free floating mats which can interfere with navigation, hydroelectric generation, and irrigation

(Harley et al. 1984). They may also harbor mosquitoes, which are vectors for diseases like

malaria and encephalitis (Holm et al. 1977).

Selective removal of hydrilla and other invasive species with aquatic herbicides is

beneficial for retention of native vegetation. Native plant density and diversity can increase

considerably if canopy forming exotic plants are removed (Getsinger et al. 1997). Non-target










damage to native species can result from submersed and foliar applications of herbicides and is a

consideration in herbicide selection

Flumioxazin is being evaluated by Valent U. S.A. Corporation for control of aquatic weeds.

The high costs associated with registering an herbicide for a new market (i.e., aquatics) may be

overcome by maximizing the market potential. Therefore, the first objective of this research was

to determine if flumioxazin has utility as a foliar and submersed treatment to control floating

aquatic weeds. Both submersed and emergent non-target aquatic plants could be impacted by

flumioxazin applications, so the second obj ective of these studies was to quantify the effects of

foliar and submersed flumioxazin treatments on submersed and emergent aquatic plants.

Materials and Methods

Floating Aquatic Plants

Water hyacinth and water lettuce were collected from Rodman Reservoir near Interlachen,

FL and established in 95 L HDPE tubs filled with 80 L of tap water (pH 8.0) in April 2006 at the

University of Florida Center for Aquatic and Invasive Plants in Gainesville, FL. Tap water was

supplemented with 1 mL of chelated ironll 12-0-0 plus Miracle-Gro@12 (150 mg L^1) prior to

herbicide treatment and added again at 1 and 2 wk after treatment. Water hyacinth (5 plants per

tub) and water lettuce (20 plants per tub) were allowed to acclimate for 3 wk before treatment.

This study was repeated in August 2006 near the University of Florida in Gainesville, FL with

water from Biven's Arm Lake (pH 7.5). Both studies were completely randomized designs with

4 replications (tubs).



'0 W. T. Haller. 2007. Personal Conununication.

11 Lesco, Inc. Cleveland, OH. 44114.

12 The Scotts Company. Marysville, OH.









Flumioxazin was applied to water lettuce and water hyacinth as a foliar treatment using a

forced air CO2-pOwered sprayer at an equivalent of 935 L ha-l diluent delivered through a single

TeeJet@13 80-0067 nozzle at 10 psi. Flumioxazin was applied at 0, 36, 72, 143, 286, 572, and

1144 g ha-l plus a non-ionic surfactant (0.25% v/v). An additional study was conducted

concurrently to determine the effects of submersed flumioxazin treatment on these floating

invasive species with collection and setup procedures as described above for foliar treatments.

Water hyacinth and water lettuce were treated with flumioxazin as a submersed treatment at 0,

100, 200, 400, 800, and 1600 Clg L^1. All studies were conducted under full sunlight.

All live water hyacinth and water lettuce biomass was harvested 34 DAT, placed in a

drying oven at 90 C for ca. 1 wk, and then weighed. Plant dry weight data were analyzed using

non-linear regression (PROC NLIN, SAS Institute 2002). Regression models were used to

determine the effective concentration 50 (ECso), which is the concentration of flumioxazin that is

required to cause a 50% reduction in dry weight compared to control plants.

A population of landoltia was collected from a pond with no history of herbicide

treatments in Alachua County, FL and maintained in 266 L fiberglass tanks in a greenhouse

(70% sunlight). The landoltia was cultured in tap water (pH 8.2) amended with topsoil and

Miracle-Gro in a greenhouse (1200 Cpmol m-2 S-1). Plants were treated with carbaryl insecticidel4

weekly and allowed to acclimate in the tanks for 2 wk before treatment. A 10 g aliquot (fresh

weight) (1.3 + 0.07 g dry weight) of landoltia was placed in each 3 L HDPE (17.1 cm diameter

by 13.3 cm deep) pot filled with tap water (pH 8.0). Plants were allowed to acclimate in the pots

for 2 d prior to herbicide treatment. All pots were amended with the same Miracle-Gro rate as



13 TeeJet Technologies. Wheaton, IL 60189.

14 Sevin insecticide label. Bayer CropScience. Research Triangle Park, NC 27709.









the previous studies 2 and 14 DAT. Landoltia was treated with a submersed treatment of

flumioxazin at 0, 10, 25, 50, 100, 200, 400, 800, and 1600 Clg L^1. As a comparison treatment,

diquat was applied as a foliar treatment at 1.1 kg ha-l a.i. using the described methods for foliar

flumioxazin treatments. The initial experiment was conducted in April 2007 and was repeated in

May 2007. This experiment was a randomized design with 5 replicates.

Due to the difficulty of removing large quantities of bleached and dead landoltia plants,

visual estimates of control (% control) were determined on a scale of 0 to 100%, where 0 = no

chlorosis/necrosis and 100 = plant death. Percent control ratings were based on nontreated

control plants. There were no differences in control between the two experiments (Fisher' s

Protected LSD, p<0.05); therefore, the data were pooled for analysis and means were separated

using 95% confidence intervals.

Foliar flumioxazin treatments were also applied to landoltia in October 2005, April 2007,

and May 2007. Flumioxazin was applied to landoltia at 0, 36, 72, 143, 286, 572, and 1144 g hal

plus a non-ionic surfactant (0.25% v/v) using the equipment described in the water lettuce and

water hyacinth study.

Submersed Aquatic Plants

The submersed aquatic plants coontail, egeria (Egqeria densa Planch.), hydrilla, southern

naiad, and vallisneria were evaluated for sensitivity to flumioxazin at a high (9.0) and low (7.0)

pH in 2006 and 2007. Hydrilla was collected from Rodman Reservoir near Interlachen, FL in

July, September, and December 2006, while all other species were purchased from a local plant

nursery. The high pH study was only conducted once (August 2006); the low pH study was

conducted in September 2006 and repeated in January 2007. The high pH and initial low pH

experiments were conducted in a shade house (70% sunlight), whereas the repeated low pH

experiment was conducted in a greenhouse with 70% sunlight. Two sprigs of each species were









planted in each 10 by 10 by 12 cm (1 L) pot which were filled with masonry sand amended with

Osmocote (15-9-12) fertilizer at a rate of 1g kg-l soil and placed in 95 L HDPE tubs filled with

tap water (pH 7.5 at planting). Each tub contained all Hyve species (2 pots/species/tub). Plants

were allowed to acclimate for 2 wk prior to herbicide application. This experiment was a

randomized design with 4 replications (tubs). Flumioxazin was applied as a submersed treatment

at 0, 50, 100, 200, 400, 800, and 1600 Clg L^1 in high and low pH water. Water pH in all tubs

was >9.0 prior to treatment so each low pH treatment tub was treated with ca. 15 mL of muriatic

acid to lower pH to 7.0. The pH was monitored to determine if and when the pH would return to

pH >9.0. Tub pH was not maintained at 7 since pH beyond 24 HAT was not relevant in this

study since flumioxazin is taken up within minutes to hours after treatment (see Chapter 2 and

3). All living plant tissue was harvested at the soil line 28 DAT, placed in a drying oven at 90 C

for ca. 1 wk and weighed.

Plant dry weight data were analyzed using non-linear regression (PROC NLINT, SAS

Institute 2002). Regression models were used to determine the effective concentration 50

(ECso), which is the concentration of flumioxazin that is required to cause a 50% reduction in dry

weight compared to control plants.

Data from both low pH experiments were pooled for coontail, egeria, and hydrilla because

there was no difference between the slopes of regression lines for both experiments at the 95%

confidence interval level. There were notable differences in the pre-treatment dry weight and

growth of naiad and vallisneria between the initial and repeated study; plants in the second study

(January) did not grow as rapidly as those in the first study (September), so data from these

experiments were analyzed separately by species.









Emergent Aquatic Plants

The sensitivity of the emergent aquatic plants eleocharis (Eleocharis interstincta (Vahl)

Roemer & J.A. Schultes), maidencane, pickerelweed, and sagittaria were evaluated in submersed

and foliar flumioxazin treatments. All plants were purchased from a local plant nursery in

August 2006 and April 2007 for the submersed and foliar studies, respectively. Two sprigs of

each species were planted in a mixture of 2: 1 potting soil:masonry sand in 3 L HDPE pots (17. 1

cm diameter by 13.3 cm deep) amended with Osmocote (15-9-12) fertilizer at a rate of 1g kg-l

soil. The submersed flumioxazin experiment was a randomized design with five replicates

(tubs). One pot of each species was grown for 4 wk in 95 L HDPE tubs placed in a shade house

(70% sunlight) for the submersed flumioxazin study. Water level in the tubs was maintained at

25 cm, and pH remained at or near 7.5. Plants were grown for 1 mo when flumioxazin was

applied at 0, 50, 100, 200, 400, 800, and 1600 Clg L1 as a submersed treatment. Prior to

treatment with a foliar application (1 mo after planting), all 5 emergent replicates (pots) were

placed in one 266 L fiberglass tank (72 cm by 82 cm by 45 cm) for a total of 7 treatments

(tanks). Flumioxazin was applied at 0, 36, 72, 143, 286, 572, and 1144 g ha-l a.i. plus a non-ion

surfactant (0.25% v/v) as a foliar treatment. Plants were treated using a forced air CO2-pOwered

sprayer at an equivalent of 935 L ha-l diluent and delivered through a single TeeJet@"S 80-0067

nozzle at 10 psi. Water level was maintained at 25 cm with a drain pipe and water was

exchanged in the tanks for 10 min after each foliar treatment and again 24 h after treatment to

ensure no herbicide would bind to the soil or remain in the water column for underwater uptake.





15 TeeJet Teclmologies. Wheaton. IL 60189.









Plants were harvested 40 DAT and plant height from the soil surface to the tip of the tallest

leaf was recorded. All emergent plants were harvested in the manner described for submersed

plants with plant height and dry weight subj ected to the same statistical procedures. Data were

pooled across experimental runs when no statistical difference between the slopes of regression

lines were observed.

Results and Discussion

Floating Aquatic Plants

Water lettuce was more sensitive to foliar applications of flumioxazin at 36 to 1 144 g ha-l

than water hyacinth as indicated by calculated ECso values of 69 and 1435 g ha- respectively

(Figure 4-1). Flumioxazin applied as a foliar treatment at rates greater than 143 g ha-l resulted in

complete control of water lettuce. Treated water lettuce plants exhibited chlorosis and necrosis

on the leaves (3 to 5 DAT) and defoliation (12 to 15 DAT); also, plants completely decayed at

herbicide rates >286 g ha-l 21 DAT. Sublethal rates resulted in regrowth of young plants

(ramets) from the meristematic region of water lettuce. The highest flumioxazin rate evaluated

(1 144 g ha- ) reduced water hyacinth biomass by only 41% of the nontreated control 34 DAT.

Treated water hyacinth plants exhibited blackening on younger leaves only, which is similar to

injury symptoms reported in water lettuce and water hyacinth treated with the PPO inhibitor

carfentrazone (Koschnick et al. 2004).

Water lettuce treated with a submersed application of flumioxazin was controlled at all

concentrations evaluated in this study (data not shown) and these results suggest that water

lettuce is more sensitive to flumioxazin applied in the water than applied to the foliage. In

contrast, flumioxazin did not reduce water hyacinth biomass by more than 30% of the nontreated

control plants at any rate evaluated in this study and confirms that water hyacinth is more









tolerant to flumioxazin than water lettuce (data not shown), similar to results with the PPO

inhibitor carfentrazone (Koschnick et al. 2004).

Water hyacinth plants treated in these studies were large and mature (45 & 8 cm in height)

and may have been more tolerant of foliar treatments than smaller and immature plants;

therefore, younger, more actively growing plants (10 & 3 cm) were also tested for flumioxazin

sensitivity, but these immature plants did not respond differently to treatments (data not shown).

Immature and mature water hyacinths treated with foliar flumioxazin treatments in field EUP

trials (data not shown) have shown tolerance levels similar to those noted in these studies.

The effects of a submersed application of flumioxazin from 10 to 1600 Clg L^1 are

presented for landoltia in Figure 4-2. Most flumioxazin treatments caused foliar bleaching

within 7 to 10 DAT, but none of the treatments resulted in complete control of landoltia. Each

flumioxazin treatment was different as indicated by no overlapping of 95% confidence interval

bars. Landoltia colonies treated at rates above 25 Clg L-1 began to separate and roots became

detached from individual fronds. Koschnick (2005) found landoltia treated in the dark with

diquat underwent root detachment without chlorosis. The primary function of roots of plants in

the Lemnaceae family is stabilization of fronds (Landolt 1986). Diquat applied as a comparison

treatment resulted in 100% control less than 5 DAT when applied at 1.1 kg hal as a foliar

treatment. Duckweed is extremely sensitive to diquat with an EC5o of 4 Clg L-1 (Peterson et al.

1997) and is the current industry standard for duckweed control.

The foliar applied flumioxazin landoltia study was conducted 3 times; treated plants were

visually similar to control plants at all rates showing no dose response and therefore were not

harvested (data not shown). The foliar treatment to landoltia and water hyacinth was

unsuccessful and high foliar rates were needed to control water lettuce. These results suggest









that flumioxazin uptake is limited by the leaf cuticle or occurs primarily through the underside or

roots of the plant.

Koschnick et al. (2004) reported that the calculated ECso for a foliar application of

carfentrazone to reduce water lettuce was 8 to 10 g ha- approximately 7 to 9 times less than

flumioxazin' s calculated ECso for water lettuce. Higher concentrations (30 and 35 g ha- ) of

carfentrazone were required to control water hyacinth (Koschnick et al. 2004), but flumioxazin

in our study failed to reduce water hyacinth biomass by more than 30%. Results from these

studies show that roots or the underside of the plant is effective at absorbing flumioxazin when

applied as a submersed treatment.

If registered as an aquatic herbicide, flumioxazin will likely be used primarily as a

treatment for submersed weed control. Although most floating aquatic weeds are controlled by

foliar herbicide applications, these results indicate that water lettuce and landoltia may be

controlled with submersed treatments. Diquat and 2,4-D are the primary aquatic herbicides used

for control of water lettuce, water hyacinth and duckweed (Westerdahl and Getsinger 1988;

Langeland et al. 2002) and are more efficacious on water lettuce, water hyacinth, and duckweed

as foliar treatments. Flumioxazin in these studies provided control only at higher application

rates (water lettuce) or provided <30% control (water hyacinth and landoltia).

Further research is needed to determine if flumioxazin as a submersed treatment is as

efficacious to water lettuce in higher pH water (9.0). Efficacy data for flumioxazin is also

needed on additional floating species such as Salvina spp. and Wolffia spp. Additionally, the

effect of flumioxazin used in conjunction with surfactants should be examined in both immature

and mature water hyacinths to determine whether surfactants increase flumioxazin uptake and

activity in these species.









Submersed Aquatic Plants

Water pH in control tanks returned to 9.0 within 24 HAT. The pH of water in the 100,

200, and 400 Clg L^1 low pH treatments returned to 9.0 ca. 36 HAT, while pH of water in the two

highest herbicide rates (800 and 1600 Clg L^1) never exceeded 8.5.

Flumioxazin applied in low (7.0) pH water at 50 to 1600 Clg L1 caused greater injury to

coontail, hydrilla, naiad, and vallisneria than flumioxazin applied in high pH (9.0) water (Figure

4-3). All species with the exception of egeria were more tolerant to flumioxazin applied in high

pH water compared to those in low pH water according to the calculated EC5o values for dry

weight (Table 4-1). Coontail treated in high pH water was the only susceptible species to

flumioxazin at the current EUP label rate of 400 Clg L^1. All other species treated with

flumioxazin in high pH water would require an estimated flumioxazin concentration of >3 194 Clg

L- to reduce biomass by 50%.

The pH of Florida lakes infested with surface matted hydrilla may be greater than 8.0 and

likely in excess of 9.0 as a result of the hydrilla utilizing free CO2 and HCO 3 during daily

photosynthesis (Van et al. 1976), so egeria, naiad, and vallisneria would be only slightly injured

from flumioxazin treatment in higher pH water based on these data. However, hydrilla was less

affected by the treatment in high pH water, in contrast to observations from the general efficacy

study (see Chapter 2). Hydrilla in this study responded immediately to flumioxazin exposure by

bleaching and undergoing rapid decline, but new apical tips sprouted <1 WAT from treated

tissue and flumioxazin application at 1600 Clg L^1 reduced dry weight by only ca. 40% of the

nontreated control plants. These data and the pond efficacy data outlined in Chapter 2 indicate

flumioxazin produces highly variable results when applied to hydrilla growing in high pH water.

Coontail, hydrilla, and naiad in were highly susceptible to flumioxazin in low pH water

and would likely be injured or controlled at the proposed label rate of 400 Clg L^1 (Table 4-1).









The calculated ECso value for naiad in experiment 1 was 10x greater than the ECso in the

repeated experiment (517 vs. 51, respectively), but flumioxazin at 400 Clg L-1 would still

significantly reduce biomass.

In comparison to these data, previous research has shown that coontail is sensitive to the

contact herbicide dipotasium salt of endothall at concentrations as low as 0.5 mg L^1 a.i. (Hofstra

and Clayton 2001) and endothall has been shown to reduce vallisneria biomass at concentrations

greater than 0.5 mg L- but plants recovered 8 WAT (Skogerboe and Getsinger 2002). In this

study, egeria and vallisneria from the 2nd low pH experiment were not affected by flumioxazin;

also, egeria was the only plant unaffected by flumioxazin treatments at either pH. Most plants

exposed to flumioxazin in the high and some in the low pH treatments, with the exception of

coontail, were beginning to regrow prior to harvest. Similarly, many of the non-target species

treated with flumioxazin in EUP ponds recovered within a few weeks after treatment (data not

shown). Non-target submersed aquatic species will only be exposed to flumioxazin for short

exposures due to the rapid degradation of this herbicide (Katagi 2003), especially when applied

to water with a pH >8.0. Those species with marginal tolerance should be able to overcome a

flumioxazin treatment in high pH water since flumioxazin will not be present in the water for

more than a couple of days. In contrast, non-target plants exposed to herbicides with a longer

half-life, such as fluridone or penoxsulam, have the potential of being severely injured or killed

because of longer exposures (Koschnick et al. 2007; Langeland and Warner 1986).

Development and use of herbicides that selectivity control non-target aquatic plants is a priority

of most state agencies involved in aquatic weed management (Anonymous 2007b; Koschnick et

al. 2007).









Symptomology of hydrilla treated with flumioxazin in previous EUP studies (see Chapters

2 and 3) was bleaching in the apical tip and reddening in the stem followed by chlorosis and

necrosis. Visual symptoms of other flumioxazin-treated plants in these studies included

bleached apical tips followed by reddening of the stem (egeria), defoliation and darkening of

tissue (naiad), defoliation and loss of stem/leaf integrity (coontail), and transparent appearing

leaves (possibly due to loss of chlorophyll) (vallisneria). Plants treated with flumioxazin at pH

7.0 were glossy and darker green in color and were treated with muriatic acid to maintain pH

<7.0 for 24 HAT but control plants were not discolored, so muriatic acid did not contribute any

injury symptoms. Flumioxazin is hydrolyzed at a much slower rate in lower pH water (Vencill

2002) having a half-life of 16. 1 h at pH 7.0 compared to 17.5 min at pH 9.0 (Katagi 2003), so the

increased exposure time of these submersed aquatic plants to flumioxazin at pH 7.0 resulted in

greater injury and reduction in biomass to all species except egeria. These data clearly show

flumioxazin in high pH water is more selective with regard to non-target plant injury than

flumioxazin in low pH water.

Emergent Aquatic Plants

Emergent aquatic plants had varying levels of sensitivity to flumioxazin concentrations

<800 Clg L^1; however, there were minimal differences among plant species at concentrations of

1600 Clg L^1 (Figure 4-5). Sagittaria dry weight was reduced by 100% at the highest

concentration compared to a 73 to 83% dry weight reduction in all other emergent plants.

Sagittaria was the most sensitive species followed by maidencane, eleocharis, and pickerelweed

based on calculated ECso values for dry weight and height (Table 4-2). Elocharis and

pickerelweed were more tolerant of a submersed flumioxazin application than sagittaria and

maidencane, but these species would likely be injured by flumioxazin at 400 Clg L^1. Although

dry weight data is often a more reliable indicator to assess herbicide effectiveness, plant height









data in this study provided additional evidence of the selectivity of submersed flumioxazin

treatments on emergent plants.

Visual injury symptoms observed 2 WAT included interveinal chlorosis (sagittaria and

pickerelweed), reddening on leaf margins (maidencane), and minor chlorosis (eleocharis).

Flumioxazin and other protox-inhibiting herbicides are absorbed primarily by plant roots with

some absorbance in the shoots, but translocation is limited once herbicides are absorbed into

foliar tissue (Fadayomi and Warren 1977; Ritter and Coble 1981; Unland et al. 1999; Vencill

2002). Pots without holes were used in these studies and few roots were visible above the soil

line, so herbicide uptake directly from the root zone was unlikely as flumioxazin was mixed

directly into the water column. Flumioxazin uptake occurred either through the underwater stem

or submersed leaves. Previous research found little flumioxazin translocation in plants, but

submersed treatment of emergent aquatic plants in this study suggested movement of

flumioxazin from the soil or lower stem into the leaves. If translocation of flumioxazin was

limited, this herbicide should have girdled the plant at the soil line and produced injury

symptoms such as necrosis throughout the stem and leaves without veinal chlorosis first

appearing in the leaves. Ferrell et al. (2007a) showed flumioxazin + MSMA (monosodium salt

of MAA) resulted in a 94% yield reduction when applied as a high post-direct treatment to 20 cm

tall cotton. Symptomology of flumioxazin treated cotton included necrotic lesions on leaves,

reddening stems, stem girdling, and eventual lodging. Previous research also found that as

cotton matures, plants become more tolerant to flumioxazin because of greater bark development

and metabolic capacity (Ferrell and Vencill 2003b).

Foliar flumioxazin treatments were less injurious to emergent aquatic plants than

submersed treatments (Figure 4-5). Maidencane and sagittaria would require foliar application









rates greater than 1320 and 6478 g ha- to reduce dry weight and height by 50% (ECso),

respectively (Table 4-3). An ECso value could not be calculated for dry weight and height for

both eleocharis and pickerelweed since increased flumioxazin concentrations resulted in an

increase in dry weight (positive regression slope). Postemergent applications of flumioxazin are

generally recommended for actively growing weeds less than 5 cm in height (Anonymous 2006),

so the minimal foliar injury and substantial lack of reduction in biomass observed in this study to

eleocharis, maidencane, pickerelweed, and sagittaria were probably due to the maturity of these

plants. Injury symptoms (including chlorotic and necrotic lesions on the leaves) were similar to

those described for other protox-inhibiting herbicides (Peterson et al. 2001). Tolerant species

have reduced or no symptoms, whereas the leaves of susceptible species rapidly desiccate and

die (Peterson et al. 2001).

Selective control of invasive weed species has always been a goal of aquatic weed

managers. Herbicide applicators target specific non-native plants through the use of specifically

formulated herbicides, seasonally timed herbicide application, and/or preemptive spot treatments

before weeds become a problem (Ceryone and Schardt 2003). Flumioxazin provided selective

weed control when applied as a foliar treatment or selectivity could be attributed to the maturity

of these plants. In contrast, submersed applications resulted in more injury to nontarget plants

especially when treatments were made in low pH water. 2,4-D selectively controls broadleaf

weed species (Ceryone and Schardt 2003), whereas fluridone selectivity is based on initial

treatment rate, length of exposure, and initial plant biomass (Netherland et al. 2007).

These data indicate that coontail, sagittaria, and maidencane are adversely affected by

flumioxazin at concentrations <400 Clg L^1 in water with a pH of 7.0. Most emergent and

submersed plants evaluated in these studies could be injured or killed if flumioxazin is applied to










low pH water (<7.5). These studies provided "worst-case" scenarios where emergent and

submersed plants were continuously exposed to flumioxazin. Herbicide concentrations in lakes

are influenced by factors such as wind, flow, dilution, and pH, which minimize direct contact of

native plants with herbicides such as flumioxazin when applied as a submersed contact

application. Direct foliar applications to these native emergent plants would occur if they grow

among targeted emergent or floating plants. Most of these non-target emergent plants will be

minimally affected by the maximum proposed foliar label ratel6 Of 286 g ha-l a.i.

Submersed aquatic plants are often found among or near hydrilla infestations, whereas

emergent plants usually grow along the shoreline. Whole lake treatments with flumioxazin are

unlikely since this product will be used primarily as a contact herbicide for submersed weed

control. Partial lake treatments allow for dilution of the herbicide to further reduce exposure of

non-target plants.

Although several submersed and emergent species were evaluated for sensitivity to

flumioxazin, further research should be conducted to determine the sensitivity of other non-target

and invasive plants at various water pHs. All emergent plants in this study were mature and

consequently more tolerant of both foliar and submersed flumioxazin treatments; however, most

flumioxazin treatments would likely occur in early spring or summer when hydrilla can be

controlled more easily due to rapid growth, lower pH, and less hydrilla biomass. Most emergent

and submersed non-target plants will be immature and actively growing and could possibly be

injured by foliar and submersed flumioxazin treatments.







16 M. S. Rifle. 2007. Personal Communication.












100 ,Water Hyacinth: y = 86.0893-0.000483, r' = 0.95
ECo = 1435 g ha' a.i.
-Water Lettuce: y = 67.2077-0.00998x, r2 = 0.92
80 ---ECo = 69 g ha' a.i.



60 -0




20-






0 200 400 600 800 1000 1200

Flumioxazin Concentration (g hal a.i.)
Figure 4-1. The effect of a foliar flumioxazin application (g ha-l a.i.) on water lettuce and water
lettuce dry weight 34 d after treatment under 100% sunlight. Flumioxazin was applied
as a single application by a CO2-pOwered sprayer at an equivalent of 379 L ha-
diluent with a non-ionic surfactant (0.25% v/v) to water lettuce and water hyacinth
grown in 95 L tubs (pH 7.5 to 8.0). Data are shown as dry weight means + standard
error (n=10). ECso = effective concentration 50, concentration of flumioxazin (g ha-l
a.i.) that is required to reduce water lettuce and water lettuce biomass by 50%.














I Diquat (kg ha' a.i.)
80 -I I Flumioxazin (mg L' a.i.)






40-



20-




1.1 10 25 50 100 200 400 800 1600

Herbicide Rate


Figure 4-2. Percent control (visual) of landoltia 21 d after a foliar diquat (g hal a.i.) and
submersed flumioxazin application (Clg L^1 a.i.). Diquat and flumioxazin each applied
as a single application to landoltia cultured in 1 L pots (water pH 8.0) under 70%
sunlight. Diquat was applied by a CO2-pOwered sprayer at an equivalent of 379 L ha-
Sdiluent with a non-ionic surfactant (0.25% v/v). Percent control + 95% confidence
interval (CI) (n=10). Overlapping CI bars indicate no significant difference.














- Egeria High pH
- -- EgeriaLow pH


Hydrilla High pH -Nalad High pH
Hydrilla Low pH Nalad Low pH Experiment 1
10 --- Nalad Low pH Experiment 2

S8-










0 -------- -
12~~ 0 200 400 600 800 1000 1200 1400 1600
Valhlsneria High pH
Valhlsneria Low pH Experiment 1 Flumioxazin Concentration (pg L-1 a.i.)
---- Valhlsneria Low pH Experiment 2













0 200 400 600 800 1000 1200 1400 1600

Flumioxazin Concentration (pg L-1 a.i.)

Figure 4-3. The effect of flumioxazin concentration on the dry weight of submersed aquatic
plants 28 d after exposure. Flumioxazin applied as a single application to submersed
aquatic species cultured in low (7.0) and high (9.0) pH water in 95 L tubs under 70%
sunlight. Data are shown as actual dry weight means a standard error (n=10 for low
pH, except for naiad and vallisneria n=5; n=5 for high pH). Dry weight means &
standard error (n=10).










Table 4-1. The effect of a single submersed flumioxazin application on dry
weight of submersed aquatic plants 28 d after exposure.
High pHb ECsoc (95% CId) Regression equation r2

Coontail 403 (248-1081) y = 7.9148e-0.00172x 0.86

Egeria 3747 (1720-23104) y = 2.6419e-0.000185x 0.94

Hydrilla 3194 (869-6931) y = 4.0822e-0.000351x 0.83
Naiad NAe y = 4.9646e0.0002x 0.90

Vallisneria 5172 (2173-13863) y = 3.6688e-0.000134x 0.95

Low pHf

Coontail 34 (27-46) y = 9.6997e-0.0204x 0.87

Egeria 3285 (1925-11179) y = 2.8606e-0.000211x 0.94

Hydrilla 77 (53-138) y = 3.8329e-0.00902x 0.86
Naiad #18 517 (338-1093) y = 1.6128e-0.00134x 0.90

Naiad #2 51 (30-204) y = 4.1424e-0.0133x 0.64

Vallisneria #1 853 (533-2120) y = 3.1724e-0.000813x 0.90

Vallisneria #2 3536 (1270-4621) y = 1.4483e-0.000196x 0.86
a Submersed aquatic species cultured under 70% sunlight.
b High pH: 9.0.
" Effective concentration 50: EC50 = concentration of flumioxazin (pLg L' a.i.) in water
required to reduce plant dry weight by 50%. Each value is a mean of two experiments with a
total of 5 replications (pots) for high pH: 10 reps for coontail, egeria, and hydrilla at low pH:
and 5 reps for naiad #1, naiad # 2, vallisneria #1, and vallisneria #2 at low pH.
d 95% CI = 95% Confidence Interval.
e NA = not applicable due to positive regression slope.
f Low pH: 7.0, pH reduced with muriatic acid.
g #1 and #2: experiment 1 and experiment 2.











I


14-


Eleocharis
r~ I t- --- Maidencane
& 10 1 -C ------ Pickerelweed
I [R~~- ~ -- -- Sagittaria














0 200 400 600 800 1000 1200 1400 1600

Flumioxazin Concentration (tpg L1 a.i.)

Figure 4-4. The effect of flumioxazin concentration on dry weight of emergent aquatic plants 40
d after exposure. Flumioxazin applied as a single application to emergent aquatic
species cultured in 95 L tubs (pH 7.5) under 70% sunlight. Data are shown as actual
dry weight means + standard error (n=5).









Table 4-2. The effect of a single submersed flumioxazin application on dry
weight of emergent aquatic species 40 d after exposure.
Dry Weight EC50b (95% CIc) Regression equation r2
Eleocharis 559 (389-1009) y =13.7460e-0.00124x 0.92
Maidencane 259 (168-564) y = 9.2236e-0.00268x 0.84

Pickerelweed 894 (598-1777) y = 9.4660e-0.000775x 0.91

Sagittaria 15 (11-26) y= 8.5266e-0.0448x 0.93

Height
Eleocharis 2295 (1513-4780) y = 77.5573e-0.000302x 0.97

Maidencane 1764 (1208-3285) y =74.5654e-0.000393x 0.96

Pickerelweed 13591 (5590-34657) y = 64.0690e-0.000051x 0.99

Sagittaria 38 (32-47) y = 64.3085e-0.0182x 0.95
a Emergent aquatic species cultured at pH 7.5 under 70% sunlight.
b Effective concentration 50: EC50 = concentration of flumioxazin (pLg L' a.i.) in water
required to reduce plant dry weight or height by 50%. Each value is a mean of two
experiments with a total of 10 replications (pots).
" 95% CI = 95% Confidence Interval.












Eleocharis
-Maindencane
16 -I -- -- Pickerehweed
-Sagittaria
h
S14-










6 io




0 200 400 600 800 1000 1200

Flumioxazin Rate (g hal a.i.)
Figure 4-5. The effect of flumioxazin rate on dry weight of emergent aquatic species 40 d after
treatment. Flumioxazin applied as a single application by a CO2-powered sprayer at
an equivalent of 379 L ha-l diluent with a non-ionic surfactant (0.25% v/v) to
emergent aquatic species cultured in 277 L tanks (pH 7.5) under 70% sunlight. Data
are shown as actual dry weight means + standard error (n=5).










Table 4-3. The effect of a single foliar flumioxazin application on dry weight
and height of emergent aquatic species 40 d after treatment.


EC50b (95% CIc)

NAd

1884 (1002-15753)

NA

1320 (859-2852)


Dry Weight
Eleochari s

Maidencane

Pickerelweed


Regression equation

y =11.5964e0.00005x

y = 12.8338e-0.000368x


0.99

0.92

0.97

0.95



0.99

0.99

0.99

0.99


9.6895e0.00013x


Sagittaria


y = 13.0027e-0.000525x


Height


Eleochari s

Maidencane

Pickerelweed


64 8843e0 00001x


6478 (3938-18734)


y = 88.3085e- 0.000107x

y = 65.9479e-0.000144x
v = 76.3892e-0.000057x


NA


Sagittaria


12160 (5501-18887)


a Flumioxazin applied by a CO,-powered sprayer at an equivalent of 379 L/ha diluent with a
non-ionic surfactant (0.25% v/v). Emergent aquatic species cultured in 277 L tanks at pH 7.5
under 70% sunlight.
b Effective concentration 50: EC5o = concentration of flumioxazin (g ha-' a.i.) to reduce plant
dry weight or height by 50%. Each value is a mean of one experiments with a total of 5
replications (pots).
" 95% CI = 95% Confidence Interval.
d NA = not applicable due to positive regression slope.









CHAPTER 5
ORNAMENTAL AND ROW CROP SUSCEPTIBILITY TO FLUMIOXAZIN INT
IRRIGATION WATER

Introduction

Homeowners, commercial nurseries, and farmers in Florida often irrigate plants from

surface waters (canals, ponds, lakes, etc.) (Hassell et al. 20004; Hodges and Haydu 2006); non-

target plants may be affected if these waters are treated with herbicides for aquatic weed control.

The use of herbicide treated irrigation water before herbicide residues dissipate below phytotoxic

levels will result in injury or death of irrigated plants. Previous research has evaluated the

phytotoxic effects of irrigation water containing copper, 2,4-D, fluridone, diquat, and endothall

on non-target turf and ornamental species (Andrew et al. 2003; Hiltibran and Turgeon 1977;

Koschnick et al. 2005a; Koschnick et al. 2005b; Mudge et al. 2007; Reimer and Motto 1980), but

similar studies have not been conducted with flumioxazin. Tolerances of flumioxazin on certain

food crops have been established by the EPA by determining the maximum amount of pesticide

residue that can remain in or on a treated food commodity to ensure food safety (EPA 2003), but

no such tolerances are required for ornamental plants (non-food crops). Phytotoxicity is a major

concern when water with aquatic herbicide residues is used for irrigation of both food and non-

food crops. This study was conducted to assist in determining the minimum time required before

treated water may be used for irrigation of row crops and ornamental species by evaluating

phytotoxicity of flumioxazin-treated irrigation water on three ornamental and four row crop

plants.

Materials and Methods

Ornamental Susceptibility

Greenhouse studies were conducted in July and August 2006 at the University of Florida

Center for Aquatic and Invasive Plants in Gainesville, FL to evaluate the sensitivity of the









ornamental plant species begonia (Begonia x sensperflorens-cultorunt 'Senator'), impatiens

(Inspatiens wallerana 'Super Elfin Red'), and snapdragon (Antirrhinunt najus 'LaBella Pink') to

flumioxazin. These three common ornamental plants were purchased from local nurseries in

Gainesville and grown in 9.0 x 9.0 x 9.0 cm pots as purchased from the growers. Pots contained

an organic commercial potting medium and were top-dressed with Osmocote (15-9-12) fertilizer

at a rate of 1g kg-l soil upon arrival. Plants were subj ected to a 14 h photoperiod with maximum

daytime temperatures of 3 1 & 2 C and minimum nighttime temperatures of 21 & 2 C.

Experimental plants were selected based on uniform height to minimize variation in initial height

and weight then grown for 1 wk to allow acclimation before treatment. At treatment, plant

height (cm a standard error) for each species was as follows: begonia 16.9 & 0.8, impatiens 17.9

& 1.1, and snapdragon 41.0 & 4.3 cm. Snapdragons were mature, hardy, and flowering at time of

treatment, while begonias and impatiens were immature with no floral production at the time of

treatment.

The experiment was a completely randomized design with five replications (pots) per

treatment. Plants were overhead irrigated with herbicide treated water once with a sprinkle can

(equivalent to 1.27 cm of irrigation water). This volume was sufficient to cover plants and

saturate the soil. The pH of the irrigation water was 7.5 and flumioxazin was mixed with the

irrigation water immediately prior to irrigation. Flumioxazin concentrations of 0, 10, 25, 50,

100, 200, 400, 800, 1600, and 3200 Clg L^1 were applied to all ornamental plant species. Plants

were subsequently irrigated daily for 14 d with 1.27 cm of well water (containing no herbicide)

applied overhead via a sprinkle can. Plant height was recorded from the soil surface to the tip of

the tallest leaf 14 d after treatment (DAT). Pants were harvested 14 DAT by collecting all









aboveground biomass excluding dead tissue. Aboveground tissue was placed in a drying oven at

90 C for ca. 1 wk then weighed.

Plant dry weight and height data were analyzed using non-linear regression (PROC NLIN,

SAS Institute 2002). Regression models were used to determine the effective concentration 10

(EClo), which is the concentration of flumioxazin in irrigation water that caused a 10% reduction

in dry weight compared to control plants. Koschnick et al. (2005a) reported this value to be

conservative but near the threshold where an observant homeowner might detect adverse effects

on plant growth. All dry weight and height data were pooled for each ornamental species as

there was no difference between the slopes of regression lines for both experiments at the 95%

confidence interval level.

Crop Susceptibility

Corn (Zea mays L. 'Garst 8346 LL'), cotton (' Stoneville 6611 B2RF'), soybean

('NG2328R) and wheat (Triticum aestivum L., 'Wakefield') were evaluated for flumioxazin

sensitivity in 2006, with the initial study conducted in January and repeated in July. All crop

seeds were planted and grown in 10 x 10 x 12 cm (1 L) pots filled with masonry sand amended

with Osmocote (15-9-12) fertilizer at a rate of 1g kg-l soil. Plants were kept in a greenhouse

under a 14 h photoperiod with maximum daytime temperatures of 29 & 2 C and minimum

nighttime temperatures of 15 & 2 C. Plants for this experiment were selected based on uniform

height to minimize variation in initial height and weight. At treatment, plant height (cm A

standard error) for each species was as follows: corn (experiment 1) 42.8 & 1.0, corn (experiment

2) 59.8 & 2.0, cotton 20.5 A 1.2, soybean 18.6 & 0.8, and wheat 26.7 & 0.4 cm. All crops were

subjected to the same herbicide rates, overhead irrigation, harvest techniques, and statistical

procedures as the ornamental species.









All row crop data (except comn) were pooled across experiments because there was no

differences between the slopes of regression lines for both experiments at the 95% confidence

interval level. Comn grew more quickly in the summer study and was ready for treatment 1 wk

after planting, but all other crop species needed an additional week to be of similar size to plants

treated in the winter study. Initial comn dry weight and height differed between summer and

winter studies so corn data were not pooled across experiments.

Results and Discussion

Ornamental Susceptibility

Dry weights of impatiens and begonia differed at flumioxazin concentrations greater than

50 Clg L^1 (Figure 5.1). Snapdragons displayed minimal necrosis and chlorosis at all flumioxazin

rates throughout the course of the study, were more tolerant to flumioxazin in irrigation water

than begonia or impatiens, and had greater EClo values for dry weight and plant height (Table

5.1). Snapdragon EClo value for dry weight (7024 Clg L^1) was 68 and 175 times more than the

begonia and impatiens, respectively and begonia was less sensitive to flumioxazin than

impatiens. Higher sensitivity levels of impatiens and begonia to flumioxazin in irrigation water

was probably related to plant size and maturity at the time of treatment. Initial mean dry weights

(g & standard error) of begonia and impatiens were 1.7 & 0.1 and 1.6 & 0.1 and impatiens and

begonia control plants increased in dry weight by as much as 119 and 135% by the conclusion of

experiments. In contrast, initial mean dry weight of snapdragon was 13.4 & 0.3 g and only

increased by 32% during the course of experiments. Both preemergence and postemergence

applications of flumioxazin are generally recommended for actively growing weeds less than 5

cm in height (Anonymous 2006), so the lack of injury to mature snapdragons was likely due to

slow growth and resulted in reduced activity by flumioxazin on these mature plants.









Flumioxazin is ideal for use as a contact herbicide in aquatic systems since the half-life is

relatively short and it is degraded by hydrolysis in 4. 1 d, 16. 1 h, and 17.5 min at pH 5.0, 7.0, and

9.0, respectively (Katagi 2003). The half-life of flumioxazin is dependent on pH and is similar

to the aquatic herbicides endothall and diquat. Endothall reduced begonia and impatiens dry

weight by 10% at concentrations of 2 to 4 mg L^1 a.i. when applied in irrigation water

(Koschnick et al. 2005b) and diquat reduced dry weight by 10% at concentrations of 5.1 and 2.8

mg L^1 a.i. for begonia and impatiens, respectively (Mudge et al. 2007). The 10% reduction in

biomass of these two ornamental species with endothall was within the labeled use rate, but the

rate required to reduce impatiens and begonia biomass with diquat was more than 7 and 13 times

the labeled use rate, respectively. The EClo dry weight values for impatiens and begonia are well

within the proposed flumioxazin label rate of 400 Clg L^1 a.i. High pH water bodies treated with

this herbicide should cause rapid breakdown of flumioxazin and consequently permit shorter

irrigation restrictions; however, medium and low pH aquatic systems may need longer irrigation

restrictions to ensure sensitive species are not injured or killed by higher herbicide rates.

Crop Susceptibility

Wheat was more sensitive to flumioxazin in irrigation water than all other crops evaluated

(Figure 5.2, Table 5.2). The sensitivity of these plants to flumioxazin is indicated by dry weight

EClo values as follows: wheat (35), corn experiment #1 (53), cotton (106), corn experiment #2

(181), and soybean (193) (Table 5.2). Similar results were also observed with plant height data.

Corn in experiment 2 and soybean were generally more tolerant of flumioxazin than other

species, which was expected since flumioxazin is registered for preemergence control of

broadleaf weeds in soybean (Anonymous 2005).

Although the immature ornamental species and row crops evaluated in this study displayed

higher levels of sensitivity to flumioxazin compared to other short-lived contact herbicides such









as endothall and diquat, the short half-life of flumioxazin in water would partially ameliorate any

damage that may occur if homeowners irrigate soon after flumioxazin application. This may be

advantageous in pond or lake situations where pH exceeds 9.0, since homeowners could irrigate

sooner than if a longer residual herbicide was applied. Flumioxazin is still being evaluated under

an EUP and must be granted a full EPA Section 3 Label before this product can be applied to

public aquatic systems, since water bodies treated with flumioxazin under the EUP may not be

used for irrigation, swimming, drinking, or fish consumption. This study indicates that

flumioxazin has the potential to injure and kill immature ornamental and crop species and that

these plants may be injured when flumioxazin is applied at the potential maximum label use rate

of 400 Clg L 1. Based on these data, if homeowners or farmers irrigate with water treated with

flumioxazin soon after treatment, 10% or more injury may occur on young, actively growing

plants. Flumioxazin is generally more effective on hydrilla in the early spring when it is

immature and actively growing and this is the same time most crops and ornamental species are

planted by farmers and homeowners. Thus, irrigation restrictions will be variable due to the

significant effect of water pH on flumioxazin half-life, application rate, and differences in non-

target plant susceptibility as a result of the stage of maturity.










- Begonia
- Impatiens


4`


500


3000


1000


1500


2000


2500


Flumioxazin Concentration (Clg L1 a.i.)


Figure 5-1. The effect of flumioxazin concentration in irrigation water (pH 7.5) on ornamental
species dry weight 14 d after treatment. Flumioxazin was applied once to the plants as
an overhead irrigation with a sprinkle can (equivalent to 1.27 cm of irrigation water).
Data are shown as actual dry weight means + standard error (n=10). Data for
snapdragon were not included as flumioxazin resulted in minimum effects at all rates.










Table 5-1. The effect of a single overhead irrigation with 1.27 cm water
containing flumioxazin on ornamental species dry weight and
height 14 d after treatment.a


EC10b (95% CIC)

103 (84-136)


Dry weight

Begonia

Impatiens


Regression equation

y = 3.9194e-0.00102x

y = 3.7097e-0.00265x

y = 13.6060e-0.000015x


y = 23.2842e-0.000731x

y = 24.3930e-0.00209x

y = 50.3696e-0.000044x


0.94

0.92

0.94


0.97

0.93

0.98


40 (32-53)


Snapdragon 7024 (2450-10536)


Height

Begonia

Impatiens


144 (125-171)


50 (40-67)


Snapdragon 2395 (1463-7024)


a Flumioxazin was applied in water (pH 7.5) with a sprinkle can.
b Effective concentration 10: EClo = concentration of flumioxazin (pLg L-' a.i.) in
irrigation water required to reduce plant dry weight or height by 10%. Each value is a
mean of two experiments with a total of 10 replications (pots).
" 95% CI = 95% Confidence Interval.


























I\
\3


0 500 1000 1500 2000 2500 3000


0 500 1000 1500 2000 2500 3000


- Wheat


0 500 1000 1500 2000 2500 3000

Flumioxazin Concentration (pLg L-1 a.i.)


0 500 1000 1500 2000 2500 3000

Flumioxazin Concentration (pLg L-1 a.i.)


Figure 5-2. The effect of flumioxazin concentration in irrigation water (pH 7.5) on crop species
dry weight 14 d after treatment. Flumioxazin was applied once to the plants as an
overhead irrigation with a sprinkle can (equivalent to 1.27 cm of irrigation water).
Data are shown as actual dry weight means 0- standard error (n=10), except for corn
(n=5, for each experiment).










Table 5-2. The effect of single overhead irrigation with 1.27 cm water
containing flumioxazin on crop species dry weight and
height 14 DAT. a


EC10b (95% CIC)

53 (45-66)

181 (135-279)

106 (84-142)

193 (158-247)

35 (30-47)



56 (49-64)

221 (160-357)

120 (100-150)

206 (176-248)

46 (40-55)


Dry weight
Corn #1d

Corn #2

Cotton

Soybean
Wheat

Height
Corn #1

Corn #2

Cotton

Soybean
Wheat


Regression equation

y = 2.8429e-0.00198x

y = 4.7306e-0.000581x

y = 3.1634e-0.000993x

y =2.7891e-0.000547x

y =1.2876e-0.00289x



y = 67.2884e-0.00189x

y = 68.2482e-0.000476x

y = 33.1105 e-0.000875x

y = 42.6972e-0.000511x

y =38. 1144e-0.00229x


r

0.98

0.97

0.94

0.97

0.95



0.99

0.97

0.97

0.98

0.97


a Flumioxazin was applied in water (pH 7.5) with a sprinkle can.
b Effective concentration 10: EClo = concentration of flumioxazin (pLg L-' a.i.) in
irrigation water required to reduce plant dry weight or height by 10%. Each value is a
mean of two experiments with a total of 10 replications (pots).
" 95% CI = 95% Confidence Interval.
d #1 and #2: experiment 1 and experiment 2.









CHAPTER 6
THE EFFECT OF FLUMIOXAZIN AND DIQUAT ON MEMBRANE PERMEABILIITY
AND CHLOROPHYLL CONTENT OF LANDOLTIA

Introduction

Species of duckweed in the Lenanaceae family are commonly used in biochemical and

toxicity tests because of their small size, high reproductive rate and the ease with which they are

cultivated (Gensemer et al. 1999; Geoffroy et al. 2004; Lewis 1995; Ma et al. 2002; Parr et al.

2002). Studies evaluating pigment content (chlorophyll a, b, and carotenoids), oxygen emission,

and ion leakage are often reliable indicators of herbicide toxicity (Koschnick et al. 2006; Wang

and Freemark 1995).

Although hydrilla is the primary target weed in flumioxazin EUP research, invasive

floating plants such as landoltia are being evaluated for sensitivity to this compound. Members

of the Lenanaceae family are extremely sensitive to diquat as common duckweed possesses an

ECso of 4 Clg L^1 (Peterson et al. 1997). It has been controlled with diquat for many years;

however, landoltia plants have been discovered in Lake County FL with a resistance factor of

50x for diquat (Koschnick et al. 2006). Herbicides such as flumioxazin possess a different mode

of action and can be utilized to help prevent further development of resistance and may control

resistant plants. Previous research has shown flumioxazin at 1, 10, and 50 Clg L-1 decreased

photosynthetic capacity of common duckweed (Lenana minor L.) by 23, 62, and 64%,

respectively (Frankart et al. 2002). Therefore, the obj ective of this research was to compare the

effects of flumioxazin and diquat on ion leakage and chlorophyll content in landoltia.

Materials and Methods

Landoltia was collected from a pond with no history of herbicide treatments in Alachua

County, FL in April 2007 and was cultured in 9.5-L aquaria containing a standard growth

medium (Wang 1990) at the University of Florida' s Center for Aquatic and Invasive Plants,









Gainesville, FL. Plants were maintained in a growth room with a 16 h photoperiod at a

temperature of 26 & 4 C. Agitation was continuously supplied to each culture with forced air via

small aquarium air pumps. Aquaria and plants were rinsed and growth media were refreshed ca.

every 3 to 6 wk and light levels were maintained at 150 & 10 Cpmol m-2 S-1 to minimize algal

g~rowth.

Ion Leakage

The effect of flumioxazin on landoltia was measured by comparing non-specific ion

leakage (conductance in Cpmhos cm l) over time using a conductivity bridgel7 since conductance

may be used to measure non-specific ion leakage resulting from loss of membrane integrity

(Koschnick et al. 2006; MacDonald et al. 1993; O'Brien and Prendeville 1978). All ion leakage

techniques were modeled after research conducted by Koschnick et al. (2006). Ten colonies of

landoltia (4 to 6 fronds per colony) were placed into individual 20-ml high-density polyethylene

(HDPE) scintillation vials containing 15 ml DI water. Flumioxazin was added to each of the

vials to achieve concentrations of 0, 10, 25, 50, 100, 200, 400, 800, and 1600 Cpg L^1. Diquat at

10 Cpg L^1 was applied as a comparison treatment. The experiment was conducted and repeated in

May 2007 as a randomized design with 5 replications. Vials were covered with Parafilm Mls

and inverted 3x after addition of herbicide. Vials containing only flumioxazin or diquat

(duplicate treatment solutions) at each herbicide concentration and no plants were immediately

measured for initial conductance (Ci) (conductance contributed by addition of herbicides) and

appropriate corrections were made to determine total ion leakage. Vials were placed on a shaker

table (100 oscillations min- ) in a growth chamber and temperature of treatment solutions was



17 Fisher Scientific Conductivity Meter. Pittsburgh, PA.

1s Trademark of Pechiney Plastic Packaging. Menasha, WI. 54952.









maintained at 27 C with a 14 h photoperiod at 380 + 10 Cpmol m-2 S-1. Conductivity of the

treatment solutions was measured at 0, 1, 3, 6, 9, 12, 24, 30, 48, and 72 h after treatment (HAT).

The study was terminated 96 HAT due to the decline in conductivity resulting from algal growth

in vials. Final conductivity measurements were recorded, then treatment vials containing

landoltia were frozen and thawed 3x to ensure 100% ion leakage (Ct). Ion leakage at each time

(Cx) is reported as percent conductivity according to the following formula used by MacDonald

et al. (1993) and Koschnick et al. (2006):

% conductivity = [(Cx-Ci)/(Ct-Ci)]*100.

A repeated measures analysis was performed and means were separated using 95%

confidence intervals (95% CI) (PROC GLM, SAS Institute 2002).

Chlorophyll

The effect of flumioxazin on chlorophyll content was determined on colonies of landoltia

placed into scintillation vials and treated as described above for the ion leakage experiments. As

a comparison treatment, landoltia was treated with diquat at 10 Cpg L^1. This experiment was

conducted and repeated in May 2007 as a randomized design with 5 replications. Treated plants

were placed in a growth chamber for a 96 h exposure period then plants were removed, excess

water was blotted with a paper towel, and fresh weights were recorded. Total chlorophyll was

extracted by placing plants from each treatment vial into polystyrene test tubes (12 x 75 mm)

containing dimethylsulfoxide (DMSO) (Hiscox and Israelstam 1979) in a water bath (65 C) for 3

h. Chlorophyll content was determined spectrophotometrically (Arnon 1949) and expressed as

mg chlorophyll kg-l of fresh weight. Data were analyzed using non-linear regression (PROC

NLIN, SAS Institute 2002) and ECso values (flumioxazin concentration required to cause a 50%

reduction in chlorophyll content) were derived.









Results and Discussion


lon Leakage

Control plants did not produce more than 10% conductivity throughout the course of these

studies (data not shown). There were no differences in conductivity between flumioxazin at any

concentration and diquat 1 and 3 HAT (Figure 6-1), but differences in conductivity were noted

between flumioxazin at 10, 25, 50 and 1600 Clg L^1 and diquat 6 HAT. Leakage due to diquat >9

HAT was greater than leakage due to flumioxazin at all concentrations. These results are similar

to those reported for landoltia treated with diquat at 10 Clg L^1 when conductivity exceeded 80%

by 18 HAT (Koschnick et al. 2006). All flumioxazin treated plants displayed bleaching to a

certain extent 24 HAT, but none of the treatments resulted in more than ca. 50% bleaching; in

contrast, diquat treated plants were 100% chlorotic 24 HAT. Conductivity in the diquat

treatment was >90% 30 HAT, while flumioxazin conductivity never exceeded 50% at any

concentration throughout the course of the experiments. At the conclusion of these studies (96

HAT), most flumioxazin treatments began to show signs of decreasing conductivity, probably

due to algal growth in the scintillation vials. If these studies were continued for a few more days,

similar to the landoltia treated with flumioxazin in Chapter 4, it is likely the plants would have

continued to bleach and completely leak 100% of the ions. Based on these studies, flumioxazin

is not as fast acting as diquat with respect to bleaching and ion leakage; however, mesocosm data

in Chapter 4 demonstrated that flumioxazin did not exhibit significant bleaching until 7-10 DAT

but provided 65% control 21 DAT when applied at 400 Clg L^1

Chlorophyll

An estimated flumioxazin concentration of 944 Cpg L^1 is required to reduce landoltia

chlorophyll content by 50% (ECso) after a 96 h exposure (Figure 6-2), but plants were not

completely bleached even at the highest flumioxazin concentration (1600 Cpg L^)~. In contrast,









diquat at 10 Clg L^1 resulted in a 99% reduction of chlorophyll content 96 HAT (data not shown).

These data provide evidence that flumioxazin may have slower activity on landoltia than diquat,

and suggests that landoltia may not have been exposed to flumioxazin for a sufficient amount of

time in these experiments. Submersed flumioxazin applications required >1 wk to cause

significant chlorosis in landoltia in mesocosm experiments (see Chapter 4) and the time allotted

in these studies (96 h) was not sufficient for flumioxazin to cause significant bleaching.

Protox-inhibiting herbicides are more active in the presence of full sunlight (Sherman et al.

1991; Wright et al. 1995) and landoltia was exposed to flumioxazin in a growth chamber under

low light conditions (380 + 10 Cpmol m-2 S-1) in these experiments. Also, these studies were

conducted under a 14 h day length and not continuous light as in previous research (Koschnick et

al. 2006). Although flumioxazin did not cause the same level of injury to landoltia as diquat,

flumioxazin treatments still resulted in significant levels of ion leakage and bleaching.

Continuous and higher light levels may result in greater leakage similar to that observed in

diquat; however, the environment in the growth chambers was similar to field conditions (14 h

day length) and not artificially altered by providing continuous light. These data indicate that

flumioxazin causes slower, less severe injury to landoltia than diquat under short exposure times.













-* Diquat 10 pg L-
-0 Flumioxazin 10 upg L-1


-* Diquat 10 pg L'
-0 Flumioxazin 50 pg L'


100 7 lmoain2 gL 7 Flumioxazin 100 pg L'


80-


60-


40-


S20-



S120
-*-~ Diquat 10 pg L' -* Diquat 10 pg L'
-0- Flumioxazin 200 pg L' -0 Flumioxazin 800 pg L'
S1 -7- Flumioxazin400 pgL L kkkkkkkkk -7- Flumioxazin 1600 pg L1


80-


60-


40-




20


1 3 6 9 12 24 30 48 72 96 1 3 6 9 12 24 30 48 72 96

Hours after treatment

Figure 6-1. The effect of diquat and flumioxazin on ion leakage from landoltia cultured in DI
water (pH 8.5) in a growth chamber for 96 h. Values are presented as means a 95%
confidence interval (CI) (n=10). Overlapping CI bars indicate no significant
difference at a given time.













0.25 -


- y = 0.1539-0 000734x, 2 = 0.83
EC,,o= 944 pg L-'


0.20



0.15



0.10



0.05


0 200 400 600 800 1000 1200 1400 1600


Flumioxazin Concentration pg L-1 a.i.

Figure 6-2. The effect of flumioxazin concentration on landoltia chlorophyll content 96 h after
treatment. Landoltia was cultured in 20 mL vials contain DI water (pH 8.5) in a
growth chamber. Data are shown as actual means + standard error (n=6). ECso =
effective concentration 50, concentration of flumioxazin in water required to reduce
landoltia chlorophyll content by 50%.









CHAPTER 7
SUMMARY AND DRAFT AQUATIC USE DIRECTIONS

Summary

These experiments provided information regarding the effectiveness of flumioxazin as an

aquatic herbicide and will aid in the registration of flumioxazin for use in aquatic ecosystems to

control invasive aquatic species such as hydrilla and water lettuce. Flumioxazin was screened

for hydrilla control in 2005 and significantly reduced hydrilla biomass. Through a cooperative

agreement with Valent U.S. A. Corporation, The Center for Aquatic and Invasive Plants at the

University of Florida began evaluation of flumioxazin as a potential aquatic herbicide.

Various EUP ponds throughout Florida ranging in pH from 6.7 to 10.0 were treated with

flumioxazin at concentrations of 100 to 400 Clg L^1. Early season treatments were successful due

to lower pH, less hydrilla biomass, and/or time of year; however, flumioxazin failed to provide

more than Ito 4 months of control in ponds with pH >8.5. These high pH ponds were generally

infested with mature hydrilla that was near the surface. Early and late season treatments are

recommended since hydrilla grows more actively and water pH is likely to be lower than in

summer treatments.

Due to the lack of efficacy in EUP ponds treated with flumioxazin in the summer when the

water pH was in excess of 8.5 or when hydrilla was near the surface of the water, greenhouse and

laboratory studies were initiated to determine why flumioxazin was not as efficacious under

these conditions. Outdoor mesocosm studies showed that flumioxazin reduces hydrilla biomass

by 90% at concentrations of 186 Clg L- but possesses activity at concentrations as low as 50 Clg

L^. Hydrilla treated with flumioxazin underwent chlorosis in apical tips followed by reddening

of lower stems. Hydrilla treated at 50 to 1600 Clg L-1 began to lose integrity and fall to the

bottom of the tanks within 5 to 7 DAT; however, new apical tips from adventitious buds in the









leaf axils soon sprouted from treated rooted and floating tissue. Flumioxazin is rapidly

hydrolyzed in high pH water and has an average half-life of 17.5 min under laboratory conditions

at pH 9.0 or greater. The effects of pH on flumioxazin half-life and hydrilla efficacy were

evaluated in a pH efficacy study. Flumioxazin reduced hydrilla dry weight by 90% in the high

(>8.5) pH treatment when hydrilla was placed into mesocosms the same day as treatment;

however, hydrilla placed in high pH water 2 to 5 DAT was reduced in biomass by no more than

50%. Biomass of hydrilla in the low (6.0 to 6.2) and medium (7.0 to7.2) pH was reduced by 93

and 68%, respectively, of the nontreated control plants 3 DAT. The half-life of flumioxazin in

low, medium, and high pH water (6.0 to 6.2, 7.0 to 7.2 and >8.5, respectively) was 39, 18.6, and

1.7 h, respectively. These data indicate flumioxazin is rapidly taken up by hydrilla and the short

half-life of flumioxazin in water with pH >9.0 can be overcome by higher application rates.

The net photosynthetic rates of apical hydrilla tips treated with flumioxazin were measured

to determine the effects of rate, pH, stem type, and light quantity. Flumioxazin applied in high

pH water (9.0) at >200 Clg L^1 or in low pH water (6.0) at >100 Clg L^1 required less than 124 h to

reduce hydrilla net photosynthesis by 50% (ETso) of the non-treated control plants and only the

100 Clg L-1 treatment applied in high pH water failed to have a significant impact on

photosynthesis. Since activity of flumioxazin is influenced by light, it was hypothesized that

hydrilla at the bottom of water bodies were not being controlled due to the low quantity of light

reaching plants. Growth chamber studies indicated 170 and 400 Clmol m-2 S-1 of light was

sufficient to reduce net photosynthesis of hydrilla by 45 and 78% of the nontreated control

plants, respectively, 168 HAT; however, hydrilla treated with flumioxazin at light levels of 20

Clmol m-2 S-1 resulted in less than a 30% reduction in net photosynthesis 168 HAT. Furthermore,









plants treated at the lowest light level did not appear to be injured and maintained a healthy

appearance throughout the course of the experiments.

Due to the high costs associated with registering a pesticide in a new market like aquatics,

other invasive species were evaluated for sensitivity to flumioxazin. Water lettuce was highly

sensitive to submersed and foliar flumioxazin applications, whereas water hyacinth was impacted

only slightly by either application technique. Landoltia was sensitive to submersed treatments,

but was non-responsive to foliar treatments.

If flumioxazin is approved for aquatic use, non-target aquatic plants may be affected by

submersed and foliar flumioxazin applications and terrestrial plants may be injured from

irrigation with treated water. The impact of a submersed flumioxazin application on submersed

aquatic plant species including coontail, egeria, hydrilla, southern naiad, and vallisneria was

evaluated at high (9.0) and low pH (7.0). Coontail, naiad, and hydrilla dry weight were reduced

by 50% (ECso) when flumioxazin was applied at 34, 51, and 77 Clg L^1 in low pH water. Only

coontail dry weight was reduced by 50% with flumioxazin concentrations near the proposed

label maximum rate of 400 Clg L~lin high pH water. The emergent aquatic plants maidencane

and sagittaria were reduced by 50% with flumioxazin application of less than 400 Clg L^1. Foliar

application of flumioxazin reduced sagittaria dry weight by 50% at 147 g ha- which is

approximately half of the proposed maximum foliar application rate of 286 g ha l. Other

emergent species (eleocharis, maidencane, and pickerelweed) were more tolerant to applications

within the proposed maximum labeled rate. Non-target ornamental and row crop plants were

overhead irrigated once with irrigation water containing flumioxazin. Immature begonia and

impatiens were highly sensitive compared to mature snapdragons and were reduced by 10% in

dry weight with flumioxazin in irrigation water at rates of 103 and 44 Clg L^1, respectively. All









row crops (corn, cotton, soybeans, and wheat) were reduced by 10% in dry weight with

flumioxazin at less than 200 Clg L1 in irrigation water. These data clearly indicate that irrigation

restrictions will be required on the label; however, since flumioxazin is rapidly degraded

(especially in high pH water), shorter water use restrictions should be possible for homeowners

and farmers who use flumioxazin treated water for irrigation; however, treated water with a pH

of less than 8.0 will likely be restricted for irrigation for several days.

These data indicate flumioxazin has potential utility as an aquatic herbicide for control of

hydrilla and other aquatic weeds. Flumioxazin possesses several desirable traits including

activity at low use rates, a short half-life, and non-target selectivity. Hydrilla control will depend

on several factors including water pH, growth stage of hydrilla, and light availability to stems

near the bottom of a water body. Summer applications or situations where hydrilla has already

surface matted (resulting in high pH) will be less effective than early and late season

applications, so treatments under these conditions should be avoided since these treatments will

result in a "burndown" or removal of the upper canopy and hydrilla will regrow quickly within 1

to 2 months.

Draft Aquatic Use Directions

General information

Flumioxazin is a 51 percent water dispersible granule that controls weeds by inhibiting

protoporphyrinogen oxidase (Protox), an essential enzyme required by plants for chlorophyll

biosynthesis. This herbicide is rapidly absorbed by aquatic plants and breaks down by pH

dependent hydrolysis. Flumioxazin is a fast-acting, contact herbicide that can be applied directly

into water for control of submersed aquatic weeds or directly to the foliage of emergent or

floating weeds. The most effective flumioxazin applications occur when applied to young,

actively growing weeds in water with a pH <8.0.









Mixing Guidelines

If the diluent water (tank mix) is pH >7.0, add an agent to lower the pH in the tank.

Agitation may be applied to the spray tank, but the addition of more than 1 lb of product per

gallon at a high agitation rate will likely result in large quantities of foam in the tank. The

herbicide solution should be applied no longer than 2 h after mixing to prevent hydrolysis.

Control of Submersed Weeds

Apply flumioxazin for control of hydrilla and other susceptible submersed weeds in lakes,

ponds, non-irrigation canals and other water bodies with limited water exchange. Total

concentration of flumioxazin in a single treatment should not exceed 400 Clg L^1 in the treated

water area. For best results, apply in spring or early summer when submersed weeds are actively

growing but have not reached the water surface. Water pH at a depth of 1 ft below the surface

should be measured at the time of application. Flumioxazin may be applied at concentrations as

low as 50 Clg L- but concentrations above 100 Clg L^1 provide better efficacy, especially in water

with a pH >8.5. Treatments should be applied as early in the morning as possible to minimize

the effect of hydrolysis in high pH water since infested waters have a tendency to cycle pH from

as low as 7.0 (6 A.M.) to as high as 10.0 (6 P.M.) in the upper 10 to 25 cm of a water body. If

the water pH is >8.5 or hydrilla is surface matted, flumioxazin should be applied at 400 ppb to

ensure best efficacy. Repeat applications of 400 ppb within 1 to 2 months after treatment may be

necessary due to the rapid breakdown of this product in water with high pH. If the water pH is

7.0 to 8.4, flumioxazin may be applied at 200 to 400 ppb for control of hydrilla. Additional

applications may be necessary for complete control at this pH range. Flumioxazin applied to

water with a lower pH (<6.9) will result in better efficacy due to slower breakdown in low pH

water. Flumioxazin may be applied at rates of 50 to 400 ppb in these lower pH waters.









Subsurface Application

Flumioxazin should be applied with long weighted hoses to ensure proper mixing of

herbicide in the water column. Thermal stratification is common in lakes with surface-matted

hydrilla and this thermal layer can create a physical barrier, isolating layers in the water column

and preventing surface-applied herbicides from reaching the target vegetation below the

thermocline. If weighted hoses are not available, every effort should be made to ensure

herbicide is uniformly mixed below the water surface to assure all plant parts are exposed to the

herbicide. Inadequately mixed flumioxazin will likely break down before it comes in sufficient

contact with plants near the bottom, allowing for more rapid regrowth.

Surface/Foliar Application

Flumioxazin may be applied up to 8 ounces of formulated product per acre. Control of

water lettuce and other floating or emergent weeds require the addition of a spray adjuvant for

foliar applications. For best results, use nonionic surfactants or methylated seed oils at

manufacturer' s recommended rates. Mix in sufficient diluent (50 to 100 GPA) to ensure

adequate coverage.

Plant Susceptibility

Submersed, emergent, and floating vascular aquatic plants as well as macrophytic algae

vary in susceptibility to flumioxazin (Table 7-1). Plants are more susceptible to submersed

flumioxazin applications in lower pH water (<8.0) where half-lives are longer. For best results,

treatments with this herbicide should be applied when plants are actively growing and before

they are surface matted. If plants are surface matted, an initial application may be required to

control the surfaced plants and a subsequent application may be necessary to provide season long

control. Lower pH, actively growing weeds, less biomass, and high light penetration into the

water colum favor increased flumioxazin efficacy on hydrilla and other submersed species.










Irrigation Restrictions

In addition to efficacy on weeds and impact on non-target plants, the focus of this research

was to determine possible irrigation restrictions following flumioxazin applications. Chapters 2,

5, and 8 provide data on the half-life of flumioxazin under various pH situations and the

sensitivity of non-target plants to flumioxazin in irrigation water. Water use restrictions for

flumioxazin will be dependent on a number of factors including herbicide placement in the water

column, herbicide rate, pH, and maturity of the plants being irrigated with flumioxazin treated

water (Table 7-2). Submersed applications will be more restrictive than foliar applications due

to higher concentrations of flumioxazin in the water. For example, ornamental species are

usually planted during the spring when hydrilla is actively growing and highly susceptible to

flumioxazin; therefore, irrigation water containing flumioxazin may also severely injure these

immature plants. Water with a pH of 6.0 to 8.0 that has received a submersed flumioxazin

application (200 to 400 Clg L 1) should not be used for irrigation for up to 7 d after herbicide

application compared to a 2 d restriction if the pH is >9.0. The shorter half-life at pH 9.0 (see

Chapter 2) prevents injurious levels of this herbicide from being present in the water beyond 1

DAT. Immature plants are more susceptible to submersed flumioxazin applications as pH

decreases and the use rate increases. On the other hand, foliar applications pose less of a threat

to these young plants. The majority of the herbicide solution will come in contact with the

foliage of the emergent or floating weed and less will be available in the water to harm irrigated

plants.










Table 7-1. Aquatic plant and algae control with flumioxazin in water with a pH of
7.0 to 9.5b
Common name Scientific name Submersede Foliard
alligatorweed Alternanthera philoxeroides F-G G-E
baby's-tears M~icranthentum spp. F NA
broadleaf arrowhead Sagittaria latifolia E NA
Carolina mosquito fern Azolla caroliniana E NA
cattail Typha spp. P P
coontail Ceratophylhtna dentersunt E NA
duck potato Sagittaria lan2cifolia E F-G
duckweed Lenana minor G-E G-E
egeria Egeria densa P NA
Eurasian water milfoil M~yriophyllunt spicatunt E NA
fanwort Cabonaba caroliniana E NA
frog' s-bit Linanobium spongia E NA
hydrilla Hydrilla verticillata F-E NA
jointed spikerush Eleocharis interstincta F F
landoltia Land'oltia punctata G-E P
maidencane Panicunt hentitonzon G F
muskgrass Chara spp. P-G NA
pennywort Hydrocotyle spp. F-G NA
pickerelweed Pontederia cord'ata F F
southern naiad Najas guadahelpensis F-E NA
stonewort Nitella spp. P-F NA
torpedo grass Panicunt repens P-F P-F
vallisneria Vallisneria americana P-F NA
variable-leaf milfoil M~yriophyllunt heterophyllunt E NA
water fern Salvinia naininta G-E NA
water hyacinth Eichhornia crassipes P P
water lettuce Pistia stratiotes E E
water meal Wolffia cohenabiana G-E NA
water shield Brasenia schreberi G-E NA
willow Salix spp. P P
"Control: NA = not applicable or not evaluated, P = poor, F= fair, G = good, E = excellent; based on
EUP field and mesocosm observations.
blmproved efficacy at lower pH for most species.
"Submersed: treatment applied by weighted hoses.
dFoliar treatment applied with 0.25% v/v non-ionic surfactant by handgun at 935 L ha diluent.









Table 7-2. Proposed water use restrictions to overhead irrigated crop and ornamental species
following submersed and foliar flumioxazin applications.
Flumnioxazin Application Use Rate Maturity of Irrigated Plantsa Water pH Daysb'
Submersed 200-400 Clg L1 Immature >8.5 1
200-400 Clg L1 Mature >8.5 1
200-400 Clg L1 Immature 7.0-8.4 7
200-400 Cig L1 Mature 7.0-8.4 1
200-400 Clg L1 Immature 16.9 14
200-400 yg L' Mature <6.9 2
<200 Clg L^ Immature >8.5 1
<200 Clg L^1 Mature >8.5 1
<200 Clg L^1 Immature 7.0-8.4 6
<200 Clg L^1 Mature 7.0-8.4 1
<200 Clg L^1 Immature 16.9 12
<200 Clg L^1 Mature 16.9 1
Foliar 143-286 g hal Immature >8.5 1
143-286 g hal Mature >8.5 0
143-286 g hal Immature 7.0-8.4 1
143-286 g hal Mature 7.0-8.4 0
143-286 g hal Immature 16.9 1
143-286 g hal Mature 16.9 0
a Growth stage of plants irrigated with water treated with flumioxazin.
b Number of days before water treated with flumioxazin may be used for irrigation of crop and ornamental species. Based
on the half-life of flumioxazin at a given pH and the growth stage of irrigated species.










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BIOGRAPHICAL SKETCH

Christopher Ray Mudge, the son of Alvin and Wanda Mudge, was born in Alexandria, LA

and was raised in the rural rice and soybean farming community of Branch, LA. Upon

graduation from lota High School in 1997, he enrolled at Louisiana State University A&M in

Baton Rouge in the fall of 1997. While attending LSU, he was active in the agronomy and

collegiate 4-H clubs.

After earning a B.S. in agronomy (crop management) in December 2001, he began his

graduate career under the direction of Dr. Eric P. Webster, working in rice weed management.

While working on his master' s degree, Chris was an active participant in the Southern Weed

Contest. He graduated from LSU in the spring of 2004 with a M.S. in agronomy and the title of

his thesis was Water-seeded Rice Response to Clomazone.

Chris moved to Gainesville, FL in 2004 to work as a biological scientist for the University

of Florida. In 2005, he enrolled at the University of Florida to study for a PhD in aquatic weed

science under the direction of Dr. William T. Haller. During his graduate career, he presented

many talks and posters at the Southern Weed Science, Aquatic Plant Management Society,

Florida Weed Science, and Florida Aquatic Plant Management Society annual meetings. In

2005, Chris married the former Miss Erin Gravois of Vacherie, LA. Upon graduation, he will

work in the field of aquatic weed management and continue his involvement in weed science and

aquatic societies.





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CHARACTERIZATION OF FLUMIOXAZIN AS AN AQUATIC HERBICIDE By CHRISTOPHER R. MUDGE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007 1

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2007 Christopher R. Mudge 2

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To my patient and loving wife, Erin, as well as my parents Alvin and Wanda whose love and support has never ended, and to my departed grandparents Houston and Hazel Doucet; I miss you both. 3

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ACKNOWLEDGMENTS First and foremost, I thank God for all he has blessed me with throughout my life. Without him, little would be possible. A thank you goes to Dr. Bill Haller for the opportunity to study under his tutelage and teach me so much about aquatic weed research. He provided me with an opportunity to learn about aquatic research by starting with a new compound and taking it close to registration. Appreciation is also extended to my committee members Drs. Jay Ferrell, Greg MacDonald, Bill Stall, and Kevin Kenworthy for your advice and guidance. It has been a pleasure collaborating with Dr. Mike Netherland of the U.S. Army Corps of Engineers on many aspects of my research, and I thank him also for his guidance and perspectives on several issues. Margaret Glenn is greatly appreciated for all her help from the moment I arrived as a student until the day I finished my dissertation. I am lucky to have worked with and learned from several outstanding weed science graduate students and post-docs including Brett Bultemeier, Dr. Tyler Koschnick, Tomas Chiconela, Dr. Lyn Gettys, Dr. Atul Puri, and Eileen Ketterer. Brett and Tyler are especially appreciated for helping me with research and class work. David Mayo, Cole Hullon, William Jordan, and Michael Aldridge were all vital in data collection. Without the unyielding and relentless support of Drs. Mike Riffle and Joe Chamberlin of Valent U.S.A. Corporation, flumioxazin would not have had the opportunity to be evaluated for use in aquatics. I appreciate their suggestions for studies and discussions over the last two years. The use of time, boats, and willingness to learn about new herbicides is acknowledged by the crews at the St. Johns River Water Management District (Johnnie Drew, Tom Boyette, Shawn Moore, Richard Krantz, and James Godfrey). Much gratitude is due to Mr. Sonny Phillips, Dr. Seigfred Fagerberg, Osceola County, and the South Florida Water Management District for use of ponds in research. The financial assistance provided by the Aquatic Ecosystem Restoration Foundation (AERF) and the Florida Department of Environmental Protection (FDEP) is greatly appreciated. 4

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Finally, a most grateful and heartfelt thank you is long overdue to my entire family for their support during this special time in my life. I am so blessed to have a wife who loved and encouraged me throughout the course of this experience. I thank her for being at my side and always loving me no matter what the circumstance. Finally, my parents taught me to never settle for less than what I could accomplish. I have learned from them how to work hard and have fun with what I love the most. 5

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TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................12 CHAPTER 1 INTRODUCTION..................................................................................................................14 2 THE EFFECT OF FLUMIOXAZIN ON HYDRILLA CONTROL IN NATURAL SYSTEMS AND THE INFLUENCE OF WATER PH ON HYDRILLA CONTROL IN MESOCOSMS........................................................................................................................24 Introduction.............................................................................................................................24 Materials and Methods...........................................................................................................24 Efficacy in Ponds.............................................................................................................24 Efficacy in Mesocosms....................................................................................................25 Impact of pH on Efficacy................................................................................................26 Results and Discussion...........................................................................................................28 Efficacy in Ponds.............................................................................................................28 Efficacy in Mesocosms....................................................................................................32 Impact of pH on Efficacy................................................................................................33 3 THE EFFECT OF FLUMIOXAZIN ON HYDRILLA PHOTOSYNTHESIS AND CHLOROPHYLL CONTENT...............................................................................................41 Introduction.............................................................................................................................41 Materials and Methods...........................................................................................................42 Photosynthesis and pH....................................................................................................42 Photosynthesis and Light.................................................................................................44 Effect of Contact Herbicides on Photosynthesis.............................................................45 Chlorophyll Content........................................................................................................45 Results and Discussion...........................................................................................................46 Photosynthesis and pH....................................................................................................46 Photosynthesis and Light.................................................................................................48 Effect of Contact Herbicides on Photosynthesis.............................................................50 Chlorophyll Content........................................................................................................51 4 THE EFFECT OF FLUMIOXAZIN ON SUBMERSED, EMERGENT, AND FLOATING AQUATIC PLANT PLANTS...........................................................................59 6

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Introduction.............................................................................................................................59 Materials and Methods...........................................................................................................60 Floating Aquatic Plants...................................................................................................60 Submersed Aquatic Plants...............................................................................................62 Emergent Aquatic Plants.................................................................................................64 Results and Discussion...........................................................................................................65 Floating Aquatic Plants...................................................................................................65 Submersed Aquatic Plants...............................................................................................68 Emergent Aquatic Plants.................................................................................................70 5 ORNAMENTAL AND ROW CROP SUSCEPTIBILITY TO FLUMIOXAZIN IN IRRIGATION WATER..........................................................................................................82 Introduction.............................................................................................................................82 Materials and Methods...........................................................................................................82 Ornamental Susceptibility...............................................................................................82 Crop Susceptibility..........................................................................................................84 Results and Discussion...........................................................................................................85 Ornamental Susceptibility...............................................................................................85 Crop Susceptibility..........................................................................................................86 6 THE EFFECT OF FLUMIOXAZIN AND DIQUAT ON MEMBRANE PERMEABILIITY AND CHLOROPHYLL CONTENT OF LANDOLTIA........................92 Introduction.............................................................................................................................92 Materials and Methods...........................................................................................................92 Ion Leakage.....................................................................................................................93 Chlorophyll......................................................................................................................94 Results and Discussion...........................................................................................................95 Ion Leakage.....................................................................................................................95 Chlorophyll......................................................................................................................95 7 SUMMARY AND DRAFT AQUATIC USE DIRECTIONS................................................99 Summary.................................................................................................................................99 Draft Aquatic Use Directions...............................................................................................102 General information.......................................................................................................102 Mixing Guidelines.........................................................................................................103 Control of Submersed Weeds........................................................................................103 Subsurface Application.................................................................................................104 Surface/Foliar Application............................................................................................104 Plant Susceptibility........................................................................................................104 Irrigation Restrictions....................................................................................................105 LIST OF REFERENCES.............................................................................................................108 BIOGRAPHICAL SKETCH.......................................................................................................120 7

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LIST OF TABLES Table page 2-1 Hydrilla infested ponds in Florida treated with flumioxazin under an Experimental Use Permit in 2006.............................................................................................................37 3-1 The effect of flumioxazin on photosynthesis of apical hydrilla tips at high (9.0) and low (6.0) pH a ......................................................................................................................55 3-2 The effect of flumioxazin (400 g L -1 a.i.) at pH 9.0 on photosynthesis of apical hydrilla tips cultured in a growth chamber for 168 h at low, medium, and high light quantities a ...........................................................................................................................57 3-3 The effect of select contact herbicides at pH 9.0 on photosynthesis of apical hydrilla tips cultured in a growth chamber for 96 h at 380 mol m -2 s -1a light quantity.................57 4-1 The effect of a single submersed flumioxazin application on dry weight of submersed aquatic plants 28 d after exposure a ..................................................................77 4-2 The effect of a single submersed flumioxazin application on dry weight of emergent aquatic species 40 d after exposure a ..................................................................................79 4-3 The effect of a single foliar flumioxazin application on dry weight and height of emergent aquatic species 40 d after treatment a ..................................................................81 5-1 The effect of a single overhead irrigation with 1.27 cm water containing flumioxazin on ornamental species dry weight and height 14 d after treatment. a .................................89 5-2 The effect of single overhead irrigation with 1.27 cm water containing flumioxazin on crop species dry weight and height 14 DAT. a ..............................................................91 7-1 Aquatic plant and algae control a with flumioxazin in water with a pH of 7.0 to 9.5 b .....106 7-2 Proposed water use restrictions to overhead irrigated crop and ornamental species following submersed and foliar flumioxazin applications...............................................107 8

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LIST OF FIGURES Figure page 2-1 The effect of flumioxazin concentration on hydrilla dry weight 21 d after exposure under 70% sunlight. Flumioxazin applied as a single application to hydrilla cultured in 95 L tubs (pH 9.0-9.5). Data are shown as dry weight means standard error (n=10). EC 50 = effective concentration 50, concentration of flumioxazin in water required to reduce hydrilla biomass by 50%......................................................................38 2-2 The effect of flumioxazin at 400 g L -1 on hydrilla dry weight as influenced by low (6.0 to 6.2), medium (7.0 to 7.2), and high (>8.5) water pH under 70% sunlight. Hydrilla plants were added to low, medium, and high pH water treated with flumioxazin 0 to 4 d after initial treatment and allowed to grow for 21 d after treatment until harvest. Data are shown as percent of nontreated control of each pH standard error (n=8). Treatment means within a particular day were separated using least square means (p<0.05)...............................................................................................39 2-3 Dissipation of flumioxazin applied at 400 g L -1 to low (6.0 to 6.2), medium (7.0 to 7.2) and high pH (>8.5) tap water in 95 L tubs under 70% sunlight. The dissipation of flumioxazin was calculated using non-linear regression (exponential decay) for the low (y = 0.0178e -0.0178x ; r 2 = 0.92; half-life 39.0 h), medium (y = 0.3074e -0.0373x ; r 2 = 0.93; half-life 18.6 h), and high (y = 0.3209e -0.3991x ; r 2 = 0.94; half-life 1.7 h) pH treatments. All residues are reported as the mean standard error (n=6).........................40 3-1 The effect of flumioxazin rate at high (9.0) and low pH (6.0) on photosynthesis of apical hydrilla tips cultured in a growth chamber for 168 h at 380 mol m -2 s -1 of light quantity. Data are normalized to the control at each respective pH and shown as means standard error (n=8).............................................................................................54 3-2 The effect of flumioxazin (400g L -1 ) at pH 9.0 on photosynthesis of apical hydrilla tips cultured in a growth chamber for 96 h at low (20 mol m 2 s 1 ), medium (170 mol m 2 s 1 ), and high light (400 mol m 2 s 1 ) quantity levels. Data are normalized to the control at each respective light quantity and shown as means standard error (n=10).................................................................................................................................56 3-3 The effect of flumioxazin concentration on hydrilla chlorophyll content (mg kg -1 fresh weight) 1 to 4 d after treatment (DAT) under 70% sunlight. Flumioxazin was applied as a single application to hydrilla cultured in 18.9 L buckets filled with tap water (pH 9.0-9.5). Data are shown as actual means standard error (n=6)....................58 4-1 The effect of a foliar flumioxazin application (g ha -1 a.i.) on water lettuce and water lettuce dry weight 34 d after treatment under 100% sunlight. Flumioxazin was applied as a single application by a CO 2 -powered sprayer at an equivalent of 379 L ha -1 diluent with a non-ionic surfactant (0.25% v/v) to water lettuce and water hyacinth grown in 95 L tubs (pH 7.5 to 8.0). Data are shown as dry weight means standard error (n=10). EC 50 = effective concentration 50, concentration of 9

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flumioxazin (g ha -1 a.i.) that is required to reduce water lettuce and water lettuce biomass by 50%.................................................................................................................74 4-2 Percent control (visual) of landoltia 21 d after a foliar diquat (g ha -1 a.i.) and submersed flumioxazin application (g L -1 a.i.). Diquat and flumioxazin each applied as a single application to landoltia cultured in 1 L pots (water pH 8.0) under 70% sunlight. Diquat was applied by a CO 2 -powered sprayer at an equivalent of 379 L ha -1 diluent with a non-ionic surfactant (0.25% v/v). Percent control 95% confidence interval (CI) (n=10). Overlapping CI bars indicate no significant difference...........................................................................................................................75 4-3 The effect of flumioxazin concentration on the dry weight of submersed aquatic plants 28 d after exposure. Flumioxazin applied as a single application to submersed aquatic species cultured in low (7.0) and high (9.0) pH water in 95 L tubs under 70% sunlight. Data are shown as actual dry weight means standard error (n=10 for low pH, except for naiad and vallisneria n=5; n=5 for high pH). Dry weight means standard error (n=10).........................................................................................................76 4-4 The effect of flumioxazin concentration on dry weight of emergent aquatic plants 40 d after exposure. Flumioxazin applied as a single application to emergent aquatic species cultured in 95 L tubs (pH 7.5) under 70% sunlight. Data are shown as actual dry weight means standard error (n=5)...........................................................................78 4-5 The effect of flumioxazin rate on dry weight of emergent aquatic species 40 d after treatment. Flumioxazin applied as a single application by a CO2-powered sprayer at an equivalent of 379 L ha -1 diluent with a non-ionic surfactant (0.25% v/v) to emergent aquatic species cultured in 277 L tanks (pH 7.5) under 70% sunlight. Data are shown as actual dry weight means standard error (n=5)..........................................80 5-1 The effect of flumioxazin concentration in irrigation water (pH 7.5) on ornamental species dry weight 14 d after treatment. Flumioxazin was applied once to the plants as an overhead irrigation with a sprinkle can (equivalent to 1.27 cm of irrigation water). Data are shown as actual dry weight means standard error (n=10). Data for snapdragon were not included as flumioxazin resulted in minimum effects at all rates....................................................................................................................................88 5-2 The effect of flumioxazin concentration in irrigation water (pH 7.5) on crop species dry weight 14 d after treatment. Flumioxazin was applied once to the plants as an overhead irrigation with a sprinkle can (equivalent to 1.27 cm of irrigation water). Data are shown as actual dry weight means standard error (n=10), except for corn (n=5, for each experiment).................................................................................................90 6-1 The effect of diquat and flumioxazin on ion leakage from landoltia cultured in DI water (pH 8.5) in a growth chamber for 96 h. Values are presented as means 95% confidence interval (CI) (n=10). Overlapping CI bars indicate no significant difference at a given time...................................................................................................97 10

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6-2 The effect of flumioxazin concentration on landoltia chlorophyll content 96 h after treatment. Landoltia was cultured in 20 mL vials containg DI water (pH 8.5) in a growth chamber. Data are shown as actual means standard error (n=6). EC 50 = effective concentration 50, concentration of flumioxazin in water required to reduce landoltia chlorophyll content by 50%................................................................................98 11

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CHARACTERIZATION OF FLUMIOXAZIN AS AN AQUATIC HERBICIDE By Christopher R. Mudge December 2007 Chair: W. T. Haller Major: Agronomy The suitability of flumioxazin as an aquatic herbicide for control of hydrilla and other invasive aquatic plant species was evaluated in field, greenhouse, and laboratories studies. Flumioxazin is a protoporphyrinogen oxidase inhibitor in plants, which is a precursor to production of chlorophyll. It is degraded by hydrolysis and has a half-life of 17.5 min in water at pH 9.0 compared to a half-life of 16.1 h and 4.1 d at pH 7.0 and 5.0, respectively. Flumioxazin efficacy was evaluated at various concentrations, pH, and light levels to determine the impact on hydrilla biomass, net photosynthesis, and chlorophyll content. The effective concentration of flumioxazin required to reduce hydrilla dry weight by 50% (EC 50 ) was 56 g L -1 in mesocosm studies; however, regrowth was noted in concentrations as high as 1600 g L -1 under high pH conditions. Apical hydrilla tips treated under high pH (>9.0) with flumioxazin at 100 g L -1 failed to reduce net photosynthesis by 20% of the nontreated control. All concentrations >100 g L -1 at high pH and 100 g L -1 at low pH reduced net photosynthesis by at least 60% 168 HAT. Under low light quantity (20 mol m -2 s -1 ), flumioxazin failed to reduce net photosynthesis of apical hydrilla tips compared to medium (170 mol m -2 s -1 ), and high light (400 mol m -2 s -1 ) quantities. Non-target emergent plants appeared to tolerate flumioxazin at high pH (>9.0), whereas selectivity decreased in waters of lower pH (7.0). Phytotoxicity to non12

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target ornamental and row crop plants irrigated with flumioxazin treated water was dependent on maturity as immature, actively growing plants were highly susceptible. Additionally, water lettuce and landoltia were more susceptible to submersed flumioxazin applications than foliar applications. These data provide evidence that flumioxazin has the potential for use as an herbicide with submersed and foliar applications to control hydrilla and other weeds and that application methods, environmental conditions, and concentrations influence non-target plant selectivity. 13

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CHAPTER 1 INTRODUCTION Currently, there are ten herbicides labeled (FIFRA-Section 3) by the U.S. Environmental Protection Agency (EPA) for aquatic use in the U.S. including 2,4-D [(2,4-dichlorophenoxy) acetic acid], carfentrazone {X,2-dichloro-5-[4-(difluromethyl)-4,5-dihydro-3-methyl-5-oxo-1H-1,2,4-triazol-1-yl]-4-fluorobenzenepropanic acid}, copper (copper sulfate or copper chelate), diquat (1,1-ethylene-2,2-bipyridylium dibromide), endothall (7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylic acid), fluridone {1-methyl-3-phenyl-5-[3-(trifluoromethyl) phenyl]-4(1 H )-pyridinone}, glyphosate N -(phosphonomethyl)glycine, imazapyr {()-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1 H -imidazol-2-yl]-3-pyridinecarboxylic acid}, penoxsulam {2-(2,2-difluoroethoxy)-6-(trifluoromethyl-N-(5,8-dimethoxy[1,2,4]triazolo[1,5-c]pyrimidin-2-yl))benzenesulfonamide}, and triclopyr {[(3,5,6-trichloro-2-pyridinyl)oxy]acetic acid}. Of these, only copper, diquat, endothall, and fluridone have historically been used for hydrilla (Hydrilla verticillata (L. f.) Royle) control (Vandiver 2002). Hydrilla is a submersed aquatic fresh-water angiosperm in the family Hydrocharitaceae native to Asia or Africa that has become a serious weed problem in the United States and many other countries (Cook 1985; Haller and Sutton 1975; USDA 2007; Van and Vandiver 1992). Once established, hydrilla readily dominates and replaces native submersed species by forming a canopy and reducing light penetration (Haller and Sutton 1975). There are two biotypes of hydrilla in the U.S., which are the dioecious (plants produce only female flowers) and monoecious (male and female flowers on the same plants) (Cook and Lnd 1982; Langeland 1996). The dioecious female biotype was introduced from Sri Lanka to the Tampa Bay, Florida area (Schmitz et al. 1990) in the early 1950s and was first observed growing outside of nursery conditions in a canal in Miami and in a spring near Crystal River, FL 14

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in 1960 (Blackburn et al. 1969). The dioecious female plant has spread throughout the southern U.S. and as far west as California (Yeo and McHenry 1977; Yeo et al. 1984). The first population of the monoecious biotype of hydrilla was discovered in Delaware in 1976 with a second discovery in 1980 in the Potomac River (Haller 1982; Steward et al. 1984). Hydrilla has been described as the perfect aquatic weed due to its specialized growth habit, physiological characteristics, and various means of asexual reproduction (Langeland 1996). The dioecious biotype is especially problematic since its response to management efforts results in rapid regrowth from vegetative propagules such as tubers, turions, and plant fragments (Van and Vandiver 1992). Turions are compact dormant buds produced in leaf axils which detach from the plant upon maturation, while tubers (subterranean turions) are formed terminally on subterranean rhizomes (Langeland 1996). The Florida Department of Natural Resources (now the Florida Department of Environmental Protection) estimated over 20,000 ha of water in Florida contained hydrilla in 1988 (Schardt and Nall 1988), with hydrilla spreading to 40,000 ha of water in 43% of public lakes in Florida by 1995 (Langeland 1996). Rapid hydrilla growth and expansion is favored by its low light and CO2 compensation points, reduced photorespiration due to a C4 like photosynthetic mechanism and its prolific reproductive capacity (Van et al. 1976; Holaday et al. 1983; Magnin et al. 1997). Hydrilla is typically rooted in the hydrosol, although fragments frequently break loose and survive in a free-floating state (Langeland 1996). The stems may be quite long especially when the plant grows in deep clear water and branching usually does not occur until the plant grows near the water surface (Langeland 1996). Upon reaching the surface, stems begin to branch profusely forming a surface mat. This density of stem biomass causes pH in the upper 0.3 m of water during the summer to increase 10.0 during the day by utilization of CO 2 and HCO 3 and to fall below 7.0 in the evening (Spencer et al. 15

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1994; Van et al. 1976). Surface matted hydrilla can also cause dissolved oxygen levels to fall below the air-saturated level during the night, but by noon levels may reach 16 mg L -1 equivalent to over 200% air saturation (Van et al. 1976). Various forms of weed control have been evaluated for hydrilla control in Florida including biological, cultural, chemical, mechanical, and preventative techniques. Biological control agents such as grass carp are usually unpredictable forms of control (Martyn 1985). Cultural management techniques such as drawdowns have had limited success, due to regrowth from subterranean turions (Haller et al. 1976). Mechanical removal of hydrilla is not practical for large lakes, costing as much as $2500 ha yr -1 (SE-EPPC 2005). Due to the high costs or limited effectiveness of mechanical, biological, and cultural techniques, early successful control of hydrilla in the 1960s and 1970s was through the use of herbicides. These included the contact herbicides diquat, endothall, and diquat plus copper combinations (Brian et al. 1958; Hiltibrand 1963; Sutton et al. 1972; Simsiman 1976; Vencill 2002). Due to the rapid activity of these herbicides, it is recommended that no more than half of an infested water body be treated at one time due to potential reduced oxygen supply and fish toxicity (Anonymous 2007a; Anonymous 2003). In addition, these herbicides possess a relatively short half-life (Simsiman and Chesters 1975; Langeland et al. 1994); this usually results in rapid control but also encourages rapid regrowth, so season long weed control has not been possible with single applications of these contact herbicides. The first residual herbicide for hydrilla control, fluridone, received a Section 3 EPA registration for aquatic use in 1986 (Dayan and Netherland 2005). Although the maximum labeled rate is 150 g L -1 active ingredient (a.i.), fluridone is commonly applied at 8 to 12 g L -1 with concentrations as low as 4 to 7 g L -1 providing hydrilla control if the dosage was 16

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maintained for several weeks (Van and Steward 1985). Large areas of hydrilla in Florida were being controlled at 6 to 10 g L -1 in the late 1980s at costs of $250 ha -1 or less, (Haller et al. 1990). Several factors, including low use rates, favorable native plant selectivity, slow activity (reduced oxygen depletion), and often more than one year of hydrilla control have resulted in the reliance on fluridone for hydrilla control in large, shallow Florida lakes (Puri et al. 2006). However, poor performance was observed in the late 1990s and the development of fluridone resistance in hydrilla was characterized in 2004 (Puri et al. 2007). Since then, fluridone resistant populations of hydrilla have expanded in the waterways of Florida. This is likely a result of continuous use of fluridone, low application rates (<20 g L -1 ) and persistent fluridone residue (MacDonald et al. 2001; Arias et al. 2005). The loss of fluridone from an already limited number of aquatic herbicides has resulted in the search for new and effective herbicides that can be applied to aquatic systems. Beginning in 2004, herbicide efficacy studies have been conducted at the Center for Aquatic and Invasive Plants at the University of Florida to evaluate and identify herbicides that are relatively non toxic to aquatic organisms, possess a short half-life, and show native plant selectivity 1 which are characteristics necessary for aquatic registration. Of the herbicides evaluated, flumioxazin {2-[7-fluoro-3,4-dihydro-3-oxo-4-(2-propynyl)-2 H -1,4-benzoxazin-6-yl]-4,5,6,7-tetrahydro-1 H -isoindole-1,3(2 H )-dione} met the desired criteria. Hydrilla dry weight was reduced by 63% and 99% in static tests when treated with 50 and 400 g L -1 a.i., respectively, in 2005 (Mudge and Haller 2006). Based upon these initial evaluations, Valent U.S.A. Corporation applied for an Experimental Use Permit (EUP) from the U.S. Environmental Protection Agency (EPA) and the Florida Dept. of Agriculture and Consumer Services (FDACS) 1 Chiconela, T. F. and W. T. Haller. 2007. Personal Communication. 17

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in 2006 to evaluate control of the submersed aquatic weed hydrilla with flumioxazin (FDACS 2006; Fishel 2006). Flumioxazin is an N-phenylphthalimide herbicide that is registered for preemergence weed control in peanut (Arachis hypogaea L.) and soybean (Glycine max L.) and for post-direct weed control in cotton (Gossypium hirsutum L.) (Anonymous 2005; Askew et al. 1999; Burke et al. 2002; Clewis et al. 2002; Grichar and Colburn 1996; Main et al. 2003). Direct foliar contact (postemergence) with flumioxazin results in unacceptable crop injury regardless of plant species (Yoshida et al. 1991). Flumioxazin is a protoporphyrinogen oxidase (PPO) (protoporphyrin IX:oxygen oxidoreductase, EC 1.3.3.4) inhibiting herbicide with both soil and foliar activity (Cranmer et al. 2000; Hartzler 2004; Price et al. 2002; Price et al. 2004) and is a strong inhibitor of chlorophyll synthesis. It prevents the transformation of protoporphyrinogen IX into protoporphyrin IX which is a precursor to heme and chlorophyll production (Aizawa and Brown 1999; Matringe et al. 1989; Cobb 1992). Protoporphyrinogen IX accumulates in plastids due to inhibition of the PPO enzyme and then diffuses through plastid membrane into the cytosol, where it is oxidized to protoporphyrin IX by a plasma membrane-bound protox (Dayan and Duke 1997; Duke et al. 1991). Protoporphyrin IX reacts with light to produce toxic singlet oxygen radicals leading to lipid peroxidation and the destruction of cellular components (Duke et al. 1991; Gupta and Tripathy 2000). Irreversible damage to the plasmalemma and tonoplast membrane lipids is followed by swelling of organelles, rupture of organelle membranes, and eventually, rupture of the cellular membranes in susceptible plants (Duke et al. 1989). Entire cell contents (both cytoplasmic and vacuolar) are released after extensive membrane destruction resulting in cell desiccation and electrolyte leakage (Becerril and Duke 1989; Duke and Kenyon 1993). 18

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Flumioxazin generally provides 4 to 6 wk of residual broadleaf control when applied to the soil and there is low potential for soil carryover to subsequent rotational crops (Vencill 2002). The potential for phytotoxicity increases with high soil moisture (Sakaki et al. 1991). The half-life of flumioxazin in a sandy clay loam soil was 17.9 and 16.0 d compared to 13.6 and 12.9 d in a loamy sand soil (Ferrell and Vencill 2003a). The flumioxazin molecule is non-ionic and has a water solubility of 1.79 mg L -1 (Vencill 2002); due to its low water solubility, flumioxazin has a greater affinity for organic matter than for silicate clay, which prevents it from binding tightly to the soil matrix and allows it to be readily removed from soil adsorption sites by soil water (Ferrell et al. 2004). Flumioxazin use in row crops is environmentally beneficial because of low use rates (71 to 107 g ha -1 a.i.) and rapid soil dissipation (Lovell et al. 2001). Flumioxazin possesses favorable characteristics for use in aquatic systems since half of the herbicide is degraded by hydrolysis in 4.1 d and 16.1 h at pH 5.0 and 7.0, respectively; however, at pH 9.0, the half-life decreases to 17.5 min (Katagi 2003) which may limit aquatic weed control in water with high pH. Heavy hydrilla infestations have been shown to raise water pH to >9.0 by utilizing free CO 2 and HCO 3 (Van et al. 1976) which has the potential to reduce the efficacy of this herbicide by causing rapid breakdown. Kwon et al. (2004) found the photolytic degradation rate of flumioxazin increased as a function of pH with a half-life of 41.5 and 4.9 h at pH 5.0 and 7.0, respectively, after correction for the effects of hydrolysis (dark conditions). Other forms of degradation can influence flumioxazin activity in the aquatic environment. Flumioxazin showed maximum absorbance at 217 and 286 nm but could absorb UV energy of sunlight at wavelengths greater than 290 nm (Kwon et al. 2004). In aquatic systems, natural sunlight would cause the direct breakdown by photolysis near the surface of the water, but degradation is more likely a function of hydrolysis and water pH. 19

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Carfentrazone received federal registration in 2004 as the only PPO inhibiting aquatic herbicide (FMC 2005; Iverson and Vandiver 2005). Carfentrazone is similar to flumioxazin because of its rapid degradation by hydrolysis at high pH (Elmarakby et al. 2001; Ngim and Crosby 2001). It is effective in controlling the submersed aquatic plant Eurasian watermilfoil (Myriophyllum spicatum L.) and floating plants such as water lettuce (Pistia stratiotes L.), water fern ( Salvinia minima ) watermeal (Wolffia spp.), and water hyacinth (Eichhornia crassipes (Mart.) Solms) (FMC 2004; Koschnick et al. 2004). Species of duckweed in the Lemnaceae family are commonly used in biochemical and toxicity tests because of their small size, high reproductive rate and ease of culture (Gensemer et al., 1999; Geoffroy et al. 2004; Lewis 1995; Ma et al. 2002; Parr et al. 2002). Studies evaluating pigment content (chlorophyll a, b, and carotenoids) and oxygen evolution are often reliable indicators of herbicide toxicity (Wang and Freemark 1995). Previous research has shown that flumioxazin at 1, 10, and 50 g L -1 decreased photosynthetic capacity of common duckweed (Lemna minor L.) by 23, 62, and 64%, respectively (Frankart et al. 2002). In these studies photosynthetic capacity of common duckweed was inhibited more when plants were exposed to 200 g L -1 of copper mixed with flumioxazin than all rates of flumioxazin applied alone. A very slight synergistic or additive effect was observed between the two chemicals. One of the primary goals of aquatic weed control in public and private waters is to eliminate invasive species while maintaining a diversity of native submersed and emergent species. Native aquatic plants have been shown to improve water clarity and quality, provide valuable fish and wildlife habitat, reduce rate of sediment resuspension, and help prevent the spread of invasive plants (Dibble et al. 1996b; Heitmeyer and Vohs 1984; Savino and Stein 1982; Smart 1995). Selective removal of invasive species is beneficial for continued existence 20

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and diversity of native vegetation. Invasive submersed aquatic species often form dense canopies that significantly increase surface water temperature, reduce dissolved oxygen, and decrease light penetration for native species (Bowes et al. 1979; Honnell et al. 1993). Native plant density and diversity has been shown to increase when canopy forming exotic plants are removed (Getsinger et al. 1997) and diversity of invertebrate and fish habitats are maintained (Dibble et al. 1996a). Damage to non-target native plants species is a major consideration in herbicide selection, with favorable aquatic herbicides being able to selectively remove unwanted plants while minimizing damage to non-target native plants. Various mesocosm studies have been conducted to evaluate the sensitivity of native plant species to registered aquatic herbicides. Netherland et al. (1997) evaluated the effects of fluridone on the submersed species elodea (Elodea Canadensis Michaux), American pondweed (Potamogeton nodosus Poiret), sago pondweed (Potamogeton pectinatus L), and vallisneria (Vallisneria americana Michaux). Emergent natives such as soft-stem bulrush (Scirpus validus Vahl.), Egyptian panicgrass (Paspalidium geminatum Forssk), maidencane (Panicum hemitomon Schult.), pickerelweed (Pontederia cordata L.), and sagittaria (Sagittaria lancifolia L.) have been evaluated for tolerance to the systemic acetolactate synthase (ALS) inhibiting herbicides bispyribac-sodium {sodium 2,6-bis[(4,6-dimethoxypyrimidin-2-yl)oxy]benzoate}, imazamox {2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1 H -imidazol-2-yl]-5-(methoxymethyl)-3-pyridinecarboxylic acid}, and penoxsulam (Koschnick et al. 2007). These ALS herbicides (as well as fluridone) usually have relatively long half-lives, while contact herbicides have a relatively short half-life. Emergent, submersed, and floating species have also been evaluated for sensitivity to the contact herbicide dipotassium salt of endothall (Skogerboe and Getsinger 2002). Those species evaluated included Eurasian watermilfoil, curlyleaf 21

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pondweed (Potamogeton crispus L.), Illinois pondweed (Potamogeton illinoensis Morong.), sago pondweed, coontail (Ceratophyllum demersum L.), elodea, vallisneria, cattail (Typha latifolia L.), smartweed (Polygonum hydropiperoides Michx.), pickerelweed and spatterdock (Nuphar advena Aiton). Homeowners, commercial nurseries, and farmers in Florida often irrigate plants from surface waters (canals, ponds, lakes, etc.) (Hassell et al. 20004; Hodges and Haydu 2006); non-target plants may be affected if these waters are treated with herbicides for aquatic weed control. The use of herbicide treated irrigation water before herbicide residues dissipate below phytotoxic levels will result in injury or death of irrigated plants. Similarly, farmers may irrigate food crops from treated water, so the aquatic herbicides must have established tolerances or acceptable levels of residue on or in food crops. Tolerances of flumioxazin on certain food crops have been established by the EPA by determining the maximum amount of pesticide residue that can remain in or on a treated food commodity to ensure food safety (EPA 2003), but no such tolerances are required for ornamental plants (non-food crops). Phytotoxicity is a major concern when water with aquatic herbicide residues is used for irrigation of both food and non-food crops. Previous research has been conducted to evaluate the phytotoxic effects of irrigation water containing 2,4-D, copper, diquat, endothall, and fluridone on non-target turf and ornamental species (Andrew et al. 2003; Hiltibran and Turgeon 1977; Koschnick et al. 2005a; Koschnick et al. 2005b; Mudge et al. 2007; Riemer and Motto 1980), but similar studies have not been reported for flumioxazin. Therefore, the objectives of this research were to determine the effect of flumioxazin on hydrilla with respect to efficacy, photosynthesis, and chlorophyll content as influenced by rates of application, water pH, and light. Further research was conducted to determine the impact of 22

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flumioxazin on other aquatic invasive plants, non-target aquatic plants, non-target row crops and non-target ornamental plants. An additional objective of this research was to judge the suitability of flumioxazin as an aquatic herbicide, establish herbicide use patterns, and determine irrigation restriction parameters through development of possible aquatic use directions. 23

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CHAPTER 2 THE EFFECT OF FLUMIOXAZIN ON HYDRILLA CONTROL IN NATURAL SYSTEMS AND THE INFLUENCE OF WATER PH ON HYDRILLA CONTROL IN MESOCOSMS Introduction Flumioxazin is fast acting herbicide which inhibits chlorophyll production and results in the production of toxic singlet oxygen radicals leading to lipid peroxidation and the destruction of cellular components (Duke et al. 1991; Gupta and Tripathy 2000). Injury symptoms may occur within 1 d after plants are treated with flumioxazin and other herbicides in the N-phenylphthalimide family (Ferrell et al. 2007b). Although flumioxazin acts rapidly to inhibit chlorophyll and destroy membranes, this herbicide is rapidly hydrolyzed with an average half-life of 4.1 d, 16.1 h, and 17.5 min at pH 5.0, 7.0, and 9.0, respectively (Katagi 2003; Vencill 2002). Hydrilla infested waters may have pH in excess of 9.0 (Van et al. 1976) and will likely influence the efficacy of flumioxazin since water with a pH >9.0 results in rapid breakdown of this herbicide by hydrolysis with an average half-life of 17.5 min under laboratory conditions (Katagi 2003). Most aquatic herbicides are degraded by either photolysis or microbial activity (Vencill 2002) so efficacy of these herbicides is unaffected by time of day during application; however, higher pH waters (due to pH cycling throughout the day) may limit when flumioxazin can be applied to successfully control hydrilla. Flumioxazin treatments at rates less than 400 g L -1 in water with a pH >8.0 may not be adequate for successful hydrilla control; therefore, the objectives of these studies were to determine flumioxazin use rates and the effect of water pH. Materials and Methods Efficacy in Ponds Eight ponds infested with hydrilla in Florida were treated in 2006 with submersed flumioxazin applications to determine herbicide efficacy under an Experimental Use Permit (EUP). Flumioxazin was applied at 100, 200, or 400 g L -1 to each pond (0.10 to 10.12 ha) 24

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having water pH ranging from 6.7 to 10.0. Two of the ponds were treated with flumioxazin (400 g L -1 ) plus copper (chelated copper, EarthTec 2 200 g L -1 ). One or more herbicide applications were made to each pond between February and October. All ponds were treated with the appropriate amount of herbicide in an equivalent of 935.0 L water ha -1 in a 378.5 L spray tank with hydraulic agitation. Most herbicide treatments were applied using a boat equipped with 3 weighted hoses mounted on the bow of the boat and connected to the spray tank. The weighted hoses were 1.2 to 3.7 m in length on the left, center, and right side of the boat, respectively. Some ponds were treated with hoses (0.3 to 0.6 m long) that trailed behind the boat, which applied the herbicide treatment to the surface of the water, while other treatments were applied using a handgun sprayed at the water surface. All data were based on visual observations (including injury symptoms, time of hydrilla regrowth and recovery time) prior to and after herbicide treatments. Efficacy of flumioxazin treatments was primarily based on how quickly hydrilla produced new apical tips from treated tissue and the amount of time hydrilla needed to reach the water surface following treatments. Efficacy in Mesocosms A mesocosm study was conducted and repeated at the Center for Aquatic and Invasive Plants at the University of Florida to determine the effect of flumioxazin on hydrilla. Hydrilla was collected from Rodman Reservoir near Interlachen, FL in June 2005 and February 2007. Four sprigs of hydrilla (15 cm) were planted in each 10 x 10 x 12 cm (1 L) pot filled with masonry sand amended with Osmocote 3 4 15-9-12 fertilizer at a rate of 1g kg -1 soil. Five pots 2 Registered trademark of Earth Sciences Laboratories. Rogers, AZ. 72756. 3 The Scotts Company. Marysville, OH. 43041. 4 Mention of a trademark or a proprietary product throughout this document does not constitute a guarantee, warranty, or endorsement of the product by the author or the University of Florida, and does not imply its approval to the exclusion of other products that may also be suitable. 25

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were placed in each 95 L HDPE (high-density polyethylene) tub filled with tap water (pH 7.5 at planting). The experiment was a completely randomized design with five replications (tubs). The initial study was conducted outside in a shade house (70% sunlight) in July 2005, while the repeated study was conducted in a greenhouse (70% sunlight) in April 2007. Hydrilla was allowed to acclimate for 2 wk in 2005 and 6 wk in 2007 before herbicide treatment. Hydrilla was immature (actively growing) and had just began to branch at the water surface (pH 8.5 to 9.5) at the time of treatment. Flumioxazin was applied as a submersed treatment at 0, 50, 100, 200, 400, 800, and 1600 g L -1 a.i. and the dipotasium salt of endothall was applied to other tubs at 3000 g L -1 a.i. as a comparison treatment. All plants were harvested 21 d after treatment (DAT) by clipping biomass at the soil line; shoots were placed in a drying oven at 90 C for ca. 1 wk and then weighed. Plant dry weight data were analyzed using non-linear regression (PROC NLIN, SAS Institute 2002) and regression models were used to determine the effective concentration 50 (EC 50 ), which is the concentration of flumioxazin in water that resulted in a 50% reduction in dry weight compared to control plants. Data from both studies were pooled as there was no difference between the slopes of regression lines for both studies at the 95% confidence interval. Impact of pH on Efficacy Hydrilla was collected from 900 L concrete mesocosm stock tanks and one 15 cm sprig was planted in a 50 mL plastic tube (13.5-cm by 4-cm in diameter). Tubes were filled with potting media 5 amended with Osmocote fertilizer at a rate of 1g kg -1 soil and topped off with a 1 cm sand cap. A total of 144 tubes were planted and hydrilla was acclimated in 900 L concrete tanks for 2 wk prior to herbicide treatment. The initial study was conducted in September 2006 5 Earthgro Topsoil is a registered trademark of The Scotts Company. Marysville, OH. 43041. 26

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and repeated in May 2007 under a shadehouse (70% sunlight). Efficacy of flumioxazin was evaluated in low (6.0 to 6.2), medium (7.0 to 7.2), and high (>8.5) pH water. Prior to herbicide treatment, one tube planted with hydrilla was placed into each 95 L HDPE tub that had been filled with 83 L of tap water (pH 8.0). Hydrilla was 45 3.6 cm long with an average dry weight of 0.51 0.3 g at treatment. Muriatic acid was added at a rate of 10 mL or 25 mL per tub to establish medium or low pH treatments as needed. The high pH treatment was achieved by placing extra pots filled with hydrilla in tubs 24 h before herbicide application. These additional plants were kept in high pH treatment tubs so that photosynthesis would maintain water pH at a level above 8.5. Flumioxazin was applied at a rate of 400 g L -1 and mixed in the water as a submersed treatment. The pH was monitored daily and muriatic acid was added as needed to maintain desired pH levels in the medium and low pH treatments. Following initial treatment, an additional tube of hydrilla was added every 24 h to the treated water at 1, 2, 3, 4, and 5 DAT. Day 0 plants were in the tubs at the time of treatment (Day 0) and removed from the treated solutions after 4 d of exposure and harvested 21 d later. Plants were placed in the tubs at 1, 2, 3, 4, and 5 days after initial treatment to assess if pH or a pH mediated degradation of flumioxazin impacted efficacy. All treated plants were given a 96 h exposure and were then removed from treatment and placed into 900 L mesocosms with flowing tap water (pH 7.5) for 3 wk. This experiment was conducted as a completely randomized design with four replications (tubs). Water samples were collected from the low and medium pH treatment at 0.5, 1, 24, 48, 72, 96, and 120 h after treatment (HAT). Due to anticipated rapid breakdown under high pH, water samples were collected at 0.25, 0.5, 1, 4, 7, 19, 24, 48, 72, 96, and 120 HAT. All water samples were immediately acidified with 0.5 mL of muriatic acid at the time of collection to a pH <4.0 to 27

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prevent further breakdown by reducing hydrolysis, frozen and shipped to the Valent U.S.A. Corporation laboratory (Walnut Creek, CA) for flumioxazin analysis by gas chromatography/mass spectrometry (GC/MS) using methods reported by Hirota et al. (1992). All biomass above the soil line was harvested 21 DAT. Shoots were placed in a drying oven at 90 C for ca. 1 wk and then weighed. Plant weight data were converted to percent of the respective non-treated plants at each pH for each day and analyzed as a mixed model (PROC MIXED, SAS Institute 2002) with experiment used as a random factor. The pH of the water was considered a fixed effect, while experiment, replication (nested within experiment), and all interactions containing either of these effects were considered random effects. Classification of experiment (or the combination of experiment and location) as an environmental or random effect, permits inferences about pH to be made over a range of environments (Carmer et al. 1989; Hager et al. 2003). Type III statistics were used to test all possible effects of fixed factors. Least square means were used for mean separation at p0.05. Data from both studies were pooled. Water residue data were analyzed using non-linear regression (PROC NLIN, SAS Institute 2002) to calculate flumioxazin half-life at each pH. Results and Discussion Efficacy in Ponds The maximum EUP rate of 400 g L -1 was chosen to treat all the ponds until results suggested lower rates could successfully control hydrilla. Preliminary studies conducted at the Center for Aquatic and Invasive Plants indicated flumioxazin controlled hydrilla at concentrations as low as 50 g L -1 but higher rates may be required in the field due to the rapid hydrolysis of flumioxazin, especially in high pH waters where hydrilla infestations commonly occur. 28

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The Micanopy pond treated in February 2006 was the most successful of all flumioxazin treatments (Table 2-1). Hydrilla began to exhibit apical tip bleaching within 3 DAT followed by tip abscission and decay within 5 to 7 DAT. The stem segment immediately below the bleached apical tip became chlorotic/necrotic and the tissue eventually reddened before the stem lost cellular integrity and buoyancy. Hydrilla height and dry weight were measured weekly for 2 mo and periodically sampled for 6 mo in the Micanopy pond (data not shown). In addition, a non-replicated residue study was conducted in the Micanopy Pond and data suggested flumioxazin possessed a 25 to 35 h half-life at pH 6.7 (data not shown). Flumioxazin provided >95% control for at least 6 mo after treatment, as few new apical tips could be found throughout the pond; however, flumioxazin was not solely responsible for the extensive hydrilla control due to very low water levels 7 mo after herbicide treatment. Factors that could have influenced this particular treatment included low water pH (6.7), clear water, lower water temperature, time of year, and actively growing plants that were 0.6 to 1.0 m from the surface (non-matted). The Apopka pond possessed a higher water pH at treatment (7.4), but flumioxazin still provided >80% reduction in hydrilla biomass for 8 wk when treated at 400 g L -1 (Table 2-1). The first flumioxazin treatments in February and March 2006 controlled hydrilla with minimal regrowth in the first two mo after treatment (MAT). Due to the success of these initial treatments at the maximum EUP rate, it appeared that flumioxazin at lower application rates could be as effective in controlling hydrilla as the higher application rates. Three ponds were treated in Kissimmee, FL with three rates of flumioxazin (100, 200, and 400 g L -1 ) in May 2006 (Table2-1). These were the first EUP ponds treated that had water with a pH > 9.0, whereas the Micanopy and Apopka ponds had <7.5 pH. Hydrilla in all Kissimmee ponds (A-C) demonstrated similar injury symptoms as the Micanopy and Apopka ponds; however, hydrilla 29

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began to sprout new shoots from treated tissue within 3 to 5 wk after treatment (WAT) and began to reach the surface by 7 WAT. These particular ponds were treated with a hand gun at the water surface, whereas the Micanopy and Apopka ponds were treated with weighted and trailing hoses, respectively. The Kissimmee ponds averaged 2.5 to 4.0 m in depth and it is possible that the majority of the herbicide treatment remained in the upper 1.0 m due to thermal stratification. Thermal stratification can occur in the summer when less dense warmer waters overly colder more dense waters (Wetzel 1975). Thermal stratification is common in surface-matted submersed plants, especially on sunny days in hydrilla stands (Getsinger et al 1990). This thermal layer can create a physical barrier, isolating layers in the water column and preventing surface-applied herbicides from immediately reaching the target vegetation. The upper layer of these infested water bodies may be in excess of 10 C more than the layers below the hydrilla mat. Consequently, the hand gun application technique may have failed to evenly distribute the herbicide into the water column. In addition, these three ponds were more turbid and heavily infested with hydrilla, permitting less light to reach the bottom. Although hydrilla in the Kissimmee ponds had not begun to form a surface mat, the hydrilla was beginning to branch near the water surface at the time of herbicide treatment. The Apopka pond was retreated in June with only 200 g L -1 flumioxazin (Table 2-1); however, hydrilla began to form new apical tips within 1 WAT of herbicide application and was surface matted 3 WAT (Table 2-1). At treatment, this pond was shallow (0.5-1.3 m deep) compared to other ponds, so hydrilla did not require a long period of time to once again reach the water surface. The failure of this treatment and the low efficacy in the Kissimmee ponds at 100 and 200 g L -1 suggested that flumioxazin may not control hydrilla when rates <400 g L -1 are 30

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used in the warmer months when hydrilla may cause pH in the water to increase to 10.0 during the day by utilization of CO 2 and HCO 3 (Spencer et al. 1994; Van et al. 1976). Flumioxazin treatments in the latter portion of June and throughout the rest of the year were applied only with weighted hoses to ensure thorough herbicide distribution in the water column. When Kissimmee pond C was retreated with flumioxazin, the pH was measured throughout the water column; the pH was 6.5 to 7.0 near the bottom (2.5 m) compared to >9.0 at and near the water surface. Weighted hose applications placed approximately one-third of the flumioxazin treatment in direct contact with the lower portion of the hydrilla stand where it would mix with low pH water and consequently degrade at a slower rate than the flumioxazin applied in the upper 1 m of the water. Due to reduced flumioxazin efficacy in the summer, this herbicide has too short of a half-life in high pH water and does not provide acceptable control of mature hydrilla. Therefore, flumioxazin treatments should be restricted to early season for hydrilla control or should be used in combination with other herbicides as a tank mixture. Combinations of herbicides can result in increased efficacy when used for aquatic weed management (Gray et al. 2007; Nelson et al. 2001). Copper was chosen because of increased efficacy when mixed with diquat for hydrilla and common duckweed control (Langeland et al. 2002; Sutton et al. 1972). This flumioxazin plus copper combination was evaluated as a fall treatment (September and October) in the Interlachen and Kissimmee C pond and provided ca. 6 mo of control. These treatments were successful because of potential synergism between copper and flumioxazin, later season applications (when hydrilla began tuber production and may have been more susceptible to flumioxazin), or lower water pH (7.2 to 8.5) (Haller et al. 1976; Sutton et al. 1972; Van et al. 1978). 31

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These field studies indicate flumioxazin efficacy in ponds is highly variable. Early season applications (February and March) provided longer hydrilla control than treatments in warmer months. The level of control observed with flumioxazin varied according to factors such as water pH, water temperature, maturity/growth stage of hydrilla, time of year, and placement of herbicide in water column. Hydrilla is exposed to higher concentrations of flumioxazin for a longer period of time at low pH than at higher pH. Flumioxazin applied to hydrilla in May through August failed to provide more than a few weeks to a couple of months of control as new apical shoots sprouted from treated tissue on most plants and began to reach the surface within 3 to 7 WAT (Table 2-1). Consequently, several of these ponds were treated with flumioxazin multiple times. Although hydrilla recovered within a few WAT, flumioxazin at 400 g L -1 usually provided an additional 2 to 4 wk of control compared to 100 and 200 g L -1 treatments. Efficacy in Mesocosms Flumioxazin applied at 50 to 1600 g L -1 to actively growing immature hydrilla in mesocosms resulted in bleaching of the upper 5 cm of all apical tips within 3 DAT and stems began to redden (probably due to anthocyanin production) from 3 cm below the bleached tip to the soil surface. Bleached apical tips began to abscise and decay within 3 to 7 DAT, when the plants began to lose cellular integrity. Despite rapid bleaching, reddening, and loss of integrity, hydrilla in all treatments began to regrow from treated tissue and formed new apical shoots at the internodes within 5 to 13 DAT depending on flumioxazin concentration. The calculated EC 50 of flumioxazin was 56 g L -1 while the EC 90 for flumioxazin was 186 g L -1 (Figure 2-1). Low herbicide use rates are desirable in aquatic ecosystems and the proposed flumioxazin labeled rate of 400 g L -1 reduced dry weight by 90% of the nontreated control plants. As a comparison, 3000 g L -1 endothall reduced hydrilla dry weight by 91% in the same time period. 32

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Impact of pH on Efficacy Dry weights of hydrilla treated at high pH differed from those treated at low and medium pH on all days except for hydrilla added 4 and 5 DAT (Figure 2-2). Although statistically different from the other treatments at 0 DAT, hydrilla placed in the high pH flumioxazin treated water was still reduced by approximately 90% of the nontreated control. There was no difference in dry weight at low and medium pH treatments except for hydrilla placed in treatment solutions 3 or 4 DAT. Hydrilla biomass generally increased daily following exposure by the plants from 1 to 5 d after exposure as percent of nontreated control. This increase in biomass corresponded with a decrease in flumioxazin residue (Figure 2-3). Plants in tubs treated with flumioxazin at medium and low pH levels were still exposed to herbicide through the 96 h exposure period, while approximately 98% of flumioxazin in the high pH treatment was hydrolyzed by 3 DAT (Figure 2-3). Although the residue analysis detected less than 10 g L -1 of flumioxazin in the high pH solution, hydrilla biomass was still reduced by ca. 10 to 20% on the last 2 d of exposure. These data indicate flumioxazin is active at low concentrations (<10 g L -1 ) and/or secondary metabolites possess activity on hydrilla as well. The half-life of flumioxazin in low, medium, and high pH water (6.0 to 6.2, 7.0 to 7.2 and >8.5, respectively) was 39, 18.6, and 1.7 h, respectively (Figure 2-3). Katagi (2003) reported that the half-life of flumioxazin at pH 5.0, 7.0, and 9.0 under controlled laboratory conditions, was 98.4, 16.1, and 0.3 h, respectively. Environmental factors in our studies (e.g., higher light levels, potting media, water quality, and plants which possibly absorbed and/or metabolized the flumioxazin molecule) most likely influenced the half-life of flumioxazin. The half-life data in these studies is not directly comparable to previous research by Katagi (2003) since the water pH evaluated differed between studies. However, flumioxazin degradation in both studies followed a similar trend, as pH increased, half-life decreased. Under field conditions, pH fluctuates and 33

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can not be controlled, whereas previously reported data (Katagi 2003) was conducted under stable conditions. Consequently flumioxazin may be exposed to different pHs within the same water body; therefore, degrading at a faster rate within the same system. As a result, some plants would be exposed to higher herbicide concentrations than other plants These field EUP trials and mesocosm studies provided further evidence of the impact of pH on flumioxazin efficacy. Water pH does not directly influence flumioxazin efficacy; pH influences degradation, which in turn reduces flumioxazin concentration and exposure time to hydrilla. The pH efficacy study data indicated high pH treatments were successful, possibly due to ample mixing and hydrilla exposure to higher concentrations for an extended period of time. Conversely, field treatments usually dont provide adequate mixing and adequate exposure at higher concentrations. Flumioxazin applied to hydrilla in ponds under high pH conditions were less effective since flumioxazin was rapidly hydrolyzed before it could control the weed; however, application of flumioxazin to water with pH levels less than 8.0 generally provided adequate to complete control of hydrilla. These data provided evidence of rapid flumioxazin uptake in hydrilla as exhibited by biomass reduction in high pH treatments. If plants treated at the high pH did not absorb flumioxazin within the first few minutes or hours after treatment, these plants would not have become chlorotic and would not have been reduced by 90% as in the 0 d treatments. Other contact herbicides such as endothall require exposures of at least 48 h at 2 mg L -1 acid equivalent (a.e.) to provide greater than 85% reduction in biomass of hydrilla (Netherland et al. 1991). Flumioxazin appears to require greatly reduced exposure times and has increased activity in lower pH water. The degradation of flumioxazin within minutes of application in the high pH treatment prevented a significant reduction in hydrilla biomass 2 to 5 DAT. Flumioxazin is not 34

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highly water soluble (1.79 mg L -1 ) (Vencill 2002), so uptake into hydrilla is probably rapid due to the lipophilic nature of this molecule. Hydrilla treated with flumioxazin in EUP ponds in Florida showed similar bleaching of apical stem segments regardless of stem length or water depth. Field treated plants followed the same bleaching pattern and reddening of the stem below the tip as noted in the preliminary mesocosm study that demonstrated flumioxazin activity on hydrilla. However, symptoms of hydrilla treated at depths greater than 1.5 m were less pronounced and stems had only 5 to 10 cm of reddened tissue below the apical tip. Submersed plants growing at these depths are often light limited (Haller and Sutton 1975) and net photosynthesis is restricted to the apical portions of the plant. Upon forming a dense mat on and just below the water surface, hydrilla can further limit light penetration. Flumioxazin and other PPO inhibitors are more active in the presence of light and may require full sunlight for optimal activity (Sherman et al. 1991; Wright et al. 1995). Additionally, oxygen-derived free radicals have very short half-lives ranging from milliseconds to microseconds (Kobayashi et al. 1989) and the short half-life of flumioxazin in higher pH water potentially reduces radical formation. By the time sufficient light reaches the lower apical tips and stem segments, flumioxazin has been degraded by hydrolysis and is no longer present to inhibit the PPO (Aizawa and Brown 1999; Matringe et al. 1989; Cobb 1992). High light conditions (>1000 mol m -2 s -1 ) in all greenhouse experiments facilitated bleaching as a result of inhibition of chlorophyll and membrane disruption through radical production (Duke et al. 1989). Lower efficacy observed in EUP pond treatments was likely due to high pH, low light intensity in these deeper waters, and growth stage. Most treatments resulted in >95% control of hydrilla within 1.5 m of shore, where water was shallow, hydrilla was less than 0.5 m in length and plants were not shaded by emergent or floating vegetation. This evidence further supports previous 35

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36 studies (Sherman et al. 1991; Wright et al. 1995 ) that show higher light levels increase flumioxazin activity.

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37 Table 2-1. Hydrilla infested ponds in Florida treated with flumioxazin under an Experimental Use Permit in 2006. Location Date pH Comment/Results Micanopy Feb 2006 6.7 WH application at 400 g L -1 ; no regrowth for 6 MAT; > 95% hydrilla control for 6-8 MAT Apopka March 2006 7.4 TH application at 400 g L -1 ; >80% hydrilla control for 8 wk; 30% hydrilla surface matted by 3 MAT Kissimmee A May 2006 9.7 HG application at 100 g L -1 ; hydrilla regrowth 3 WAT and near surface 7 WAT Kissimmee B May 2006 9.7 HG application at 200 g L -1 ; hydrilla regrowth 3 WAT and near surface 7 WAT Kissimmee C May 2006 9.7 HG application at 400 g L -1 on approximately one third of pond; bleaching and control in whole pond but better control in treated area; hydrilla regr owth 5 WAT and near surface 7 WAT Apopka June 2006 7.2 TH application at 200 g L -1 ; hydrilla regrowth at 1 WAT was surface matted 3 WAT Eustis June 2006 9.5 TH application at 400 g L -1 ; minimum injury symptoms and hydrilla reached surface 1 MAT Interlachen June 2006 7.2 HG application at 200 g L -1 ; hydrilla controlled for 1 mont h but began to regrow and reached surface 12 WAT Kissimmee C June 2006 9.4 WH application at 400 g L -1 ; hydrilla regrowth 3 WAT, ha d not reached surface by 10 WAT Gainesville June 2006 10.0 WH application at 400 g L -1 ; minimum hydrilla injury a nd reached surface 1 MAT Interlachen July 2006 7.2 WH application at 400 g L -1 ; hydrilla regrowth at 1M AT and surfaced 3 MAT Kissimmee C Sept 2006 8.5 WH application at 400 g L -1 plus 200 g L -1 copper; hydrilla regrowth 2 MAT, but had not reached surface by March 2007 Interlachen October 2006 7.2 WH application at 400 g L -1 on whole pond, but half of pond received copper at 200 g L -1 ; >6 months of control; hydrilla be gan to reach surf ace in March 2007 Abbreviations: WAT, weeks after treatment; MA T, months after treatment; WH = weighted hose, 1.2 to 3.7 m l ong; TH = trailing ho se, 0.3 to 0.6 m long injected at rear of boat; HG = hand gun, using pressure to inject herbicide 0.6-0.9 m into the water ahead of boat.

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Flumioxazin Concentration (g L-1 a.i.) 02004006008001000120014001600 Mean dry weight (g/tank) 0246810 y = 8.4710-0.0124x, r2 = 0.89EC50 = 56g L-1 a.i. Figure 2-1. The effect of flumioxazin concentration on hydrilla dry weight 21 d after exposure under 70% sunlight. Flumioxazin applied as a single application to hydrilla cultured in 95 L tubs (pH 9.0-9.5). Data are shown as dry weight means standard error (n=10). EC 50 = effective concentration 50, concentration of flumioxazin in water required to reduce hydrilla biomass by 50%. 38

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pH Replacement, Days After Treatment (DAT) % of Nontreated Control 020406080100120 Low pH Medium pH High pH 0 1 2 3 4 5 abbcbabababaaabbabb Figure 2-2. The effect of flumioxazin at 400 g L -1 on hydrilla dry weight as influenced by low (6.0 to 6.2), medium (7.0 to 7.2), and high (>8.5) water pH under 70% sunlight. Hydrilla plants were added to low, medium, and high pH water treated with flumioxazin 0 to 4 d after initial treatment and allowed to grow for 21 d after treatment until harvest. Data are shown as percent of nontreated control of each pH standard error (n=8). Treatment means within a particular day were separated using least square means (p<0.05). 39

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Hours After Treatment (HAT) 020406080100120 Flumioxazin Concentration (g L-1) 0100200300400 High pH Water Medium pH Water Low pH water Figure 2-3. Dissipation of flumioxazin applied at 400 g L -1 to low (6.0 to 6.2), medium (7.0 to 7.2) and high pH (>8.5) tap water in 95 L tubs under 70% sunlight. The dissipation of flumioxazin was calculated using non-linear regression (exponential decay) for the low (y = 0.0178e -0.0178x ; r 2 = 0.92; half-life 39.0 h), medium (y = 0.3074e -0.0373x ; r 2 = 0.93; half-life 18.6 h), and high (y = 0.3209e -0.3991x ; r 2 = 0.94; half-life 1.7 h) pH treatments. All residues are reported as the mean standard error (n=6). 40

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CHAPTER 3 THE EFFECT OF FLUMIOXAZIN ON HYDRILLA PHOTOSYNTHESIS AND CHLOROPHYLL CONTENT Introduction Flumioxazin was evaluated for hydrilla control in EUP field trials throughout Florida in 2006 and 2007 in water bodies ranging in pH from 6.7 to 10.0 at rates of 100 to 400 g L -1 (see Chapter 2). Despite early season success, summer applications of flumioxazin failed to provide adequate season-long control. Most summer EUP trials resulted in rapid injury to the hydrilla canopy followed by re-infestation within 1 to 4 months. Higher water pH (either due to hydrilla infestations or pH cycling throughout the day) may limit when flumioxazin can be applied for successful hydrilla control. Flumioxazin applied to water with a pH >9.0 is rapidly degraded by hydrolysis and submersed aquatic plants growing at greater depths are often light limited (Haller and Sutton 1975). Flumioxazin and other PPO inhibitors require full sunlight for optimal activity (Sherman et al. 1991; Wright et al. 1995), so the limited quantity of light at greater water depths may reduce flumioxazin activity on hydrilla. Measurement of the net photosynthetic rate of submersed aquatic plants has been utilized to study the effects of aquatic herbicides on submersed plants (MacDonald et al. 2003; Netherland and Getsinger 1995a; Netherland and Getsinger 1995b). Carbon dioxide is absorbed by plants and fixed as one of the primary products of photosynthesis, while O 2 is evolved from the splitting of water (Oja et al. 2007). Submersed aquatic plants treated with herbicides that interfere with photosynthesis or the production of light capturing pigments (chlorophyll and carotenoids) typically evolve less O 2 compared to nontreated plants (MacDonald et al. 2002; Netherland and Getsinger 1995a). Measuring total chlorophyll content in aquatic plants provides further evidence of the impact of these herbicides on plant status (Doong et al. 1993; Netherland and Getsinger 1995a). Such studies provide information regarding the health of plants treated 41

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with herbicides that may not show visual damage for several days. Therefore, the objective of these studies was to determine the effect of water pH, light, and flumioxazin rate on net photosynthesis and chlorophyll content of hydrilla. Materials and Methods Photosynthesis and pH The effects of flumioxazin at low (6.0) and high (9.0) pH on the net photosynthetic rates of hydrilla were determined by measuring oxygen evolution (dissolved oxygen, DO) over time. Hydrilla was collected at Rodman Reservoir near Interlachen, FL in January 2007. All DO techniques were similar to those reported by Netherland and Lembi (1992) and Netherland and Getsinger (1995b). The plants evaluated in those previously cited studies remained continuously in biological oxygen demand (BOD) bottles as static exposures, whereas plants in these studies were removed every 24 h from herbicide treatments to prevent oxygen saturation. Apical hydrilla tips (4 cm) were excised from freshly collected plants and placed in clear plastic cups (473 mL) with 350 mL deionized water (DI) and allowed to acclimate for 24 h before treatment. Tips were acclimated and treated in a growth chamber 6 at a constant temperature of 27 C with a 14 h light/10 h dark photoperiod. The daytime light intensity was 380 mol m -2 s -1 Preliminary studies were conducted to determine appropriate concentrations of nutrients and buffer (data not shown) to attain active growth and hold pH. Nutrients were supplied by 1% v/v Hoaglands solution and 4.7mM NaHCO 3 was added as a carbon source. The experimental design was a randomized design with 4 replications. The culture solution of plants treated at the low pH also received 10 L of muriatic acid (diluted HCl) and MES buffer (5 mM) to maintain a pH of 6.0. Hydrilla tips were treated with flumioxazin at 0, 100, 200, 400, and 800 g L -1 in the 6 Percival Scientific, Inc. Perry, IA. 50220. 42

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same plastic culture cups and the same hydrilla tip was reused daily to measure DO over time. DO was measured pre-treatment (0), 24, 48, 72, 96, 120, 144, and 168 h after treatment (HAT) using a dissolved oxygen meter 7 To eliminate possible oxygen saturation, treated and nontreated tips were removed from the plastic cups, rinsed twice in DI water to ensure no herbicide residue transfer and placed in 300 mL BOD bottles 8 filled with fresh DI water and the same nutrient solution as the plastic culture cups for DO daily measurements. Initial DO measurements were recorded for each bottle prior to tip placement in the bottles, then tips were placed in BOD bottles and allowed to incubate for 1 h. Final DO was measured in each bottle and tips were removed, gently blotted with a dry paper towel, and weighed (fresh weight). Tips were immediately returned to their original cups after fresh weights were obtained. BOD bottles were emptied each day and rinsed after completion of DO measurements. The formula for calculating the net photosynthetic rate was: (Final DO-Initial DO)/weight(g)/time(min)*1000 = g O 2 g fresh weight -1 min -1 Many of the treated hydrilla tips lost turgor and became defoliated during the course of the experiments and disintegrated by the conclusion of the study. Decayed tips were discarded when they occurred and a value of 0 for net photosynthesis was assigned to that treatment for the remainder of the experiment. All data were normalized to the control to account for differences in photosynthetic rates at each pH. Percent data were analyzed using non-linear regression (exponential decay) (PROC NLIN, SAS Institute 2002) and regression models were used to determine an ET 50 which is the amount of time hydrilla was exposed to flumioxazin before a 50% reduction in photosynthesis 7 Accumet Excel XL40 Dissolved Oxygen/BOD/OUR/SOUR Temperature Meter. Fisher Scientific. Pittsburgh, PA 15275. 8 Wheaton Science Products. Millville, NJ. 08332. 43

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was reached for each rate and pH. The ET 50 values were calculated using the slope of the line based on the following formula: ln(0.5)/-b 1 Additionally, SAS produced upper and lower 95% confidence intervals (CI) for each line. When placing an upper and lower 95% confidence interval on the slope of a non-linear equation, calculated ET 50 values 95% CI will not be a single value. This is because a non-linear equation will not always fit exactly between the range of data values. This can then require differing 95% CI values for the upper or lower limits, depending on which direction the equation trends toward. For these experiments, data were pooled because were no difference between the slopes of regression equations for both experiments at the 95% level of confidence. Photosynthesis and Light The effects of light quantity on net photosynthetic rates of apical hydrilla tips treated with flumioxazin were evaluated in May and June 2007. Hydrilla was collected from the Withlacoochee River near Dunnellon, FL in May 2007. Methods used were as described above for the DO pH experiment except plants were only cultured at pH 9.0. Apical tips were acclimated for 24 h and treated at low (20), medium (170), and high (400 mol m -2 s -1 ) light quantities in a growth chamber. The low light quantity chosen in this study is near the upper threshold of the light compensation point for hydrilla and is typical of the quantity of light found near the bottom of a pond/lake, whereas the high light quantity is less than the light saturation point of hydrilla and is near the light quantity found near the surface of matted hydrilla (Van et al. 1976; Bowes et al. 1979; Steward 1991). Hydrilla DO was measured every 24 h and the experiment was concluded 168 HAT. Light levels in the chambers were adjusted by removing incandescent and fluorescent bulbs to obtain desired light levels. Each light quantity treatment consisted of 10 hydrilla tips (cups); 5 control plants and 5 receiving flumioxazin at 400 g L -1 for 44

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a total of 30 tips (5 reps/treatment). Cups were placed in a randomized design in each of the growth chambers. All data were normalized to the control to account for differences in photosynthetic rates at each light level. Percent data were analyzed using non-linear regression (exponential decay) (PROC NLIN, SAS Institute 2002) and regression models were used to calculate ET 50 values at each light quantity. Effect of Contact Herbicides on Photosynthesis The effect of flumioxazin on hydrilla photosynthesis was compared to the effect of other registered contact aquatic herbicides in April 2007. Most culture and treatment techniques used in this experiment were the same as those described above in the DO pH study but this experiment had 5 replications, plants were treated at pH 9.0, and this experiment was concluded 96 HAT. Hydrilla was cultured and treated with a light quantity of 380 mol m -2 s -1 Herbicide treatments applied to hydrilla apical tips in this experiment included flumioxazin at 400 g L -1 (all flumioxazin treatment combinations were applied at this rate), copper (copper-ethylenediamine complex, Komeen 9 50 and 200 g L -1 a.i., hereinafter referred to as K50 and K200), flumioxazin plus K50, flumioxazin plus K200, and the dipotassium salt of endothall (5000 g L -1 ). All data were normalized to the control and analyzed using non-linear regression (exponential decay) (PROC NLIN, SAS Institute 2002) and regression models were used to calculate ET 50 values for each herbicide treatment. Chlorophyll Content The effect of flumioxazin on chlorophyll content of hydrilla was determined using hydrilla collected from the Withlacoochee River near Dunnellon, FL in April and May 2007. Four 15 cm 9 Registered trademark of SePRO Corporation. Carmel, IN. 46032. 45

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sprigs were planted in each 10 x 10 x 12 cm (1 L) pot filled with masonry sand amended with Osmocote (15-9-12) fertilizer at a rate of 1g kg -1 soil. Two pots of hydrilla were placed in each 18.9 L bucket (28 cm diameter by 31 cm deep) filled with tap water (pH 7.5 at planting) under 70% sunlight. Hydrilla was allowed to acclimate for 4 and 3 wk for the initial and repeated study, respectively, at which time plants had reached the water surface and begun to branch. The pH ranged from 9.0 to 9.5 when flumioxazin was applied at 0, 50, 100, 200, 400, 800, and 1600 g L -1 This experiment was a randomized design with 3 replicates. Apical tips (ca. 2.5 cm, 0.09-0.11 g fresh weight) were collected from each treatment 1, 2, 3, and 4 DAT for chlorophyll analysis. Bleaching of hydrilla continued beyond 4 DAT, but no apical tips could be used for chlorophyll analysis as they had abscised the stems and were disintegrating. Excess water was removed by gentle blotting with paper towels, then tips were immediately weighed (fresh weight), placed in 20 mL scintillation vials, and frozen until chlorophyll analysis. Total chlorophyll was extracted by placing the apical tips in tubes containing dimethylsulfoxide (DMSO) (Hiscox and Israelstam 1979) in a water bath at 60 C for 6 h. Chlorophyll content was determined spectrophotometrically (Arnon 1949) and expressed as mg chlorophyll kg -1 of fresh weight. Non-linear regression (PROC NLIN, SAS Institute 2002) was used to determine the effect of flumioxazin on chlorophyll content. Results and Discussion Photosynthesis and pH All flumioxazin treatments of 100 to 800 g L -1 showed similar trends with respect to reduction in photosynthesis except 100 g L -1 at high pH (Figure 3-1) which only reduced photosynthesis by approximately 20% of the nontreated control plants 168 HAT. The amount of time required to reduce photosynthesis by 50% (ET 50 ) of the control plants was 737 h for flumioxazin applied at 100 g L -1 in the high pH solution (Table 3-1). All other treatments 46

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reduced photosynthesis by 50% of the nontreated control between 68 and 118 HAT (2 to 5 DAT). Flumioxazin applied at 800 g L -1 in low pH was different from 200 g L -1 in the high pH treatment and from 100 g L -1 in low pH treatment based on ET 50 ( 95% confidence interval) values. No other treatment differences were observed at low or high pH. Photosynthesis was reduced more rapidly as a function of flumioxazin concentration at both high and low pH. Net photosynthesis of nontreated control plants prior to herbicide treatment (0 HAT) was 174.6 16.9 (low pH) and 155.9 8.7 g O 2 g fresh weight -1 min -1 (mean standard error) (high pH), respectively, compared to 61.2 10.9 (low pH) and 65.3 10.4 g O 2 g fresh weight -1 min -1 (high pH) 168 HAT. The low and high pH nontreated control plants decreased in net photosynthesis by 64.9 and 58.1%, respectively. This gradual decline in net photosynthesis over the course of the experiment was possibly attributed to decline in NaHCO 3 and Hoaglands solution in the plastic cups in addition to stress caused by blotting them dry to obtain fresh weight. These data indicate a minimal pH effect on photosynthesis except for flumioxazin applied at 100 g L -1 to apical tips in high pH treatment solutions. Based upon these data, flumioxazin should be applied at rates higher than 100 g L -1 in high pH waters to over come the effects of rapid breakdown. Regardless of pH, flumioxazin at 400 and 800 g L -1 had similar ET 50 values and DO measurements at 168 HAT. The light quantity in this study (380 mol m -2 s -1 ) in combination with low pH or flumioxazin at rates above 100 g L -1 appears to be sufficient to reduce photosynthesis and kill apical tips exposed for 168 h. Apical hydrilla tips in this particular study began to bleach at ca. 48 HAT followed by whole tip disintegration within 120 HAT in all treatments except high pH 100 and 200 g L -1 treatments (data not shown). These data suggest that flumioxazin is rapidly absorbed into hydrilla despite the half-life being very 47

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short in high pH water (9.0) and is similar to the pH efficacy data (see Chapter 2). However, these studies provide evidence that flumioxazin concentration greater than 200 g L -1 may be required to overcome the rapid degradation when applied in high pH water. Flumioxazin does not halt hydrilla photosynthesis as quickly as other herbicides such as diquat. MacDonald et al. (2002) demonstrated diquat at 344 g L -1 decreased net photosynthesis in apical hydrilla tips by 44% 10 min after treatment and completely inhibited net photosynthesis 2 HAT. Although diquat quickly inhibits photosynthesis under controlled conditions, it is typically applied with copper to improve efficacy, due to difficulty of controlling hydrilla when applied alone (Sutton et al. 1970; Sutton et al. 1972; Anonymous 2003). Photosynthesis and Light The effect of 400 g L -1 flumioxazin on photosynthesis of apical hydrilla tips at low (20), medium (170), and high (400 mol m -2 s -1 ) light levels showed apical hydrilla tips treated at medium and high light levels followed a similar trend (Figure 3-2). The photosynthetic rates of hydrilla tips treated under high light conditions were different from those at the medium light by the conclusion of the study. Low light treated tips were still photosynthesizing at approximately 73% of the nontreated control plants by the conclusion of the study. The amount of time required by flumioxazin to reduce photosynthesis by 50% (ET 50 ) at medium light was not different from the low or high light quantities (Table 3-2). The high and low light quantities differed with respect to the calculated ET 50 values. Low light treated hydrilla would require an estimated 303 h to achieve a 50% reduction, while high light plants only required 99 h. Low light treated tips did not visually appear to be different from the low light nontreated control plants. There was no bleaching or chlorosis of the apical tips in either treatment. In addition, low light treated apical tips elongated more than 5 cm during the course of the study, whereas those treated at medium and high light quantities elongated less than 1 cm (data not shown). 48

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Low light conditions such as those used in this study are similar to those found at the mid-depths or bottom of water bodies infested with hydrilla. Previous research demonstrated that the light compensation point of hydrilla ranged from 7 to 20 mol m -2 s -1 (Van et al. 1976; Bowes et al. 1979; Steward 1991). The low light quantities in these studies were ample for hydrilla to produce O 2 but were not sufficient enough for flumioxazin to completely halt photosynthesis or bleach the apical tips. Conversely, the high light levels reduced photosynthesis to about 30% of the nontreated control plants by the conclusion of the study. Flumioxazin is a strong inhibitor of chlorophyll synthesis and prevents the transformation of protoporphyrinogen IX into protoporphyrin IX. (Aizawa and Brown 1999; Matringe et al. 1989; Cobb 1992). Protoporphyrinogen IX accumulates in plastids due to inhibition of the PPO enzyme and then diffuses through the plastid membrane into the cytosol, where it is oxidized to protoporphyrin IX by a plasma membrane-bound protox (Dayan and Duke 1997; Duke et al. 1991). Protoporphyrin IX reacts with light to produce toxic singlet oxygen radicals leading to lipid peroxidation and the destruction of cellular components (Duke et al. 1991; Gupta and Tripathy 2000). The low light treated plants did not exhibit bleaching within the course of these studies. However, chlorophyll turnover is continuously occurring as the average half-life of chlorophyll is relatively short (minutes to days) (Grumbach et al. 1978; Hendry and Stobart 1986); therefore, protoporphyrinogen IX should have been inhibited during the course of the experiment and result in bleaching of the apical tip. Apparently, chlorophyll turnover in hydrilla tips grown under low light was reduced, but not rapid enough to result in bleaching. Hydrilla infested lakes form dense canopies and restrict light penetration to the lower portions of the plant (Haller and Sutton 1975). This study provides a possible explanation for the 49

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observations noted at EUP treatment sites where the surface canopy of hydrilla was injured, the lower stems received minimal injury, and rapid regrowth occurred from the lower stem segments (see Chapter 2). These data provide evidence that low light levels hinder flumioxazin activity and explains why rapid regrowth occurs from apical tips growing under the hydrilla canopy. Effect of Contact Herbicides on Photosynthesis Flumioxazin applied alone to apical hydrilla tips at 400 g L -1 required 156 h to reduce photosynthesis by 50% (ET 50 ) of the nontreated control plants (Table 3-3). Copper applied alone at 50 g L -1 had minimal impact on photosynthesis of hydrilla, whereas copper at 200 g L -1 reduced DO by 50% within 35 HAT. Flumioxazin plus K200 reduced photosynthesis by 50% more rapidly than any other treatments evaluated; however, this increase in activity was minimal compared to K200 applied alone. The combination of flumioxazin and copper may be beneficial for control of surface matted hydrilla. Flumioxazin activity has been variable in EUP field research (see Chapter 2) and the combination of these products could aid in overcoming the rapid breakdown in high pH water. Copper alone has activity on submersed and floating aquatic weeds such as elodea (Elodea densis), coontail, giant salvinia (Salvinia molesta D. S. Mitchell), and duckweed (Angelo et al. 1998; Filbin and Hough 1979, Nelson et al. 2001; Ware 1966). In addition, copper has been used successfully in combination with diquat for hydrilla control (Sutton et al. 1972; Langeland et al. 2002) and overcomes diquat resistance in landoltia [Landoltia punctata (G. Meyer) D.H. Les and D.J. Crawford] (Koschnick and Haller 2006). Previous research has shown that flumioxazin at 1, 10, and 50 g L -1 decreased photosynthetic capacity of common duckweed (Lemna minor L.) by 23, 62, and 64%, respectively (Frankart et al. 2002). In these studies photosynthetic capacity of common duckweed was inhibited more when plants were exposed to 200 g L -1 of copper mixed with flumioxazin than all rates of flumioxazin applied alone. 50

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Photosynthesis in this study was only reduced to 60% of the nontreated control with 5000 g L -1 of endothall after a 96 h exposure. MacDonald et al. (2002) reported technical grade endothall acid applied at 372 mg L -1 stopped photosynthesis 120 min after treatment; however, this rate of endothall is approximately 75x higher than the maximum label rate of the dipotassium salt of endothall (Anonymous 2007a). Previous research has shown that this formulation of endothall kills various terrestrial and aquatic plants by inhibiting respiration (MacDonald et al. 1993), photosynthesis (Turgeon et al. 1972), lipid synthesis (Mann and Pu 1968), and protein synthesis (Mann et al. 1965) as well as causing cellular disruption (Keckemet 1968; Keckemet and Nelson 1968). According to the calculated photosynthesis ET 50 values, flumioxazin alone is slower than the dipotassium salt of endothall at reducing DO by 50%. Flumioxazin and endothall were applied at the maximum labeled rate compared to 5 and 20% of maximum labeled rate for copper at 50 and 200 g L -1 respectively. These data indicate that copper at 200g L -1 reduced hydrilla photosynthesis faster than flumioxazin or the dipotasium salt of endothall. The addition of 50 g L -1 copper to 400 g L -1 flumioxazin did not decrease the calculated ET 50 value from 400 g L -1 flumioxazin alone. However, the addition of 200 g L -1 copper to the flumioxazin did provide a faster reduction in photosynthesis. Chlorophyll Content Flumioxazin applied to hydrilla in a mesocosm study at rates of 50 to 1600 g L -1 showed that chlorophyll content was reduced as function of increased flumioxazin concentrations (Figure 3-3). Hydrilla chlorophyll content 3 and 4 d after the flumioxazin treatment reflected more of a flumioxazin rate response than 1 and 2 DAT, as indicated by the trend of the regression lines. These data demonstrate the ability of flumioxazin to rapidly bleach apical hydrilla tips due to the inhibition of protoporphyrinogen oxidase which is a precursor to chlorophyll (Aizawa and Brown 1999; Matringe et al. 1989; Cobb 1992). Hydrilla treated with flumioxazin in EUP trials 51

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(see Chapter 2) showed similar apical tip bleaching in the field within 1 to 3 DAT followed by reddening of the stem 5 cm below the apical tip. These results are similar to those observed in these studies. Some of the red coloration in treated plants was likely due to anthocyanin formation or destruction of other secondary chlorophyll protecting pigments which were visible because of chlorophyll inhibition or were produced in response to the stress of the herbicide treatment (Hrazdina 1982; Sandmann et al. 1991; Spencer and Ksander 1990). Susceptible terrestrial crops and weeds exposed to other PPO inhibiting herbicides rapidly show injury symptoms including chlorosis, leaf crinkling, and stunting followed by necrosis (Lovell et al. 2001; Wilcut et al. 2001; Vencill 2002). Hydrilla may grow as much as an inch per day (Langeland 1996) and new tissue and chlorophyll is being produced in the tip much more quickly than in the lower stem segments. PPO inhibitors would inhibit chlorophyll and consequently produce more toxic radicals in the tip region than in the more mature and shaded lower stem segments. Lower stem segments of plants treated with flumioxazin were not tested for chlorophyll content in these studies since flumioxazin primarily causes bleaching in the upper apical tip (1 to 3 cm) where chlorophyll turnover is occurring more rapidly. In these chlorophyll studies, flumioxazin was applied to hydrilla cultured in high pH water. Further studies could be conducted to evaluate the effects of flumioxazin on chlorophyll content in low pH water; however, regardless of pH, only the apical portion of the plant will bleach since chlorophyll is being produced more rapidly in this region and consequently flumioxazin toxicity symptoms are more pronounced. In conclusion, these data once again show flumioxazin is rate dependent up to 400 g L -1 when applied to hydrilla in high pH solutions. Most of the treatments applied to apical hydrilla tips reduced photosynthesis before the conclusion of the experiments. Approximately half of 52

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hydrillas biomass occurs in the upper 0.5 m of the water column when the plant forms a dense surface mat (Haller and Sutton 1975). Most of the apical tips can be found in the upper surface of these mats; hence, flumioxazin treatments tend to work exceptionally well on the upper canopy because of ample light. Treated apical tips growing >1.5 m from the surface under low light conditions at and near the lake bottom are bleached, but the lower stem sections typically dont become chlorotic and disintegrate in the same manner as the upper stem segments which receive high light intensity. Stem segments treated with flumioxazin responded by reduced photosynthesis but didnt disintegrate similar to the apical tips. The light study confirmed the lack of flumioxazin efficacy at light levels near hydrillas light compensation point which is typically found below hydrilla mats in greater water depths. These studies provide further evidence that pH is not the only factor influencing flumioxazin efficacy at 400 g L -1 Light quantity and tissue type can reduce efficacy in field treatments. If flumioxazin received a Section 3 label for aquatic use, treatments applied to high pH water (>9.0) may result in failure. Based on these data and EUP research, applications should be made early in the morning before the pH is cycled to above 9.0, early in the year, or before hydrilla forms dense canopies. Flumioxazin is most effective in rapidly growing tissue where chlorophyll turnover is high. Lower pH water results in a longer half-life and increases exposure of plants to higher flumioxazin concentrations for a longer period of time. Non-matted hydrilla allows more light to penetrate to the lower canopy, where flumioxazin will more injury to lower portions of the hydrilla plant. Thus, optimum conditions for aquatic weed control with flumioxazin included rapid growth, low pH, and high light intensity. 53

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Flumioxazin Exposure Time (h) 020406080100120140160180 020406080100120 high pH (9.0), 800 g L-1 low pH (6.0), 800 g L-1 020406080100120140160180 % of Nontreated Control 020406080100120 high pH (9.0), 400 g L-1 low pH (6.0), 400 g L-1 020406080100120140160180 020406080100120 high pH (9.0), 200 g L-1 low pH (6.0), 200 g L-1 020406080100120140160180 % of Nontreated Control 020406080100120 high pH (9.0), 100 g L-1 low pH (6.0), 100 g L-1 Flumioxazin Exposure Time (h) Figure 3-1. The effect of flumioxazin rate at high (9.0) and low pH (6.0) on photosynthesis of apical hydrilla tips cultured in a growth chamber for 168 h at 380 mol m -2 s -1 of light quantity. Data are normalized to the control at each respective pH and shown as means standard error (n=8). 54

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Table 3-1. The effect of flumioxazin on photosynthesis of apical hydrilla tips at high (9.0) and low (6.0) pH a pH 9.0 b ET 50 c (95% CI d ) Regression equation r 2 100 737 (315-2166) y = 91.3356e-0.000940x 0.93 200 118 (90-175) y = 87.9257e-0.00585x 0.88 400 87 (61-148) y = 90.3356e-0.00801x 0.75 800 81 (57-144) y = 92.0237e-0.00852x 0.71 pH 6.0 e 100 123 (89-197) y = 92.3011e-0.00564x 0.85 200 92 (70-134) y = 108.8e-0.00753x 0.85 400 69 (55-91) y = 94.0648e-0.0101x 0.86 800 68 (56-87) y = 113.4e-0.0102x 0.89 a Hydrilla cultured in a growth chamber for 168 h at 380 mol m -2 s -1 of light quantity at high and low pH a Data are normalized to the control at each respective pH. b High pH: 9.0. c Effective time 50: ET 50 = time required by flumioxazin (h) to reduce hydrilla photosynthesis by 50%. Each value is a mean of two experiments with a total of 8 replications. d 95% CI = 95% Confidence Interval. e Low pH: 6.0, pH reduced with muriatic acid. 55

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Flumioxazin Exposure Time (h) 020406080100120140160180 % of Nontreated Control 020406080100120 Low Light Med Light High Light Figure 3-2. The effect of flumioxazin (400g L -1 ) at pH 9.0 on photosynthesis of apical hydrilla tips cultured in a growth chamber for 96 h at low (20 mol m 2 s 1 ), medium (170 mol m 2 s 1 ), and high light (400 mol m 2 s 1 ) quantity levels. Data are normalized to the control at each respective light quantity and shown as means standard error (n=10). 56

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Table 3-2. The effect of flumioxazin (400 g L -1 a.i.) at pH 9.0 on photosynthesis of apical hydrilla tips cultured in a growth chamber for 168 h at low, medium, and high light quantities a Light Quantity b ET 50 c (95% CI d ) Regression equation r 2 Low 303 (198-648) y = 95.4156e-0.00229x 0.92 Medium 140 (105-209) y = 95.9852e-0.00495x 0.88 High 99 (81-130) y = 102.4e-0.00697x 0.90 a Data are normalized to the control at each respective light quantity. b Low: 20mol m 2 s 1 ; Medium 170mol m 2 s 1 ; High 400mol m 2 s 1 c Effective time 50: ET 50 = time required by flumioxazin (h) to reduce hydrilla photosynthesis by 50%. Each value is a mean of two experiments with a total of 10 replications (sprigs). d 95% CI = 95% Confidence Interval. Table 3-3. The effect of select contact herbicides at pH 9.0 on photosynthesis of apical hydrilla tips cultured in a growth chamber for 96 h at 380 mol m -2 s -1a light quantity. Herbicide b % Label rate ET 50 c (95% CI d ) Regression equation r 2 Flumi 400 e 100 156 (117-233) y = 88.9311e-0.00444x 0.99 K50 5 1568 (291-6931) y = 103.8e-0.000442x 0.98 K200 20 35 (26-53) y = 93.2636e-0.0198x 0.92 Flumi 400 + K50 100 + 5 126 (100-171) y = 93.0193e-0.00549x 0.99 Flumi 400 + K200 100 + 20 20 (17-25) y = 96.3408e-0.0340x 0.97 Endothall 5000 100 71 (62-82) y = 101.4e-0.00978x 0.98 a Data are normalized to the control. b Herbicide rate: g L -1 a.i. c Effective time 50: ET 50 = time required by the herbicide (h) to reduce hydrilla photosynthesis by 50%. Each value is a mean of two experiments with a total of 10 replications (sprigs). d 95% CI = 95% Confidence Interval. e Abbreviations: Flumi, flumioxazin; K, Komeen (copper chelate); endothall = dipotasium salt. 57

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Flumioxazin Concentration (g L-1 a.i.) 02004006008001000120014001600 Chlorophyll (mg kg-1 fresh weight) 0.10.20.30.4 1 DAT 2 DAT 3 DAT 4 DAT Figure 3-3. The effect of flumioxazin concentration on hydrilla chlorophyll content (mg kg -1 fresh weight) 1 to 4 d after treatment (DAT) under 70% sunlight. Flumioxazin was applied as a single application to hydrilla cultured in 18.9 L buckets filled with tap water (pH 9.0-9.5). Data are shown as actual means standard error (n=6). 58

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CHAPTER 4 THE EFFECT OF FLUMIOXAZIN ON SUBMERSED, EMERGENT, AND FLOATING AQUATIC PLANT PLANTS Introduction One of the primary goals of aquatic weed control in public and private waters is to eliminate invasive plants while maintaining a diversity of native submersed and emergent species. Native aquatic plants may improve water clarity and quality, provide valuable fish and wildlife habitat, reduce rates of sediment resuspension, and help prevent the spread of invasive plants (Savino and Stein 1982; Heitmeyer and Vohs 1984; Smart 1995; Dibble et al. 1996b). Hydrilla is a submersed aquatic fresh water angiosperm that is considered native to Asia or Africa and has become a serious weed problem in the United States and many other countries (Cook 1985; Haller and Sutton 1975; USDA 2007; Van and Vandiver 1992). Once established in a body of water, hydrilla readily dominates and replaces native submersed species by forming a canopy that reduces light penetration, increases surface water temperature, and reduces dissolved oxygen (Bowes et al. 1979; Haller and Sutton 1975; Honnell et al. 1993). Floating aquatic plants such as water hyacinth, water lettuce, and duckweed form dense, free floating mats which can interfere with navigation, hydroelectric generation, and irrigation (Harley et al. 1984). They may also harbor mosquitoes, which are vectors for diseases like malaria and encephalitis (Holm et al. 1977). Selective removal of hydrilla and other invasive species with aquatic herbicides is beneficial for retention of native vegetation. Native plant density and diversity can increase considerably if canopy forming exotic plants are removed (Getsinger et al. 1997). Non-target 59

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damage to native species can result from submersed and foliar applications of herbicides and is a consideration in herbicide selection 10 Flumioxazin is being evaluated by Valent U.S.A. Corporation for control of aquatic weeds. The high costs associated with registering an herbicide for a new market (i.e., aquatics) may be overcome by maximizing the market potential. Therefore, the first objective of this research was to determine if flumioxazin has utility as a foliar and submersed treatment to control floating aquatic weeds. Both submersed and emergent non-target aquatic plants could be impacted by flumioxazin applications, so the second objective of these studies was to quantify the effects of foliar and submersed flumioxazin treatments on submersed and emergent aquatic plants. Materials and Methods Floating Aquatic Plants Water hyacinth and water lettuce were collected from Rodman Reservoir near Interlachen, FL and established in 95 L HDPE tubs filled with 80 L of tap water (pH 8.0) in April 2006 at the University of Florida Center for Aquatic and Invasive Plants in Gainesville, FL. Tap water was supplemented with 1 mL of chelated iron 11 12-0-0 plus Miracle-Gro 12 (150 mg L -1 ) prior to herbicide treatment and added again at 1 and 2 wk after treatment. Water hyacinth (5 plants per tub) and water lettuce (20 plants per tub) were allowed to acclimate for 3 wk before treatment. This study was repeated in August 2006 near the University of Florida in Gainesville, FL with water from Bivens Arm Lake (pH 7.5). Both studies were completely randomized designs with 4 replications (tubs). 10 W. T. Haller. 2007. Personal Communication. 11 Lesco, Inc. Cleveland, OH. 44114. 12 The Scotts Company. Marysville, OH. 60

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Flumioxazin was applied to water lettuce and water hyacinth as a foliar treatment using a forced air CO 2 -powered sprayer at an equivalent of 935 L ha -1 diluent delivered through a single TeeJet 13 80-0067 nozzle at 10 psi. Flumioxazin was applied at 0, 36, 72, 143, 286, 572, and 1144 g ha -1 plus a non-ionic surfactant (0.25% v/v). An additional study was conducted concurrently to determine the effects of submersed flumioxazin treatment on these floating invasive species with collection and setup procedures as described above for foliar treatments. Water hyacinth and water lettuce were treated with flumioxazin as a submersed treatment at 0, 100, 200, 400, 800, and 1600 g L -1 All studies were conducted under full sunlight. All live water hyacinth and water lettuce biomass was harvested 34 DAT, placed in a drying oven at 90 C for ca. 1 wk, and then weighed. Plant dry weight data were analyzed using non-linear regression (PROC NLIN, SAS Institute 2002). Regression models were used to determine the effective concentration 50 (EC 50 ), which is the concentration of flumioxazin that is required to cause a 50% reduction in dry weight compared to control plants. A population of landoltia was collected from a pond with no history of herbicide treatments in Alachua County, FL and maintained in 266 L fiberglass tanks in a greenhouse (70% sunlight). The landoltia was cultured in tap water (pH 8.2) amended with topsoil and Miracle-Gro in a greenhouse (1200 mol m -2 s -1 ). Plants were treated with carbaryl insecticide 14 weekly and allowed to acclimate in the tanks for 2 wk before treatment. A 10 g aliquot (fresh weight) (1.3 0.07 g dry weight) of landoltia was placed in each 3 L HDPE (17.1 cm diameter by 13.3 cm deep) pot filled with tap water (pH 8.0). Plants were allowed to acclimate in the pots for 2 d prior to herbicide treatment. All pots were amended with the same Miracle-Gro rate as 13 TeeJet Technologies. Wheaton, IL 60189. 14 Sevin insecticide label. Bayer CropScience. Research Triangle Park, NC 27709. 61

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the previous studies 2 and 14 DAT. Landoltia was treated with a submersed treatment of flumioxazin at 0, 10, 25, 50, 100, 200, 400, 800, and 1600 g L -1 As a comparison treatment, diquat was applied as a foliar treatment at 1.1 kg ha -1 a.i. using the described methods for foliar flumioxazin treatments. The initial experiment was conducted in April 2007 and was repeated in May 2007. This experiment was a randomized design with 5 replicates. Due to the difficulty of removing large quantities of bleached and dead landoltia plants, visual estimates of control (% control) were determined on a scale of 0 to 100%, where 0 = no chlorosis/necrosis and 100 = plant death. Percent control ratings were based on nontreated control plants. There were no differences in control between the two experiments (Fishers Protected LSD, p0.05); therefore, the data were pooled for analysis and means were separated using 95% confidence intervals. Foliar flumioxazin treatments were also applied to landoltia in October 2005, April 2007, and May 2007. Flumioxazin was applied to landoltia at 0, 36, 72, 143, 286, 572, and 1144 g ha -1 plus a non-ionic surfactant (0.25% v/v) using the equipment described in the water lettuce and water hyacinth study. Submersed Aquatic Plants The submersed aquatic plants coontail, egeria (Egeria densa Planch.), hydrilla, southern naiad, and vallisneria were evaluated for sensitivity to flumioxazin at a high (9.0) and low (7.0) pH in 2006 and 2007. Hydrilla was collected from Rodman Reservoir near Interlachen, FL in July, September, and December 2006, while all other species were purchased from a local plant nursery. The high pH study was only conducted once (August 2006); the low pH study was conducted in September 2006 and repeated in January 2007. The high pH and initial low pH experiments were conducted in a shade house (70% sunlight), whereas the repeated low pH experiment was conducted in a greenhouse with 70% sunlight. Two sprigs of each species were 62

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planted in each 10 by 10 by 12 cm (1 L) pot which were filled with masonry sand amended with Osmocote (15-9-12) fertilizer at a rate of 1g kg -1 soil and placed in 95 L HDPE tubs filled with tap water (pH 7.5 at planting). Each tub contained all five species (2 pots/species/tub). Plants were allowed to acclimate for 2 wk prior to herbicide application. This experiment was a randomized design with 4 replications (tubs). Flumioxazin was applied as a submersed treatment at 0, 50, 100, 200, 400, 800, and 1600 g L -1 in high and low pH water. Water pH in all tubs was 9.0 prior to treatment so each low pH treatment tub was treated with ca. 15 mL of muriatic acid to lower pH to 7.0. The pH was monitored to determine if and when the pH would return to pH 9.0. Tub pH was not maintained at 7 since pH beyond 24 HAT was not relevant in this study since flumioxazin is taken up within minutes to hours after treatment (see Chapter 2 and 3). All living plant tissue was harvested at the soil line 28 DAT, placed in a drying oven at 90 C for ca. 1 wk and weighed. Plant dry weight data were analyzed using non-linear regression (PROC NLIN, SAS Institute 2002). Regression models were used to determine the effective concentration 50 (EC 50 ), which is the concentration of flumioxazin that is required to cause a 50% reduction in dry weight compared to control plants. Data from both low pH experiments were pooled for coontail, egeria, and hydrilla because there was no difference between the slopes of regression lines for both experiments at the 95% confidence interval level. There were notable differences in the pre-treatment dry weight and growth of naiad and vallisneria between the initial and repeated study; plants in the second study (January) did not grow as rapidly as those in the first study (September), so data from these experiments were analyzed separately by species. 63

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Emergent Aquatic Plants The sensitivity of the emergent aquatic plants eleocharis (Eleocharis interstincta (Vahl) Roemer & J.A. Schultes ), maidencane, pickerelweed, and sagittaria were evaluated in submersed and foliar flumioxazin treatments. All plants were purchased from a local plant nursery in August 2006 and April 2007 for the submersed and foliar studies, respectively. Two sprigs of each species were planted in a mixture of 2:1 potting soil:masonry sand in 3 L HDPE pots (17.1 cm diameter by 13.3 cm deep) amended with Osmocote (15-9-12) fertilizer at a rate of 1g kg -1 soil. The submersed flumioxazin experiment was a randomized design with five replicates (tubs). One pot of each species was grown for 4 wk in 95 L HDPE tubs placed in a shade house (70% sunlight) for the submersed flumioxazin study. Water level in the tubs was maintained at 25 cm, and pH remained at or near 7.5. Plants were grown for 1 mo when flumioxazin was applied at 0, 50, 100, 200, 400, 800, and 1600 g L -1 as a submersed treatment. Prior to treatment with a foliar application (1 mo after planting), all 5 emergent replicates (pots) were placed in one 266 L fiberglass tank (72 cm by 82 cm by 45 cm) for a total of 7 treatments (tanks). Flumioxazin was applied at 0, 36, 72, 143, 286, 572, and 1144 g ha -1 a.i. plus a non-ion surfactant (0.25% v/v) as a foliar treatment. Plants were treated using a forced air CO 2 -powered sprayer at an equivalent of 935 L ha -1 diluent and delivered through a single TeeJet 15 80-0067 nozzle at 10 psi. Water level was maintained at 25 cm with a drain pipe and water was exchanged in the tanks for 10 min after each foliar treatment and again 24 h after treatment to ensure no herbicide would bind to the soil or remain in the water column for underwater uptake. 15 TeeJet Technologies. Wheaton, IL 60189. 64

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Plants were harvested 40 DAT and plant height from the soil surface to the tip of the tallest leaf was recorded. All emergent plants were harvested in the manner described for submersed plants with plant height and dry weight subjected to the same statistical procedures. Data were pooled across experimental runs when no statistical difference between the slopes of regression lines were observed. Results and Discussion Floating Aquatic Plants Water lettuce was more sensitive to foliar applications of flumioxazin at 36 to 1144 g ha -1 than water hyacinth as indicated by calculated EC 50 values of 69 and 1435 g ha -1 respectively (Figure 4-1). Flumioxazin applied as a foliar treatment at rates greater than 143 g ha -1 resulted in complete control of water lettuce. Treated water lettuce plants exhibited chlorosis and necrosis on the leaves (3 to 5 DAT) and defoliation (12 to 15 DAT); also, plants completely decayed at herbicide rates 286 g ha -1 21 DAT. Sublethal rates resulted in regrowth of young plants (ramets) from the meristematic region of water lettuce. The highest flumioxazin rate evaluated (1144 g ha -1 ) reduced water hyacinth biomass by only 41% of the nontreated control 34 DAT. Treated water hyacinth plants exhibited blackening on younger leaves only, which is similar to injury symptoms reported in water lettuce and water hyacinth treated with the PPO inhibitor carfentrazone (Koschnick et al. 2004). Water lettuce treated with a submersed application of flumioxazin was controlled at all concentrations evaluated in this study (data not shown) and these results suggest that water lettuce is more sensitive to flumioxazin applied in the water than applied to the foliage. In contrast, flumioxazin did not reduce water hyacinth biomass by more than 30% of the nontreated control plants at any rate evaluated in this study and confirms that water hyacinth is more 65

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tolerant to flumioxazin than water lettuce (data not shown), similar to results with the PPO inhibitor carfentrazone (Koschnick et al. 2004). Water hyacinth plants treated in these studies were large and mature (45 8 cm in height) and may have been more tolerant of foliar treatments than smaller and immature plants; therefore, younger, more actively growing plants (10 3 cm) were also tested for flumioxazin sensitivity, but these immature plants did not respond differently to treatments (data not shown). Immature and mature water hyacinths treated with foliar flumioxazin treatments in field EUP trials (data not shown) have shown tolerance levels similar to those noted in these studies. The effects of a submersed application of flumioxazin from 10 to 1600 g L -1 are presented for landoltia in Figure 4-2. Most flumioxazin treatments caused foliar bleaching within 7 to 10 DAT, but none of the treatments resulted in complete control of landoltia. Each flumioxazin treatment was different as indicated by no overlapping of 95% confidence interval bars. Landoltia colonies treated at rates above 25 g L -1 began to separate and roots became detached from individual fronds. Koschnick (2005) found landoltia treated in the dark with diquat underwent root detachment without chlorosis. The primary function of roots of plants in the Lemnaceae family is stabilization of fronds (Landolt 1986). Diquat applied as a comparison treatment resulted in 100% control less than 5 DAT when applied at 1.1 kg ha -1 as a foliar treatment. Duckweed is extremely sensitive to diquat with an EC 50 of 4 g L -1 (Peterson et al. 1997) and is the current industry standard for duckweed control. The foliar applied flumioxazin landoltia study was conducted 3 times; treated plants were visually similar to control plants at all rates showing no dose response and therefore were not harvested (data not shown). The foliar treatment to landoltia and water hyacinth was unsuccessful and high foliar rates were needed to control water lettuce. These results suggest 66

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that flumioxazin uptake is limited by the leaf cuticle or occurs primarily through the underside or roots of the plant. Koschnick et al. (2004) reported that the calculated EC 50 for a foliar application of carfentrazone to reduce water lettuce was 8 to 10 g ha -1 approximately 7 to 9 times less than flumioxazins calculated EC 50 for water lettuce. Higher concentrations (30 and 35 g ha -1 ) of carfentrazone were required to control water hyacinth (Koschnick et al. 2004), but flumioxazin in our study failed to reduce water hyacinth biomass by more than 30%. Results from these studies show that roots or the underside of the plant is effective at absorbing flumioxazin when applied as a submersed treatment. If registered as an aquatic herbicide, flumioxazin will likely be used primarily as a treatment for submersed weed control. Although most floating aquatic weeds are controlled by foliar herbicide applications, these results indicate that water lettuce and landoltia may be controlled with submersed treatments. Diquat and 2,4-D are the primary aquatic herbicides used for control of water lettuce, water hyacinth and duckweed (Westerdahl and Getsinger 1988; Langeland et al. 2002) and are more efficacious on water lettuce, water hyacinth, and duckweed as foliar treatments. Flumioxazin in these studies provided control only at higher application rates (water lettuce) or provided <30% control (water hyacinth and landoltia). Further research is needed to determine if flumioxazin as a submersed treatment is as efficacious to water lettuce in higher pH water (9.0). Efficacy data for flumioxazin is also needed on additional floating species such as Salvina spp. and Wolffia spp. Additionally, the effect of flumioxazin used in conjunction with surfactants should be examined in both immature and mature water hyacinths to determine whether surfactants increase flumioxazin uptake and activity in these species. 67

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Submersed Aquatic Plants Water pH in control tanks returned to 9.0 within 24 HAT. The pH of water in the 100, 200, and 400 g L -1 low pH treatments returned to 9.0 ca. 36 HAT, while pH of water in the two highest herbicide rates (800 and 1600 g L -1 ) never exceeded 8.5. Flumioxazin applied in low (7.0) pH water at 50 to 1600 g L -1 caused greater injury to coontail, hydrilla, naiad, and vallisneria than flumioxazin applied in high pH (9.0) water (Figure 4-3). All species with the exception of egeria were more tolerant to flumioxazin applied in high pH water compared to those in low pH water according to the calculated EC 50 values for dry weight (Table 4-1). Coontail treated in high pH water was the only susceptible species to flumioxazin at the current EUP label rate of 400 g L -1 All other species treated with flumioxazin in high pH water would require an estimated flumioxazin concentration of >3194 g L -1 to reduce biomass by 50%. The pH of Florida lakes infested with surface matted hydrilla may be greater than 8.0 and likely in excess of 9.0 as a result of the hydrilla utilizing free CO 2 and HCO 3 during daily photosynthesis (Van et al. 1976), so egeria, naiad, and vallisneria would be only slightly injured from flumioxazin treatment in higher pH water based on these data. However, hydrilla was less affected by the treatment in high pH water, in contrast to observations from the general efficacy study (see Chapter 2). Hydrilla in this study responded immediately to flumioxazin exposure by bleaching and undergoing rapid decline, but new apical tips sprouted <1 WAT from treated tissue and flumioxazin application at 1600 g L -1 reduced dry weight by only ca. 40% of the nontreated control plants. These data and the pond efficacy data outlined in Chapter 2 indicate flumioxazin produces highly variable results when applied to hydrilla growing in high pH water. Coontail, hydrilla, and naiad in were highly susceptible to flumioxazin in low pH water and would likely be injured or controlled at the proposed label rate of 400 g L -1 (Table 4-1). 68

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The calculated EC 50 value for naiad in experiment 1 was 10x greater than the EC 50 in the repeated experiment (517 vs. 51, respectively), but flumioxazin at 400 g L -1 would still significantly reduce biomass. In comparison to these data, previous research has shown that coontail is sensitive to the contact herbicide dipotasium salt of endothall at concentrations as low as 0.5 mg L -1 a.i. (Hofstra and Clayton 2001) and endothall has been shown to reduce vallisneria biomass at concentrations greater than 0.5 mg L -1 but plants recovered 8 WAT (Skogerboe and Getsinger 2002). In this study, egeria and vallisneria from the 2nd low pH experiment were not affected by flumioxazin; also, egeria was the only plant unaffected by flumioxazin treatments at either pH. Most plants exposed to flumioxazin in the high and some in the low pH treatments, with the exception of coontail, were beginning to regrow prior to harvest. Similarly, many of the non-target species treated with flumioxazin in EUP ponds recovered within a few weeks after treatment (data not shown). Non-target submersed aquatic species will only be exposed to flumioxazin for short exposures due to the rapid degradation of this herbicide (Katagi 2003), especially when applied to water with a pH >8.0. Those species with marginal tolerance should be able to overcome a flumioxazin treatment in high pH water since flumioxazin will not be present in the water for more than a couple of days. In contrast, non-target plants exposed to herbicides with a longer half-life, such as fluridone or penoxsulam, have the potential of being severely injured or killed because of longer exposures (Koschnick et al. 2007; Langeland and Warner 1986). Development and use of herbicides that selectivity control non-target aquatic plants is a priority of most state agencies involved in aquatic weed management (Anonymous 2007b; Koschnick et al. 2007). 69

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Symptomology of hydrilla treated with flumioxazin in previous EUP studies (see Chapters 2 and 3) was bleaching in the apical tip and reddening in the stem followed by chlorosis and necrosis. Visual symptoms of other flumioxazin-treated plants in these studies included bleached apical tips followed by reddening of the stem (egeria), defoliation and darkening of tissue (naiad), defoliation and loss of stem/leaf integrity (coontail), and transparent appearing leaves (possibly due to loss of chlorophyll) (vallisneria). Plants treated with flumioxazin at pH 7.0 were glossy and darker green in color and were treated with muriatic acid to maintain pH <7.0 for 24 HAT but control plants were not discolored, so muriatic acid did not contribute any injury symptoms. Flumioxazin is hydrolyzed at a much slower rate in lower pH water (Vencill 2002) having a half-life of 16.1 h at pH 7.0 compared to 17.5 min at pH 9.0 (Katagi 2003), so the increased exposure time of these submersed aquatic plants to flumioxazin at pH 7.0 resulted in greater injury and reduction in biomass to all species except egeria. These data clearly show flumioxazin in high pH water is more selective with regard to non-target plant injury than flumioxazin in low pH water. Emergent Aquatic Plants Emergent aquatic plants had varying levels of sensitivity to flumioxazin concentrations 800 g L -1 ; however, there were minimal differences among plant species at concentrations of 1600 g L -1 (Figure 4-5). Sagittaria dry weight was reduced by 100% at the highest concentration compared to a 73 to 83% dry weight reduction in all other emergent plants. Sagittaria was the most sensitive species followed by maidencane, eleocharis, and pickerelweed based on calculated EC 50 values for dry weight and height (Table 4-2). Elocharis and pickerelweed were more tolerant of a submersed flumioxazin application than sagittaria and maidencane, but these species would likely be injured by flumioxazin at 400 g L -1 Although dry weight data is often a more reliable indicator to assess herbicide effectiveness, plant height 70

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data in this study provided additional evidence of the selectivity of submersed flumioxazin treatments on emergent plants. Visual injury symptoms observed 2 WAT included interveinal chlorosis (sagittaria and pickerelweed), reddening on leaf margins (maidencane), and minor chlorosis (eleocharis). Flumioxazin and other protox-inhibiting herbicides are absorbed primarily by plant roots with some absorbance in the shoots, but translocation is limited once herbicides are absorbed into foliar tissue (Fadayomi and Warren 1977; Ritter and Coble 1981; Unland et al. 1999; Vencill 2002). Pots without holes were used in these studies and few roots were visible above the soil line, so herbicide uptake directly from the root zone was unlikely as flumioxazin was mixed directly into the water column. Flumioxazin uptake occurred either through the underwater stem or submersed leaves. Previous research found little flumioxazin translocation in plants, but submersed treatment of emergent aquatic plants in this study suggested movement of flumioxazin from the soil or lower stem into the leaves. If translocation of flumioxazin was limited, this herbicide should have girdled the plant at the soil line and produced injury symptoms such as necrosis throughout the stem and leaves without veinal chlorosis first appearing in the leaves. Ferrell et al. (2007a) showed flumioxazin + MSMA (monosodium salt of MAA) resulted in a 94% yield reduction when applied as a high post-direct treatment to 20 cm tall cotton. Symptomology of flumioxazin treated cotton included necrotic lesions on leaves, reddening stems, stem girdling, and eventual lodging. Previous research also found that as cotton matures, plants become more tolerant to flumioxazin because of greater bark development and metabolic capacity (Ferrell and Vencill 2003b). Foliar flumioxazin treatments were less injurious to emergent aquatic plants than submersed treatments (Figure 4-5). Maidencane and sagittaria would require foliar application 71

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rates greater than 1320 and 6478 g ha -1 to reduce dry weight and height by 50% (EC 50 ), respectively (Table 4-3). An EC 50 value could not be calculated for dry weight and height for both eleocharis and pickerelweed since increased flumioxazin concentrations resulted in an increase in dry weight (positive regression slope). Postemergent applications of flumioxazin are generally recommended for actively growing weeds less than 5 cm in height (Anonymous 2006), so the minimal foliar injury and substantial lack of reduction in biomass observed in this study to eleocharis, maidencane, pickerelweed, and sagittaria were probably due to the maturity of these plants. Injury symptoms (including chlorotic and necrotic lesions on the leaves) were similar to those described for other protox-inhibiting herbicides (Peterson et al. 2001). Tolerant species have reduced or no symptoms, whereas the leaves of susceptible species rapidly desiccate and die (Peterson et al. 2001). Selective control of invasive weed species has always been a goal of aquatic weed managers. Herbicide applicators target specific non-native plants through the use of specifically formulated herbicides, seasonally timed herbicide application, and/or preemptive spot treatments before weeds become a problem (Cervone and Schardt 2003). Flumioxazin provided selective weed control when applied as a foliar treatment or selectivity could be attributed to the maturity of these plants. In contrast, submersed applications resulted in more injury to nontarget plants especially when treatments were made in low pH water. 2,4-D selectively controls broadleaf weed species (Cervone and Schardt 2003), whereas fluridone selectivity is based on initial treatment rate, length of exposure, and initial plant biomass (Netherland et al. 2007). These data indicate that coontail, sagittaria, and maidencane are adversely affected by flumioxazin at concentrations <400 g L -1 in water with a pH of 7.0. Most emergent and submersed plants evaluated in these studies could be injured or killed if flumioxazin is applied to 72

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low pH water (<7.5). These studies provided worst-case scenarios where emergent and submersed plants were continuously exposed to flumioxazin. Herbicide concentrations in lakes are influenced by factors such as wind, flow, dilution, and pH, which minimize direct contact of native plants with herbicides such as flumioxazin when applied as a submersed contact application. Direct foliar applications to these native emergent plants would occur if they grow among targeted emergent or floating plants. Most of these non-target emergent plants will be minimally affected by the maximum proposed foliar label rate 16 of 286 g ha -1 a.i. Submersed aquatic plants are often found among or near hydrilla infestations, whereas emergent plants usually grow along the shoreline. Whole lake treatments with flumioxazin are unlikely since this product will be used primarily as a contact herbicide for submersed weed control. Partial lake treatments allow for dilution of the herbicide to further reduce exposure of non-target plants. Although several submersed and emergent species were evaluated for sensitivity to flumioxazin, further research should be conducted to determine the sensitivity of other non-target and invasive plants at various water pHs. All emergent plants in this study were mature and consequently more tolerant of both foliar and submersed flumioxazin treatments; however, most flumioxazin treatments would likely occur in early spring or summer when hydrilla can be controlled more easily due to rapid growth, lower pH, and less hydrilla biomass. Most emergent and submersed non-target plants will be immature and actively growing and could possibly be injured by foliar and submersed flumioxazin treatments. 16 M. S. Riffle. 2007. Personal Communication. 73

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Flumioxazin Concentration (g ha-1 a.i.) 020040060080010001200 Mean Dry Weight (kg/tub) 020406080100 Water Hyacinth: y = 86.0893-0.000483, r2 = 0.95EC50 = 1435 g ha-1 a.i. Water Lettuce: y = 67.2077-0.00998x, r2 = 0.92EC50 = 69 g ha-1 a.i. Figure 4-1. The effect of a foliar flumioxazin application (g ha -1 a.i.) on water lettuce and water lettuce dry weight 34 d after treatment under 100% sunlight. Flumioxazin was applied as a single application by a CO 2 -powered sprayer at an equivalent of 379 L ha -1 diluent with a non-ionic surfactant (0.25% v/v) to water lettuce and water hyacinth grown in 95 L tubs (pH 7.5 to 8.0). Data are shown as dry weight means standard error (n=10). EC 50 = effective concentration 50, concentration of flumioxazin (g ha -1 a.i.) that is required to reduce water lettuce and water lettuce biomass by 50%. 74

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Herbicide Rate 1.11025501002004008001600 % Control 020406080100 Diquat (kg ha-1 a.i.) Flumioxazin (mg L-1 a.i.) Figure 4-2. Percent control (visual) of landoltia 21 d after a foliar diquat (g ha -1 a.i.) and submersed flumioxazin application (g L -1 a.i.). Diquat and flumioxazin each applied as a single application to landoltia cultured in 1 L pots (water pH 8.0) under 70% sunlight. Diquat was applied by a CO 2 -powered sprayer at an equivalent of 379 L ha -1 diluent with a non-ionic surfactant (0.25% v/v). Percent control 95% confidence interval (CI) (n=10). Overlapping CI bars indicate no significant difference. 75

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024681012 Coontail High pH Coontail Low pH Egeria High pH Egeria Low pH Mean Dry Weight (g/pot) 024681012 Hydrilla High pH Hydrilla Low pH Flumioxazin Concentration (g L-1 a.i.) 02004006008001000120014001600 Naiad High pH Naiad Low pH Experiment 1 Naiad Low pH Experiment 2 Flumioxazin Concentration (g L-1 a.i.) 02004006008001000120014001600 024681012 Vallisneria High pH Vallisneria Low pH Experiment 1 Vallisneria Low pH Experiment 2 Figure 4-3. The effect of flumioxazin concentration on the dry weight of submersed aquatic plants 28 d after exposure. Flumioxazin applied as a single application to submersed aquatic species cultured in low (7.0) and high (9.0) pH water in 95 L tubs under 70% sunlight. Data are shown as actual dry weight means standard error (n=10 for low pH, except for naiad and vallisneria n=5; n=5 for high pH). Dry weight means standard error (n=10). 76

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Table 4-1. The effect of a single submersed flumioxazin application on dry weight of submersed aquatic plants 28 d after exposure a High pH b EC 50 c (95% CI d ) Regression equation r 2 Coontail 403 (248-1081) y = 7.9148e-0.00172x 0.86 Egeria 3747 (1720-23104) y = 2.6419e-0.000185x 0.94 Hydrilla 3194 (869-6931) y = 4.0822e-0.000351x 0.83 Naiad NA e y = 4.9646e0.0002x 0.90 Vallisneria 5172 (2173-13863) y = 3.6688e-0.000134x 0.95 Low pH f Coontail 34 (27-46) y = 9.6997e-0.0204x 0.87 Egeria 3285 (1925-11179) y = 2.8606e-0.000211x 0.94 Hydrilla 77 (53-138) y = 3.8329e-0.00902x 0.86 Naiad #1 g 517 (338-1093) y = 1.6128e-0.00134x 0.90 Naiad #2 51 (30-204) y = 4.1424e-0.0133x 0.64 Vallisneria #1 853 (533-2120) y = 3.1724e-0.000813x 0.90 Vallisneria #2 3536 (1270-4621) y = 1.4483e-0.000196x 0.86 a Submersed aquatic species cultured under 70% sunlight. b High pH: 9.0. c Effective concentration 50: EC 50 = concentration of flumioxazin (g L -1 a.i.) in water required to reduce plant dry weight by 50%. Each value is a mean of two experiments with a total of 5 replications (pots) for high pH; 10 reps for coontail, egeria, and hydrilla at low pH; and 5 reps for naiad #1, naiad # 2, vallisneria #1, and vallisneria #2 at low pH. d 95% CI = 95% Confidence Interval. e NA = not applicable due to positive regression slope. f Low pH: 7.0, pH reduced with muriatic acid. g #1 and #2: experiment 1 and experiment 2. 77

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Flumioxazin Concentrationg L-1 a.i.) 02004006008001000120014001600 Dry Weight (g/pot) 0246810121416 Eleocharis Maidencane Pickerelweed Sagittaria Figure 4-4. The effect of flumioxazin concentration on dry weight of emergent aquatic plants 40 d after exposure. Flumioxazin applied as a single application to emergent aquatic species cultured in 95 L tubs (pH 7.5) under 70% sunlight. Data are shown as actual dry weight means standard error (n=5). 78

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Table 4-2. The effect of a single submersed flumioxazin application on dry weight of emergent aquatic species 40 d after exposurea. Dry Weight EC 50 b (95% CIc) Regression equation r2 Eleocharis 559 (389-1009) y = 13.7460e-0.00124x 0.92 Maidencane 259 (168-564) y = 9.2236e-0.00268x 0.84 Pickerelweed 894 (598-1777) y = 9.4660e-0.000775x 0.91 Sagittaria 15 (11-26) y = 8.5266e-0.0448x 0.93 Height Eleocharis 2295 (1513-4780) y = 77.5573e-0.000302x 0.97 Maidencane 1764 (1208-3285) y =74.5654e-0.000393x 0.96 Pickerelweed 13591 (5590-34657) y = 64.0690e-0.000051x 0.99 Sagittaria 38 (32-47) y = 64.3085e-0.0182x 0.95 a Emergent aquatic species cultured at pH 7.5 under 70% sunlight. b Effective concentration 50: EC 50 = concentration of flumioxazin (g L-1 a.i.) in water required to reduce plant dry weight or height by 50%. Each value is a mean of two experiments with a total of 10 replications (pots). c 95% CI = 95% Confidence Interval. 79

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Flumioxazin Rate (g ha-1 a.i.) 020040060080010001200 Mean Dry Weight (g/pot) 681012141618 Eleocharis Maindencane Pickerelweed Sagittaria Figure 4-5. The effect of flumioxazin rate on dry weight of emergent aquatic species 40 d after treatment. Flumioxazin applied as a single application by a CO2-powered sprayer at an equivalent of 379 L ha-1 diluent with a non-ionic surfactant (0.25% v/v) to emergent aquatic species cultured in 277 L tanks (pH 7.5) under 70% sunlight. Data are shown as actual dry weight means standard error (n=5). 80

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Table 4-3. The effect of a single foliar flumioxazin application on dry weight and height of emergent aquatic species 40 d after treatmenta. Dry Weight EC 50 b (95% CIc) Regression equation r2 Eleocharis NAd y = 11.5964e0.00005x 0.99 Maidencane 1884 (1002-15753) y = 12.8338e-0.000368x 0.92 Pickerelweed NA y = 9.6895e0.00013x 0.97 Sagittaria 1320 (859-2852) y = 13.0027e-0.000525x 0.95 Height Eleocharis NA y = 64.8843e0.00001x 0.99 Maidencane 6478 (3938-18734) y = 88.3085e0.000107x 0.99 Pickerelweed NA y = 65.9479e-0.000144x 0.99 Sagittaria 12160 (5501-18887) y = 76.3892e-0.000057x 0.99 a Flumioxazin applied by a CO 2 -powered sprayer at an equivalent of 379 L/ha diluent with a non-ionic surfactant (0.25% v/v). Emergent aquatic species cultured in 277 L tanks at pH 7.5 under 70% sunlight. b Effective concentration 50: EC 50 = concentration of flumioxazin (g ha-1 a.i.) to reduce plant dry weight or height by 50%. Each value is a mean of one experiments with a total of 5 replications (pots). c 95% CI = 95% Confidence Interval. d NA = not applicable due to positive regression slope. 81

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CHAPTER 5 ORNAMENTAL AND ROW CROP SUSCEPTIBILITY TO FLUMIOXAZIN IN IRRIGATION WATER Introduction Homeowners, commercial nurseries, and farmers in Florida often irrigate plants from surface waters (canals, ponds, lakes, etc.) (Hassell et al. 20004; Hodges and Haydu 2006); non-target plants may be affected if these waters are treated with herbicides for aquatic weed control. The use of herbicide treated irrigation water before herbicide residues dissipate below phytotoxic levels will result in injury or death of irrigated plants. Previous research has evaluated the phytotoxic effects of irrigation water containing copper, 2,4-D, fluridone, diquat, and endothall on non-target turf and ornamental species (Andrew et al. 2003; Hiltibran and Turgeon 1977; Koschnick et al. 2005a; Koschnick et al. 2005b; Mudge et al. 2007; Reimer and Motto 1980), but similar studies have not been conducted with flumioxazin. Tolerances of flumioxazin on certain food crops have been established by the EPA by determining the maximum amount of pesticide residue that can remain in or on a treated food commodity to ensure food safety (EPA 2003), but no such tolerances are required for ornamental plants (non-food crops). Phytotoxicity is a major concern when water with aquatic herbicide residues is used for irrigation of both food and non-food crops. This study was conducted to assist in determining the minimum time required before treated water may be used for irrigation of row crops and ornamental species by evaluating phytotoxicity of flumioxazin-treated irrigation water on three ornamental and four row crop plants. Materials and Methods Ornamental Susceptibility Greenhouse studies were conducted in July and August 2006 at the University of Florida Center for Aquatic and Invasive Plants in Gainesville, FL to evaluate the sensitivity of the 82

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ornamental plant species begonia (Begonia x semperflorens-cultorum 'Senator'), impatiens (Impatiens wallerana Super Elfin Red), and snapdragon (Antirrhinum majus LaBella Pink) to flumioxazin. These three common ornamental plants were purchased from local nurseries in Gainesville and grown in 9.0 x 9.0 x 9.0 cm pots as purchased from the growers. Pots contained an organic commercial potting medium and were top-dressed with Osmocote (15-9-12) fertilizer at a rate of 1g kg-1 soil upon arrival. Plants were subjected to a 14 h photoperiod with maximum daytime temperatures of 31 2 C and minimum nighttime temperatures of 21 2 C. Experimental plants were selected based on uniform height to minimize variation in initial height and weight then grown for 1 wk to allow acclimation before treatment. At treatment, plant height (cm standard error) for each species was as follows: begonia 16.9 0.8, impatiens 17.9 1.1, and snapdragon 41.0 4.3 cm. Snapdragons were mature, hardy, and flowering at time of treatment, while begonias and impatiens were immature with no floral production at the time of treatment. The experiment was a completely randomized design with five replications (pots) per treatment. Plants were overhead irrigated with herbicide treated water once with a sprinkle can (equivalent to 1.27 cm of irrigation water). This volume was sufficient to cover plants and saturate the soil. The pH of the irrigation water was 7.5 and flumioxazin was mixed with the irrigation water immediately prior to irrigation. Flumioxazin concentrations of 0, 10, 25, 50, 100, 200, 400, 800, 1600, and 3200 g L-1 were applied to all ornamental plant species. Plants were subsequently irrigated daily for 14 d with 1.27 cm of well water (containing no herbicide) applied overhead via a sprinkle can. Plant height was recorded from the soil surface to the tip of the tallest leaf 14 d after treatment (DAT). Pants were harvested 14 DAT by collecting all 83

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aboveground biomass excluding dead tissue. Aboveground tissue was placed in a drying oven at 90 C for ca. 1 wk then weighed. Plant dry weight and height data were analyzed using non-linear regression (PROC NLIN, SAS Institute 2002). Regression models were used to determine the effective concentration 10 (EC 10 ), which is the concentration of flumioxazin in irrigation water that caused a 10% reduction in dry weight compared to control plants. Koschnick et al. (2005a) reported this value to be conservative but near the threshold where an observant homeowner might detect adverse effects on plant growth. All dry weight and height data were pooled for each ornamental species as there was no difference between the slopes of regression lines for both experiments at the 95% confidence interval level. Crop Susceptibility Corn (Zea mays L. Garst 8346 LL), cotton (Stoneville 6611 B2RF), soybean (NG2328R) and wheat (Triticum aestivum L., Wakefield) were evaluated for flumioxazin sensitivity in 2006, with the initial study conducted in January and repeated in July. All crop seeds were planted and grown in 10 x 10 x 12 cm (1 L) pots filled with masonry sand amended with Osmocote (15-9-12) fertilizer at a rate of 1g kg-1 soil. Plants were kept in a greenhouse under a 14 h photoperiod with maximum daytime temperatures of 29 2 C and minimum nighttime temperatures of 15 2 C. Plants for this experiment were selected based on uniform height to minimize variation in initial height and weight. At treatment, plant height (cm standard error) for each species was as follows: corn (experiment 1) 42.8 1.0, corn (experiment 2) 59.8 2.0, cotton 20.5 1.2, soybean 18.6 0.8, and wheat 26.7 0.4 cm. All crops were subjected to the same herbicide rates, overhead irrigation, harvest techniques, and statistical procedures as the ornamental species. 84

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All row crop data (except corn) were pooled across experiments because there was no differences between the slopes of regression lines for both experiments at the 95% confidence interval level. Corn grew more quickly in the summer study and was ready for treatment 1 wk after planting, but all other crop species needed an additional week to be of similar size to plants treated in the winter study. Initial corn dry weight and height differed between summer and winter studies so corn data were not pooled across experiments. Results and Discussion Ornamental Susceptibility Dry weights of impatiens and begonia differed at flumioxazin concentrations greater than 50 g L-1 (Figure 5.1). Snapdragons displayed minimal necrosis and chlorosis at all flumioxazin rates throughout the course of the study, were more tolerant to flumioxazin in irrigation water than begonia or impatiens, and had greater EC 10 values for dry weight and plant height (Table 5.1). Snapdragon EC 10 value for dry weight (7024 g L-1) was 68 and 175 times more than the begonia and impatiens, respectively and begonia was less sensitive to flumioxazin than impatiens. Higher sensitivity levels of impatiens and begonia to flumioxazin in irrigation water was probably related to plant size and maturity at the time of treatment. Initial mean dry weights (g standard error) of begonia and impatiens were 1.7 0.1 and 1.6 0.1 and impatiens and begonia control plants increased in dry weight by as much as 119 and 135% by the conclusion of experiments. In contrast, initial mean dry weight of snapdragon was 13.4 0.3 g and only increased by 32% during the course of experiments. Both preemergence and postemergence applications of flumioxazin are generally recommended for actively growing weeds less than 5 cm in height (Anonymous 2006), so the lack of injury to mature snapdragons was likely due to slow growth and resulted in reduced activity by flumioxazin on these mature plants. 85

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Flumioxazin is ideal for use as a contact herbicide in aquatic systems since the half-life is relatively short and it is degraded by hydrolysis in 4.1 d, 16.1 h, and 17.5 min at pH 5.0, 7.0, and 9.0, respectively (Katagi 2003). The half-life of flumioxazin is dependent on pH and is similar to the aquatic herbicides endothall and diquat. Endothall reduced begonia and impatiens dry weight by 10% at concentrations of 2 to 4 mg L-1 a.i. when applied in irrigation water (Koschnick et al. 2005b) and diquat reduced dry weight by 10% at concentrations of 5.1 and 2.8 mg L-1 a.i. for begonia and impatiens, respectively (Mudge et al. 2007). The 10% reduction in biomass of these two ornamental species with endothall was within the labeled use rate, but the rate required to reduce impatiens and begonia biomass with diquat was more than 7 and 13 times the labeled use rate, respectively. The EC 10 dry weight values for impatiens and begonia are well within the proposed flumioxazin label rate of 400 g L-1 a.i. High pH water bodies treated with this herbicide should cause rapid breakdown of flumioxazin and consequently permit shorter irrigation restrictions; however, medium and low pH aquatic systems may need longer irrigation restrictions to ensure sensitive species are not injured or killed by higher herbicide rates. Crop Susceptibility Wheat was more sensitive to flumioxazin in irrigation water than all other crops evaluated (Figure 5.2, Table 5.2). The sensitivity of these plants to flumioxazin is indicated by dry weight EC 10 values as follows: wheat (35), corn experiment #1 (53), cotton (106), corn experiment #2 (181), and soybean (193) (Table 5.2). Similar results were also observed with plant height data. Corn in experiment 2 and soybean were generally more tolerant of flumioxazin than other species, which was expected since flumioxazin is registered for preemergence control of broadleaf weeds in soybean (Anonymous 2005). Although the immature ornamental species and row crops evaluated in this study displayed higher levels of sensitivity to flumioxazin compared to other short-lived contact herbicides such 86

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as endothall and diquat, the short half-life of flumioxazin in water would partially ameliorate any damage that may occur if homeowners irrigate soon after flumioxazin application. This may be advantageous in pond or lake situations where pH exceeds 9.0, since homeowners could irrigate sooner than if a longer residual herbicide was applied. Flumioxazin is still being evaluated under an EUP and must be granted a full EPA Section 3 Label before this product can be applied to public aquatic systems, since water bodies treated with flumioxazin under the EUP may not be used for irrigation, swimming, drinking, or fish consumption. This study indicates that flumioxazin has the potential to injure and kill immature ornamental and crop species and that these plants may be injured when flumioxazin is applied at the potential maximum label use rate of 400 g L-1. Based on these data, if homeowners or farmers irrigate with water treated with flumioxazin soon after treatment, 10% or more injury may occur on young, actively growing plants. Flumioxazin is generally more effective on hydrilla in the early spring when it is immature and actively growing and this is the same time most crops and ornamental species are planted by farmers and homeowners. Thus, irrigation restrictions will be variable due to the significant effect of water pH on flumioxazin half-life, application rate, and differences in non-target plant susceptibility as a result of the stage of maturity. 87

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Flumioxazin Concentration (g L-1 a.i.) 050010001500200025003000 Mean Dry Weight (g/pot) 01234 Begonia Impatiens Figure 5-1. The effect of flumioxazin concentration in irrigation water (pH 7.5) on ornamental species dry weight 14 d after treatment. Flumioxazin was applied once to the plants as an overhead irrigation with a sprinkle can (equivalent to 1.27 cm of irrigation water). Data are shown as actual dry weight means standard error (n=10). Data for snapdragon were not included as flumioxazin resulted in minimum effects at all rates. 88

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Table 5-1. The effect of a single overhead irrigation with 1.27 cm water containing flumioxazin on ornamental species dry weight and height 14 d after treatment.a Dry weight EC 10 b (95% CIc) Regression equation r2 Begonia 103 (84-136) y = 3.9194e-0.00102x 0.94 Impatiens 40 (32-53) y = 3.7097e-0.00265x 0.92 Snapdragon 7024 (2450-10536) y = 13.6060e-0.000015x 0.94 Height Begonia 144 (125-171) y = 23.2842e-0.000731x 0.97 Impatiens 50 (40-67) y = 24.3930e-0.00209x 0.93 Snapdragon 2395 (1463-7024) y = 50.3696e-0.000044x 0.98 a Flumioxazin was applied in water (pH 7.5) with a sprinkle can. b Effective concentration 10: EC 10 = concentration of flumioxazin (g L-1 a.i.) in irrigation water required to reduce plant dry weight or height by 10%. Each value is a mean of two experiments with a total of 10 replications (pots). c 95% CI = 95% Confidence Interval. 89

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050010001500200025003000 Mean Dry Weight (g/pot) 0123456 Corn # 1 Corn # 2 050010001500200025003000 0123456 Cotton Flumioxazin Concentration (g L-1 a.i.) 050010001500200025003000 Mean Dry Weight (g/pot) 0123456 Soybean Flumioxazin Concentration (g L-1 a.i.) 050010001500200025003000 0123456 Wheat Figure 5-2. The effect of flumioxazin concentration in irrigation water (pH 7.5) on crop species dry weight 14 d after treatment. Flumioxazin was applied once to the plants as an overhead irrigation with a sprinkle can (equivalent to 1.27 cm of irrigation water). Data are shown as actual dry weight means standard error (n=10), except for corn (n=5, for each experiment). 90

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Table 5-2. The effect of single overhead irrigation with 1.27 cm water containing flumioxazin on crop species dry weight and height 14 DAT. a Dry weight EC 10 b (95% CIc) Regression equation r2 Corn #1d 53 (45-66) y = 2.8429e-0.00198x 0.98 Corn #2 181 (135-279) y = 4.7306e-0.000581x 0.97 Cotton 106 (84-142) y = 3.1634e-0.000993x 0.94 Soybean 193 (158-247) y = 2.7891e-0.000547x 0.97 Wheat 35 (30-47) y = 1.2876e-0.00289x 0.95 Height Corn #1 56 (49-64) y = 67.2884e-0.00189x 0.99 Corn #2 221 (160-357) y = 68.2482e-0.000476x 0.97 Cotton 120 (100-150) y = 33.1105 e-0.000875x 0.97 Soybean 206 (176-248) y = 42.6972e-0.000511x 0.98 Wheat 46 (40-55) y = 38.1144e-0.00229x 0.97 a Flumioxazin was applied in water (pH 7.5) with a sprinkle can. b Effective concentration 10: EC 10 = concentration of flumioxazin (g L-1 a.i.) in irrigation water required to reduce plant dry weight or height by 10%. Each value is a mean of two experiments with a total of 10 replications (pots). c 95% CI = 95% Confidence Interval. d #1 and #2: experiment 1 and experiment 2. 91

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CHAPTER 6 THE EFFECT OF FLUMIOXAZIN AND DIQUAT ON MEMBRANE PERMEABILIITY AND CHLOROPHYLL CONTENT OF LANDOLTIA Introduction Species of duckweed in the Lemnaceae family are commonly used in biochemical and toxicity tests because of their small size, high reproductive rate and the ease with which they are cultivated (Gensemer et al. 1999; Geoffroy et al. 2004; Lewis 1995; Ma et al. 2002; Parr et al. 2002). Studies evaluating pigment content (chlorophyll a, b, and carotenoids), oxygen emission, and ion leakage are often reliable indicators of herbicide toxicity (Koschnick et al. 2006; Wang and Freemark 1995). Although hydrilla is the primary target weed in flumioxazin EUP research, invasive floating plants such as landoltia are being evaluated for sensitivity to this compound. Members of the Lemnaceae family are extremely sensitive to diquat as common duckweed possesses an EC 50 of 4 g L-1 (Peterson et al. 1997). It has been controlled with diquat for many years; however, landoltia plants have been discovered in Lake County FL with a resistance factor of 50x for diquat (Koschnick et al. 2006). Herbicides such as flumioxazin possess a different mode of action and can be utilized to help prevent further development of resistance and may control resistant plants. Previous research has shown flumioxazin at 1, 10, and 50 g L-1 decreased photosynthetic capacity of common duckweed (Lemna minor L.) by 23, 62, and 64%, respectively (Frankart et al. 2002). Therefore, the objective of this research was to compare the effects of flumioxazin and diquat on ion leakage and chlorophyll content in landoltia. Materials and Methods Landoltia was collected from a pond with no history of herbicide treatments in Alachua County, FL in April 2007 and was cultured in 9.5-L aquaria containing a standard growth medium (Wang 1990) at the University of Floridas Center for Aquatic and Invasive Plants, 92

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Gainesville, FL. Plants were maintained in a growth room with a 16 h photoperiod at a temperature of 26 4 C. Agitation was continuously supplied to each culture with forced air via small aquarium air pumps. Aquaria and plants were rinsed and growth media were refreshed ca. every 3 to 6 wk and light levels were maintained at 150 10 mol m-2 s-1 to minimize algal growth. Ion Leakage The effect of flumioxazin on landoltia was measured by comparing non-specific ion leakage (conductance in mhos cm-1) over time using a conductivity bridge 17 since conductance may be used to measure non-specific ion leakage resulting from loss of membrane integrity (Koschnick et al. 2006; MacDonald et al. 1993; OBrien and Prendeville 1978). All ion leakage techniques were modeled after research conducted by Koschnick et al. (2006). Ten colonies of landoltia (4 to 6 fronds per colony) were placed into individual 20-ml high-density polyethylene (HDPE) scintillation vials containing 15 ml DI water. Flumioxazin was added to each of the vials to achieve concentrations of 0, 10, 25, 50, 100, 200, 400, 800, and 1600 g L-1. Diquat at 10 g L-1 was applied as a comparison treatment. The experiment was conducted and repeated in May 2007 as a randomized design with 5 replications. Vials were covered with Parafilm M 18 and inverted 3x after addition of herbicide. Vials containing only flumioxazin or diquat (duplicate treatment solutions) at each herbicide concentration and no plants were immediately measured for initial conductance (Ci) (conductance contributed by addition of herbicides) and appropriate corrections were made to determine total ion leakage. Vials were placed on a shaker table (100 oscillations min-1) in a growth chamber and temperature of treatment solutions was 17 Fisher Scientific Conductivity Meter. Pittsburgh, PA. 18 Trademark of Pechiney Plastic Packaging. Menasha, WI. 54952. 93

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maintained at 27 C with a 14 h photoperiod at 380 10 mol m-2 s-1. Conductivity of the treatment solutions was measured at 0, 1, 3, 6, 9, 12, 24, 30, 48, and 72 h after treatment (HAT). The study was terminated 96 HAT due to the decline in conductivity resulting from algal growth in vials. Final conductivity measurements were recorded, then treatment vials containing landoltia were frozen and thawed 3x to ensure 100% ion leakage (Ct). Ion leakage at each time (Cx) is reported as percent conductivity according to the following formula used by MacDonald et al. (1993) and Koschnick et al. (2006): % conductivity = [(CxCi)/(Ct-Ci)]*100. A repeated measures analysis was performed and means were separated using 95% confidence intervals (95% CI) (PROC GLM, SAS Institute 2002). Chlorophyll The effect of flumioxazin on chlorophyll content was determined on colonies of landoltia placed into scintillation vials and treated as described above for the ion leakage experiments. As a comparison treatment, landoltia was treated with diquat at 10 g L-1. This experiment was conducted and repeated in May 2007 as a randomized design with 5 replications. Treated plants were placed in a growth chamber for a 96 h exposure period then plants were removed, excess water was blotted with a paper towel, and fresh weights were recorded. Total chlorophyll was extracted by placing plants from each treatment vial into polystyrene test tubes (12 x 75 mm) containing dimethylsulfoxide (DMSO) (Hiscox and Israelstam 1979) in a water bath (65 C) for 3 h. Chlorophyll content was determined spectrophotometrically (Arnon 1949) and expressed as mg chlorophyll kg-1 of fresh weight. Data were analyzed using non-linear regression (PROC NLIN, SAS Institute 2002) and EC 50 values (flumioxazin concentration required to cause a 50% reduction in chlorophyll content) were derived. 94

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Results and Discussion Ion Leakage Control plants did not produce more than 10% conductivity throughout the course of these studies (data not shown). There were no differences in conductivity between flumioxazin at any concentration and diquat 1 and 3 HAT (Figure 6-1), but differences in conductivity were noted between flumioxazin at 10, 25, 50 and 1600 g L-1 and diquat 6 HAT. Leakage due to diquat 9 HAT was greater than leakage due to flumioxazin at all concentrations. These results are similar to those reported for landoltia treated with diquat at 10 g L-1 when conductivity exceeded 80% by 18 HAT (Koschnick et al. 2006). All flumioxazin treated plants displayed bleaching to a certain extent 24 HAT, but none of the treatments resulted in more than ca. 50% bleaching; in contrast, diquat treated plants were 100% chlorotic 24 HAT. Conductivity in the diquat treatment was 90% 30 HAT, while flumioxazin conductivity never exceeded 50% at any concentration throughout the course of the experiments. At the conclusion of these studies (96 HAT), most flumioxazin treatments began to show signs of decreasing conductivity, probably due to algal growth in the scintillation vials. If these studies were continued for a few more days, similar to the landoltia treated with flumioxazin in Chapter 4, it is likely the plants would have continued to bleach and completely leak 100% of the ions. Based on these studies, flumioxazin is not as fast acting as diquat with respect to bleaching and ion leakage; however, mesocosm data in Chapter 4 demonstrated that flumioxazin did not exhibit significant bleaching until 7-10 DAT but provided 65% control 21 DAT when applied at 400 g L-1. Chlorophyll An estimated flumioxazin concentration of 944 g L-1 is required to reduce landoltia chlorophyll content by 50% (EC 50 ) after a 96 h exposure (Figure 6-2), but plants were not completely bleached even at the highest flumioxazin concentration (1600 g L-1). In contrast, 95

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diquat at 10 g L-1 resulted in a 99% reduction of chlorophyll content 96 HAT (data not shown). These data provide evidence that flumioxazin may have slower activity on landoltia than diquat, and suggests that landoltia may not have been exposed to flumioxazin for a sufficient amount of time in these experiments. Submersed flumioxazin applications required >1 wk to cause significant chlorosis in landoltia in mesocosm experiments (see Chapter 4) and the time allotted in these studies (96 h) was not sufficient for flumioxazin to cause significant bleaching. Protox-inhibiting herbicides are more active in the presence of full sunlight (Sherman et al. 1991; Wright et al. 1995) and landoltia was exposed to flumioxazin in a growth chamber under low light conditions (380 10 mol m-2 s-1) in these experiments. Also, these studies were conducted under a 14 h day length and not continuous light as in previous research (Koschnick et al. 2006). Although flumioxazin did not cause the same level of injury to landoltia as diquat, flumioxazin treatments still resulted in significant levels of ion leakage and bleaching. Continuous and higher light levels may result in greater leakage similar to that observed in diquat; however, the environment in the growth chambers was similar to field conditions (14 h day length) and not artificially altered by providing continuous light. These data indicate that flumioxazin causes slower, less severe injury to landoltia than diquat under short exposure times. 96

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020406080100120 Diquat 10 g L-1 Flumioxazin 10 g L-1 Flumioxazin 25 g L-1 Diquat 10 g L-1 Flumioxazin 50 g L-1 Flumioxazin 100 g L-1 Hours after treatment 1369122430487296 Ion leakage (% total) 020406080100120 Diquat 10 g L-1 Flumioxazin 200 g L-1 Flumioxazin 400 g L-1 1369122430487296 Diquat 10 g L-1 Flumioxazin 800 g L-1 Flumioxazin 1600 g L-1 Figure 6-1. The effect of diquat and flumioxazin on ion leakage from landoltia cultured in DI water (pH 8.5) in a growth chamber for 96 h. Values are presented as means 95% confidence interval (CI) (n=10). Overlapping CI bars indicate no significant difference at a given time. 97

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Flumioxazin Concentration g L-1 a.i. 02004006008001000120014001600 Chlorophyll (mg kg-1 fresh weight) 0.000.050.100.150.200.25 y = 0.1539-0.000734x, r2 = 0.83EC50 = 944 g L-1 Figure 6-2. The effect of flumioxazin concentration on landoltia chlorophyll content 96 h after treatment. Landoltia was cultured in 20 mL vials containg DI water (pH 8.5) in a growth chamber. Data are shown as actual means standard error (n=6). EC 50 = effective concentration 50, concentration of flumioxazin in water required to reduce landoltia chlorophyll content by 50%. 98

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CHAPTER 7 SUMMARY AND DRAFT AQUATIC USE DIRECTIONS Summary These experiments provided information regarding the effectiveness of flumioxazin as an aquatic herbicide and will aid in the registration of flumioxazin for use in aquatic ecosystems to control invasive aquatic species such as hydrilla and water lettuce. Flumioxazin was screened for hydrilla control in 2005 and significantly reduced hydrilla biomass. Through a cooperative agreement with Valent U.S. A. Corporation, The Center for Aquatic and Invasive Plants at the University of Florida began evaluation of flumioxazin as a potential aquatic herbicide. Various EUP ponds throughout Florida ranging in pH from 6.7 to 10.0 were treated with flumioxazin at concentrations of 100 to 400 g L-1. Early season treatments were successful due to lower pH, less hydrilla biomass, and/or time of year; however, flumioxazin failed to provide more than 1to 4 months of control in ponds with pH >8.5. These high pH ponds were generally infested with mature hydrilla that was near the surface. Early and late season treatments are recommended since hydrilla grows more actively and water pH is likely to be lower than in summer treatments. Due to the lack of efficacy in EUP ponds treated with flumioxazin in the summer when the water pH was in excess of 8.5 or when hydrilla was near the surface of the water, greenhouse and laboratory studies were initiated to determine why flumioxazin was not as efficacious under these conditions. Outdoor mesocosm studies showed that flumioxazin reduces hydrilla biomass by 90% at concentrations of 186 g L-1, but possesses activity at concentrations as low as 50 g L-1. Hydrilla treated with flumioxazin underwent chlorosis in apical tips followed by reddening of lower stems. Hydrilla treated at 50 to 1600 g L-1 began to lose integrity and fall to the bottom of the tanks within 5 to 7 DAT; however, new apical tips from adventitious buds in the 99

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leaf axils soon sprouted from treated rooted and floating tissue. Flumioxazin is rapidly hydrolyzed in high pH water and has an average half-life of 17.5 min under laboratory conditions at pH 9.0 or greater. The effects of pH on flumioxazin half-life and hydrilla efficacy were evaluated in a pH efficacy study. Flumioxazin reduced hydrilla dry weight by 90% in the high (>8.5) pH treatment when hydrilla was placed into mesocosms the same day as treatment; however, hydrilla placed in high pH water 2 to 5 DAT was reduced in biomass by no more than 50%. Biomass of hydrilla in the low (6.0 to 6.2) and medium (7.0 to7.2) pH was reduced by 93 and 68%, respectively, of the nontreated control plants 3 DAT. The half-life of flumioxazin in low, medium, and high pH water (6.0 to 6.2, 7.0 to 7.2 and >8.5, respectively) was 39, 18.6, and 1.7 h, respectively. These data indicate flumioxazin is rapidly taken up by hydrilla and the short half-life of flumioxazin in water with pH >9.0 can be overcome by higher application rates. The net photosynthetic rates of apical hydrilla tips treated with flumioxazin were measured to determine the effects of rate, pH, stem type, and light quantity. Flumioxazin applied in high pH water (9.0) at 200 g L-1 or in low pH water (6.0) at 100 g L-1 required less than 124 h to reduce hydrilla net photosynthesis by 50% (ET 50 ) of the non-treated control plants and only the 100 g L-1 treatment applied in high pH water failed to have a significant impact on photosynthesis. Since activity of flumioxazin is influenced by light, it was hypothesized that hydrilla at the bottom of water bodies were not being controlled due to the low quantity of light reaching plants. Growth chamber studies indicated 170 and 400 mol m-2 s-1 of light was sufficient to reduce net photosynthesis of hydrilla by 45 and 78% of the nontreated control plants, respectively, 168 HAT; however, hydrilla treated with flumioxazin at light levels of 20 mol m-2 s-1 resulted in less than a 30% reduction in net photosynthesis 168 HAT. Furthermore, 100

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plants treated at the lowest light level did not appear to be injured and maintained a healthy appearance throughout the course of the experiments. Due to the high costs associated with registering a pesticide in a new market like aquatics, other invasive species were evaluated for sensitivity to flumioxazin. Water lettuce was highly sensitive to submersed and foliar flumioxazin applications, whereas water hyacinth was impacted only slightly by either application technique. Landoltia was sensitive to submersed treatments, but was non-responsive to foliar treatments. If flumioxazin is approved for aquatic use, non-target aquatic plants may be affected by submersed and foliar flumioxazin applications and terrestrial plants may be injured from irrigation with treated water. The impact of a submersed flumioxazin application on submersed aquatic plant species including coontail, egeria, hydrilla, southern naiad, and vallisneria was evaluated at high (9.0) and low pH (7.0). Coontail, naiad, and hydrilla dry weight were reduced by 50% (EC 50 ) when flumioxazin was applied at 34, 51, and 77 g L-1 in low pH water. Only coontail dry weight was reduced by 50% with flumioxazin concentrations near the proposed label maximum rate of 400 g L-1in high pH water. The emergent aquatic plants maidencane and sagittaria were reduced by 50% with flumioxazin application of less than 400 g L-1. Foliar application of flumioxazin reduced sagittaria dry weight by 50% at 147 g ha-1, which is approximately half of the proposed maximum foliar application rate of 286 g ha-1. Other emergent species (eleocharis, maidencane, and pickerelweed) were more tolerant to applications within the proposed maximum labeled rate. Non-target ornamental and row crop plants were overhead irrigated once with irrigation water containing flumioxazin. Immature begonia and impatiens were highly sensitive compared to mature snapdragons and were reduced by 10% in dry weight with flumioxazin in irrigation water at rates of 103 and 44 g L-1, respectively. All 101

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row crops (corn, cotton, soybeans, and wheat) were reduced by 10% in dry weight with flumioxazin at less than 200 g L-1 in irrigation water. These data clearly indicate that irrigation restrictions will be required on the label; however, since flumioxazin is rapidly degraded (especially in high pH water), shorter water use restrictions should be possible for homeowners and farmers who use flumioxazin treated water for irrigation; however, treated water with a pH of less than 8.0 will likely be restricted for irrigation for several days. These data indicate flumioxazin has potential utility as an aquatic herbicide for control of hydrilla and other aquatic weeds. Flumioxazin possesses several desirable traits including activity at low use rates, a short half-life, and non-target selectivity. Hydrilla control will depend on several factors including water pH, growth stage of hydrilla, and light availability to stems near the bottom of a water body. Summer applications or situations where hydrilla has already surface matted (resulting in high pH) will be less effective than early and late season applications, so treatments under these conditions should be avoided since these treatments will result in a burndown or removal of the upper canopy and hydrilla will regrow quickly within 1 to 2 months. Draft Aquatic Use Directions General information Flumioxazin is a 51 percent water dispersible granule that controls weeds by inhibiting protoporphyrinogen oxidase (Protox), an essential enzyme required by plants for chlorophyll biosynthesis. This herbicide is rapidly absorbed by aquatic plants and breaks down by pH dependent hydrolysis. Flumioxazin is a fast-acting, contact herbicide that can be applied directly into water for control of submersed aquatic weeds or directly to the foliage of emergent or floating weeds. The most effective flumioxazin applications occur when applied to young, actively growing weeds in water with a pH <8.0. 102

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Mixing Guidelines If the diluent water (tank mix) is pH >7.0, add an agent to lower the pH in the tank. Agitation may be applied to the spray tank, but the addition of more than 1 lb of product per gallon at a high agitation rate will likely result in large quantities of foam in the tank. The herbicide solution should be applied no longer than 2 h after mixing to prevent hydrolysis. Control of Submersed Weeds Apply flumioxazin for control of hydrilla and other susceptible submersed weeds in lakes, ponds, non-irrigation canals and other water bodies with limited water exchange. Total concentration of flumioxazin in a single treatment should not exceed 400 g L-1 in the treated water area. For best results, apply in spring or early summer when submersed weeds are actively growing but have not reached the water surface. Water pH at a depth of 1 ft below the surface should be measured at the time of application. Flumioxazin may be applied at concentrations as low as 50 g L-1, but concentrations above 100 g L-1 provide better efficacy, especially in water with a pH >8.5. Treatments should be applied as early in the morning as possible to minimize the effect of hydrolysis in high pH water since infested waters have a tendency to cycle pH from as low as 7.0 (6 A.M.) to as high as 10.0 (6 P.M.) in the upper 10 to 25 cm of a water body. If the water pH is >8.5 or hydrilla is surface matted, flumioxazin should be applied at 400 ppb to ensure best efficacy. Repeat applications of 400 ppb within 1 to 2 months after treatment may be necessary due to the rapid breakdown of this product in water with high pH. If the water pH is 7.0 to 8.4, flumioxazin may be applied at 200 to 400 ppb for control of hydrilla. Additional applications may be necessary for complete control at this pH range. Flumioxazin applied to water with a lower pH (6.9) will result in better efficacy due to slower breakdown in low pH water. Flumioxazin may be applied at rates of 50 to 400 ppb in these lower pH waters. 103

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Subsurface Application Flumioxazin should be applied with long weighted hoses to ensure proper mixing of herbicide in the water column. Thermal stratification is common in lakes with surface-matted hydrilla and this thermal layer can create a physical barrier, isolating layers in the water column and preventing surface-applied herbicides from reaching the target vegetation below the thermocline. If weighted hoses are not available, every effort should be made to ensure herbicide is uniformly mixed below the water surface to assure all plant parts are exposed to the herbicide. Inadequately mixed flumioxazin will likely break down before it comes in sufficient contact with plants near the bottom, allowing for more rapid regrowth. Surface/Foliar Application Flumioxazin may be applied up to 8 ounces of formulated product per acre. Control of water lettuce and other floating or emergent weeds require the addition of a spray adjuvant for foliar applications. For best results, use nonionic surfactants or methylated seed oils at manufacturers recommended rates. Mix in sufficient diluent (50 to 100 GPA) to ensure adequate coverage. Plant Susceptibility Submersed, emergent, and floating vascular aquatic plants as well as macrophytic algae vary in susceptibility to flumioxazin (Table 7-1). Plants are more susceptible to submersed flumioxazin applications in lower pH water (<8.0) where half-lives are longer. For best results, treatments with this herbicide should be applied when plants are actively growing and before they are surface matted. If plants are surface matted, an initial application may be required to control the surfaced plants and a subsequent application may be necessary to provide season long control. Lower pH, actively growing weeds, less biomass, and high light penetration into the water colum favor increased flumioxazin efficacy on hydrilla and other submersed species. 104

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Irrigation Restrictions In addition to efficacy on weeds and impact on non-target plants, the focus of this research was to determine possible irrigation restrictions following flumioxazin applications. Chapters 2, 5, and 8 provide data on the half-life of flumioxazin under various pH situations and the sensitivity of non-target plants to flumioxazin in irrigation water. Water use restrictions for flumioxazin will be dependent on a number of factors including herbicide placement in the water column, herbicide rate, pH, and maturity of the plants being irrigated with flumioxazin treated water (Table 7-2). Submersed applications will be more restrictive than foliar applications due to higher concentrations of flumioxazin in the water. For example, ornamental species are usually planted during the spring when hydrilla is actively growing and highly susceptible to flumioxazin; therefore, irrigation water containing flumioxazin may also severely injure these immature plants. Water with a pH of 6.0 to 8.0 that has received a submersed flumioxazin application (200 to 400 g L-1) should not be used for irrigation for up to 7 d after herbicide application compared to a 2 d restriction if the pH is 9.0. The shorter half-life at pH 9.0 (see Chapter 2) prevents injurious levels of this herbicide from being present in the water beyond 1 DAT. Immature plants are more susceptible to submersed flumioxazin applications as pH decreases and the use rate increases. On the other hand, foliar applications pose less of a threat to these young plants. The majority of the herbicide solution will come in contact with the foliage of the emergent or floating weed and less will be available in the water to harm irrigated plants. 105

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Table 7-1. Aquatic plant and algae controla with flumioxazin in water with a pH of 7.0 to 9.5b Common name Scientific name Submersedc Foliard alligatorweed Alternanthera philoxeroides F-G G-E babys-tears Micranthemum spp. F NA broadleaf arrowhead Sagittaria latifolia E NA Carolina mosquito fern Azolla caroliniana E NA cattail Typha spp. P P coontail Ceratophyllum demersum E NA duck potato Sagittaria lancifolia E F-G duckweed Lemna minor G-E G-E egeria Egeria densa P NA Eurasian water milfoil Myriophyllum spicatum E NA fanwort Cabomba caroliniana E NA frogs-bit Limnobium spongia E NA hydrilla Hydrilla verticillata F-E NA jointed spikerush Eleocharis interstincta F F landoltia Landoltia punctata G-E P maidencane Panicum hemitomon G F muskgrass Chara spp. P-G NA pennywort Hydrocotyle spp. F-G NA pickerelweed Pontederia cordata F F southern naiad Najas guadalupensis F-E NA stonewort Nitella spp. P-F NA torpedo grass Panicum repens P-F P-F vallisneria Vallisneria americana P-F NA variable-leaf milfoil Myriophyllum heterophyllum E NA water fern Salvinia minima G-E NA water hyacinth Eichhornia crassipes P P water lettuce Pistia stratiotes E E water meal Wolffia columbiana G-E NA water shield Brasenia schreberi G-E NA willow Salix spp. P P aControl: NA = not applicable or not evaluated, P = poor, F= fair, G = good, E = excellent; based on EUP field and mesocosm observations. bImproved efficacy at lower pH for most species. cSubmersed: treatment applied by weighted hoses. dFoliar treatment applied with 0.25% v/v non-ionic surfactant by handgun at 935 L ha-1 diluent. 106

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Table 7-2. Proposed water use restrictions to overhead irrigated crop and ornamental species following submersed and foliar flumioxazin applications. Flumioxazin Application Use Rate Maturity of Irrigated Plantsa Water pH Daysb Submersed 200-400 g L-1 Immature 8.5 1 200-400 g L-1 Mature 8.5 1 200-400 g L-1 Immature 7.0-8.4 7 200-400 g L-1 Mature 7.0-8.4 1 200-400 g L-1 Immature 6.9 14 200-400 g L-1 Mature 6.9 2 <200 g L-1 Immature 8.5 1 <200 g L-1 Mature 8.5 1 <200 g L-1 Immature 7.0-8.4 6 <200 g L-1 Mature 7.0-8.4 1 <200 g L-1 Immature 6.9 12 <200 g L-1 Mature 6.9 1 Foliar 143-286 g ha-1 Immature 8.5 1 143-286 g ha-1 Mature 8.5 0 143-286 g ha-1 Immature 7.0-8.4 1 143-286 g ha-1 Mature 7.0-8.4 0 143-286 g ha-1 Immature 6.9 1 143-286 g ha-1 Mature 6.9 0 a Growth stage of plants irrigated with water treated with flumioxazin. b Number of days before water treated with flumioxazin may be used for irrigation of crop and ornamental species. Based on the half-life of flumioxazin at a given pH and the growth stage of irrigated species. 107

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LIST OF REFERENCES Aizawa, H. and H. M. Brown. 1999. Metabolism and degradation of porphyrin biosynthesis herbicides. In: Bger, P. and Wakabayashi, K., Editors, 1999. Peroxidizing Herbicides, Springer-Verlag, Berlin, pp. 348. Andrew, W., W. T. Haller, and D. G. Shilling. 2003. Response of St. augustinegrass to fluridone in irrigation water. J. Aquat. Plant Manage. 41:61-63. Angelo, D. J., M. Hartle, and W. K. Hock. 1998. Pond management and aquatic plant control. http://pubs.cas.psu.edu/FreePubs/pdfs/agrs76.pdf Accessed October 25, 2007. Anonymous. 2003. Reward Landscape and Aquatic Herbicide product label. Greensboro NC: Syngenta Crop Protection, Inc. Anonymous. 2005. Valor SX herbicide product label. Walnut Creek, CA: Valent U.S.A. Anonymous. 2006. SureGuard herbicide product label. Walnut Creek, CA: Valent U.S.A. Anonymous. 2007a. Aquathol K herbicide product label. King of Prussia, PA: Cerexagri-Nisso LLC. Anonymous. 2007b. Hydrilla management in Florida: current and future chemical management practice. http://plants.ifas.ufl.edu/osceola/hydrilla_mngmt_fl/management _practices.html Accessed October 22, 2007. Arias, R. S., M. D. Netherland, B. E. Scheffler, A. Puri, and F. E. Dayan. 2005. Molecular evolution of herbicide resistance to phytoene desaturase inhibitors in Hydrilla verticillata and its potential use to generate herbicide-resistant crops. Pest Manage. Sci. 61:258-268. Arnon, D. I. Copper enzymes in isolated chloroplasts. 1949. Polyphenoloxidase in Beta vulgaris. Plant Physiology. 24:1-15. Askew, S. D., J. W. Wilcut, and J. R. Cranmer. 1999. Weed management in peanut (Arachis hypogaea) with flumioxazin and postemergence herbicides. Weed Technol. 13:594. Becerril, J. M. and S. O. Duke. 1989. Protoporphyrin IX content correlates with activity of photobleaching herbicides. Plant Physiol. 90:1175. Blackburn, R. D., L. W. Weldon, R. R. Yeo, and T. M. Taylor. 1969. Identification and distribution of certain similar-appearing submerged aquatic weeds in Florida. Hyacinth Control J. 8:12-21. Bowes, G. A., A. S. Holaday, and W. T. Haller. 1979. Seasonal variation in the biomass, tuber density, and photosynthetic metabolism of hydrilla in three Florida lakes. J. Aquat. Plant Manage. 9:55-58. 108

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Brian, R. C., R. F. Homer, J. Stubbs, and R. L. Jones. 1958. A new herbicide 1:1-ethylene-2:2dipyridylium dibromide. Nature 181:446-447. Burke, I. C., S. D. Askew, and J. W. Wilcut. 2002. Flumioxazin systems for weed management in North Carolina peanut (Arachis hypogaea). Weed Technol. 16:743. Carmer, S. G., W. E. Nyquist, and W. M. Walker. 1989. Least significant differences for combined analysis of experiments with two or three-factor treatment designs. Agron. J. 81:665-672. Cervone, S. and J. Schardt. 2003. Selective application of aquatic herbicides. http://plants.ifas.ufl.edu/guide/selecherb.html Accessed October 26, 2007. Clewis, S. B., S. D. Askew, and J. W. Wilcut. 2002. Economic assessment of diclosulam and flumioxazin in stripand conventionaltillage peanut. Weed Sci. 50:378. Cobb A. 1992. Herbicides that inhibits photosynthesis. In: Cobb, A., Editor, Herbicides and Plant Physiology, Chapman and Hall, London, pp. 46. Cook, C. D. K. 1985. Range extensions of aquatic vascular plant species. J. Aquat. Plant Manage. 30:15-20. Cook, C. D. K. and R. Lnd, 1982. A revision of the genus Hydrilla (Hydrocharitaceae). Aquat. Bot. 13:485-504. Cranmer, J. R., J. V. Altom, J. C. Braun, and J. A. Pawlak. 2000. Valor herbicide: a new herbicide for weed control in cotton, peanuts, soybeans, and sugarcane. Proc. South. Weed Sci. Soc. 53:158. Dayan, F. E. and S. O. Duke. 1997. Phytotoxicity of protoporphyrinogen oxidase inhibitors: phenomenology, mode of action and mechanisms of resistance: Pages 11-35 in R. M. Roe, J. D. Burton, R. J. Kuhr, Ed. Herbicide Activity: Toxicology, Biochemistry and Molecular Biology. I.O.S. Press, Amsterdam, The Netherlands. Dayan, F. E. and M. D. Netherland. 2005. Hydrilla, the perfect aquatic weed, becomes more noxious than ever. Outlooks on Pest Management. 16:277-282. Dibble, E. D., K. J. Killgore, and S. L. Harrel. 1996a. Assessment of fish-plant interactions. Amer. Fish. Soc. Symp. 16:357-372. Dibble, E. D., K. J. Killgore, and S. L. Harrel. 1996b. Assessment of fish-plant interactions. In L. E. Miranda and D. R. DeVries (eds.) Multidimensional Approaches to Reservoir Fisheries Management. Amer. Fish. Soc. Symp. 16: 347-356. Doong, R. L, G. E. MacDonald, and D. G. Shilling. 1993. Effect of fluridone on chlorophyll, carotenoid and anthocyanin content of hydrilla. J. Aquat. Plant Manage. 31:55-59. 109

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Duke, S. O. and W. H. Kenyon. 1993. Peroxidizing activity determined by cellular leakage. In Target Assays for Modern Herbicides and Related Phytotoxic Compounds, edt. by P. Boger and G. Sandmann, CRC Press, Boca Raton, FL, pp. 61-66. Duke, S. O., J. Lydon, J. M. Becerril, T. D. Sherman, L. P. Lehnen Jr., and H. Matsumoto. 1991. Protoporphrinogen oxidase-inhibiting herbicides. Weed Sci. 39:465. Duke, S. O., J. Lydon, and R. N. Paul. 1989. Oxadiazon activity is similar to that of p-nitrodiphenyl ether herbicides. Weed Sci. 37:152. Elmarakby, S. A., D. Supplee, and R. Cook. 2001. Degradation of (14C) carfentrazone-ethyl under aerobic aquatic conditions. J. Agric. Food Chem. 49:5285-5293. [EPA] U.S. Environmental Protection Agency. 2003. Setting Tolerances for Pesticide Residues in Food. (search: food safety tolerance). Web page: http://www.epa.gov Accessed March 24, 2007. Fadayomi, O. and G. F. Warren. 1977. Uptake and translocation of nitrofen and oxyfluorfen. Weed Sci. 25:111. [FDACS] Florida Department of Agricultural and Consumer Services. 2006. http://www.flaes.org/pdf/PREC_2006_04_AG.pdf Accessed March 15, 2007. Ferrell, J. A., W. H. Faircloth, B. J. Brecke, and G. E. MacDonald. 2007a. Influence of cotton height on injury from flumioxazin and glyphosate applied post-directed. Weed Tech. 21:709-713. Ferrell, J. A., W. M. Stall, and G. E. MacDonald. 2007b. Diagnosing herbicide injury. University of Florida, Institute of Food and Agricultural Sciences. SS-AGR-15. http://edis.ifas.ufl.edu/WG053 Accessed October 12, 2007. Ferrell, J. A. and W. K. Vencill. 2003a. Flumioxazin soil persistence and mineralization in laboratory experiments. J. Agric. Food Chem. 51:4719-4721. Ferrell, J. A. and W. K. Vencill. 2003b. Impact of adjuvants and nozzle types on cotton 250 injury from flumioxazin applied post-directed. J. Cotton Sci. 7:242-247. Ferrell, J. A., W. K. Vencill, K. Xia, and T. L. Grey. 2004. Sorption and desorption of flumioxazin to soil, clay minerals and ion-exchange resin. Pest. Manage. Sci. 61:40-46. Filbin, G.J. and R.A. Hough. 1979. The effects of excess copper sulfate on the metabolism of the duckweed Lemna minor. Aquat. Bot. 7:79-86. Fishel, F. 2006. Pesticide registration and actions. Chemically Speaking. http://pested.ifas.ufl.edu/newsletters/april2006/pestreg.html Accessed: March 15, 2007. 110

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FMC. 2004. Stingray Aquatic Herbicide. Philadelphia, PA: FMC Corporation, Agricultural Products Group. FMC. 2005. FMC receives federal approval for new herbicide stingray. http://pestsolutions.fmc.com/Home/NewsEvents/tabid/1185/Default.aspx?itemId=440 FMC Corporation. Philadelphia, PA. Accessed April 9, 2007. Frankart, C., P. Eullaffroy, and G. Vernet. 2002. Photosynthetic responses of Lemna minor exposed to xenobiotics, copper, and their combinations. Ecotoxicol. Environ. Saf. 40:194-200. Gensemer, R. W., D. G. Dixon, and B. M. Greenberg. 1999. Using chlorophyll a fluorescence to detect the onset of anthracene photoinduced toxicity in Lemna gibba, and the mitigating effects of a commercial humic acid. Limnol. Oceanogr. 44:878. Geoffroy, L., C. Frankart, and P. Eullaffroy. 2004. Comparison of different physiological parameter responses in Lemna minor and Scenedesmus obliquus exposed to herbicide flumioxazin. Environ. Pollut. 131: 233-241. Getsinger, K. D., A. M. Fox, and W. T. Haller. 1990. Understanding water exchange characteristics to improve the control of submersed plants. Aquatic Plant Control Research Program. Vol. A-90-2. Getsinger, K. D., J. D. Madsen, E. G. Turner, and M. D. Netherland. 1997. Restoring native vegetation in a Eurasian watermilfoil-dominated plant community using the herbicide triclopyr. Regul. Rivers Res. and Manage. 13:357-375. Gray, C. J., J. D. Madsen, R. M. Wersal, and K. D. Getsinger. 2007. Eurasian watermilfoil and parrotfeather control using carfentrazone-ethyl. J. Aquat. Plant Manage. 45:43-46. Grichar, J. W. and A. E. Colburn. 1996, Flumioxazin for weed control in Texas peanut (Arachis hypogaea L): Peanut Sci. 23:30. Grumbach, K. H., H. V. Lichtenthaler, and K. H. Erismann. 1978. Incorporation of l4CO 2 in photosynthetic pigments of Chlorella pyrenoidosa. Planta. 140:37-43. Gupta, I. and B. C. Tripathy. 2000. Oxidative stress in cucumber (Cucumis sativus L.) seedlings treated with acifluorfen. Indian J. Biochem. Biophys. 37:498-505. Hager, A. G., L. M. Wax, G. A. Bollero, and E. W. Stoller. 2003. Influence of diphenylether herbicide application rate and timing on common waterhemp (Amaranthus rudis) control in soybean (Glycine max). Weed Technol. 17:14-20. Haller, W. T. 1982. Hydrilla goes to Washington. Aquatics. 4(4):6-7. 111

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BIOGRAPHICAL SKETCH Christopher Ray Mudge, the son of Alvin and Wanda Mudge, was born in Alexandria, LA and was raised in the rural rice and soybean farming community of Branch, LA. Upon graduation from Iota High School in 1997, he enrolled at Louisiana State University A&M in Baton Rouge in the fall of 1997. While attending LSU, he was active in the agronomy and collegiate 4-H clubs. After earning a B.S. in agronomy (crop management) in December 2001, he began his graduate career under the direction of Dr. Eric P. Webster, working in rice weed management. While working on his masters degree, Chris was an active participant in the Southern Weed Contest. He graduated from LSU in the spring of 2004 with a M.S. in agronomy and the title of his thesis was Water-seeded Rice Response to Clomazone. Chris moved to Gainesville, FL in 2004 to work as a biological scientist for the University of Florida. In 2005, he enrolled at the University of Florida to study for a PhD in aquatic weed science under the direction of Dr. William T. Haller. During his graduate career, he presented many talks and posters at the Southern Weed Science, Aquatic Plant Management Society, Florida Weed Science, and Florida Aquatic Plant Management Society annual meetings. In 2005, Chris married the former Miss Erin Gravois of Vacherie, LA. Upon graduation, he will work in the field of aquatic weed management and continue his involvement in weed science and aquatic societies. 120